Mouse Model Of Polycystic Ovary Syndrome Research Articles

Beneficial effects of minocycline on the ovary of polycystic ovary syndrome mouse model: Molecular docking analysis and evaluation of TNF-α, TNFR2, TLR-4 gene expression

Polycystic ovary syndrome (PCOS) is the most common cause of ovulatory infertility. Inflammation may be involved in the pathogenesis and development of PCOS. We investigated the anti-inflammatory effect of minocycline on TNF-α, TNFR2, and TLR4 expression levels and the key features of PCOS in a mouse model. Molecular docking was performed by Molecular Operating Environment software. PCOS was induced by estradiol valerate injection (EV) (2 mg/kg/day) in 40 mice. After 28 days, the mice were divided into five groups, including control, PCOS, minocycline control, minocycline PCOS model (50 mg/kg), and letrozole PCOS (0.5 mg/kg). The Levels of FSH, LH, E2, and testosterone were determined by ELISA. H&E staining was used for histological analysis in the ovarian tissues. Docking scores were −10.35, −10.57, and −12.45 kcal/mol for TNFα, TLR-4, and TNFR2, respectively. The expression levels of TNF-α, TNFR2, and TLR4 were detected by Real-Time PCR. PCOS models exhibited acyclicity, a significant increase in E2 levels (P < 0.01), and no difference in FSH, LH, and testosterone. The expression levels of TNF-α, TNFR2, and TLR-4 significantly increased in PCOS (2.70, 7.90, and 14.83-fold, respectively). EV treatment significantly increased graafian follicles (P < 0.001) and decreased corpus luteum (CL) (P < 0.01). Minocycline treatment in PCOS led to a significant decrease in E2 (P < 0.01) and graafian follicles (P < 0.001) and a significant increase in the CL numbers (P < 0.05). Our findings showed the positive effects of minocycline on estradiol level, CL and graafian follicles counts, suggesting that minocycline might inhibit these proteins and improve ovulation in our mouse model of PCOS.

Prenatal androgen exposure causes a sexually dimorphic transgenerational increase in offspring susceptibility to anxiety disorders

If and how obesity and elevated androgens in women with polycystic ovary syndrome (PCOS) affect their offspring’s psychiatric health is unclear. Using data from Swedish population health registers, we showed that daughters of mothers with PCOS have a 78% increased risk of being diagnosed with anxiety disorders. We next generated a PCOS-like mouse (F0) model induced by androgen exposure during late gestation, with or without diet-induced maternal obesity, and showed that the first generation (F1) female offspring develop anxiety-like behavior, which is transgenerationally transmitted through the female germline into the third generation of female offspring (F3) in the androgenized lineage. In contrast, following the male germline, F3 male offspring (mF3) displayed anxiety-like behavior in the androgenized and the obese lineages. Using a targeted approach to search for molecular targets within the amygdala, we identified five differentially expressed genes involved in anxiety-like behavior in F3 females in the androgenized lineage and eight genes in the obese lineage. In mF3 male offspring, three genes were dysregulated in the obese lineage but none in the androgenized lineage. Finally, we performed in vitro fertilization (IVF) using a PCOS mouse model of continuous androgen exposure. We showed that the IVF generated F1 and F2 offspring in the female germline did not develop anxiety-like behavior, while the F2 male offspring (mF2) in the male germline did. Our findings provide evidence that elevated maternal androgens in PCOS and maternal obesity may underlie the risk of a transgenerational transmission of anxiety disorders in children of women with PCOS.

Defining the impact of dietary macronutrient balance on PCOS traits

Lifestyle, mainly dietary, interventions are first-line treatment for women with polycystic ovary syndrome (PCOS), but the optimal diet remains undefined. We combined a hyperandrogenized PCOS mouse model with a systematic macronutrient approach, to elucidate the impact of dietary macronutrients on the development of PCOS. We identify that an optimum dietary macronutrient balance of a low protein, medium carbohydrate and fat diet can ameliorate key PCOS reproductive traits. However, PCOS mice display a hindered ability for their metabolic system to respond to diet variations, and varying macronutrient balance did not have a beneficial effect on the development of metabolic PCOS traits. We reveal that PCOS traits in a hyperandrogenic PCOS mouse model are ameliorated selectively by diet, with reproductive traits displaying greater sensitivity than metabolic traits to dietary macronutrient balance. Hence, providing evidence to support the development of evidence-based dietary interventions as a promising strategy for the treatment of PCOS, especially reproductive traits.

Regulatory Functions of L-Carnitine, Acetyl, and Propionyl L-Carnitine in a PCOS Mouse Model: Focus on Antioxidant/Antiglycative Molecular Pathways in the Ovarian Microenvironment.

Polycystic ovary syndrome (PCOS) is a complex metabolic disorder associated with female infertility. Based on energy and antioxidant regulatory functions of carnitines, we investigated whether acyl-L-carnitines improve PCOS phenotype in a mouse model induced by dehydroepiandrosterone (DHEA). CD1 mice received DHEA for 20 days along with two different carnitine formulations: one containing L-carnitine (LC) and acetyl-L-carnitine (ALC), and the other one containing also propionyl-L-carnitine (PLC). We evaluated estrous cyclicity, testosterone level, ovarian follicle health, ovulation rate and oocyte quality, collagen deposition, lipid droplets, and 17ß-HSD IV (17 beta-hydroxysteroid dehydrogenase type IV) expression. Moreover, we analyzed protein expression of SIRT1, SIRT3, SOD2 (superoxide dismutase 2), mitochondrial transcriptional factor A (mtTFA), RAGE (receptor for AGEs), GLO2 (glyoxalase 2) and ovarian accumulation of MG-AGEs (advanced glycation end-products formed by methylglyoxal). Both carnitine formulations ameliorated ovarian PCOS phenotype and positively modulated antioxidant molecular pathways in the ovarian microenvironment. Addition of PLC to LC-ALC formulation mitigated intraovarian MG-AGE accumulation and increased mtTFA expression. In conclusion, our study supports the hypothesis that oral administration of acyl-L-carnitines alleviates ovarian dysfunctions associated with this syndrome and that co-administration of PLC provides better activity. Molecular mechanisms underlying these effects include anti-oxidant/glycative activity and potentiation of mitochondria.

EFFECTS OF VITAMIN D ADMINISTRATION IN THE MOUSE MODEL OF POLYCYSTIC OVARIAN SYNDROME

To investigate effects of vitamin .D(Vit.D) administration in patients with polycystic ovary syndrome(PCOS) Experimental animal study. Forty female pre-pubertal mice were randomly divided into 4 groups: the control, PCOS, PCOS+low dose Vit.D and PCOS+high dose Vit.D groups(N=10 per group). The PCOS mouse model was developed by 6mg/kg/day dehydroepiandrosterone(DHEA) administration with subcutaneously injections and high fat diet feeding. After 30 days, Vit.D was administrated by intraperitoneal injection in the following 40 days, 130ng/100g/week 1,25(OH)2D3 in low dose Vit.D group, and 1300ng/100g/week 1,25(OH)2D3 in high dose Vit.D group. Controls were injected with vehicle alone and fed with normal diet. At the end of the 70 days, blood samples were collected and the ovarian and liver tissues were taken. All the mice in PCOS+high dose Vit.D group died in two weeks after Vit.D administration. In the other three group, the weight of the PCOS mice was significantly higher than the weight of the controls before Vit.D administration(31.10±2.52, 31.76±2.54 VS 28.82±1.83g, PCOS group, PCOS+low dose Vit.D group VS control group, P=0.022). However, at the end of the study, the weight of the mice in PCOS group was significantly higher than those in control group and PCOS+low dose Vit.D group (41.41±3.90 VS 35.50±2.50, 34.55±2.31 g, P=0.000). The serum 25(OH)D concentration was significantly higher in PCOS+low dose Vit.D group than in control group and PCOS group (99.29±18.31 VS 19.55±4.10,18.04±6.51 ng/ml,P=0.000). The testosterone levels in PCOS group were significantly higher than those of control group and PCOS+low dose Vit.D group (1.27±0.27 VS 0.95±0.15 ,0.90±0.17 ng/ml, P=0.001). Furthermore, total cholesterol levels in control group were lower than in PCOS and PCOS+low dose Vit.D group (3.08±0.44 VS 4.55±0.47, 4.42±0.45mmol/L, P=0.011). Moreover, the ratio of liver weight to body weight was significantly different among the three groups (0.045±0.0046 VS 0.036±0.0043 VS 0.041±0.0031, control group VS PCOS group VS PCOS+low dose Vit.D group, P=0.000). Our results indicate that low dose Vit.D has positive effects on the hormonal changes and obesity observed in PCOS, maybe through liver metabolism regulation, and high dose Vit.D administration may be harmful.

Erratum. Metformin inhibits testosterone-induced endoplasmic reticulum stress in ovarian granulosa cells via inactivation of p38 MAPK.

Study question Does metformin inhibit excessive androgen-induced endoplasmic reticulum (ER) stress in mouse granulosa cells (GCs) in vivo and in vitro? Summary answer Metformin inhibits testosterone-induced ER stress and unfolded protein response (UPR) activation by suppressing p38 MAPK phosphorylation in ovarian GCs. What is known already Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism. Excessive testosterone induces ER stress and UPR activation in human cumulus cells, leading to cell apoptosis. Metformin has potential inhibitory effects on ER stress and UPR activation, as demonstrated in human pancreatic beta cells and obese mice. Study design, size, duration Cumulus cells and follicular fluid were collected from 25 women with PCOS and 25 controls at our IVF centre. A dihydrotestosterone (DHT)-induced PCOS mouse model was constructed and treated with or without metformin. Primary mouse GCs and cumulus-oocyte complexes (COCs) were cultured with testosterone, metformin, a p38 MAPK inhibitor, or p38 MAPK small interfering RNA. Participants/materials, setting, methods The levels of UPR sensor proteins and UPR-related genes were measured in cumulus cells from PCOS and control patients by real-time quantitative PCR (qPCR) and western blot. The ovaries, oocytes, GCs and COCs were collected from PCOS mice treated with metformin and controls. The expressions of ER stress markers and p38 MAPK phosphorylation were assessed by qPCR, western blot and immunofluorescence. A subsequent in vitro analysis with primary cultured GCs and COCs was used to confirm the influence of metformin on ER stress activation by qPCR and western blot. Finally, the effects of ER stress activation on GCs and COCs in relation to LH responsiveness were examined by qPCR and COC expansion. Main results and the role of chance The expression of the ER stress markers GRP78, CHOP and XBP1s in the cumulus cells was higher in PCOS patients than in control patients, as were the levels of the UPR sensor proteins p-IRE1α, p-EIF2α and GRP78. Compared to those of control mice, the ovaries, GCs and COCs of DHT-treated PCOS mice showed increased levels of ER stress marker genes and proteins. Hyperandrogenism in PCOS mouse ovaries also induced p38 MAPK phosphorylation in COCs and GCs. Metformin inhibited ER stress activation was associated with decreased p-p38 MAPK levels. In vitro experiments, testosterone-induced ER stress was mitigated by metformin or p38 MAPK inhibition in primary cultured GCs and COCs. COCs expanded rapidly in the presence of testosterone during LH administration, and ovulation-related genes, namely, Areg, Ereg, Ptgs2, Sult1e1, Ptx3 and Tnfaip6, were strongly expressed in the COCs and GCs. These effects were reversed by treatment with metformin, an ER stress inhibitor or by knockdown of p38 MAPK. Limitations, reasons for caution The number of PCOS patients in this study was small. Wider implications of the findings This study provides further evidence for metformin as a PCOS treatment. Study funding/competing interest(s) This study was funded by the National Key Research and Developmental Program of China (2018YFC1004800), the Key Research and Development Program of Zhejiang Province (2017C03022), the Zhejiang Province Medical Science and Technology Plan Project (2017KY085, 2018KY457), the National Natural Science Foundation of China (31701260, 81401264, 81701514), and the Special Funds for Clinical Medical Research of the Chinese Medical Association (16020320648). The authors report no conflict of interest in this work and have nothing to disclose. Trial registration number N/A.

Assessing the effect of MitoQ10 and Vitamin D3 on ovarian oxidative stress, steroidogenesis and histomorphology in DHEA induced PCOS mouse model

Polycystic ovary syndrome (PCOS) continues to be one of the most complex reproductive and endocrine disorder among women of reproductive age. Recent reports have identified close interaction of Vitamin D deficiency and oxidative stress (OS) in exacerbating the pathophysiology of PCOS. This current study aims at assessing the combine effect of MitoQ10 and Vitamin D3 on dehydroepiandrosterone (DHEA) induced PCOS. Following successful induction of PCOS using DHEA, mice were organized into five groups (n = 8) namely: Negative Control (NC), Vitamin D3 Vehicle (VDV), Vitamin D3 (VD), MitoQ10 (MQ), Vitamin D3 plus MitoQ10 (V+M) and DHEA, ethanol and distilled water, Vitamin D3, MitoQ10 and Vitamin D3 plus MitoQ10 were respectively administered for 20 consecutive days. The study also included positive control (PC) group (n = 8) in which no treatment was applied. Treatment effects were assessed using hormonal assays, biochemical assays, Real-Time PCR, western blotting and histological analysis. Combination of Vitamin D3 and MitoQ10 significantly reduced levels of estradiol, progesterone, FSH, LH, LH/FSH, SOD and MDA. The expression rate of mRNAs of 3β-HSD, Cyp19a1, Cyp11a1, StAR, Keap1, HO-1 and Nrf2 were also significantly low in V+M group. Moreover, the histomorphological inspection of ovaries from this group revealed many healthy follicles at various stages of development including few atretic follicles, pre-antral and antral follicles and many corpora lutea. The characteristics observed in this group were in many ways similar to that of the PC group. The combination of MitoQ10 and Vitamin D3 may be potential candidate to ameliorate PCOS.

Androgen signaling pathways driving reproductive and metabolic phenotypes in a PCOS mouse model.

As the mechanistic basis of polycystic ovary syndrome (PCOS) remains unknown, current management relies on symptomatic treatment. Hyperandrogenism is a major PCOS characteristic and evidence supports it playing a key role in PCOS pathogenesis. Classically, androgens can act directly through the androgen receptor (AR) or, indirectly, following aromatization, via the estrogen receptor (ER). We investigated the mechanism of androgenic actions driving PCOS by comparing the capacity of non-aromatizable dihydrotestosterone (DHT) and aromatizable testosterone to induce PCOS traits in WT and Ar-knockout (ARKO) mice. DHT and testosterone induced the reproductive PCOS-like features of acyclicity and anovulation in WT females. In ARKO mice, DHT did not cause reproductive dysfunction; however, testosterone treatment induced irregular cycles and ovulatory disruption. These findings indicate that direct AR actions and indirect, likely ER, actions of androgens are important mediators of PCOS reproductive traits. DHT, but not testosterone, induced an increase in body weight, body fat, serum cholesterol and adipocyte hypertrophy in WT mice, but neither androgen induced these metabolic features in ARKO mice. These data infer that direct AR-driven mechanisms are key in driving the development of PCOS metabolic traits. Overall, these findings demonstrate that differing PCOS traits can be mediated via different steroid signaling pathways and indicate that a phenotype-based treatment approach would ensure effective targeting of the underlying mechanisms.

The therapeutic effect of interleukin-22 in high androgen-induced polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a complex syndrome involving both endocrine and metabolic disorders. Gut microbiota and the intestinal immune factor IL-22 play an important role in the pathogenesis of PCOS. However, the therapeutic role of IL-22 in high androgen-induced PCOS mice is not clear. We aimed to determine the therapeutic effects of IL-22 on the DHEA-induced PCOS mouse model and to explore the possible mechanism of IL-22 in regulating hyperandrogenism-associated PCOS. Insulin resistance levels and ovarian functions were investigated in DHEA-induced PCOS mice with or without additional IL-22 treatment. We found that IL-22 could reverse insulin resistance, disturbed estrous cycle, abnormal ovary morphology, and decreased embryo number in DHEA mice. Mechanistically, IL-22 upregulated the browning of white adipose tissue in DHEA mice. This study demonstrated that IL-22-associated browning of white adipose tissue regulated insulin sensitivity and ovarian functions in PCOS, suggesting that IL-22 may be of value for the treatment of PCOS with a hyperandrogenism phenotype.

Decreased microRNA-125b-5p disrupts follicle steroidogenesis through targeting PAK3/ERK1/2 signalling in mouse preantral follicles

BackgroundHyperandrogenism is one of the major characteristics of polycystic ovary syndrome (PCOS). Abnormal miR-125b-5p expression has been documented in multiple diseases, but whether miR-125b-5p is associated with aberrant steroidogenesis in preantral follicles remains unknown. MethodsSteriod hormone concentrations and miR-125b-5p expression were measured in clinical serum samples from PCOS patients. Using a mouse preantral follicle culture model and a letrozole-induced PCOS mouse model, we investigated the mechanism underlying miR-125b-5p regulation of androgen and oestrogen secretion. ResultsThe decreased miR-125b-5p expression was observed in the sera from hyperandrogenic PCOS (HA-PCOS) patients. In mouse preantral follicles, inhibiting miR-125b-5p increased the expression of androgen synthesis-related genes and stimulated the secretion of testosterone, while simultaneously downregulating oestrogen synthesis-related genes and decreasing oestradiol release. Ectopically expressed miR-125b-5p reversed the effects on steroidogenesis-related gene expression and hormone release. Mechanistic studies identified Pak3 as a direct target of miR-125b-5p. Furthermore, inhibiting miR-125b-5p facilitated the activation of ERK1/2 in mouse preantral follicles, while inhibiting Pak3 abrogated this activating effect. These results were recapitulated in letrozole-induced PCOS mouse ovaries. Of note, inhibiting PAK3 antagonised the positive effect of miR-125b-5p siRNA on the expressions of androgen synthesis-related enzymes and testosterone secretion. Luteinizing hormone (LH) inhibited miR-125b-5p expression, and stimulated Pak3 expression. ConclusionHigh serum LH concentrations in PCOS patients repress miR-125b-5p expression, which further increases Pak3 expression, leading to activation of ERK1/2 signalling, thus stimulating the expression of androgen synthesis-related enzymes and testosterone secretion in HA-PCOS.

IN-UTERO ANDROGEN EXPOSURE CHANGES NEONATAL OVARIAN GENE EXPRESSION AND MITOCHONDRIA IN A LEAN PCOS MODEL

Polycystic ovary syndrome (PCOS) affects up to 10% of women worldwide, has a strong hereditary pattern with a heterogeneous phenotype. While no candidate genes have been found, a shared disease predisposing environment, such as intrauterine hyperandrogenemia, may explain an epigenetic inheritance pattern. A lean PCOS mouse model, eliminating the obesity confounder, with maternal androgen administration prenatally, provides an opportunity to study the effect of androgens on fetal programming.

Potential of Auraptene in Improvement of Oocyte Maturation, Fertilization Rate, and Inflammation in Polycystic Ovary Syndrome Mouse Model.

Polycystic ovary with poor-quality oocytes has remained problematic in polycystic ovary syndrome (PCOS) patients. It is well documented that the inflammation and production of reactive oxygen species (ROS) in PCOS ovaries are significantly higher than normal voluntaries. In this study, we hypothesized that auraptene (AUR), as a coumarin derivative with anti-inflammatory properties, may be effective in improvement of oocyte maturation and fertilization rate in PCOS patients. For this purpose, PCOS model was induced in NMRI mice and confirmed by ovarian histopathology observations and hormonal assays. PCOS-induced mice were administrated with AUR (PCOS-AUR) and metformin (PCOS-MET), and their effects on inflammation, apoptosis rate, oocyte maturation, and in vitro fertilization capacity were determined and compared with those normal and PCOS animals treated with sesame oil (PCOS-sesame oil) and no treatment (PCOS). Treatment with AUR and MET decreased the inflammation and apoptosis rates in PCOS mice compared with PCOS animals with no treatment. PCOS-AUR and PCOS-MET oocytes also showed higher intracellular glutathione and lower ROS concentrations compared with PCOS mice, indicating improved oocyte maturation rate. PCOS-AUR and PCOS-MET groups showed higher percentages of expansion rate and MII stage oocytes, and lower rate of abnormal oocytes compared with PCOS with no treatment. The rate of fertilization in the oocytes isolated from PCOS-AUR and PCOS-MET groups was higher than PCOS-sesame oil and PCOS groups. Our findings suggest that AUR can be considered as a potential candidate for improvement of oocyte maturation and fertilization capacity in PCOS patients, comparable to MET.

Vitamin D3 affects mitochondrial biogenesis through mitogen‐activated protein kinase in polycystic ovary syndrome mouse model

Polycystic ovarian syndrome (PCOS) is a disorder characterized by oligomenorrhea, anovulation, and hyperandrogenism. Altered mitochondrial biogenesis can result in hyperandrogenism. The goal of this study was to examine the effect of vitamin D3 on mitochondrial biogenesis of the granulosa cells in the PCOS-induced mouse model. Vitamin D3 applies its effect via the mitogen-activated pathway kinase-extracellular signal-regulated kinases (MAPK-ERK1/2) pathway. The PCOS mouse model was induced by the injection of dehydroepiandrosterone (DHEA). Isolated granulosa cells were subsequently treated with vitamin D3, MAPK activator, and MAPK inhibitor. Gene expression levels were measured using real-time polymerase chain reaction. MAPK proteins were investigated by western blot analysis. We also determined reactive oxygen species (ROS) levels with 2', 7'-dichlorofluorescein diacetate. Mitochondrial membrane potential (mtMP) was also measured by TMJC1. Mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator 1-α and nuclear respiratory factor), antioxidant (superoxide dismutase, glutathione peroxidase, and catalase), and antiapoptotic (B-cell lymphoma-2) genes were upregulated in the PCOS mice that treated with vitamin D3 compared with the PCOS mice without any treatment. Vitamin D3 and MAPK activator-treated groups also reduced ROS levels compared with the nontreated PCOS group. In summary, vitamin D3 and MAPK activator increased the levels of mitochondrial biogenesis, MAPK pathway, and mtMP markers, while concomitantly decreased ROS levels in granulosa cells of the PCOS-induced mice. This study suggests that vitamin D3 may improve mitochondrial biogenesis through stimulation of the MAPK pathway in cultured granulosa cells of DHEA-induced PCOS mice which yet to be investigated.

Prenatal androgen induced lean PCOS impairs mitochondria and mRNA profiles in oocytes.

Polycystic ovary syndrome (PCOS) is the most common ovulatory defect in women. Although most PCOS patients are obese, a subset of PCOS women are lean but show similar risks for adverse fertility outcomes. A lean PCOS mouse model was created using prenatal androgen administration. This developmentally programmed mouse model was used for this study. Our objective was to investigate if mitochondrial structure and functions were compromised in oocytes obtained from lean PCOS mouse. The lean PCOS mouse model was validated by performing glucose tolerance test, HbA1c levels, body weight and estrous cycle analyses. Oocytes were isolated and were used to investigate inner mitochondrial membrane potential, oxidative stress, lipid peroxidation, ATP production, mtDNA copy number, transcript abundance and electron microscopy. Our results demonstrate that lean PCOS mice have similar weight to that of the controls but exhibit glucose intolerance and hyperinsulinemia along with dysregulated estrus cycle. Analysis of their oocytes show impaired inner mitochondrial membrane function, elevated reactive oxygen species (ROS) and increased RNA transcript abundance. Electron microscopy of the oocytes showed impaired mitochondrial ultrastructure. In conclusion, the lean PCOS mouse model shows a decreased oocyte quality related to impaired mitochondrial ultrastructure and function.

Methylglyoxal-Dependent Glycative Stress and Deregulation of SIRT1 Functional Network in the Ovary of PCOS Mice.

Advanced glycation end-products (AGEs) are involved in the pathogenesis and consequences of polycystic ovary syndrome (PCOS), a complex metabolic disorder associated with female infertility. The most powerful AGE precursor is methylglyoxal (MG), a byproduct of glycolysis, that is detoxified by the glyoxalase system. By using a PCOS mouse model induced by administration of dehydroepiandrosterone (DHEA), we investigated whether MG-dependent glycative stress contributes to ovarian PCOS phenotype and explored changes in the Sirtuin 1 (SIRT1) functional network regulating mitochondrial functions and cell survival. In addition to anovulation and reduced oocyte quality, DHEA ovaries revealed altered collagen deposition, increased vascularization, lipid droplets accumulation and altered steroidogenesis. Here we observed increased intraovarian MG-AGE levels in association with enhanced expression of receptor for AGEs (RAGEs) and deregulation of the glyoxalase system, hallmarks of glycative stress. Moreover, DHEA mice exhibited enhanced ovarian expression of SIRT1 along with increased protein levels of SIRT3 and superoxide dismutase 2 (SOD2), and decreased peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC1α), mitochondrial transcriptional factor A (mtTFA) and translocase of outer mitochondrial membrane 20 (TOMM20). Finally, the presence of autophagy protein markers and increased AMP-activated protein kinase (AMPK) suggested the involvement of SIRT1/AMPK axis in autophagy activation. Overall, present findings demonstrate that MG-dependent glycative stress is involved in ovarian dysfunctions associated to PCOS and support the hypothesis of a SIRT1-dependent adaptive response.

Effect of Vitamin D3 on Mitochondrial Biogenesis in Granulosa Cells Derived from Polycystic Ovary Syndrome.

BackgroundPolycystic ovary syndrome (PCOS) is an endocrine disorder diagnosed by anovulation hyperandro- genism. Hyperandrogenism increases apoptosis, which will eventually disturb follicular growth in PCOS patients. Since mitochondria regulate apoptosis, they might be affected by high incidence of follicular atresia. This may cause infertility. Since vitamin D3 has been shown to improve the PCOS symptoms, the aim of study was to investigate the effects vitamin D3 on mtDNA copy number, mitochondrial biogenesis, and membrane integrity of granulosa cells in a PCOS-induced mouse model.Materials and MethodsIn this experimental study, the PCOS mouse model was induced by dehydroepiandrosterone (DHEA). Granulosa cells after identification by follicle-stimulating hormone receptor (FSHR) were cultured in three groups: 1. granulosa cells treated with vitamin D3 (100 nM for 24 hours), 2. granulosa cells without any treatments, 3. Non-PCOS granulosa cells (control group). Mitochondrial biogenesis gene (TFAM) expression was compared between different groups using real-time PCR. mtDNA copy number was also investigated by qPCR. The mitochon- drial structure was evaluated by transmission electron microscopy (TEM). Hormonal levels were measured by an enzymelinked immunosorbent assay (ELISA) kit.ResultsThe numbers of pre-antral and antral follicles increased in PCOS group in comparison with the non-PCOS group. Mitochondrial biogenesis genes were downregulated in granulosa cells of PCOS mice when compared to the non-PCOS granulosa cells. However, treatment with vitamin D3 increased mtDNA expression levels of these genes compared to PCOS granulosa cells with no treatments. Most of the mitochondria in the PCOS group were spherical with almost no cristae. Our results showed that in the PCOS group treated with vitamin D3, the mtDNA copy number increased significantly in comparison to PCOS granulosa cells with no treatments.ConclusionAccording to this study, we can conclude, vitamin D3 improves mitochondrial biogenesis and membrane integrity, mtDNA copy number in granulosa cells of PCOS mice which might improve follicular development and subsequently oocyte quality.

Letrozole treatment of pubertal female mice results in activational effects on reproduction, metabolism and the gut microbiome.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder in reproductive-aged women that is comprised of two out of the following three features: hyperandrogenism, oligo- or amenorrhea, or polycystic ovaries. In addition to infertility, many women with PCOS have metabolic dysregulation that increases the risk of developing type 2 diabetes, hypertension, and non-alcoholic fatty liver disease. Changes in the gut microbiome are associated with PCOS and gut microbes may be involved in the pathology of this disorder. Since PCOS often manifests in the early reproductive years, puberty is considered to be a critical time period for the development of PCOS. Exposure to sex steroid hormones during development results in permanent, organizational effects, while activational effects are transient and require the continued presence of the hormone. Androgens exert organizational effects during prenatal or early post-natal development, but it is unclear whether androgen excess results in organizational or activational effects during puberty. We recently developed a letrozole-induced PCOS mouse model that recapitulates both reproductive and metabolic phenotypes of PCOS. In this study, we investigated whether letrozole treatment of pubertal female mice exerts organizational or activational effects on host physiology and the gut microbiome. Two months after letrozole removal, we observed recovery of reproductive and metabolic parameters, as well as diversity and composition of the gut microbiome, indicating that letrozole treatment of female mice during puberty resulted in predominantly activational effects. These results suggest that if exposure to excess androgens during puberty leads to the development of PCOS, reduction of androgen levels during this time may improve reproductive and metabolic phenotypes in women with PCOS. These results also imply that continuous letrozole exposure is required to model PCOS in pubertal female mice since letrozole exerts activational rather than organizational effects during puberty.

Mesenchymal stem cells derived exosomal miR-323-3p promotes proliferation and inhibits apoptosis of cumulus cells in polycystic ovary syndrome (PCOS)

Polycystic ovary syndrome (PCOS) is a heterogeneous reproductive disease. Adipose mesenchymal stem cells (AMSCs) can produce a mass of exosomes. The objective of this study was to determine the effects of exosomal miR-323-3p on cumulus cells (CCs) of PCOS patients. Exosomal miR-323-3p were collected from modified AMSCs. Real-time PCR, western blots, MTT assays, flow cytometry, luciferase reporter assays and a letrozole-induced PCOS mouse model were used to identify mechanisms of exosomal miR-323-3p on CCs. The results revealed that miR-323-3p expression was upregulated in AMSCs, exosomes and CCs. Upregulated miR-323-3p promoted cell proliferation and suppressed apoptosis in CCs, while miR-323-3p inhibitor exerted opposite roles in exosome-treated CCs. Moreover, PDCD4 was upregulated in PCOS CCs, displayed an inverse expression pattern to those of miR-323-3p, and was a direct target of miR-323-3p. Overexpression of PDCD4 reversed the effects of upregulated miR-323-3p on CCs. Serum FSH, LH and testosterone were upregulated while E2 levels were downregulated in the PCOS mice. Upregulation of miR-323-3p alleviated PCOS by suppressing CCs’ apoptosis through targeting PDCD4 in vivo. The results demonstrated that exosomal miR-323-3p promoted cell proliferation and inhibited apoptosis in CCs through targeting PDCD4 in PCOS. This study provides insight into developing new therapeutic strategies for PCOS.

Towards cell therapy of polycystic ovary syndrome (PCOS): human mesenchymal stem cells secretome inhibits androgen production by pcos theca cells

Background & Aim Background Polycystic Ovarian Syndrome (PCOS) is a metabolic disorder characterized by inflammation, infertility and excess ovarian androgen production by theca cells. Though its etiology is not fully understood. Women with PCOS exhibit increased expression of CYP17A1, and CYP11A1 genes involved in androgen production. Due to the robust anti-inflammatory ability of human bone marrow Mesenchymal stem cells (hMSCs), we evaluated its utility as a novel cell-therapy by injecting hMSCs directly into the ovaries of PCOS mouse model. It showed a significant improvement in hyperandrogenemia. hMSCs also secrete various factors in media, known as secretome. The literature shows that the secretome of hMSCs has the same regenerative and reparative function likes hMSCs. Hypothesis We hypothesize that hMSCs secretome contains the factors that are able to inhibit the androgen biosynthesis in PCOS theca cells. Methods, Results & Conclusion Material and Methods Theca cells, collected from normal cycling and PCOS patient underwent ovariectomy, were seeded on culture dishes pre-coated with the extracellular matrix at a density of 6 × 104 cells/ml and cultured for 24 hours. Cells were then treated with either 3T3 conditioned media as a control (C) or Mesenchymal stem cells conditioned media (CM). The C and CM were prepared through collecting the serum-free supernatant of 3T3 and hMSCs at an optimal confluence, 48 hours later. The mRNA and protein expression of CYP17A1, CYP11A1, and DENND1A.V2 were quantified by RT-PCR and Western blot, while testosterone level in media was measured by RIA assay. Results Human PCOS theca cells treated with hMSCs CM for 48 hours secreted significantly lower level of testosterone (146 ± 13.6 ng/dl) compared to control C (215 ± 11.3 ng/dl) (P Conclusion The therapeutic capabilities of hMSCs in PCOS are partially mediated by its secretome which contains various growth factors and anti-inflammatory cytokines both free and within extracellular vesicles. Stem cell therapy, either by cell implantation or via secretome injection might potentially be a novel approach for effective treatment of PCOS patients.

Low-Dose Tacrolimus Prevents Dysregulated Peri-Conceptional Ovarian and Systemic Immune Cellular Homeostasis in Subjects with PCOS

Polycystic ovary syndrome (PCOS) is characterized by failure of ovulation and is associated with obesity and chronic inflammation. Recent evidence suggests that anomalous activation of ovarian macrophages and numerical and functional deficits in the Th17 (CD4+IL17A+) and the CD4+CD25+CD127low Tregs plays crucial role in PCOS. We have shown that the pre-pregnancy use of tacrolimus prevents adverse reproductive outcomes in a mouse model of PCOS. Here we used the HFD-NONcNZO mice to test a hypothesized beneficial use of tacrolimus relative to metformin in favorably influencing the ovarian and systemic immune milieux conducive to gestational success in subjects with PCOS. Compared to normative controls, our data revealed an aberrant peri-conceptional suppression of the CD4+CD25+CD127low Tregs together with an overexpression of the Th17 T cells and lack of coordinated activation of ovarian macrophages in untreated HFD-dNONcNZO mice. Significant variances in treatment outcomes favoured the use of tacrolimus over metformin in treated mice. Consistent with the human fertility studies, this investigation reveals a beneficial systemic use of tacrolimus (0.1 mg/kg) in promoting early pregnancy in individuals with PCOS and suggests the need for further research into the selective inhibition of IL17A as a plausibly alternative immunotherapeutic approach in the clinical management of infertile individuals with PCOS.