Introduction
Polycystic ovary syndrome (PCOS) is a common and intricate endocrine disorder and metabolic condition, making it one of the most prevalent endocrine disorders among women of reproductive age. It affects approximately 8–13% of this demographic worldwide [
1]. Current treatment strategies for PCOS patients are often unsatisfactory and this syndrome accounts for primary infertility in women [
2]. Typical symptoms that concern patients with PCOS include hirsutism, irregular and chronic ovulation, acne, oily skin, oligomenorrhea, alopecia, insulin resistance, dyslipidemia and hyperandrogenism [
3]. Irregular and chronic ovulation is a critical reason for infertility in patients with PCOS. Even though the pathogenesis and etiology of PCOS are not yet fully understood [
4], studies suggest that its pathogenesis is influenced by various mechanisms including, genetic factors, oxidative stress, inflammation, lipid imbalance, and insulin resistance (IR) [
5].
PCOS, one of the considerable diseases that restrict reproductive outcome, has been vigorously characterized by increased inflammation, oxidative stress, mitochondrial dysfunction, detrimental modification in cell signaling pathways (such as phosphoinositide-3-kinase-protein kinase B/AKT or PI3K-AKT), hyperinsulinemia and infertility [
6]. The women with PCOS often have complication in metabolism of androgen and estrogen. This results in enhanced concentration of androgens (hyperandrogenism) and that means high levels of androgens account for ovulation disruption and effects of quality and fertility of oocyte in women with PCOS [
7]. The PI3K/AKT signaling pathway is a critical component of insulin signaling, contributing significantly to not only insulin resistance but also playing a crucial role in various processes such as cell proliferation, growth, migration, invasion, inhibition of apoptosis, and angiogenesis [
8,
9]. Modulating PI3K-AKT signaling pathway may cause significant changes in granulosa cell proliferation, follicular development and oocyte fertility [
10]. Recent research indicates that inhibiting the PI3K-AKT pathway may impact insulin functionality and glucose metabolism [
11]. Consequently, the PI3K-AKT signaling pathway has essential role in the pathogenesis PCOS disorders [
12].
Mitochondria is a dynamic organelle associated with a delicate balance between fission and fusion [
13]. Disturbance in mitochondrial dynamics, genes expression, enzymes and mitochondrial membrane potential (MMP) can lead to inordinate between fusion and fission, and occurrence various disorders such as neurodegenerative, cardiovascular, renal and reproductive diseases [
14]. There is growing evidence showing that dysregulation of mitochondrial dynamic function is an essential cause of PCOS development and decreased fertility in women [
15]. It has been reported that tissues exhibiting IR are affected by a decline mitochondrial content, gene expression and enzyme activity. In this context, hyperandrogenism, insulin resistance, oxidative stress, and glucose intolerance are exerted by alteration in mitochondrial function and biogenesis [
16]. Oxidative stress, established by an imbalance between antioxidant and pro-oxidant factors, can cause abnormal granulosa cells function and negative fertility outcome [
16].
Nowadays, the therapeutic options for PCOS patients are limited. The guidelines of PCOS by the World Health Organization (WHO) recommend lifestyle changes, including dietary modification, exercise and use of drugs such as metformin to alleviate hyperandrogenism and metabolic symptoms [
17]. Using novel techniques, such as gene or cell therapy, may present an achievable way to treat PCOS.
Over the last decade, stem cell-based treatments have garnered considerable interest as a potential new therapeutical strategy for different endocrine disorders, such as PCOS [
18‐
20]. Among the stem cells used, mesenchymal stem cells have garnered growing attention due to their potential therapeutic applications in various disorders of the female reproductive system. MSCs have received notable interest in research investigations due to their specific biological function, ease of isolation, anti-inflammatory, anti-apoptotic, antioxidant, and immunomodulatory effects, making them good candidate for cell therapy [
21,
22]. The positive effects of MSCs are attributed to their ability to differentiate into epithelial, endothelial and stromal cells, regulate immune responses, and modulate paracrine signaling pathways [
23]. Interestingly, recent findings have revealed that MSCs restore injured tissues and cells by delivering their mitochondria [
24]. Xie et al., demonstrate that MSCs by activating PI3K-AKT pathway significantly can improve adrenal cells proliferation and differentiation [
25]. The survival of follicles in the ovary related to balanced PI3K-AKT pathway [
26]. MSCs can improve ovarian function by activating the PI3K-AKT pathway via a paracrine mechanism [
27]. In a study conducted by Ataf et al., only fission and fusion parameters were evaluated, and mitochondrial membrane potential and biogenesis were not assessed [
28]. Safai's et al., research evaluated the effect of vitamin D only on mitochondrial biogenesis within a model of polycystic ovary syndrome induced by DHEA, without assessing the complexities of mitochondrial dynamics [
29]. In another study, only mitochondrial dynamics were investigated in the polycystic ovary syndrome rat model [
30]. Xie et al. demonstrated that transplantation of mesenchymal stem cells could enhance ovarian function by decreasing systemic and local inflammation. However, their study did not assess the effect of mesenchymal stem cells on the mitochondrial function of ovarian tissue [
31]. The novelty aspect of our study is evaluating effects of MSCs transplantation on mitochondrial dynamics, biogenesis, and function in ovaries via the PI3K-AKT pathway in letrozole-induced polycystic ovary syndrome. In this current investigation, we assessed the effects of MSCs transplantation on mitochondrial dynamic, biogenesis, oxidative stress, inflammation, IR, hormonal assay and histopathological change of ovarian tissue via PI3K-AKT pathway in letrozole induced PCOS rats. We hypothesized that PCOS induction impairs mitochondria dynamics in the ovary, and transplanted MSCs are capable of attenuating the complication of this syndrome and rescue ovarian cells by restore mitochondria dynamic and biogenesis, mainly through the PI3K-AKT pathway.
Discussion
Based on recent studies, a defect in mitochondrial dynamic (by fission and fusion) and biogenesis could lead to consequences such as insulin resistance, hyperandrogenism, oxidative stress and glucose intolerance which are considered key features in PCOS [
29] as well as female infertility [
45]. Restoring mitochondrial dynamics and promoting mitochondrial biogenesis by mitigating oxidative stress could lead to ameliorating polycystic ovary syndrome [
46] and are crucial for maintaining healthy follicular development [
47]. Mitochondrial fission is regulated by expression levels of Drp1 gene, while mitochondrial fusion is mediated by expression levels of MFN2 genes. Mitochondrial biogenesis is modulated by the expression levels of genes such as PGC-1α, NRF1 and TFAM [
48]. The study conducted by XIE et al., demonstrated that expression level of mentioned genes altered within the ovaries of patients with PCOS compared to the control group. Furthermore, this research has suggested that ameliorating ovarian mitochondrial function (including dynamic and biogenesis) could improve the quality of oocyte and follicular development in patients with PCOS. Therefore, mitochondrial fission, fusion and biogenesis genes could be key players in clinical outcome of PCOS patients [
49]. Our findings, as demonstrated by JC-1 staining, revealed a reduction in mitochondrial membrane potential (MMP) in PCOS rats. This reduction in MMP can lead to substantially decreased efficiency of oxidative phosphorylation and respiratory chain-mediated proton pumping from the matrix, which can account for reactive oxygen species (ROS) production and various mutants in mitochondrial genes and malfunction, as well as damage to oocyte development potential [
50]. MSCs administration restored mitochondrial dynamic balance, increased CS activity and MMP. MSCs, with their specific features, could rescue damaged ovarian tissue and cells, possibly by transferring their own mitochondria through membrane thin channels [
51]. Mitochondrial transfer restores oxidative phosphorylation, dynamic balance, MMP and CS activity. One study demonstrated that a calcium-binding mitochondria Rho GTPase is a key mediator of MSC-derived organelle that enables the movement of mitochondria along microtubules [
52]. Furthermore, the transplantation of MSCs has the potential to regulate the balance of mitochondrial dynamics through posttranslational alterations to Drp1 and enhance mitochondrial fission.
PCOS often appears with insulin resistance (IR) and hyperandrogenism [
53]. IR can cause overproduction of androgen levels, which increases the risk of cardiovascular diseases, tumors and metabolic disorders [
54]. In our study the concentration of testosterone, insulin levels and serum glucose levels were elevated in PCOS rats. PCOS induction led to ovarian hyperandrogenism and significantly increased basal insulin secretion. The occurrence of IR and hyperandrogenism is crucial for mitochondrial malfunctions to impair follicular development and progression of PCOS [
55]. Mitochondria plays a key role in normal insulin function, and conversely, insulin secretion is an important mediator of oxidative phosphorylation and mitochondrial function [
56]. In addition to the higher serum glucose, elevated HOMA-IR and lower ISI in our results, mitochondrial dysfunction led to IR, excessive androgens and oxidative stress. The decrease in HOMA-IR, insulin level and glucose concentration, as well as an increase in ISI, were observed with the MSCs transplantation via their antioxidant effects. This positive effect may rescue the function of mitochondria, leading to improvements in these parameters. In the present study, the induction of PCOS and inhibition of the PI3K-AKT pathway caused a reduction of estradiol and an enhancement of testosterone concentration. MSCs treatment significantly increased estradiol and decreased testosterone concentration in the PCOS + MSCs group. Several studies, including Park et al., have demonstrated that MSCs transplantation could decrease androgen synthesis pathways through paracrine secretion via exosomes [
57].
In our study, MSCs transplantation restore the oxidant anti-oxidant balance. The induction of PCOS caused significant elevation in MDA levels, which can affect oocyte differentiation. Elevated MDA levels may cause a decrease in ISI and an increase in HOMA-IR, as well as decreased glucose uptake in insulin-sensitive cells and increased blood glucose concentration [
56]. One study reported that MSCs, with their secretion of HSPs and regulation of free calcium and ATP in mitochondria, can rescue oxidative stress conditions. Therefore, HSPs secreted from MSCs could lead to phosphorylation of intracellular tyrosine kinase and activation of PI3K-AKT pathway to restore oxidative balance [
58]. The pathogenesis of PCOS is significantly influenced by the inflammatory response, which can cause metabolic irregularities, deteriorate oxidative stress status and ovarian dysfunction in PCOS patients [
59]. It has been demonstrated in recent studies that PCOS patients have elevated levels of inflammatory markers, such as TNF-α and IL-6 [
31,
60]. Consistent with previous studies on this case, we demonstrated that induction of PCOS could increase inflammatory markers such as TNF-α and IL-6 in ovarian tissue. In this study, we demonstrated that administration of MSCs could regulate inflammation by decreasing the levels of two pro-inflammatory factors, including TNF-α and IL-6. The current findings suggested that MSCs have anti-inflammatory effect, capable of converting a pro-inflammatory to an anti-inflammatory state and reduced the pathological changes in local ovarian tissue of PCOS rats.
Letrozole is a non-steroidal aromatase inhibitor that blocks the conversion of testosterone to estradiol, leading to increased levels of androgens in the blood and ovaries [
61]. The PCOS model induced by letrozole exhibited various facets of PCOS, including reproductive and metabolic features, making it a valuable model for PCOS studies [
62]. The Rotterdam Criteria define PCOS by the presence of at least two of these three conditions: 1- elevated androgen levels, either clinically or biochemically, 2- oligo- or amenorrhea, and 3- polycystic ovaries [
63]. Considering the fact that we observed a significant increase in serum testosterone levels and an increase in the number of cystic follicles in the PCOS group compared to the sham rats, PCOS model was certainly established. Zhang et al., have shown that overproduction of ROS (reactive oxygen species) in mitochondria can lead to an increase in atresia of antral follicles, corpus luteum, preovulatory and graafian follicles. Additionally, increased levels of ROS have been shown to cause ovarian cell mitophagy and destruction [
56]. In present study, our results showed an elevation in pre-antral and cystic follicle, while there was a reduction in antral, preovulatory and graafian follicles, which may be related to mitochondrial dysfunction. It seems that MSCs restore follicular development by inhibiting oxidative stress and improving mitochondrial function. There is little controversy about the differentiation of mesenchymal stem cells, and the mentioned feature in the introduction is one of the general effects of mesenchymal stem cells [
64]. Recent research indicated that the positive effects on tissue repair following mesenchymal stem cell transplantation resulted from releasing soluble factors through the paracrine mechanism [
65]. In our recent studies, MSCs transplantation via intraperitoneal injection have shown that MSCs may have beneficial therapeutic effects in septic shock and cerebral ischemia reperfusion, primarily through paracrine pathways [
20,
66,
67]. Several studies, using animal model and transitional research, have demonstrated that MSCs could home to sites of injury, such as the ovary in PCOS. Furthermore, studies have shown that the positive effects of MSCs on ovarian function are mainly exerted via the paracrine pathway [
68,
69].
Recent studies have widely reported that the PI3K/AKT signaling pathway is dysregulated in both patients with PCOS and animal models of PCOS, additionally the critical role of the PI3K-AKT signaling pathway in regulating of the growth, development, differentiation, and survival of ovarian follicles were documented [
12,
70]. Therefore, maintaining optimal PI3K-AKT signaling pathway activity appears to be essential for the normal development and physiological functions of the ovary, as a result, disruption of this pathway plays an important role in the pathogenesis of PCOS [
70]. Dysfunctional PI3K-AKT pathway has been associated with insulin resistance, anovulation, decreased granulosa cell proliferation, reduced number of mature follicles, increased atresia follicles, and ultimately results in decreased fertility [
71]. Previous studies have recommended further research on the PI3K-AKT pathway and its inhibitor (LY294002), that not only enhance understanding of the pathogenesis of PCOS but also offer a novel approach so as to treat PCOS [
72]. In the present manuscript, the administration of LY294002 as a PI3K-AKT pathway inhibitor could exacerbate the pathogenesis of PCOS by diminishing the survival and differentiation of ovarian tissue. Our study exhibited that activation of the PI3K-AKT signaling pathway is primary pathway for preserving follicles survival and determining female reproductive life span. Our results revealed the protein expression of pAKt and pPI3K was decreased in PCOS rats. Several studies have confirmed PI3K-AKT signaling pathway key role in insulin resistance and have shown MSCs are involved in the activation of this intracellular signaling pathway [
6,
27]. It was also reported that ovarian cells proliferation and apoptosis are regulated by PI3K-AKT pathway [
27]. In our study, MSCs transplantation led to an increase in protein expression of pAKT and pPI3K. The ability of MSCs transplantation to enhance the activation of the PI3K-AKT signaling pathway could improve the pathogenesis of PCOS and insulin resistance. Additionally, to support the role of PI3K-AKT pathway in MSCs function, a PI3K-AKT inhibitor (LY294002) was applied. Interestingly, in the PCOS + MSCs + LY294002 group, all parameters deteriorated compared to the PCOS + MSCs rats, indicating that inhibition of the PI3K-AKT signaling pathway completely blocked the beneficial effects of MSCs on PCOS ovaries. These findings demonstrate the effectiveness of MSCs in ameliorating PCOS ovaries is mainly attributed to the PI3K-AKT pathway and paracrine mechanism. Notably, the PI3K/AKT signaling pathways are recognized for their pivotal role in regulating multiple cellular behavior of MSCs, such as survival, proliferation, growth and mobilization [
73,
74]. Huang et al., demonstrated that inhibition of PI3K-AKT pathway with LY294002 could disrupt intracellular homeostasis by inducing mitochondrial dysfunction [
73]. Recent studies indicate that disruption of the PI3K-AKT pathway can lead to hypoxia and apoptosis in MSCs [
75]. Inhibiting this pathway with LY294002 could potentially reverse the protective effects of MSCs and affect the transfer and restoration of mitochondria [
76]. Therefore, optimal activity of the PI3K-AKT signaling pathway is essential for better outcomes in stem cell therapy [
77]. By regulating the status signaling using LY294002, we exhibited that inhibiting the PI3K-Akt signaling pathway reduced the expression of mitochondrial dynamic genes, MMP and CS activity. Thus, we suggest this pathway may be related to mitochondrial dysfunction and the pathogenesis of PCOS. In accordance with previous studies, in our study there was no significant difference in all parameters between the Sham and Sham + LY294002 groups [
78]. In this study, no notable difference was observed between the PCOS + MSCs + LY294002 and PCOS + LY294002 groups. Recent studies have shown that the LY294002 could affect the activity and survival of MSCs. It effectively prevented the impact of MSCs on ovaries [
79]. The administration of LY294002 leads to increased ROS production [
80], raised apoptosis [
81,
82], reduced proliferation [
83], decreased the gene expression of cyclin D1 and E1 and finally diminished the activity of MSCs [
84]. One of the limitations of the present study was the lack of localization of MSCs in ovarian tissue. It is also recommended to investigate the effect of MSCs on apoptosis and autophagy from the PI3K-AKT pathway in future studies.
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