MOLECULAR INVESTIGATION AND DETECTION OF PROTEIN …

48

MOLECULAR INVESTIGATION AND DETECTION

OF PROTEIN BIOMARKER/S IN POLYCYSTIC

OVARY SYNDROME

A THESIS SUBMITTED TO

THE UNIVERSITY OF THE PUNJAB

IN FULFILMENT OF THE REQUIREMNETS FOR THE DEGREE

OF DOCTOR OF PHILOSOPHY IN MEDICINE (PHYSIOLOGY)

BY

DR. MUHAMMAD AKRAM

MBBS (Pb), MPHIL (Pb)

SUPERVISOR

DR. NABILA ROOHI

UNIVERSITY OF THE PUNJAB

LAHORE, PAKISTAN

2015

49

Contents

Title Pages

SUMMARY

i

ACKNOWLEDGEMENTS

iii

LIST OF ABBREVIATIONS

v

LIST OF TABLES

xv

LIST OF FIGURES

xvi

CHAPTER 1

1

INTRODUCTION

1

CHAPTER 2

6

LITERATURE REVIEW

6

2.1Diagnostic Criteria

8

2.2 Onset of PCOS

9

2.3 Pathophysiology of PCOS

10

2.4 Inappropriate Gonadotrophin Secretion

11

2.5 Premature Action of LH in PCOS

13

2.6 Why Follicular Arrest is Inconstant

14

2.7 Antimullerian Hormone in PCOS

15

2.8 Hyperandrogenism

16

50

2.9 Physiological Hyperandrogenism of Puberty

16

2.10 Role of Androgens

18

2.11 BMI in PCOS

21

2.12 Ovarian Morphology in PCOS

24

CHAPTER 3

28

MATERIALS AND METHODS

28

3.1 Study Sample

28

3.2 Blood Sampling

29

3.3 Analytical Procedures

30

3.4 Handling of Samples

30

3.5 Statistical Analysis

31

HORMONAL ASSAYS

32

3.6 Measurements of LH, FSH, Insulin, Prolactin, and Growth

Hormone

32

3.6.1 Principle

32

3.6.2 Reagents Provided 32

3.6.3 Preparation of reagents 33

3.6.4 Assay procedure: 33

3.7 TESTOSTERONE AND ANDROSTEINDIONE 34

3.7.1 Principle 34

51

3.7.2 Reagents used 34

3.7.3 Assay procedure 35

3.8 ONE DIMENSIONAL POLYACRYLAMIDE GEL

ELECTROPHORESIS

36

3.8.1 Reagents Preparation 36

Acrylamide-Bis-acrylamide (30%) 36

1.5 M Tris-HCl (pH 8.8) 37

1.0 M Tris-HCl (pH 6.8) 37

SDS (10%) 37

Electrophoresis Buffer 37

Fixative Solution 37

Colloidal Coomassie Staining Solution 37

Destaining Solution 38

Low Molecular Weight Markers For 12% Gel 38

Gel Assembly 38

Casting Polyacrylamide Gel 39

Electrophoresis Module Assembly 39

Gel Buffer System 39

Placement of Electrode Assemblies in Mini-Protean Tetra Tank 40

Sample Loading 40

52

Mini-Protean Tetra Tank Assembly 40

Power Conditions 40

Gel Removal 40

Fixation 41

Staining 41

Destaining 41

Image Storing and Photography 41

Quantification of Protein Fractions 41

Statistical Analysis 41

3.9 TWO DIMENSIONAL GEL ELECTROPHORESIS (2-DE) 42

3.9.1 Reagents Preparation 42

Rehydration Buffer 42

Equilibration buffer 42

SDS-PAGE Equilibration Buffer 1 (with DTT) 43

SDS-PAGE Equilibration Buffer 2 (with IAA) 43

0.5% Agarose Sealing Solution 43

3.9.2 ISO-ELECTRIC FOCUSING (IEF) 43

Sample Preparation 44

IPG Strip Rehydration and Sample Application 44

3.9.3 SECOND DIMENSION/SDS-PAGE 45

Glass Plates Assembling 45

53

Preparation and Casting the Gel 45

IPG Strip Equilibration 46

Transferring the Strip on SDS-PAGE 46

Electrophoresis 47

Fixation 47

Staining 47

Destaining 47

Analysis 47

CHAPTER 4 48

RESULTS 48

4.1 BODY MASS INDEX (BMI) 48

4.2 HORMONAL ANALYSES 49

4.2.1 LUTEINIZING HORMONE (LH) 49

4.2.2 FOLLICLE STIMULATING HORMONE (FSH) 49

4.2.3 LH: FSH RATIO 50

4.2.4 INSULIN 51

4.2.5 TESTOSTERONE 51

4.2.6 ANDROSTENEDIONE 52

4.2.7 GROWTH HORMONE (GH) 53

4.2.8 PROLACTIN 53

54

4.2.9 CORRELATION OF LH WITH TESTOSTERONE AND 54

ANDROSTENEDIONE

4.3 ONE DIMENSIONAL POLYACRYLAMIDE GEL

ELECTROPHORESIS

57

4.3.1 CONTROL GROUP 57

4.3.2 PCOS PATIENTS GROUP 58

4.3.3 GROUP COMPARISON 71

181kDa Protein Fraction 71












55

4.4 TWO DIMENSIONAL POLYACRYLAMIDE GEL

ELECTROPHORESIS

78

4.4.1 CONTROL GROUP 78

4.4.2 PCOS PATIENTS GROUP 78

4.4.3 GROUP COMPARISON 79

CHAPTER 5 85

DISCUSSION 85

CHAPTER 6 99

REFERENCES 99

ANNEXURES

Annexure I xviii

Annexure II xxi

Annexure III xxv

Annexure IV xxvii

Annexure V xxviii

xv

LIST OF TABLES

Table Title Page No. No.

4.1 Summary statistics of categorical variables in different cohorts of 55

study

56

4.2 BMI and hormonal variables in control group, PCOS patients and 56

their first degree relatives (FDR)

4.3 Band percentages as indicated by various protein fractions (181-13 65

kDa) in control (C1-C8) subjects and PCOS patients (P9-P27)

4.4 Average band percentages of various protein fractions in control and 77

PCOS patients groups showing group comparison

4.5 Protein fractions showing variable expressions in women with PCOS 84

as compared to controls

i

ii

ACKNOWLEDGEMENTS “Praise be to Allah The Cherisher and Sustainer of the Worlds”

First, and foremost, I pay all my praises to Al- Mighty ALLAH, the Most Merciful, the most

beneficent, for enabling me to carry out and complete this project.

I wish to express my deepest gratitude to my supervisor, Dr. Nabila Roohi for taking a keen

interest in this PhD project which is an International/global problem (Infertility/PCOS). Dr. Nabila

Roohi, is a dynamic and devoted personality. She has been extremely sincere in solving technical

and administrative hurdles and diligently crossed all the obstacles.The proteomic analysis of

plasma samples of PCOS by 2-DPAGE could not have been materialized without personal interest

and painstaking efforts of Dr Nabila Roohi in the Physiology Lab of the Department. She proved

to be an excellent supervisor and morale booster at times of frustration on account of technical

hurdles.

I am grateful to Prof Dr Zafar Ullah Khan, Vice Chancellor, King Edward Medical University,

Lahore who approved the project for funding and purchase of kits for hormonal analysis. The ex-

Director of Centre of Nuclear Medicine (CENUM), Dr. Muhammad Naeem

Qureshi at Mayo Hospital, Lahore, provided all the laboratory facilities specially access to RIA and

IRMA equipments for hormonal estimations at CENUM. Dr. Shan Elahi was extremely helpful in

storage of samples, and expert technical assistance in performing the hormonal measurements.

The selection of patients, collection of blood samples and ultrasound diagnosis of PCOS patients

was only possible by the strong facilitation of Professor Dr. Farah, Professor of Obstetrics and

Gynaecology at Lady Wallingdon Hospital, Lahore and Dr Saima MS of Lady

Aitchison Hospital, Lahore at their Infertility Clinics of these Hospitals. Dr Imran Waheed,

Director Abdullah Diagnostic Clinic, who is a foreign qualified Sonologist took keen interest in

selection of infertile patients on account of PCOS. He was quite instrumental in following the

First Degree Relatives (FDR) of PCOS, thus helped the author to develop FDR group to study the

early diagnosis of PCOS and detection of protein biomarkers in this group of normal teens of

relatives of PCOS. My thanks are owed to Dr Syed Muhammad Zubair, who physically labored

and toiled hard to go to Infertility Clinics at the above mentioned Hospitals and convinced the

patients to give samples of blood to investigate their problem of being infertile. Dr. Imrana Ehsan

with a high degree of ethics took the history of female patients, filled the proforma, drew the

57

blood samples after their consent; and also provided the results of hormonal analysis to the

normal/subjects at the Physiology Department of King Edward Medical University Lahore.

iv

In fact, the whole faculty of Physiology Department of King Edward Medical University, Lahore

coordinated with devotion and sincerity in achieving completion with perfection of this project,

specially Dr Muhammad Shafique, and Dr. Imrana Ehsan.

I am highly obliged to the Fellows and Scholars of Physiology lab, University of the Punjab who

extended every facility, help and guidance, especially in 2 D PAGE experiments. The faculty of

Physiology was found to be free of professional bias and instrumental in the completion of this

study/project.

Mr Maqsood Hussain, Lab Attendant deserves full marks for his services for the guest scholars in

addition to the regular scholars and fellows.

I am greatly indebted to Mr Babar Ali, Laboratory Assistant of the Physiology Department of King

Edward Medical University, Lahore for his extraordinary care in storage and preservation of

blood samples drawn for both hormonal analysis and subsequent investigations of 2-

Dimensional Polyacrylamide Gel Electrophoresis (2-D PAGE).

I have all the regards for Mr. Javaid Iqbal, stenographer to the undersigned for the speed and

efficiency with which he typed this manuscript; he is an expert in computer typing and has good

knowledge of medical terminology. I am extremely thankful for his in time completion of this

report.

In the end, I cannot forget the Information Technology services of Mr. Nasir Aziz, Data Entry

Operator (DEO) of Mohtarma Benazir Bhutto Shaheed Medical College (M-B-B-S MC), Mirpur,

Azad State of Jammu and Kashmir (AJ&K). He has revised, spell-checked and used his skills in

finalizing this manuscript. He deserves “Shabash” for his devotion, dedication and truthfulness. I

am highly obliged for his sense of commitment.

(Dr. Muhammad Akram)

58

CHAPTER 1

INTRODUCTION

Polycystic Ovary Syndrome (PCOS) is the commonest female endocrine entity/disorder in the

developed world, Pakistan being no exception to it. It is a multifactorial and most debatable

reproductive endocrinal disorder in the young females with highly controversial pathophysiology

(Norman et al., 2007).

The National Institutes of Health (NIH) criteria 1990 include excess of androgens and oligo-

ovulation, as the diagnostic criteria. However, 20-25% of regularly ovulating women have PCOS

on ultrasound examination. The abnormal ovarian morphology that resembles with PCOS but it is

not necessary for diagnosis. Moreover, recent reports indicate that ovarian morphology is no

longer an indispensable diagnostic criterion of PCOS (Artini et al., 2010).

The endocrine Society, however, recommended that PCOS can be diagnosed if the adult women

presents with two of the following features i.e., i) excess production of androgens, ii)

anovulation and iii) pearl-sized cysts found in the ovaries (Legro et al., 2013).

PCOS was initially recognised in women who presented with irregular menstrual cycle and

excessive hair growth on the body. These women had different degrees of infertility. It was

hypothesized that failure of release of ovum from the ovarian follicle resulted in the rise of

precurssor hormones like testosterone, and androstenedione (Stein and Leventhal, 1935). It was

presumed that on account of thickened ovarian capsule, the ovum was not released from the

follicle causing anovulation and resultant amenorrhoea.

The surgeons resorted to a surgical procedure called as ovarian wedge resection. A part of the

capsule is resected for release of the ovum. As a result of ovarian surgery all of the hormones of

ovary decreased suddenly. Moreover, rise in pituitary gonadotrophins was also observed, thus

restoring the normal feed-back system. Consequently, the normal folliculogenesis was

encountered (Hendrik’s et al., 2007; Blank et al., 2006). This surgical resection is not done

routinely these days. Nevertheless, some surgeons/researchers are still performing ovarian

resection via laparoscope to induce ovulation.

The process of ovulation depends upon coordination between hypothalamus, hypophysis

cerebri/pituitary gland and ovaries. Nevertheless, the hypothalamo-hypophyseal system

integration is the primary determinant of ovulation in women (Blank et al., 2006). It has been

reported that women who suffer from PCOS have constantly abnormal gonadotrophin secretion

(Taylor et al., 1997). The PCOS is related with increased luteinizing hormone (LH) secretion from

pituitary gland. The follicle stimulating hormone (FSH) levels are found to be on the lower side

(Rebar et al., 1976).

The fully developed PCOS patient has a history of less than 6 ovulations per year. The cycles are

prolonged usually greater than 35 days. The typical male type hair distribution along with hair

loss is characteristic feature of PCOS. There is accompanying obesity, hyperandrogenemia and

acne. On ultrasound examination there are multiple immature follicles. Morphologically,

ultrasound examination of the ovary in PCOS may present only few small immature follicles

(cysts). Some patients have cysts that may give rise to garland type of appearance to the ovary.

Whereas, in other patients immature follicles (cysts) are so numerous in the ovary that it

59

resembles a honeyecomb. On account of the typical PCOS heterogeneity, the the syndrome is

yet not exactly known (Belosi et al., 2006). It has, however, been reported that the number and

size of immature ovarian follicles is quite significant. It has been concluded by some authors that

2-5 mm immature follicles are probably involved in the arrest of follicular growth. Moreover, the

age of the patient and fasting insulin levels have important bearing on the terminal-follicle

growth. It has been conjectured to be on account of exaggerated physiological inhibition from

the pool of 2-5 mm follicular number and size (Dewailly et al., 2007).

The recent understanding of the mechanism of PCOS includes insulin resistance besides

alterations in menstrual cycle, disturbed LH:FSH ratio and anovulation. Banaszewska et al. (2003)

have reported that insulin resistance and consequent hyperinsulinaemia as occurs in maturity

onset diabetes mellitus, is an important factor that may develop PCOS in women. At present the

hypothalamo-hypophyseal-ovarian (H-H-O) axis and its relationship with insulin resistance is yet

to be understood. It is relevant to know the role of gonadotrophins and their relationship with

insulin resistance.

The underlying mechanism of genesis of polycystic ovary syndrome is unclear. Recently it has

been interpreted that the principal cause of PCOS may be hyperinsulinaemia that occurs on

account of insulin resistance. It has been hypothesized that ovarian tissue has insulin receptors

and insulin itself stimulates ovarian steroids which lead to hyperandrogenism (Barbeiri 1986;

Ehrman et al., 1995). Some workers blame that insulin resistance and accompanying

hyperinsulinism leads to increase in intra-ovarian hyperandrogenism. The hyperandrogenism

prevents elevation of FSH that finally results in anovulation (Jonard and Dewailly, 2004).

Insulin resistance has been linked with excess of androgens in PCOS patients. Moreover, insulin

resistance is labeled as one of the important diagnostic criteria of PCOS. On the contrary some

PCOS patients do not have features of insulin resistance. As a matter of fact insulin resistance

has been blamed to be a vital factor in the causation of (Ojaniemi and Pugeat, 2006).

The basic mechanism of development of PCOS in women is not known. However, features like

insulin resistance, obesity, excess of androgens in the blood and derranged gonadotrophin

secretions are concerned in its causation. Hyperinsulinaemia has been reported to cause

hyperandrogenism. Moreover, insulin may stimulate androgen level by the follicular cells in

collaboration with LH. It has been reported that the main cause of failure to ovulate is

inadequate release of FSH (Guzick, 2004).

Recarbarren et al. (2008) have reported that obesity and insulin resistance are heritable.

Moreover, PCOS has been labeled as a familial disease (Azziz, 2008). According to Laven (2007),

genes have little role to play in the aetiology of PCOS. Some authors have reported that both

genes and environment may be implicated in the aetiology of PCOS. Furthermore, poor diet and

sedentary habits may lead to obesity that can deteriorate PCOS in some patients. However, the

role of infectious agents and toxins is doubtful (DiamantiKandarkis et al., 2006). Despite the

discrepancies in definition, diagnostic criteria, the role of gonadotrophins in the pathogenesis of

PCOS, ethnic and climatic/environmental predisposition of the syndrome; the bulk of etiological

and clinical data available in the literature serves to provide only a framework for studying

genesis of PCOS in Pakistani women. Therefore, original investigations of various endocrine and

presentation parameters are warranted to understand the patho-physiology of the syndrome in

local women. Over the last few years there has been an increased recognition and importance of

hormonal changes in the manifestation of PCOS. The current work was, therefore, planned to

60

systematically examine the specific endocrinal changes and hormonal interactions in Pakistani

cohorts of PCOS patients.

Clinical application of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is

important for identifying specific proteins in healthy individuals and in patients with selected

known diseases. Electrophoretograms exposed certain reference protein profiles in the selected

clinical disorders. Abnormal profiles were detected in the form of “abnormal spots”. It has been

found that some diseases are associated with specific ‘spot patterns’ on the protein maps of the

patients. Similarly, regulatory proteins have been identified in diseases like leukaemia, Ewing

sarcoma and Alzheimer’s disease (Chrambach et al., 1993).

The electrophoretic protein patterns have been recognized for different human fluids such as

plasma, amniotic fluid and cerebro-spinal fluid respectively (Anderson and Anderson, 1991;

Hughes et al., 1992; Yun et al., 1992). Recently, seminal plasma of men was studied by 2-D PAGE.

These authors reported that electrophoresis can be helpful for studying seminal plasma proteins

in azospermia. They suggested further research for identification of these markers in the

diagnosis of impaired spermatogenesis (Starita-Geribaldi, 2001).

On account of rising incidence of PCOS, unknown aetiology and pathogenesis; and scarcity of

work in Pakistani women, it was considered imperative to investigate the syndrome in Pakistani

population, and to find out any correlations amongst various parameters in PCOS. The study has

been planned:

1. To find out the role of gonadotrophins, LH:FSH ratio, hyperinsulinaemia and

hyperandrogenism in PCOS.

2. To determine whether or not there exists any correlation between hyperinsulinaemia and

hyperandrogenism in PCOS.

3. To detect/investigate the presence of any specific protein(s) in sera of patients with PCOS.

The author, therefore, considered imperative to probe appropriate molecular diagnosis by

applying 2-Dimensional Polyacrylamide Gel Electrophoresis (2-D PAGE) that may lead to

discover novel proteins associated with PCOS. This might be helpful in understanding the

underlying etiology and facilitating early diagnosis to prevent early life complications like

cardio-vascular disorders and endometrial cancer.

61

CHAPTER 2

LITERATURE REVIEW

PCOS is described as a heterogenous condition and got evolved from polycystic ovary disease to

polycystic ovary syndrome. It is an ovarian disorder characterized by hyperandrogenism, oligo-

ovulation and polycystic ovaries The classical features of PCOS however, are hirsutism, irregular

menstrual cycles, obesity and typical immature, small ovarian follicles. The diagnostic criteria for

PCOS is chronic anovulation/oligo-ovulation with evidence of clinical hyperandrogenism i.e.

hirsutism, acne and male type baldness. The biochemical criterion is raised serum androgens.

The other causes of hyperandrogenemia must be ruled out for the diagnosis of PCOS. Abnormal

ovarian morphology/folliculogenesis is inadequate to make a diagnosis of PCOS. It is on account

of the fact that 20-25% of regularly ovulating women have PCOS on ultrasound examination.

Therefore; abnormal ovarian morphology is consistent with PCOS but not essential for diagnosis.

It is the most common reproductive endocrinal disorder in the females with highly controversial

pathophysiology (Legro, 2009).

Recent reports indicate that ovarian morphology is no longer an essential requirement for

diagnosis of PCOS (Ferry, 2010). There is no doubt that PCOS is a syndrome i.e. collection of signs

and symptoms. Furthermore, there is no single test that can be considered as essentially

diagnostic. It has been mentioned that a just and comprehensive definition is essential to

enhance future research (Azziz et al., 2005). PCOS is continuously generating controversy in its

definition and pathophysiology (Balen and Michelmore, 2002; Azziz, 2006; Franks, 2006).

It has been reported that ambiguities in the definition of PCOS and diagnostic criteria are still

generating controversies and a clear-cut definition is indispensable to successfully probe the

syndrome for knowing the pathogenesis (Azziz, 2006).

PCOS is described as a heterogenous disorder and comprises a variety of potential signs and

symptoms. Uptil now, there is no universally accepted clinical definition of PCOS (Legro, 2003).

Therefore, the attention is shifting towards assessment of increased health risks of PCOS in the

later life (Fauser, 2008).

It was observed that major factors responsible for generating features of PCOS are

hyperandrogenism and oligo-ovulation. Others have defined PCOS as a disorder of androgen

excess (Zawadski and Dunaif, 1992). Subsequently PCOS is recognized as having two of the

following features (ESHRE/ASRM, 2004):

1. oligo-ovulation or anovulation

2. clinical or biochemical signs of hyperandrogenism

3. polycystic ovaries.

Some researchers described PCOS on the appearance of immature follicles on ultrasound

examination. These multiple immature pre-antral follicles having a size of 2-8 mm that makes a

“black-pearl necklace” sign (Franks, 1995). It has been proposed that typical ovarian picture on

ultrasound examination along with menstrual disorders and signs of hyperandrogenism are good

indicators of PCOS. However, hormonal parameters are not essential for the diagnosis

62

(Homburg, 2002). According to Balen et al. (2003), the echographic pattern of ovary for the

diagnosis of PCOS is defined as presence of 12 or more follicles in which ovary measuring 2-9

mm in diameter and /or increased ovarian volume more than10 cm3. It was also recognized that

other androgen excess disorders should be excluded before declaring the diagnosis of PCOS

(ESHRE/ASRM, 2004).

2.1 DIAGNOSTIC CRITERIA

The diagnostic criteria for PCOS as suggested in the Rotterdam conference sponsored by

European Society for Human Reproduction and Embryology and the American Society for

Reproductive Medicine (ESHRE/ASRM) in 2004, include oligo-ovulation/or anovulation, androgen

excess, and clinical features of high androgen levels based on polycystic ovary (PCO) morphology.

The diagnosis is made by anovulation, hyperandrogenism and multiple cysts in ovary. The experts

yet differ about the importance of each feature (Athanasia et al, 2008). The endocrine Society,

however, recommended that PCOS can be diagnosed if the adult women presents with two of

the following features i.e., i) excess production of androgens, ii) anovulation and iii) pearl-sized

cysts found in the ovaries (Legro et al., 2013).

Thus if all the three criteria are present the PCOS is regarded as severe (Norman et al., 2007).

When there is positive history of anovulation and clinical signs of hyperandrogenemia was the

criteria for the diagnosis of PCOS before 2003 (Zawadski and Dunaif, 1992).

With advancement of sensitive hormonal assays, hyper-androgenemia became the most

important diagnostic criterion (Raj, 1978). Moreover, the ultrasound examination made easier

the study of ovarian morphology as the diagnostic feature (Adams et al., 1986). As the insulin

resistance is being labelled as one of the etiologic factor, insulin levels have become a part of the

diagnostic evaluation of PCOS (Legro et al., 1998).

The classical presentation of PCOS comprises of amenorrhoea or oligomenorrhoea with

hyperandrogenism. However, presenting symptoms vary widely; including anovulation without

hirsutism and hirsutism with regular cycles. The diagnosis of PCOS is made principally on

hormonal analysis i.e. serum testosterone, LH and FSH. It has been suggested that on account of

high risk of glucose tolerance or frank diabetes, and women complaining of

irregular/anovulation/oligo-ovulation along with PCOS should undergo glucose tolerance test.

The researcher further suggested that circulating insulin levels are not necessarily required

(Franks, 2010).

According to Rotterdam ESHRE sponsored PCOS consensus workshop group 2003, PCOS remains

a syndrome. There is no single criterion for clinical diagnosis.

In order to establish the diagnosis of PCOS, it is essential to exclude other disorders that

resemble PCOS. It is also necessary to exclude hypogonadotrophic hypogonadism which is a

central ovarian dysfunction. Moreover, the Rotterdam 2003 group participants emphasized that

routine measurement of prolactin should be performed in hyperandrogenic patients. This is

necessary to exclude hyperprolactinaemia.

It has been mentioned that ovarian morphology is not diagnostic of PCOS because this

morphologic pattern is also found in childhood and adolescence (Bridges et al., 1993; Battaglia et

al., 2002). The same pattern of ovary is also found in menopausal women (Birdsall and Farquhar,

63

1996; Dahlgren et al., 1992). Moreover, this ovarian pattern is also reported in

hyperandrogenism in the absence of regular menstrual history (Ibanez et al., 2008; Norman et

al., 1995; Carmina and Lobo, 2001).

2.2 ONSET OF PCOS

The features of PCOS begin around menarche (Franks, 2002), but may appear after puberty as a

result of environmental factors like weight gain. Premature puberty may result on account of

early secretion of adrenal steroids, may be a predictor of the disease (Ibanez et al., 2001). In

addition, the abnormal intrauterine environment may be responsible in the pathogenesis of the

syndrome (Eisner et al., 2003; Eisner et al., 2002; Abbot et al., 2002).

It has been experimented that ovarian theca cells in affected women convert androgenic

precursors to testosterone as compared with normal theca cells (Nelson et al., 1999).

The frequency of the stimulus of hypothalamic gonadotrophin-releasing hormone (GnRH)

determines, in part, the relative proportion of luteinizing hormone and follicle-stimulating

hormone synthesized within the gonadotropes. Increased pulse frequency of hypothalamic

gonadotrophin releasing hormone (GnRH) favours transcription of the β -subunit of luteinizing

hormone over the β-subunit of follicle-stimulating hormone; conversely,

decreased pulse frequency of GnRH favours transcription of the β-subunit of folliclestimulating

hormone, which decreases the ratio of luteinizing hormone to folliclestimulating hormone

(Hasenloder et al., 1991). The women with the polycystic ovary syndrome appear to have an

increased luteinizing hormone pulse frequency, it has been inferred that the pulse frequency of

GnRH must be accelerated in the syndrome. It is not clear whether this accelerated pulse

frequency is due to an intrinsic abnormality in the GnRH pulse generator or is caused by the

relatively low levels of progesterone resulting from infrequent ovulatory events. Since progestins

slow the GnRH pulse generator, low circulating progestin levels in women with the polycystic

ovary syndrome may lead to an acceleration in the pulsatility of GnRH, increased levels of

luteinizing hormone, and overproduction of ovarian androgens (Eagleson et al., 2000).

The polycystic ovary syndrome remains one of the most common hormonal disorders in women,

with a prevalence estimated between 5 and 10 percent. Variance in prevalence among

populations may reflect the effect of ethnic origin, race, and other environmental factors on the

phenotype (Knochenhauer, 1998; Asuncion et al., 2000).

2.3 PATHOPHYSIOLOGY OF PCOS

The fundamental abnormality in PCOS remains unknown, but growing evidence indicates that it

includes insulin resistance, androgen excess and abnormal gonadotrophin dynamics (Guzick,

1998). Recent data suggest that the principal disorder in PCOS is insulin resistance, that results

into hyperinsulinemia that stimulates ovarian androgen production

(Nestler and Strauss, 1991; Nestler, 1994.)

In patients of PCOS the conversion of follicular androstenedione to estradiol suggests disordered

aromatization. Moreover, aromatase gene mutation has been reported to cause a form of the

syndrome (Conter et al., 1994).

2.4 INAPPROPRIATE GONADOTROPHIN SECRETION

64

LH plays an important role in the development and functioning of the reproductive system. The

measurement of LH is significant for detecting the integrity of pituitaryovarian axis, diagnosis of

reproductive disorders and monitoring the anti-fertility programmes (Vandan et al., 2005).

Gonadotrophin abnormalities in PCOS include elevated levels of testosterone and LH; or high

levels of LH-to-FSH ratio, an augmented LH pulse frequency and deranged diurnal rhythm of LH.

The prominently elevated serum LH levels are present in a considerable proportion of women

with the syndrome but are not crucial for its diagnosis (Dunaif et al., 1992).

It has been reported that inappropriate secretion of LH, is a characteristic feature of PCOS

(Rebar et al., 1976). Initially an excess of LH was first detected in the urine (Keettle et al.,

1957). Abnormal levels of LH secretion were later on found in the blood (McArthur et al.,

1958). The subsequent studies documented an elevated level of LH: FSH ratio (Yen et al., 1970).

One of the factors implicated in the genesis of PCOS is impairment of the action of FSH.

Moreover, LH that rises prematurely has been predicted to prevent the selection of dominant

follicle (Jonard and Dewailly, 2004).

Abnormal levels of pituitary gonadotrophins i.e. follicle stimulating hormone (FSH) and

luteinizng (LH) has also been labeled to participate in the pathophysiology of PCOS (Guzick,

1998). In addition to abnormal gonadotrophins, elevated levels of testosterone and abnormal

LH:FSH ratio have been reported in women suffering from PCOS. Further, LH:FSH ratio of 3:1 is

diagnostic feature of PCOS. It has been mentioned by Ehrmann, (2005) that FSH may regulate

production of aromatase activity of granulosa cells of ovarian follicles. Thus, FSH determines the

amount of synthesis of oestrogen from the androgen precussors. The elevated LH levels as

compared to FSH may stimulate the ovary to start synthesizing excess of androgens. It has been

summarized by some researchers that LH:FSH ratio is not a characteristic feature of all the

patients of PCOS. Rather, it is true for a small group of women i.e. less than 50% PCOS patients

have altered LH:FSH ratio (Banaszewska et al., 2003).

A recent study on LH:FSH ratio regarding pathophysiology of PCOS reports that this ratio should

not be considered as the diagnostic criteria These authors have suggested that diagnosis of

PCOS is based upon clinical picture and concomitant hyperandrogenaemia. Nevertheless, other

causes of hyperandrogenaemia should be ruled out (Alnakash and AlTae’e, 2007).

Moran et al. (2003) have hypothesized that LH, FSH, LH:FSH ratio are not related with insulin

resistance. Nevertheless, insulin resistance can influence gonadotrophins with long-standing

stimulation, especially in obese patients.

In another study it has been reported that insulin resistance has been observed in nonobese

patient suffering from PCOS. Moreover, insulin resistance is also related with raised levels of LH

hormone and free and unbound testosterone. It has therefore, been suggested that insulin

resistance needs to be treated at an early stage. Consequently, it may decrease the risks of

potential complications at a later stage of life in these women having PCOS. Furthermore, these

authors described/observed a strong relationship between excessive insulin, excess of androgen

and low sex hormone binding globulin (SHBG). In addition, new vessel growth in the ovarian

stroma has been considered to be on account of hyperinsulinaemia (Toprak et al., 2001).

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It has been blamed by certain authors that several factors in PCOS disturb the normal

folliculogenesis. The theca interna cells and granulosa cells dysregulation play main role in

abnormal follicular growth. The oocyte may be involved in causing increased formation of

follicles. FSH is important in proliferation and survival of granulosa cells (GC). Moreover, FSH

recruits the dominant follicle in normal folliculogenesis. It has been hypothesized that lofty

levels of inhibin B are released by the candidate follicles. While dominant follicle produces

estradiol and inhibin A, that consequents to inhibition of FSH release from anterior pituitary

(Zelznik and Fairchild-Benyo, 1994). In PCOS the intercycle rise of FSH is prevented on account of

excessive release of inhibin B. Inhibin B ultimately suppresses FSH production by negative feed-

back system.

2.5 THE PREMATURE ACTION OF LH IN PCOS

It is reported that granulosa cells (GC) build up their own receptors for LH in the middle to late

follicular phase (Erickson et al., 1979). It has been hypothesized that premature action of

luteinizing hormone on granulosa cells of the follicle may arrest its growth. (Franks et al., 1998).

Insulin enhances the ability of granulosa cells to respond to luteinizing hormone. These authors

have suggested that raised insulin level may be labeled for arrest of maturation and subsequent

anovulation (Willis et al., 1996). It has been reported that insulin resistance at the level of GC is

the actual culprit (Coffler et al., 2003a). Nevertheless hyperinsulinism may be a secondary cause

of anovulation in PCOS that can deteriorate the follicular arrest. However, hyperinsulinism may

be a secondary cause of anovulation in PCOS that nonspecifically worsens the follicular arrest

(Jonard and Dewailly, 2004).

2.6 WHY FOLLICULAR ARREST IS INCONSTANT

In fact, oligo-ovulation rather than anovulation is the real feature of PCOS. At times, a dominant

follicle escapes all inhibitory influences and matures to release the ovum. Moreover, some

patients of PCOS ovulate regularly despite simultaneous presence of clinical features and excess

of androgens (Carmina and Lobo, 2001). It has been reported that premature LH receptors

developed in GC of immature follicles are found only in those patients of PCOS who don’t

ovulate (Willis et al.,1998). It has been further hypothesized that excess of small immature

follicles exert relatively less blocking effect on the pool of selectable follicles (Jonard and

Dewailly, 2004).

The LH:FSH ratio of more than 2 presents the best combination of sensitivity and specificity. It

was concluded that LH:FSH ratio is a significant and important diagnostic criterion in detecting

PCOS women presenting with oligomenorrhea or anovulation. The authors have further

hypothesized that BMI and LH may provide insights in the etiopathogenesis of PCOS (Ming,

2009).

It has been reported that LH is elevated in PCOS in relation to FSH. However, LH in turn is

influenced by BMI. Researchers have investigated to determine the effect of BMI on neuro-

endocrine disorder in PCOS. These authors have recorded that both LH and GnRH are raised in

PCOS; but BMI does not influence hypothalamic function. The response of pituitary to

administration of GnRH is found to be inversely related to BMI in PCOS. It was hence, concluded

that BMI can influence the secretion/elevation of LH through pituitary. The rise in LH on account

of increased BMI is attributed to pituitary in women suffering from PCOS (Pagan et al., 2006).

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Blank et al., (2006) reported that excess of LH enhances hyperandrogenism via ovarian

stimulation. It was considered on account of the known effect of LH on theca cells of the ovarian

follicles; thus LH has the primary role in PCOS. Some researchers have concluded that LH

dysregulation is not primary; but it may be secondary to some other ovarian factor. It has been

claimed to be due to abnormal secretion of androstenedione produced by ovarian theca cells in

PCOS (Suhail, 2008).

It has been reported that androstenedione may be the cause of LH rise in patients of PCOS. It is

known that absence of physiological levels of estradiol facilitates elevation of gonadotrophins in

both men and women (Carani et al., 1997). It was thus suggested that estradiol rather than

androgens modify GnRH feed-back. It has been hypothesized that some ovarian factor, probably

androstenedione may enhance un-coupling of hypothalamic GnRH secretion from the effect of

estradiol; thus accelerating the activity of this pulse generator. It has also been reported that LH

level was raised alongwith androstenedione in all the women studied. Consequently,

androstenedione may be responsible for uncoupling of GnRH from inhibition by estradiol (Suhail,

2008).

2.7 ANTI-MULLERIAN HORMONE IN PCOS

Anti-Mullerian Hormone (AMH) is produced by granulosa cells (GC) of the ovary. The hormone

makes its appearance in the blood in the 36th week of gestation and remains positive until

cessation of menstrual cycles in the women. The highest expression of the hormone is from

small antral follicles. The concentration of AMH gradually falls with the progression of follicular

growth. AMH has been observed to play an inhibitory role in the growth of ovarian follicles. It

not only decreases primordial follicle initiation but also decreases follicle senstivity to FSH by

inhibiting aromatase. AMH has therefore, been a focus of study in PCOS. It has thus been

hypothesized that it may contribute towards anovulation, a charachteristic finding in PCOS

(Pellat et al., 2010). AMH values were compared amongst overweight and normal weight

women with four different phenotypes of PCOS and healthy control subjects. AMH levels were

significantly raised in all the four PCOS groups compared with the control group. Elevated LH

concentration has been found to be the most significant link between PCOS-associated disorders

of ovulation and raised AMH concentration in these patients (Athanasia et al., 2008). A positive

correlation has been observed between serum AMH levels and the number of antral follicles in

women with PCOS. The raised AMH concentration in the blood may be associated with PCOS

and its diagnosis. Moreover, AMH can also be helpful in establishing sub-classification of PCOS

that is the most controversial and heterogenous syndrome (Jenny et al., 2006).

2.8 HYPERANDROGENISM

The adrenals and ovaries add to circulating androgens in women. The adrenal androgens, like

dehydro-epiandrosterone (DHEA), are relatively weak in action as compared to ovarian

testosterone. The adrenal DHEA and ovarian androstenedione may serve as prohormones and

heightens the androgens in the blood. Androstenedione is the solitary circulating androgen that

is higher in premenopausal women (Legro, 2003). It has been mentioned that circulating

testosterone was found to be the best representative of hyperandrogenism, chronic anovulation

and polycystic ovaries (Robinson et al., 1992). Hyperandrogenemia is not accepted universally as

an indicator of androgen excess. It has been reported that hirsutism was fomd in 50% women

who have PCOS (Moran et al., 1994). Chronic anovulation on account of hyperandrogenemia

was found only in 50-60% of women (Azziz et al., 2001). Hirsutism which is a clinical

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manifestation of hyperandrogenemia is accompanied by normal circulating androgen levels

(Lobo et al., 1983).

2.9 PHYSIOLOGICAL HYPERANDROGENISM OF PUBERTY

At the onset of puberty, the gonadotrophin hormones especially LH is only found to be raised

during sleep. Subsequently, it is found increased in the day time; accompanied by appearance of

increased androgens in plasma. There is a concomitant enlargement of ovaries along with

visible multiple follicles on ultrasound examination.

The initial menstrual cycles in adolescent girls have been found to be without ovulation. These

cycles have a characteristically raised serum levels of testosterone, androstenedione and LH. At

this stage there is slight degree of hyperandrogenic tendency in these adolescent girls. Within a

passage of 1-2 years, the menstrual cycles become regular. However, in some girls these

abnormalities and menstrual cycles get worsened, and start mimicing features of PCOS; along

with hirsutism, anovulation, increased androgens, raised LH levels and increased ovarian

volume. It has been commented that development of acne and irregular periods in adolescent

girls was a part of normal ovarian development along with hyperandrogenism of PCOS.

Moreover, lean subjects were found to be more prone to develop insulin resistance and

dyslipidaemia. The researchers concluded that adolescent girls with hyperandrogenism were at

risk of developing metabolic disturbances. They further reported that these girls should be

investigated for metabolic abnormalities irrespective of their age, or BMI (Huppert et al., 2004).

PCOS is commonly accompanied by infertility and insulin resistance; which may be associated

with frank type 2 diabetes mellitus or metabolic syndrome. The metabolic syndrome is

concerned with a variety of findings like central obesity, hypertension, glucose intolerance and

dyslipidemia (Eckel et al., 2005). The metabolic syndrome may also present with an increased

risk of endometrial cancer (Ehrmann, 2005; Rotterdam

ESHRE/ARSM, 2004)

It has been observed that most of the patients suffering from PCOS have obesity and insulin

resistance. Obesity is presumed to cause insulin resistance. In order to determine whether or

not insulin resistance, in the absence of obesity and acanthosis nigricans can generate the

features of PCOS; non-obese patients of PCOS were selected for study.The results were

compared with healthy control subjects matched for age and weight. It was concluded that

insulin resistance in non-obese patients of PCOS is positively related with the syndrome. It was

further hypothesized that insulin resistance is positively related with serum LH and testosterone

levels in the blood. It is, therefore, imperative to treat the features developed on account of

insulin-resistance. It seems mandatory to decrease the insulin-resistance at an early stage to

prevent subsequent cardiovascular risks in the later part of life of the patients of PCOS (Toprak et

al., 2001).

Metabolic syndrome has been reported to be closely related to PCOS on account of a common

underlying etiologic factor i.e. insulin resistance. It has been postulated that metabolic

syndrome is characterized by visceral obesity and insulin resistance. These two characteristics

are further associated with atherosclerosis and cardio-vascular disease (Doalle, 2004; Isomaa,

2001). It has been reported that cardiovascular risk factors are invariably present in women with

PCOS (Tallbot et al., 2004; Carmina et al., 2005).

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The major criterion for the diagnosis of metabolic syndrome in PCOS is that adopted by the

American National Cholesterol Panel/Adult Treatment Panel III (ATP-III). These criteria are

presence of increased waist circumference, high blood pressure, raised fasting blood glucose,

low serum HDL cholesterol and increased triglycerides. Applying these criteria, the prevalence of

metabolic syndrome (MBS) in PCOS has been described to be very high i.e., 43-46% (Gluek et al.,

2003; Apridonidze, 2005). This prevalence of MBS in PCOS is based upon abnormal lipids and

increased waist circumference. In conclusion, it appears that MBS is much more common in

PCOS as compared with general population in women of the same age group (Carmina et al.,

2006).

2.10 ROLE OF ANDROGEN

The luteinizing hormone has been reported to regulate the formation of androgens by theca

cells of ovary. While FSH is blamed to control the aromatase activity in granulosa cells of ovarian

follicles. Consequently, aromatase activity leads to enhanced biosynthesis of estrogens from

precursor hormone i.e, androgens. It has thus been hypothesized that rise in LH level as

compared to FSH culminates into excessive synthesis of androgens by the ovary (Ehrmann,

2005).

Hyperandrogenism is also related with PCOS. It has been mentioned that in PCOS,

17hydroxyprogesterone (17 OHP) levels are raised above normal. This is on account of

administration of GnRH (gonadotrophin releasing hormone) agonists like nafarelin. It has been

concluded that at least 50% of women who have oligomenorrhea, hirsutism, and acne give

abnormal response to GnRH. This clearly shows that these features of PCOS are closely related

with excess of androgens that are produced by ovary (Erhmann et al., 1992).

PCOS has been related with excess of insulin in the blood/hyperinsulinaemia and is currently

being regarded as central pathogenic feature of the syndrome. Hyperinsulinaemia is also related

with androstenedione. Moreover, androstenedione was found to be significantly elevated and

sex hormone binding globulin (SHBG) was significantly lesser in patients of PCOS, who had

hyperinsulinaemia. Consequently, it is concluded that hyperinsulinaemia, hyperandrogenemia

and low SHBG are strong correlates of each other on account of a common factor i.e. insulin

resistance. (Loverro et al., 2001).

Hyperinsulinism and hyperandrgenism are associated with PCOS. Insulin resistance has been

found to co-exist in a significant number of patients with hyperandrogeninaemia. This is

specifically true in patients with positive family history of type 2 diabetes mellitus. Nevertheless,

hyperinsulinaemia may occur in type 1 diabetes mellitus. It may be on account of the fact that

these patients of type-1 diabetes are treated with insulin. It has also been observed that type-1

diabetics also manifest hyperandrogenaemia (Garcia –Romero et al., 2006).

It has been reported that obesity is associated with hyperactivity of hypothalamo-

pituitaryadrenal (HPA) axis (Ivana et al., 2010). It was further hypothesized that PCOS may have

multiple pathologies. The PCOS is a highly complicated disorder and there is no single pathologic

factor (Oussama, 2008).

It has been experimentally studied in rats that ovulation can be induced with bilateral section of

superior ovarian nerve (SON). Moreover, cutting the SON results in asymmetric hormonal

secretion by both ovaries in the animal. The typical PCOS was induced in rat by administration of

estradiol valerate (EV) with a single dose. EV-induced PCOS is related with increased peripheral

sympathetic discharge. It was observed that none of the EVtreated animals ovulated; while oil-

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treated animals ovulated normally. All EV-treated animals developed features of PCOS. In oil-

treated animals, incidence of ovulation was higher in the unilaterally sectioned and/or

denervated ovary. The experiment supports the notion of neural interaction between the

ovaries. It was consequently suggested that some neural mechanism develops the control of

ovulation in the innervated ovary (Dominguez et al., 2008).

It has most recently been suggested that chronic infections may increase the risk of infertility. In

this regard Helicobacter pylori (H Pylori) have been blamed as one of the newest pathological

factor. The PCOS and H Pylori infection has been found to be coincidental. It has, therefore, been

added as one of the latest microorganism that may cause PCOS (Irfan et al., 2009).

Recently it has been hypothesized that the function of resistance vessels can be modified by

PCOS. The study was to determine the role of excess of androgens, obesity and insulin resistance

in the modulation of vascular function. It was concluded that PCOS women who were obese; had

greatly decreased vascular smooth muscle compared to non-obese subjects. Finally, it was

reported that obesity and insulin have significant influence on smooth muscle of blood vessels as

compared with hyperandrogenism (Dokras et al., 2006).

Differences in anthropometry, hormone levels and insulin resistance have been recognized in

PCOS in the Korean Women. It has been concluded that patients diagnosed as PCOS do not

suffer from concomitant hyperandrogenism. It is further reported that these Korean women are

not prone to develop metabolic syndrome and cardiovascular complications. Therefore, PCOS in

Korean women population may prove to be mild phenotype of PCOS (Chae et al., 2008).

PCOS is a multifactorial and controversial endocrine disorder in women in young age. It is being

strongly associated with type 2 diabetes mellitus and hyper-insulinaemia.

The mode of presentation of PCOS was studied and it has been reported that ovarian

overproduction of androgen is on account of hyperinsulinism. Furthermore, it has been labelled

that raised insulin levels are described as an important etiologic factor in the production of

PCOS. (Ayisha et al., 2005).

2.11 BMI IN PCOS

Women with PCOS are at risk of impaired glucose tolerance (IGT), type 2 diabetes mellitus, and

gestational diabetes (Ehrmann, 2005 and Ehrmann et al., 1995). The specific defect is increased

serine and decreased tyrosine phosphorylation of the insulin receptor.

The women suffering from PCOS are hyperinsulinemic (Ehrmann, 2005). Hyperinsulinemia

worsens and insulin sensitizers improve ovarian dysfunction and hyperandrogenemia in PCOS.

Therefore, assessment of glucose homeostasis by an oral glucose tolerance test (OGTT) has

become a regular practice (Qin and Rosenfield, 1998). The interpretation of glucose intolerance

has changed in recent years. The fasting glucose cut off for diabetes has reduced from 140 to

126 mg/dl and the new term, impaired fasting glucose (IFG), has been introduced for the values

between 110 and 125 mg/dl. In 2003, the normal fasting glucose was reduced to 100 mg/dl

(Genuth et al., 2003).

It has been concluded that patients of PCOS develop impaired fasting glucose tolerance (IFG).

Moreover, these patients are also at risk of impaired glucose tolerance (IGT).The increase in

cardiac risk factors and free androgen levels precede the overt glucose intolerance. (Kasim-

Karakas et al., 2004).

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Insulin resistance increases with weight gain as observed by stepwise increase in BMI in the

normal glucose tolerance test (NGT).

The relationship between hyperandrogenism and hyperinsulinaemia was studied in PCOS. The

body mass index (BMI) was higher, and free testosterone index was raised. Similarly, the values

of dehydroepiandrosterone sulphate (DHEAS), testosterone, 17hydroxyprogesterone (17 OHP)

and androstenedione were all found to be raised as compared with normal controls. All

parameters concerned with insulinaemia both basal and maximum peak values were on the

higher side. Importantly, in patients with hyperinsulinaemia, free testosterone index was highly

raised. Nevertheless, gonadotrophin levels were not affected and were found to be within the

normal range. These researchers presented a typical hormonal picture in PCOS on account of

markedly elevated hyperi nsulinaemia (Vidal-Ping and Munoz-Torres, 1994).

It has been reported that excess of insulin in the blood can be considered as one of the

pathogenic agent in PCOS (Dunaif, 1997). Other studies do not coincide and the role of

hyperinsulinaemia in PCOS remains controversial (Webber et al., 2002).

Despite the factors blamed so far in the pathogenesis of PCOS; there is no definite criterion for

the diagnosis of syndrome. The diagnosis is based on a group of clinical signs and symptoms.

These clinical features vary from patient to patient and from investigator to investigator. PCOS

was strictly defined for the first time in 1990 in the National Institute of Health conference.

Zawadzki and Dunaif, (1992) nominated a triad comprising of hyperandrogenism, oligo-ovulation

and ruling out other conditions that mimic PCOS.

Nevertheless, Welt et al. (2006) have reported that even these strict criteria may vary in severity

and presentation of clinical features. These authors have mentioned that the clinical features

also vary with ethnicity and environment. They have suggested cross population studies to verify

the main differences in different ethnic groups in the syndrome. In order to standardize the

diagnosis, number of studies were carried out, consequently leading to greater insight into the

pathology of the disease (Nestler et al., 1998; Azziz et al., 2001).

It has been reported that hyperandrogenism and multiple immature follicles in the ovary are the

cardinal signs of PCOS The clinical features include oligo-ovulation, signs of excessive androgens

in blood and obesity. Further, it has been observed that some women who have characteristic

features of PCOS but may have regular menstrual cycles along with hyperandrogenism (Adams et

al., 1986; Carmina and Lobo, 2001). The Rotterdam ESHRE/ASRM-PCOS consensus workshop

2004 concluded that PCOS remains a syndrome and no single parameter like

hyperandrogenaemia is a diagnostic feature. Furthermore, the PCOS regarding its definition is

still creating controversies (Balen and Michelmore, 2002; Azziz, 2006; Franks, 2006).

Clinically speaking, the patients of PCOS may present with wide variety of features like infertility,

obesity, type 2 diabetes, dyslipidaemia and other systemic features e.g. cardiovascular disease

and endometrial cancer (Azziz et al., 2005).

The androgen excess guideline (AEG) task force has mentioned four key features of PCOS. These

are ovulatory dysfunction, hyperandrogenemia, signs and symptoms of hyperandrogenism and

typical cysts are found on on ultrasound examination of ovaries. The androgen excess in PCOS

patients has been found in 60-80 %, mainly in the form of free testosterone.

Rresearchers have reported menstrual dysfunction in PCOS. At the same time 20% patients of

PCOS present with normal menstrual cycles and oligo-ovulation (Azziz et al., 2004).

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Many authors have reported that polycystic ovaries were found in approximately 75% of women

who are clinically diagnosed as suffering from polycystic ovarian syndrome (Jonard et al., 2003).

Nevertheless, the disease cannot be diagnosed merely on the basis of appearance of the ovary

(Jonard et al., 2005). It has been mentioned that PCOS can be diagnosed only when one ovary

has volume of more than 10 cm3 or more than 12 follicles having a diameter of 2-9 mm (The

ROTTERDAM ESHRE/ASRM-PCOS Consensus workshop group, 2004). However, Balen et al., 2003

have reported that 10-30% patients do not manifest polycystic ovaries on ultrasound. Moreover,

20% women do not consent for transvaginal ultrasonography. Furthermore, the clinicians

depend upon radiologists who may not be familiar with the clinical diagnosis (Farquhar et al.,

1994).

2.12 OVARIAN MORPHOLOGY IN PCOS

The ovarian picture on ultrasound is not consistent with clinical disorder of PCOS. It is also found

in childhood, adolescent, and in menopausal women (Battaglia et al., 2002; Bridges et al., 1993;

Dahlgren et al., 1992).

The evidence of polycystic ovarian morphology is not essential for the diagnosis of the

syndrome. Nevertheless, if signs and symptoms of infertility and menstrual disturbance do not

exist, the ovarian morphology that is consistent with PCOS becomes a criterion for diagnosis

(Rotterdam ESHRE/ARSM, 2004; Azziz, 2005).

It has been reported that volume and follicle number in the ovaries decrease with age in

women. The decrease was observed longitudinally and found to be more pronounced in PCOS

than controls. This age-based criterion can be used for defining polycystic ovarian morphology. It

is possible to use these criteria to distinguish PCOS in women over the age of 40 years

(Alsamarai et al., 2009).

PCOS has been described as one of the most widely studied and disputed/controversial topic in

reproductive endocrinology. The signs of hyperandrogenism in lean/non-obese women with

normal ovulatory cycles contrasting with severe hirsutism, obesity, oligomenorrhea or

amenorrhea represent somersault of the clinical spectrum of PCOS. The heterogeneity in the

clinical and endocrinal features remains a confounding factor in the diagnostic investigations of

the syndrome. A large amount of literature review and experimental data could not guide to the

pathogenesis of PCOS (Battaglia et al., 2002). Some 30% women who have normal menstrual

cycles and normal androgen levels in the blood have polycystic ovaries (Legro, 2003; Farquhar et

al., 1994; Koivunen et al., 1999). It has been reported that the polycystic ovaries can be

independent risk factor to develope ovarian hyperstimulation syndrome, especially after

ovulation induction (Enskog et al., 1999). It is important to note that on the basis of morphology

of the ovary, the patient should not be labelled as a case of PCOS (Louks et al., 2000). It has,

therefore, been suggested that ovarian morphology cannot be employed as gold standard for

the diagnosis of PCOS (Koskinen et al., 1996).

“The excess of small follicles appears as the salient and constant feature of PCOS. Pathological

studies have shown that the pool of growing primary and secondary follicles is 2-3 fold that of

normal ovaries, while the pool of primordial follicle is normal. The hyperandrogenism of PCOS is

designated as the main culprit for this follicle excess” (Jonard and Dewailly, 2004).

It has been reported that ethnicity also determines the ovarian morphology. Maximum ovarian

volumes and the number of follicles observed in a single plane on ultrasound were smaller in

Icelandic than Boston subjects with PCOS; and were even smaller in Icelandic control than

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Icelandic PCOS subjects. There was no difference in the proportion of ovaries in the Icelandic

and Boston PCOS groups, meeting the criteria for PCO morphology. However, there were more

normal ovaries in the Icelandic group when compared with

Boston group. (Farquhar et al.,1994; Carmina et al., 1997; Gilling -Smith, 1997)

Insulin resistance (IR) and hyperinsulinemia are common characteristics of PCOS; and are

associated with the pathophysiology of the condition (Diamanti-Kandarakis and Papavassiliou,

2006, Poretsky et al., 1999).

Evidence exists for altered adipocyte function in PCOS with altered secretion of adipokines such

as adiponectin. The high molecular weight (HMW) adiponectin has been hypothesized to

influence PCOS (Carmina et al., 2005). A large group of women with PCOS was compared with

BMI–matched controls. In order to explore the variables other than IR and BMI that may

regulate HMW adiponectin in PCOS. These authors reported that HMW adiponectin is selectively

decreased in women suffering from PCOS irrespective of BMI and insulin resistance (O’Connor et

al., 2010).

The diagnosis of PCOS is highly controversial (Azziz 2006, Franks 2006). Many authors over the

world have made attempts to evolve standard criteria to develop unanimity of diagnosis of PCOS

(Zawadzki and Dunaif, 1992; Rotterdam ESHRE/ASRM-sponsored PCOS Consensus Workshop

Group 2004; Azziz et al., 2006; Catherine et al., 2006). The study of plasma proteomics of PCOS

by 2-Dimensional Gel Electrophoresis (2-D PAGE) is becoming popular in establishing molecular

diagnosis of PCOS. This is on account of the fact that proteomic analysis of plasma provides

direct pictures of cellular activities and changes in various organs of the body including ovaries.

Moreover plasma or serum sampling is the easiest approach for proteomic analysis of PCOS

(Catherine et al., 2006). Further, plasma proteomics of PCOS can be of great help in getting each

individual’s molecular profile. It has been reported that plasma proteomic biomarkers of PCOS

are highly significant to understand the mechanism of genesis of PCOS. A biomarker explains and

indicates underlying pathological/inflammatory process (Biomarkers Definitions Working Group,

2001). According to some authors proteomic analysis of PCOS for molecular diagnosis is in very

preliminary stage; yet it has the potential to detect and explore proteins that can guide us to the

underlying pathology (Atiomio et al., 2009). Moreover, proteomic techniques applied to

investigate PCOS have revealed number of processes related to oxidative stress, inflammatory

changes and iron metabolism involved in the pathogenesis of features characteristic of PCOS

(Insensner and Escobar-Morreale, 2011). Neverthless, reproducibility of proteomic studies,

analysis of data its interpretation and biomarkers specific to PCOS is yet to be developed (Zhao

et al., 2008).

CHAPTER 3

MATERIALS AND METHODS

3.1 STUDY SAMPLE

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The study recruited a total of 204 subjects and patients. Fifty normal, healthy subjects with age

ranging 18-45 years were randomly selected as a reference population for comparative purposes

(Control). They were healthy, non-pregnant, non-lactating and non-smoking for the previous two

years and had regular menstrual cycles.

One hundred and five patients of PCOS were selected from the infertility clinics at Lady

Wallingdon Hospital, Lady Aitchison Hospital, Abdullah diagnostic Clinic and Private Community

based Family Planning Clinic in the city of Lahore. A third group comprising of 25 subjects who

were the first degree relatives (FDR) of the patients of PCOS, were also selected for investigating

hormonal profile and electrophoretic protein patterns. The participation rate was 77% in

controls (n=50), 62% in PCO patients (n=65) and 50% (n=25) in FDR of the patients. All of the

participants, i.e., controls, PCO patients and FDR of patients belonged to the middle socio-

economic status. The study proposal was approved by the Ethical Review Committee (ERC) of

King Edward Medical University and Mayo Hospital, Lahore.

The objective of the study was explained to each women (including controls, PCOS patients and

FDR of the patients), and a written consent was obtained. All of the participants were provided

with the results of all the investigations i.e., ovarian morphology (sonograms) records and

hormonal estimations.

A detailed history of past gynecological events of each patient was noted on a proforma devised

for this study (Annex I). History of hypertension, diabetes mellitus, menstrual irregularities,

weight gain, malignancy of genito-urinary tract was recorded. Detailed menstrual history

including date of onset of first menstrual period, last menstrual period, duration of length of

cycles, history of missed periods, or complete loss of cycles was recorded. Each woman was

examined for the presence of signs of acne and hair growth on the body and face region. Women

with hepatic, renal or with evident endocrine disorders, history of immunosuppresive therapy or

on any form of drug treatment were excluded from the study.

The ultrasound diagnosis of PCOS was made on the basis of ESHRE/ASRM criteria, 2004 i.e., 10 or

more cystic follicles/ovary, varying in size from 2-9 mm or ovarian volume of 10 cms in maximal

diameter.

The women were interviewed (history–taking) and examined for the signs and symptoms of

PCOS. The clinical diagnosis was based on history, general physical examination and trans-

abdominal in young, unmarried girls and vaginal ultrasound assessment in the married women.

The height and weight were recorded for calculation of body mass index. Each woman’s weight

(Kg) was recorded by digital weighing machine (Tanita weight balance, model:0170) and height

(in centimeters) were measured without shoes and light clothings in the presence of two female

doctors of the clinic at room temperature. The weight of average clothing was substracted from

the actual body weight of the controls, PCOS patients and FDR of women suffering from PCOS.

3.2 BLOOD SAMPLING

Blood sample (5 - 7 ml) of each patient was drawn from cubital vein puncture, with least possible

stress, by registered technicians; using good quality sterilized disposable syringes (Becton

Dickinson Private Limited). The blood samples were drawn at 10.00 a.m. to 11.00 a.m in the

morning considering the diurnal hormonal variations. The collected blood samples were

immediately transferred from the syringe to the centrifuge tubes after removal of the needle to

avoid possibility of haemolysis and allowed to coagulate at room temperature. Subsequently the

74

sera were separated by centrifugation (2000xg) for 5 minutes at room temperature with a

centrifuge (Model NF 1215, Nuve, Turkey). The collected sera were divided into multiple aliquots

and stored in the labeled plastic vials at 80oC until used for analyses.

3.3 ANALYTICAL PROCEDURES

Serum LH, FSH, insulin, testosterone, androstenedione, prolactin, growth hormone (GH) were

analysed by hormonal assay kits in all of the samples. Electrophoretic protein profiles were

determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS- PAGE) in blood

samples selected on the basis of hormonal variations. The hormonal analyses were performed at

radio-immuno-assay (RIA) laboratories, Centre for Nuclear Medicine (CENUM), Mayo Hospital,

Lahore. The protein profiles of controls, PCOS patients and FDRs were performed at University of

the Punjab. Serum androstenedione and testosterone were determined by competitive

radioimmunoassay (RIA) while serum LH, FSH, prolactin, insulin and GH were determined by a

‘sandwich type’ immunoradiometric assay (IRMA) by using commercial kits of Immunotech Inc.

(Beckman Coulter, Czech Republic and France).

Commercially derived control sera of low, medium and high concentrations were included in

every run for determination of assay reliability. All assays were carried out in duplicate, and were

performed in batches to eliminate variability within assays, and RIA and IRMA results were

expressed at less than 10% cumulative variation (CV) of imprecision profile. The electrophoretic

protein profiles were studied by using the method of Laemli (1970) for one dimensional and

Garfin (2003) for two dimensional gel electrophoresis.

3.4 HANDLING OF SAMPLES

• All precautionary measures were taken considering as the serum samples were potentially

capable of transmitting hepatitis or AIDs. All waste was discarded.

• Eating, drinking, smoking and use of cosmetics were not allowed during the process of

sample handling. The pipeting of radio-active material/solutions was strictly not done by

mouth. The gloves and lab overalls were used to avoid contact with radio-active material.

3.5 STATISTICAL ANALYSIS

Statistical analysis was performed using Graph Pad Prism version 5.00 for windows Graph Pad

software, San Diego, California, USA. Data obtained from each parameter of the study was

presented as mean±SEM. The data was analysed statistically using one-way analysis of variance

(ANOVA). Tukey’s Post-Hoc Multiple comparison test was employed for comparing variations

amongst different groups of the study.

75

HORMONAL ASSAYS

3.6 MEASUREMENT OF LH, FSH, INSULIN, PROLACTIN AND GROWTH

HORMONE

These hormones were measured by immune-radiometric assay (IRMA) with the help of

respective kits provided by Immunotech SAS (Czech Republic) on gamma camera model PC-

RIA.MAS (Stratec Inc, Germany).

3.6.1 Principle The immuno-radio-metric assay (IRMA) is a sandwich-type assay. The principle of the assay is to

use mouse monoclonal antibodies directed against two different epitopes of respective

hormones. Samples or calibrators are incubated in tubes, which are coated with the first

monoclonal antibody in the presence of the second 125I-labeled monoclonal antibody. The

contents of tubes were aspirated and rinsed after incubation and finally the bound radioactivity

was measured. The values of relevant hormonal estimations were calculated by interpolation

from the standard curve. The radioactivity bound is directly proportionate to the concentrations

of concerned hormones in the samples.

3.6.2 Reagents provided The following reagents were provided in the kits used for hormonal analysis:

Anti-LH monoclonal antibody coated tubes: 2 X 50 tubes, 125I-labeled monoclonal antiLH, FSH,

Insulin, Prolactin and Growth hormone antibodies for each hormone: one 5.5 ml vial.The vial

contains 370 KBq (Kilo Buequral), at the date of manufacture, of 125I-labeled immunoglobulins in

buffer with bovine serum albumin, sodium azide and a dye. Calibrators: six vials, the calibrator

vials contain from 0 - 180 IU/L of LH in horse serum with sodium azide. The exact concentration is

indicated on each vial.Control serum: one vial, the vial contains relevant hormones lyophilized in

human serum.Wash solution: one 50 ml vial, concentrated solution has to be diluted before use.

All reagents of the kit were stored at 2-8°C.

3.6.3 Preparation of reagents All reagents were allowed to acquire the room temperature. The calibrators and control serum

were reconstituted with distilled water according to the instruction on the label. After 30 minutes

of reconstitution, the contents of the vials were mixed gently and stored at 2-8°C. The wash

solution was prepared by pouring the contents of the vial into 950 ml of distilled water. It was

homogenized and stored at 2-8°C.

3.6.4 Assay procedure:

Series of antibody coated tubes for the respective hormones were numbered in duplicate. A

measured quantity i.e. 100 ml of calibrator control or sample was added in respective tubes. 50 µl

76

of the tracer was added to each tube and contents were thouroughly mixed. Two tubes were

prepared separately containing only 50 µl of tracer in order to obtain total cpm (T). The tubes

were incubated at 18-25°C while shaking ( at >350 rpm) for 90 minutes. The contents of the tubes

were aspirated carefully and washed with 2 ml of wash solution twice except the 2 tubes for total

cpm.

The radioactivity of calibrator, control and each sample tube (bound counts) and separated 2

tubes (total counts) was counted for 1 minute by gamma counts. The results were recorded, as

obtained from the standard curve by interpolation in case of each hormone i.e. LH, FSH, Insulin,

Prolactin and Growth hormone. The standard curve serves to determine the concentration of

each hormone in samples measured at the same time as the calibrators.

3.7 TESTOSTERONE AND ANDROSTENEDIONE

These two hormones were measured by Radio-Immuno-Assay (RIA) with the help of RIA

testosterone and androstenedione kits provided by Immunotech SAS (France) on gamma camera

model PC-RIA.MAS (Stratec Inc, Germany).

3.7.1 Principle Testosterone and androstenedione were measured by radio-immuno-assay which is a

competitive assay. The samples or calibrators are incubated with 125I-labeled testosterone and

androstenedione respectively in antibody-coated tubes. The liquid contents of tubes were

aspirated after incubation, and the bound radioactivity was determined with a gamma counter.A

standard curve is drawn and unknown values are obtained from curve by interpolation.

3.7.2 Reagents used • Anti-testosterone and anti-androstenedione antibody-coated tubes: 2 X 50 tubes for each

hormone, 125I-labeled testosterone tracer: one 55 ml vial.The vial contains 185KBq (Kilo

Bacqural) of 125I-labeled testosterone in liquid form containing gelatin and a dye.

• Calibrators: six 0.5 ml vials.The calibrator vials contain from 0 - 20 ng/ml ( 0 - 69 nM) of

testosterone and androstenedione from 0-15 ng/ml (0-52 nM) of ∆4androstenedione in

buffer with bovine serum albumin and sodium azide respectively.

• Human serum with sodium azide.

77

• Control serum: one 0.5 ml vial

All reagents of the kit were stored at 2-8°C.

3.7.3 Assay procedure All reagents and samples were allowed to acquire room temperature used for the

measurement of testosterone and androstenedione. The series of antibody coated tubes

were numbered in duplicate. 50 µl of calibrator, control or sample was added in

respective tubes. 500 µl of tracer was added to each tube and was thoroughly mixed.Two

tubes were prepared separately containing only 500 µl of tracer in order to obtain total

cpm (T). Tubes were covered and incubated at 37°C in water bath for 3 hours. The

contents of the tubes were aspirated carefully except the 2 tubes separately prepared for

total cpm (T). The radioactivity of each calibrator, control and sample tubes (bound

counts) and separately prepared two tubes (total counts) was counted for 1 minute by

gamma camera.The results were obtained from the standard curve by interpolation. The

standard curve was employed to determine the concentrations of testosterone and

androstenedione in samples as well as the calibrators.

3.8 ONE DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS Taking into account the importance of Gel Electrophoresis in the detection of proteins that may

be associated with patients of PCOS, SDS-PAGE was performed.

Serum protein fractions were analyzed by SDS-PAGE using the method of Laemmli (1970). The gel

is prepared by polymerizing acrylamide (CH2=CH.CO.NH2) and a small quantity of cross linking

reagent, methylene-bisacrylamide (CH2=CH.CO.NH2). CH2 (bis), in the presence of a catalyst

ammonium persulfate. Tetramethyl-ethylene diamine (TEMED) is also present to initiate and

control the polymerization. Sodium dodecyl sulfate [CH3(CH2)10CH2OSO-3Na+] abbreviated as SDS

is added as a detergent that readily binds to proteins. At pH 7.0, in the presence of 1% W/V SDS

and dithiothreitol (DTT), proteins dissociate into their subunits and bind large quantities of the

detergent. This binding masks the natural charge of the protein giving a constant charge to mass

ratio. The larger the molecule, the greater is the charge. Thus the electrophoretic mobility of the

complex depends on the size (molecular weight) of the protein. Molecular weights of unknown

proteins can therefore are determined by comparing their mobilities with a series of protein

standards.

The sieving effect of polyacrylamide is important in this technique and the range of

molecular weights that can be separated on a particular gel depends on the pore size of the gel.

The amount of cross linking and hence the pore size in a gel can be varied by simply altering the

amount of acrylamide-bisacrylamide solution to make different percentages of gels.

3.8.1 Reagents Preparation

Acrylamide-Bis-acrylamide (30%) After weighing 29 g of acrylamide and 1 g of bis-acrylamide in a flask and dissolved in distilled

water with magnetic stirrer. The volume was made up to 100 ml. It was stored in a reagent bottle

at 4oC, and wrapped with aluminium foil to avoid exposure to light thus preventing any reaction.

1.5 M Tris-HCl (pH 8.8)

78

Then 18.17 g of trizma base [Tris (hydroxymethyl) amino methane] was measured in a flask and

dissolved in distilled water on a magnetic stirrer. The pH was adjusted to 8.8 with 1 N HCl. The

final volume was made up to 100 ml by adding distilled water and was stored at 4oC in a reagent

bottle.

1.0M Tris-HCl (pH 6.8) In order to prepare 1.0M Tris-HCl, 12.11 g of trizma base was weighed and dissolved in distilled

water on a magnetic stirrer. The pH was brought to 6.8 with 1 N HCl. Final volume was made up

to 100 ml and stored at 4oC in a reagent bottle.

SDS (10%) Ten gram (10 g) of SDS was weighed and dissolved in distilled water on a magnetic stirrer. The

final volume was made up to 100 ml and stored in a reagent bottle at room temperature.

Electrophoresis Buffer Took 3.02 g of Tris, 18.8 g of glycine and 1 g of SDS and dissolved in distilled water. The volme

was made up to 1 liter and stored at room temperature.

Fixative Solution In order to make fixative solution 30 ml of absolute ethanol, 10 ml of glacial acetic acid were

taken in a graduated cylinder and 60 ml of distilled water was added to it. Final volume was thus

made up to 100 ml. Colloidal Coomassie Staining Solution

To prepare Colloidal Coomassie Staining Solution (CCSS) 100 g of ammonium sulfate

(NH4)2SO4 was dissolved in 200 ml distilled water usig magnetic stirrer. Then 100 ml of 85%

orthophosphoric acid was added in ammonium sulfate solution then 1.2 g of Coomassie Brilliant

Blue G-250 was added to it. The solution thus prepared was thoroughly dissolved on magnetic

stirrer for one hour. The final volume was made up to 800 ml with distilled water and stored in a

reagent bottle at room temperature. Methanol was added in 1:4 ratios to stain the gel.

Destaining Solution A measured amount of 50 ml methanol and 70 ml of acetic acid was taken in a graduated cylinder

and distilled water was added to make the final volume up to 1 litre. The solution was stored

after mixing at room temperature.

Low Molecular Weight Markers For 12% Gel PageRuler™ Unstained Protein Ladder (Fermentas, Catalog#SM0661 (5μl) was used as standard.

It was a mixture of 14 recombinant, highly purified proteins from 10 kDa to 200 kDa.

Approximately 0.02-0.05 mg/ml of each protein was present in the following storage buffer: 62.5

mM Tris-H3PO4 (pH 7.5 at 25°C), 1 mM EDTA, 2% SDS, 0.1 M DTT, 1 mM NaN3, 0.01%

bromophenol blue and 33% glycerol. It was stored at -20°C.

Gel Assembly Mini protean tetra electrophoresis cell (Bio-Rad, Catalog#165-3301) was used to run the SDS-

PAGE with PowerPacTM HV Power Supply (Bio-Rad, Catalog#164-5056). Casting frame was

placed upright with the pressure cams in the open position and facing forward on a flat surface. A

short plate was placed on top of the spacer oriented upside. The two glass plates were slided into

the casting frame with short plate facing the front of the frame. When the glass plates were in

place, pressure cams were engaged to secure the glass cassette sandwich in the casting frame.

79

Casting frame was placed in the casting stand by positioning the casting frame onto the casting

gasket while engaging the spring-loaded lever of the casting stand onto the spacer plate.

Casting Polyacrylamide Gel The glass plate was marked at the level at which the resolving gel is to be poured and a comb was

placed completely into the assembled gel cassette and marked 1cm below the comb teeth.

Resolving gel monomer solution was prepared by combining all reagents except APS and TEMED

which were added at the time of pouring. After adding APS and TEMED, solution was poured to

the mark using disposable plastic pipette. Immediately, monomer solution was overlaid with

water; added slowly to prevent mixing. Gel was polymerized within 45 minutes to1 hour. Gel

surface was rinsed completely with distilled water and dried the top of the resolving gel with

filter paper before pouring the stacking gel. Stacking gel monomer solution was prepared and

poured between the glass plates until the top of the short plate was reached. Comb was inserted

between the spacers and stacking gel was allowed to polymerize for 30-45 minutes. After

polymerization, comb was removed and wells were rinsed thoroughly with distilled water or

running buffer.

Electrophoresis Module Assembly Clamping frame was set to the open position on a clean flat surface and gel cassette was placed

(with the short plate facing inward) onto the gel support at an angle of 30º tilting away from the

center of clamping frame. Second gel was placed on the other side of the clamping frame and

both gels were gently pulled towards each other creating a functioning assembly. Electrode

assembly was used to run two gels simultaneously. Gel Buffer System

In this experiment, discontinuous buffer system was used i.e. different buffer ions were present

in the gel and electrode reservoirs. Proteins were denatured by heating in buffer containing SDS

and DTT. The resultant denatured polypeptides appeared as a rod-like shape and a uniform

charge-to-mass ratio proportional to their molecular weights.

Placement of Electrode Assemblies in Mini-Protean Tetra Tank Electrode assembly was placed in the back position of the cell and lower chamber of the tank was

filled with buffer to the indicated level (550 ml for 2 gels and 680 ml for 4 gels).

Required total buffer volume in the upper and lower chamber was 700 ml for 2 gels.

Sample Loading Upper chamber of the assembly was filled with buffer up to just under the edge of the outer gel

plate. Samples were loaded slowly to settle evenly on the bottom of the wells with a Hamilton

syringe or a pipette using gel loading tips.

Mini-Protean Tetra Tank Assembly Mini-protean tetra tank lid was placed and aligned with the color coded banana plugs and jacks.

The correct orientation was made by matching the jacks on the lid with the banana plugs on the

electrode assembly. At this point, lid was firmly pressed down with thumbs using even pressure,

till the lid securely and tightly positioned on the tank.

Power Conditions Power was applied to the mini-protean tetra cell through PowerPacTM HV power supply. The

process of electrophoresis was started and gel was run at 60 V for 1 hour for best stacking. The

80

voltage was increased to 120 V for resolving gel. For mini protean tetra cell, the minimum run

time was approximately 2:30 hours for 12% SDS-PAGE.

Gel Removal After electrophoresis was completed, power supply was turned off and electric leads were

disconnected. Tank lid was removed and electrode assembly was lifted carefully. The running

buffer was poured and the arms of the assembly were opened to remove the gel cassettes. The

gel was removed from gel cassette by separating the two glass plates.

Finally the gel was picked carefully and placed in the fixative solution.

Fixation

Gel was fixed in fixative solution for 4 hours to overnight.

Staining After fixation, gel was transferred to staining solution. Then methanol was added afresh to the

staining solution for characteristic granules formation. The gel was stained overnight with

constant agitation on an orbital shaker.

Destaining After staining the gel, it was transferred to destaining solution. Once the gel was destained with

constant agitation, a clear background became visible. Consequently, the protein fractions

appeared to the naked eye in the form of blue colored bands.

Image Storing and Photography Once the protein fractions appeared in the form of blue coloured bands, the gel was

photographed and its image was saved with Gene Genius Bio-imaging gel documentation system.

Quantification of Protein Fractions Total Lab Quant that provided the data of molecular weight and density of each protein fraction

was used to quantify the separated proteins. Ultimately, the band percentage displayed by each

protein fraction was recorded.

Statistical Analysis The protein fractions in the gels were presented as Mean±SEM (standard error of mean).

The data obtained was analyzed statistically using one-way analysis of variance (ANOVA). P-

values of less than 0.05 (p< 0.05) were considered statistically significant.

3.9 TWO DIMENSIONAL GEL ELECTROPHORESIS (2-DE)

The samples were further analyzed through two-dimensional gel electrophoresis to determine

the expression of protein fractions that could employ as biomarkers. The technique consisted of

two steps, first iso-electric focusing (IEF) and second SDS-PAGE. In IEF, soluble proteins were

81

separated according to their iso-electric point (pI) and then migrated into the SDS-PAGE where

they resolved according to their molecular weights.

The experimental sequence of 2-Dimensional Electrophoresis (2-DE) is as under:

1. Sample preparation

2. IPG strip rehydration

3. Iso-electric focusing

4. IPG strip equilibration

5. SDS-PAGE

6. Visualization

7. Analysis

3.9.1 Reagents Preparation

Rehydration Buffer

Description Final Concentrations Amount Urea 7 M 4.20 g Thiourea 2 M 1.52 g CHAPS 1.2% (w/v) 0.12 g DTT 20 mM 0.031 g Ampholyte (Bio-Lyte 3/10

ampholyte, 100X, 1 ml) 0.25% (v/v) 25 μl

Bromophenol blue 0.005% (w/v) 0.005 g Deionized water up to 10 ml

It was stored in 2 ml aliquots at -20°C.

Equilibration buffer

Description Final Concentrations Amount Urea 6 M 18.02 g Tris-HCl, pH 8.8 50 Mm 2.5 ml SDS 2% (w/v) 1 g Glycerol 30% (v/v) 15 ml Bromophenol blue 0.002% (w/v) 5 mg Final volume was made up to 50 ml with deionized water.

SDS-PAGE Equilibration Buffer 1 (with DTT) Equilibration buffer 1 was made by dissolving 0.08 g DTT in 4 ml of equilibration buffer achieving

final 2% (w/v) DTT concentration. SDS-PAGE Equilibration Buffer 2 (with IAA)

Equilibration buffer 2 was made by dissolving 0.1 g IAA in 4 ml of equilibration buffer achieving

final 2.5% (w/v) IAA concentration.

82

0.5% Agarose Sealing Solution Description Concentration Amount SDS electrophoresis

buffer 1X (0.302 g Tris, 1.88 g

glycine and 0.1 g SDS

in 100 ml deionized

water)

100 ml

Agarose 0.5% 0.5 g Bromophenol blue 0.002% (w/v) 200 μl

Agarose and bromophenol blue were dissolved in Tris-Glycine-SDS buffer on heating stirrer. The

solution was not allowed to boil over. It was stored at room temperature.

3.9.2 ISO-ELECTRIC FOCUSING (IEF) The first-dimension separation procedure involved the following steps:

Sample preparation

IPG strip rehydration

Sample application

Iso-electric focusing

As the IPG strips were provided dry, rehydration was required with appropriate additives prior to

IEF. For a well-focused first-dimension separation, sample proteins were completely

disaggregated and fully solubilized. IEF was performed on a flat-bed system at very high voltages

with active temperature control. Strip was rehydrated in PROTEAN® IEF focusing tray (Bio-Rad,

Catalog#163-4010). The ReadystripTM IPG strip(Bio-Rad, Catalog#163-2007) with a strip length of

17 cm (strip length 17.8 cm, gel length 17.1 cm, strip width 3.3 mm and gel thickness 0.5 mm) and

linear pH interval of 3-10 was used for an overview of total protein distribution. The PROTEAN®

IEF cell (Bio-Rad, Catalog#165-4000) was programmed for active rehydration and transition

automatically to focusing run.

Sample Preparation Plasma sample was thawed; vortexed and protein concentration was determined by

Bradford assay. Protein concentration of 250 μg (sample volume 300 μl) was loaded on 17 cm IPG

strip for proper protein separation.

IPG Strip Rehydration and Sample Application The sample was prepared in rehydration buffer. The sample volume of 300 μl was dispensed as a

line along the periphery of a channel in an IEF focusing tray. After protein sample had been

loaded, the cover sheet from the IPG strip was peeled off using forceps and strip was gently

placed with gel side down onto the sample in IEF focusing tray so that the acidic (marked with

“+”) end was at the anode of the IEF cell. It was ensured that the gel made contact with the

electrodes and no air bubble was trapped beneath the IPG strip. The IPG strip was soaked with

samples at room temperature and the mineral oil (2-3 ml) was overlaid on the strip to prevent

evaporation during rehydration process. The focusing tray was covered with lid and placed in the

PROTEAN IEF cell. Rehydration was done under active condition at 50 V for at least 12 hours.

Focusing conditions were set according to sample composition, complexity and IPG pH range.

83

Following rehydration, the voltage was maintained at 250 V for 15 minutes (S1) and rapidly

attained 10,000 V in three hours (S2). During Step 3, the voltage was linearly increased for

another 3 hours attaining the final 40-60000 V/hr, determining the ramp of the process.

After IEF had been completed, the IPG strip was removed from the focusing tray and transferred

with gel side up into a new clean, dry disposable equilibration tray according to the length of the

IPG strip. The IPG strip was held vertically with forceps to let the mineral oil be drained for at

least 5 seconds before transfer.

3.9.3 SECOND DIMENSION/SDS-PAGE SDS-PAGE for second dimension consisted of four steps:

Assembling the gel plates and preparation of the gel

Equilibrating the IPG strip in SDS equilibration buffer

Placing the equilibrated IPG strip on the SDS gel

Electrophoresis

Glass Plates Assembling PROTEAN II XL Cell (Bio-Rad, Catalog#165-3188) was used for second dimension SDS-PAGE. The

longer glass plate was laid down and two spacers of equal thickness were placed along the

rectangular plate. The shorter glass plate was placed on top of the spacers so that the bottom

ends of the spacers and glass plates were aligned. The screws on the clamp assembly were

loosened and glass plate sandwich was gently slided into the clamp assembly. The screws of the

clamp assembly were tightened and clamp assembly was placed into the alignment slot of the

casting stand. The screws were loosened to allow the plates and spacers to set firmly against the

casting stand base. All the screws were gently tightened and clamp assembly was transferred to

one of the casting slots in the casting stand.

Preparation and Casting the Gel By using the following reagents, 50 ml of 12% resolving gel was prepared.

12% Resolving Gel

Distilled Water (ml)

30% AcrylamideBisacrylamide

(ml)

1.5M Tris (pH 8.8) (ml)

10% SDS

(μl)

10% Ammonium persulfate

(APS) (μl)

TEMED

(μl)

Final

volume

(ml)

16.5 20 12.5 500 500 20 50

The gel mixture was poured into the assembled glass plates on its top, avoiding air bubbles and a

comb was inserted in the gel. The gel was allowed to polymerize for one hour. The comb was

removed and gel surface was rinsed with de-ionized water.

IPG Strip Equilibration The IPG strip was equilibrqted in SDS equilibration buffers prior to the second dimension. The

two step equilibrations ensured the reduction and alkylation of cysteine which

minimized/eliminated vertical streaking. The equilibration buffer 1 and 2 were prepared with 2%

84

DTT and 2.5% IAA respectively. Mineral oil was removed from the IPG strip by placing it (the gel is

upside) on to a piece of dry filter paper and blotting with a second piece of wet filter paper. The

equilibration buffer 1 was placed in an equilibration tray and the blotted IPG strip was transferred

(gel side should be up) in it. The strip was equilibrated with DTT for 10 minutes with constant

shaking. IPG strip was blotted after incubation, holding vertically and transferred to equilibration

buffer 2 and the tray was returned to orbital shaker for 10 minutes. During the process of

incubation, 0.5% agarose solution was melted in a microwave oven. At the end of incubation

period, strip was blotted again and was washed briefly by dipping in 100 ml glass cylinder with 1X

TGS buffer.

Transferring the Strip on SDS-PAGE Excess of buffer was blotted from equilibrated IPG strip and the strip was placed on the PAGE

with the plastic side towards the back plate. The IPG strip was carefully pushed into the well by

using forceps, avoiding any air bubbles beneath the IPG strip. Agarose solution was overlaid into

the IPG well over the strip and allowed to solidify for 5 minutes. The gel was mounted into the

electrophoresis cell and marker proteins (15 to 20

μl) were loaded on the “+” end of the IPG strip.

Electrophoresis The reservoir was filled with 1X TGS running buffer and lid was placed on the electrophoresis cell;

and power was turned on. The migration of the bromophenol blue, present in the overlay

agarose solution, was used to monitor the progress of the electrophoresis run. The gel was

initially run at 16 mA for one hour and increased to 35 mA for the rest of gel.

Fixation Following the completion of electrophoresis, the electric supply was switched off and gel

assembly was lifted out of the chamber. The plates forming the gel assembly were carefully

separated from each other with the help of spatula. The gel was carefully picked up and kept in

fixative solution overnight.

Staining The staining solution was prepared by mixing 80 ml of staining solution and 20 ml of methanol.

Subsequently the gel was kept in it after fixation. Characteristic granules were appeared. Gel was

stained overnight with constant shaking.

Destaining The stained gel was shifted to to the destaining solution and destained with constant agitation

until protein spots became visible with a transparent background.

Analysis The gels were analyzed through Ludesi REDFIN and Swiss 2-D page database. Different proteins

were identified in patients of PCOS and their expression was compared with controls group.

CHAPTER 4

85

RESULTS

Table 4.1 presents the analysis of basic parameters in age-matched groups comprising of normal

control subjects (n=50), patients of PCOS (n=65) and first degree relatives (FDR, n=25) of PCOS

patients. Significant alterations were observed in comparable groups as indicated by one way

ANOVA at P<0.0001 and confidence interval of 99.9% (Table 4.2).

For multiple comparisons Tukey’s post-HOC analysis was performed.

4.1 BODY MASS INDEX (BMI)

The body mass index in the control group had a mean value of 20.81±0.42 Kg/m2 The BMI in PCO

group was significantly high and showed a 26% rise (26.23±0.55) as compared with control group

(P<0.0001 and CI of 99.9%). The FDR group depicted 23% significant decrease (20.11±0.43) in the

BMI as compared with PCO group (P<0.0001 and CI of 99.9%). There was no significant difference

in the control Vs FDR group (Fig. 4.1).

Fig. 4.1. Average body mass index (kg/m2) of comparable groups. *** Significance at P<0.001 (CI 99.9 %) in comparison to control *** Significance at P<0.001 (CI 99.9 %) in comparison to PCO

4.2 HORMONAL ANALYSES

4.2.1 LUTEINIZING HORMONE (LH)

The average levels (mean± SEM) of LH in the control, PCO and FDR groups were 6.39±0.88,

38.11±3.11 and 5.09±0.69 respectively. The LH was elevated significantly in the PCO group as

compared with control group indicating 60% rise (P<0.0001 and CI of 99.9%).The LH level fell

significantly in the FDR group demonstrating 87% decrease compared with PCO group (P<0.0001

and CI of 99.9%).The FDR group LH values were insignificant as compared with normal control

group (Fig. 4.2).

86

Fig. 4.2. Average levels of luteinizing hormone in comparable groups. *** Significance at P<0.001 (CI 99.9 %) in comparison to control

*** Significance at P<0.001 (CI 99.9 %) in comparison to PCO

4.2.2 FOLLICLE STIMULATING HORMONE (FSH)

The mean FSH values were 5.54± 0.38, 3.74± 0.25, and 3.62± 0.27 in the control, PCO and FDR

groups respectively. The FSH estimations were significantly lowered in PCO and FDR groups as

compared with controls. There was 33% fall in PCO group and 35% fall in the FDR group

respectively (P<0.0001 and CI of 99.9%) in the FSH values (Fig.

4.3).

Fig. 4.3. Average levels of follicle stimulating hormone in comparable groups. *** Significance at P<0.001 (CI 99.9 %) in comparison to control

** Significance at P<0.01 (CI 99.9 %) in comparison to PCO

4.2.3 LH: FSH RATIO

87

The ratio of LH: FSH was observed to be 1.12± 0.11, 21.53±5.83 and 1.68± 0.26 in the control,

PCO and FDR groups respectively. The LH: FSH ratio in PCO Vs controls was

Fig. 4.4. Average levels of LH/FSH ratio in comparable groups. ** Significance at P<0.01 (CI 99.9 %) in comparison to control * Significance at P<0.05 (CI 95 %) in comparison to PCO

observed to be significantly higher (P<0.01 and CI of 99%). The FDR group Vs PCO group also

showed significantly lower ratio (P<0.05 and CI of 95%).The ratio of LH: FSH was non-significant in

the FDR Vs control group (4.4).

4.2.4 INSULIN

The estimated mean levels of insulin were 7.22±0.61 in the control group, 43.48±4.09 in the PCO

patients and 4.82±0.46 in the FDR group. The insulin in the PCO Vs control was significantly

elevated. The PCO group manifested a very high mean value of 43.48± 4.09 indicating five-fold

rise in this group having (P<0.001 and CI of 99.9%). The FDR group showed a significant fall

(P<0.001 and CI of 99.9%) depicting 89% decrease compared with PCO group. The FDR group Vs

control group demonstrated no significant difference (Fig. 4.5).

Fig. 4.5. Average levels of insulin in comparable groups.

Control PCO FDR 0

10

20

30 **

*

Groups

Control PCO FDR 0

10

20

30

40

50 ***

* ***

Groups

88

*** Significance at P<0.001 (CI 99.9 %) in comparison to control *** Significance at P<0.001 (CI 99.9 %) in comparison to PCO

* Significance at P<0.05 (CI 95 %) in comparison to PCO

4.2.5 TESTOSTERONE

Testosterone estimations in control, PCO and FDR groups were found to be 0.64±0.04, 1.19±0.05

and 0.38±0.04 respectively. The testosterone levels in the PCO group were highly significantly

raised, depicting 86% elevation as compared with control group (P<0.001 and CI of 99.9%). There

was 68% decrease in the testosterone values in the FDR group in comparison with PCO group.

This indicated 96% significant fall (P<0.001 and CI of 99.9%) in the FDR group. The testosterone

levels also showed a significant fall in the FDR group in comparison with control group (P<0.01

and CI of 99%). It demonstrated 40% decrease in the testosterone (Fig. 4.6).

Fig. 4.6. Average levels of testosterone in comparable groups. *** Significance at P<0.001 (CI 99.9 %) in comparison to control *** Significance at P<0.001 (CI 99.9 %) in comparison to PCO **

Significance at P<0.01 (CI 99.9 %) in comparison to PCO

4.2.6 ANDROSTENEDIONE

The average androstenedione estimation was found to be 2.11±0.09, 5.55±0.28 and 1.29±

0.17 in the control, PCO and FDR groups respectively. The androstenedione was greatly

Control PCO FDR 0.0

0.5

1.0

1.5

* **

* * ***

Groups

89

Fig. 4.7. Average levels of androstenedione in comparable groups. *** Significance at P<0.001 (CI 99.9 %) in comparison to control


elevated in the PCO group as compared to the controls indicating a very highly significant rise of

163% (P<0.0001 and CI of 99.9%). There was significant decrease of 76% (P<0.0001 and CI of

99.9%) in the FDR group as compared with PCO group. There was no significant difference in

normal controls and FDR group (Fig. 4.7). A significant correlation was observed between LH and

androstenedione (P<0.05 and CI of 95%).

4.2.7 GROWTH HORMONE (GH)

The mean GH estimations were found to be 3.20±0.52, 3.46±0.83 and 1.86±0.37 in the control,

PCO and FDR groups respectively. These values in the controls Vs PCO group were non significant

at 11%. Similarly mean values in FDR Vs PCO and FDR Vs control groups were non-significant

respectively at 46% and 42% (Fig. 4.8).

Groups

Fig. 4.8. Average levels of growth hormone in comparable groups.

4.2.8 PROLACTIN

Control PCO FDR 0

2

4

6

8

* **

***

Groups

90

The prolactin measurements revealed to be 3.99±0.51, 9.75±0.71 and 2.71±0.22 in the control,

PCO and FDR groups. The values of prolactin in control Vs PCO group was significantly raised in

PCO patients indicating 45% elevation of prolactin in this group(P<0.001 and CI of 99.9%). The

FDR group showed significantly higher values of prolactin Vs controls manifesting 48% rise (Fig.

4.9).

Fig. 4.9. Average levels of prolactin in comparable groups. *** Significance at P<0.001 (CI 99.9 %) in comparison to control


4.2.9 CORRELATION OF LH WITH TESTOSTERONE AND ANDROSTENEDIONE

The LH and testosterone correlation was found to be non-significant (r=0.068, P=0.637

NS). The LH and androstenedione was highly significantly correlated (r=0.375, P=0.007,CI of 99%).

91

Table 4.1. Summary statistics of categorical variables in different cohorts of study. Variables Control group PCOS group FDR group

Mean age (yrs) 19.67 26.71 19.86

Amenorrhea (%) Nil 75.38 Nil

Acne (%) 18.00 32.31 16.00

Hair growth (%) 10.00 63.08 05.71

Obesity (%) 06.00 81.54 08.00

Hypertension (%) Nil 10.77 Nil

Mean age of menarche (yrs) 13.05 13.85 16.00

Family H/O infertility (%) Nil 26.15 100.0

Family H/O hypertension (%) 34.00 44.62 36.00

Family H/O diabetes (%) 32.00 32.31 32.00

FDR First degree relatives of PCOS patients. H/O History of.

92

Table 4.2. BMI and hormonal variables in control group, PCOS patients and their first degree

relatives (FDR). Variables Controls PCOS group FDR group P Value

BMI (kg/m2) 20.81±0.42 26.23±0.55* 20.11±0.43∆ P<0.001

LH (IU/L) 6.39±0.88 38.11±3.11* 5.09±0.69∆

P<0.001

FSH (IU/L) 5.54±0.38 3.74±0.25* 3.62±0.27* P<0.001

LH:FSH 1.12±0.11 21.53±5.83* 1.68±0.26∆ P<0.01

Insulin (µIU/mL) 7.22±0.61 43.48±4.09* 4.82±0.46∆ P<0.001

Testosterone (ng/mL) 0.64±0.04 1.19±0.05* 0.38±0.04*∆ P<0.001

Androstendione (ng/mL) 2.11±0.09 5.54±0.28* 1.28±0.16∆

P<0.001

Growth hormone(mIU/L) 3.20±0.52 3.46±0.83 1.86±0.37 P>0.05

Prolactin (ng/mL) 3.99±0.51 9.75±0.71* 2.71±0.22∆

P<0.001

* Significant in comparison to control group ∆ Significant in comparison to PCOS group Values are mean ± SEM

4.3 ONE DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS

The electrophoretic results in the form of appearance/disappearance of protein fractions in

healthy subjects and PCOS patients are presented (Fig. 4.10-4.29). Band percentages expressed

by each of the fractions are analyzed in comparable subjects (Tables 4.3).

93

4.3.1 CONTROL GROUP

An overall view of protein profile resulted in the detection of seven high molecular weight

protein fractions ranging between 181-13kDa.

Among these, protein fractions of 181kDa (ranging between 3.88-6.74%) was first in expression.

Its band percent was 6.74, 5.59, 6.60, 5.74, 6.10, 3.88, 4.52 % in subject no. 18 (Fig. 4.10).

Protein fraction of 131kDa (ranging between 1.04-3.67%). This fraction exhibited a band percent

of 1.19, 1.87, 1.04, 1.90, 1.95, 1.81, 3.67% in subject no. 1-8 (Fig. 4.11).

Protein fraction of 100kDa (ranging between 3.53-6.46%) was next in expression. In subject no. 1-

8, the fraction covered 5.32, 6.83, 6.54, 7.28, 6.04, 6.04, 5.92 band percent, respectively (Fig.

4.12).

Protein fraction of 80kDa (ranging between 7.24-9.86%) was next to 100kDa in expression. This

fraction exhibited a respective band percent of 9.15, 7.24. 8.52, 8.36, 8.43, 9.00, 9.86, in subject

no. 1-8 (Fig. 4.13).

Protein fraction of 66kDa (ranging between 3.41-5.45%) was next in expression. This protein

fraction exhibited band percentage of 35.66, 33.74, 36.65, 39.74, 37.89, 35.77, 38.88% in subject

no. 1-8 (Fig. 4.14).

51kDa protein fraction (ranging between 11.32-16.02%) was next in expression. This fraction

covered band percent of 11.32, 16.02, 15.40, 18.46, 15.99, 14.51, 11.65% in subject no.1-8 (Fig.

4.15).

Protein fraction of 32kDa (ranging between 4.70-9.98%) was next in expression. It covered 8.99,

4.70, 8.38, 7.73, 6.05, 7.06, 9.98 fraction percent in subject no. 1-8, respectively (Fig. 4.16).

Protein fraction of 29kDa (ranging between 3.10-4.63%) was next in expression. This fraction

covered 4.50, 3.10, 4.48, 3.99, 3.33, 3.11, 4.63 band % in subject no. 1-8 (Fig. 4.17).


covered 15.46, 15.73, 14.51, 14.98, 15.91, 15.47 band % in subject no. 1-8 (Fig. 4.18).

Protein fraction of 17kDa (ranging between 3.38-5.87%) was next to 23kDa in expression. This

fraction covered 4.01, 5.87, 4.98, 3.38, 5.78, 4.37, 4.09 respective band % in subject no. 1-8 (Fig.

4.19).

Protein fraction of 15kDa (ranging between 0.83-1.61%) was next in expression. It covered,

respectively, 1.37, 1.35, 1.52, 1.55, 1.61, 1.20, 0.83 band % in subject no. 1-8 (Fig. 4.20).

Protein fraction of 13kDa (ranging between 0.97-1.59%) was next in expression. Its fraction

percent was 1.30, 1.13, 0.97, 1.59, 1.37, 1.33, 1.52, 1.13 in subject no. 1-8, respectively (Fig.

4.21).

4.3.2 PCOS PATIENTS GROUP

The serum protein profile analysis in PCOS patients resulted in the detection of twelve protein

fractions resolved on 10 and 15% gel ranging between 181-13kDa.

94

Among these, protein fraction of 181kDa (ranging between 4.70-9.71%) was first in expression.. It

exhibited fraction percent of 9.71, 8.79, 7.13, 6.38, 6.97, 5.27, 7.34, 8.53, 7.52 in subject no. 9-18,

respectively. Whereas, 6.22, 7.94, 4.75, 6.23, 5.78, 6.84, 6.26,

5.95, 4.70 fraction percent was observed in subject no. 19-27, respectively (Fig. 4.10).

The fraction covered by 131kDa proteins was not expressed in any PCOS patient (Fig. 4.11).


exhibited a band percent of 8.01, 8.36, 7.95, 7.73, 8.35, 7.16, 6.15, 6.53, 6.90 in subject no. 9-18,

respectively. The same fraction occupied 8.55, 8.29, 7.97, 7.32, 8.02, 7.27, 9.15, 8.75, 8.47 band

% in subject no. 19-27, respectively (Fig. 4.12).


exhibited 9.61, 10.63, 11.71,10.15, 9.75, 9.08, 10.70, 12.17, 9.51 band % in subject no. 9-18,

respectively. A fraction % of 10.86, 11.91, 7.14, 9.42, 9.51, 7.98, 7.37, 8.96, 10.34 was found in

subject no. 19-27, respectively (Fig. 4.13).

Protein fraction of 66kDa (ranging between 32.13-39.92%) was next in expression. This protein

fraction occupied 32.13, 39.86, 35.00, 35.56, 36.84, 33.25, 34.17, 37.41, 33.33% in subject no. 9-

18, respectively. Moreover, a band % of 38.95, 39.92, 39.40, 39.68, 37.39, 37.69, 35.24, 38.04,

33.09 was observed in subject no. 19-27, respectively (Fig. 4.14). Protein fraction of 51kDa

(ranging between 9.50-15.11%) was next in expression. This fraction exhibited a percentage of

15.11, 12.90, 12.48, 13.11, 14.24, 14.82, 10.66, 12.52, 11.58 in subject no. 9-18, respectively.

Subject no. 19-27 showed 12.79, 14.41, 10.76,

13.66, 11.27, 11.49, 12.79, 11.63, 9.50 band percent, respectively (Fig. 4.15).


exhibited a band % of 7.67, 6.61, 8.19, 6.50, 7.01, 5.66, 7.47, 8.75, 9.47% in subject no. 9-18,

respectively. Subject no. 19-27 showed 7.20, 7.17, 7.18, 5.23, 7.64, 7.95, 7.18 fraction percent,

respectively (Fig. 4.16).


exhibited %age of 4.36, 3.52, 4.21, 4.43, 4.14, 3.37, 4.62, 4.41, 4.50 in subject no. 9-18,

respectively. The same fraction occupied 3.59, 4.44, 4.50, 6.07, 5.53, 4.92, 5.66, 5.48, 4.38 band

% in subject no. 19-27, respectively (Fig. 4.17).


exhibited 12.62, 13.70, 12.58, 13.49, 11.33, 13.83, 12.31, 11.56, 12.34 band %age in subject no. 9-

18, respectively. A fraction percent of 12.65, 12.28, 11.70, 12.74, 12.99, 11.09, 11.23, 11.59,

12.30 was found in subject no. 19-27, respectively (Fig. 4.18). Protein fraction of 17kDa (ranging

between 0.45-3.17%) was next in expression. This protein fraction occupied 0.45, 2.52, 2.09, 2.77,

3.62, 3.00, 2.01, 2.29, 3.17 band %age in subject no. 9-18, respectively. A band percent of 2.19,

2.51, 2.28, 2.82, 2.61, 2.23, 2.80, 2.74, 2.18% was observed in subject no. 19-27, respectively (Fig.

4.19).


covered 1.46, 1.66, 1.57, 2.65, 2.68, 1.07, 1.01, 1.13, 1.31% in subject no. 9-18, respectively.

Subject no. 19-27 showed 0.99, 1.11, 1.16, 1.52, 1.05, 1.11, 1.18, 1.24, 1.59 fraction percent,

respectively (Fig. 4.20).

95


exhibited a percentage of 1.67, 2.09, 1.94, 2.94, 2.30, 2.03, 1.32, 1.67, 1.61 in subject no. 9-18,

respectively. Subject no. 19-27 showed 1.35, 0.91, 1.36, 2.01, 2.78, 1.68,

1.51, 2.69, 2.32 band percent, respectively (Fig. 4.21).

96

Fig. 4.10. Band %age exhibited by 181kDa protein fraction in Control subjects and PCOS Patients

showing individual variations.



Fig. 4.12. Band %age exhibited by 100kDa protein fraction in Control subjects and PCOS Patients showing individual variations.

0

5

10

15

Controls Patients

0

1

2

3

4

Controls Patients

0

2

4

6

8

10

Controls Patients

97






0

5

10

15

Controls Patients

0

10

20

30

40

50

Controls Patients

98





0

5

10

15

20

Controls Patients

0

5

10

15

Controls Patients

99





0

2

4

6

8

Controls Patients

0

5

10

15

20

Controls Patients

100


Fig. 4.19. Band %age exhibited by 17kDa protein fraction in Control subjects and PCOS Patients showing individual variations.



0

2

4

6

8

C ont rols P atien ts

0

1

2

3

Controls Patients

101



0

1

2

3

4

Controls Patients

102


103


104


105


4.3.3 GROUP COMPARISON

181kDa Protein Fraction (Table 4.4, Fig. 4.30) The average band percentage covered by 181kDa protein fraction was found to be 5.6 ±

0.40 in control group and 6.8 ± 0.32 in PCOS patients group indicating significant

(p=0.0480) increase of 21.42 % in patients when compared to control subjects.

106


Fig. 4.30. Average band %age exhibited by 181kDa protein fraction in controls and patients group. Values

are Mean ± SEM, * representing significance at p<0.05.

131kDa Protein Fraction (Table 4.4, Fig. 4.31) Average band percentage covered by 131kDa protein fraction was found to be 1.9 ±0.32 in

control group and 0.00 ± 0.00 in PCOS patients group indicating highly significant (p=<0.0001)

decrease of 100 % in patients when compared to control subjects.

Fig. 4.31. Average band %age exhibited by 131kDa protein fraction in controls and patients group. Values are Mean ± SEM, *** representing significance at p<0.001.

100kDa Protein Fraction (Table 4.4, Fig. 4.32) The average band percentage covered by 100kDa protein fraction was found to be 6.3 ± 0.25 in

control group, whereas, patients group, the value increased to 7.8 ± 0.19 indicating highly

significant (p=0.0001) increase of 23.8% in patients when compared to control subjects.

107



are Mean ± SEM, *** representing significance at p<0.001.

80kDa Protein Fraction (Table 4.4, Fig. 4.33) The average band percentage covered by 80kDa protein fraction was found to be 8.7 ±

0.31 in control group, whereas, 9.8 ± 0.34 in patients group indicating non significant (p=0.0542)

increase of 12.64% in patients group when compared to control group subjects.

Fig. 4.33. Average band %age exhibited by 80kDa protein fraction in controls and patients group. Values are Mean ± SEM.

66kDa Protein Fraction (Table 4.4, Fig. 4.34) The average band percentage covered by 66kDa protein fraction was found to be 37 ± 0.78 in

control group, whereas, in exposed worker group, the value increased to 36 ± 0.61 indicating

non significant (p=0.7148) decrease of 2.70% in patients when compared to control subjects.

Fig. 4.34. Average band %age exhibited by 66kDa protein fraction in controls and patients group. Values are Mean ± SEM.


control group, whereas, in patients group, the value decreased to 13 ± 0.36 indicating

significant (p=0.0130) decrease of 13.34% in patients when compared to control subjects.

108


Fig. 4.35. Average band %age exhibited by 51kDa protein fraction in controls and patients group. Values are Mean ± SEM, * representing significance at p<0.05.


control group, whereas, in patients group, the value decreased to 7.4 ± 0.25 indicating a non

significant (p=0.7714) decrease of 2.64% in patients as compared to control subjects.


are Mean ± SEM.


control group whereas in patients group, the value increased to 4.6 ± 0.17

*

109


Fig. 4.37. Average band %age exhibited by 29kDa protein fraction in controls and patients group.

Values are shown as Mean ± SEM, * representing significance at p<0.05.

indicating a significant (p=0.0448) increase of 17.94% in patients when compared to control

subjects.


control group, whereas, in patients, the value decreased to 12 ± 0.19 indicating highly significant

(p=<0.0001) decrease of 20% in patients when compared to control subjects.

Fig. 4.38. Average band %age exhibited by 23kDa protein fraction in controls and patients group. . Values

are Mean ± SEM, *** representing significance at p<0.001.


control group, whereas, in patients group, the value decreased to 2.5 ± 0.15 indicating highly

significant (p=<0.0001) decrease of 45.66% in patients when compared to control subjects.

Fig. 4.39. Average band %age exhibited by 17kDa protein fraction in controls and patients group. . Values are Mean ± SEM, *** representing significance at p<0.001.

15kDa Protein Fraction (Table 4.4, Fig. 4.40) The average band percentage presented by 15kDa protein fraction was found to be 1.3 ± 0.10 in

control group, whereas, in patients, the value increased to 1.4 ± 0.12 indicating a non significant

(p=0.7353) decrease of 7.69% in patients as compared to control subjects.

110



are Mean ± SEM.


control group whereas in patients group, the value increased to 1.9 ± 0.14 indicating a

significant (p=0.0096) increase of 46.15% in patients when compared to control subjects.


are shown as Mean ± SEM, * representing significance at p<0.05.

Table 4.4. Average band percentages of various protein fractions in control and PCOS patients

groups showing group comparison. Values are Mean±SEM.

Molecular weigh(kDa) t ControlsAverage band percentage Patients

Percentage ↑ or ↓

P value

181 5.6±0.40 6.8±0.32 *21.42↑ 0.0480

131 1.9±0.32 0.0±0.00 ***100↓ <0.0001

100 6.3±0.25 7.8±0.19 ***23.8↑ 0.0001

80 8.7±0.31 9.8±0.34 12.64↑ 0.0542

66 37.0±0.78 36.0±0.61 2.7↓ 0.7148

51 15.0±0.96 13.0±0.36 *13.34↓ 0.013

111


32 7.6±0.68 7.4±0.25 2.6↓ 0.7714

29 3.9±0.26 4.6±0.17 17.94*↑

0.0448

23 15.0±0.20 12.0±0.19 ***20↓ <0.0001

17 4.6±0.35 2.5±0.15 ***45.66↓ <0.0001

15 1.3±0.10 1.4±0.12 0.08↑ 0.7353 13 1.3±0.085 1.9±0.14 *46.15↑ 0.0096

* Significance level in relations to control at p<0.05 *** Significance level in relations to control at

p<0.001 ↑ Increase

↓

Decrease

4.4 TWO DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS 4.4.1

CONTROL GROUP

On 2D gel, first spot in expression was Serotransferrin (Transferrin) with molecular weight in the

range of 76-87kDa, pI (6.14-6.55) and accession number P02787. The next band/spot was serum

albumin with molecular weight in the range of 66-68kDa, pI (5.567.38) and accession number

P02768, was detected. Spot number 3 was recognized as

Immunoglobulin heavy chain gamma (IGHG) with molecular weight in the range of 5155kDa, pI

(6.15-9.02) and accession number P99006. Spot number 4 was identified as Haptoglobin Beta

chain with molecular weight 37-44kDa, pI (4.81-5.86) and accession number P00738. Spot

number 5 was identified as Alpha-1 Antitrypsin with molecular weight in the range of 53-58kDa,

http://expasy.org/swiss-2dpage/ac=P02787

112


pI (4.87-5.10) and accession number P01009. Spot number 6 was recognized as Alpha-1-acid

glycoprotein with molecular weight 43-46kDa, pI (4.11-4.29) and accession

number P02763. Spot number 5 was identified as Alpha-2HS-glycoprotein (Fetuin-A) with

molecular weight in the range of 52-58kDa, pI (4.564.71) and accession number P02765. Spot

number 8 was an unidentified protein which was present in control group. Spot number 9 was

recognized as Apolipoprotein A-1 had molecular weight 23-24kDa, pI (4.99-5.48) and accession

number P02647. Spot number 10 was identified as Immunoglobulin light chain (IGLC) having

molecular weight 2329kDa, pI (5.02-8.56) and accession number P99007. Band number 11 was

said to be Haptoglobin Alpha chain had molecular weight 16-17kDa, pI (5.40-6.07) and accession

number P00738. Band number 12 was recognized as Transthyretin with molecular weight 13-

14kDa, pI (5.02-5.52) and accession number P02766 (Table 4.5, Fig. 4.42).

4.4.2 PCOS PATIENTS GROUP

In PCOS patients, the first spot, on 2D gel, in expression was Serotransferrin (Transferrin) with

molecular weight in the range of 76-87kDa, pI (6.14-6.55) and accession number P02787. The

next band/spot was serum albumin with molecular weight in the range of 6668kDa, pI (5.56-

7.38) and accession number P02768, was detected. Spot number 3 was recognized as

Immunoglobulin heavy chain gamma (IGHG) with molecular weight in the range of 51-55kDa, pI

(6.15-9.02) and accession number P99006. Spot number 4 was identified as Haptoglobin Beta

chain with molecular weight 37-44kDa, pI (4.81-5.86) and accession number P00738. Spot

number 5 was identified as Alpha-1 Antitrypsin with molecular weight in the range of 53-58kDa,

pI (4.87-5.10) and accession number P01009.

Spot number 6 was recognized as Alpha-1-acid glycoprotein with molecular weight 4346kDa, pI

(4.11-4.29) and accession number P02763. Spot number 5 was identified as

Alpha-2-HS-glycoprotein (Fetuin-A) with molecular weight in the range of 52-58kDa, pI (4.56-

4.71) and accession number P02765. Spot number 8 was an unidentified protein which was

present in control group. Spot number 9 was recognized as Apolipoprotein A-1 had molecular

weight 23-24kDa, pI (4.99-5.48) and accession number P02647. Spot number 10 was identified

as Immunoglobulin light chain (IGLC) having molecular weight 23-29kDa, pI (5.02-8.56) and

accession number P99007. Band number 11 was said to be Haptoglobin Alpha chain had

molecular weight 16-17kDa, pI (5.40-6.07) and accession number P00738. Band number 12 was

recognized as Transthyretin with molecular weight 13-14kDa, pI (5.02-5.52) and accession

number P02766 (Table 4.5, Fig. 4.43-4.44).

4.4.3 GROUP COMPARISON

Comparison was done between PCO’s patient and healthy women (control group) through the

comparison of qualitative expression of different spots in both groups. Up to 12 different

protein bands/spots were identified and their expressions were compared with that of control.

Some of these bands showed an up regulation and some of them exhibited an expression of

down regulation as compared to control. The bands/spots that showed up regulation in their

expression were Serotransferrin (spot 1), Haptoglobin alpha chain (spot 11) and Alpha-2-HS-

glycoprotein (Fetuin-A) (spot 7) showed up regulated expression in PCO’s patients as compared

to control healthy females. Whereas Immunoglobulin heavy chain gamma (IGHG) (spot 3),

Haptoglobin beta chain (spot 4), Alpha-1 antitrypsin (spot 5), and Alpha-1-acidic glycoprotein

(spot 6), Apolipoprotein A-1 (spot 9), Immunoglobulin light chain (IGLC) (spot 10) and

Transthyretin (spot 12) showed down regulation in their expression when compared with

http://expasy.org/swiss-2dpage/ac=P02787

113


control. An unidentified protein (spot 8) was also down-regulated in PCO,s as compared with

control group (Table 4.5, Fig.

4.42-4.44).

114


Fig. 4.42. Gel image of healthy control woman indicating variedly expressed protein spots (1-12) as resolved by 2D PAGE.

1: Serotransferrin (STF); 2: Albumin (ALB); 3: Immunoglobulin heavy chain (IGHG); 4:

Haptoglobin β chain; 5: α-1 antitrypsin (ATR); 6: Alpha-1 acidic glycoprotein (AGP); 7: Alpha-2-HS-glycoprotein (FETUA); 8: Unidentified protein; 9: Apolipoprotein A-1 (ApoAI); 10:

Immunoglobulin light chain (IGLC); 11: Haptoglobin α chain, 12: Transthyretin (TTR)

115


Fig. 4.43. Gel image of Polycystic Ovary Syndrome patient indicating variedly expressed protein spots (1-12) as resolved by 2D PAGE.


Haptoglobin β chain; 5: α-1 antitrypsin (ATR); 6: Alpha-1 acidic glycoprotein (AGP); 7:

Alpha-2-HS-glycoprotein (FETUA); 8: Unidentified protein; 9: Apolipoprotein A-1 (ApoAI); 10:


116


Fig. 4.44. Gel image of Polycystic Ovary Syndrome patient indicating variedly expressed protein spots (1-12) as resolved by 2D PAGE.


Haptoglobin β chain; 5: α-1 antitrypsin (ATR); 6: Alpha-1 acidic glycoprotein (AGP); 7:

Alpha-2-HS-glycoprotein (FETUA); 8: Unidentified protein; 9: Apolipoprotein A-1 (ApoAI); 10:


117


Table 4.5. Protein fractions showing variable expressions in women with PCOS as compared to

controls.

Spot No. Protein Names Accession Numbers

Molecular

Weights pI Expression in PCOS

1 Serotransferrin (STF) P02787 76-87kDa 6.14-6.55 Slightly Up-Regulated 2 Albumin (ALB) P02768 66-68kDa 5.56-7.38 ---------

3 Immunoglobulin P99006 heavy chain (IGHG)

51-55kDa 6.15-9.02 Down-Regulated

4 Haptoglobin β chain P00738 33-44kDa 4.81-5.86 Down-Regulated

5 α-1 antitrypsin (ATR) P01009 53-58kDa 4.87-5.1 Down-Regulated

6 Alpha-1 acidic

glycoprotein (AGP) P02763 43-46kDa 4.11-4.29 Down-Regulated

7 Alpha-2-HSglycoprotein (FETUA)

P02765 52-58kDa 4.56-4.71 Up-Regulated

8 Unidentified Protein ------- 5.8 Down-Regulated

9 Apolipoprotein A-1

(ApoAI) P02647 23-24kDa 4.99-5.48 Down-Regulated

10 Immunoglobulin light P99007 chain

(IGLC) 23-29kDa 5.02-8.56 Down-Regulated

11 Haptoglobin α chain, P00738 16-17kDa 5.4-6.07 Up-Regulated

12 Transthyretin (TTR) P02766 13-14kDa 5.02-5.52 Down-Regulated

118


CHAPTER 5

DISCUSSION

PCOS is an entity which is generating controversy regarding its pathogenesis, diagnostic criteria

and therapeutic procedures (Banaszewska et al., 2003). The diagnosis of PCOS becomes difficult

on account of discrepancies in diagnostic criteria; especially because of controversy in

interpretation of ovarian ultrasound imaging (Cajdler-luba et al., 2010).

Over the current years, hormonal analyses are being regarded as the principal diagnostic criteria

of PCOS. It has been reported that insulin appears to disrupt all components of

hypothalamohypophyseal-ovarian axis; and that hyperandrogenemia is the major factor

responsible for the clinical features and complications of PCOS (Rojas et al., 2014).

Nevertheless, classical presentation of PCOS comprises of oligomenorhea with clinical and

biochemical evidence of hyperandrogenism; however, presenting symptoms vary widely;

including anovulation with hirsutism or without hirsutism and regular cycles. Interestingly the

principal criterion of diagnosis of PCOS is hormonal analysis i.e. serum LH, FSH, insulin and

testosterone levels (Franks, 2010). The present study was, therefore, carried out to determine

endocrine attributes of PCOS in the back drop of local, ethnic, socioeconomic and environmental

elements in Pakistani population.

The results of the hormonal analysis in the present study demonstrated significantly higher

levels of LH, FSH, LH:FSH ratio, insulin, testosterone, androstenedione and body mass index.

Nevertheless, the hormonal profile is supportive of international studies. Similarly the LH:FSH

ratio was found to be significantly higher as compared to the reported ratios. Serum LH levels

were significantly elevated (P<0.001) in PCOS group as compared to control group, indicating

60% rise. The elevated LH in PCOS has also been reported earlier

(Suhail, 2008). However, the elevation in LH level, in our study, was significantly intense than the

reported values possibly on account of ethnic variations and environmental factors in our

population. The LH levels fell significantly in the FDR group demonstrating 87% decrease

compared to PCO group (P<0.001).The LH levels in FDR group were insignificant as compared to

normal control group.

It has most recently been investigated that LH, FSH, testosterone and androstenedione can

prove to be predictive markers of ovulation after laparoscopic ovarian drilling (LOD). The serum

LH and androstenedione levels were found to be independent markers of ovulation after LOD.

The PCOS patients who have elevated levels of LH prior to LOD, demonstrated good

response in terms of postoperative induction of ovulation (Johannes et al., 2009). LOD markedly

decreased postoperative LH and androstenedione levels depicting the effectivity of laparoscopic

ovarian drilling. It was especially true for the patients who had higher androstenedione values of

more than 3.26 ng/ml; and for patients with both LH concentration of 12.1 IU/L, or greater and

androstenedione exceeding these cut off values. Moreover, ovarian surgery has been reported

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to cause rapid reduction in serum levels of all ovarian hormones i.e. initiation of folliculogenesis,

and restoration of normal feed-back to hypothalamus and pituitary, resulting in

appropriate gonadotrophin secretion and normal follicular development. This reversal towards

normal situation may be due to increasing FSH levels that occur on account of restoration of

normal feed back system as a consequence of ovarian surgery (Hendriks et al., 2007).

The LH: FSH ratio in PCOS in comparison to control subjects was observed to be significantly

higher (P<0.001) in the present study. This ratio was raised by 18–folds in the PCOS group. It has

been reported that most of the women (75%) with PCOS have an elevated

LH level in the early follicular phase; and 94% have an enhanced LH to FSH ratio13 (Taylor et al,

1997). It has, therefore, been hypothesized that gonadotrophin dysfunction plays a key role in

generating anovulatory amenorrhoea characteristic of PCOS women14 (Pagan et al, 2006).

Moreover, raised LH:FSH ratio may prove to be beneficial in revealing PCOS by gonadotrophin

releasing hormone (GnRH) stimulation test. This test is suggested to be an important additional

investigation in the diagnosis of PCOS based on elevated LH in serum (Cajdler-Luba, 2010).

The LH:FSH ratio in PCOS demonstrates greater accuracy in prediction of PCOS than total

testosterone and average ovarian volume in these women presenting with oligomenorrhea or

anovulation. Further, an LH:FSH ratio of more than one presented the best combination of

senstivity and specificity (Ming et al., 2009). It, therefore, appears that hormonal analysis

especially LH levels and LH:FSH ratio are reliable predictors of PCOS as observed in the current

study.

On the other hand PCOS group compared to FDR group also showed significantly lower ratio

(P<0.05) indicating 91% decrease in the ratio. The ratio of LH:FSH varied nonsignificantly in the

FDR group in comparison to control group. The highly raised ratio may be on account of very

high LH values in PCOS patients. Conversely, the FSH concentrations were significantly lower

(P<0.01), indicating 33% fall in the PCOS group, contributing towards a very high LH:FSH ratio as

observed in this study. The FSH also decreased in FDR group compared to controls indicating

35% fall in this group. The raised LH:FSH ratio has been regarded by most authors an important

diagnostic feature in PCOS. The highly significantly raised levels of LH, and LH:FSH ratio in our

study is in agreement with most of the international reports.

Moreover, raised LH:FSH ratio may prove to be beneficial in revealing PCOS by gonadotrophin

releasing hormone (GnRH) stimulation test. This test is suggested to be an important

additional investigation in the diagnosis of PCOS based on elevated LH in serum (Cajdler-Luba et

al., 2010). In a retrospective study conducted at a tertiary academic centre, it was concluded

that LH:FSH ratio is a valuable diagnostic tool in evaluating women having PCOS specially

complaining of oligomenorrhea or anovulation. An LH:FSH ratio of more than one (>1) may be

used as a decision threshold. It has been observed that LH:FSH ratio is a better predictor of

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PCOS. This ratio demonstrates greater accuracy in prediction of PCOS than total testosterone

and average ovarian volume in these women presenting with oligomenorrhea or

anovulation. Further, an LH:FSH ratio of more than one (>1) presented the best combination of

senstivity and specificity. It has been further reported that body mass index was negatively

correlated with LH in PCOS, and no correlation was depicted in non-PCOS subjects. It was hence

concluded that the link between body mass index and LH may provide clues for deeper insight in

the pathophysiology of PCOS (Ming et al., 2009).

On the contrary, our study indicates a positive role of LH. The raised LH levels in PCOS patients

also depicted a significantly raised BMI as compared to the control subjects. The BMI in PCOS

group of patients showed a significant difference (P<0.001), indicating 26% increase in BMI. The

BMI decreased sifnificantly (P<0.001) indicating 23 % fall in the FDR group as compared to PCOS

group. There was no significant difference in BMI in comparison to control group.

It has been reported that women suffering from PCOS have consistent abnormalities in the

gonadotrphin secretion. About 75% of these patients have elevated LH levels and 94% have

elevated LH:FSH ratio (Blank et al., 2006). The overproduction of LH and consequently disturbed

LH:FSH ratio is detected in 94% of PCOS patients. Until 1980s, LH:FSH ratio was considered a

gold standard for the diagnosis of PCOS. Later, it was found that most of PCOS women with

normal LH:FSH ratio suffered from hyperinsulunemia and obesity. Moreover, the elevated levels

of LH occur more rarely in a group of patients with insulin resistance and hyperinsulinemia. It

was suggested that LH:FSH ratio is not a characteristic sign of all PCOS patients (Banaszewska et

al., 2003).

Most recently, raised leptin levels have been found to be associated with obesity; it has also

been implicated to influence FSH, testosterone (T), obesity and PCOS (Sunita et al., 2014). It has

also been observed that PCOS when associated with obesity leads to elevation of proinsulin

concentrations, that is a known indicator of fertility outcomes in PCOS women. Moreover,

treatment with metformin antagonises proinsulin and decreases the fertility outcomes in these

patients of PCOS (Kruszyńska et al., 2014).

The LH:FSH ratio was correlated with elevated serum LH levels, insulin resistance, menstrual

irregularities and obesity. It was reported that characteristic increase in LH relative to FSH,

which is the commonest finding in PCOS is not a dependable variable/parameter. This is because

LH is released in pulses, and a single test estimation of LH may not represent a real

concentration of LH in serum. Consequently it was suggested that LH:FSH ratio may not be

considered as a criterion for diagnosis of PCOS. These authors failed to find any correlation

between LH:FSH ratio, BMI, menstrual pattern and hirsutism. Ultimately they disproved the

notion that heavier the patient, higher the LH value; and conjectured that loss of correlation of

LH with disease manifestations may add more to this mysrerious syndrome (Alnakash

and Al-Tae, 2007).

Sheehan (2004), in a review article has mentioned that LH:FSH ratio of more than two

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(>2.0) is suggestive of PCOS; but is not highly sensitive or specific. The diagnosis of PCOS in

Europe does not require hormonal estimations in routine. This is in absolute contrast to

the National Institute of Health conference held in 1990, which did not include ovarian

morphology on pelvic ultrasound examination as a criterion for diagnosis of PCOS (Dunaif and

Thomas, 2001). This is on account of the fact that 20% patients of PCOS do not have typical

ultrasound picture consistent with its diagnosis. Nevertheless, pelvic ultrasonography is a useful

evaluation tool for diagnosis of PCOS, but polycystic ovarian morphology is not specific for PCOS.

The diagnosis of PCOS is made largely by ovarian morphology on ultrasound examination. It has

been proposed that if four classic features of PCOS are present i.e. menstrual disturbance,

hirsutism, acne or anovulatory infertility; an ultrsound assessment of ovaries is suggested. If

polycystic ovarian morphology (PCOM) is detected, the diagnosis is confirmed (Homburgh,

2002). It therefore appears that hormonal analysis especially LH levels and LH: FSH ratio are

reliable and good predictor of PCOS as indicated in our study.

In addition, we have observed highly significantly raised insulin levels. The PCOS group

manifested a very high mean value of 43.48± 4.09 indicating 5-fold rise in this group (P<0.001).

The FDR group showed a significant fall (P<0.001) depicting 89% decrease compared with PCO

group. The increase in insulin levels in our study may be attributed to high levels of LH. It has

been reported that insulin exerts a synergistic action along with LH. This in turn stimulates

ovarian androgen synthesis (Nestler et al., 1998a).

Insulin also decreases hepatic production of sex hormone binding globulin (SHBG) resulting in

higher levels of free testosterone in the blood (Dunkel et al., 1985). Insulin resistance and

consequent hyperinsulinemia is a characteristic finding associated with PCOS reported by most

workers. Moreover, insulin resistance is a common metabolic abnormality seen in obese and to

a lesser degree in lean women with PCOS (Dunaif et al., 1989; Carmina et al., 1992; Legro et al.,

1998; DeUgrate et al., 2005). It has been reported that treatment of patients of PCOS improves

not only insulin resistance but also corrects hyperandrogenemia and restores normal ovarian

function (Fleming et al., 2002; Ghazeeri et al., 2003; Lord et al., 2003). It has been further

observed that correction of hyperandrogenemia is on account of decrease in circulating insulin

levels. Moreover, it has been studied in vitro, that insulin directly influences steroid biosynthesis

in the ovary (Attia et al., 2001; Mitwally et al., 2002; Mansfield et al., 2003). The highly

significantly raised serum insulin levels in our study strongly support these international reports

of hyperinsulinemia that contribute towards pathophysiology of PCOS. Moreover, the

significantly raised LH concentrations in our study in PCOS may be on account of

neuroendocrine dysfunction. It is evidenced by animal studies that hyperinsuliemia raises basal

and GnRH-stimulated LH and FSH secretion from pituitary cells in vitro (Soldani et al., 1994).

Nevertheless infusions of exogenous insulin doesnot alter LH secretion amongst the women

suffering from PCOS (Patel et al., 2003; Mehta et al., 2005). Moreover, it has been found that

short-term treatment with anti-diabetic drugs like metformin does not inhibit or slow down the

LH pulse frequency; despite the fact that significant improvement in hyperinsulinemia is

observed in these patients (Eagleson et al., 2003). Similarly, thiazoladinedione/pioglitazone

improves insulin sensitivity but does not influence or alter LH pulse patterns (Mehta et al.,

2005). The body of international literature cited, suggests that hyperinsulinemia is no

doubt, associated with PCOS, but does not appear to be directly linked with neuroendocrine

abnormalities. Nevertheless, hyperinsulinemia may modify hypothalamic function indirectly by

increasing androgen levels.The androgen levels especially testosterone and androstenedione

were also significantly raised in our experiments. The testosterone levels in the PCOS group

were highly significantly raised, depicting 86% elevation as compared with control group

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(P<0.001). There was 68% decrease in the testosterone values in the FDR group in comparison

with PCOS group. This indicated 96% significant fall (P<0.001) in the FDR group. The

testosterone levels also showed a significant fall in FDR group in comparison to control group

(P<0.01). It demonstrated 40% decrease in the testosterone. The rise in testosterone in our

study is in accordance with reported androgen excess in PCOS. The significant fall in the

testosterone levels in FDR group as compared to control group couldn’t be explained.

The androstenedione was greatly elevated in the PCO group as compared to the controls

indicating a very highly significant rise of 163% (P<0.001). There was significant decrease of 76%

(P<0.001) in FDR group as compared to PCO group. There was no significant difference in normal

controls and FDR group.

A significant correlation (P<0.05) was observed between LH and androstenedione levels in this

study. The highly significant rise in LH may potentiate hyperinsulinemia, which in turn can

stimulate hyperandrogenemia. Nevertheless, hyperandrogenemia is an important variable

associated with PCOS; the raised androgen level in PCOS appears to have multiple influences like

raised LH levels, insulin-based stimulation of ovaries that may lead to hypersecretion of

androgens (Nestler et al., 1998a; Nelson et al., 1999).

Irrespective of the cause of hyperandrogenemia in PCOS, the elevated androgens in the PCOS

group in our study may be the reason of neuroendocrine disturbance. It was presumed initially

that neither androgen infusions in women with PCOS nor short term therapy (01 month) with

flutamide brought any change in the LH concentration and its pulsatility. Subsequently, it was

found that short term treatment with flutamide, which is an androgen receptor blocking drug,

restores hypothalamic senstivity to progesterone mediated slowing of LH pulsatility in women

with PCOS (Eagleson et al., 2000). Futhermore, long term treatment (06 months) with flutamide

may alleviate many of the reproductive manifestations of PCOS (De Leo et al., 1998). This shows

that elevated androgens have significant neuroendocrine effects. Moreover, rapid GnRH pulse

frequency in PCOS is the factor that promotes excessive LH feed-forward mechanism leading to

high androgen synthesis. It also affects the relative FSH deficiency that culminates into ovulatory

dysfunction. It is thus summarized that elevated serum LH, elevated LH:FSH ratio and rapid

GnRH pulse frequency in our study may have lead to insulin resistance. Insulin in synergy with

elevated LH may increase the androgens in the blood that is characteristic of adult PCOS and

adolescent hyperandrogenemia (Blank et al., 2006). We therefore, postulate that LH elevation in

the blood is the central feature of PCOS. The elevated LH concentration subsequently leads to

other clinical and biochemical events of the syndrome. Henceforth, it is suggested that raised LH

levels, high LH:FSH ratio and hyperinsulinemia are the best indicators of PCOS.

The elevation of gonadotrophin especially LH in the patients of PCOS has been related with

endocrine disruption of the reproductive neuro-endocrine system. This regulation of this system

involves GnRH. GnRH has been cited as the primary stimulus for the reproductie axis i.e.,

hypothalamo-hypophyseal-gonadal (H-H-G) axis. LH and FSH act on the ovary and generate and

generate steroid hormones (Gore, 2002).

Another important hypothesis states that sex steroid also regulate the GnRH in an

indirect manner. The GnRH cells/neurons cannot express receptors for steroid hormones. It has,

therefore, been hypothesized that some other neurons of the brain that can express receptors

for steroid hormones are the target of the endocrine disrupting cells (EDCs). It further explains

that EDCs can modify the brain neurotransmitter systems. The EDCs can modify or toxify the

nor-adrenergic, serotonergic, dopaminergic and other neuroendocrine cells of the brain

123


(Dickerson and Gore, 2007; Walker and Gore, 2007). It has also been observed that all these

neurons endocrine cells can express steroid hormone receptors. Moreover, all these

neurons can project to and regulate GnRH neurons. Thus, EDCs act on this linkage between

neural and endocrine systems and exert their effects on the hypothalamo-pictuar-gonadal axis

(Gore, 2002).

Polychlorinated biphenyls (PCBs), organochlorine pesticides like methoxychlor and chlorpyrifos

have been blamed to influence GnRH cells, and cause significant changes in GnRH gene

expression (Gore et al., 2002; Gore, 2002).

It has recently been reported that disposable plastic cups, cooking oil fumes and indoor

decoration are environmental risk factors associated with PCOS (Huang et al., 2007).

All these data cited above, is suggestive of direct influence on GnRH cell lines. It appears that

this complex and enigmatic disorder originates from within and outside the H-P-O axis. It may be

conjectured/concluded that EDCs as environmental factors contribute in the pathogenesis of

PCOS.

Hyperinsulinaemia is reported in women with polycystic ovary syndrome. It has been reported

that administration of supplements of essential amino-acids significantly decreased total

testosterone, LH and FSH. This consequently improves the clinical features of hyperandrogenism

in PCOS. It appears that hyperandrogenism is related with and a consequence of

hyperinsulinaemia (Unfer et al., 2008). .

It has been demonstrated that environmental chemicals called as endocrine-disrupting

chemicals (EDC) can modify reproductive function (Gore, 2007). The hypothalamopituitary-

ovarian (HPO) axis has been reported to be influenced by environmental EDCs. (Gore, 2008).

Moreover, it has been found that polychlorinated biphenyls (PCBs) and organochlorine

pesticides such as methoxychlor and chlorpyrifas altered the GnRH gene expression, GnRH

peptide release and morphology of the gonadotrophin cells (Gore et al., 2002; Gore, 2002).

Animal models also exhibited that GnRH neurons are targets of both natural environmental

estrogens such as coumestrol, and industrial contaminants and pesticides (McGarvey et al.,

2001; Gore, 2001). It has been concluded that EDCs can impair the reproductive physiology

specially H-P-O-axis (Gore, 2008).

The use of oral contraceptives in the hyperandrogenic non-ovulating women, slows the

LH pulse frequency but not to the extent observed in normal controls (Daniels and Berga, 1997).

Most recently, raised leptin levels have been found to be associated with obesity; it has also

been implicated to influence FSH, testosterone (T), obesity and PCOS (Sunita et al., 2014). It has

also been observed that PCOS when associated with obesity leads to elevation of proinsulin

concentrations, that is a known indicator of fertility outcomes in PCOS women.

Moreover, treatment with metformin antagonises proinsulin and decreases the fertility

outcomes in these patients of PCOS (Kruszynska et al., 2014).

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This part of the present study of PCOS patients is focused on gonadotrophins, androgens and

insulin levels mainly. Our data supported the renewed interest in the importance of endocrine

parameters in the diagnosis of PCOS. However, attempts to predict PCOS risk in asymptomatic

subjects could not be successful as the FDR group was small in number (n = 25). This was on

account of decreased participation (18%) of the first degree relatives of PCOS patients who

avoided investigations on account of being labeled as “infertile family”.

In the present study we have further investigated PCOS specific proteins and determined

expression pattern with 2-D PAGE in order to understand the pathogenesis of PCOS. Poteomic

techniques applied to investigate biomarkers of PCOS have revealed a number of processes

related to oxidative stress, inflammatory changes and iron metabolism to be involved in the

pathogenesis of PCOS (Insenser and Escobar-Morreale, 2012). For this purpose we analysed

plasma protein profiles in normal subjects and patients of PCOS in Pakistani women.

Consequently we have recognized 12 proteins, out of which 08 proteins were under-expressed

and down-regulated in PCOS patients as shown in PCO table. The proteins like sero-transferrin,

alpha 2-heat specific (HSP) glycoprotein and haptoglobin alpha chain were increased and

upregulated in our population of patients of PCOS as compared with controls. All these proteins

along with their accession number, molecular weights and isoelectric points (pI) are listed in

PCO table.

Serotransferrin is known to be stress related, acute phase response molecule. Its over

expression in patients of PCOS in our study can be elaborated on the basis of underlying

inflammatory process (Galazi et al., 2013). Serotransferrin is a glycoprotein that transports iron

to the ovarian follicles (Briggs et al., 1999). Its up-regulation seems to be legitimate and

reasonable in the present study conducted in Pakistani patients of PCOS. Moreover,

overexpression of serotransferrin appears to be a compensatory change on account of anaemia

of chronic inflammation. Upregulation of transferrin has also been reported in human follicular

fluid (Gua and Gungxia, 2012).

Haptoglobin is a plasma alpha-2 glycoprotein formed by hepatocytes in the liver and

subsequently thrown in the circulation (Bowman, 1993). The anti-inflammatory and antioxidant

properties of haptoglobin justify its up-regulation and over-expression in our study. It has been

suggested that haptoglobin alpha chain over-expression is a sign of reactive response to iron

loss and renal injury; as a result oxidative stress gets improved and alleviated (Insenser et al.,

2010). It has further been reported that haptoglobin apha chain is over expressed in patients of

PCOS as well as in type-2 diabetes mellitus (T2 DM) patients. It has been mentioned that both

PCOS and T2DM are inflammatory conditions (Galaziz et al., 2013); it has, therefore, been

hypothesized that haptoglobin interacts with haemoglobin and is found to be elevated on

account of inflammation and oxidative stress (Zhang et al., 2004). Studies have suggested

that oxidative stress, in women with PCOS, stimulates androgen-producing

steroidogenic enzymes thus leading to hyperandrogenism, most notable characteristic of PCOS

(Gonzalez et al., 2006).

Alpha-2-heat specific glycoprotein (alpha-2 HSG) has been reported to be over-expressed in

preterm birth and PCOS in response to inflammation (Galazi et al., 2013). Alpha-2

125


HSG is a receptor signaling protein and tyrosine kinase inhibitor. The levels of α-2 HSG are

increased by the mechanisms such as oxidative stress, heat shock exposure,

infection, inflammation and anaemia (Lindquist and Craig, 1988; Ciocca et al., 1992). Alpha-2

HSG is upregulated in our presented proteomic identification of PCOS which further potentiates

the notion that PCOS is an inflammatory condition.

The proteomic map of our study in PCOS group of patients indicates under-expression of

haptoglobin beta chain, alpha -1 antitrypsin (α-1 ATR), acidic glycoprotein (AGP), apolipoprotein

A-1(Apo A-1), immunoglobulin light chain (IGLC) and transthyretin (TTR).

Apolipoprotein A-1(Apo A-1), in our study, is down regulated in PCOS patients as compared to

healthy controls. The proteomic identification in granulosa cells of patients of PCOS also shows

significant under-expression of Apo A-1. It has been claimed that the reduced level of Apo A-1 in

granulosa cells may disturb normal steroidogenesis (Choi et al., 2010). It has been proposed that

heat specific proteins (HSP) and Apo A-1 may be labeled as Disease Specific Proteins (DSPs) of

PCOS. It is important to note that ApoA-1 is down-regulated in both PCOS and T2 DM (Galaziz et

al., 2013).Hence, it is concluded that down-regulated ApoA-1 in women with PCOS is closely

associated with aetiology of T2 DM and its cardiovascular complications. Transthyretin (TTR) is

down-regulated in PCOS patients of the present study. TTR is a serum and cerebro-spinal fluid

carrier of thyroxine and retinol. Its reduced expression may indicate the future risks of diabetes,

insulin resistance and cardiovascular manifestations in these women.

Another important protein identified to be down-regulated in PCOS women of our study was

alpha 1-antitrypsin (α-1 ATR). Alpha 1-antitrypsin is protease inhibitor, generally called as trypsin

inhibitor. It has been reported that α-1 ATR inhibits wide range of proteases (Gettins, 2002). It

protects the enzymes released from inflammatory cells like neutrophil elastase whose

concentration rises many times during acute inflammation. The enzyme neutrophil elastase

causes breakdown of elastic tissue thus affects elasticity in tissues. α-1 ATR was also found to be

underexpressed in mice fed on dietary supplements of branched chain amino acids and analysed

by 2-D PAGE (Brocca et al., 2013). The results of proteomic identification, in our study suggest

the potential role of immuneregulation, inflammation and anti-oxidant activity involved in the

pathogenesis of PCOS. Most recently it has been reported that PCOS gets exaggerated in

pregnancy on account of low grade chronic inflammation. The Endocrine Society’s Journal of

Clinical Endocrinology and Metabolism (JCEM) described; as studied by one of the authors in

May, 2014 that the chronic low grade inflammation deteriorates during pregnancy.The author

has blamed that PCOS is associated with hormonal alteration which may trigger imflammatory

process (Palomba et al., 2014) This report further supports our impression that PCOS is chronic

low grade inflammatory process. In nut-shell, the scientific community is now in a better

position to comprehend the differential expressions of these biomarkers in the causation of

PCOS (Galazi et al., 2013).The biomarkers of our study can/may provide a useful framework for

early diagnosis of PCOS and for screening and prevention of complications in women with PCOS.

Conclusions

The endocrinological responses, in PCOS women of the present study, reinforce that

the criteria of endocrinal diagnosis of the syndrome, especially gonadotrophins and LH to

FSH ratio are important aids in the description of PCOS. It, however, seems mandatory that

molecular investigations like protein biomarkers in PCOS, in addition to endocrinal correlations,

be studied to arrive at exact and early diagnosis of the syndrome.

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The altered expressions of ApoA-I, Haptoglobin and transthyretin predict that PCOS women are

at a higher risk of future progression towards diabetes, insulin resistance and

cardiovascular disorders, particularly, atherosclerosis. Further, the over-expressions of

Serotransferrin and Haptoglobin α chain, whereas, declined expression of Haptoglobin β chain

demonstrate that variations in iron metabolism, oxidative stress and low grade chronic

inflammation might be the culprits in molecular pathogenesis of PCOS in women.

i

SUMMARY

Incidence of PCOS in Pakistani women is considerably increasing, however, bulk of available

clinical and aetiological data serves to provide only a framework for studying its genesis. We, in

our study, found that the gonadotrophins, LH to FSH ratio and androgens are dependable

diagnostic parameters of PCOS. The LH:FSH ratio in PCOS group, was raised by 18-folds in this

study.This highly raised ratio may be on account of very high LH and significantly lower FSH

values in PCOS patients. It has, therefore, been hypothesized that gonadotrophin dysfunction

plays a key role in generating anovulatory amenorrhoea characteristic of PCOS women.

Moreover, raised LH:FSH ratio may prove to be beneficial in revealing PCOS by Gonadotrophin

Releasing Hormone (GnRH) stimulation test. The LH:FSH ratio in PCOS demonstrates

greateraccuracy in prediction of PCOS than total testosterone and average ovarian volume in

these women presenting with oligomenorrhea or anovulation. Further, an LH:FSHratio of more

than one presented the best combination of sensitivity and specificity. The elevated LH in PCOS

has also been reported earlier. However, the elevation in LH level, in our study, was

significantlyintense. Henceforth, LH appears to be the most important in this regard,

consequenting to hyperandrogenism.In addition, we have observed highly significantly raised

insulin levels in PCOS patients indicating a 5-fold rise in this group of patients; that may be

attributed to high levels of LH. Moreover, the women suffering from PCOS have higher rates of

obesity and hyperinsulinemia. The higher levels of insulin can be a consequence ofdifferent

ethnic backgrounds.Our data supported the renewed interest in the importance of endocrine

parameters in the diagnosis of PCOS.

In the present study we have further investigated PCOS specific proteins and determined

expression pattern with 2-D PAGE in order to understand the pathogenesis of PCOS.

ii

Poteomic techniques applied to investigate biomarkers of PCOS have revealed a number

of processes related to oxidative stress, inflammatory changes and iron metabolism to be

involved in the pathogenesis of PCOS. Moreover, proteomic identification of serum signatures

seems to be a valid tool for predicting and tracking the progression of comorbidities in women

with PCOS.

127


For this purpose we analysed plasma protein profiles in normal subjects and patients of PCOS in

Pakistani women. Consequently we have recognized 12 proteins, out of which 08

proteins were under-expressed and down-regulated in PCOS patients. The proteins like sero-

transferrin, alpha 2-heat specific (HSP) glycoprotein and haptoglobin alpha chain were increased

and upregulated in our population of patients of PCOS as compared with controls.

Serotransferrin is known to be stress related, acute phase response molecule. Its over

expression in patients of PCOS in our study can be elaborated on the basis of underlying

inflammatory process.

The altered expressions of ApoA-I, Haptoglobin and transthyretin predict that PCOS women are

at a higher risk of future progression towards diabetes, insulin resistance and cardiovascular

disorders, particularly, atherosclerosis. Moreover, the over-expressions of Serotransferrin and

Haptoglobin α chain, whereas, declined expression of Haptoglobin β chain demonstrate that

variations in iron metabolism, oxidative stress and low grade chronic inflammation might be the

culprits in molecular pathogenesis of PCOS in women. Haptoglobin is a plasma alpha-2

glycoprotein formed by hepatocytes in the liver and subsequently thrown in the circulation. The

anti-inflammatory and anti-oxidant properties of haptoglobin justify its up-regulation and over-

expression in our study. It is important to note that ApoA-1 is down-regulated in both PCOS and

T2 DM. Hence, it is concluded that down-regulated ApoA-1 in women with PCOS is closely

associated with aetiology of T2 DM and its cardiovascular complications.

CHAPTER 6

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