Abnormal Estrous Cycles Research Articles

#324 : Gasdermin D-Mediated Pyroptosis Underlies Dehydroepiandrosterone-Induced Polycystic Ovary Syndrome

Background and Aims: Intestinal microflora disturbance is one of the important etiologies of polycystic ovary syndrome (PCOS). The purpose of this study was to investigate the mechanism of macrophage pyroptosis induced by intestinal microbiota disturbance in PCOS. Method: The PCOS mouse model was established using dehydroepiandrosterone, and was studied by 16S rDNA sequencing, western blot, ELISA, GSDMD gene knockout/oral pyroptotic inhibitors, single cell transcriptomics and translatomics analysis. Results: PCOS mice showed abnormal estrous cycle, elevated serum testosterone and polycystic ovaries. Fecal 16S rDNA sequencing showed that Muribaculacea (26.76%), and Lactobacillus (40.98%) were the most abundant microbe in the intestinal tracts of control mice and PCOS mice, respectively. Difference analysis showed a significant decrease in the abundance of intestinal probiotics Akkermansia and a significant increase in Gram-negative bacteria (Desulfovibrio and Burkholderia) in PCOS mice. Intestinal microbial disturbance in PCOS mice was associated with increased serum lipopolysaccharide (LPS). LPS intraperitoneal injection can induce pyroptosis of ovarian macrophages, which also exists in the ovaries of PCOS mice. The cleavage of GSDMD protein is the final key step in pyroptosis of macrophages. GSDMD knockout ameliorated PCOS in mice. Oral administration of pyroptotic inhibitors disulfiram and metformin can increase the abundance of intestinal Akkermansia, inhibit pyroptosis of ovarian macrophages and ameliorated PCOS in PCOS group mice. Mechanically, transcriptome and translatome sequencing of primary granulosa cells showed that pyroptosis of macrophages disrupted estrogen production and promoted granulosa cell apoptosis. Conclusion: This study revealed that macrophage pyroptosis induced by intestinal flora disorder can lead to the occurrence of PCOS through granulosa cell apoptosis and estrogen synthesis dysfunction. Macrophage pyroptotic inhibitors may be a potential therapeutic strategy for PCOS.

LIPUS combined with TFSC alleviates premature ovarian failure by promoting autophagy and inhibiting apoptosis

Objective Premature ovarian failure (POF), also known as primary ovarian insufficiency, is a major cause of infertility in female worldwide. Excessive apoptosis and impaired autophagy in ovarian granulosa cells are the main pathological mechanisms of POF. The total flavonoids from semen cuscutae (TFSC) are often used in the treatment of gynecological endocrine disorders. In addition, low intensity pulsed ultrasound (LIPUS) is report as an effective method to improve ovarian function. This study aims to investigate the protective effect of POF by the combined use of TFSC and LIPUS. Methods POF rats model and granulosa cell model were successfully induced by tripterygium glycosides and cyclophosphamide, respectively. After that, model rats and cells received TFSC plus LIPUS administration. Then ovarian histomorphology, senescence, estrus cycle, and serum sex hormone levels were detected in rats. Ovarian tissue and granulosa cells autophagy and apoptosis levels were also assessed. Results Disturbed sex hormone levels, atrophied and senescent ovaries, and abnormal estrous cycle were found in POF rats. Meanwhile, cell autophagy was inhibited and cell apoptosis was activated in POF ovarian tissue and granulosa cells. However, TFSC combined with LIPUS improved these changes, and this combination treatment exhibited synergistic effects. The abnormal expression of the cell apoptosis-, autophagy-, and PI3K/AKT/mTOR signaling pathway-related proteins were also improved by combination treatment. Conclusion The study found that the combination of TFSC and LIPUS can alleviate POF by modulating cell autophagy and apoptosis. The findings may provide a viable scientific basis for POF treatment.

Promising drug candidates for the treatment of polycystic ovary syndrome (PCOS) as alternatives to the classical medication metformin

Polycystic ovary syndrome (PCOS) is a prevalent hormonal disorder that affects women of reproductive age. It is characterized by abnormal production of androgens, typically present in small quantities in females. This study aimed to investigate the therapeutic potential of Irosustat (STX64), STX140, and compound 1G as new drug candidates for the treatment of letrozole-induced PCOS in female Wistar rats. 36 rats were divided into six groups of equal size. PCOS was induced in all groups, except the normal control group, by administering letrozole orally (1 mg/kg/day for 35 days). The onset of abnormal estrous cycle was confirmed by examining daily vaginal smears under a microscope. Subsequently, each rat group was assigned to a different treatment regimen, including one control group, one letrozole group, one metformin group (500 mg/kg/day) as a reference drug, and the other groups received a different drug candidate orally for 30 days. After treatment, blood collection was performed for biochemical measurements and determination of oxidative stress markers. The rats were dissected to separate ovaries and uterus for morphological, histological, and western blotting studies. Treatment with the drug candidates improved the ovaries and uterus weight measurements compared to the untreated PCOS group. The three tested drug candidates demonstrated promising improvements in lipid profile, blood glucose level, testosterone, progesterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estradiol levels. In addition, western blotting confirmed their promising effects on Akt, mTOR, and AMPK-α pathways. This study led to the discovery of three promising drug candidates for the management of PCOS as alternatives to metformin.

SAT386 Constitutive Overexpression Of Microrna-93 As A Cause Of Metabolic Dysfunction And Hyperandrogenism In Polycystic Ovary Syndrome (PCOS): Evidence From A Transgenic (Mir-93+) Murine Model

Abstract Disclosure: Y. Chen: None. H. Wu: None. B.A. Gower: None. R. Azziz: Advisory Board Member; Self; Arora Forge. Consulting Fee; Self; Fortress Biotech, Spruce Biosciences, Core Access Surgical Technologies, Rani Therapeutics. Grant Recipient; Self; Ferring Pharmaceuticals, Foundation for Research and Education Excellence. Stock Owner; Self; Martin Imaging. Background: PCOS appears to result from enhanced stimuli, either increased LH or insulin, and exaggerated responsiveness of ovarian theca cell androgen biosynthesis to these stimuli. However, the etiology of the enhanced stimuli remains unclear. We previously reported that a specific microRNA, miR-93, regulates GLUT4 content in adipocytes; and that in PCOS miR-93 overexpression in adipocytes results in suppressed GLUT4 expression and insulin-meditated glucose uptake in these cells, and is associated with whole-body insulin resistance (IR). These data suggest that constitutive miR-93 overexpression may underly the metabolic dysfunction and, possibly, the hyperandrogenism of PCOS. Objective: To test the hypothesis in a transgenic murine model that miR-93 overexpression results in the metabolic and hyperandrogenic features of PCOS. Materials and methods: We generated a novel miR-93 overexpressed (miR-93+) mouse model in collaboration with InGenious Targeting Laboratory (Ronkonkoma, NY). The mouse miR-93 sequence with the R26/CAG promoter was inserted immediately downstream of loxP flanked stop cassette in intron 1 of ROSA26 locus. miR-93 insertion was not expected to cause unwanted phenotypes resulting from modification of the Rosa26 locus. When mating miR-93+ transgenic mice to whole body Cre mice (The Jackson Lab) the stop code is removed, and miR-93 is overexpressed systemically. Results: In miR-93+ mice, miR-93 was overexpressed in adipose tissue (AT), muscle, liver, heart, kidneys, and ovaries. Phenotypically, miR-93+ mice had reduced GLUT4 expression in AT, and higher fasting serum insulin, higher fasting serum glucose, and higher testosterone levels. In PCOS women, increased CYP17A1 expression in theca cells is associated with excess androgen production. Consistent with this PCOS phenotype, Cyp17a1 expression in miR-93+ mice ovaries were also increased. In addition, the ovaries of miR-93+ mice had reduced expression of Cdkn1a, similar to that observed in granulosa cells from PCOS patients. miR-93+ mice gained more weight and epididymal AT than controls. miR-93+ mice also demonstrated a lesser response to insulin during an insulin tolerance test (ITT) and exhibited altered estrous cycles as compared to wild type. Conclusion: We successfully developed a miR-93 overexpressed (miR93+) murine model, which demonstrates IR, hyperandrogenemia, exaggerated ovarian Cyp17a1 expression, increased weight gain, and abnormal estrous (ovulatory) cyclicity, mimicking the PCOS phenotype. These results provide further evidence to support the role for constitutive overexpression of miR-93 in the metabolic and hyperandrogenic features of PCOS. These data also suggest a potential model to study the mechanisms mediating the effect of miR-93 overexpression on IR and on the PCOS phenotype in tissues and conditions not readily accessible in human subjects. Presentation Date: Saturday, June 17, 2023

Effect of Hypoglycemic Drugs on Kisspeptin Expression in the Hypothalamic Arcuate Nucleus of PCOS Rats.

To investigate the change in hypothalamic kisspeptin-1 (Kiss1) expression during the development of polycystic ovary syndrome (PCOS) and hypoglycemic drug intervention. Letrozole lavage was used to construct a polycystic ovary rat model. After successful modeling, we treated PCOS rats with metformin, pioglitazone, and acarbose, and we then observed changes in weight, estrus, glucose tolerance, insulin resistance, sex hormones, and hypothalamic kiss1 expression. PCOS rats exhibited increased body weight, abnormal estrous cycle, impaired glucose tolerance, insulin resistance, increased testosterone level, increased luteinizing hormone level, and increased Kiss1 expression in the hypothalamus. However, intervention with metformin, pioglitazone, and acarbose improved the reproductive and metabolic disorders as well as reduced hypothalamic Kiss1 expression. The expression of hypothalamic Kiss1 may play an important role in the pathogenesis of PCOS. Metformin, pioglitazone, and acarbose may reduce the expression of hypothalamic Kiss1 by improving insulin resistance, thereby improving reproductive and metabolic disorders in PCOS rats.

O-090 Endocrine disruptors

Abstract Female mammals, including women, are exposed to endocrine disrupting chemicals (EDCs) on a daily basis. This is of concern because many EDCs inhibit ovarian follicle growth and steroidogenesis, processes that are critical for maintaining female fertility. Our research focuses on the effects of EDCs known as phthalates on female fertility. Phthalates are of interest because they are ubiquitous synthetic chemicals used as plasticizers and stabilizers in a myriad of consumer products, including everything from shower curtains to children’s toys to cosmetics and personal care products such as perfumes, nail polish, deodorants, and lotions. Phthalates are also used in pesticides, wood finishes, adhesives, solvents, lubricants, defoaming agents, and in medical devices including tubing, blood bags, surgical gloves, and dialysis equipment. Despite the widespread use of phthalates and ubiquitous human exposure, limited information is available about the effects of environmentally relevant phthalate mixtures on female reproduction. Thus, we tested the hypothesis that prenatal exposure to an environmentally relevant phthalate mixture affects female reproduction in the F1, F2, and F3 generations of mice. To test this hypothesis, pregnant CD-1 mice were orally dosed with vehicle control (corn oil) or a phthalate mixture (20µg/kg/day-500mg/kg/day) daily from gestational day 10 to birth. The mixture was based on urinary phthalate metabolite levels measured pregnant women in the United States, and consisted of 35% diethyl phthalate, 21% di(2-ethylhexyl) phthalate, 15% dibutyl phthalate, 15% di-isononyl phthalate, 8% diisobutyl phthalate, and 5% benzylbutyl phthalate. Adult F1 females born to these dams were used to create the F2 generation by mating them with unexposed males and F2 females were used to create the F3 generation by mating them with unexposed males. At selected times, estrous cyclicity and fertility indices were monitored and ovaries and sera were collected for analysis. Our data indicate that prenatal exposure to the phthalate mixture significantly decreases anogenital distance, testosterone levels, and litter size, but it significantly increases abnormal estrous cyclicity, uterine weight, the number of cystic ovaries, follicle-stimulating hormone levels, and luteinizing hormone levels compared to control in the F1 generation. Further, our data indicate that prenatal exposure to the phthalate mixture increases body weight, uterine weight, the number of cystic ovaries, and time to pregnancy and it decreases testosterone levels compared to control in the F2 generation. In addition, our data indicate that prenatal exposure to the phthalate mixture increases body weight, uterine weight, the number of cystic ovaries, and abnormal cyclicity and it decreases anogenital distance and the number of live pups compared to control in the F3 generation. Collectively, these data suggest that prenatal exposure to an environmentally relevant mixture of phthalates adversely affects reproductive function in a multi- and transgenerational manner in female mice. These findings have increased our understanding of the mechanisms by which EDCs cause female reproductive toxicity. This information eventually may lead to the development of novel targets for the prevention or treatment of infertility induced by EDCs.

Calcium chloride diluted in ethanol 95% as female sterilizing agent: effect of transcutaneous delivery in rats

BackgroundDifferent fertility control methods are investigated as a tool for population control of free-roaming animals. Chemical castration using calcium chloride has been widely studied over the years in males, but there are few studies related to its use in females. Therefore, we aimed to evaluate the local effects, as a potential chemosterilant, of two concentrations of calcium chloride diluted in 95% ethanol when administered by transcutaneous ultrasound-guided intraovarian injection in rats. In this study, 30 female Wistar rats were randomly divided into three treatment groups, which consisted of transcutaneous ultrasound-guided intraovarian injection of: 0.9% sodium chloride solution (GC); 10% calcium chloride diluted in 95% ethanol (G10); 20% calcium chloride diluted in 95% ethanol (G20). The animals were subdivided into two evaluation times, 15 days (n = 5 of each group) and 30 days (n = 5 of each group) after the intraovarian injection. The ovarian diameter was measured using ultrasound image prior and immediately after the injection and after the treatment period. Furthermore, animals’ clinical evaluation, estrous cycles assessment, macroscopic examination of the abdominal cavity and histological evaluation of the ovaries were performed.ResultsOvarian ultrasound measurement revealed changes (p < 0.05) between ovarian diameters before and immediately after the injection in all treatments. Three animals in G20 had a small focal skin lesion at the injection site that evolved to total healing. Extended and abnormal estrous cycles were observed in G10 and G20. At gross examination, adhesions and ovarian cysts were noticed in both groups, G10 and G20. Also, the histopathology analysis revealed changes in ovarian architecture and vessel congestion in G10 and G20, but ovarian tissue damage was greater in the ovaries treated with the highest concentration (G20).ConclusionsThe results indicate that 20% calcium chloride diluted in 95% ethanol may be a potential agent for inducing sterilization in females and was possible to be minimally invasively delivered.

Hypo-Hydroxymethylation of Nobox is Associated with Ovarian Dysfunction in Rat Offspring Exposed to Prenatal Hypoxia

Prenatal hypoxia (PH) is a common feature of a suboptimal intrauterine environment affecting the development of fetuses. Whether PH leads to abnormal ovary development is not yet clear. This study investigated ovarian function in offspring exposed to PH and the potential underlying molecular mechanisms. SD female rats (n = 12 per group) at 9 weeks of age were housed in individual cages (21% O2). After the pregnant rats were exposed to hypoxia (10.5% oxygen) from embryonic day (E) 5 to E21, PH offspring were generated. All animals maintained normoxia during lactation. The number of follicles was counted in female offspring at 3 months under an optical microscope. The expression of Nobox, Gdf9, and Tets was detected by quantitative real-time polymerase chain reaction (PCR) and Western blot. Global DNA hydroxymethylation was measured by dot blot. The hydroxymethylation level of the Nobox gene was evaluated with an NGS-based multiple targeted CpG hydroxymethylation analysis method. Body weight and ovary weight were significantly decreased in the PH group compared with the control group. PH offspring have abnormal estrous cycle, decreased serum anti-Mullerian hormone (AMH), and increased serum follicle-stimulating hormone (FSH), and follicular atresia, which are consistent with the clinical manifestations in patients with ovarian dysfunction. In terms of mechanism, the expression of Nobox was significantly decreased in the PH group. Subsequent high-throughput sequencing results showed that the level of hydroxymethylation in the candidate region of the Nobox gene was reduced. Cultured cells treated with hypoxia exhibited lower levels of both 5hmC and Nobox, while vitamin C, a coactivator of Tets, rescued hypo-hydroxymethylation and increased the expression level of Nobox. This study indicated that PH could cause hypo-hydroxymethylation of Nobox through epigenetic regulation and may consequently contribute to ovarian dysfunction in adult rat offspring.

Assessment of 28-day oral exposure to Pueraria candollei var. mirifica (Fabaceae) roots on pituitary-ovarian axis function and selected metabolic parameters in ovary-intact rats

Pueraria candollei var. mirifica (Fabaceae) root (PMR) has recently been developed as a potential selective estrogen receptor modulator (SERM) in menopausal women. Nowadays, many premenopausal women also take dietary PMR supplements, however, the exact biological effects of PMR have not been evaluated. This study included the application of the OECD guideline 407 for the assessment of 28-day oral exposure to PMR on pituitary-ovarian (PO) axis function and metabolic parameters in the premenopausal rat model. Ovary-intact adult rats were orally administrated with 10, 100, 750, 1000, and 1500 mg/kg body weight (BW)/day of PMR powder. The positive estrogenic group was given 2 mg 17β-estradiol (E2)/kg BW/day. Serum levels of reproductive hormones, lipid and thyroid parameters, estrous cycle determination, and histomorphometric and histopathological evaluations of the anterior pituitary, ovary, uterus, vagina, mammary gland, and liver were investigated. PMR displayed neutral effects on uterine, vaginal, and body weights, and circulating E2 and prolactin levels. PMR exerted E2-like effects by i) reducing ovarian and increasing hepatic weights, ii) decreasing serum gonadotropins, iii) lowering serum lipids without altering thyroid parameters, iv) increasing the prevalence of abnormal estrous cycles with prolonged estrus, v) increasing nuclear diameter of anterior pituitary cells, vi) decreasing ovarian size and follicular numbers and increasing follicular degeneration, vii) thickening of uterine myometrium and luminal epithelium, and vaginal epithelium, and viii) induction of mammary alveolar hyperplasia and ductal secretion. Unlike E2, the appearance of very small numbers of focal microvesicular steatosis in hepatocytes demonstrated mild toxicity at high PMR doses. This is the first report that high-dose PMR exerted actions exactly like E2 on gonadotrope-ovarian axis function and histology, lipid, and thyroid parameters without affecting uterine and vaginal growth in ovary-intact rats according to OECD guidelines.

IL-22 and its interaction with amino acid and glycolipid metabolite in polycystic ovary syndrome (PCOS) patients

IL-22 and its interaction with amino acid and glycolipid metabolite in polycystic ovary syndrome (PCOS) patients

P–589 The effects of vanillic acid on production of testosterone and function of insulin in adipose and ovarian tissues in rat model of polycyctic ovary syndrome

Abstract Study question What is the possible therapeutic effects of vanillic acid (VA) on the treatment of Polycystic Ovary Syndrome (PCOS)? Summary answer Vanillic acid successfully alleviated metabolic and endocrine abnormalities in adipose and ovarian tissues in a rat model of PCOS. What is known already Polycystic Ovary Syndrome (PCOS), as a common endocrine disorder, is accompanied by hyperandrogenism, insulin resistance, ovulation problems, and infertility. In this study, the effects of vanillic acid (VA) on metabolic and endocrine abnormalities were evaluated in the adipose and ovarian tissues of a letrozole-induced rat model of PCOS. Study design, size, duration In this study, a letrozole-induced rat model was established and then the experimental groups were treated by VA and MET through oral gavage for one month. Before and after the treatment with drugs, the vaginal smear was carried out . Ovarian and adipose tissues were collected and frozen at − 80 for further analysis following euthanasia of the rats. Participants/materials, setting, methods Thirty Six -week- old Wistar rats were divided into four groups (six rats per each group). Twenty-four rats were received letrozole for five weeks. Then, two experimental groups were treated by metformin (MET), and VA . Serum lipid profile, fasting glucose levels, and hormonal assays were done by corresponding commercial kits. The phosphorylation of AMP-activated protein kinase (AMPK) was detected by western blotting and the expression of studied genes were measured by Real-time PCR. Main results and the role of chance Our results showed that both VA and MET successfully reversed the abnormal estrous cycles of PCOS rats and reduced the serum testosterone levels and Steroid 17-alpha-hydroxylase/17,20 lyase (CYP17A1) gene expression. Furthermore, they improved Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) and decreased the serum glucose and triglyceride levels, and gene expression of resisitin. The phosphorylation of AMPK was significantly decreased in adipose and ovarian tissues in PCOS group and both therapeutic factors successfully activated the AMPK in these tissues. However, VA had not such a significant effect in adipose tissue. Limitations, reasons for caution The main limitation of the current study was its design as a rat model, which may have limitation in the translation of knowledge to the clinics. Wider implications of the findings: In summary, to the best of our knowledge, this study is the first study reported beneficial effects of VA on the treatment of PCOS. The findings of the current study shed light on an urgent need for discovering novel therapeutic pharmaceuticals regarding the treatment of the PCOS. Trial registration number 0

The fertility assessment of normal cyclic Wistar rats following the administration of methanolic extract of Portulaca oleracea: an experimental study

BackgroundPurslane is a widely distributed shrub used for the treatment of different ailments. The increasing reproductive complications associated with herbal treatments have led to the need to critically evaluate the safety and/or reproductive potentials of commonly used plant extracts. This study investigated the reproductive effect of methanolic extracts of Portulaca oleracea (MEPO) in adult female Wistar rats.ResultsGroup C showed a significant decrease both in relative ovarian weight (p = 0.000), and relative uterine weight (p = 0.037), when compared with the control. There were no significant (p ˃ 0.05) changes in the levels of follicle-stimulating hormone, luteinizing hormone, progesterone, and estradiol. When compared to the control, groups B and C showed abnormal estrous cycle and cycle arrest especially at the metestrus phase with mild congestion of a few blood vessels in the ovary and uterus.ConclusionsMEPO may possess some anti-fertility effect, as it disrupts the estrous cycle of adult female Wistar rats; although it has no major effect on the reproductive hormones, uterus, and ovarian histology of adult female Wistar rats. However, high dose consumption should be taken with precaution.

Overaccumulation of Fat Caused Rapid Reproductive Senescence but not Loss of Ovarian Reserve in ob/ob Mice

Ovarian reserve and fertility are reduced by aging and a poor energy balance. To date, the relationships of high energy accumulation and aging with the ovarian reserve have not been elucidated. Here, the effects of obesity on the aging ovarian reserve were evaluated in a leptin-deficient (ob/ob) mouse model. Abnormal estrous cyclicity appeared as early as 6 weeks and worsened with aging. The blood level patterns of 17β-estradiol (E2), testosterone (T), and progesterone (P4) with aging were similar between lean and ob/ob mice. The blood level of E2 but not P4 or T was similar at 24 weeks. Many more atretic follicles but fewer corpora lutea were observed in ob/ob mice than in lean mice within all age groups. Anti-Müllerian hormone (Amh) mRNA levels were similar between genotypes. Dazl, Stra8, and ZP3 mRNAs were highly expressed in ob/ob mice after 12 weeks. Sohlh1 and Ybx2 mRNAs were highly expressed at 24 weeks in ob/ob compared with lean mice. In addition, SOHLH1-positive primordial follicle counts were significantly increased in ob/ob mice at 24 weeks. The proportions of AMH-positive secondary and small antral follicles were similar between genotypes. Together, these results show that the ovarian reserve lasts longer in ob/ob mice than in lean mice, suggesting that the loss of normal physiological or physical status causes decreased fertility at a young age in ob/ob mice and that an increase in adipocytes without leptin, as in ob/ob mice, can improve the ovarian reserve. Such knowledge can be applied to understanding reproductive dysfunction.

The Protective Effect of Pre-Moxibustion on Reproductive Hormones Profile of Rats with Tripterygium Glycosides-Induced Ovarian Damage

Background: Acupuncture and moxibustion have been proven to be conducive to improving the ovarian reserve. However, the mechanism of pre-moxibustion on Tripterygium glycosides (TG)-induced ovarian damage has not been previously reported. Method: Female rats were randomly divided into 5 groups: control, model (75 mg/kg TG, 14 days), preventive moxibustion 1 (PM1, receiving moxibustion for 4 weeks before TG administration), preventive moxibustion 2 (PM2, receiving moxibustion for 2 weeks before TG administration and another 2 weeks during TG administration), and preventive moxibustion 3 (PM3, receiving 4 weeks of moxibustion during TG administration). The estrous cycle of the animal was recorded after TG administration. Rats were sacrificed 14 days after TG administration. The reproductive hormones profiles in serum, ovary, and hypothalamic tissues were analyzed. Result: Pre-moxibustion could revert abnormal estrous cycles, relieve follicle damage, and improve abnormal secretion of reproductive hormones resulting from ovarian damage. However, both PM2 and PM3 were more effective than PM1. In addition, PM2 disclosed more advantages in regulating reproductive hormones abnormalities, while PM3 performed better in follicular development. Conclusion: In combination, the findings of this study suggest that pre-moxibustion is effective in protecting the ovary from damage in TG-induced ovarian damage rats. But different time points correspond to different modulation targets and mechanisms.

Dietary Advanced Glycation End Products (AGEs) could alter ovarian function in mice

Nutrition is an important source of exogenous AGEs and thermally processed foods present in western-style diets contain a large amount of these pro-inflammatory AGEs. Additionally, the intake of dietary AGEs could upregulate ovarian gene expression of inflammatory macrophage markers. The objective of this study was to investigate the effect of diet rich in AGEs on estrous cyclicity and ovarian function in a mouse model. Six-week old C57BL/6 J female mice were randomly subjected to either a diet low in AGEs (L-AGE) or a diet high in AGEs (H-AGE) for a total of 13 weeks. Experiments performed included daily vaginal smears to assess estrous cyclicity, ovarian superovulation with gonadotropins to assess the number of oocytes released, whole ovarian tissue mRNA quantification by RT-PCR to quantify genes involved in folliculogenesis, steroidogenesis, and macrophage markers, and ovarian morphology for follicle count. Outcome measures included estrous cyclicity, number of oocytes following superovulation, expression of genes involved in folliculogenesis, steroidogenesis, and macrophage infiltration as well as the number of primordial, primary, secondary, antral follicles and corpora lutea. Compared to mice on L-AGE diet, mice on H-AGE spent significantly longer time in the diestrus phase, had similar number of oocytes released following ovarian superovulation, and showed significant alterations in genes involved in steroidogenesis (increase in Star mRNA expression levels) and folliculogenesis (increase in Gdf-9 and Fshr mRNA expression levels). Mouse macrophage marker F4/80 mRNA expression was upregulated in mice on H-AGE diet compared to mice on L-AGE diet. Finally, mice on H-AGE diet had significantly fewer corpora lutea in their ovaries. These results indicate that the ingestion of high amounts of dietary AGEs could disrupt folliculogenesis and steroidogenesis that might lead to abnormal estrous cyclicity. Intake of dietary AGEs could also upregulate ovarian gene expression of inflammatory macrophage markers.

Nicotinamide and its metabolite N1-Methylnicotinamide alleviate endocrine and metabolic abnormalities in adipose and ovarian tissues in rat model of Polycystic Ovary Syndrome

Polycystic Ovary Syndrome (PCOS), as a common endocrine disorder is accompanied by hyperandrogenism, insulin resistance, ovulation problems, and infertility. Various types of off-label drugs like metformin have been used for the management of targeted problems caused by PCOS such as insulin resistance and hyperandrogenism. Nicotinamide (NAM) acts as a substrate of visfatin and Nicotinamide N-Methyltransferase (NNMT) leading to the generation of Nicotinamide Adenine Dinucleotide (NAD) and N1-Methylnicotinamide (MNAM), respectively. MNAM is known as an anti-inflammatory, anti-thrombosis, and anti-diabetic agent. In this study, the effects of NAM and MNAM on metabolic and endocrine abnormalities were evaluated in the adipose and ovarian tissues of a letrozole-induced rat model of PCOS. Our results showed that MNAM and NAM reversed abnormal estrous cycle and reduced the serum testosterone levels and CYP17A1 gene expression. Furthermore, all therapeutic factors improved HOMA-IR after treatment and NAM significantly increased the expression of GLUT4 and decreased the gene expression of visfatin. Also, MNAM diminished the gene expression of visfatin and resistin. It is noteworthy that all the therapeutic factors successfully activated the AMPK. In summary, this study is the first study reported beneficial effects of NAM and MNAM on the treatment of PCOS. Additionally, the alleviative effects of our therapeutic factors may be partially mediated by the AMPK-dependent manner due to the contribution of the AMPK in the expression of CYP17A1, visfatin, resistin, and GLUT4. Although more studies are required to unravel the exact mode of actions of MNAM and NAM in the PCOS, the findings of the current study shed light on an urgent need for discovering novel therapeutic pharmaceuticals regarding the treatment of PCOS.

Characterization on gut microbiome of PCOS rats and its further design by shifts in high-fat diet and dihydrotestosterone induction in PCOS rats.

Polycystic ovary syndrome (PCOS) is associated with gut microbiota disturbance. Emerging evidence has shown that gut microbiota plays a major role in the development of PCOS. To better understand how the gut microbiota contributes to the development of PCOS, we investigated the influences of high-fat diet and hyperandrogenism, independently or synergistically, have on the gut microbiota in rats. Furthermore, we explored the associations between gut microbiota and hyperandrogenism or other hallmarks of PCOS. Twenty female SD rats were randomized at aged 3weeks into 4 groups (n = 5, each); HA: PCOS rats fed with ordinary diet; HF: rats with high-fat diet (HFD); HA-HF: PCOS rats fed with HFD; and C: control rats with ordinary diet. PCOS rat model was induced by 5α-dihydrotestosterone (DHT) injection for 6weeks. The fasting blood glucose (FBG), plasma insulin, testosterone, free testosterone, TNF-α, MDA, SOD, LPS, TLR4, TG, TC, HDL-C, and LDL-C levels were measured. The molecular ecology of the fecal gut microbiota was analyzed by 16S rDNA high-throughput sequencing. The results showed that rats in the HA and HA-HF group displayed abnormal estrous cycles with increasing androgen level and exhibited multiple large cysts with diminished granulosa layers in ovarian tissues. Compare with the C group, relative abundance of the Bacteroidetes phylum decreased significantly in the other groups (P < 0.05). The Chao1 was the highest in the group C and significantly higher than the HA-HF group (P < 0.05). T, FT, insulin, MDA, LPS, and TNF-α levels had the negative correlation with the richness of community (Chao1 index) in the gut. The rats in the HF and HA-HF groups tended to have lower Shannon and Simpson indices than the C group (P < 0.01, respectively). However, there were no significant differences between C group and the HA group in the Shannon and Simpson values. Beta diversity analysis was then performed based on a weighted UniFrac analysis. The PCoA plots showed a clear separation of the C group from the other groups. ANOSIM analysis of variance confirmed that there were statistically significant separations between the C group and the HA, HA-HF, and HF groups (P < 0.01, respectively). These results showed that DHT with HFD could lower diversity of the gut microbial community. Both HFD and DHT could shift the overall gut microbial composition and change the composition of the microbial community in gut. Furthermore, our analyses demonstrated that the levels of TG, MDA, TNF-α, LPS, TLR4, T, FT, FINS, and HDL-C were correlated with the changes of in the gut microbiome. HFD and DHT were associated with the development and pathology of PCOS by shaping gut microbial communities.

FSH/AMH Ratio and Adipocyte Size are Linked to Ovarian Dysfunction

ABSTRACT Background and Aim While the relationship between obesity and reproductive dysfunction is well known, the physiological mechanism behind obesity-related infertility remains unclear. Previous work suggests that follicle development prior to ovulation is disrupted in obese individuals. Follicle-stimulating hormone (FSH) and anti-Mullerian hormone (AMH) are two key regulators of follicle development, and the poorest reproductive outcomes have been recorded when these hormones are imbalanced. In order to understand how obesity impacts the reproductive axis, the present study induces reproductive dysfunction in female rats using a high-fat, high-sugar diet (HFHS). Results: In our study, several animals on the HFHS diet displayed abnormal estrous cycles. The HFHS diet also resulted in an increased prevalence of ovarian cysts and decreased formation of corpora lutea. Across all groups, the FSH/AMH ratio displayed a strong negative correlation with pre-antral, antral, and total follicle counts. Moreover, rats on the HFHS diet displayed larger adipocytes and produced higher levels of leptin than controls. When combined with average adipocyte size in multiple regression, the FSH/AMH ratio was strongly associated with cyst formation in the ovary. Conclusions: These findings provide strong evidence for the potential relevance of a combined FSH/AMH ratio as a marker of ovarian health and follicular status. Therefore, this ratio reflects a complex interaction between the reproductive and metabolic systems.

Ascorbic acid and curcumin alleviate abnormal estrous cycle and morphological changes in cells induced by repeated ultraviolet B radiations in female Wistar rats

Objective: To study the protective effect of ascorbic acid and curcumin against the abnormal estrous cycle and morphological changes in cells induced by repeated ultraviolet B (UVB) radiation in female Wistar rats.Methods: Sixteen female sexually mature Wistar rats weighing 130-150 g and aged 12-16 weeks were randomly divided into four groups. The control group received normal food and water ad libitum. The UVB group was exposed to a dose of 280 nm of UVB radiation for 2 h daily. The UVB+curcumin group received a dose of 280 nm of UVB radiation for 2 h daily and also an oral dose of curcumin (25 mg/kg body weight) daily. The UVB+ascorbic acid group received a dose of 280 nm of UVB radiation for 2 h daily and also an oral dose of ascorbic acid (250 mg/kg body weight) daily. All the treatments last for 15 consecutive days. Body and ovary weight and gonadosomatic index were measured. The stages (proestrus, estrus, metaestrus and diestrus) of the estrous cycle were determined by the cell types observed in the vaginal smear.Results: UVB radiation caused irregular alterations on the estrous cycle and morphological changes of the female Wistar rat as compared with the control group. Ascorbic acid and curcumin protected UVB-induced estrous phases and their cells. But curcumin showed greater protection than ascorbic acid.Conclusions: Ascorbic acid and curcumin at low doses can alleviate abnormal estrous cycle and morphological changes in cells induced by UVB radiations in female Wistar rats.

High-fat diet causes dysregulation of ovarian endothelin-2 expression across the estrous cycle

We have previously shown that high-fat diet (HFD) feeding in female mice leads to abnormal estrous cyclicity, subfertility, and aberrant ovarian expression of genes important in ovulatory function, regardless of obese phenotype1,2. We found that a gene critical to normal ovulation, endothelin-2 (Edn2), is significantly downregulated in animals exposed to HFD. Edn2’s ovarian expression increases sharply right before ovulation. However, it is unknown how Edn2 is expressed in the ovary across the estrous cycle and how that expression is impacted by HFD. We aimed to evaluate ovarian Edn2 expression throughout the estrous cycle in HFD exposed mice and compare it with chow fed controls. Prospective laboratory animal study. 5-week-old C57Bl/6J mice were fed a standard chow or 60% HFD for 10 weeks. Estrous cyclicity was evaluated daily for the last two weeks of feeding and ovaries were collected in each of the four estrous cycle stages (N = 9/group/stage). T-test and chi-square tests were used for statistical analysis, as appropriate. After 10 weeks of diet, HFD mice weighed more than chow controls (28.8 ± 0.7g, 21.1 ± 0.2g p < 0.0001). HFD mice also had a higher prevalence of abnormal estrous cycles compared to chow controls (58.3% and 21.6% p = 0.0018). In chow controls, Edn2 was expressed as expected with basal levels during diestrus and proestrus, increased 11.6-fold during estrus, and decreased back to basal levels during metestrus. In the HFD mice, Edn2 was dysregulated across the entire estrous cycle (table 1), and when Edn2 expression was examined across all cycle stages in HFD mice, there was no characteristic peak of Edn2 expression in estrus with the lowest levels of Edn2 observed. Endothelin converting enzyme (Ece, cleaves Edn2 pre-peptide to active form) transcript expression levels were found to be uniformly upregulated in the HFD exposed mice across all stages of the estrous cycle (table 1).Tabled 1Estrous StageEdn2 Ovarian ExpressionEce Ovarian ExpressionHFD Compared to Chow Controls (fold change)p-valueHFD Compared to Chow Controls (fold change)p-valueDiestrus6.20.0136.6<0.0001Proestrus10.90.0394.20.0002Estrus-6.250.267.9<0.0001Metestrus-4.760.216.10.0002 Open table in a new tab Our data suggest that Edn2 and its post-translational regulation is dysregulated across the estrous cycle in HFD-fed mice. Work is currently underway to examine ovarian protein Edn2 levels across the estrous cycle to confirm our gene expression data. Future research should investigate mechanisms behind dysregulated Edn2 expression with HFD feeding. Collectively, this work will allow us to better understand how HFD leads to ovulatory dysfunction and to develop strategies targeting HFD-induced ovulation defects.