New Insights into Mechanisms of Berberine in Alleviating Reproductive Disorders of Polycystic Ovary Syndrome: Anti-in�ammatory Properties

Polycystic ovary syndrome (PCOS) is a complex reproductive disorder which seriously harms female reproductive health and decreases their quality of life. Although spontaneous or assisted ovulation, women with PCOS suffer from poor-quality oocytes and embryos, lower fertilization and �nal pregnancy rate. Therefore, it is urgent to reveal new pathological mechanisms and discover the underlying therapeutic targets for the reproductive disorders in PCOS. Berberine, one of the famous traditional Chinese medicines, has been shown to improve ovulation and live birth rates in women with PCOS. The effects of berberine on insulin resistance and abnormal glucose and lipid metabolism for restoring reproductive health of PCOS are well recognized and widely studied, but much less attention has been paid to its anti-in�ammatory properties. Chronic low-grade in�ammation as the unifying feature of PCOS may contribute to reproductive disorders in PCOS. Berberine can tune the in�ammatory state of ovaries and uterus in PCOS. The anti-in�ammatory properties of berberine may provide new insight into mechanisms of berberine in alleviating reproductive disorders of PCOS. Here, we summarize the most recent insights into anti-in�ammatory properties of berberine in reproductive disorders of PCOS, inspiring researchers to go in new study directions of berberine.


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Background Polycystic ovary syndrome (PCOS) is a complex reproductive disorder affecting 5-10% of women with childbearing age [1,2].
Although spontaneous or assisted ovulation, there are still some questions regarding the paucity of high-quality oocyte, low fertilization and nal pregnancy rate in women with PCOS, which seriously harms women's reproductive health and affects their quality of life [3,4]. Therefore, revealing the new pathological mechanism of reproduction in women with PCOS and discovering new therapeutic targets are very urgent for the treatment of reproductive disorder in PCOS.
In ammation is an innate defense responses that can cause activated immune cells to release various pro-in ammatory cytokines which are involved in stimulating chemokines and cytokines, as well as regulating gene expression in cell meiosis and apoptosis to devour damaged tissue and induce remodeling of the tissue [5,6]. In ammation is related to multiple aspects of female reproductive physiology, such as oocyte maturation and embryo implantation [7][8][9]. However, in terms of reproduction related diseases, in ammatory response subject to underlying pathology may be pushed to present a negative impact in female reproduction [10][11][12]. Chronic low-grade in ammation has been proposed as the unifying feature in PCOS, although the phenotype of PCOS is diverse [13]. Chronic low-grade in ammation may contribute, at least in part, to reproductive disorders in PCOS. Therefore, anti-in ammatory drugs may provide potential strategies for treating impaired oocyte quality and early pregnancy loss in PCOS.
Berberine has been used as an anti-in ammatory drug in the clinical treatment of many diseases, such as gastroenteritis [1,14]. It is a bioactive alkaloid isolated from various medicinal herbs such as Coptis chinensis Franch. and Hydrastis canadensis L. [15,16]. The molecular formula of berberine is C 20 H 18 NO 4 with a molar mass of 336.36 g/mol and four major metabolites have been identi ed, including berberrubine (M1), thalifendine (M2), demethylene-berberine (M3) and jatrorrhizine (M4) [17]. Berberine has also been empirically used in PCOS treatment alone or combined with other drugs improving reproductive disorders. However, only a few studies have preliminarily explored the mechanisms of berberine in restoring reproductive health of PCOS. More importantly, the anti-in ammatory properties of berberine are commonly ignored, though are possibly signi cant [18][19][20]. This paper summarizes the most recent insights of relationship among berberine, chronic low-grade in ammation and reproductive disorders in PCOS, and discusses the anti-in ammatory properties of berberine on reproductive disorders of PCOS. Furthermore, discussing suppression of chronic low-grade in ammation using berberine may provide new potential therapeutic approaches for reproductive disorders.

E cacy And Safety Of Berberine For Reproductive Disorders Of PCOS
Basic scienti c researches and clinical trials had clearly established the therapeutic bene t of berberine on reproductive disorders in PCOS but with different treatment regimens. Wang, et al. administered berberine 100 mg/kg•d or 200 mg/kg•d for 8 weeks. The ovulation and endometrial receptivity in rat model of PCOS were ameliorated after administration by regulating LHCGR, CYP19A1, LPAR3 and αvβ3 [19]. Besides, Li et al. administered berberine 400 mg three times daily for 16 weeks. After treatment, 51 of 98 PCOS women had at least 1 ovulatory cycle [21]. An et al. administered berberine 500 mg three times daily for 12 weeks.
Compared with placebo, the biochemical pregnancy rate (54.1%), clinical pregnancy rates (59.5%) and live birth rates (48.6%) were signi cantly higher in PCOS women who received berberine. However, there were no obvious differences in the mean number of oocytes collected, diploid fertilization rate and embryo utilization rate between placebo and berberine group [22]. Wu et al. administered berberine in a daily dose of 1500 mg for 24 weeks. There were 36.3% ovulation, 28.5% conception, 22.4% clinical pregnancy and 22.0% live birth rates in PCOS women who received berberine. Besides, the ovulation (61.0%), conception (48.8%), clinical pregnancy (37.7%) and live birth (34.4%) rates in PCOS women who received berberine combined with letrozole were higher than berberine group but similar to letrozole group [23] (Table 1). Berberine did not add fecundity in reproductive disorders with PCOS when used in combination with letrozole. Nevertheless, this nding suggests new directions for future research concerning the combination of berberine with other agents to support the potential applicability of berberine in restoring reproductive health of PCOS. Furthermore, traditional Chinese medicines as complementary and alternative medicines have gradually gained researchers' attention due to their powerful therapeutic effects and minimal or no side effects. Berberine as one of the famous traditional Chinese medicines with standard dose has been shown to be well tolerated and no apparent side effects, with the exception of a weak side effect of on the gastrointestinal system [15,17,24]. However, despite the side effect of berberine in the female reproductive system have yet to be discovered, further assess the safety of berberine is still necessary.
Berberine treatment signi cantly affected oocyte maturation and subsequent development in a dose-dependent manner. In vitro studies revealed that pretreatment of mouse oocytes preincubated with 5 or 10 µM of berberine for 24 h exerted an increase in apoptosis compared with the control group, whereas oocytes with 2.5 µM berberine showed no signi cant differences.
Preincubation of oocytes with 5 or 10 µM of berberine negatively impacted mouse oocyte development, fertilization, embryonic development and even implantation. In contrast, preincubation with 2.5 µM berberine caused signi cantly enhanced oocyte development, embryonic development, and no deleterious effects on oocyte fertilization and implantation [25]. Similar dosedependent effects of berberine were also demonstrated in mouse blastocysts [26]. Moreover, intravenous injection of berberine via the tail vein with 3 or 5 mg/kg exerted a signi cant decrease in oocyte development and caused harmful effects on fertilization and early embryonic development [25]. Instead, there was a signi cant positive impact in oocyte development and embryos that developed to the blastocyst stages with intravenous injection of berberine at a dose of 1 mg/kg. Dual effects of berberine appeared to be attributable to promotion or inhibition of reactive oxygen species induced apoptosis, which dependent on the dose of berberine [25]. The safety of berberine on female reproductive system is particularly important ( Table 2). Notably, bioavailability of berberine may be affected through co-administration with other drugs because of potential interactions among drugs. Clinical dose adjustment based on drug monitoring is recommended. Further evaluation to con rm the safety and e cacy of berberine for PCOS is warranted.  The anti-in ammatory properties of berberine on reproductive disorders of PCOS In ammation is related to multiple aspects of female reproductive physiology. However, in terms of reproduction related diseases, in ammatory response subject to underlying pathology may be pushed to present a negative impact in female reproduction, such as the paucity of high-quality oocyte, low fertilization and nal pregnancy rate. Chronic low-grade in ammation as the unifying feature of PCOS may contribute, at least in part, to reproductive disorders in PCOS. Anti-in ammatory strategies may become a priority for the prevention and treatment of reproductive disorders in PCOS. However, most of studies exploring the mechanisms of berberine in restoring reproductive health of PCOS focus on its effects on insulin resistance and abnormal glucose and lipid metabolism, and less attention has been paid to its anti-in ammatory properties [19,[27][28][29].
In ammatory Response Involved In Female Reproductive Physiology In female reproductive system, reproductive function such as oocyte quality and implantation are thought to be attributable, at least in part, to an in ammatory response, which includes direct and indirect actions to cause vasodilation, hyperemia, edema, collagenolysis and cell proliferation [7][8][9][10][11]. Follicle as the fundamental unit of the ovary has key physiologic roles in female reproductive function. The development of follicles depends heavily on the microenvironment constituted by follicular uid, which is an exudate from blood and exhibits positive correlations with a series of different serum in ammatory factors.
Proin ammatory cytokines are also produced throughout folliculogenesis [30][31][32]. The quality of oocytes is important for embryonic development. In mammals, the developing oocyte undergoes germinal vesicle breakdown (GVBD), spindle assembly, and polar body extrusion. Each month, only one dominant oocyte undergo directly to meiosis resumption. During meiosis, microtubules are organized into barrel-shaped bipolar spindles, with chromatin condensed and all chromosomes arranged neatly. inhibitory antioxidants make this process possible [33,34]. During ovulation, in ammation stimulate the follicle wall weakening and connective tissue remodeling, which induce normal ovarian tissue rupture and the release of mature oocytes [35,36]. Then, oocytes arrest at metaphase II (MII) waiting for fertilization, which was promoted by antioxidants [34]. Any error in this process will affect female fertility, which is the main cause of poor oocyte and embryo qualities, even abortion. It has been reported that over 75% of embryonic loss is due to abnormal chromosomal separation during oocyte maturation [37][38][39][40]. Embryo implantation for healthy female reproduction can only occur during the mid-secretory phase of the menstrual cycle which is commonly referred to as the window of implantation (WOI). During this period, a fully prepared and responsive uterus occurs. Fibroblast-like endometrial stromal cells proliferate and differentiate into larger and rounded decidua cells. Large apical protrusions and microvilli emerge on the luminal epithelium [41,42]. In parallel, the gradient of chemokines and cytokines produced by endometrial cells guides the blastocyst to the endometrial surface epithelium, allowing its invasion break through the epithelial and stromal cells [43]. After that, the 'wound healing-like' process is triggered to repaired and remodeled endometrial tissue. Proin ammatory effects mediated by Th1 cytokines such as IL-6 and TNF-α recruit immune cells to the decidua [44]. A large number of decidual leukocytes also in ltrated the implantation site, such as uterine-speci c natural killer (uNK) cells, macrophages and dendritic cells [45]. Moreover, maternal tolerance to fetal alloantigens is established and maintained by the delicate balance of Th1/Th2 immune response, which is an important factor for implantation and maintenance of pregnancy [46]. Once the balance is broken, the defense mechanisms of immune system protecting against invading organisms will be activated, leading to implantation failure and pregnancy loss.

Impact of in ammation in reproductive disorders of PCOS
The in ammatory response engaged in repetitive cyclic changes in the ovaries and uterus are notably disrupted in PCOS (Table  3). Increasing evidence demonstrates that women with PCOS, even if matched for body mass index (BMI), showed signi cantly higher levels of C-reactive protein (CRP), interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), interleukin-17 (IL-17), interleukin-18 (IL-18), monocyte chemotactic protein-1 as well as soluble endothelial leukocyte adhesion molecule and soluble intercellular adhesion molecule than normal women [47,48]. This series of in ammatory factors coupled with the changing follicular microenvironment can directly affect oocyte and embryo development, ovulation, and fertilization. IL-6 as a pro-in ammatory cytokine can directly affect the chromatin structure and spindle morphology in oocytes [49][50][51]. TNF-α can control ovarian repair and remodeling by affecting apoptosis and autophagy of unruptured follicles. This pro-in ammatory cytokine can also stimulate hyperplasia of follicles and affect steroidogenesis and luteolysis of thecal and granulosa cells [52][53][54]. CRP is a marker of systemic in ammation in response to TNF-α and IL-6 produced by hepatocytes. Systemic CRP levels uctuate in follicular dynamics. IL-1 induced production of prostaglandins in the dominant follicle can mimic the effect of the LH surge and trigger ovulation. However, IL-1 can inhibit plasminogen activators, which in turn interfere with normal ovulation. Despite not required for successful ovulation, IL-1 has negative effects on oocyte maturation and the number of fertilized oocytes [55]. Of note, there is a close connection between in ammation and oxidative stress (OS of granulosa cells and reduce nutrient supply to oocytes, and eventually in uencing oocyte quality and maturation [37]. Considerable evidence indicates in the ovulation process, adequate amounts of ROS regulate the normal physiologic process, but an increase in ROS production alters the physiologic ovarian dynamics, and adversely affects reproductive functions [10]. The mitochondrial caspase pathway induced by OS can also lead to the deterioration of oocyte quality after ovulation [67]. Therefore, we argue that aberrant concentration of in ammatory factors in PCOS may result in predisposition to poor oocyte and embryo qualities, ovulatory dysfunction and fertilization failure. IL-1 Elevated l Affect oocyte maturation and the number of fertilized oocytes l Increase postovulatory progesterone production which is pivotal for maintenance of the pregnancy [55] ROS Elevated l Affect the recovery of meiotic division in diploid terminated oocyte l Attack microtubules and interfere with spindle formation, eventually leading to abnormal chromosome arrangement which increase the risk for oocyte aneuploidy l Contribute to telomere shortening and chromosome segregation disorder, eventually leading to oocyte fragmentation and fertilization failure l Mediate apoptosis of granulosa cells and reduce nutrient supply to oocytes, and eventually in uencing oocyte quality and maturation l Lead to the deterioration of oocyte quality after ovulation l Lead to impaired implantation and loss of embryos Growing evidence suggests that an in ammatory milieu in PCOS endometrium with concomitant changes in immune cell chemoattraction, pro-in ammatory cytokine and matrix metalloproteinase (MMP) release may contribute to embryo implantation failure and pregnancy loss [69 -72]. As a multifunctional cytokine, IL-6 expressed in endometrial stromal cells and decidua play an important role in coordinating trophoblast invasion and communication between endometrial stromal broblasts (eSF) and endometrial epithelium or leukocytes. However, when aberrant production of IL-6 occurs, paracrine signaling throughout the endometrium is altered, which in turn interrupt the signal of lumen epithelium and affect the crosstalk of maternal fetal interface [71,73,74]. TNF-α synthesized by decidual and trophoblast cells also plays an important role in embryo implantation and pregnancy maintenance [75,76]. TNF-α can signi cantly inhibit trophoblast cell motility, but not alter trophoblast cell adhesion [77]. Moreover, aberrant production of TNF-α can prevent the endometrial epithelial cells from adhering to mesothelial cells, which in turn impair the expression of adhesion molecule complex cadherin/ beta-catenin, and ultimately cause the dyscohesion of the endometrial epithelial cells [78,79]. IL-15 and IL-18 can induce uNK cells activation during implantation [80]. IL-1 is pivotal for maintenance of the pregnancy as it is thought to increase the production of postovulatory progesterone [55]. Furthermore, OS can contribute to changes in local immune function in the uterus, leading to impaired implantation and loss of embryos [81, 82].
Therefore, we argue that implantation failure and early pregnancy loss in PCOS are partly attributable to an aberrant in ammatory milieu in PCOS endometrium.
The anti-in ammatory properties of berberine for the in ammatory state of PCOS The evidence obtained thus far indicates that anti-in ammatory strategies can be a valuable choice for the prevention and therapy of reproductive disorders in PCOS. Berberine with anti-in ammatory properties can reduce the expression of IL-6, TNF-α and other in ammatory factors, so as to tune the in ammatory state of ovaries and uterus in PCOS [29,83,84]. Antiin ammatory mechanisms of berberine through several cell kinases and signal transduction pathways such as PI3K/AKt and AMPK pathway have also been described [20,27]. Berberine can inhibit activation of NF-κB signaling pathways and its subsequent upregulation of the in ammatory response in PCOS by regulating the expression of TLR4 and LYN [29] (Table 4).
Moreover, berberine can regulate the generation of ROS and the exhaustion of antioxidant system associated with in ammation [27]. Recent studies have found that elevated AMPK mRNA level and protein amounts as well as phosphorylated FOXO3a protein levels were observed paralleling increased expression of antioxidant enzymes (SOD, CAT and GSR) in berberine treated KGN  . SIRT3 is a soluble protein deacetylase located in the mitochondrial matrix [90]. As a key molecule regulating autophagy, FOXO3a is not only regulated by phosphorylation but also by deacetylation [91]. SIRT3 deacetylated FOXO3a and phosphorylated AMPK, thus up-regulating antioxidant enzymes in response to oxidative stress and regulating autophagy [85,92] (Table 4). [93] Berberine used as an anti-in ammatory drug may provide potential strategies for reproductive disorders in PCOS (Fig. 1). Importantly, several studies have already directly or indirectly demonstrated this conjecture. Miao

Conclusions
Chronic low-grade in ammation may contribute to reproductive disorders in PCOS. As an anti-in ammatory strategy, berberine can be valuable for restoring reproductive health of PCOS. Moreover, PCOS is a chaotic disease with multiple factors and various clinical manifestations, multi-target drug or multi-drug combination is also needed. Berberine as an alternative and complementary strategy with anti-in ammatory properties may provide strong future insights regarding controlling reproductive disorders in PCOS. We hope to present the impact of in ammation on reproductive disorders in PCOS and the bene cial effects Possible impact of berberine on in ammation-mediated reproductive disorders in PCOS The perpetuating cycle between in ammation and OS can affect oocyte and embryo development, ovulation, fertilization and implantation in PCOS. Berberine can inhibit activation of NF-κB signaling pathways and its subsequent upregulation of the in ammatory response. Berberine may upregulate antioxidant enzymes in response to OS through the AMPK/FOXO3a pathway. Sirt3 deacetylated Foxo3a and phosphorylated AMPK, thus up-regulating antioxidant enzymes in response to OS and regulating autophagy. Berberine can decrease SIRT3 protein amounts and enhance SIRT3 protein ubiquitination-degradation. NF-κB, nuclear factor-κB; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; IL-6, interleukin 6; AMPK, adenosine monophosphate activated protein kinase; mTOR, mammalian target of rapamycin; SIRT3, sirtuin 3; FOXO3a, forkhead box O3a; OS, oxidative stress