Ulipristal (UPA) effects on rat ovaries: Unraveling follicle dynamics, ovulation inhibition, and safety implications for prolonged use.

Ulipristal (UPA), a selective progesterone receptor modulator, has both agonistic and antagonistic effects on progesterone receptors. UPA suppresses ovulation by inhibiting the luteinizing hormone (LH) surge from the pituitary gland; however, the direct effect of UPA on ovarian tissue remains poorly studied. In the present study, we examined the effects of UPA on the ovaries of rats. Rats were treated for 28 days with UPA, and the effects of UPA on ovarian tissue were examined histologically and the expression of antioxidant genes and cell death markers were also investigated. UPA treatment increased the number of primordial follicles at each treatment group, primordial follicles increased at all dose levels, but the size/magnitude of the effect decreased with the increasing dose. The number of primary and antral follicles tended to increase with increasing UPA levels. Furthermore, the decrease in primary follicle number could be attributed to the exhaustion of follicles, but the examination of proliferation markers, oxidative stress markers, and cell death markers revealed no remarkable toxic effects on ovarian tissues. These results suggest that UPA treatment promotes follicle development at each stage but inhibits ovulation by suppressing the LH surge, resulting in an increase in atretic follicles or unruptured luteinized cysts. These results suggest that UPA may not have both toxic effects on the ovary and a direct local effect on ovarian follicles, but we should be careful about the effects of prolonged UPA treatment in patients with uterine fibroids on their future fecundity.

Validation of an on-chip p16ink4a/Ki-67 dual immunostaining cervical cytology system using microfluidic device technology.

More specific screening systems for cervical cancer may become necessary as the human papillomavirus (HPV) vaccine becomes more widespread. Although p16/Ki-67 dual-staining cytology has several advantages, it requires advanced diagnostic skills. Here, we developed an automated on-chip immunostaining method using a microfluidic device. An electroactive microwell array (EMA) microfluidic device with patterned thin-film electrodes at the bottom of each microwell was used for single-cell capture by dielectrophoresis. Immunostaining and dual staining for p16/Ki-67 were performed on diagnosed liquid cytology samples using the EMA device. The numbers of p16/Ki-67 dual-stained cells captured by the EMA device were determined and compared among the cervical intraepithelial neoplasia (CIN) lesion samples. Seven normal, fifteen CIN grade 3, and seven CIN grade 2 samples were examined. The percentage of dual-positive cells was 18.6% in the CIN grade 2 samples and 23.6% in the CIN grade 3 samples. The percentages of dual-positive staining increased significantly as the severity of the cervical lesions increased. p16/Ki67 dual immunostaining using the EMA device is as sensitive as the conventional method of confirming the histopathological diagnosis of cervical samples. This system enables a quantified parallel analysis at the individual cell level.

Prediction of the final menstrual period in women taking Dienogest using estradiol and follicle-stimulating hormone values: a case-control study.

Dienogest (DNG) is widely used to treat dysmenorrhea associated with estrogen-dependent diseases such as endometriosis and adenomyosis. DNG becomes unnecessary after menopause when estrogen secretion declines drastically. However, there are no clear criteria for when to halt DNG in perimenopausal patients. Menstruation and dysmenorrhea often resume after discontinuation due to approaching menopause. This case-control study used serum estradiol and follicle-stimulating hormone (FSH) levels to predict whether menstruation would resume in perimenopausal women after discontinuation of DNG. The study enrolled patients aged ≥40 years with endometriosis and/or adenomyosis and who had either completed oral DNG therapy (DNG group) or had spontaneous menopause without hormone therapy (control group). We assessed estradiol and FSH values before DNG termination or the final menstrual period. DNG group members that resumed menstruation after DNG termination (D (+) group, n = 17) had significantly higher estradiol and lower FSH levels than those who did not (D (-) group, n = 22) up to four months before DNG termination but not from four to 12 months. Estradiol and FSH levels were not significantly different between the D (-) and control groups. Receiver operating characteristic curves created from the estradiol and FSH values indicated that menstruation resumed when levels were ≥17 pg/mL and <100 mIU/mL, respectively. In contrast, menstruation did not resume in cases of estradiol ≤20 pg/mL and FSH >80 mIU/mL. The study results provide useful criteria for deciding when to terminate DNG in perimenopausal patients that consider their tolerance for resuming menstruation. Applications to menopause-inducing therapy for uterine fibroids and other conditions are anticipated. Further large-scale studies are needed.

Evaluation of atherosclerosis-related biomarkers during perimenopause: A prospective cohort study in women with endometriosis.

AIM: Endometriosis is linked to asymptomatic atherosclerosis and increases the risk of cardiovascular disease (CVD). Vascular function tests are used to assess atherosclerosis, an important indicator of CVD development. In this study, we aimed to evaluate atherosclerosis-related biomarkers, such as vascular function tests and laboratory data, in perimenopausal women with endometriosis. METHODS: This prospective cohort study enrolled 207 women (≥40 years old) with endometriosis. Laboratory data, ankle brachial index (ABI), and cardio-ankle vascular index (CAVI) were evaluated. RESULTS: The mean age, CAVI, and ABI of the participants at the initial examination were 45.02 years, 6.9 ± 0.6, and 1.07 ± 0.06, respectively. In multiple regression analysis, no factor was associated with CAVI, but ABI was significantly correlated with elevated total cholesterol (TC), low-density lipoprotein cholesterol, and estradiol (partial regression coefficient [ß] = -0.00074, p = 0.017; ß = -0.00075, p = 0.033; and ß = -0.00022, p = 0.015, respectively). The annual rate of change in CAVI showed a positive correlation with TC and pentosidine and a negative correlation with history of hormone therapy. Neither correlation was strong. In a comparison of three groups classified by the annual rate of change in CAVI, the group with the severe change had a higher level of pentosidine. In adjusted multinomial logistic regression analysis, high pentosidine levels were a risk factor for increased rate of change in CAVI. CONCLUSIONS: The association between CAVI and serum markers related to lipid metabolism and ovarian function was mild. Longitudinal analysis of CAVI showed an association with pentosidine, which may be helpful in assessing atherosclerosis risk in women with endometriosis during perimenopause.

Relationship between bone mineral density and ovarian function and thyroid function in perimenopausal women with endometriosis: a prospective study.

BACKGROUND: In women with endometriosis, the association between ovarian function, hormones, and bone mineral density (BMD) is unclear. Therefore, this study aimed to elucidate the association between changes in bone mineral density (BMD) and clinical data, such as ovarian reserves, in perimenopausal women with endometriosis. METHODS: In this prospective study, we evaluated 207 female patients who visited the Department of Obstetrics and Gynecology at the University of Tokyo Hospital between December 2015 and December 2020. We included patients aged ≥ 40 years with a history of endometriosis or who presented with endometriosis lesions. Patients with a history of smoking, steroid administration, autoimmune diseases, dyslipidaemia, and heart disease were excluded. During the study period, patients who underwent two tests, an initial and a follow-up test (n = 142, average age: 45.02 years, average BMD: 1.16 g/cm2), were evaluated at regular intervals based on the annual rate of change in BMD. RESULTS: There was a weak negative correlation between the follicle-stimulating hormone (FSH) and BMD and a weak positive correlation between the anti-Müllerian hormone (AMH) and BMD. The annual rate of change in BMD showed a very weak correlation with thyroid-stimulating hormone (TSH) levels. A large decline in BMD was associated with high TSH levels and higher average age at menopause. Patients with higher TSH exhibited a higher rate of decrease in BMD than those without. CONCLUSIONS: High FSH or low AMH levels are associated with decreased BMD. Decreased ovarian reserve is associated with decreased BMD in perimenopausal women with endometriosis. High TSH levels increase the risk of BMD loss. This finding may suggest that women with endometriosis should undergo bone scanning to rule out the possibility of reduced bone mass and subsequent increased risk of fracture.

Case report: Posterior reversible encephalopathy syndrome, an adverse effect of lenvatinib and pembrolizumab combination therapy, in a patient with advanced endometrial cancer.

Background: Lenvatinib-pembrolizumab combination (LEAP) is an approved therapy in Japan for advanced endometrial cancer, based on the data from the KEYNOTE-775 clinical trial. We report a case of posterior reversible encephalopathy syndrome (PRES) in a patient who received LEAP therapy for advanced endometrial cancer. Case presentation: A 53-year-old patient with stage IVB endometrial cancer having rectal metastases, after four cycles of paclitaxel-carboplatin therapy, was found to have increased rectal invasion, peritoneal dissemination, and multiple paraaortic lymph node metastases. She was treated with LEAP therapy and discharged on day 12 without adverse events, except for mild anemia on day 11 of treatment. She was carefully managed in the outpatient department, but on day 18, she was admitted to the emergency department with severely impaired consciousness and generalized seizures. Computed tomography of the head and lumbar tap showed no abnormal findings, and the seizures resolved with anticonvulsant medication alone. Based on a thorough physical examination and findings on magnetic resonance imaging (MRI), which showed high signal intensity in the left occipital lobe, encephalopathy, rather than encephalitis, was the likely diagnosis. Symptomatic improvement was observed, and pembrolizumab monotherapy was resumed. Conclusions: If consciousness is impaired during LEAP treatment, it is necessary to differentiate between immunogenic encephalitis caused by pembrolizumab or encephalopathy caused by lenvatinib. MRI and lumbar tap can help in distinguishing between the two and diagnosing the responsible drug.

Examining the association between menstrual symptoms and health-related quality of life among working women in Japan using the EQ-5D.

BACKGROUND: Menstrual symptoms have been identified as a substantial burden among women of reproductive age, affecting their health status and quality of life globally. A range of menstrual symptoms have been studied as they affect the health-related quality of life (HRQoL), showing variations across specific menstrual symptoms and study settings. A major concern is demonstrated due to menstrual symptoms in women's professional and social life, and consequently societal and economic loss for women and the society at large. Yet evidence is scarce that estimates the index form HRQoL score related to menstrual symptoms that is needed for health economic evaluations. METHODS: This study aims to investigate the association between menstrual symptoms and the HRQoL among working women in Japan in an index form, using a self-reporting questionnaire (n = 6048). The EQ-5D-3L (EuroQoL 5-dimension 3-level) is used that is a widely used tool to measure health outcomes for health economic evaluations globally. Multivariate regression analysis is conducted to assess the association between the HRQoL score and specific nineteen physical and mental conditions related to menstruation (e.g., pain, heavy bleeding, concentration, negative affect). RESULTS: The index form HRQoL score for menstrual symptoms is estimated as 0.682 in the study population (where a score one suggests perfect health). The association of the HRQoL score varies substantially across the menstrual symptoms. Several of the physical conditions and disorders show a substantial negative association with the HRQoL score. Also, most of the mental and psychological issues are significantly and negatively related to the HRQoL score. CONCLUSIONS: This study suggests that HRQoL is substantially and negatively affected by menstruation among working women in Japan. Distinct variations of negative influences across menstrual symptoms underscore the multi-dimensional nature of menstruation and consequently the need of collective interventions to address these difficulties. The evidence of HRQoL continues to be an important area for future research on women's health and health economic evaluations to inform effective and efficient resource allocations for relevant health policies and financing strategies.

Enhanced fallopian canal as a potential marker for temporal bone vasculitis.

OBJECTIVES: This study aimed to test the hypothesis that contrast-enhanced 3D MRI with gradient-echo sequences (CE-3D-GRE) can detect signs of vasculitis in the fallopian canal, which may cause otologic involvement, in four patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV). METHODS: CE-3D-GRE acquired at 3.0 Tesla was performed on four patients diagnosed with granulomatosis with polyangiitis or eosinophilic granulomatosis with polyangiitis, at onset or relapse of the disease, and in remission. Clinical correlations between otologic symptoms and radiological findings were examined for each patient. Furthermore, signal intensity of the mastoid segment of the fallopian canal was compared between the ears with active disease (n = 3) and those in remission or without vasculitis (n = 3). RESULTS: Intense enhancement in the tympanic and mastoid segments of the fallopian canal was associated with development of external otitis, otitis media, and sensorineural hearing loss, and was unrelated to the presence of facial paresis. Maximal intensity projection images visualized the close relationship between the enhanced fallopian canal and middle ear inflammation. The findings were absent in remission. Signal intensity of the mastoid segment of the fallopian canal was higher in ears with active disease than in normal ears (P < .001) and decreased to normal levels during remission (P = .597). CONCLUSION: CE-3D-GRE can demonstrate vasculitis in the temporal bone, reflecting disease activity and the severity of otologic manifestations, including cochlear involvement, in AAV patients. Intense enhancement of the fallopian canal on CE-3D-GRE can be a potential marker for vasculitis of the temporal bone. LEVEL OF EVIDENCE: 5.

A case of functioning gonadotroph adenoma in a reproductive aged woman.

A 34-year-old woman presented our hospital with complaint of irregular menstruation and abnormal uterine bleeding lasting for a month. After her second parturition at the age of 27, her menstrual cycle had been regular, but it suddenly became irregular at the age of 30. Transvaginal ultrasound revealed the presence of ovarian mass, and the patient underwent diagnostic laparoscopic surgery. Bilateral ovaries temporally shrink after puncture but the size soon resumed. Gonadotropins were almost normal, but estradiol and PRL levels turned out to be elevated, and cabergoline treatment was initiated. After referral to our hospital, we found that the ovaries showed multifollicular appearance. Brain magnetic resonance imaging showed an 18-mm macroadenoma in the suprasellar area. To suppress the secretion of endogenous gonadotropins and estrogen, low-dose estrogen-progestin was prescribed. Surprisingly, the treatment temporarily reduced the size of the ovaries. The patient was referred to a neurosurgeon, and a functioning gonadotroph adenoma was suspected. After the resection of the pituitary tumor, her menstrual cycle became regular, and the size of bilateral ovaries became normal. We also noticed that her ovarian reserve judged by anti-Müllerian hormone had been almost diminished after the surgical treatment, probably reflecting the exhaustion of follicular pool. Women with multifollicular ovaries and elevated estradiol levels may have functioning gonadotroph adenomas, although the level of FSH is relatively normal, and ovarian reserve can be followed by measuring anti-Müllerian hormone.

Activation of Nrf2/Keap1 pathway by oral Dimethylfumarate administration alleviates oxidative stress and age-associated infertility might be delayed in the mouse ovary.

BACKGROUND: Age-associated infertility is a problem worldwide, and management of oxidative stress is known to be essential. Nuclear factor-E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1)-antioxidant response element (ARE) signaling pathway works as an essential defense mechanism against oxidative stress, and an oral drug Dimethylfumarate (DMF) is known to activate the pathway. METHODS: We tested the hypothesis that oral DMF could alleviate oxidative stress in the ovary, resulting in salvation of age-associated infertility in a mouse model of reproductive age, and we examined the effects of DMF administration. 20 mg/kg DMF was administrated to female mice from 32 to 48 weeks, and Nrf2 levels, antioxidant levels, ovarian reserve, DNA damage, and oxidative stress were examined. RESULTS: DMF administration resulted in elevated mRNA and protein levels of Nrf2, antioxidants, and telomere, and serum levels of Nrf2 and anti-mullerian hormone were also elevated. Results of TUNEL assay and Immunohistochemistry of mice ovarian tissues showed that DNA damage and oxidative stress were decreased by DMF administration, and significantly more oocytes were collected along with preservation of 60% more primordial follicles. CONCLUSIONS: Our data suggest that DMF administration activates the Nrf2/Keap1 pathway, elevate levels of antioxidants, and decrease DNA damage and oxidative stress, resulting in improved ovarian reserve in the mouse ovary.

Activation of Nrf2 might reduce oxidative stress in human granulosa cells.

Nuclear factor-E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1)-antioxidant response element (ARE) signaling pathway is one of the most important defense mechanisms against oxidative stress (OS). It is well documented that equilibration status of OS plays fundamental roles in human reproductive medicine, and the physiological role of Nrf2 in ovarian granulosa cells (GCs) has not been determined yet. Herein we aimed to study the function of Nrf2 in GCs. Human ovarian tissues were subjected to immunohistochemistry to localize Nrf2 and Keap1 and we detected the expression of Nrf2 and Keap1 in the human GCs. Human luteinized GCs were isolated and cultured, and hydrogen peroxide (H2O2) or Dimethylfumarates (DMF), an activator of Nrf2, were added to GCs to analyze the relationship between Nrf2 and antioxidants by quantitative RT-PCR. The mRNA levels of Nrf2, catalase, superoxide dismutase 1 (SOD1), and 8-Oxoguanine DNA glycosylase (OGG1) were elevated by H2O2, and DMF treatment showed similar but pronounced effects through activation of Nrf2. To determine the relationship of Nrf2 and the generation of antioxidants, siRNAs were used and quantitative RT-PCR were conducted. Decreased expression of Nrf2 resulted in a decreased level of these antioxidant mRNA. Intracellular levels of ROS were investigated by fluorescence of 8-hydroxy-2'-deoxyguanosine and fluorescent dye, 2',7'-dichlorodihydrofluorescein diacetate after H2O2 and/or DMF treatment, and DMF treatment quenched intracellular ROS generation by H2O2. These results show that activation of Nrf2 might lead to alleviate OS in human GCs, and this could provide novel insight to conquer the age-related fertility decline that is mainly attributed to the accumulation of aberrant OS.

The Emerging Role of FOXL2 in Regulating the Transcriptional Activation Function of Estrogen Receptor ß: An Insight Into Ovarian Folliculogenesis.

Germline mutations of the fork-head transcriptional factor forkhead box L2 (FOXL2) predispose embryos to autosomal-dominant blepharophimosis-ptosis-epicanthus inversus syndrome with primary ovarian insufficiency in female patients, but the mechanisms of FOXL2 in ovarian follicular development remain elusive. Estrogens produced by ovarian granulosa cells and estrogen receptor (ER) α and ERß play fundamental roles in ovarian pathophysiology, and a previous study revealed that ERα and ERß physically interact with FOXL2. However, the underlying functions of these interactions have not been investigated. Herein, we report an ERß-specific repressive function of FOXL2. Histological examination demonstrated that FOXL2 expression tends to be intense during early follicular development. Immunoprecipitation revealed that ERß and FOXL2 interact in a ligand-independent manner. In vitro pull-down assays revealed a direct interaction between FOXL2 and the activation function (AF)-1/2 domain of ERß. The expression of FOXL2 represses the ligand-dependent transcriptional activation of ERß, but FOXL2 does not influence the ligand-dependent transcriptional activation of ERα. Consistent with these results, RNA interference-mediated depletion of FOXL2 stimulates the expression of the ERß-downstream gene p450 aromatase. The convergence between FOXL2 functions and ERß-mediated transcription in the ovary suggests the putative mechanism of FOXL2 in early-phase follicular development, which may be partially attributed to the regulation of ERß-dependent gene expression.

HAND2-mediated proteolysis negatively regulates the function of estrogen receptor α.

A previous study demonstrated that the progesterone­inducible HAND2 gene product is a basic helix­loop­helix transcription factor and prevents mitogenic effects of estrogen receptor α (ERα) by inhibiting fibroblast growth factor signalling in mouse uteri. However, whether HAND2 directly affects the transcriptional activation function of ERα remains to be elucidated. In the present study, the physical interaction between HAND2 and ERα was investigating by performing an immunoprecipitation assay and an in vitro pull­down assay. The results demonstrated that HAND2 and ERα interacted in a ligand­independent manner. The in vitro pull­down assays revealed a direct interaction between HAND2 and the amino­terminus of ERα, termed the activation function­1 domain. To determine the physiological significance of this interaction, the role of HAND2 as a cofactor of ERα was investigated, which revealed that HAND2 inhibited the ligand­dependent transcriptional activation function of ERα. This result was further confirmed and the mRNA expression of vascular endothelial growth factor, an ERα­downstream factor, was decreased by the overexpression of HAND2. This inhibition of ligand­dependent transcriptional activation function of ERα was possibly attributed to the proteasomic degradation of ERα by HAND2. These results indicate a novel anti­tumorigenic function of HAND2 in regulating ERα­dependent gene expression. Considering that HAND2 is commonly hypermethylated and silenced in endometrial cancer, it is hypothesized that HAND2 may serve as a possible tumor suppressor, particularly in uterine tissue.

CCAR2 negatively regulates nuclear receptor LXRα by competing with SIRT1 deacetylase.

Liver X receptors (LXRs) monitor endogenous sterol levels to maintain whole-body cholesterol levels and regulate inflammatory responses. Recent studies have demonstrated that LXRs may inhibit cellular proliferation, but the underlying mechanism remains unclear. Cell cycle and apoptosis regulator 2 (CCAR2), previously known as DBC1/KIAA1967, is a transcriptional regulator that regulates cellular proliferation and energy metabolism by inhibiting sirtuin 1 (SIRT1) deacetylase. Based on the findings that CCAR2 regulates several nuclear receptors, including the estrogen receptors and androgen receptor, we aimed to identify the underlying mechanism of CCAR2 regulation of LXRα. We found that CCAR2 formed a complex with LXRα in a ligand-independent manner in HepG2 cells, and in vitro pull-down assays, it revealed a direct interaction between the amino terminus of CCAR2 and the AF-2 domain of LXRα. Thereby, CCAR2 attenuates the ligand-dependent transcriptional activation function of LXRα. RNA interference-mediated depletion of endogenous CCAR2 potentiated the expression of the LXRα target genes ATP-binding cassette transporter A1 and G1, and the abrogation of CCAR2 resulted in decreased cellular proliferation. Moreover, competitive immunoprecipitation studies revealed that the LXRα downregulation involves the inhibition of SIRT1-LXRα complex formation. Therefore, these results clearly indicate a novel mechanism in which CCAR2 may regulate the transcriptional activation function of LXRα due to its specific inhibition of SIRT1 and serve to regulate cellular proliferation.

SIRT3 positively regulates the expression of folliculogenesis- and luteinization-related genes and progesterone secretion by manipulating oxidative stress in human luteinized granulosa cells.

SIRT3 is a member of the sirtuin family and has recently emerged as a vital molecule in controlling the generation of reactive oxygen species (ROS) in oocytes. Appropriate levels of ROS play pivotal roles in human reproductive medicine. The aim of the present study was to investigate SIRT3 expression and analyze the SIRT3-mediated oxidative response in human luteinized granulosa cells (GCs). Human ovarian tissues were subjected to immunohistochemical analysis to localize SIRT3 expression. Hydrogen peroxide and human chorionic gonadotropin were used to analyze the relationship between ROS and SIRT3 by quantitative RT-PCR and Western blotting. Intracellular levels of ROS were investigated by fluorescence after small interfering RNA-mediated knockdown of SIRT3 in human GCs. To uncover the role of SIRT3 in folliculogenesis and luteinization, mRNA levels of related genes and the progesterone concentration were analyzed by quantitative RT-PCR and immunoassays, respectively. We detected the expression of SIRT3 in the GCs of the human ovary. The mRNA levels of SIRT3, catalase, and superoxide dismutase 1 were up-regulated by hydrogen peroxide in both COV434 cells and human GCs and down-regulated by human chorionic gonadotropin. Knockdown of SIRT3 markedly elevated ROS generation in human GCs. In addition, SIRT3 depletion resulted in decreased mRNA expression of aromatase, 17ß-hydroxysteroid dehydrogenase 1, steroidogenic acute regulatory protein, cholesterol side-chain cleavage enzyme, and 3ß-hydroxysteroid dehydrogenase in GCs and thus resulted in decreased progesterone secretion. These results have the important clinical implication that SIRT3 might play a positive role in the folliculogenesis and luteinization processes in GCs, possibly by sensing and regulating the generation of ROS. Activation of SIRT3 function might help to sustain human reproduction by maintaining GCs as well as oocytes.

Regulation of SIRT1 determines initial step of endometrial receptivity by controlling E-cadherin expression.

Sirtuin 1 (SIRT1), originally found as a class III histone deacetylase, is a principal modulator of pathways downstream of calorie restriction, and the activation of SIRT1 ameliorates glucose homeostasis and insulin sensitivity. We examined the role of SIRT1 in the regulation of uterine receptivity using Ishikawa and RL95-2 endometrial carcinoma cell lines. Exogenous expression of SIRT1 significantly enhanced E-cadherin expression, while small interfering RNA-mediated depletion of endogenous SIRT1 resulted in a significant reduction of E-cadherin expression. A SIRT1 activator resveratrol elevated E-cadherin expression in a dose dependent manner, while SIRT1 repressors nicotinamide and sirtinol exhibited a dose dependent reduction of E-cadherin expression. We also showed that both forced expression of SIRT1 and activation of SIRT1 promote E-cadherin-driven reporter gene constructs, and SIRT1 is localized at E-cadherin promoter containing E-box elements in Ishikawa cells. Using an in vitro model of embryo implantation, we demonstrate that exogenous expression of SIRT1 and stimulation of SIRT1 activity resulted in the Ishikawa cell line becoming receptive to JAR cell spheroid attachment. Furthermore, resveratrol enhanced E-cadherin and Glycodelin protein expression at sites of intercellular contact, suggesting an additive role of resveratrol in promoting implantation. The initial step of human reproduction depends on the capacity of an embryo to attach and implant into the endometrial wall, and these results revealed the novel mechanism that activation and increased expression of SIRT1 play an important role in uterine receptivity.

Resveratrol promotes expression of SIRT1 and StAR in rat ovarian granulosa cells: an implicative role of SIRT1 in the ovary.

BACKGROUND: Resveratrol is a natural polyphenolic compound known for its beneficial effects on energy homeostasis, and it also has multiple properties, including anti-oxidant, anti-inflammatory, and anti-tumor activities. Recently, silent information regulator genes (Sirtuins) have been identified as targets of resveratrol. Sirtuin 1 (SIRT1), originally found as an NAD+-dependent histone deacetylase, is a principal modulator of pathways downstream of calorie restriction, and the activation of SIRT1 ameliorates glucose homeostasis and insulin sensitivity. To date, the presence and physiological role of SIRT1 in the ovary are not known. Here we found that SIRT1 was localized in granulosa cells of the human ovary. METHODS: The physiological roles of resveratrol and SIRT1 in the ovary were analyzed. Immunohistochemistry was performed to localize the SIRT1 expression. SIRT1 protein expression of cultured cells and luteinized human granulosa cells was investigated by Western blot. Rat granulosa cells were obtained from diethylstilbestrol treated rats. The cells were treated with increasing doses of resveratrol, and subsequently harvested to determine mRNA levels and protein levels. Cell viability was tested by MTS assay. Cellular apoptosis was analyzed by caspase 3/7 activity test and Hoechst 33342 staining. RESULTS: SIRT1 protein was expressed in the human ovarian tissues and human luteinized granulosa cells. We demonstrated that resveratrol exhibited a potent concentration-dependent inhibition of rat granulosa cells viability. However, resveratrol-induced inhibition of rat granulosa cells viability is independent of apoptosis signal. Resveratrol increased mRNA levels of SIRT1, LH receptor, StAR, and P450 aromatase, while mRNA levels of FSH receptor remained unchanged. Western blot analysis was consistent with the results of quantitative real-time RT-PCR assay. In addition, progesterone secretion was induced by the treatment of resveratrol. CONCLUSIONS: These results suggest a novel mechanism that resveratrol could enhance progesterone secretion and expression of luteinization-related genes in the ovary, and thus provide important implications to understand the mechanism of luteal phase deficiency.