1

DIETARY GLYCOTOXINS AFFECT SCAVENGER RECEPTORS EXPRESSION 1

AND THE HORMONAL PROFILE OF FEMALE RATS. 2

3

Antonios Chatzigeorgiou1,2

, Eleni Kandaraki3, Christina Piperi

3, Sarantis Livadas

4, 4

Athanasios G. Papavassiliou3, Michael Koutsilieris

1, Apostolos Papalois

5, Evanthia Diamanti-5

Kandarakis4 6

7

1Department of Experimental Physiology, University of Athens Medical School, Athens, 8

Greece 9

2Department of Internal Medicine III and Institute of Physiology, Dresden University of 10

Technology, Dresden, Germany 11

3Department of Biological Chemistry, University of Athens Medical School, Athens, Greece 12

4Endocrine Unit, Third Department of Internal Medicine, University of Athens Medical 13

School, Sotiria Hospital, Greece 14

5Experimental Research Centre, ELPEN Pharmaceuticals, Greece 15

16

17

18

19

Address correspondence to: 20

Evanthia Diamanti-Kandarakis 21

Professor of Internal Medicine and Endocrinology 22

Endocrine Unit, Third Department of Internal Medicine, University of Athens Medical 23

School, Sotiria Hospital, Greece 24

Tel: +30-210-7706410 25

Email:e.diamanti.kandarakis@gmail.com 26

27

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RUNNING TITLE 29

AGEs’ scavenger receptors and endocrine dysregulation 30

31

KEYWORDS 32

AGEs, RAGE, SR-A, PCOS, insulin resistance, endocrine dysregulation 33

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Word Count: 2561 36

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Page 1 of 18 Accepted Preprint first posted on 3 July 2013 as Manuscript JOE-13-0175

Copyright © 2013 by the Society for Endocrinology.

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ABSTRACT 38

Advanced glycation end-products (AGEs) are increased under conditions of impaired glucose 39

metabolism and/or oxidative stress, promoting insulin resistance and other endocrine 40

abnormalities. AGEs play a major role in the pathogenesis of several diseases such as 41

diabetes, atherosclerosis, PCOS and Alzheimer’s disease, contributing to progressive ageing. 42

Receptor-based clearance of AGEs by the Receptor for AGE (RAGE) and/or the Macrophage 43

Scavenger receptor-A (SR-A) is considered as main factor for the regulation of AGEs' 44

concentration in these conditions. This study aims to investigate the expression of RAGE 45

and SR-A under high/low dietary AGE conditions in vivo and their potential 46

contribution to the metabolic and sex hormone profile of female rats. 47

Female Wistar rats were fed a low- or high-AGEs diet for 3months. Serum samples were 48

taken at baseline and at the completion of 3months period for measurements of metabolic and 49

hormonal parameters. Peripheral blood mononuclear cells were isolated for determination of 50

RAGE and SR-A expression. The high-AGEs fed animals showed increased glucose, insulin 51

and testosterone levels, as well as decreased estradiol and progesterone levels compared to the 52

low-AGEs fed ones, thus indicating a metabolic and hormonal dysregulation attributed to 53

high-AGEs dietary exposure. The expression of RAGE was significantly downregulated in 54

PBMCs of high-AGEs fed rats (p=0.041) and it was negatively correlated to insulin and 55

testosterone levels, while positively correlated to progesterone levels. SR-A expression was 56

also decreased in high-AGEs fed rats to marginal significance. Decreased monocytic 57

expression of scavenger receptors such as RAGE or SR-A may result in higher deposition of 58

AGEs in peripheral endocrine tissues, thus promoting endocrine-related abnormalities and 59

diseases. 60

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INTRODUCTION 64

Advanced glycation end-products (AGEs) are a heterogeneous group of cross-linking 65

molecules, formed from non-enzymatic reaction of reducing sugars with the amino groups of 66

proteins and lipids via the Maillard reaction. AGEs are highly reactive molecules, which may 67

induce many injuries in cells and tissue structures by the formation of insoluble cross-links, 68

reduction of enzymatic activity, induction of oxidative stress, damage to nucleic acids and 69

activation of cytotoxic pathways (Niwa 2006; Tan, et al. 2011). AGEs are accumulated in 70

conditions of impaired glucose metabolism or oxidative stress and thus are predominantly 71

increased during ageing and diabetes mellitus (DM), while they seem to play a pivotal role in 72

the pathogenesis of other diseases such as atherosclerosis, chronic renal failure, polycystic 73

ovary syndrome (PCOS) and Alzheimer’s disease (Mukhopadhyay and Mukherjee 2005; Tan 74

et al. 2011). Furthermore, data in humans and experimental animals favour that exogenous 75

food-ingested AGEs, present in highly thermolyzed fat-containing Westernized diets, result in 76

elevated serum levels and increased tissue deposition of AGEs that can provoke the initiation 77

or progression of insulin resistance and other endocrine abnormalities (Cai, et al. 2008; de 78

Assis, et al. 2009; Diamanti-Kandarakis, et al. 2007a). Similarly, low glycotoxin-fed mice 79

exhibit extended lifespan and reduced oxidative stress, while restriction of AGEs improves 80

insulin resistance in T2DM patients (Cai, et al. 2007; Uribarri, et al. 2011). 81

RAGE, a member of the immunoglobulin superfamily, which interacts with numerous 82

ligands including AGEs, is expressed on the surface of a variety of cell types, including 83

endothelial cells, smooth muscle cells, lymphocytes, monocytes and neurons (Mukhopadhyay 84

and Mukherjee 2005), although it also exists in a soluble form, as the product of proteolytic 85

cleavage of RAGE from the cell surface or alternative splicing (Sourris and Forbes 2009). 86

The interaction of RAGE with AGEs generates ROS via the activation of NADPH oxidase 87

and pro-inflammatory transcription factor NF-κB that in turn engages pro-inflammatory and 88

pro-oxidative signaling events in cells. NF-κB may contribute to the development of a pro-89

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inflammatory and pro-oxidative state by stimulating the expression of numerous target genes, 90

such as tissue factor (TF), vascular cell adhesion molecule-1 (VCAM-1), p21Ras, 91

extracellular signal-regulated kinase (ERK) 1/2 and RAGE itself (Mukhopadhyay and 92

Mukherjee 2005; Tanaka, et al. 2000). Furthermore, AGE-RAGE signalling through NF-κB 93

activation has been reported to regulate expression of inflammatory cytokines (such as TNF-α 94

and interleukin-1), PAI-1 and endothelin-1, contributing to the development of several 95

inflammation-related conditions and diseases such as atherosclerosis, diabetic nephropathy 96

and retinopathy (Diamanti-Kandarakis, et al. 2007b; Murphy, et al. 2005). 97

AGEs being generated through long-term exposure of proteins to glucose, also behave 98

as active ligands for scavenger receptors, including class A scavenger receptor (SR-A) and 99

class B scavenger receptors such as CD36 and class B, type I scavenger receptor (SR-BI) 100

(Miyazaki, et al. 2002). Macrophage scavenger receptor A (SR-A), the product of the MSR-1 101

gene, is a multifunctional, multiligand pattern recognition receptor with roles in innate 102

immunity, apoptotic cell clearance, and age-related degenerative pathologies, such as 103

atherosclerosis and Alzheimer's disease. Endogenous SR-A ligands are commonly 104

polyanionic and include modified lipoproteins, AGEs, and extracellular matrix proteins 105

(Neyen, et al. 2009). Therefore the potential role of SR-A as a defence mechanism of AGEs 106

regulation during AGEs accumulating pathologies should be defined (Cai, et al. 2012). 107

We have previously shown that high-AGEs diets had an impact on metabolic as well 108

as on the hormonal profile of female rats, in addition to AGEs deposition in the ovarian tissue 109

(Diamanti-Kandarakis et al. 2007a). The aim of the present study was to investigate the 110

expression of the scavenger receptors RAGE and SR-A under high AGE environment and 111

their potential involvement or contribution to the deregulation of insulin and female hormone 112

levels. 113

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MATERIALS AND METHODS 115

Animals and diets 116

Female Wistar rats (10 per group, aged 21 days at feeding’s start) were fed a low- or 117

high-AGEs containing diet as previously described (Kandaraki, et al. 2012). 118

A single standard rat chow (AIN-93G, Bioserve, Frenchtown, NJ), containing 18% 119

protein, 58% carbohydrate, 7.5% fat, and 3.73 kcal/g and prepared normally at 190°C for 30 120

min was used for the preparation of the diets as previously described (Diamanti-Kandarakis et 121

al. 2007a). For the high-in-AGEs diet (H-AGE) preparation, the diet contained 76.0±15.3 mg 122

carboxymethyllysine (CML)/100 g sample (or 436.9±88.1 mg CML/100 g protein), 123

205.32±22.25 mg fructoselysine/100 g sample (or 1,179.98±127.90 mg fructoselysine/100 g 124

protein), and 52.68±5.71 mg furosine/ 100 g sample (or 302.78±32.82 mg furosine/100 g 125

protein). The same diet mixture was also prepared without heating. This preparation, that was 126

considered as a low-in-AGE diet (L-AGE), was consisted by equivalent macro- and 127

micronutrient and energy content but contained 1.3±0.4 mg CML/100 g sample (or 7.7±2.2 128

mg CML/100 g protein), 104.58±3.08 mg fructoselysine/100 g sample (or 601.01± 17.7 mg 129

fructoselysine/100 g protein), and 26.83±0.79 mg furosine/100 g sample (or 154.22±4.54 mg 130

furosine/100 g protein). 131

The animals were housed under controlled conditions (21–22 oC, 55–65% humidity, 132

12-h light/12-h dark cycle) four to five per cage at ELPEN S.A, Experimental Research 133

Centre, Athens, Greece and were given pelleted food and water ad libitum. Animal care and 134

experimental procedures were approved by the Institutional Animal Care and Use Committee 135

and conformed to the “Guide for the Care and Use of Laboratory Animals” (Department of 136

Health, Education and Welfare, Athens, Greece). 137

The study was completed at 3 months period. The rats were sacrificed with 138

administration of 20 mg/mL xylazine hydrochloride and 100 mg/mL ketamine hydrochloride, 139

under anesthesia with ether, followed by blood sample collection. 140

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141

Laboratory procedures and assays 142

Blood samples from the animals were collected and immediately centrifuged at 4°C. 143

Serum samples were used immediately or stored at −80°C until analysis. The buffy coat of 144

each sample was collected and diluted in a balanced salt solution (BSS) and platelet-free 145

peripheral blood mononuclear cells (PBMCs) were isolated by using a Ficoll-plus gradient 146

(STEMCELL Technologies, France) and stored at −80°C for later use. 147

Insulin and progesterone were measured by using ELISA immunoassays (Biovendor 148

Laboratory Medicine, Czech Republic and Neogen Corporation, Lexington, KY, USA 149

respectively). Rat estradiol (E2) and testosterone were quantified by commercially available 150

ELISA kits (Calbiotech Inc., California, USA), while serum AGE levels (U/ml) were 151

evaluated by competitive CML-specific ELISA as described previously (Diamanti-152

Kandarakis, et al. 2005). Serum glucose concentrations (mg/dl) were measured with the 153

glucose oxidase technique (Linear Diagnostics, Spain). 154

155

Preparation of cell lysates 156

PBMCs were lysed according to Chatzigeorgiou et al. (Chatzigeorgiou, et al. 2010), in 157

1 % Triton (50 mmol / L Tris, pH 7.4, 150 mmol / L NaCl, 0.02 % sodium azide, and 1 % 158

Triton X-100) supplemented with protease inhibitor cocktail (Sigma, Missouri, USA) for 30 159

min on ice. After centrifugation at 13.000 rpm for 20 min, the supernatants were collected and 160

stored at −80°C. Protein content was determined by Bradford protein assay. 161

162

Western blot 163

Samples were separated by SDS-PAGE (10%) and transferred (100V, 3h) to a 164

nitrocellulose membrane (Bio-Rad, UK). The blot was blocked with 5% (w/v) milk, 0.1% 165

(v/v) Tween 20 in TBS at 4°C overnight followed by incubation with 0.5 µg/ml goat anti-rat 166

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RAGE polyclonal antibody (Santa Cruz Biotechnology, Heidelberg, Germany) or 0.5 µg/ml 167

goat anti-rat SR-A polyclonal antibody (Santa Cruz Biotechnology, Heidelberg, Germany) in 168

1% (w/v) milk, 0.1% Tween 20 in TBS for 2 hours at room temperature. 169

The membrane was washed 3 times with 0.1% Tween 20 in TBS and incubated with 170

0.1 µg/ml anti-goat IgG-HRP antibody (Santa Cruz Biotechnology, Heidelberg, Germany) in 171

5% (w/v) milk, 0.1% (v/v) Tween 20 in TBS for 1.5 hour at room temperature. The 172

membrane was again washed thrice with 0.1% Tween 20 in TBS and once with TBS. Relative 173

quantity loading was confirmed by using goat anti-rat beta-actin antibody (Santa Cruz 174

Biotechnology, Heidelberg, Germany) followed by the same secondary antibody as above. 175

The bands were visualized by Chemiluminescent Substrate (Thermo Scientific, Rockford, 176

USA), exposured to X-ray film (Thermo Scientific, Rockford, USA) and quantified by the 177

NIH Image / Gel Plotting analysis program (National Institutes of Health, Bethesda, MD) 178

(Rasband 1997-2004). The signals were standardized to the expression of beta-actin. 179

180

Statistical analysis 181

The data are expressed as means ± standard error of mean and data analysis was 182

performed using the SPSS 18.0 software for windows. A two-tailed Student’s t-test and the 183

Mann-Whitney test were used for parametric and non-parametric parameters respectively. The 184

Spearman rank correlation test was performed to examine the associations between 185

parameters tested. Statistically significant difference was defined as a P value <0.05. 186

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RESULTS 188

Metabolic and Hormonal Parameters 189

High-AGEs diet provoked several alterations in the metabolic and hormonal profile 190

between the two groups. Although the low-AGEs fed female rats displayed increased weight 191

gain compared to the high-AGEs fed group (153.11±6.48 vs 99.88±12.36 grams, p=0.002), 192

the latter were more insulin resistant at the end of the feeding as defined by the increased 193

glucose and insulin levels in these animals compared to the Low-AGEs group (141.00±10.66 194

vs 103.12±5.91 mg/dl, p=0.008 and 2.20±0.286 vs 0.98±0.139 µIU/ml, p=0.002 respectively). 195

Furthermore the dietary exposure to high AGEs levels provoked an hormonal deregulation of 196

the female reproductive system of the rats as shown by the increased testosterone 197

(0.330±0.023 vs 0.165±0,011 nmol/L, p<0.001) and the decreased estradiol (10.94±0.56 vs 198

20.44±1.38 pmol/L, p<0.001) and progesterone (16.01±2.21 vs 37.81±2.69 nmol/L, p<0.001) 199

levels in High-AGEs fed rats compared to Low-AGEs ones. This metabolic phenotype can be 200

attributed to the accumulation of AGEs in the body as shown by the higher serum levels of 201

AGEs in the high-AGEs fed rats (6.70 ±0.22 vs 4.71±0.36 U/ml, p<0.001). 202

203

RAGE and SR-A expression in PBMCs 204

To evaluate the role of RAGE and SR-A as scavenger receptors during exposure in 205

dietary AGEs we performed western blot for the detection of these receptors on PBMCs 206

isolated from Low- or High-AGEs fed rats. Indeed, the levels of both receptors were 207

downregulated in animals fed with a high-AGEs diet. More specifically, RAGE expression is 208

decreased in the High-AGEs fed group as compared to the Low-AGEs fed group (55.04 ± 209

3.71% vs 100 ± 29.08 %, p=0.041). Additionally, SR-A expression showed a tendency to 210

lower levels in the High-AGEs group in comparison to the Low-AGEs fed group (37.12 ± 211

3.49% vs 100 ± 46.20%, p=0.11) (Figure 1). 212

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Correlation analysis 214

The negative impact of the high AGEs concentrations on the metabolic and hormonal 215

regulation of the high-AGEs fed rats is indicated by the strong negative correlation of serum 216

AGEs levels to the levels of estradiol (rs=-0.800, p<0.001) and progesterone (rs=-0.521, 217

p=0.019) and the parallel positive correlation to insulin (rs=0.584, p=0.005) and testosterone 218

(rs=0.640, p=0.002) levels. The contribution of the scavenger receptors to this phenotype 219

could be demonstrated by the fact that RAGE expression showed negative correlation to 220

insulin and testosterone levels (rs=-0.581, p=0.009 and rs=-0.432, p=0.047 respectively) and 221

positive correlation to progesterone levels (rs=0.473, p=0.044). Similarly, SR-A expression 222

levels showed a trend to negative correlation to insulin levels (rs=-0.480, p=0.057) (Figure 2). 223

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DISCUSSION 225

Current evidence indicates that the accumulation of AGEs in the body either via 226

endogenous pathways or exogenous intake is associated with the development of insulin 227

resistance even in non-diabetic or non-obese individuals (de Assis et al. 2009; Tan et al. 228

2011). Apart from the metabolic deregulation, AGEs are also able to induce further hormone-229

related malfunctions such as functional alterations of the female reproductive system 230

(Diamanti-Kandarakis et al. 2005; Diamanti-Kandarakis et al. 2007a). However, the 231

mechanism by which these endocrine phenomena, due to AGEs accumulation, are taking 232

place is still unsettled. The scavenger receptors, such as RAGE, SR-A, CD36 or LOX-1, can 233

recognize AGEs and activate several cellular pathways promoting or dampening 234

inflammatory and oxidative processes. Therefore, they have been emerged as a major 235

regulatory mechanism of AGEs levels during AGEs-accumulating pathophysiological 236

conditions including ageing, diabetes, PCOS and Alzheimer’s disease (Miyazaki et al. 2002; 237

Murphy et al. 2005). 238

In this study we demonstrate that the levels of RAGE and SR-A were decreased in 239

peripheral monocytes of female rats fed with a high-AGEs diet compared to the low-AGEs 240

fed ones. Furthermore, the expression of RAGE was negatively correlated with insulin 241

and testosterone levels and positively with progesterone levels, while the expression of 242

SR-A showed a tendency to negative correlation with insulin levels. These findings 243

suggest a possible link between scavenger receptors expression with the metabolic 244

deregulation and the hormonal dysfunction in the female reproductive system that is taking 245

place under dietary exposure to high levels of AGEs. This is the first study demonstrating the 246

downregulation of SR-A expression upon AGEs accumulation due to dietary glycotoxins and 247

also the first study that correlates scavenger receptors expression with the hormonal 248

imbalance of the female reproductive system. 249

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Our data regarding RAGE expression agree with the results of Miura et al. and Sourris 250

et al. that presented decreased monocytic expression of RAGE in AGE-related diseases such 251

as diabetes, as a result of negative feedback loop in RAGE expression (Miura, et al. 2004; 252

Sourris, et al. 2010). Similarly, Leuner et al. have shown increased insulin resistance in 253

RAGE-deficient mice (Leuner, et al.). On the other hand, the concentrations of soluble forms 254

of the RAGE receptor have been found elevated in and correlated to increased concentrations 255

of serum AGEs, indicating that the decreased cellular expression of RAGE could be attributed 256

to increased shedding of the receptor or to a change of the alternative splicing process of the 257

gene (Nakamura, et al. 2007; Tan, et al. 2006). Therefore, the decreased antioxidant defence 258

due to lower cellular RAGE expression together with the increased levels of the soluble form 259

can probably explain the endocrine or vascular complications present in AGE-related 260

pathologies. In parallel, the expression of SR-A which also showed a tendency to decreased 261

levels in the high-AGEs fed group seems to contribute to the decreased clearance of AGEs, 262

promoting thus AGEs-related complications. Although SR-A related published data are still 263

controversial since high glucose or modified LDL are able to increase the expression of SR-A 264

(Fukuhara-Takaki, et al. 2005; Lam, et al. 2004), while TNF-α is able to decrease it in 265

macrophages (Hsu, et al. 1996), even these in vitro or in vivo data can not be compared to the 266

peripheral monocytic SR-A expression and have a lot of differences to the AGEs receptors’ 267

regulation in the dietary glycotoxins model that we performed here. 268

Another novel finding of this study is the possible link of scavenger receptors 269

expression, such as RAGE, to the hormonal deregulation of the female reproductive 270

system, since the monocytic expression of RAGE was negatively correlated to 271

testosterone levels and positively correlated to progesterone levels. This association could 272

imply either a direct interfering role of AGEs in ovarian steroidogenesis with increased 273

testosterone and ovulatory deregulation with poor progesterone production, or an indirect role 274

via aggravation of the insulin resistance which in turn deteriorates the ovarian dysfunction 275

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(Diamanti-Kandarakis et al. 2005). Additionally, the increased levels of AGEs and RAGE 276

expression, demonstrated by our group in rat and human ovaries (Diamanti-Kandarakis et al. 277

2007a) could be explained by the decreased expression of scavenger receptors in circulating 278

monocytes that in turn lead to an increased deposition of AGEs in endocrine related tissued 279

such as the ovaries. 280

In conclusion, here we demonstrate that dietary AGEs besides their implication to the 281

development of insulin resistance can also contribute to the hormonal deregulation of the 282

female reproductive system, as indicated by increased testosterone and decreased estradiol 283

and progesterone levels. This phenomenon is probably linked to the reduced monocytic 284

expression of scavenger receptors of AGEs such as RAGE and SR-A which in turn results in 285

higher deposition of AGEs in peripheral endocrine tissues and ends up to endocrine-related 286

abnormalities and diseases. 287

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DECLARATION OF INTEREST 289

There is no conflict of interest to declare. 290

291

FUNDING 292

This research did not receive any specific grant from any funding agency in the public, 293

commercial or not-for-profit sector. 294

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FIGURE LEGENDS 395

Figure 1 396

A. Western immunoblot analysis of peripheral blood mononuclear cells (PBMCs) lysates 397

from Low- and High-AGEs fed rats (LA and HA respectively) using specific antibodies 398

targeting SR-A, RAGE and actin proteins. 399

B, C. The western blot signal of the SR-A and RAGE bands was normalized to the intensity 400

of the corresponding beta-actin. In the graphs of panels B and C, the mean normalized RAGE 401

or SR-A signal amount of Low AGEs fed rats was defined as 100% and related to the 402

normalized signal intensity of High AGEs fed rats bands. Data are expressed as mean±SEM 403

and p≤0.05 was considered significant (*). 404

405

Figure 2 406

RAGE expression on PBMCs was negatively correlated to insulin and testosterone levels 407

(rs=-0.581, p=0.009 and rs=-0.432, p=0.047 respectively) and positively correlated to 408

progesterone levels (rs=0.473, p=0.044). 409

410

Page 16 of 18

A. Western immunoblot analysis of peripheral blood mononuclear cells (PBMCs) lysates from Low- and High-AGEs fed rats (LA and HA respectively) using specific antibodies targeting SR-A, RAGE and actin proteins.

B, C. The western blot signal of the SR-A and RAGE bands was normalized to the intensity of the

corresponding beta-actin. In the graphs of panels B and C, the mean normalized RAGE or SR-A signal amount of Low AGEs fed rats was defined as 100% and related to the normalized signal intensity of High AGEs fed rats bands. Data are expressed as mean±SEM and p£0.05 was considered significant (*).

167x328mm (300 x 300 DPI)

Page 17 of 18

RAGE expression on PBMCs was negatively correlated to insulin and testosterone levels (rs=-0.581, p=0.009

and rs=-0.432, p=0.047 respectively) and positively correlated to progesterone levels (rs=0.473, p=0.044).

152x289mm (300 x 300 DPI)

Page 18 of 18