Local Cortisol Elevation Contributes to Endometrial Insulin Resistance in Polycystic Ovary Syndrome

Local Cortisol Elevation Contributes to Endometrial Insulin Resistance in Polycystic Ovary Syndrome Abstract Context Endometrial insulin resistance (IR) may account for the endometrial dysfunction in polycystic ovary syndrome (PCOS). The underlying mechanism remains to be elucidated. Objective To investigate whether the abundance of 11β-hydroxysteroid dehydrogenases (11β-HSDs) 1 and 2 and cortisol as well as the insulin signaling pathway are altered in PCOS endometrium and to clarify the relationship between endometrial IR and local cortisol. Design We measured cortisol and cortisone concentrations, 11β-HSD1 and 11β-HSD2, and core insulin signaling molecules in endometrial biopsies collected from non-PCOS and PCOS with or without IR patients on the seventh day after human chorionic gonadotropin injection. We also studied the effects of cortisol on glucose uptake and the insulin signaling pathway in primary cultured endometrial epithelial cells (EECs). Results The cortisol concentration was elevated, whereas 11β-HSD2 expression was diminished in endometrial biopsies obtained from PCOS with IR patients compared with those from non-PCOS and PCOS without IR patients. The implantation rate was relatively impaired and the endometrial insulin signaling pathway was defective in PCOS with IR patients. In addition, cortisol attenuated insulin-stimulated glucose uptake in EECs, which was mediated by inhibition of Akt phosphorylation and glucose transporter type 4 translocation via induction of phosphatase and tensin homolog deleted on chromosome ten (PTEN). Conclusions Decreased oxidation of cortisol and defects of insulin signaling in endometrium were observed in PCOS with IR patients. The excessive cortisol level, derived from the reduction of 11β-HSD2, might contribute to the development of endometrial IR by inhibiting the insulin signaling pathway via induction of PTEN expression in EECs. Polycystic ovary syndrome (PCOS), a common and complex endocrine disorder, affects 5% to 20% of reproductive-age women with short- and long-term effects (1). PCOS is characterized by polycystic ovaries, ovulatory dysfunction, and hyperandrogenism (2). In addition, a high percentage of patients with PCOS have symptoms of insulin resistance (IR) (2–4). The abnormal endocrine and metabolic characteristics of PCOS might be detrimental to endometrial function, manifesting as endometrial hyperplasia or cancer and reduction of receptivity (5–7). Patients with PCOS have been reported to have adverse reproductive outcomes, including higher abortion rates compared with the unaffected population (7). The reduction in fertility might not only be attributed to ovulatory dysfunction, but also to endometrial defects. When compared with fertile endometrium, several alterations of the insulin signaling pathway in PCOS endometrium have been reported, such as increased phosphatase and tensin homolog deleted on chromosome ten (PTEN) as well as decreased insulin receptor substrate 1 (IRS-1) (8, 9). Some studies have linked endometrial IR to decreased endometrial receptivity (10) and tumorigenesis (11, 12). However, the etiology of local endometrial IR remains to be elucidated. Endogenous glucocorticoids play a crucial role in many areas, including in the pathophysiology of IR. The conversion of inactive glucocorticoids and active glucocorticoids is catalyzed by 11β-hydroxysteroid dehydrogenases (11β-HSDs). There are two types of 11β-HSDs: 11β-HSD1 and 11β-HSD2 (13, 14). 11β-HSD1 has both reductase and oxidase functions, bidirectionally converting biologically inactive cortisone and active cortisol; 11β-HSD2 only has an oxidase function, converting active cortisol to inactive cortisone (15). Several studies have demonstrated that cortisol concentration and 11β-HSDs were changed in serum, adipose tissue, and granulosa cells in PCOS patients (16–18). In addition, our previous study showed that cortisol generated locally by 11β-HSD1 contributed to IR in granulosa cells in PCOS (19). However, it is unclear whether the local generation of cortisol in endometrium exerts a role in the process of endometrial IR in PCOS. Considering the ovarian 11β-HSD alterations in women with PCOS, our primary aim in this study was to clarify whether there is an imbalanced state of glucocorticoid and its metabolic enzymes in endometria from patients with PCOS with or without IR. We also sought to establish a possible correlation between cortisol and local IR. A secondary aim of this study was to clarify in vitro whether cortisol could abolish insulin-stimulated glucose uptake and insulin signaling pathway in primary cultured endometrial epithelial cells (EECs). Materials and Methods Patients and tissue collection The endometrial biopsies were collected with endometrial suction curettes (Runting) from PCOS and non-PCOS patients undergoing gonadotropin-releasing hormone antagonist stimulation cycle without fresh embryo transfer. The biopsies were collected on the seventh day after human chorionic gonadotropin (hCG) injection, known as the window of implantation (WOI) phase. The diagnosis of PCOS was established according to the revised Rotterdam consensus (20). The subgroups of PCOS with IR and PCOS without IR were subdivided according to the homeostasis model assessment of IR index (HOMA-IR [fasting serum insulin (μIU/mL) × fasting serum glucose (mmol/L)/22.5]), with 3.15 selected as a cutoff point (21). Non-PCOS patients were women with regular menstrual cycles, normal body mass index (18.5 to 23.9 kg/m2), and only tubal infertile conditions without IR. The endometrial biopsies from non-PCOS (n = 18), PCOS without IR (n = 18), and PCOS with IR (n = 18) were snap frozen in liquid nitrogen for extraction of glucocorticoids, messenger RNA (mRNA), and protein to detect cortisol, cortisone, 11β-HSD mRNA and protein, phosphorylated IRS-1 and IRS-1, phosphorylated Akt and Akt, and PTEN mRNA and protein. The EECs were isolated from other patients including non-PCOS (n = 29) and PCOS (n = 21). All procedures were performed at the Center for Reproductive Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University. Written informed consent was obtained, and approval of the ethics protocol was granted from the Ethics Committee of Renji Hospital (2017081109). Demographic features and clinical outcomes Baseline serum hormonal profiles, including follicle-stimulating hormone, luteinizing hormone, testosterone, estradiol, and anti-Müllerian hormone, were determined using chemiluminescence assay kits (Beckman Access Health Co.). Serum fasting insulin and fasting glucose were measured using a chemiluminescence assay kit (Beckman Access Health Co.) and a standard glucose oxidase method (Roche), respectively. Quantitative insulin sensitivity check index was calculated as 1/[logI0(μIU/mL) + logG0(mg/dL)]. The implantation rate was defined as the number of gestational sacs per number of embryos transferred in every frozen embryo transfer cycle. Extraction and measurement of cortisol and cortisone in endometrium The endometrium was ground in liquid nitrogen and extracted with ethyl acetate. After evaporation, the extract was resuspended in phosphate-buffered saline. The suspension was equally divided and reconstituted in the assay buffer provided by the manufacturers and then measured using a cortisol assay kit (R&D Systems) and a cortisone chemiluminescent immunoassay kit (Innovative Research) following manufacturer instructions. Immunohistochemical and immunofluorescent staining Protein expression of 11β-HSD2 was assessed in paraffin-embedded endometrial tissue sections. Immunostaining was performed on 5-μm-thick tissue sections as previously described (22). Briefly, the endogenous peroxidase activity was quenched with 3% H2O2, and then 11β-HSD2 or preimmune serum at 1:500 dilution was used as a primary or negative control for overnight incubation at 4°C followed by secondary antibody for 30 minutes at 37°C, respectively. The colorimetric reactions were developed using a standard diaminobenzidine kit (ZSGB-BIO). Immunofluorescent staining was performed on cultured cells fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. After blocking and incubation with primary antibodies, cells were incubated with Alexa Fluor 488- and 594-labeled secondary antibodies (Proteintech). Nuclei were stained with 4´,6´-diamino-2-phenylindole (1 μg/mL). Images were obtained with a microscope and camera connected to a computer with an image analysis system (Zeiss). The primary antibodies are listed in Supplemental Table 1. Cell culture and treatment EECs were enzymatically isolated from human endometrium curettage samples according to a selective attachment method (23) with minor modifications. Briefly, endometrial samples were digested with collagenase type I and deoxyribonuclease and then sequentially size fractionated with 180- and 40-μm sieves. Epithelial glands were retained on the 40-μm sieve and were collected by backwashing the 40-μm filter paper and resuspended in Dulbecco’s modified Eagle medium/Hams F12 containing 10% fetal bovine serum (Gibco) and 1% antibiotic-antimycotic solution (Gibco). Cell purity was tested routinely by immunofluorescence staining for cytokeratin and vimentin. To compare the glucose uptake and glucose transporter type 4 (GLUT4) translocation among non-PCOS, PCOS without IR, and PCOS with IR patients, the cells were cultured in phenol red-free and serum-free medium 1 day after plating and stimulated with insulin (100 nM; Sigma) for 20 and 30 minutes. To study the role of cortisol on the insulin-stimulated glucose uptake, Akt phosphorylation, and GLUT4 translocation, EECs were treated in phenol red-free and serum-free culture medium after plating for 3 days. After treatment with cortisol (1 μM; Sigma) for 24 hours, the cells were stimulated with insulin for 20, 15, and 30 minutes, respectively. The measurement of glucose uptake is described below. To detect the effect of cortisol on PTEN expression, cells were treated with cortisol (0.1 and 1 μM) for 24 hours before analysis with Western blotting or quantitative real-time polymerase chain reaction (PCR) as described below. To determine the involvement of PTEN, the PTEN inhibitor bpV (phen) (1 μM; Sigma) was added to EECs for 30 minutes before insulin stimulation. Glucose uptake Glucose uptake in EECs was measured after insulin stimulation (100 nM, 20 minutes) using Glucose Uptake-Glo Assay (Promega) according to manufacturer instructions. Quantitative real-time PCR Total RNA from cells and endometrial biopsies was extracted using a total RNA Kit (Omega Bio-Tek) according to manufacturer instructions and was reverse transcribed to complementary DNA using the PrimeScript reverse transcription kit (TaKaRa) with appropriate controls. Quantitative real-time PCR was performed and analyzed with ABI Prism System (Applied Biosystems) using SYBR Premix (TaKaRa) in triplicate. Relative mRNA expression was calculated by the comparative cycle threshold method with ACTB as the housekeeping gene. The primer sequences are listed in Supplemental Table 2. Protein extraction and Western blotting Total protein was extracted from cells and endometrial biopsies using ice-cold radio-immunoprecipitation assay lysis buffer (CWBIO) containing protease inhibitor cocktail (Roche) and phosphatase inhibitor (Active Motif). Membrane and cytoplasmic proteins were extracted using the Membrane and Cytoplasmic Protein Extraction Kit (Sangon Biotech). Protein was quantified with a Bradford assay, and 20 μg protein of each sample was electrophoresed in 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to a nitrocellulose blot. After blocking and incubation with primary antibodies, the membranes were incubated with the respective secondary antibody conjugated with horseradish peroxidase (Proteintech) for 1 hour. Bands with peroxidase activity were detected by an enhanced chemiluminescent detection kit (Merck Millipore) and visualized with a G-Box chemiluminescence image capture system (Syngene). Primary antibodies are listed in Supplemental Table 1. Statistical analysis All data are reported as the mean ± standard deviation (SD). Analyses were performed using the Statistical Package for Social Science (version 16.0; SPSS) and Graphpad Prism statistical software (version 5.0, Graphpad). The data were initially subjected to Kolmogorov-Smirnov tests to assess deviation from Gaussian distribution. For normally distributed data, we applied unpaired t test and one-way analysis of variance followed by Bonferroni tests. For data not normally distributed, we applied Kruskal-Wallis test followed by Dunn’s multiple comparison test. Correlation between variables was performed using Pearson correlation analysis. P < 0.05 was considered to be statistically significant. Results Clinical characteristics and implantation outcomes The demographic characteristics and implantation outcomes of recruited participants are displayed in Table 1. Fifty-four patients were classified into three groups: non-PCOS (n = 18), PCOS without IR (n = 18), and PCOS with IR (n = 18). Patient age, basal follicle-stimulating hormone, estradiol, and hormones on hCG injection day were comparable among the three groups. Basal levels of anti-Müllerian hormone, luteinizing hormone, and testosterone were significantly higher in PCOS with or without IR than in non-PCOS patients. The fasting insulin, HOMA-IR, and quantitative insulin sensitivity check index were significantly higher in PCOS with IR patients than in non-PCOS and PCOS without IR patients. This is a characteristic of IR syndrome. Although there was not a significant difference, there was a tendency for the implantation rate in PCOS with IR women to be lower than that of non-PCOS (P = 0.06). Table 1. Demographic Features and Clinical Outcomes of Recruited Patients Non-PCOS (n = 18) PCOS Without IR (n = 18) PCOS With IR (n = 18) Age, y 27.67 ± 2.74 28.28 ± 3.39 28.56 ± 2.20 BMI, kg/m2 20.84 ± 2.19 23.54 ± 4.48 25.79 ± 3.84a Basal FSH, mIU/mL 7.13 ± 1.71 6.04 ± 1.23 6.13 ± 1.17 Basal LH, mIU/mL 4.87 ± 1.67 6.88 ± 5.77a 9.66 ± 7.36a Basal E2, pg/mL 42.40 ± 17.94 38.68 ± 20.68 42.24 ± 17.45 Basal T, nmol/L 1.01 ± 0.58 1.66 ± 0.67a 1.84 ± 0.83a Hormones on hCG day  LH, mIU/mL 2.04 ± 1.69 1.48 ± 0.85 2.13 ± 1.52  E2, pg/mL 2988.44 ± 1613.16 3992.64 ± 1825.50 3157.94 ± 1436.53  P4, ng/mL 0.92 ± 0.27 1.29 ± 0.67 1.21 ± 1.14 Fasting glucose, mmol/L 4.46 ± 0.37 4.88 ± 0.38 5.42 ± 1.44a Fasting insulin, μIU/mL 6.49 ± 1.99 8.91 ± 2.00 17.31 ± 5.91a,b HOMA-IR 1.30 ± 0.45 1.91 ± 0.39 4.18 ± 1.90a,b QUICKI 0.37 ± 0.03 0.35 ± 0.01a 0.31 ± 0.01a,b AMH, ng/mL 5.29 ± 1.44 12.99 ± 4.15a 10.94 ± 5.04a Frozen embryo transfer cycle 27 24 24 Implantation rate 50.0% ± 46.0% 37.5% ± 39.7% 27.1% ± 39.0% Non-PCOS (n = 18) PCOS Without IR (n = 18) PCOS With IR (n = 18) Age, y 27.67 ± 2.74 28.28 ± 3.39 28.56 ± 2.20 BMI, kg/m2 20.84 ± 2.19 23.54 ± 4.48 25.79 ± 3.84a Basal FSH, mIU/mL 7.13 ± 1.71 6.04 ± 1.23 6.13 ± 1.17 Basal LH, mIU/mL 4.87 ± 1.67 6.88 ± 5.77a 9.66 ± 7.36a Basal E2, pg/mL 42.40 ± 17.94 38.68 ± 20.68 42.24 ± 17.45 Basal T, nmol/L 1.01 ± 0.58 1.66 ± 0.67a 1.84 ± 0.83a Hormones on hCG day  LH, mIU/mL 2.04 ± 1.69 1.48 ± 0.85 2.13 ± 1.52  E2, pg/mL 2988.44 ± 1613.16 3992.64 ± 1825.50 3157.94 ± 1436.53  P4, ng/mL 0.92 ± 0.27 1.29 ± 0.67 1.21 ± 1.14 Fasting glucose, mmol/L 4.46 ± 0.37 4.88 ± 0.38 5.42 ± 1.44a Fasting insulin, μIU/mL 6.49 ± 1.99 8.91 ± 2.00 17.31 ± 5.91a,b HOMA-IR 1.30 ± 0.45 1.91 ± 0.39 4.18 ± 1.90a,b QUICKI 0.37 ± 0.03 0.35 ± 0.01a 0.31 ± 0.01a,b AMH, ng/mL 5.29 ± 1.44 12.99 ± 4.15a 10.94 ± 5.04a Frozen embryo transfer cycle 27 24 24 Implantation rate 50.0% ± 46.0% 37.5% ± 39.7% 27.1% ± 39.0% All data are mean ± standard deviation values. Abbreviations: AMH, anti-Müllerian hormone; BMI, body mass index; E2, estradiol; FSH, follicle-stimulating hormone; LH, luteinizing hormone; P4, progesterone; QUICKI, quantitative insulin sensitivity check index; T, testosterone. a P < 0.05 vs non-PCOS. b P < 0.05 vs PCOS without IR. View Large Table 1. Demographic Features and Clinical Outcomes of Recruited Patients Non-PCOS (n = 18) PCOS Without IR (n = 18) PCOS With IR (n = 18) Age, y 27.67 ± 2.74 28.28 ± 3.39 28.56 ± 2.20 BMI, kg/m2 20.84 ± 2.19 23.54 ± 4.48 25.79 ± 3.84a Basal FSH, mIU/mL 7.13 ± 1.71 6.04 ± 1.23 6.13 ± 1.17 Basal LH, mIU/mL 4.87 ± 1.67 6.88 ± 5.77a 9.66 ± 7.36a Basal E2, pg/mL 42.40 ± 17.94 38.68 ± 20.68 42.24 ± 17.45 Basal T, nmol/L 1.01 ± 0.58 1.66 ± 0.67a 1.84 ± 0.83a Hormones on hCG day  LH, mIU/mL 2.04 ± 1.69 1.48 ± 0.85 2.13 ± 1.52  E2, pg/mL 2988.44 ± 1613.16 3992.64 ± 1825.50 3157.94 ± 1436.53  P4, ng/mL 0.92 ± 0.27 1.29 ± 0.67 1.21 ± 1.14 Fasting glucose, mmol/L 4.46 ± 0.37 4.88 ± 0.38 5.42 ± 1.44a Fasting insulin, μIU/mL 6.49 ± 1.99 8.91 ± 2.00 17.31 ± 5.91a,b HOMA-IR 1.30 ± 0.45 1.91 ± 0.39 4.18 ± 1.90a,b QUICKI 0.37 ± 0.03 0.35 ± 0.01a 0.31 ± 0.01a,b AMH, ng/mL 5.29 ± 1.44 12.99 ± 4.15a 10.94 ± 5.04a Frozen embryo transfer cycle 27 24 24 Implantation rate 50.0% ± 46.0% 37.5% ± 39.7% 27.1% ± 39.0% Non-PCOS (n = 18) PCOS Without IR (n = 18) PCOS With IR (n = 18) Age, y 27.67 ± 2.74 28.28 ± 3.39 28.56 ± 2.20 BMI, kg/m2 20.84 ± 2.19 23.54 ± 4.48 25.79 ± 3.84a Basal FSH, mIU/mL 7.13 ± 1.71 6.04 ± 1.23 6.13 ± 1.17 Basal LH, mIU/mL 4.87 ± 1.67 6.88 ± 5.77a 9.66 ± 7.36a Basal E2, pg/mL 42.40 ± 17.94 38.68 ± 20.68 42.24 ± 17.45 Basal T, nmol/L 1.01 ± 0.58 1.66 ± 0.67a 1.84 ± 0.83a Hormones on hCG day  LH, mIU/mL 2.04 ± 1.69 1.48 ± 0.85 2.13 ± 1.52  E2, pg/mL 2988.44 ± 1613.16 3992.64 ± 1825.50 3157.94 ± 1436.53  P4, ng/mL 0.92 ± 0.27 1.29 ± 0.67 1.21 ± 1.14 Fasting glucose, mmol/L 4.46 ± 0.37 4.88 ± 0.38 5.42 ± 1.44a Fasting insulin, μIU/mL 6.49 ± 1.99 8.91 ± 2.00 17.31 ± 5.91a,b HOMA-IR 1.30 ± 0.45 1.91 ± 0.39 4.18 ± 1.90a,b QUICKI 0.37 ± 0.03 0.35 ± 0.01a 0.31 ± 0.01a,b AMH, ng/mL 5.29 ± 1.44 12.99 ± 4.15a 10.94 ± 5.04a Frozen embryo transfer cycle 27 24 24 Implantation rate 50.0% ± 46.0% 37.5% ± 39.7% 27.1% ± 39.0% All data are mean ± standard deviation values. Abbreviations: AMH, anti-Müllerian hormone; BMI, body mass index; E2, estradiol; FSH, follicle-stimulating hormone; LH, luteinizing hormone; P4, progesterone; QUICKI, quantitative insulin sensitivity check index; T, testosterone. a P < 0.05 vs non-PCOS. b P < 0.05 vs PCOS without IR. View Large Cortisol and cortisone concentrations in human endometrial tissues No statistically significant differences were found in the summed concentrations of cortisol plus cortisone among the three groups [Fig. 1(a)]. Concentrations of cortisol in PCOS with IR patients were significantly higher compared with non-PCOS and PCOS without IR patients [Fig. 1(b)]. Concentrations of cortisone in PCOS with IR patients were significantly lower than those in non-PCOS patients and PCOS without IR patients [Fig. 1(c)]. Ratios of cortisol to cortisone were significantly elevated in PCOS with IR patients compared with non-PCOS and PCOS without IR patients [Fig. 1(d)]. The quantitative detection showed an imbalanced metabolic state between cortisol and cortisone in the endometria of PCOS with IR patients. Figure 1. View largeDownload slide The abundance of cortisol, cortisone, and 11β-HSDs in endometrium of non-PCOS, PCOS without IR, and PCOS with IR. (a–d) Endometrial concentrations of (a) cortisol plus cortisone, (b) cortisol, (c) cortisone, and (d) ratio of cortisol to cortisone in endometrial biopsies obtained from non-PCOS patients (n = 18), PCOS without IR patients (n = 18), and PCOS with IR patients (n = 18). *P < 0.05; **P < 0.01; ***P < 0.001. (e) The representative blot of 11β-HSD1 and 11β-HSD2 in endometrial biopsies from non-PCOS patients (n = 7), PCOS without IR patients (n = 7), and PCOS with IR patients (n = 7). (f and g) Quantification of 11β-HSD 1 and 11β-HSD2 in endometrial biopsies from non-PCOS patients (n = 18), PCOS without IR patients (n = 18), and PCOS with IR patients (n = 18). *P < 0.05; ***P < 0.001 vs non-PCOS; #P < 0.05 vs PCOS without IR. (h and i) Correlation of the abundance of 11β-HSD2 mRNA with (h) local cortisone and (i) cortisol in endometrial biopsies. Circles represent data points for non-PCOS (n = 18); squares represent data points for PCOS without IR (n = 18); triangles represent data points for PCOS with IR (n = 18). Data are mean ± SD values. Figure 1. View largeDownload slide The abundance of cortisol, cortisone, and 11β-HSDs in endometrium of non-PCOS, PCOS without IR, and PCOS with IR. (a–d) Endometrial concentrations of (a) cortisol plus cortisone, (b) cortisol, (c) cortisone, and (d) ratio of cortisol to cortisone in endometrial biopsies obtained from non-PCOS patients (n = 18), PCOS without IR patients (n = 18), and PCOS with IR patients (n = 18). *P < 0.05; **P < 0.01; ***P < 0.001. (e) The representative blot of 11β-HSD1 and 11β-HSD2 in endometrial biopsies from non-PCOS patients (n = 7), PCOS without IR patients (n = 7), and PCOS with IR patients (n = 7). (f and g) Quantification of 11β-HSD 1 and 11β-HSD2 in endometrial biopsies from non-PCOS patients (n = 18), PCOS without IR patients (n = 18), and PCOS with IR patients (n = 18). *P < 0.05; ***P < 0.001 vs non-PCOS; #P < 0.05 vs PCOS without IR. (h and i) Correlation of the abundance of 11β-HSD2 mRNA with (h) local cortisone and (i) cortisol in endometrial biopsies. Circles represent data points for non-PCOS (n = 18); squares represent data points for PCOS without IR (n = 18); triangles represent data points for PCOS with IR (n = 18). Data are mean ± SD values. 11β-HSD1 and 11β-HSD2 mRNA and protein abundance in endometrial tissues No significant difference in 11β-HSD1 mRNA and protein was observed among the three groups [Fig. 1(e) and 1(f)], but both mRNA and protein abundance of 11β-HSD2 in the endometria of PCOS with IR patients were significantly decreased in comparison with those in non-PCOS and PCOS without IR patients [Fig. 1(e) and 1(g)]. Consistently, Pearson analysis showed that 11β-HSD2 mRNA level in endometrium was positively correlated with endometrial cortisone level but negatively correlated with endometrial cortisol level [Fig. 1(h) and 1(i)]. These data suggested that the decrease in 11β-HSD2 expression may account for the increased cortisol level and decreased cortisone level in the endometria obtained from PCOS with IR patients. The abundance of IRS-1, phosphorylated IRS-1, Akt, phosphorylated Akt, and PTEN in endometrium Quantitative Western blotting revealed that IRS-1, p-IRS-1 Ser307, and Akt protein level were comparable among three groups [Fig. 2(a)–2(c) and 2(f)]. The phosphorylation of IRS-1 at Ser318 was significantly higher in the PCOS with IR group compared with non-PCOS [Fig. 2(a) and 2(d)], and the phosphorylation of IRS-1 at Ser612 was significantly higher in PCOS with IR group compared with non-PCOS and PCOS without IR [Fig. 2(a) and 2(e)]. Akt phosphorylation at Ser473 was significantly lower in PCOS with IR patients compared with non-PCOS and PCOS without IR [Fig. 2(a) and 2(g)]. Furthermore, PTEN mRNA and protein levels were significantly increased in PCOS with IR patients [Fig. 2(h) and 2(i)]. These data suggested that the insulin signaling pathway was abolished in the endometria of PCOS with IR patients. Figure 2. View largeDownload slide The abundance of IRS-1, p-IRS-1 Ser307, p-IRS-1 Ser318, p-IRS-1 Ser612, Akt, p-Akt Ser473, and PTEN in endometrium. (a) The representative blot of the protein abundance of IRS-1, p-IRS-1 Ser307, p-IRS-1 Ser318, p-IRS-1 Ser612, Akt, p-Akt Ser473, and PTEN in endometria from non-PCOS (n = 7), PCOS without IR (n = 7), and PCOS with IR patients (n = 7). (b–h) Quantification of the Western blotting assays of (b) IRS-1, (c) p-IRS-1 Ser307, (d) p-IRS-1 Ser318, (e) p-IRS-1 Ser612, (f) Akt, (g) p-Akt Ser473, and (h) PTEN in endometria from non-PCOS (n = 18), PCOS without IR (n = 18), and PCOS with IR patients (n = 18). (i) The mRNA level of PTEN in endometria from non-PCOS patients (n = 18), PCOS without IR patients (n = 18), and PCOS with IR patients (n = 18). *P < 0.05; **P < 0.01, ***P < 0.001. Data are mean ± SD values. Figure 2. View largeDownload slide The abundance of IRS-1, p-IRS-1 Ser307, p-IRS-1 Ser318, p-IRS-1 Ser612, Akt, p-Akt Ser473, and PTEN in endometrium. (a) The representative blot of the protein abundance of IRS-1, p-IRS-1 Ser307, p-IRS-1 Ser318, p-IRS-1 Ser612, Akt, p-Akt Ser473, and PTEN in endometria from non-PCOS (n = 7), PCOS without IR (n = 7), and PCOS with IR patients (n = 7). (b–h) Quantification of the Western blotting assays of (b) IRS-1, (c) p-IRS-1 Ser307, (d) p-IRS-1 Ser318, (e) p-IRS-1 Ser612, (f) Akt, (g) p-Akt Ser473, and (h) PTEN in endometria from non-PCOS (n = 18), PCOS without IR (n = 18), and PCOS with IR patients (n = 18). (i) The mRNA level of PTEN in endometria from non-PCOS patients (n = 18), PCOS without IR patients (n = 18), and PCOS with IR patients (n = 18). *P < 0.05; **P < 0.01, ***P < 0.001. Data are mean ± SD values. Impaired glucose uptake and GLUT4 translocation in EECs derived from PCOS-IR patients Immunohistochemistry revealed strong staining of 11β-HSD2 on surface and glandular epithelial cells and low-intensity staining in stromal cells [Fig. 3(a)]. This suggested that the reduction of cortisol occurred mainly in EECs. The isolated EECs were identified by immunofluorescence staining with cytokeratin 7 before further experiments [Fig. 3(b)]. To clarify the glucose uptake capacity in EECs in PCOS patients, we obtained EECs from non-PCOS, PCOS without IR, and PCOS with IR patients and measured the GLUT4 translocation and glucose uptake under insulin stimulation. Insulin could stimulate both GLUT4 translocation from cytoplasm to membrane [Fig. 3(c) and 3(d)] and glucose uptake [Fig. 3(e)] among three groups, but the GLUT4 translocation and glucose uptake were diminished in EECs from PCOS with IR patients compared with EECs from non-PCOS patients [Fig. 3(c)–3(e)]. These data suggested that glucose uptake and GLUT4 translocation capacity were impaired in PCOS with IR patients. Figure 3. View largeDownload slide The GLUT4 translocation and glucose uptake in EECs derived from non-PCOS, PCOS without IR, and PCOS with IR patients. (a) Immunohistochemical staining of 11β-HSD2 in human uterine endometrium. (b) Immunofluorescence staining of cytokeratin 7 (CK7) (red) and Vimentin (green). The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). (c) The representative blot of GLUT4 translocation from cytoplasm to membrane in EECs derived from non-PCOS, PCOS without IR, and PCOS with IR endometria. Na+K+ATPase is used as a housekeeping protein located in membrane. (d) Quantification of the Western blotting assays of GLUT4 translocation from cytoplasm to membrane (membrane GLUT4/cytoplasm GLUT4) in non-PCOS (n = 4), PCOS without IR (n = 4), and PCOS with IR (n = 4). (e) Fold change of glucose uptake in non-PCOS (n = 4), PCOS without IR (n = 4), and PCOS with IR (n = 4). *P < 0.05; **P < 0.01. Data are mean ± SD values. Figure 3. View largeDownload slide The GLUT4 translocation and glucose uptake in EECs derived from non-PCOS, PCOS without IR, and PCOS with IR patients. (a) Immunohistochemical staining of 11β-HSD2 in human uterine endometrium. (b) Immunofluorescence staining of cytokeratin 7 (CK7) (red) and Vimentin (green). The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). (c) The representative blot of GLUT4 translocation from cytoplasm to membrane in EECs derived from non-PCOS, PCOS without IR, and PCOS with IR endometria. Na+K+ATPase is used as a housekeeping protein located in membrane. (d) Quantification of the Western blotting assays of GLUT4 translocation from cytoplasm to membrane (membrane GLUT4/cytoplasm GLUT4) in non-PCOS (n = 4), PCOS without IR (n = 4), and PCOS with IR (n = 4). (e) Fold change of glucose uptake in non-PCOS (n = 4), PCOS without IR (n = 4), and PCOS with IR (n = 4). *P < 0.05; **P < 0.01. Data are mean ± SD values. Cortisol attenuates insulin-stimulated glucose uptake by inhibition of Akt phosphorylation and GLUT4 translocation via induction of PTEN expression in EECs Prior treatment with cortisol (1 μM, 24 hours) could attenuate the insulin-induced glucose uptake [Fig. 4(a)], Akt phosphorylation [Fig. 4(b)], and GLUT4 translocation from cytoplasm to membrane [Fig. 4(c)] in EECs derived from non-PCOS patients. The cortisol-mediated impairment of glucose uptake, Akt phosphorylation, and GLUT4 translocation were also observed in EECs derived from PCOS patients [Fig. 4(d)–4(f)]. Figure 4. View largeDownload slide The effects of cortisol on glucose uptake and insulin signaling in EECs derived from non-PCOS and PCOS patients. (a) Insulin-stimulated glucose uptake, and the effects of cortisol on insulin-stimulated glucose uptake in EECs from non-PCOS patients (n = 4). (b) Effects of cortisol on Akt phosphorylation (Ser473) in EECs from non-PCOS patients (n = 5). (c) Effects of cortisol on insulin-stimulated GLUT4 translocation in EECs from non-PCOS patients (n = 4). (d–f) Effects of cortisol on insulin-stimulated glucose uptake (n= 4), Akt phosphorylation (n = 5), and GLUT4 translocation (n = 4) in EECs from PCOS patients. *P < 0.05; **P < 0.01; ***P < 0.001 vs control without insulin and cortisol; #P < 0.05; ##P < 0.01; ###P < 0.001 vs insulin alone. Data are mean ± SD with representative blots. Figure 4. View largeDownload slide The effects of cortisol on glucose uptake and insulin signaling in EECs derived from non-PCOS and PCOS patients. (a) Insulin-stimulated glucose uptake, and the effects of cortisol on insulin-stimulated glucose uptake in EECs from non-PCOS patients (n = 4). (b) Effects of cortisol on Akt phosphorylation (Ser473) in EECs from non-PCOS patients (n = 5). (c) Effects of cortisol on insulin-stimulated GLUT4 translocation in EECs from non-PCOS patients (n = 4). (d–f) Effects of cortisol on insulin-stimulated glucose uptake (n= 4), Akt phosphorylation (n = 5), and GLUT4 translocation (n = 4) in EECs from PCOS patients. *P < 0.05; **P < 0.01; ***P < 0.001 vs control without insulin and cortisol; #P < 0.05; ##P < 0.01; ###P < 0.001 vs insulin alone. Data are mean ± SD with representative blots. In addition, PTEN mRNA and protein levels were induced by cortisol in cultured EECs obtained from non-PCOS patients [Fig. 5(a)]. Treatment of EECs with the PTEN inhibitor bPV (phen) rescued the cortisol-induced suppression of Akt phosphorylation [Fig. 5(b)], as well as GLUT4 translocation [Fig. 5(c)]. Furthermore, Pearson analysis showed that cortisol levels positively, but 11β-HSD2 abundance negatively, correlated with PTEN mRNA [Fig. 5(d) and 5(e)]. These data suggested that cortisol attenuated insulin-stimulated glucose uptake via the suppression of Akt phosphorylation and GLUT4 translocation in EECs from non-PCOS and PCOS patients. PTEN was involved in cortisol-induced attenuation of Akt phosphorylation and GLUT4 translocation. Figure 5. View largeDownload slide The involvement of PTEN in the effects of cortisol in EECs. (a) Effects of cortisol on PTEN mRNA and protein abundance in EECs obtained from non-PCOS patients (n = 4). *P < 0.05 and ***P < 0.001 vs control (cortisol = 0). (b) The amount of insulin-stimulated phosphorylated Akt in response to cortisol in the presence or absence of bPV(phen), the PTEN inhibitor (n = 4). **P < 0.01 vs control without cortisol and bPV; ###P < 0.001 vs cortisol alone. (c) The amount of insulin-stimulated GLUT4 in membrane and cytoplasm in response to cortisol in the presence or absence of bPV(phen) (n = 4). *P < 0.05 vs membrane control without insulin and bPV; #P < 0.05 vs membrane cortisol alone. (d and e) Correlation of PTEN mRNA with (d) cortisol and (e) 11β-HSD2 in non-PCOS (n = 18), PCOS without IR (n = 18), and PCOS with IR patients (n = 18). Circles represent data points for non-PCOS; squares represent data points for PCOS without IR; triangles represent data points for PCOS with IR. Data are mean ± SD with representative blots. Figure 5. View largeDownload slide The involvement of PTEN in the effects of cortisol in EECs. (a) Effects of cortisol on PTEN mRNA and protein abundance in EECs obtained from non-PCOS patients (n = 4). *P < 0.05 and ***P < 0.001 vs control (cortisol = 0). (b) The amount of insulin-stimulated phosphorylated Akt in response to cortisol in the presence or absence of bPV(phen), the PTEN inhibitor (n = 4). **P < 0.01 vs control without cortisol and bPV; ###P < 0.001 vs cortisol alone. (c) The amount of insulin-stimulated GLUT4 in membrane and cytoplasm in response to cortisol in the presence or absence of bPV(phen) (n = 4). *P < 0.05 vs membrane control without insulin and bPV; #P < 0.05 vs membrane cortisol alone. (d and e) Correlation of PTEN mRNA with (d) cortisol and (e) 11β-HSD2 in non-PCOS (n = 18), PCOS without IR (n = 18), and PCOS with IR patients (n = 18). Circles represent data points for non-PCOS; squares represent data points for PCOS without IR; triangles represent data points for PCOS with IR. Data are mean ± SD with representative blots. Discussion To our knowledge, this study is the first to evaluate endometrial cortisol, cortisone, and 11β-HSDs levels in PCOS patients. Endometria from PCOS with IR patients had increased cortisol, decreased cortisone, and diminished 11β-HSD2 in the WOI phase compared with those from non-PCOS and PCOS without IR patients. PCOS with IR patients also exhibited endometrial insulin signaling pathway defects and relatively lower implantation rates. Further in vitro studies clarified that excessive cortisol might attenuate insulin sensitivity by decreasing the phosphorylation of Akt (Ser473) and the translocation of GLUT4 via induction of PTEN expression. Thus, this study uncovered the imbalanced state of cortisol and cortisone in endometrium of PCOS with IR patients and established a correlation between the elevated local cortisol and endometrial IR. With a prevalence of 44% to 70%, IR is common among PCOS patients. IR is traditionally defined as the insensitivity or unresponsiveness to insulin and thus requires an increased insulin level (4). In addition to systemic dysfunction of hyperinsulinemia, IR also exhibits as peripheral IR affecting insulin target tissue including endometrium. Li et al. (24) found that maternal hyperinsulinemia impaired mouse endometrial receptivity in early pregnancy. Chang et al. (10) found that PCOS patients with IR had compromised implantation rates, suggesting the effects of IR on endometrial function and receptivity. Consistently, we also observed a lower implantation rate in PCOS with IR patients, although it was not statistically significant, possibly due to the small number of patients in our study. All the evidence suggested that IR might play an important role on human endometrial receptivity in PCOS. Successful implantation requires the endometrium to undergo changes and be receptive to embryos in a short period known as the WOI phase (25). This demands a large amount of energy, mainly from glucose uptake, for the endometrium to properly differentiate to a receptive state (26, 27). Therefore, local endometrial IR might diminish the glucose uptake in endometrial cells and interfere with endometrial receptivity. The metabolic actions of insulin are mainly mediated through activation of IRS-1, phosphatidylinositol-3,4,5-trisphosphate, and Akt, resulting in the translocation of GLUT4 from intracellular vesicles to the plasma membrane. Defects in this signaling pathway could induce IR (4). Previous studies have reported the diminished endometrial IRS-1 in PCOS with hyperinsulinemia and increased endometrial PTEN expression in PCOS (8, 9, 28). In this study, we also revealed significantly elevated PTEN and relatively decreased IRS-1 in PCOS with IR patients. Furthermore, we demonstrated that IRS-1 phosphorylation at Ser318 and Ser612 was increased and Akt phosphorylation at Ser473 was diminished in the endometria from PCOS with IR patients. These evidences further clarified the defective insulin signaling pathway in the endometria from PCOS with IR patients. Because serine phosphorylation of IRS-1 acts as a negative feedback signal for insulin effect, we provided supporting evidence for the former study of Fornes et al. (9) that the IRS-1 activating tyrosine phosphorylation (Y612) was decreased in endometria of PCOS. In addition, the diminished GLUT4 translocation and glucose uptake in the EECs of PCOS with IR patients further clarified the local IR in endometrium. Excessive glucocorticoid exposure has correlated with whole-body IR for decades, and the local effect of cortisol is exaggerated in insulin-target tissue (29, 30). It has been documented that excessive cortisol generated by 11β-HSD1 contributes to the development of IR in adipose, skeletal muscle, and granulosa cells (19, 31, 32). However, quantitative measurements of cortisol and its metabolic enzymes in the endometria of PCOS women have not been performed previously. In endometrium, 11β-HSD1 expression was low except during the menstrual and decidua phases, whereas 11β-HSD2 was the codominant metabolic glucocorticoid enzyme in the secretory phase (13, 14). Here we detailed the elevated cortisol and ratios of cortisol to cortisone in the endometria obtained from PCOS with IR patients. This might reflect either an increase in local reduction of cortisone or a decrease in local oxidation of cortisol. Upon further exploration, we found a significant decrease of 11β-HSD2 in the endometria from PCOS with IR patients, whereas 11β-HSD1 was comparable among the three groups. Furthermore, 11β-HSD2 was negatively correlated with cortisol and positively correlated with cortisone levels. Based on these data, we attributed the elevated ratios of cortisol to cortisone in the endometria of PCOS with IR patients mainly to the decreased local cortisol oxidation caused by 11β-HSD2. This was consistent with the codominant role for 11β-HSD2 in secretory phase. An optimal amount of cortisol is indispensable to embryo implantation (33). However, excessive cortisol caused by decreased oxidation of 11β-HSD2 may disrupt glucocorticoid homeostasis and impair insulin sensitivity in endometrium. As embryo implantation is initiated by embryo attachment to endometrial epithelium (34) and 11β-HSD2 is mainly expressed in endometrial epithelium, we focused on the effects of cortisol on insulin sensitivity in EECs. In the insulin-stimulated glucose uptake pathway, GLUT4 translocation is critical and mediated by Akt phosphorylation. Defects in GLUT4 translocation and Akt phosphorylation indicated IR (35, 36). In this study, we demonstrated that glucose uptake, Akt phosphorylation, and exocytosis of GLUT4 to the plasma membrane were inhibited by cortisol in EECs from both non-PCOS and PCOS patients. This suggested that cortisol was involved in endometrial IR. It is understood that PTEN negatively regulates Akt activity by dephosphorylating phosphatidylinositol-3,4,5-trisphosphate (37, 38) and the induction of PTEN expression could attenuate insulin sensitivity (37, 39). Our in vitro study revealed that PTEN was induced by cortisol in EECs. Additionally, the inhibitor of PTEN restored the cortisol-caused depression of Akt phosphorylation and GLUT4 translocation, suggesting a crucial role of PTEN in cortisol-stimulated endometrial IR. Because PTEN expression in endometrium was also closely correlated with cortisol level, PTEN might be recognized as an important contributor to cortisol-induced IR in endometrium. In this study, we intended to compare the endometria from non-PCOS, PCOS without IR, and PCOS with IR, but we did not exclude the possibility that glucocorticoid alteration might also exist in non-PCOS IR patients. Furthermore, the causes of diminished 11β-HSD2 and the specific underlying molecular mechanisms involving PTEN require further investigation. Our study provides vital preliminary evidence for direct future research on cortisol-induced endometrial IR. In conclusion, we demonstrated that PCOS with IR patients had increased cortisol, diminished 11β-HSD2, and impaired insulin sensitivity in endometrium in the WOI phase compared with non-PCOS and PCOS without IR patients. This indicated that a decreased local inactivation of cortisol by 11β-HSD2 might be a possible cause of endometrial IR in PCOS. Our in vitro study suggested a detrimental role of cortisol in insulin sensitivity in endometrium. We propose that maintaining cortisol at an optimal level in PCOS patients might be beneficial for embryo implantation. Abbreviations: Abbreviations: 11β-HSD 11β-hydroxysteroid dehydrogenase EEC endometrial epithelial cell GLUT4 glucose transporter type 4 hCG human chorionic gonadotropin HOMA-IR homeostasis model assessment of insulin resistance index IR insulin resistance IRS-1 insulin receptor substrate 1 mRNA messenger RNA PCOS polycystic ovary syndrome PCR polymerase chain reaction PTEN phosphatase and tensin homolog deleted on chromosome ten WOI window of implantation Acknowledgments We thank Huiliang Xie, Xiaoming Zhao, Yan Hong, and Minzhi Gao for help with patient recruitment and endometrial biopsies collection and Jianjun Liu, Xiaoping Zhao, Li Zhao, and Panli Li for skillful technical support. Financial Support: This work was supported by National Natural Science Foundation of China Grant 81771648 (to Y.S.), National Key R&D Program of China Grant 2017YFC1001403 (to Y.S.), National Natural Science Foundation of China Grant 81571499 (to Y.S.), Chinese National Key Basic Research Projects Grant 2014CB943300 (to Y.S.), Program of Shanghai Academic Research Leader in Shanghai Municipal Commission of Health and Family Planning Grant 2017BR015 (to Y.S.), Clinical Skills Improvement Project of Major Disorders Hospital Development Center of Shanghai Grant 16CR1022A (to Y.S.), Shanghai Technological Innovation Plan Grant 18140902400 (to Y.S.), and Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant 20161413 (to Y.S.). Disclosure Summary: The authors have nothing to disclose. References 1. Azziz R , Carmina E , Chen Z , Dunaif A , Laven JS , Legro RS , Lizneva D , Natterson-Horowtiz B , Teede HJ , Yildiz BO . Polycystic ovary syndrome . 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He X , Saji M , Radhakrishnan D , Romigh T , Ngeow J , Yu Q , Wang Y , Ringel MD , Eng C . PTEN lipid phosphatase activity and proper subcellular localization are necessary and sufficient for down-regulating AKT phosphorylation in the nucleus in Cowden syndrome . J Clin Endocrinol Metab . 2012 ; 97 ( 11 ): E2179 – E2187 . 39. Gupta A , Dey CS . PTEN, a widely known negative regulator of insulin/PI3K signaling, positively regulates neuronal insulin resistance . Mol Biol Cell . 2012 ; 23 ( 19 ): 3882 – 3898 . Copyright © 2018 Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Clinical Endocrinology and Metabolism Oxford University Press

Local Cortisol Elevation Contributes to Endometrial Insulin Resistance in Polycystic Ovary Syndrome

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Oxford University Press
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Copyright © 2018 Endocrine Society
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0021-972X
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1945-7197
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10.1210/jc.2017-02459
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Abstract

Abstract Context Endometrial insulin resistance (IR) may account for the endometrial dysfunction in polycystic ovary syndrome (PCOS). The underlying mechanism remains to be elucidated. Objective To investigate whether the abundance of 11β-hydroxysteroid dehydrogenases (11β-HSDs) 1 and 2 and cortisol as well as the insulin signaling pathway are altered in PCOS endometrium and to clarify the relationship between endometrial IR and local cortisol. Design We measured cortisol and cortisone concentrations, 11β-HSD1 and 11β-HSD2, and core insulin signaling molecules in endometrial biopsies collected from non-PCOS and PCOS with or without IR patients on the seventh day after human chorionic gonadotropin injection. We also studied the effects of cortisol on glucose uptake and the insulin signaling pathway in primary cultured endometrial epithelial cells (EECs). Results The cortisol concentration was elevated, whereas 11β-HSD2 expression was diminished in endometrial biopsies obtained from PCOS with IR patients compared with those from non-PCOS and PCOS without IR patients. The implantation rate was relatively impaired and the endometrial insulin signaling pathway was defective in PCOS with IR patients. In addition, cortisol attenuated insulin-stimulated glucose uptake in EECs, which was mediated by inhibition of Akt phosphorylation and glucose transporter type 4 translocation via induction of phosphatase and tensin homolog deleted on chromosome ten (PTEN). Conclusions Decreased oxidation of cortisol and defects of insulin signaling in endometrium were observed in PCOS with IR patients. The excessive cortisol level, derived from the reduction of 11β-HSD2, might contribute to the development of endometrial IR by inhibiting the insulin signaling pathway via induction of PTEN expression in EECs. Polycystic ovary syndrome (PCOS), a common and complex endocrine disorder, affects 5% to 20% of reproductive-age women with short- and long-term effects (1). PCOS is characterized by polycystic ovaries, ovulatory dysfunction, and hyperandrogenism (2). In addition, a high percentage of patients with PCOS have symptoms of insulin resistance (IR) (2–4). The abnormal endocrine and metabolic characteristics of PCOS might be detrimental to endometrial function, manifesting as endometrial hyperplasia or cancer and reduction of receptivity (5–7). Patients with PCOS have been reported to have adverse reproductive outcomes, including higher abortion rates compared with the unaffected population (7). The reduction in fertility might not only be attributed to ovulatory dysfunction, but also to endometrial defects. When compared with fertile endometrium, several alterations of the insulin signaling pathway in PCOS endometrium have been reported, such as increased phosphatase and tensin homolog deleted on chromosome ten (PTEN) as well as decreased insulin receptor substrate 1 (IRS-1) (8, 9). Some studies have linked endometrial IR to decreased endometrial receptivity (10) and tumorigenesis (11, 12). However, the etiology of local endometrial IR remains to be elucidated. Endogenous glucocorticoids play a crucial role in many areas, including in the pathophysiology of IR. The conversion of inactive glucocorticoids and active glucocorticoids is catalyzed by 11β-hydroxysteroid dehydrogenases (11β-HSDs). There are two types of 11β-HSDs: 11β-HSD1 and 11β-HSD2 (13, 14). 11β-HSD1 has both reductase and oxidase functions, bidirectionally converting biologically inactive cortisone and active cortisol; 11β-HSD2 only has an oxidase function, converting active cortisol to inactive cortisone (15). Several studies have demonstrated that cortisol concentration and 11β-HSDs were changed in serum, adipose tissue, and granulosa cells in PCOS patients (16–18). In addition, our previous study showed that cortisol generated locally by 11β-HSD1 contributed to IR in granulosa cells in PCOS (19). However, it is unclear whether the local generation of cortisol in endometrium exerts a role in the process of endometrial IR in PCOS. Considering the ovarian 11β-HSD alterations in women with PCOS, our primary aim in this study was to clarify whether there is an imbalanced state of glucocorticoid and its metabolic enzymes in endometria from patients with PCOS with or without IR. We also sought to establish a possible correlation between cortisol and local IR. A secondary aim of this study was to clarify in vitro whether cortisol could abolish insulin-stimulated glucose uptake and insulin signaling pathway in primary cultured endometrial epithelial cells (EECs). Materials and Methods Patients and tissue collection The endometrial biopsies were collected with endometrial suction curettes (Runting) from PCOS and non-PCOS patients undergoing gonadotropin-releasing hormone antagonist stimulation cycle without fresh embryo transfer. The biopsies were collected on the seventh day after human chorionic gonadotropin (hCG) injection, known as the window of implantation (WOI) phase. The diagnosis of PCOS was established according to the revised Rotterdam consensus (20). The subgroups of PCOS with IR and PCOS without IR were subdivided according to the homeostasis model assessment of IR index (HOMA-IR [fasting serum insulin (μIU/mL) × fasting serum glucose (mmol/L)/22.5]), with 3.15 selected as a cutoff point (21). Non-PCOS patients were women with regular menstrual cycles, normal body mass index (18.5 to 23.9 kg/m2), and only tubal infertile conditions without IR. The endometrial biopsies from non-PCOS (n = 18), PCOS without IR (n = 18), and PCOS with IR (n = 18) were snap frozen in liquid nitrogen for extraction of glucocorticoids, messenger RNA (mRNA), and protein to detect cortisol, cortisone, 11β-HSD mRNA and protein, phosphorylated IRS-1 and IRS-1, phosphorylated Akt and Akt, and PTEN mRNA and protein. The EECs were isolated from other patients including non-PCOS (n = 29) and PCOS (n = 21). All procedures were performed at the Center for Reproductive Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University. Written informed consent was obtained, and approval of the ethics protocol was granted from the Ethics Committee of Renji Hospital (2017081109). Demographic features and clinical outcomes Baseline serum hormonal profiles, including follicle-stimulating hormone, luteinizing hormone, testosterone, estradiol, and anti-Müllerian hormone, were determined using chemiluminescence assay kits (Beckman Access Health Co.). Serum fasting insulin and fasting glucose were measured using a chemiluminescence assay kit (Beckman Access Health Co.) and a standard glucose oxidase method (Roche), respectively. Quantitative insulin sensitivity check index was calculated as 1/[logI0(μIU/mL) + logG0(mg/dL)]. The implantation rate was defined as the number of gestational sacs per number of embryos transferred in every frozen embryo transfer cycle. Extraction and measurement of cortisol and cortisone in endometrium The endometrium was ground in liquid nitrogen and extracted with ethyl acetate. After evaporation, the extract was resuspended in phosphate-buffered saline. The suspension was equally divided and reconstituted in the assay buffer provided by the manufacturers and then measured using a cortisol assay kit (R&D Systems) and a cortisone chemiluminescent immunoassay kit (Innovative Research) following manufacturer instructions. Immunohistochemical and immunofluorescent staining Protein expression of 11β-HSD2 was assessed in paraffin-embedded endometrial tissue sections. Immunostaining was performed on 5-μm-thick tissue sections as previously described (22). Briefly, the endogenous peroxidase activity was quenched with 3% H2O2, and then 11β-HSD2 or preimmune serum at 1:500 dilution was used as a primary or negative control for overnight incubation at 4°C followed by secondary antibody for 30 minutes at 37°C, respectively. The colorimetric reactions were developed using a standard diaminobenzidine kit (ZSGB-BIO). Immunofluorescent staining was performed on cultured cells fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. After blocking and incubation with primary antibodies, cells were incubated with Alexa Fluor 488- and 594-labeled secondary antibodies (Proteintech). Nuclei were stained with 4´,6´-diamino-2-phenylindole (1 μg/mL). Images were obtained with a microscope and camera connected to a computer with an image analysis system (Zeiss). The primary antibodies are listed in Supplemental Table 1. Cell culture and treatment EECs were enzymatically isolated from human endometrium curettage samples according to a selective attachment method (23) with minor modifications. Briefly, endometrial samples were digested with collagenase type I and deoxyribonuclease and then sequentially size fractionated with 180- and 40-μm sieves. Epithelial glands were retained on the 40-μm sieve and were collected by backwashing the 40-μm filter paper and resuspended in Dulbecco’s modified Eagle medium/Hams F12 containing 10% fetal bovine serum (Gibco) and 1% antibiotic-antimycotic solution (Gibco). Cell purity was tested routinely by immunofluorescence staining for cytokeratin and vimentin. To compare the glucose uptake and glucose transporter type 4 (GLUT4) translocation among non-PCOS, PCOS without IR, and PCOS with IR patients, the cells were cultured in phenol red-free and serum-free medium 1 day after plating and stimulated with insulin (100 nM; Sigma) for 20 and 30 minutes. To study the role of cortisol on the insulin-stimulated glucose uptake, Akt phosphorylation, and GLUT4 translocation, EECs were treated in phenol red-free and serum-free culture medium after plating for 3 days. After treatment with cortisol (1 μM; Sigma) for 24 hours, the cells were stimulated with insulin for 20, 15, and 30 minutes, respectively. The measurement of glucose uptake is described below. To detect the effect of cortisol on PTEN expression, cells were treated with cortisol (0.1 and 1 μM) for 24 hours before analysis with Western blotting or quantitative real-time polymerase chain reaction (PCR) as described below. To determine the involvement of PTEN, the PTEN inhibitor bpV (phen) (1 μM; Sigma) was added to EECs for 30 minutes before insulin stimulation. Glucose uptake Glucose uptake in EECs was measured after insulin stimulation (100 nM, 20 minutes) using Glucose Uptake-Glo Assay (Promega) according to manufacturer instructions. Quantitative real-time PCR Total RNA from cells and endometrial biopsies was extracted using a total RNA Kit (Omega Bio-Tek) according to manufacturer instructions and was reverse transcribed to complementary DNA using the PrimeScript reverse transcription kit (TaKaRa) with appropriate controls. Quantitative real-time PCR was performed and analyzed with ABI Prism System (Applied Biosystems) using SYBR Premix (TaKaRa) in triplicate. Relative mRNA expression was calculated by the comparative cycle threshold method with ACTB as the housekeeping gene. The primer sequences are listed in Supplemental Table 2. Protein extraction and Western blotting Total protein was extracted from cells and endometrial biopsies using ice-cold radio-immunoprecipitation assay lysis buffer (CWBIO) containing protease inhibitor cocktail (Roche) and phosphatase inhibitor (Active Motif). Membrane and cytoplasmic proteins were extracted using the Membrane and Cytoplasmic Protein Extraction Kit (Sangon Biotech). Protein was quantified with a Bradford assay, and 20 μg protein of each sample was electrophoresed in 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to a nitrocellulose blot. After blocking and incubation with primary antibodies, the membranes were incubated with the respective secondary antibody conjugated with horseradish peroxidase (Proteintech) for 1 hour. Bands with peroxidase activity were detected by an enhanced chemiluminescent detection kit (Merck Millipore) and visualized with a G-Box chemiluminescence image capture system (Syngene). Primary antibodies are listed in Supplemental Table 1. Statistical analysis All data are reported as the mean ± standard deviation (SD). Analyses were performed using the Statistical Package for Social Science (version 16.0; SPSS) and Graphpad Prism statistical software (version 5.0, Graphpad). The data were initially subjected to Kolmogorov-Smirnov tests to assess deviation from Gaussian distribution. For normally distributed data, we applied unpaired t test and one-way analysis of variance followed by Bonferroni tests. For data not normally distributed, we applied Kruskal-Wallis test followed by Dunn’s multiple comparison test. Correlation between variables was performed using Pearson correlation analysis. P < 0.05 was considered to be statistically significant. Results Clinical characteristics and implantation outcomes The demographic characteristics and implantation outcomes of recruited participants are displayed in Table 1. Fifty-four patients were classified into three groups: non-PCOS (n = 18), PCOS without IR (n = 18), and PCOS with IR (n = 18). Patient age, basal follicle-stimulating hormone, estradiol, and hormones on hCG injection day were comparable among the three groups. Basal levels of anti-Müllerian hormone, luteinizing hormone, and testosterone were significantly higher in PCOS with or without IR than in non-PCOS patients. The fasting insulin, HOMA-IR, and quantitative insulin sensitivity check index were significantly higher in PCOS with IR patients than in non-PCOS and PCOS without IR patients. This is a characteristic of IR syndrome. Although there was not a significant difference, there was a tendency for the implantation rate in PCOS with IR women to be lower than that of non-PCOS (P = 0.06). Table 1. Demographic Features and Clinical Outcomes of Recruited Patients Non-PCOS (n = 18) PCOS Without IR (n = 18) PCOS With IR (n = 18) Age, y 27.67 ± 2.74 28.28 ± 3.39 28.56 ± 2.20 BMI, kg/m2 20.84 ± 2.19 23.54 ± 4.48 25.79 ± 3.84a Basal FSH, mIU/mL 7.13 ± 1.71 6.04 ± 1.23 6.13 ± 1.17 Basal LH, mIU/mL 4.87 ± 1.67 6.88 ± 5.77a 9.66 ± 7.36a Basal E2, pg/mL 42.40 ± 17.94 38.68 ± 20.68 42.24 ± 17.45 Basal T, nmol/L 1.01 ± 0.58 1.66 ± 0.67a 1.84 ± 0.83a Hormones on hCG day  LH, mIU/mL 2.04 ± 1.69 1.48 ± 0.85 2.13 ± 1.52  E2, pg/mL 2988.44 ± 1613.16 3992.64 ± 1825.50 3157.94 ± 1436.53  P4, ng/mL 0.92 ± 0.27 1.29 ± 0.67 1.21 ± 1.14 Fasting glucose, mmol/L 4.46 ± 0.37 4.88 ± 0.38 5.42 ± 1.44a Fasting insulin, μIU/mL 6.49 ± 1.99 8.91 ± 2.00 17.31 ± 5.91a,b HOMA-IR 1.30 ± 0.45 1.91 ± 0.39 4.18 ± 1.90a,b QUICKI 0.37 ± 0.03 0.35 ± 0.01a 0.31 ± 0.01a,b AMH, ng/mL 5.29 ± 1.44 12.99 ± 4.15a 10.94 ± 5.04a Frozen embryo transfer cycle 27 24 24 Implantation rate 50.0% ± 46.0% 37.5% ± 39.7% 27.1% ± 39.0% Non-PCOS (n = 18) PCOS Without IR (n = 18) PCOS With IR (n = 18) Age, y 27.67 ± 2.74 28.28 ± 3.39 28.56 ± 2.20 BMI, kg/m2 20.84 ± 2.19 23.54 ± 4.48 25.79 ± 3.84a Basal FSH, mIU/mL 7.13 ± 1.71 6.04 ± 1.23 6.13 ± 1.17 Basal LH, mIU/mL 4.87 ± 1.67 6.88 ± 5.77a 9.66 ± 7.36a Basal E2, pg/mL 42.40 ± 17.94 38.68 ± 20.68 42.24 ± 17.45 Basal T, nmol/L 1.01 ± 0.58 1.66 ± 0.67a 1.84 ± 0.83a Hormones on hCG day  LH, mIU/mL 2.04 ± 1.69 1.48 ± 0.85 2.13 ± 1.52  E2, pg/mL 2988.44 ± 1613.16 3992.64 ± 1825.50 3157.94 ± 1436.53  P4, ng/mL 0.92 ± 0.27 1.29 ± 0.67 1.21 ± 1.14 Fasting glucose, mmol/L 4.46 ± 0.37 4.88 ± 0.38 5.42 ± 1.44a Fasting insulin, μIU/mL 6.49 ± 1.99 8.91 ± 2.00 17.31 ± 5.91a,b HOMA-IR 1.30 ± 0.45 1.91 ± 0.39 4.18 ± 1.90a,b QUICKI 0.37 ± 0.03 0.35 ± 0.01a 0.31 ± 0.01a,b AMH, ng/mL 5.29 ± 1.44 12.99 ± 4.15a 10.94 ± 5.04a Frozen embryo transfer cycle 27 24 24 Implantation rate 50.0% ± 46.0% 37.5% ± 39.7% 27.1% ± 39.0% All data are mean ± standard deviation values. Abbreviations: AMH, anti-Müllerian hormone; BMI, body mass index; E2, estradiol; FSH, follicle-stimulating hormone; LH, luteinizing hormone; P4, progesterone; QUICKI, quantitative insulin sensitivity check index; T, testosterone. a P < 0.05 vs non-PCOS. b P < 0.05 vs PCOS without IR. View Large Table 1. Demographic Features and Clinical Outcomes of Recruited Patients Non-PCOS (n = 18) PCOS Without IR (n = 18) PCOS With IR (n = 18) Age, y 27.67 ± 2.74 28.28 ± 3.39 28.56 ± 2.20 BMI, kg/m2 20.84 ± 2.19 23.54 ± 4.48 25.79 ± 3.84a Basal FSH, mIU/mL 7.13 ± 1.71 6.04 ± 1.23 6.13 ± 1.17 Basal LH, mIU/mL 4.87 ± 1.67 6.88 ± 5.77a 9.66 ± 7.36a Basal E2, pg/mL 42.40 ± 17.94 38.68 ± 20.68 42.24 ± 17.45 Basal T, nmol/L 1.01 ± 0.58 1.66 ± 0.67a 1.84 ± 0.83a Hormones on hCG day  LH, mIU/mL 2.04 ± 1.69 1.48 ± 0.85 2.13 ± 1.52  E2, pg/mL 2988.44 ± 1613.16 3992.64 ± 1825.50 3157.94 ± 1436.53  P4, ng/mL 0.92 ± 0.27 1.29 ± 0.67 1.21 ± 1.14 Fasting glucose, mmol/L 4.46 ± 0.37 4.88 ± 0.38 5.42 ± 1.44a Fasting insulin, μIU/mL 6.49 ± 1.99 8.91 ± 2.00 17.31 ± 5.91a,b HOMA-IR 1.30 ± 0.45 1.91 ± 0.39 4.18 ± 1.90a,b QUICKI 0.37 ± 0.03 0.35 ± 0.01a 0.31 ± 0.01a,b AMH, ng/mL 5.29 ± 1.44 12.99 ± 4.15a 10.94 ± 5.04a Frozen embryo transfer cycle 27 24 24 Implantation rate 50.0% ± 46.0% 37.5% ± 39.7% 27.1% ± 39.0% Non-PCOS (n = 18) PCOS Without IR (n = 18) PCOS With IR (n = 18) Age, y 27.67 ± 2.74 28.28 ± 3.39 28.56 ± 2.20 BMI, kg/m2 20.84 ± 2.19 23.54 ± 4.48 25.79 ± 3.84a Basal FSH, mIU/mL 7.13 ± 1.71 6.04 ± 1.23 6.13 ± 1.17 Basal LH, mIU/mL 4.87 ± 1.67 6.88 ± 5.77a 9.66 ± 7.36a Basal E2, pg/mL 42.40 ± 17.94 38.68 ± 20.68 42.24 ± 17.45 Basal T, nmol/L 1.01 ± 0.58 1.66 ± 0.67a 1.84 ± 0.83a Hormones on hCG day  LH, mIU/mL 2.04 ± 1.69 1.48 ± 0.85 2.13 ± 1.52  E2, pg/mL 2988.44 ± 1613.16 3992.64 ± 1825.50 3157.94 ± 1436.53  P4, ng/mL 0.92 ± 0.27 1.29 ± 0.67 1.21 ± 1.14 Fasting glucose, mmol/L 4.46 ± 0.37 4.88 ± 0.38 5.42 ± 1.44a Fasting insulin, μIU/mL 6.49 ± 1.99 8.91 ± 2.00 17.31 ± 5.91a,b HOMA-IR 1.30 ± 0.45 1.91 ± 0.39 4.18 ± 1.90a,b QUICKI 0.37 ± 0.03 0.35 ± 0.01a 0.31 ± 0.01a,b AMH, ng/mL 5.29 ± 1.44 12.99 ± 4.15a 10.94 ± 5.04a Frozen embryo transfer cycle 27 24 24 Implantation rate 50.0% ± 46.0% 37.5% ± 39.7% 27.1% ± 39.0% All data are mean ± standard deviation values. Abbreviations: AMH, anti-Müllerian hormone; BMI, body mass index; E2, estradiol; FSH, follicle-stimulating hormone; LH, luteinizing hormone; P4, progesterone; QUICKI, quantitative insulin sensitivity check index; T, testosterone. a P < 0.05 vs non-PCOS. b P < 0.05 vs PCOS without IR. View Large Cortisol and cortisone concentrations in human endometrial tissues No statistically significant differences were found in the summed concentrations of cortisol plus cortisone among the three groups [Fig. 1(a)]. Concentrations of cortisol in PCOS with IR patients were significantly higher compared with non-PCOS and PCOS without IR patients [Fig. 1(b)]. Concentrations of cortisone in PCOS with IR patients were significantly lower than those in non-PCOS patients and PCOS without IR patients [Fig. 1(c)]. Ratios of cortisol to cortisone were significantly elevated in PCOS with IR patients compared with non-PCOS and PCOS without IR patients [Fig. 1(d)]. The quantitative detection showed an imbalanced metabolic state between cortisol and cortisone in the endometria of PCOS with IR patients. Figure 1. View largeDownload slide The abundance of cortisol, cortisone, and 11β-HSDs in endometrium of non-PCOS, PCOS without IR, and PCOS with IR. (a–d) Endometrial concentrations of (a) cortisol plus cortisone, (b) cortisol, (c) cortisone, and (d) ratio of cortisol to cortisone in endometrial biopsies obtained from non-PCOS patients (n = 18), PCOS without IR patients (n = 18), and PCOS with IR patients (n = 18). *P < 0.05; **P < 0.01; ***P < 0.001. (e) The representative blot of 11β-HSD1 and 11β-HSD2 in endometrial biopsies from non-PCOS patients (n = 7), PCOS without IR patients (n = 7), and PCOS with IR patients (n = 7). (f and g) Quantification of 11β-HSD 1 and 11β-HSD2 in endometrial biopsies from non-PCOS patients (n = 18), PCOS without IR patients (n = 18), and PCOS with IR patients (n = 18). *P < 0.05; ***P < 0.001 vs non-PCOS; #P < 0.05 vs PCOS without IR. (h and i) Correlation of the abundance of 11β-HSD2 mRNA with (h) local cortisone and (i) cortisol in endometrial biopsies. Circles represent data points for non-PCOS (n = 18); squares represent data points for PCOS without IR (n = 18); triangles represent data points for PCOS with IR (n = 18). Data are mean ± SD values. Figure 1. View largeDownload slide The abundance of cortisol, cortisone, and 11β-HSDs in endometrium of non-PCOS, PCOS without IR, and PCOS with IR. (a–d) Endometrial concentrations of (a) cortisol plus cortisone, (b) cortisol, (c) cortisone, and (d) ratio of cortisol to cortisone in endometrial biopsies obtained from non-PCOS patients (n = 18), PCOS without IR patients (n = 18), and PCOS with IR patients (n = 18). *P < 0.05; **P < 0.01; ***P < 0.001. (e) The representative blot of 11β-HSD1 and 11β-HSD2 in endometrial biopsies from non-PCOS patients (n = 7), PCOS without IR patients (n = 7), and PCOS with IR patients (n = 7). (f and g) Quantification of 11β-HSD 1 and 11β-HSD2 in endometrial biopsies from non-PCOS patients (n = 18), PCOS without IR patients (n = 18), and PCOS with IR patients (n = 18). *P < 0.05; ***P < 0.001 vs non-PCOS; #P < 0.05 vs PCOS without IR. (h and i) Correlation of the abundance of 11β-HSD2 mRNA with (h) local cortisone and (i) cortisol in endometrial biopsies. Circles represent data points for non-PCOS (n = 18); squares represent data points for PCOS without IR (n = 18); triangles represent data points for PCOS with IR (n = 18). Data are mean ± SD values. 11β-HSD1 and 11β-HSD2 mRNA and protein abundance in endometrial tissues No significant difference in 11β-HSD1 mRNA and protein was observed among the three groups [Fig. 1(e) and 1(f)], but both mRNA and protein abundance of 11β-HSD2 in the endometria of PCOS with IR patients were significantly decreased in comparison with those in non-PCOS and PCOS without IR patients [Fig. 1(e) and 1(g)]. Consistently, Pearson analysis showed that 11β-HSD2 mRNA level in endometrium was positively correlated with endometrial cortisone level but negatively correlated with endometrial cortisol level [Fig. 1(h) and 1(i)]. These data suggested that the decrease in 11β-HSD2 expression may account for the increased cortisol level and decreased cortisone level in the endometria obtained from PCOS with IR patients. The abundance of IRS-1, phosphorylated IRS-1, Akt, phosphorylated Akt, and PTEN in endometrium Quantitative Western blotting revealed that IRS-1, p-IRS-1 Ser307, and Akt protein level were comparable among three groups [Fig. 2(a)–2(c) and 2(f)]. The phosphorylation of IRS-1 at Ser318 was significantly higher in the PCOS with IR group compared with non-PCOS [Fig. 2(a) and 2(d)], and the phosphorylation of IRS-1 at Ser612 was significantly higher in PCOS with IR group compared with non-PCOS and PCOS without IR [Fig. 2(a) and 2(e)]. Akt phosphorylation at Ser473 was significantly lower in PCOS with IR patients compared with non-PCOS and PCOS without IR [Fig. 2(a) and 2(g)]. Furthermore, PTEN mRNA and protein levels were significantly increased in PCOS with IR patients [Fig. 2(h) and 2(i)]. These data suggested that the insulin signaling pathway was abolished in the endometria of PCOS with IR patients. Figure 2. View largeDownload slide The abundance of IRS-1, p-IRS-1 Ser307, p-IRS-1 Ser318, p-IRS-1 Ser612, Akt, p-Akt Ser473, and PTEN in endometrium. (a) The representative blot of the protein abundance of IRS-1, p-IRS-1 Ser307, p-IRS-1 Ser318, p-IRS-1 Ser612, Akt, p-Akt Ser473, and PTEN in endometria from non-PCOS (n = 7), PCOS without IR (n = 7), and PCOS with IR patients (n = 7). (b–h) Quantification of the Western blotting assays of (b) IRS-1, (c) p-IRS-1 Ser307, (d) p-IRS-1 Ser318, (e) p-IRS-1 Ser612, (f) Akt, (g) p-Akt Ser473, and (h) PTEN in endometria from non-PCOS (n = 18), PCOS without IR (n = 18), and PCOS with IR patients (n = 18). (i) The mRNA level of PTEN in endometria from non-PCOS patients (n = 18), PCOS without IR patients (n = 18), and PCOS with IR patients (n = 18). *P < 0.05; **P < 0.01, ***P < 0.001. Data are mean ± SD values. Figure 2. View largeDownload slide The abundance of IRS-1, p-IRS-1 Ser307, p-IRS-1 Ser318, p-IRS-1 Ser612, Akt, p-Akt Ser473, and PTEN in endometrium. (a) The representative blot of the protein abundance of IRS-1, p-IRS-1 Ser307, p-IRS-1 Ser318, p-IRS-1 Ser612, Akt, p-Akt Ser473, and PTEN in endometria from non-PCOS (n = 7), PCOS without IR (n = 7), and PCOS with IR patients (n = 7). (b–h) Quantification of the Western blotting assays of (b) IRS-1, (c) p-IRS-1 Ser307, (d) p-IRS-1 Ser318, (e) p-IRS-1 Ser612, (f) Akt, (g) p-Akt Ser473, and (h) PTEN in endometria from non-PCOS (n = 18), PCOS without IR (n = 18), and PCOS with IR patients (n = 18). (i) The mRNA level of PTEN in endometria from non-PCOS patients (n = 18), PCOS without IR patients (n = 18), and PCOS with IR patients (n = 18). *P < 0.05; **P < 0.01, ***P < 0.001. Data are mean ± SD values. Impaired glucose uptake and GLUT4 translocation in EECs derived from PCOS-IR patients Immunohistochemistry revealed strong staining of 11β-HSD2 on surface and glandular epithelial cells and low-intensity staining in stromal cells [Fig. 3(a)]. This suggested that the reduction of cortisol occurred mainly in EECs. The isolated EECs were identified by immunofluorescence staining with cytokeratin 7 before further experiments [Fig. 3(b)]. To clarify the glucose uptake capacity in EECs in PCOS patients, we obtained EECs from non-PCOS, PCOS without IR, and PCOS with IR patients and measured the GLUT4 translocation and glucose uptake under insulin stimulation. Insulin could stimulate both GLUT4 translocation from cytoplasm to membrane [Fig. 3(c) and 3(d)] and glucose uptake [Fig. 3(e)] among three groups, but the GLUT4 translocation and glucose uptake were diminished in EECs from PCOS with IR patients compared with EECs from non-PCOS patients [Fig. 3(c)–3(e)]. These data suggested that glucose uptake and GLUT4 translocation capacity were impaired in PCOS with IR patients. Figure 3. View largeDownload slide The GLUT4 translocation and glucose uptake in EECs derived from non-PCOS, PCOS without IR, and PCOS with IR patients. (a) Immunohistochemical staining of 11β-HSD2 in human uterine endometrium. (b) Immunofluorescence staining of cytokeratin 7 (CK7) (red) and Vimentin (green). The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). (c) The representative blot of GLUT4 translocation from cytoplasm to membrane in EECs derived from non-PCOS, PCOS without IR, and PCOS with IR endometria. Na+K+ATPase is used as a housekeeping protein located in membrane. (d) Quantification of the Western blotting assays of GLUT4 translocation from cytoplasm to membrane (membrane GLUT4/cytoplasm GLUT4) in non-PCOS (n = 4), PCOS without IR (n = 4), and PCOS with IR (n = 4). (e) Fold change of glucose uptake in non-PCOS (n = 4), PCOS without IR (n = 4), and PCOS with IR (n = 4). *P < 0.05; **P < 0.01. Data are mean ± SD values. Figure 3. View largeDownload slide The GLUT4 translocation and glucose uptake in EECs derived from non-PCOS, PCOS without IR, and PCOS with IR patients. (a) Immunohistochemical staining of 11β-HSD2 in human uterine endometrium. (b) Immunofluorescence staining of cytokeratin 7 (CK7) (red) and Vimentin (green). The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). (c) The representative blot of GLUT4 translocation from cytoplasm to membrane in EECs derived from non-PCOS, PCOS without IR, and PCOS with IR endometria. Na+K+ATPase is used as a housekeeping protein located in membrane. (d) Quantification of the Western blotting assays of GLUT4 translocation from cytoplasm to membrane (membrane GLUT4/cytoplasm GLUT4) in non-PCOS (n = 4), PCOS without IR (n = 4), and PCOS with IR (n = 4). (e) Fold change of glucose uptake in non-PCOS (n = 4), PCOS without IR (n = 4), and PCOS with IR (n = 4). *P < 0.05; **P < 0.01. Data are mean ± SD values. Cortisol attenuates insulin-stimulated glucose uptake by inhibition of Akt phosphorylation and GLUT4 translocation via induction of PTEN expression in EECs Prior treatment with cortisol (1 μM, 24 hours) could attenuate the insulin-induced glucose uptake [Fig. 4(a)], Akt phosphorylation [Fig. 4(b)], and GLUT4 translocation from cytoplasm to membrane [Fig. 4(c)] in EECs derived from non-PCOS patients. The cortisol-mediated impairment of glucose uptake, Akt phosphorylation, and GLUT4 translocation were also observed in EECs derived from PCOS patients [Fig. 4(d)–4(f)]. Figure 4. View largeDownload slide The effects of cortisol on glucose uptake and insulin signaling in EECs derived from non-PCOS and PCOS patients. (a) Insulin-stimulated glucose uptake, and the effects of cortisol on insulin-stimulated glucose uptake in EECs from non-PCOS patients (n = 4). (b) Effects of cortisol on Akt phosphorylation (Ser473) in EECs from non-PCOS patients (n = 5). (c) Effects of cortisol on insulin-stimulated GLUT4 translocation in EECs from non-PCOS patients (n = 4). (d–f) Effects of cortisol on insulin-stimulated glucose uptake (n= 4), Akt phosphorylation (n = 5), and GLUT4 translocation (n = 4) in EECs from PCOS patients. *P < 0.05; **P < 0.01; ***P < 0.001 vs control without insulin and cortisol; #P < 0.05; ##P < 0.01; ###P < 0.001 vs insulin alone. Data are mean ± SD with representative blots. Figure 4. View largeDownload slide The effects of cortisol on glucose uptake and insulin signaling in EECs derived from non-PCOS and PCOS patients. (a) Insulin-stimulated glucose uptake, and the effects of cortisol on insulin-stimulated glucose uptake in EECs from non-PCOS patients (n = 4). (b) Effects of cortisol on Akt phosphorylation (Ser473) in EECs from non-PCOS patients (n = 5). (c) Effects of cortisol on insulin-stimulated GLUT4 translocation in EECs from non-PCOS patients (n = 4). (d–f) Effects of cortisol on insulin-stimulated glucose uptake (n= 4), Akt phosphorylation (n = 5), and GLUT4 translocation (n = 4) in EECs from PCOS patients. *P < 0.05; **P < 0.01; ***P < 0.001 vs control without insulin and cortisol; #P < 0.05; ##P < 0.01; ###P < 0.001 vs insulin alone. Data are mean ± SD with representative blots. In addition, PTEN mRNA and protein levels were induced by cortisol in cultured EECs obtained from non-PCOS patients [Fig. 5(a)]. Treatment of EECs with the PTEN inhibitor bPV (phen) rescued the cortisol-induced suppression of Akt phosphorylation [Fig. 5(b)], as well as GLUT4 translocation [Fig. 5(c)]. Furthermore, Pearson analysis showed that cortisol levels positively, but 11β-HSD2 abundance negatively, correlated with PTEN mRNA [Fig. 5(d) and 5(e)]. These data suggested that cortisol attenuated insulin-stimulated glucose uptake via the suppression of Akt phosphorylation and GLUT4 translocation in EECs from non-PCOS and PCOS patients. PTEN was involved in cortisol-induced attenuation of Akt phosphorylation and GLUT4 translocation. Figure 5. View largeDownload slide The involvement of PTEN in the effects of cortisol in EECs. (a) Effects of cortisol on PTEN mRNA and protein abundance in EECs obtained from non-PCOS patients (n = 4). *P < 0.05 and ***P < 0.001 vs control (cortisol = 0). (b) The amount of insulin-stimulated phosphorylated Akt in response to cortisol in the presence or absence of bPV(phen), the PTEN inhibitor (n = 4). **P < 0.01 vs control without cortisol and bPV; ###P < 0.001 vs cortisol alone. (c) The amount of insulin-stimulated GLUT4 in membrane and cytoplasm in response to cortisol in the presence or absence of bPV(phen) (n = 4). *P < 0.05 vs membrane control without insulin and bPV; #P < 0.05 vs membrane cortisol alone. (d and e) Correlation of PTEN mRNA with (d) cortisol and (e) 11β-HSD2 in non-PCOS (n = 18), PCOS without IR (n = 18), and PCOS with IR patients (n = 18). Circles represent data points for non-PCOS; squares represent data points for PCOS without IR; triangles represent data points for PCOS with IR. Data are mean ± SD with representative blots. Figure 5. View largeDownload slide The involvement of PTEN in the effects of cortisol in EECs. (a) Effects of cortisol on PTEN mRNA and protein abundance in EECs obtained from non-PCOS patients (n = 4). *P < 0.05 and ***P < 0.001 vs control (cortisol = 0). (b) The amount of insulin-stimulated phosphorylated Akt in response to cortisol in the presence or absence of bPV(phen), the PTEN inhibitor (n = 4). **P < 0.01 vs control without cortisol and bPV; ###P < 0.001 vs cortisol alone. (c) The amount of insulin-stimulated GLUT4 in membrane and cytoplasm in response to cortisol in the presence or absence of bPV(phen) (n = 4). *P < 0.05 vs membrane control without insulin and bPV; #P < 0.05 vs membrane cortisol alone. (d and e) Correlation of PTEN mRNA with (d) cortisol and (e) 11β-HSD2 in non-PCOS (n = 18), PCOS without IR (n = 18), and PCOS with IR patients (n = 18). Circles represent data points for non-PCOS; squares represent data points for PCOS without IR; triangles represent data points for PCOS with IR. Data are mean ± SD with representative blots. Discussion To our knowledge, this study is the first to evaluate endometrial cortisol, cortisone, and 11β-HSDs levels in PCOS patients. Endometria from PCOS with IR patients had increased cortisol, decreased cortisone, and diminished 11β-HSD2 in the WOI phase compared with those from non-PCOS and PCOS without IR patients. PCOS with IR patients also exhibited endometrial insulin signaling pathway defects and relatively lower implantation rates. Further in vitro studies clarified that excessive cortisol might attenuate insulin sensitivity by decreasing the phosphorylation of Akt (Ser473) and the translocation of GLUT4 via induction of PTEN expression. Thus, this study uncovered the imbalanced state of cortisol and cortisone in endometrium of PCOS with IR patients and established a correlation between the elevated local cortisol and endometrial IR. With a prevalence of 44% to 70%, IR is common among PCOS patients. IR is traditionally defined as the insensitivity or unresponsiveness to insulin and thus requires an increased insulin level (4). In addition to systemic dysfunction of hyperinsulinemia, IR also exhibits as peripheral IR affecting insulin target tissue including endometrium. Li et al. (24) found that maternal hyperinsulinemia impaired mouse endometrial receptivity in early pregnancy. Chang et al. (10) found that PCOS patients with IR had compromised implantation rates, suggesting the effects of IR on endometrial function and receptivity. Consistently, we also observed a lower implantation rate in PCOS with IR patients, although it was not statistically significant, possibly due to the small number of patients in our study. All the evidence suggested that IR might play an important role on human endometrial receptivity in PCOS. Successful implantation requires the endometrium to undergo changes and be receptive to embryos in a short period known as the WOI phase (25). This demands a large amount of energy, mainly from glucose uptake, for the endometrium to properly differentiate to a receptive state (26, 27). Therefore, local endometrial IR might diminish the glucose uptake in endometrial cells and interfere with endometrial receptivity. The metabolic actions of insulin are mainly mediated through activation of IRS-1, phosphatidylinositol-3,4,5-trisphosphate, and Akt, resulting in the translocation of GLUT4 from intracellular vesicles to the plasma membrane. Defects in this signaling pathway could induce IR (4). Previous studies have reported the diminished endometrial IRS-1 in PCOS with hyperinsulinemia and increased endometrial PTEN expression in PCOS (8, 9, 28). In this study, we also revealed significantly elevated PTEN and relatively decreased IRS-1 in PCOS with IR patients. Furthermore, we demonstrated that IRS-1 phosphorylation at Ser318 and Ser612 was increased and Akt phosphorylation at Ser473 was diminished in the endometria from PCOS with IR patients. These evidences further clarified the defective insulin signaling pathway in the endometria from PCOS with IR patients. Because serine phosphorylation of IRS-1 acts as a negative feedback signal for insulin effect, we provided supporting evidence for the former study of Fornes et al. (9) that the IRS-1 activating tyrosine phosphorylation (Y612) was decreased in endometria of PCOS. In addition, the diminished GLUT4 translocation and glucose uptake in the EECs of PCOS with IR patients further clarified the local IR in endometrium. Excessive glucocorticoid exposure has correlated with whole-body IR for decades, and the local effect of cortisol is exaggerated in insulin-target tissue (29, 30). It has been documented that excessive cortisol generated by 11β-HSD1 contributes to the development of IR in adipose, skeletal muscle, and granulosa cells (19, 31, 32). However, quantitative measurements of cortisol and its metabolic enzymes in the endometria of PCOS women have not been performed previously. In endometrium, 11β-HSD1 expression was low except during the menstrual and decidua phases, whereas 11β-HSD2 was the codominant metabolic glucocorticoid enzyme in the secretory phase (13, 14). Here we detailed the elevated cortisol and ratios of cortisol to cortisone in the endometria obtained from PCOS with IR patients. This might reflect either an increase in local reduction of cortisone or a decrease in local oxidation of cortisol. Upon further exploration, we found a significant decrease of 11β-HSD2 in the endometria from PCOS with IR patients, whereas 11β-HSD1 was comparable among the three groups. Furthermore, 11β-HSD2 was negatively correlated with cortisol and positively correlated with cortisone levels. Based on these data, we attributed the elevated ratios of cortisol to cortisone in the endometria of PCOS with IR patients mainly to the decreased local cortisol oxidation caused by 11β-HSD2. This was consistent with the codominant role for 11β-HSD2 in secretory phase. An optimal amount of cortisol is indispensable to embryo implantation (33). However, excessive cortisol caused by decreased oxidation of 11β-HSD2 may disrupt glucocorticoid homeostasis and impair insulin sensitivity in endometrium. As embryo implantation is initiated by embryo attachment to endometrial epithelium (34) and 11β-HSD2 is mainly expressed in endometrial epithelium, we focused on the effects of cortisol on insulin sensitivity in EECs. In the insulin-stimulated glucose uptake pathway, GLUT4 translocation is critical and mediated by Akt phosphorylation. Defects in GLUT4 translocation and Akt phosphorylation indicated IR (35, 36). In this study, we demonstrated that glucose uptake, Akt phosphorylation, and exocytosis of GLUT4 to the plasma membrane were inhibited by cortisol in EECs from both non-PCOS and PCOS patients. This suggested that cortisol was involved in endometrial IR. It is understood that PTEN negatively regulates Akt activity by dephosphorylating phosphatidylinositol-3,4,5-trisphosphate (37, 38) and the induction of PTEN expression could attenuate insulin sensitivity (37, 39). Our in vitro study revealed that PTEN was induced by cortisol in EECs. Additionally, the inhibitor of PTEN restored the cortisol-caused depression of Akt phosphorylation and GLUT4 translocation, suggesting a crucial role of PTEN in cortisol-stimulated endometrial IR. Because PTEN expression in endometrium was also closely correlated with cortisol level, PTEN might be recognized as an important contributor to cortisol-induced IR in endometrium. In this study, we intended to compare the endometria from non-PCOS, PCOS without IR, and PCOS with IR, but we did not exclude the possibility that glucocorticoid alteration might also exist in non-PCOS IR patients. Furthermore, the causes of diminished 11β-HSD2 and the specific underlying molecular mechanisms involving PTEN require further investigation. Our study provides vital preliminary evidence for direct future research on cortisol-induced endometrial IR. In conclusion, we demonstrated that PCOS with IR patients had increased cortisol, diminished 11β-HSD2, and impaired insulin sensitivity in endometrium in the WOI phase compared with non-PCOS and PCOS without IR patients. This indicated that a decreased local inactivation of cortisol by 11β-HSD2 might be a possible cause of endometrial IR in PCOS. Our in vitro study suggested a detrimental role of cortisol in insulin sensitivity in endometrium. We propose that maintaining cortisol at an optimal level in PCOS patients might be beneficial for embryo implantation. Abbreviations: Abbreviations: 11β-HSD 11β-hydroxysteroid dehydrogenase EEC endometrial epithelial cell GLUT4 glucose transporter type 4 hCG human chorionic gonadotropin HOMA-IR homeostasis model assessment of insulin resistance index IR insulin resistance IRS-1 insulin receptor substrate 1 mRNA messenger RNA PCOS polycystic ovary syndrome PCR polymerase chain reaction PTEN phosphatase and tensin homolog deleted on chromosome ten WOI window of implantation Acknowledgments We thank Huiliang Xie, Xiaoming Zhao, Yan Hong, and Minzhi Gao for help with patient recruitment and endometrial biopsies collection and Jianjun Liu, Xiaoping Zhao, Li Zhao, and Panli Li for skillful technical support. Financial Support: This work was supported by National Natural Science Foundation of China Grant 81771648 (to Y.S.), National Key R&D Program of China Grant 2017YFC1001403 (to Y.S.), National Natural Science Foundation of China Grant 81571499 (to Y.S.), Chinese National Key Basic Research Projects Grant 2014CB943300 (to Y.S.), Program of Shanghai Academic Research Leader in Shanghai Municipal Commission of Health and Family Planning Grant 2017BR015 (to Y.S.), Clinical Skills Improvement Project of Major Disorders Hospital Development Center of Shanghai Grant 16CR1022A (to Y.S.), Shanghai Technological Innovation Plan Grant 18140902400 (to Y.S.), and Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant 20161413 (to Y.S.). Disclosure Summary: The authors have nothing to disclose. References 1. Azziz R , Carmina E , Chen Z , Dunaif A , Laven JS , Legro RS , Lizneva D , Natterson-Horowtiz B , Teede HJ , Yildiz BO . Polycystic ovary syndrome . 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Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Mar 30, 2018

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