Placental Biomarkers in Gestational Diabetes Mellitus-

(1)

1

2 3

4 5

Placental Biomarkers in Gestational Diabetes Mellitus-

6

an immune histochemical study

7 8

Atosa Zangene Skärgård

9

Kull V11

10

Oslo, 19.4.2016

11 12 13

(2)

1 14

Abstract

52

Introduction:The prevalence of gestational diabetes mellitus (GDM) is increasing worldwide.

53

During a complicated pregnancy with diabetes, the human placenta undergoes a number of 54

functional and structural changes. In this study, selected immune stains related to glucose and 55

lipid metabolism were tested in placentas from mothers with and without GDM with the aim, 56

to detect biomarkers as a possible diagnostic tool for GDM.

57

Methods: From a multiethnic population-based STORK Groruddalen cohort, 24 placentas 58

were purposively selected from mothers with and without detected GDM, under prenatal 59

control in gestational week 15. After delivery, the placentas were performed as Tissue 60

microarrays (TMA) and immune stained. Semi-quantitatively, immune stains were 61

histologically evaluated on the villous trophoblast, stroma cell and fetal endothelial cell for 62

FGF2, IGF; PLA2, TNFα and VEGF.

63

Results: VEGF, FGF2, PLA2, IGF and TNF did not show clear identificable traces of GDM.

64

Further studies on more patients and controls are needed, included statistical analysis and 65

clinical correlation.

66

1 Introduction

67

1.1 Epidemiology and classification of Diabetes Mellitus 68

Diabetes mellitus is a worldwide burden with rapidly increasing prevalence. According recent 69

studies estimated 347 million people worldwide are diagnosed with diabetes(1), increasing to 70

471 million in 2035 following World Health Organization (WHO). Insulin secreted from 71

pancreas is responsible for adequate control of blood sugar. Is insulin secretion reduced or un- 72

effectively utilized by the target cells, a diabetogenic metabolism results with hyperglycemia, 73

which has effects on fat and protein metabolism (2).

74 75

Hyperglycemia over time leads to serious damage to many systems of the body such as blood 76

vessels and nerves. In 2004, estimated 3.4 million people died from consequences of high 77

fasting blood sugar (3). More than 80% of diabetes deaths occur in low- and middle-income 78

countries (4). WHO emphasizes diabetes as the 7th leading cause of death in 2030(5).

79

Diabetes prevalence in Norway is not much different from the rest of the world. According to 80

the Norwegian Institute of Public Health, in 2011 nearly 135,000 people used diabetes related 81

medication of type 1 and type 2 diabetes. However the exact number of cases of diabetes is 82

unknown due to different diagnostic criteria and treatment (6).

83

(5)

4 In Diabetes, two different conditions are distinguished, as Diabetes mellitus type 1 (T1DM) 84

and Diabetes mellitus type 2 (T2DM). Both types are included in gestational diabetes mellitus 85

(GDM) diagnosed during pregnancy.

86

1.2 Type 1 Diabetes Mellitus (T1DM) 87

Most diabetics exhibit either an absolute or relative deficiency of insulin. The deficiency 88

varies from a complete lack of insulin, such as in T1DM, to relative insulin deficiency seen in 89

most individuals with T2DM. In T1DM the patient is not able to produce insulin due to 90

autoimmune destruction of beta cells in the pancreatic Langerhans Islets. It is one of the most 91

common endocrine and metabolic conditions in childhood and the number of children 92

developing T1DM is increasing. Worldwide, about 78,000 children under 15 years are 93

estimated to develop T1DM annually(7).

94 95

1.3 Type 2 Diabetes Mellitus (T2DM) 96

T2DM is found in 90% of all diabetes cases worldwide (7), and is characterized by 97

hyperglycemia, insulin resistance and relative impairment of insulin secretion. Insulin is 98

secreted, but the sensitivity of the target cells is reduced. Up to 50% of individuals with 99

T2DM are unaware of their condition (7). The prevalence of T2DM has increased alarmingly 100

in the past decade, related to obesity and a low-activity lifestyle (8), accompanied by 101

hypertension, high serum low-density-lipoprotein (LDL) and low serum high-density- 102

lipoprotein (HDL) concentrations. The corresponding increased prevalence of cardiovascular 103

disease is referred to as metabolic syndrome (9). The diagnosis of T2DM is complicated, 104

because the patients often present a combination of varying degrees of insulin resistance and 105

relative insulin deficiency (10).

106 107

Approximately 80% of those with T2DM are obese and have variable degrees of insulin 108

resistance. In clinical practice, insulin resistance is defined by an insulin concentration 109

associated with reduced glucose uptake of target tissue liver, muscle and adipose tissue (9).

110

The fatty acid transport into the β-cells is increased and ATP transport into the target cells is 111

reduced. GLUT-4 mobilization to the cell membrane in muscle cells decreases, lipogenesis 112

increases and β-oxidation of fatty acids is downregulated. Hyperinsulinemia as a cause of 113

insulin resistance may play an important role in the development of metabolic syndrome.

114

Increased free fatty acid (FFA) levels, inflammatory cytokines from fat and oxidative factors, 115

(6)

5 have been implicated in the pathogenesis of metabolic syndrome T2DM and cardiovascular 116

complications (8-10).

117

Even though there is no linkage between the autoimmunity and T2DM, an interaction 118

between genes and environment (epigenetics), and lifestyle could be shown (8). The 119

prevalence of T2DM varies between ethnic groups living in the same environment. The gene 120

influence is evident through several observations such as higher prevalence among some 121

ethnics groups (south Asians) compared to ethnic Norwegians or whites in the Unites States 122

(11). Norwegian immigrants from India, Pakistan and Sri Lanka, show in 20 – 35% of men 123

and women between 40 to 60 years diabetes (12). Furthermore, the incidence of T2DM shows 124

a social gradient with the highest incidence among ethnic minorities and people with lower 125

social status (13).

126 127

1.4 Gestational Diabetes Mellitus (GDM) 128

Diabetes diagnosed in pregnancy is defined as gestational diabetes mellitus (GDM), including 129

several subgroups:

130 131

1) Patients with onset of T1DM in pregnancy.

132

2) Patients with previously undiagnosed T2DM diagnosed in pregnancy.

133

3) Patients developing diabetes in pregnancy and 134

4) Impaired glucose intolerance in pregnancy.

135

Gestational diabetes mellitus is seen in 3% to 5% of pregnant women and is increasing (7, 136

14), in selected populations with obese women the prevalence increases up to 20% (14).

137

According to the Medical Birth Registry, the rate of gestational diabetes including T1DM and 138

T2DM and impaired glucose intolerance, has increased by more than two fold from 13 per 139

1000 pregnant women in 1999 to 29 per 1000 pregnant women in 2011. Data from the 140

Medical Birth Registry from 1988 to 1998 show, that GDM frequency was more than twice 141

among immigrants from North Africa and South Asia than among ethnic Norwegian pregnant 142

women. The risk for developing T2DM after pregnancy is high among women with a history 143

of GDM (15).

144

(7)

6

2 Placenta

145

The placenta is part of the conceptional product and functions as a mediator organ, implanted 146

in the interface of the mother and the fetus. Structurally, the human placenta is classified as 147

hemochorial, since the placental villi are bathed directly in the maternal blood (16, 17).

148

Several studies have shown that the placenta plays a central role in diabetic environment as an 149

active co-player with effects on the mother and the fetus.

150

2.1 Placental function 151

Functionally, the placenta is a complex organ, as it performs gas exchange, nutrition and 152

exchange of waste products, hormone production and immunological protection (16). Thus 153

the location, the human placenta not only handles the maternal- fetal transport of nutrients and 154

gasses, also itself is affected by intrauterine conditions. However, to fulfill all the tasks the 155

placenta must develop and function optimally.

156

2.2 Placental development and maturation 157

The fertilized egg develops from the zygote into the morula and blastocyst with inner cell 158

mass (embryoblast) that becomes the fetus and an outer cellayer (trophoblast) developing into 159

the placenta. The blastocyst implants with the embryonic pole in the uterine wall at day seven.

160

(Fig. 1) Trophoblastcells differentiate into the outer invasive syncytiotrophoblast and the 161

inner cytotrophoblast. The endometrium, influenced by estrogen and progesterone, changes 162

into decidua with enlarged stroma cells surrounded by edema fluid; gland cells are filled with 163

glycogen(18). The syncytiotrophoblast cells erode the endometrial blood vessels and form 164

lacunes with an arterio-venous system. 17th or 18th day after conception the 165

syncytiotrophoblast invade the spiral arterioles of the uterine wall. The lacunes form the 166

intervillous space with freely circulating maternal blood in direct contact to the 167

trophoblast(19).

168

169

(8)

7

Fig 1: Development of the zygote through migration in the fallopian tube, and implantation into the endometrial wall.

170 171

Fetal circulation starts at approximately 21 days. Between the 11th and 13th 172

day cytotrophoblast cells penetrate into the syncytiotrophoblast creating the primary villi, on 173

the entire surface of the chorionic sac and appear as local masses of the cytotrophoblast 174

forming fingerlike extensions into maternal decidua (20). Proliferation of mesenchyme into 175

the primary villi from the chorionic plate (extraembryonic mesenchyme) characterize the 176

secondary villi, while vessel proliferation into secondary villi from the chorionic plate 177

characterize the tertiary villi at day 18 to 21 post conceptionem (Fig. 2).

178

179

Fig. 2: Development of the villi, primary and secondary and tertiary villi. Maturation of the diffusion barrier from four

180

(syncytium, cytotrophoblast, connective tissue, fetal endothelium) to two cell layers (syncytium and endothelium) from 1rst

181

to 3^rd trimester.

182

In further pregnancy from first to third trimester, the villous tree matures into stem-, 183

intermediate - and terminal villi, through longitudinal growth and ramification.

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Cytotrophoblast, stroma and fetal vessels undergo a maturation process, with a thin 185

trophoblast and endothelium barrier between mother and fetus, to shorten and optimize the 186

blood exchange. (Fig. 2) 187

188

At term, the placentaconsists of cotyledons as structural and functional units. Each unit 189

consists of anchoring stem villi with proliferated intermediate and terminal villi. Fetal blood 190

enters the placenta through the two umbilical arteries, which branches into capillaries of the 191

chorionic villi, and oxygenated fetal blood leaves the placenta the fetus in one umbilical vein.

192

(9)

8 193

Fig. 3: A placenta in the second trimester. Cotyledons are partially separated by the decidual septa. The intervillous space is

194

lined by the syncytium, where most of the maternal blood returns into the maternal veins.

195 196

Maternal blood circulates from the spiral arteries into the intervillous space and passes over 197

the villous surfaces toward the chorionic plate. (Fig. 3) The full term human placenta is a 198

discoid, 15-25 cm i diameter, 2-3 cm thick, netto weight 500 to 600 g.

199 200

3 Pregnancy, glucose metabolism and GDM

201

GDM pathophysiology is not fully understood, but characteristic findings of T2DM and the 202

metabolic syndrome, such as reduced maternal insulin sensitivity, hyperlipidemia and 203

hyperglycemia are described (14). Thus literature in part considers GDM as a pre-diabetic 204

T2DM, unmasked in pregnancy and manifested as T2DM in later life (14).

205 206

3.1 The glucose metabolism during pregnancy 207

During pregnancy a physiological state of insulin resistance is required to preferentially direct 208

maternal nutrients toward the fetal-placental unit, allowing adequate growth of the fetus (21).

209

Maternal glucose can freely pass the placenta, whereas the maternal insulin cannot (22). In 210

case of excessive glucose levels the fetal β-cells will increase the insulin secretion leading to 211

increased growth and adiposity in the fetus as insulin is a growth hormone (23).

212 213

Placental hormones cause a diabetic effect of insulin resistance in the mother (24). Fasting 214

blood glucose is lower (0.5-1 mmol/l), likewise HbA1c decreases slightly (HbA1c (0.5-1%).

215

Hence after a carbohydrate rich meal, glucose levels will be higher than in non-pregnant 216

(10)

9 women. Maximum value of plasma glucose is higher and it will take longer before it returns 217

to normal levels (24).

218 219

In healthy pregnancies compensatory insulin secretion leads to normal blood sugar levels. If 220

insulin production does not increase sufficiently, blood glucose increases above the reference 221

value with hyperglycemia imitating insulin resistance (24). Unlike women with T1DM, 222

women with GDM have plenty of insulin. However, their insulin effect is partially blocked by 223

a variety of hormones secreted by the placenta, such as estrogen, cortisol and human placental 224

lactogen. Resistance to insulin usually starts from gestational week 20 to 24 and increases 225

with placental development (25). The earlier gestational diabetes occurs, the greater the risk 226

for complications in pregnancy (13).

227 228

The placenta expresses virtually all known cytokines including tumor necrosis factor (TNF- 229

α), resistin, and leptin. Some of these adipokines are key players in the regulation of insulin 230

function (26). The secretion of adipokines from adipose tissue and the placenta will in 231

pregnant women multiply the effect, which results in pregnancy induced insulin resistance.

232 233

3.2 Diagnosing GDM 234

The current WHO diagnostic criteria for diabetes are fasting plasma glucose ≥ 7.0mmol/l 235

(126mg/dl) or 2–h plasma glucose ≥ 11.1mmol/l (200mg/dl). The current criteria distinguish a 236

group with significantly increased premature mortality and increased risk of microvascular 237

and cardiovascular complications(27).

238 239

WHO definition of normal glucose levels is insufficient, therefore ‘normoglycemia’ should 240

refer to glucose levels below intermediate hyperglycemia, associated with low risk diabetes or 241

cardiovascular disease(27).

242 243

Fasting plasma glucose cut-point for Impaired Fasting Glucose (IFG) is 6.1mmol/l.

244

It does not exclude the possibility that unrecognized glucose intolerance may have antedated 245

or begun concomitantly with the pregnancy(2).

246 247

The hyperglycemia and Adverse Pregnancy Outcome Study (HAPO) conducted in nine 248

countries to map associations between maternal hyperglycemia and pregnancy outcomes, 249

(11)

10 showed a strong association between maternal hyperglycemia and adverse pregnancy

250

outcomes even in the subclinical ranges(28). Based on these results, the International 251

Association of Diabetes and Pregnancy Study Group (IADPSG) suggested new criteria for 252

GDM which are more strict concerning fasting plasma glucose (FPG>5,1 mmmol/l) or 2 hour 253

plasma glucose after oral tolerance test (OGTT> 8,5 mmol/ l)(29). This indicates that the 254

number of GDM cases will arise worldwide by using these criteria.

255 256

Norwegian pregnancy healthcare still use the WHO criteria in diagnosing GDM. The Stork 257

Groruddalen cohort study material compared their participants with these two different 258

criteria and found a prevalence of 13% with WHO criteria compared to 31.5% with modified 259

IADPSG criteria. This finding was strongly associated with South Asians and pregnancy in 260

obese women (30, 31).

261

3.3 Screening 262

The Directorate of Health and Social Affairs in Norway recommends OGTT for women with ; 263

morning glucosuria, age >38 years, BMI> 27 kg/m2, a history of GDM, first degree relatives 264

with diabetes or woman with ethnic origin from outside Europe with high prevalence of 265

diabetes(32). There is no international consensus on the time point of GDM diagnosis, and the 266

practice of OGTT varies internationally (22).

267

3.4 Management and treatment of GDM 268

When GDM is diagnosed, closely follow up of the patient is important to prove the perinatal 269

outcome, and to avoid preeclampsia, macrosomia, shoulder dystocia and minimize mothers 270

and childs risk to develop T2DM after delivery.

271 272

Nutrition therapy is the initial approach, with meals at a regular basis composed of about 40 273

percent carbohydrate, 20 percent protein, and 40 percent fat. Pregnant women often require 274

1800 to 2500 kcal per day (33). The caloric requirement for pregnant women with ideal body 275

weight is 30 kcal/kg/day; for women who are overweight is 22 to 25 kcal/kg/day; and for 276

morbidly obese women is 12 to 14 kcal/kg/day (present pregnant weight) (33). These patients 277

blood glucose should be monitored to evaluate the effectiveness of medical nutritional 278

therapy. Moderate exercise as part of the treatment for women with no medical or obstetrical 279

contraindications is highly recommended (33). Anti-hyperglycemic treatment is 280

recommended for women who do not achieve adequate glycemic control with nutritional 281

(12)

11 therapy and exercise. Insulin is recommended rather than oral anti-hyperglycemic agents 282

during pregnancy (33).

283 284

3.5 The risk factors for acquiring GDM 285

The genetic component to develop GDM is one of the main risk factors, confirmed by 286

STORK Groruddalen study, including family history of T2DM or GDM, obesity (BMI>30) 287

and womens age more than 34 years (22, 30) Obese woman have more than 3-fold increased 288

risk of gestational diabetes compared with non-obese woman (22).

289 290

3.6 Complications of GDM 291

Placenta is a metabolic active organ. It produces growth factors and cytokines with paracrine 292

and endocrine effect. It is exposed to intrauterine environment, which can adversely affect 293

placental and fetal development (14, 26, 34). Pregnancies complicated by GDM have been 294

associated with alternations in the macroscopic placenta and histology, the expression of 295

cytokines and signaling molecules (14, 20). The extend and the nature of the changes depend 296

on the onset of GDM, the quality of the glycemic control achieved during the critical periods 297

in placental development (14, 26).

298 299

Placenta adapted to the diabetic environment, such as buffering excess maternal glucose or 300

increased vascular resistance to prevent limited fetal growth. If maternal hyperglycemia, 301

hyperinsulinemia or dyslipidemia exceeds the placental capacity, excessive fetal growth 302

occurs (21, 26). The abnormal maternal metabolic environment may stimulate mother’s 303

adipose tissue, resulting in increased production of inflammatory cytokines by the placental 304

cells, enhancing further insulin resistance in the mother (26). Circulating TNF-α, leptin, 305

resistin and adiponectin is assumed to function as a link between inflammation and induced 306

insulin resistance in the mother (26).

307 308

3.6.1 Placental insufficiency 309

Diabetic insult at the beginning of gestation may have long-term effects on placental 310

development and may cause placental insufficiency.

311

(13)

12 3.6.2 Fetal malformation and adverse outcome of the offspring

312

Maternal lifestyle and prenatal factors are linked to serious health consequences and diseases 313

in later life. The intrauterine environmental or nutritional status seem to be involved in fetal 314

programming (35). Epidemiological studies have identified a number of factors such as diet, 315

stress, gestational diabetes, exposure to tobacco and alcohol during gestation, which influence 316

fetal development (36). Epigenetic mechanisms such as alteration of DNA methylation, 317

chromatin modifications and modulation of gene expression during gestation are believed as 318

factors contributing to various malformations as neural tube defects, autism spectrum 319

disorder, congenital heart defects, oral clefts, allergies and cancer (36).

320

Poor diabetic control and high blood sugar levels during the second and third trimester may 321

predispose for large for gestational age (LGA) babys, over 4000 grams body weight at term 322

(21, 25), but also growth restricted and normal weight children occur with maternal GDM 323

(37).

324

Hence, GDM during pregnancy is associated with increased risk of early fetal obesity, T2DM 325

in adolescence and the development of metabolic syndrome in early childhood may be 326

programmed by uterine environment (38).

327

According to the paradigm of Developmental Origins of Health and Disease (DOHaD), the 328

long-term consequences for the offspring of a GDM pregnancy is an increased risk for 329

developing endothelial and vascular dysfunction associated with obesity, hypertension, T2DM 330

and metabolic syndrome in later life (14, 39).

331 332

4 Placental pathology in diabetes

333

4.1 Placental morphology 334

The literature is not consistent about changes in the placenta which is exposed to 335

hyperglycemia (40), but there is consensus, that the onset of GDM is one of the determining 336

factors on placental development (14, 40).

337 338

However, pregnancies complicated with GDM have been associated with alternations in 339

placental anatomy and physiology. These alternations are changes on the micro/molecular 340

and/or macro-anatomical level (14, 21, 23). On a macro-anatomical level the placenta can be 341

enlarged, thickened and plethoric in poorly controlled diabetic women, but also normal 342

weighted or growth restricted placentas are documented (14, 37, 40). There have not been 343

detected other significant differences in macro-anatomy such in eccentricity index; indicating 344

(14)

13 the placentas shape ranging from circular to elliptical, nor in cord centrality index and cord 345

coiling index; describing the umbilical cord insertion from the chorionic plate margin and the 346

coiling direction and number of coils (14).

347 348

Histological studies have revealed apparent fibrinoid necrosis and vascular lesions such as 349

chorangiosis, and thickening of the basement membrane more frequently in placentas from 350

pregnancies complicated by GDM when compared with normal placentas (14, 40, 41). In 351

other studies, poorly controlled GDM placentas have shown villous edema, fibrin deposit in 352

the syncytiotrophoblast, and hyperplasia of cytotrophoblast (14, 42). These changes have been 353

less distinct in placentas from well monitored diabetic mothers and not observed in normal 354

cases (14).

355

The most common placental findings in GDM are increased villous immaturity and increased 356

angiogenesis (40). Placental villi undergo increased angiogenesis, vascularization and 357

branching in the second half of gestation, influenced by a diabetic environment (14). An early 358

onset of GDM has been associated with impaired placental development, showing villous 359

immaturity or alteration in villous branching (14, 41, 43), whereas a late onset of GDM has 360

shown short-term effects on placental function (14).

361

362

Fig. 4: Hematoxylin-Eosin stained normal histology on the left, compared to the histology of a placenta from a patient with

363

clinically diagnosed GDM on the right with identical gestational age

364 365 .

4.2 Biomarkers and their role in placental tissue 366

The placenta expresses virtually all known cytokines (26). The cytokines that play a key role 367

in regulating insulin are suggested to play a role in the interaction between the placenta, 368

adipose tissue and GDM.

369 370

(15)

14 This study has investigated Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth 371

Factor (FGF), Phospholipase A2 (PLA2), Insulin like Growth Factor (IGF) and Tumor 372

Necrosis Factor α (TNFα).

373 374

4.2.1 Vascular Endothelial Growth Factor (VEGF) 375

VEGF family molecules and FGF family molecules have been identified as the most potent 376

angiogenesis factors promoting vascularization and angiogenesis in the placenta (14, 44).

377

Vasculogenesis is the de novo formation of new blood vessels and angiogenesis is the 378

formation of blood vessels by sprouting from preexisting vessels (40, 45).

379

Endothelial cells are major target for VEGF, where VEGF acts as a survival factor for newly 380

formed capillaries and has antiapoptotic effect on vascular endothelial integrity (16). Any 381

disbalance of angiogenetic factors in the placental microenviroment may lead to aberrant 382

villous vascularization. (14, 44). A systematic review of placental pathology in maternal 383

diabetes mellitus suggests an increase in angiogenesis and placental villous immaturity (40).

384

The latter abnormality has been independently associated with an increased risk of perinatal 385

mortality and may serve as a connection between maternal diabetes and an increased risk of 386

intrauterine fetal death (IUFD) (40).

387 388

4.2.2 Fibroblast Growth Factor (FGF) 389

FGFs are multifunctional proteins stimulating a variety of biological effects including 390

pluripotency and angiogenesis. FGFs are key players in the processes of proliferation and 391

differentiation of the endothelium (16). FGFs are stored within basement membrane and their 392

availability to target tissues is dependent on liberation by proteolysis(16, 25). The placenta is 393

a rich source of FGFs particularly FGF-2 which is induced early in the development of the 394

embryo (46). FGFs are essential for trophoblast stem cell function, requiring FGF for self- 395

renewal and prevention of terminal differentiation. Studies have shown that FGF-2 expression 396

is seen in the villous syncytiotrophoblasts and in cytotrophoblast cells of first-trimester 397

placentas. At term, the FGF-2 gene expression is detected in syncytiotrophoblasts and in fetal 398

membranes. FGF-2 is increased in pregnancies complicated with diabetes (25). There is 399

evidence that the concentrations in maternal serum, cord serum, and amniotic fluid at term are 400

increased, and the amounts of FGF-2 in maternal serum and cord serum correlate with fetal 401

and placental size (25).

402 403

(16)

15 4.2.3 Phospholipase A2(PLA2)

404

Phospholipase A2 (PLA2), is a family of lipolytic enzymes which catalyses the hydrolysis of 405

membrane phospholipids, releasing arachidonic acid (AA), docosahexaenoic acid (DHA), and 406

other polyunsaturated fatty acids (PUFAs) (47). PUFAs are precursors of a variety of 407

eicosanoids, prostaglandins, thromboxanes, leukotrienes, and lipoxins, which interact in acute 408

inflammatory reactions. PLA2 located in the trophoblast membrane catalyses the hydrolysis 409

of membrane phospholipids, activated by cytokines, providing the link between inflammatory 410

pathways and lipid metabolism (14, 47).

411 412

4.2.4 Insulin like growth factor (IGF) 413

IGFs are proteins with sequence similarity to insulin and they participate in the growth and 414

function of almost every organ in the body. IGFs are part of a complex system in cell-cell 415

communication. IGF-1 is mainly secreted in the liver, stimulated by growth hormone (GH).

416

IGF-1 is important for the regulation of normal physiology and growth and is required for 417

promotion of cell proliferation and the inhibition of cell apoptosis (48). IGF-2 is thought to be 418

a growth factor in early development, regulating fetal and placental growth. Fetal insulin may 419

stimulate endothelial cell proliferation, vascular branching and glycogen deposition around 420

the placental vessels (49).

421 422

4.2.5 Tumor necrosis factor α (TNFα) 423

TNF-α is a multifunctional cytokine, expressed in placental stroma cells, known to 424

downregulate insulin sensibility. It regulates inflammation and possesses week apoptic ability 425

(49).

426 427

5 Aim of this placental study

428 429

The aim of this placental study was to test immune stains related to GDM and lipid 430

metabolism in the placenta, as a possible diagnostic tool for GDM.

431 432

(17)

16

6 Material and methods

433

6.1 Material 434

The material of this study is part of the STORK Grorudalen cohort Research Program on 435

gestational diabetes, physical activity and obesity in pregnancy in a multi-ethnic population.

436

The population-based cohort study includes totally 823 pregnant women recruited in 437

gestational week 14 in the Oslo region Groruddalen in GP offices from 2008 to 2010.

438

Ultrasound was performed, blood samples were taken, from fasting blood, urine samples, oral 439

glucose tolerance test, and several ultrasound measurements the umbilical venous blood were 440

collected, sampled and stored after birth for further investigation. The deliveries were 441

assigned to three different hospitals in Oslo, where the placentas be examined and sample 442

sectioned.

443 444

In this study, 24 placentas were microscopically examined: 8 placentas from women clinically 445

diagnosed with GDM and 16 placentas from women without GDM. Gestational age of all 446

selected placentas was approximately 38 weeks. All clinical data have been blinded prior to 447

examination.

448 449

6.2 Methods 450

For histological examination 3,5 μm sections were cut using a Microm HM 355 microtome 451

(Thermo Fisher Scientific Inc., Waltham, Massechusetts, USA, places on Super Frost slides 452

(Menzel-Gläser, Braunschweig, Germany) and stained with Hematoxylin-Eosin (HE). Two 453

tissue microarrays (TMAs) were selected and sampled from microscopic normal looking 454

parenchyma.

455

The immune stains were performed on 3,5 μm. Two tissue microarrays (TMAs) serial 456

sections using BenchMark XT (Ventana Medical System Inc., Tucson, Arizona, USA), an 457

automated immune stain system based on the ABC avidin-biotin-peroxidase method. The 458

method includes deparaffinization, antigen retrieval, and incubation with primary and 459

secondary antibodies respectively. Enzyme-mediated detection with horseradish peroxidase 460

(HRP) labelled to diamino-benzidine (DAB) reporter molecules was used for color detection.

461

Amplification was achieved by secondary antibody conjugation to biotin molecules and by 462

avidin protein bound enzymes. All sections were counter-stained with hematoxylin. Optimal 463

antigen retrieval, antibody concentrations and incubation times were pre-tested with positive 464

and negative controls. The sections were immune staind with the selected panel of VEGF, 465

(18)

17 FGF, PLA2, IGF and TNFα. The immune histochemical reactivity was registered in all

466

selected cell types; stroma cells, fetal endothelial cells and trophoblasts. Reactivity was 467

documented for each cell compartment, as membrane, cytoplasm and nucleus semi- 468

quantitatively, graded from negative (0) to slight (1), medium (2) and intense positive (3).

469 470

7 Results

471 472

Of the 24 individuals included in this study, 8 were diagnosed with GDM.

473 474

T E St No Patient M C N M C N M C N

1 s-008 0 0 0 0 0 0 0 0 0 2 s-007 0 1 0 0 1 0 0 1 0 3 s-059 1 1 0 0 2 0 0 1 1 4 s-086 1 0 0 1 1 1 0 1 0 5 s-137 1 0 0 1 1 0 0 1 0 6 s-160 2 1 0 1 1 2 1 1 1 7 s-177 1 1 0 0 1 0 0 1 1 8 s-513 1 1 0 1 1 1 0 1 0 9 s-543 0 1 0 0 1 0 0 0 0 10 s-622 1 1 0 0 0 0 0 0 0 11 s-656 2 1 0 2 1 0 0 1 0 12 s-721 2 1 0 0 1 0 0 1 0 13 s-730 2 1 0 0 1 0 0 1 0 14 s-745 1 1 0 1 1 1 0 1 0 15 s-1009 1 0 0 1 1 1 0 1 0 16 s-1016 1 0 0 2 2 3 0 0 0 17 s-1022 1 0 0 1 2 0 0 1 0 18 s-1056 0 1 0 0 1 0 0 1 0 19 s-1107 0 1 0 1 1 1 0 1 0 20 s-1141 0 1 0 1 1 0 1 1 0 21 s-1147 1 0 0 0 1 0 0 1 0 22 s-1187 1 0 0 1 1 1 0 1 2 23 s-1241 1 0 0 2 0 0 0 1 0 24 s-1270 1 0 0 2 0 0 0 0 1

475

Tab.1: VEGF immune stain in the trophoblast (T), endothelial cells and stroma cells, in the compartments of membrane (M),

476

cytoplasma (C) and nucleus (N). Yellow marks cases with clinical GDM.

477 478

Fig. 5: Placenta tissue immune stained with VEGF (20x)

(19)

18 479

Two cases (12 and 13) showed high score in the trophoblast in combination with clinically 480

diagnosed GDM. Annother two cases (17, 23) show increased positivity in the endothelium.

481

Case 16 (not-GDM) showed increased positivity in the endothelial cells. Other cases are close 482

to negativity.

483 484 485

T E St No Patient M C N M C N M C N

1 s-008 2 1 1 0 0 0 0 1 1 2 s-007 0 1 0 1 1 2 0 0 0 3 s-059 1 1 0 1 1 2 0 1 0 4 s-086 0 1 0 0 0 0 0 0 1 5 s-137 0 1 0 0 0 0 0 0 1 6 s-160 1 1 0 1 1 2 0 1 0 7 s-177 2 1 2 0 0 0 0 0 2 8 s-513 0 0 0 0 0 1 0 0 0 9 s-543 1 1 0 0 1 0 0 1 0 10 s-622 0 1 2 1 1 2 0 2 3 11 s-656 0 0 0 0 0 1 0 1 0 12 s-721 1 2 1 0 0 0 0 1 0 13 s-730 1 1 0 0 0 0 0 0 1 14 s-745 1 1 0 0 0 1 0 1 0 15 s-1009 1 1 0 1 1 2 0 0 1 16 s-1016 0 1 0 0 0 1 0 0 1 17 s-1022 0 1 0 0 0 0 0 1 0 18 s-1056 1 1 2 0 0 0 0 1 2 19 s-1107 1 1 0 0 1 1 0 0 1 20 s-1141 0 1 0 1 0 0 0 1 2 21 s-1147 0 1 1 1 0 0 2 0 1 22 s-1187 0 1 0 0 0 0 1 0 0 23 s-1241 0 1 0 0 0 0 0 1 0 24 s-1270 0 1 0 0 0 0 1 0 0

486

Tab. 2: FGF-2 immune stain in the trophoblast (T), endothelial cells and stroma cells, in the compartments of membrane

487

(M), cytoplasma (C) and nucleus (N). Yellow marks cases with clinical GDM.

488 489

FGF2 immune reactivity scores in trophoblast nucleus and trophoblast membrane were higher 490

in three women with GDM (8, 10, 12) and two of the women without GDM (7, 18). One case 491

Fig. 6: TMA immune stained with FGF-2 (20x)

(20)

19 with GDM showed increased positivity in the stroma cells. Other cases without GDM showed 492

low scores.

493 494

T E St

No Patient M C N M C N M C N 1 s-008 3 2 2 1 1 2 1 1 2 2 s-007 3 2 1 1 1 2 1 2 2 3 s-059 3 2 3 2 1 2 2 1 2 4 s-086 2 2 2 1 2 3 1 1 2 5 s-137 3 2 3 3 2 3 1 2 2 6 s-160 3 3 3 2 1 2 1 2 3 7 s-177 2 2 3 1 1 2 1 2 3 8 s-513 1 2 2 1 1 2 1 2 3 9 s-543 3 2 3 2 2 3 0 0 0 10 s-622 2 1 2 2 2 2 2 1 3 11 s-656 2 2 2 2 1 3 0 0 0 12 s-721 3 2 2 1 2 2 2 1 3 13 s-730 3 3 3 1 1 2 1 1 3 14 s-745 3 3 3 2 1 2 1 1 2 15 s-1009 2 1 3 1 1 2 2 1 3 16 s-1016 3 1 1 2 2 2 1 1 2 17 s-1022 2 2 3 1 1 2 1 2 3 18 s-1056 1 1 2 1 1 2 2 2 3 19 s-1107 1 1 2 2 1 3 1 1 3 20 s-1141 1 1 2 1 2 3 2 2 3 21 s-1147 3 2 1 2 1 1 3 2 1 22 s-1187 3 2 1 2 1 1 2 1 1 23 s-1241 3 3 3 2 2 3 3 3 3 24 s-1270 3 2 1 2 1 3 2 1 1

495

Tab. 3: PLA-2 immune stain in the trophoblast (T), endothelial cells and stroma cells, in the compartments of membrane

496

(M), cytoplasma (C) and nucleus (N). Yellow marks cases with clinical GDM.

497 498

PLA2 showed high score outcomes in the trophoblast, endothelial cells and stroma cells, 499

diffuse distributed to GDM and not-GDM placentas. Two placentas showed negativity in the 500

stroma cells, one with GDM (9), one without GDM (11).

501 502 503 504

Fig. 7: TMA immune stained with PLA 2 (20x)

(21)

20 505

T E St

506

Tab. 4: IGF immune stain in the trophoblast (T), endothelial cells and stroma cells, in the compartments of membrane (M),

507

508 509

IGF showed high score (3) in the trophoblasts, in both GDM and not-GDM cases. Case one 510

and 17 differ with low intensity scores among the GDM placentas. Not-GDM placentas show 511

diffuse activity in all cells and all cell compartments.

512 513 514 515 516 517

Fig. 8: IGF immune stain in TMA (20x)

(22)

21

T E St

518

Tab. 5: TNF immune stain in the trophoblast (T), endothelial cells and stroma cells, in the compartments of membrane (M),

519

520 521 522

TNF was positive in the trophoblast cytoplasma in three of the GDM placentas (9, 13, 23), 523

two GDM cases (14, 23) showed score 2 in the endothelial cytoplasma. Not-GDM cases 524

showed predominantly positivity in the trophoblast and endothelial cytoplasma, and in nuclear 525

and cytoplasma compartment of the stroma cells.

526 527

8 Discussion

528

In early placental development, trophoblast proliferation and differentiation is altered by 529

maternal hyperglycemia, hyperinsulinemia and dyslipidemia with long-term effects, leading 530

Fig. 9: TMA immune stained with TNF (20x)

(23)

22 to increased fetal growth (26). Late onset of GDM in second half of gestation may have short 531

term effects mainly on placental function, including villous immaturity with alternations in 532

villous branching and increased angiogenesis and vascularization (14). Placental angiogenesis 533

is mainly regulated by angiogenic factors, such as VEGF, FGF2, and placental growth factor 534

(PGF) (14, 16, 44). Any disbalance of angiogenetic factors in the placental microenvironment 535

may lead to aberrant villous vascularization (14). FGF2 is expressed in term placentas in the 536

syncytiotrophoblast, and is increased in GDM (25). Placental oxygen demand is elevated by 537

increased collagen synthesis in the placental stroma and moderate thickening of the basement 538

membrane of the syncytiotrophoblast in GDM placentas (14, 42). VEGF is secreted by 539

Hofbauercells with major target in endothelial cells.

540

Increased angiogenesis and vascularization would confirm enhanced expression of VEGF and 541

FGF2, which promotes vasculogenesis and angiogenesis. In this study, only two cases showed 542

high score of VEGF in the trophoblast in combination with clinically diagnosed GDM.

543

Annother two cases (17, 23) showed increased positivity in the endothelium. Case 16 (not- 544

GDM) showed increased positivity in the endothelial cells. Other cases are close to negativity.

545

FGF2 immune reactivity scores in trophoblast nucleus and trophoblast membrane were higher 546

in three women with GDM (8, 10, 12) and two of the women without GDM (7, 18). One case 547

with GDM showed increased positivity in the stroma cells. Other cases without GDM showed 548

low scores. Totally, some results fit to literature, with increased VEGF and FGF2 intensity in 549

the trophoblast and the endothelial cells, but diversity indicates, that other factors may 550

contribute, such as obesity and compensatory changes for decreased vascular branching, as in 551

preeclampsia.

552

PLA2 is located in the trophoblast membrane functioning as a catalysator of the hydrolysis of 553

membrane phospholipids, activated by cytokines (14, 47). In this study, PLA2 showed high 554

score outcomes in the trophoblast, endothelial cells and stroma cells, diffuse distributed to 555

GDM and not-GDM placentas. Two placentas showed negativity in the stroma cells, one with 556

GDM (9), one without GDM (11).

557

IGF regulates fetal and placental growth and development with receptor location in the 558

placental endothelium at term. Fetal insulin stimulates endothelial cell proliferation and 559

vascular branching and glycogen deposition around the placental vessels.

560

IGF showed high score in the trophoblasts, in both GDM and not-GDM cases. Case one and 561

17 differ with low intensity scores among the GDM placentas. Not-GDM placentas show 562

diffuse activity in all cells and all cell compartments. IGF intensity on the placental sections 563

was different, indicating no coherent trace of GDM in this study material.

564

(24)

23 TNF-α is a multifunctional cytokine, expressed in placental stroma cells (14), known to

565

downregulate insulin sensitivity, regulate inflammation, and to have weak apoptotic function.

566

TNF-α and leptin stimulate the activation of PLA2 in the placenta (50). This may indicate that 567

TNF is increased in individuals with controlled GDM and normal BMI.

568

TNF was positive in the trophoblast cytoplasma in three of the GDM placentas (9, 13, 23), 569

two GDM cases (14, 23) showed score 2 in the endothelial cytoplasma. Not-GDM cases 570

showed predominantly positivity in the trophoblast and endothelial cytoplasma, and in nuclear 571

and cytoplasma compartment of the stroma cells.

572 573 574

9 Conclusion

575

In this study of 24 cases with and without clinically diagnosed GDM, the tested biomarkers 576

VEGF, FGF2, PLA2, IGF and TNF did not show clear identificable traces of GDM. One of 577

the causes may be that the study population was tightly controlled for GDM. The selected 578

pregnant women in a total number of 24, controls included, may be too little in order to 579

represent the whole study population.

580

This study may work as part of a baseline study, but further studies on more patients and 581

controls are needed, included statistical analysis on these and more different biomarkers 582

related to GDM. Immune scores should be correlated to macroscopical placental findings as 583

placental netto weight, as well as to clinical data, as fetal birth weight and mothers body mass 584

index.

585

(25)

24 586

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695 696 697 698

Placental Biomarkers in Gestational Diabetes Mellitus-

Placental Biomarkers in Gestational Diabetes Mellitus-

an immune histochemical study

Atosa Zangene Skärgård

Kull V11

Oslo, 19.4.2016

TABLE OF CONTENTS

Abstract

1 Introduction

2 Placenta

3 Pregnancy, glucose metabolism and GDM

4 Placental pathology in diabetes

5 Aim of this placental study

6 Material and methods

7 Results

8 Discussion

9 Conclusion

10 References