The impact of obesity and bariatric surgery on the immune microenvironment of the endometrium.

BACKGROUND: The incidence of endometrial cancer is rising in parallel with the obesity epidemic. Obesity increases endometrial cancer risk and weight loss is protective, but the underlying mechanisms are incompletely understood. We hypothesise that the immune microenvironment may influence susceptibility to malignant transformation in the endometrium. The aim of this study was to measure the impact of obesity and weight loss on the immunological landscape of the endometrium. METHODS: We conducted a prospective cohort study of women with class III obesity (body mass index, BMI ≥ 40 kg/m2) undergoing bariatric surgery or medically-supervised low-calorie diet. We collected blood and endometrial samples at baseline, and two and 12 months after weight loss intervention. Serum was analysed for inflammatory markers CRP, IL-6 and TNF-α. Multiplex immunofluorescence was used to simultaneously identify cells positive for immune markers CD68, CD56, CD3, CD8, FOXP3 and PD-1 in formalin-fixed paraffin-embedded endometrial tissue sections. Kruskal-Wallis tests were used to determine whether changes in inflammatory and immune biomarkers were associated with weight loss. RESULTS: Forty-three women with matched serum and tissue samples at all three time points were included in the analysis. Their median age and BMI were 44 years and 52 kg/m2, respectively. Weight loss at 12 months was greater in women who received bariatric surgery (n = 37, median 63.3 kg) than low-calorie diet (n = 6, median 12.8 kg). There were significant reductions in serum CRP (p = 3.62 × 10-6, r = 0.570) and IL-6 (p = 0.0003, r = 0.459), but not TNF-α levels, with weight loss. Tissue immune cell densities were unchanged except for CD8+ cells, which increased significantly with weight loss (p = 0.0097, r = -0.323). Tissue CD3+ cell density correlated negatively with systemic IL-6 levels (p = 0.0376; r = -0.318). CONCLUSION: Weight loss is associated with reduced systemic inflammation and a recruitment of protective immune cell types to the endometrium, supporting the concept that immune surveillance may play a role in endometrial cancer prevention.

PROgesterone Therapy for Endometrial Cancer Prevention in Obese Women (PROTEC) Trial: A Feasibility Study.

Obesity is the major etiologic driver for endometrial cancer. The levonorgestrel intrauterine system (LNG-IUS) reduces the risk of endometrial cancer and its precursor, atypical hyperplasia. We assessed feasibility and uptake of the LNG-IUS for primary prevention of endometrial cancer in high-risk women and its impact on endometrial tissue biomarkers. Women with class-III obesity [body mass index (BMI) > 40 kg/m2] and histologically normal endometrium were invited to participate in a clinical trial of the LNG-IUS for endometrial protection. Recruitment, successful LNG-IUS insertion, and adherence to trial procedures were recorded. We measured impact of the LNG-IUS on circulating biomarkers of endometrial cancer risk, endometrial proliferation (Ki-67, pAKT, PTEN), endometrial hormone receptor status [estrogen receptor and progesterone receptor (PR)], mental wellbeing, and menstrual function. At 6 months, women chose to keep their LNG-IUS or have it removed. In total, 103 women were approached, 54 were offered a participant information sheet, 35 agreed to participate, and 25 received a LNG-IUS. Their median age and BMI were 54 years [interquartile range (IQR) 52-57] and 47 kg/m2 (IQR 44-51), respectively. Three women (3/35, 9%) were ineligible due to atypical hyperplasia/endometrial cancer on their baseline biopsy. The LNG-IUS was well tolerated and had a positive overall effect on bleeding patterns and mental wellbeing. The LNG-IUS was associated with endometrial morphologic change, reduced Ki-67, and PR expression, but circulating biomarkers of endometrial cancer risk were unchanged. All but one woman (96%) kept her LNG-IUS. The LNG-IUS appears to be acceptable to some women with class-III obesity for primary prevention of endometrial cancer, which could provide a strategy for a prevention trial.Prevention Relevance: Novel strategies are urgently needed to prevent the rise in endometrial cancer diagnoses predicted by escalating obesity rates. Here, we show that women with class III obesity are willing to engage in risk reduction with a levonorgestrel intrauterine system, which could provide a strategy for an endometrial cancer prevention trial.

Baseline Serum HE4 But Not Tissue HE4 Expression Predicts Response to the Levonorgestrel-Releasing Intrauterine System in Atypical Hyperplasia and Early Stage Endometrial Cancer.

The levonorgestrel-releasing intrauterine system (LNG-IUS) is a conservative management option for atypical hyperplasia (AH) and low grade early stage endometrial cancer (EEC), but around 1 in 3 patients fail to respond to treatment. The aim of this study was to investigate if serum and/or tissue HE4 expression could predict response to LNG-IUS therapy. Patients with AH or presumed Stage I EEC had serum and endometrial samples taken at baseline and at 3-month intervals over 12 months post-insertion of LNG-IUS. 74 patients were recruited and baseline demographics recorded. Of 57 patients for whom response was histologically determinable, 39 (68%) were responders and 18 (32%) non-responders. Mean baseline serum HE4 was significantly lower in responders (62.1 ± 1.1 pM, 95% confidence interval (CI) 52.7-73.2), compared to non-responders (125.6 ± 1.3 pM, 95% CI 74.5-211.7, p = 0.014), including when considering age, BMI, menopausal status, smoking status, and histological grade as covariables (p = 0.005). Baseline tissue HE4 expression was not significantly different in responders compared to non-responders (p = 0.999). Responders showed a significant mean reduction (-9.8 ± 3.4%, 95% CI -16.7 to -2.8%, p = 0.008) in serum HE4 between baseline and 3 months (p = 0.008), whereas non-responders showed no significant change (p = 0.676). Neither responders nor non-responders showed a significant percentage change in serum HE4 from baseline beyond 3 months (p > 0.05). Change in serum HE4 between baseline and 3 and 6 months and tissue HE4 tissue expression between baseline and 3, 6, and 12 months was not significantly different in responders compared to non-responders (p > 0.05). This study suggests that baseline serum HE4, but not baseline tissue HE4 expression, is independently predictive of response to the LNG-IUS and could be used to guide management decisions.

PRE-surgical Metformin In Uterine Malignancy (PREMIUM): a Multi-Center, Randomized Double-Blind, Placebo-Controlled Phase III Trial.

PURPOSE: Endometrioid endometrial cancer is strongly associated with obesity and insulin resistance. Metformin, an insulin sensitizer, reduces endometrial tumor growth in vitro. Presurgical window studies allow rapid in vivo assessment of antitumor activity. Previous window studies found metformin reduced endometrial cancer proliferation but these lacked methodological rigor. PREMIUM measured the anti-proliferative effect of metformin in vivo using a robust window study design.Patients and Methods: A multicenter, double-blind, placebo-controlled trial randomized women with atypical hyperplasia or endometrioid endometrial cancer to receive metformin (850 mg daily for 3 days, and twice daily thereafter) or placebo for 1 to 5 weeks until surgery. The primary outcome was posttreatment IHC expression of Ki-67. Secondary outcomes investigated the effect of metformin on markers of the PI3K-Akt-mTOR and insulin signaling pathways and obesity. RESULTS: Eighty-eight women received metformin (n = 45) or placebo (n = 43) and completed treatment. There was no overall difference in posttreatment Ki-67 between the metformin and placebo arms, in an ANCOVA analysis adjusting for baseline Ki-67 expression (mean difference -0.57%; 95% CI, -7.57%-6.42%; P = 0.87). Metformin did not affect expression of markers of the PI3K-Akt-mTOR or insulin signaling pathways, and did not result in weight loss. CONCLUSIONS: Short-term treatment with standard diabetic doses of metformin does not reduce tumor proliferation in women with endometrioid endometrial cancer awaiting hysterectomy. This study does not support a biological effect of metformin in endometrial cancer and casts doubt on its potential application in the primary and adjuvant treatment settings.

The impact of obesity and bariatric surgery on circulating and tissue biomarkers of endometrial cancer risk.

Obesity is the strongest risk factor for endometrial cancer (EC). To inform targeted screening and prevention strategies, we assessed the impact of obesity and subsequent bariatric surgery-induced weight loss on endometrial morphology and molecular pathways implicated in endometrial carcinogenesis. Blood and endometrial tissue were obtained from women with class III-IV obesity (body mass index ≥40 and ≥50 kg/m2 , respectively) immediately prior to gastric bypass or sleeve gastrectomy, and at two and 12 months' follow up. The endometrium underwent pathological examination and immunohistochemistry was used to quantify proliferation (Ki-67), oncogenic signaling (PTEN, pAKT, pERK) and hormone receptor (ER, PR) expression status. Circulating biomarkers of insulin resistance, reproductive function and inflammation were also measured at each time point. Seventy-two women underwent bariatric surgery. At 12 months, the mean change in total and excess body weight was -32.7 and -62.8%, respectively. Baseline endometrial biopsies revealed neoplastic change in 10 women (14%): four had EC, six had atypical hyperplasia (AH). After bariatric surgery, most cases of AH resolved (5/6) without intervention (3/6) or with intrauterine progestin (2/6). Biomarkers of endometrial proliferation (Ki-67), oncogenic signaling (pAKT) and hormone receptor status (ER, PR) were significantly reduced, with restoration of glandular PTEN expression, at 2 and 12 months. There were reductions in circulating biomarkers of insulin resistance (HbA1c, HOMA-IR) and inflammation (hsCRP, IL-6), and increases in reproductive biomarkers (LH, FSH, SHBG). We found an unexpectedly high prevalence of occult neoplastic changes in the endometrium of women undergoing bariatric surgery. Their spontaneous reversal and accompanying down-regulation of PI3K-AKT-mTOR signaling with weight loss may have implications for screening, prevention and treatment of this disease.

Serum cystatin C is independently associated with renal impairment and high sensitivity C-reactive protein in systemic lupus erythematosus.

OBJECTIVES: In systemic lupus erythematosus (SLE) patients, glomerular filtration rate (GFR) is usually estimated using the modified Cockcroft-Gault (mCG) and Modification of Diet in Renal Disease (MDRD) equations. We aimed to study cystatin C (sCysC) in SLE to assess its agreement with standard renal indices and investigate factors affecting sCysC in SLE. METHODS: SLE patients (≥4 ACR criteria) and healthy women from Greater Manchester were recruited and clinical assessments were undertaken. SCysC was measured using R & D Systems' ELISA. Agreement between renal measures was assessed using Deming plots and factors associated with sCysC in SLE were examined by multiple linear regression analyses. RESULTS: 178 patients and 68 controls had median (IQR) ages of 53 (46-61) and 50 (39-60) years, respectively. In an age-adjusted analysis, SLE patients had higher sCysC (1.16 [0.98-1.36] vs. 0.950 [0.73-1.13] mg/l; p<0.0001) and within SLE those with a history of lupus nephritis had higher sCysC (1.31 [1.10-1.66] vs. 1.11 [0.95-1.29] mg/l; p<0.005). SCysC correlated positively with serum creatinine, and inversely to renal measures (r=-0.530; p<0.0001 [mCG], and r=-0.620; p<0.0001 [MDRD]). There was closer agreement between the two eGFR measures than between either eGFR measures and sCysC. In addition to age and serum creatinine, a multivariate analysis (ß, p) found that high-sensitivity C-reactive protein (hs-CRP) (0.03, 0.026) was also independently associated with sCysC in SLE. CONCLUSIONS: In SLE, sCysC may be influenced by low grade inflammation as well as by renal dysfunction. Therefore, SCysC should not supplant current assessment of renal dysfunction in SLE.

Explanations for the lower rates of diabetic neuropathy in Indian Asians versus Europeans.

OBJECTIVE: Risks of diabetes and cardiovascular disease are elevated worldwide in Indian Asians. However, risks of other diabetes-related complications, i.e., foot ulceration and amputation, also with a vascular basis, are substantially lower in Asians than in white Europeans in the U.K., possibly due to less neuropathy. We therefore compared signs, symptoms, and objective quantitative measures of diabetic neuropathy and their risk factors in Indian Asians and Europeans. RESEARCH DESIGN AND METHODS: This was a cross-sectional study of a population-based sample of age- and sex-matched adults with type 2 diabetes of European (95 male and 85 female) and Asian (96 male and 84 female) descent in the U.K. Patients were assessed for neuropathic symptoms, signs, nerve conduction, autonomic function, and quantitative sensory testing. Peripheral vascular function and other potential risk factors for neuropathy were measured. RESULTS Mean nerve conduction velocity Z scores were better in Asians (mean +/- SD 0.07 +/- 0.62) than in Europeans (-0.11 +/- 0.60; P = 0.007) and were explained by the shorter height, fewer pack-years smoked, and higher transcutaneous oxygen levels (TCpO(2)) in Indian Asians (P value for ethnic comparison attenuated to 0.2). Small fiber neuropathy was less prevalent in Indian Asians compared with Europeans (odds ratio 0.58 [95% CI 0.37-0.93]; P = 0.02) and was primarily accounted for by better TCpO(2) (0.70 [0.40-1.21]; P = 0.2). CONCLUSIONS: Asians with diabetes have substantially less large and small fiber neuropathy than Europeans, despite comparable traditional risk factors. Independent from smoking, the lower risk of neuropathy in Asians is due to better skin microvascularization and may help explain the substantially reduced Asian foot ulcer risk.

Biomarkers of oxidant stress, insulin sensitivity and endothelial activation in rheumatoid arthritis: a cross-sectional study of their association with accelerated atherosclerosis.

BACKGROUND: Women with rheumatoid arthritis (RA) have increased morbidity and mortality due to coronary heart disease. Chronic systemic inflammation is known to accelerate atherosclerosis and increase arterial stiffness in patients, but other mechanisms may also be involved. Biomarkers of oxidant stress, inflammation, insulinaemia and endothelial dysfunction were measured in blood and urine from 46 RA patients and 48 age-matched controls. Plaque formation and intima-medial thickness (IMT) were measured using B-mode carotid Doppler scan. FINDINGS: The prevalence of plaque was increased (p = 0.042) in RA patients between 50-59 years old compared to the same age group in controls. 8-isoprostane (p = 0.004), C-reactive protein (p < 0.001), interleukin-6 (p < 0.001), insulin (p = 0.035), adiponectin (p = 0.012), vascular cell adhesion molecule (VCAM) (p = 0.029) and E-selectin (p < 0.001) were all increased while selenium (p = 0.003) and LDL-cholesterol (p = 0.025) were both decreased in all RA patients. 8-isoprostane correlated with 10 year cardiac risk (r = 0.55, p < 0.001), VCAM with IMT (r = 0.37, p = 0.012) and E-selectin with rheumatoid factor titre (r = 0.43, p = 0.003) in RA patients. In the control group, age, carotid IMT, VCAM, systolic blood pressure and smoking status were all associated with plaque development whereas in RA patients only age was associated with plaque. CONCLUSION: The burden of atherosclerosis is particularly increased in middle-aged women with RA. Patients with RA have increased levels of oxidant stress, inflammation, insulin and soluble adhesion molecules. As the association between classical risk factors was much weaker in RA patients compared to controls, these additional factors may be more important in the accelerated development of atheroma in RA.

Local inflammation and hypoxia abolish the protective anticontractile properties of perivascular fat in obese patients.

BACKGROUND: Inflammation in adipose tissue has been implicated in vascular dysfunction, but the local mechanisms by which this occurs are unknown. METHODS AND RESULTS: Small arteries with and without perivascular adipose tissue were taken from subcutaneous gluteal fat biopsy samples and studied with wire myography and immunohistochemistry. We established that healthy adipose tissue around human small arteries secretes factors that influence vasodilation by increasing nitric oxide bioavailability. However, in perivascular fat from obese subjects with metabolic syndrome (waist circumference 111+/-2.8 versus 91.1+/-3.5 cm in control subjects, P<0.001; insulin sensitivity 41+/-5.9% versus 121+/-18.6% in control subjects, P<0.001), the loss of this dilator effect was accompanied by an increase in adipocyte area (1786+/-346 versus 673+/-60 mum(2), P<0.01) and immunohistochemical evidence of inflammation (tumor necrosis factor receptor 1 12.4+/-1.1% versus 6.7+/-1%, P<0.001). Application of the cytokines tumor necrosis factor receptor-alpha and interleukin-6 to perivascular fat around healthy blood vessels reduced dilator activity, resulting in the obese phenotype. These effects could be reversed with free radical scavengers or cytokine antagonists. Similarly, induction of hypoxia stimulated inflammation and resulted in loss of anticontractile capacity, which could be rescued by catalase and superoxide dismutase or cytokine antagonists. Incubation with a soluble fragment of adiponectin type 1 receptor or inhibition of nitric oxide synthase blocked the vasodilator effect of healthy perivascular adipose tissue. CONCLUSIONS: We conclude that adipocytes secrete adiponectin and provide the first functional evidence that it is a physiological modulator of local vascular tone by increasing nitric oxide bioavailability. This capacity is lost in obesity by the development of adipocyte hypertrophy, leading to hypoxia, inflammation, and oxidative stress.

Ursodeoxycholic acid in primary biliary cirrhosis improves glutathione status but fails to reduce lipid peroxidation.

BACKGROUND: Ursodeoxycholic acid (UDCA) may slow progression in primary biliary cirrhosis (PBC), but its effect on survival is controversial. We have previously demonstrated that oxidant stress, with severely depressed plasma glutathione, is a feature of untreated PBC; this study examines the effect of UDCA on lipid peroxidation, antioxidant status and associated processes. PATIENTS AND METHODS: Markers of lipid peroxidation, antioxidant status, hepatic fibrogenesis, inflammation, cholestasis and synthetic function were measured at 0, 3, 6, 9 and 12 months in blood and urine from 35 PBC patients receiving UDCA. RESULTS: Plasma glutathione, reflecting intrahepatic levels, climbed steadily on UDCA; although still subnormal, the median value at 12 months was 2.4-fold higher than the untreated level. Liver enzyme markers and C-reactive protein also improved, whilst PIIINP improved steadily, but the change did not attain statistical significance. Serum bilirubin remained unchanged and total antioxidant capacity, albumin and vitamin E decreased after 12 months' UDCA treatment. 8-Isoprostane increased and malondialdehyde was unchanged. CONCLUSIONS: UDCA treatment partially corrected plasma glutathione status and some other biomarkers greatly improved, but lipid peroxidation was not reduced. UDCA may, therefore, require supplementation with glutathione precursors and/or antioxidant cocktails to reduce oxidant stress and thus delay disease progression to cirrhosis.

Oxidant stress is a significant feature of primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is a chronic cholestatic disorder characterised by an immunological, and often granulomatous, attack on bile ducts leading to fibrosis, cirrhosis, liver failure and death. Animal and human studies suggest that oxidant stress plays a key role in progression of other liver diseases, but no comprehensive investigation has been performed previously in PBC. A wide range of lipid peroxidation and antioxidant markers were measured in the blood and urine of 41 patients with histologically confirmed PBC. Lipid peroxidation markers were significantly elevated [plasma and urinary 8-isoprostane, P<0.001; plasma malondialdehyde (MDA), P=0.007] compared to age- and sex-matched controls. The most striking antioxidant depletion occurred with plasma total glutathione where levels were significantly reduced (30% of controls). Total serum antioxidant levels were decreased (P=0.013) and serum selenium and vitamin A were also lower (both P<0.001); vitamins C and E were normal. Most patients had early disease biochemically and were Child-Pugh grade A. Urinary 8-isoprostane correlated positively with Ludwig stage and markers of hepatic injury and cholestasis. This study clearly demonstrates that oxidant stress, as reflected in a comprehensive spectrum of lipid peroxidation and antioxidant markers, is a significant feature of early-stage PBC.

Oxidative stress in chronic hepatitis C: not just a feature of late stage disease.

BACKGROUND/AIMS: Chronic hepatitis C infection is a major world-wide problem, frequently progressing to cirrhosis, liver failure or hepatoma. The pathological mechanisms of disease progression are unclear but oxidant stress may play a role. METHODS: Markers of lipid peroxidation, antioxidant status, hepatic fibrogenesis and liver function were measured in blood or urine from 42 chronic hepatitis C patients. Fibrosis was graded histologically in a subgroup of 33 patients. RESULTS: The lipid peroxidation marker 8-isoprostane and the ratio of oxidized to reduced glutathione were significantly elevated (P<0.001, P=0.006). The antioxidants glutathione, selenium and vitamins A, C and E were significantly decreased (all P<0.001) compared to age and sex matched controls. Abnormal values were more marked in cirrhotics, but significant changes were also observed in the non-cirrhotic group. The fibrosis score correlated positively with urinary 8-isoprostane and type III procollagen peptide and negatively with vitamin A. CONCLUSIONS: Oxidant stress, as reflected in blood and urine by a wide range of pro- and antioxidant markers, is a significant feature of hepatitis C infection. Although more severe in the cirrhotic group, there was clear evidence of oxidant stress in non-cirrhotic patients. Antioxidant therapy may therefore have a role in slowing disease progression to cirrhosis.