Kurdistan Regional Government Ministry of Higher Education and Scientific Research University of Sulaimani College of Pharmacy Clinical Biochemistry Department
Evaluation of the Serum Level of Interleukin-18 and Homocysteine in Polycystic Ovarian Syndrome Patients in Sulaimani Governorate A Thesis Submitted to the Council of the College of Pharmacy University of Sulaimani in Partial Fulfillment of Requirements for the Degree of Master Science in Clinical Biochemistry / Clinical Analysis By Sakar Karem Abdulla B.Sc. Pharmacy / University of Sulaimani-2010 Supervised by Dr. Ban Mousa Rashid (Ph.D. in Clinical Biochemistry)
December 2016
2716 Kurdi
Supervisor's Certification I certify that this thesis entitled “Evaluation of the Serum Level of Interleukin-18 and Homocysteine in Polycystic Ovarian Syndrome Patients in sulaimani Governorate” accomplished by Sakar Karem Abdulla, was prepared under my supervision at Department of Clinical Biochemistry/College of pharmacy/University of Sulaimani in partial fulfillment of the requirements for the degree of Master of Science in Clinical Biochemistry. Signature:
Dr. Ban Mousa Rashid PhD. in Clinical Biochemistry Date: Head of the Department of Clinical Biochemistry Approval In view of available recommendations, I forward this thesis for debate by examining committee. Signature:
Dr. Basima Sadiq Ahmad Assistant Professor Date: Approval of Dean of College of Pharmacy /University of Sulaimani Signature
Dr. Hiwa Khidhir Saaed PhD. in Pharmacology and Toxicology Date:
Examining Committee Certification We, the examining committee after reading this thesis entitled “Evaluation of the Serum Level of Interleukin-18 and Homocysteine in Polycystic Ovarian Syndrome Patients in sulaimani Governorate” and examining the student Sakar Karem Abdulla in its content. We believe, it meets the basic requirements for the degree of Masters of Science in Clinical Biochemistry.
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Name: Dr.Taha Othman Mahwi
Name: Dr. Basima Sadiq Ahmad
Title: Professor
Title: Assistant Professor
Date:
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/2017
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Signature:
Signature
Name: Dr.Chro Najmaldin Fatah
Name: Dr.Ban Mousa Rashid
Title: Assistant Professor
Title: Lecturer
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Dedicated to:
The memory of my father My lovely and merciful mother My dearest husband Rebin My supportive supervisor All my dear sisters and brothers All my helpful and best frien
Acknowledgments First of all, I am grateful to ALLAH for giving me the strength and power for completing this research. I would like to show my gratitude to my supervisor Dr. Ban Mousa Rashid for her kindness, guidance and support throughout the course of the study, her enthusiasm and interest in my project made me complete my thesis easily. Special thanks to College of Pharmacy and Biochemistry Department. My special thanks are extended to the staff of Central Medical Laboratory one, to gynecologist in private clinic also for female in school of Sulaimani for helping me to collect sample for my research. Thanks to all staff of Sarezh lab. Especially Mr. Soran for academic support. Thanks for Mr.Shaho Muhammad for his help in statistical analysis. Thanks a lot to entire patients who participated in this study. My special thanks to my parents for making the person I am today and for their constant support during my study. My special gratitude for my lovely husband for giving me unflinching encouragement, confidence and indispensable help in my life and work.
I
Abstract Background Polycystic ovary syndrome (PCOS) is one of the most common endocrinopathy in women during their fertile years. It is characterized by: polycystic ovarian morphology, menstrual irregularity, clinical or biochemical hyperandrogenism, increasing the risk for developing type 2 diabetes and obesity. PCOS is a complex disorder influenced by both environmental and genetic factors. Diagnosis of PCOS can be established when at least two of the three following criteria are present: clinical and/ or biochemical signs of hyperandrogenism, menstrual irregularity and polycystic ovaries observed in an ultrasound examination.
Aim of the Study The aim of this study is to evaluate the serum level of interleukin-18 and homocysteine in polycystic ovary syndrome patients and compares these values with healthy control group.
Methods This study was case-control study and serum samples randomly taken from 300 individuals (150 samples from patients with PCOS and 150 samples from healthy controls). Five milliliters of venous blood has been taken from each individual and the samples were analyzed for: interleukin-18 and homocysteine by using enzyme linked immunosorbent assay, blood sugar, HbA1c, serum uric acid, and hormones profile include: LH, FSH, LH/FSH ratio, TSH, PRL and Testosterone.
Results The results of hormone level for all cases were as follows: the serum LH level in PCOS was found to be (10.87 ± 7.36 mIU/ml) which was significantly higher than that of the control group (5.73 ± 2.25 mIU/ml), the serum FSH level in PCOS was found to be (5.84 ± 4.60mIU/ml) which was significantly lower than that of the control group (6.86 ± 2.43mIU/ml), the serum LH/FSH ratio in PCOS was found to be (2.09 ± 1.57) which was significantly higher than that of the control group (0.96 ± 0.74), the serum prolactin level in PCOS was found to be (19.67± 10.65 ng/ml) which was significantly higher than that of the control group (16.42 ± 4.52 ng/ml), the serum testosterone level in PCOS was found to be (0.90 ± 2.01 ng/ml) which was II
significantly higher than that of the control group (0.35 ± 0.24 ng/ml) and there was no significant difference in serum TSH level in PCOS (2.40 ± 1.02 μIU/ml) compare to control (2.47 ± 1.18 μIU/ml).on the other hand, The serum level of HbA1c and serum uric acid in PCOS were (5.53 ± 0.97, 252.72 ± 54.96 μmol/L ) respectively which was significantly higher than that of the control group (5.33 ± 0.73, 237.65 ± 47.04 μmol/L). Blood sugar level in PCOS and control was (5.67 ± 1.34 mmole/L, 5.51 ± 0.75mmole/L) respectively, which of no significant difference. The serum IL-18 was found to be (378.3 ±181.21 pg/ml) in PCOS and (224.98± 131.885 pg/ml) in control group which of highly significant difference (p-value < 0.001), Furthermore, There was weak correlation of IL-18 with BMI, FSH, PRL, Testosterone, moderate correlation of IL-18 with LH level and no correlation of IL-18 concentration with age in PCOS. The serum level of homocysteine was (10.36 ± 5.98 nmol/ml) in PCOS and (5.17± 5.24 nmol/ml) in control group which of highly significant differences (P-value < 0.001), furthermore, There was a significant correlation of serum Hcy with age, LH and Testosterone level, while no correlation of serum Hcy concentration with BMI, FSH and PRL level.
Conclusions Serum IL-18 and Hcy concentration significantly higher in PCOS patients compared with control group. There was correlated of IL-18 with BMI, LH, FSH, PRL and testosterone levels, but not correlated with age in PCOS. There was correlated of homocysteine with age, LH and Testosterone level, while not correlated with BMI, FSH and PRL in PCOS.
III
List of Contents Subject Titles
Page NO.
List of Contents ………………………………………………………….……………....IV List of Figures …………………………………………………………....................... IX List of Tables ……………………………………………….……………………….. X List of Abbreviations …………………………………….......................................... XI
Chapter One: Introduction and Literature Review 1.1
Polycystic ovary syndrome …………………………………………………... …….…1
1.2
The Clinical Manifestation of Polycystic Ovarian Syndrome ………………..…….....1
1.2.1
Hyperandrogenism …………………………………………………………………….1
1.2.1.1
Hirsutism ……………………………………………………………………...............1
1.2.1.2
Acne …………………………………………………………………………...............2
1.2.1.3
Alopecia …………………………………………………………………….………....2
1.2.1.4
Consequences of hyperandrogenism …………………………………………………..2
1.2.1.5
Steroidogenesis …………………………………………………………..........………3
1.2.1.5.1 Ovarian steroidogenesis ………………………………………………….………........3 1.2.1.5.2 Adrenal steroidogenesis …………...………………………………………..…...…....5 1.2.2
Menstrual Irregularity …….…………………………………………………..…….....7
1.2.3
Polycystic Ovarian Morphology ……………….…………………………………...…7
1.2.4
Obesity …………………………………………………………………….……..........7
1.2.5
Insulin Resistance ……………………………………………………..………............8
1.2.6
Infertility in PCOS ………………………………………………………………….....8
1.2.7
Risk of Cardiovascular Disease ……………………………………….……………....8
1.3
Etiology of PCOS ……………………………...………………………………….…...9
1.4
The Phenotypic Spectrum of Polycystic Ovary Syndrome …………………………...10
1.5
Pathophysiology of Polycystic Ovarian Syndrome ………………………..................10
1.5.1
Abnormal Pituitary Function ……………………………………….………………...10 IV
1.5.2
Abnormal Steroidogenesis ………………………………………………………........11
1.5.3
Insulin Resistance and Hyperinsulinemia …………………………………………....13
1.5.4
Oxidative Stress …………………………………………………………………..…..13
1.5.5
Sympathetic Nerve Activity ………………………………………………………......14
1.6
Diagnosis of Polycystic Ovarian Syndrome ……………………………….................14
1.7
Diagnostic Approach for Polycystic Ovarian Syndrome ………………….................15
1.7.1
Screening for polycystic ovary syndrome …………………………………………….15
1.7.2
Laboratory Evaluations ………………………………………………………….........16
1.7.3
Ultrasound ………………………………………….……………...............................16
1.8
Interleukin-18 ………………………………………………………………………....16
1.8.1
Structure of Interleukin-18 ……………………………………………………....…....17
1.8.2
Biological Functions of IL-18 …………………………………………………….......17
1.8.3
Pro IL-18 and activation by caspase-1 …………………………………………….....18
1.8.4
Interleukin-18 Receptors ………………………………………………………...…...20
1.8.5
Elevate Circulating Level of IL-18 ………………………………………………......20
1.8.6
Mechanism for IL-18 in the Development of Cardiovascular Disease ……………....20
1.8.7
Pathology of IL-18 in Some Disease ………………………………………………...21
1.9
Homocysteine ……………………………………………………………………...…21
1.9.1
Pathways of Methionine and Hcy Metabolism ………………………………...….....22
1.9.2
Homocysteine in Polycystic Ovarian Syndrome ……………………………………..25
1.9.3
Role of Homocysteine in the Development of Cardiovascular Disease ……………..25
1.9.4
Mechanism for Homocysteine in the Development of Cardiovascular Disease …….25 Aim of the Study ……………………………………………………………….........27
V
Chapter Two: Materials and Methods 2.1.
Materials ………………………………………………………...…………………..29
2.1.1.
Laboratory Equipment /Instruments …………………………………….…………..29
2.1.2.
Reagents and Kits ……………………………………………..................................30
2.2.
Study Design ………………………………………………………………………...31
2.3.
Methods ………………………………………………………………......................32
2.3.1.
Sample collection …………………………………………………………………....32
2.3.2.
Human Serum IL-18 ……………………………………………………………...…32
2.3.3.
Human Serum Homocysteine ………………………………....................................36
2.3.4.
Blood Sugar Measurements …………………………………………………............39
2.3.5.
HbA1c Measurements …………………………………………………………….....39
2.3.6.
Uric Acid Measurements ……………………………………………………………40
2.3.7.
Measurement of Follicle Stimulating Hormones …………………………………....41
2.3.8.
Measurement of Luteinizing Hormone ...…………………………………………...42
2.3.9.
Measurement of Testosterone …………………………………………………...…..43
2.3.10.
Measurement of Prolactin ………………………………………………………...…44
2.3.11.
Measurement of Thyroid Stimulating Hormone ………………………………….....45
2.4.
Statistical Analysis ……………………………………………………………...…...47
VI
Chapter Three: Results 3.1.
Clinical Symptoms of Polycystic Ovarian Syndrome ………………………………….49
3.2.
Hormones Level in PCOS and Control Group……………………………………….....50
3.3.
BMI and Age Distribution among Cases and Control group ………………………..…52
3.4.
Age of Menarche ……………………………………………………………................53
3.5.
Family History of PCOS …………………………………………………………….....53
3.6.
Concentration of Blood Sugar, HbA1c and Serum Uric Acid in PCOS Patients and Control Group……………………………………………………….……………........54
3.7.
Concentration of Serum IL-18 in PCOS Patients and Control Group …………………55
3.8.
Concentration of Serum Homocysteine in PCOS Patients and Control Groups ……....56
3.9.
Correlation between Serum IL-18 with age, BMI, LH, FSH, Prolactin and Testosterone Level in PCOS ………………………………………………………………....………57
3.10.
Correlation between Serum Homocysteine with age, BMI, LH, FSH, Prolactin and Testosterone Level in PCOS …………………………………………………….……58
VII
Chapter Four: Discussion and Conclusion 4.1.
Prevalence Symptoms of PCOS in PCOS Patients …………………………….............60
4.2.
Hormones Level in PCOS and Control Group ……………………………………....…60
4.3.
Age and BMI in PCOS and Control Group …………………………………………….61
4.6.
Age of Menarche ……………………………………………………………….……....62
4.7.
Family History of Polycystic Ovarian Syndrome …………………………………..….62
4.6.
HbA1c and Blood Sugar Level in PCOS Patients and Control Groups ……………..…62
4.7.
Serum Uric Acid in PCOS and Control group ……………………………………….....63
4.8.
Concentration of Serum IL-18 in PCOS Patients and Control Group ……………….....64
4.9.
Concentration of Serum Homocysteine in PCOS and Control Group ………………….64
4.10.
Correlation between Serum IL-18 with age, BMI, LH, FSH, Prolactin and Testosterone Level in PCOS ……………………………………………………..….………………...65
4.11.
Correlation between Serum Homocysteine with age, BMI, LH, FSH, Prolactin and Testosterone Level in PCOS ……………………………………………………..……...65
4.12.
Conclusions ………………………………………………………………………….…..66 Recommendations ……………………………………………………………………….67 References ………………………………………………………………………..……...68
VIII
List of Figures
Figure
Figure Title
No.
Page No.
1.1
production of androgen hormones by ovarian follicular cells
4
1.2
Steroidogenesis occurring both in gonads and adrenal gland of human origin
6
1.3
Abnormal Steroidogenesis in PCOS
12
1.4
Structure of interleukin-18
17
1.5
Interleukin-18 processing stimuli
19
1.6
Structure of homocysteine
22
1.7
A representation of the methionine cycle, remethylation pathway and transsulfuration pathway
24
2.1
Illustration of the study design and case numbers
31
2.2
Principle of human serum IL-18 measurements by ELISA
33
2.3
External standard dilution of IL-18
35
3.1
Concentration of serum IL-18 in PCOS patients and control group
56
3.2
Concentration of serum homocysteine in PCOS patients and control group
56
IX
List of Tables Table
Table Title
No.
Page No.
1.1
Four major clinical phenotypes of polycystic ovary syndrome
10
1.2
Guidelines for the diagnosis of polycystic ovary syndrome
15
2.1
List of laboratory equipment and instrument
29
2.2
List of reagents and kits
30
2.3
Test protocol for human serum IL-18
35
2.4
Procedure for Hcy ELISA test
37
2.5
Dilution of standard solution of Hcy
38
3.1
Frequency distribution of symptoms among polycystic ovarian syndrome
49
cases 3.2
Levels of LH, FSH, LH/FSH ratio, TSH, PRL and testosterone in serum of
51
PCOS and control group. 3.3
Levels of LH and testosterone in PCOS patients.
51
3.4
BMI and Age among cases and control group
52
3.5
Age of menarche in PCOS cases and control group.
53
3.6
Family history of PCOS in first degree relative of control and PCOS cases
53
3.7
Shows mean, standard deviation and p-value for blood Sugar, HbA1c and
54
s.uric acid in PCOS and control group 3.8
Serum uric acid concentration in PCOS cases and control group according
55
to BMI 3.9
Correlation of IL-18 with Age, BMI, LH, LH and FSH, prolactin and
57
testosterone level in PCOS. 3.10
Correlation of homocysteine with Age, BMI, LH, FSH, prolactin and testosterone level in PCOS
X
58
List of Abbreviations Abbreviations Meaning ACTH
Adrenocorticotropic Hormone
AES
Androgen Excess and Polycystic Ovarian Syndrome Society
ASRM
American Society for Reproductive Medicine
BMI
Body Mass Index
CAP
Congenital Adrenal Hyperplasia
CBS
Cystathionine-B-Synthase
CRP
C-Reactive Protein
CVD
Cardiovascular Disease
DAMPS
Danger-Associated Molecular Patterns
DHEA
Dehydroepiandrosterone
DHEAS
Dehydroepiandrosterone Sulfate
EDTA
Ethylene-Diamine-Tetraacetic Acid
ELISA
Enzyme-Linked Immunosorbent Assay
ESHRE
European Society for Human Reproduction and Embryology
FAH
Functional Adrenal Hyperandrogenism
Fas ligand
Type II transmembrane protein, belongs to tumor necrosis factor (TNF) family
FOH
Functional Ovarian Hyperandrogenism
FSH
Follicle Stimulating Hormone
GnRH
Gonadotropin-Releasing Hormone
HbA1c
Glycated Haemoglobin
HDL
High Density Lipoprotein
HMGCOA
3-Hydroxy-3-Methyl-Glutaryl-COA Reductase
Reductase HSD
Hydroxysteroid Dehydrogenase
HT
Hypertension
IFN-γ
Interferon-Gamma XI
IgE
Immunoglobulin E Antibody
IGF-1
Insulin-Like Growth Factor One
IL
Interleukin
IL-18BP
Interleukin-18 Binding Protein
IL-18R- β
Interleukin-18 Receptor-Beta
IL-18R-α
Interleukin-18 Receptor-Alpha
IRAK
Interleukin-1 Receptor-Associated Kinase
LDL
Low Density Lipoprotein
LH
Luteinizing Hormone
PRL
Prolactin
MAPK
Mitogen-Activated Protein Kinase
MetS
Metabolic Syndrome
MMP
Matrix Metalloproteinases
MTHF
Methyl tetrahydrofolate
MTS
Methyltransferases
MYD88
Myeloid Differentiation Factor-88
NFKB
Nuclear Transcription Factor-kB
NGF
Nerve Growth Factor
NICHD
National Institute of Child Health and Human Development
NIH
National Institutes of Health
NK Cell
Natural Killer Cell
NLR
NOD-Like Receptor
NLRP3
NOD-Like Receptor Protein-3
NOD
Nucleotide Oligomerization Domain
OHP
Hydroxyprogesterone
PAMPS
Pathogen-Associated Molecular Patterns
PCOM
Polycystic Ovarian Morphology
PCOS
Polycystic Ovarian Syndrome
PLP
Pyridoxal phosphate
PRRS
Pattern-Recognition Receptor
PWV
Pulse Wave Velocity
XII
SAH
S-Adenosyl Homocysteine
SAM
S-Adenosyl methionine
SES
Socio-economic Status
SHBG
Sex Hormone Binding Globulin
SR-B1
Scavenger Receptor-B1
STAR
Steroid Acute Regulatory Protein
T cells
T lymphocytes
T2DM
Type 2 Diabetes Mellitus
Th1
Type 1 T helper
Th2
Type 2 T helper
TLRS
Toll-Like Receptors
TNF
Tumor Necrosis Factor
TRAF6
Tumor Necrosis Receptor-Associated Factor-6
US
Ultrasound
VLDL
Very Low Density Lipoprotein
XIII
CHAPTER ONE INTRODUCTION AND LITERATURE REVIEW
Chapter one
Introduction and Literature Review
1. Introduction 1.1. Polycystic ovary syndrome Polycystic ovary syndrome is the most common endocrine disease in women of childbearing age. The prevalence ranges from 9% when the NIH (National Institutes of Health) criteria are used to 18% when use the guidelines of the Rotterdam consensus (March et al., 2010,). It is characterized by hyperandrogenism, menstrual irregularity, polycystic ovarian morphology (PCOM), insulin resistance with compensatory hyperinsulinemia, obesity and increase the risk for developing type 2 diabetes, endometrial carcinoma and cardiovascular disease (Ali, 2015).
1.2. The Clinical Manifestation of Polycystic Ovarian Syndrome The clinical manifestation of Polycystic Ovarian Syndrome includes:
1.2.1. Hyperandrogenism Clinical hyperandrogenism is defined by the presence of hirsutism, acne, or androgenic alopecia. NIH (National Institute of Health) and AES (Androgen Excess and PCOS Society) guidelines both require clinical and/or biochemical hyperandrogenism for the diagnosis of PCOS in adults, while the Rotterdam guideline recognize a phenotype of PCOS without androgen excess (Azziz et al., 2006). The actual prevalence of hyperandrogenemia among women with PCOS is debatable since there is no definitive agreement on which androgen should be measured, when and how often they should be measured, normal androgen levels in women, and which analytical techniques should be used (Barth et al., 2007). Approximately 60% to 80% of women with PCOS demonstrate elevated circulating androgen levels and symptom of hyperandrogenemia (Azziz et al., 2006). Serum levels of free testosterone are more frequently elevated in women with PCOS. Therefore, it is considered as the most sensitive biochemical marker for diagnosis of PCOS (Escobar-Morreale et al., 2001).
1.2.1.1. Hirsutism Hirsutism is the most common clinical manifestation of hyperandrogenism in women, which is defined as excessive terminal hair growth that takes on a male pattern distribution, hair 1
Chapter one
Introduction and Literature Review
growth in PCOS tends to be more gradual and commonly occurs following cessation of longterm hormonal contraceptive use (Rosenfield, 2005). Approximately 60% to 70% of women with PCOS have hirsutism (Azziz et al., 2006). Age and ethnicity significantly also influence hair growth due to genetic variances in 5 α-reductase activity which is enzyme in adipose tissue and convert testosterone to more potent androgen dihydrotestosterone (Williamson et al., 2001). Excessive hair growth in women is generally quantified by the Ferriman-Gallwey scoring system, this system grades terminal hair growth on a scale from 0 to 4 on 11 anatomical sites and uses the sum of nine areas to generate an overall hirsutism score. Scores of ≥ 8 or ≥ 5 have been commonly accepted as abnormal, while recently a score as low as 3 was proposed as the upper limit of normal (DeUgarte et al., 2006).
1.2.1.2. Acne One third of women with PCOS particularly younger women demonstrate acne (Azziz et al., 2006). Androgens participate in the development of acne by stimulating sebum production, thereby providing optimal conditions for bacterial colonization with organisms such as Propionibacterium acnes (Burkhart and Gottwald 2003).
1.2.1.3. Alopecia Prevalence of alopecia in women with PCOS approximately 5% which is relatively low compared with other androgenic symptoms (Azziz et al., 2006). Historically, alopecia was recognized as a symptom of PCOS because it is an androgen-mediated process (Lujan et al., 2008), however, it is a poor predictor of biochemical hyperandrogenemia, and low serum iron levels and aging are more common causes of hair loss in women (Barth et al., 2007).
1.2.1.4. Consequences of Hyperandrogenism Androgen excess may also contribute to the cardiovascular risks associated with PCOS, For instance, the dyslipidemia of PCOS correlates with hyperandrogenemia, also hyperandrogenemia independent risk factor for the development of hypertension among women with PCOS (Chen et al., 2007).
2
Chapter one
Introduction and Literature Review
1.2.1.5. Steroidogenesis Since androgen excess is the main feature of PCOS so great importance to clearly define how these androgens are normally produced. In healthy women 25% of androgen production is of ovarian origin, 25% is of adrenal origin and 50% is produced in peripheral tissues such as adipose tissue and skin (Vink et al., 2006). Androgens are part of the steroid hormone family. In both human tissues ovary and adrenal gland cholesterol is the precursor for pregnenolone being then converted to steroid hormones following a series of enzymatic processes (Figure 1.2). Cholesterol can be delivered either by circulating lipoproteins (mostly low-density lipoproteins) or by de novo biosynthesis via the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoA reductase). The cholesterol side-chain cleavage catalyzed by cytochrome P450 enzyme, this step considered as the rate-limiting step in steroidogenesis ( Wood and Strauss, 2002).
1.2.1.5.1. Ovarian steroidogenesis In the ovary the resulting pregnenolone is metabolized in two pathways: First pathway androgen formation are performed in LH-stimulated thecal cells, as these cells express the cytochrome P450c17 gene which exhibits both hydroxylase and lyase activity and catalyst the first two steps which is Hydroxylation of pregnenolone at the C17a position yields 17hydroxypregnenolone, and subsequent removal of the acetyl group forms the androgen precursor dehydroepiandrosterone
(DHEA),
Then
dehydroepiandrosterone
(DHEA)
convert
to
androstenedione by 3β-hydroxysteroid dehydrogenase after that androstenedione synthesized converted to testosterone by the action of the enzyme 17β-hydroxysteroid dehydrogenase in theca cells under the LH stimulus, and then most of these precursors will be passively transported to the granulose cells where it is converted in estrogen by the action of P450aromatase under the FSH stimulus (Drummond, 2006) (Figure 1.1), But ovaries will also directly secrete androgens in the circulation mainly as androstenedione and testosterone. Ovarian androgens will not significantly feedback on LH production, such that an excess in free testosterone or androstenedione will not reduce ovarian production of these androgens in women (Mendelson and Kamat, 2007). The second pathways for pregnenolone metabolism in the ovary include conversion of pregnenolone to progesterone by the action of 3β-HSD. This conversion is essentially irreversible, Progesterone can then be converted to 17-hydroxyprogesterone by 3
Chapter one
Introduction and Literature Review
CYP17 then very little 17-hydroxyprogesterone is converted to androstenedione (Sanderso, 2006).
Figure 1.1: production of androgen hormones by ovarian follicular cells (Drummond, 2006)
4
Chapter one
Introduction and Literature Review
1.2.1.5.2. Adrenal steroidogenesis The cortex of the adrenal gland is composed of three layers and each has distinct enzymatic cascades, the outer part (zona glomerulosa) has the capacity to secrete mineralocorticoids, such as aldosterone. In humans, the inner parts (zona fasciculata and zona reticularis) produce androgen such as DHEA and androstenedione. The zona fasciculata is relatively less efficient in producing androgen and thus secretes mainly glucocorticoids, namely cortisol (Miller, 2008).The most potent stimulus of adrenocortical cells is adrenocorticotropic hormone (ACTH), which induces a substantial increase in all steroids, adrenal androgens do not significantly feedback on ACTH production, which is mainly under the control of cortisol (Sewer and Waterman, 2003). Free cholesterol is transferred from the outer mitochondrial membrane to the inner mitochondrial membrane by Steroid Acute Regulatory protein (STAR) then followed by the conversion of cholesterol to pregnenolone by P450scc enzyme which is present in the inner mitochondrial membrane of all steroidogenic cells (Payne and Hales, 2004). Pregnenolone by 17α-hydroxylase convert to 17α- hydroxypregnenolone, which is converted to 17α-OH-progesterone by 3β-hydroxysteroid dehydrogenase. Thereafter, 17αhydroxyprogresterone is converted to 11-deoxycortisol by the enzyme 21-hydroxylase and finally 11-deoxycortisol is converted to cortisol by the enzyme 11β-hydroxylase. Another pathway is the conversion of 17α-hydroxyprogesterone to androstenedione by the enzyme 17, 20 lyase. In addition, 17α-hydroxypregnenolone can be converted to dehydroepiandrosterone (DHEA) by the action of the enzyme 17, 20 lyase. DHEA is then converted to DHEAS sulfate by a reversible adrenal sulfokinase. DHEA is also converted to androstenedione by 3βhydroxysteroid dehydrogenase (Payne and Hales, 2004) (Figure 1.2). Androstenedione, DHEA and DHEA sulfate are secreted from the adrenal cortex and bind mainly to albumin. after entering the circulation adrenal androgens can lead to two different pathways: First pathway degradation and inactivation through conjugation of androgens to glucuronate or sulfate residues to produce hydrophilic glucuronides or sulfates that are excreted in the urine, second pathway peripheral conversion of androgen to their more potent derivatives testosterone and dihydrotestosterone by the enzymes 17β -hydroxysteroid dehydrogenase (17βHSD) and 5-reductase, respectively (Fassnacht et al., 2003).
5
Chapter one
Introduction and Literature Review
Figure 1.2: Steroidogenesis occurring both in gonads and adrenal gland of human origin (Payne and Hales, 2004).
6
Chapter one
Introduction and Literature Review
1.2.2. Menstrual Irregularity Menstrual irregularity is found in about two thirds of adolescents with PCOS. These girls may present with oligo menorrhea (means menstrual bleeding that occurs at intervals over 40 days or fewer than 9 periods yearly ), primary amenorrhea (the absence of menarche by 16 years of age), secondary amenorrhea (the absence of menses for at least 3 months), dysfunctional uterine bleeding (excessive and irregular vaginal bleeding). Furthermore, monthly menstrual cycles may still be an ovulatory, which is suggested when there is a paucity of menstrual cramps, absence of premenstrual molimina (breast tenderness, lower abdominal bloating, or moodiness), and menorrhagia (excessive menstrual bleeding) (Nair et al., 2012). There is evidence that greater menstrual irregularity is associated with a more severe PCOS phenotype and higher androgen levels, while adolescents with primary amenorrhea and PCOS have increased features of metabolic syndrome and higher androstenedione compared with those with oligo menorrhea (Rachmiel et al., 2008).
1.2.3. Polycystic Ovarian Morphology PCOM define in both the AES-PCOS and Rotterdam diagnostic criteria for PCOS as 12 or more follicles of 2 to 9 mm or ovarian volume > 10 cm3 in at least one ovary (Rotterdam workshop group, 2004), but Recently Dewailly et al suggested that the former threshold of greater than 12 follicles per ovary may even be too low and a count of 19 follicles would be needed for more accurate diagnosis (Dewailly et al., 2011), in married patients transvaginal US is the standard imaging method used to evaluate ovarian morphology, while trans abdominal US (TA-US) is appropriate method for use in adolescents (Hardy and Norman, 2013).
1.2.4. Obesity Obesity is present in approximately half of patients with PCOS which is android in type (Baumann E et al., 2002), but there is unclear whether PCOS predisposes women to obesity or whether obesity exacerbates PCOS (Hoeger and Oberfield, 2012). Independent of BMI, women with PCOS have been reported to have a high prevalence of upper-body obesity (android) as demonstrated by increased waist circumference and waist-hip ratio compared to BMI-matched control women (Kirchengast and Huber, 2001), This related to chronic exposure to higher testosterone levels which modify body fat distribution in these 7
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women (Sam, 2007). In PCOS patients levels of the sex hormone-binding globulin (SHBG) tend to linearly decrease with increasing body fat, and this may lead to an increased fraction of free androgens delivered to target sensitive tissues (Gambineri et al., 2002).
1.2.5. Insulin Resistance Insulin resistance and hyperinsulinemia appears in both obese and no obese women with PCOS, in a prevalence of (30%) in no obese women and (70%) in obese women. These insulin abnormalities play a significant role in the pathogenesis of PCOS (Chandrika N. Wijeyaratne et al., 2002), Insulin resistance appear to stem from intrinsic abnormalities of post-receptor insulin signaling (e.g. excess serine phosphorylation), abnormal insulin secretion or polymorphic genes controlling insulin action (Tucci et al., 2001), There are several factors influence insulin sensitivity, including ethnicity, history of diabetes mellitus and BMI (Wilcox, 2005), In PCOS increased abdominal adiposity is a common feature that impairs insulin sensitivity and it is largely responsible for the increased insulin resistance (Escobar-Morreale and Millán, 2007).
1.2.6. Infertility in PCOS Infertility most common in PCOS patients, 90% of women with PCOS and infertility are overweight means obesity independently exacerbates infertility, reduces efficacy of infertility treatment and induces a greater risk of miscarriage (Brassard et al., 2008). Infertility is the consequence of chronic anovulation, being usually accompanied by menstrual abnormalities, also the presence of regular menstrual bleeding doesn't exclude anovulation because twenty one percent of the patients that are anovulatory have regular menstrual cycles (March et al., 2010) . PCOS patients also have more frequently pregnancy associated pathology for example increased risk of spontaneous abortion, gestational diabetes, and pregnancy induced hypertension, the major causes for this complication are hyperandrogenism and obesity (Kousta et al., 2000).
1.2.7. Risk of Cardiovascular Disease Women with PCOS are at increased risk for dyslipidemia, hypertension, and related cardiovascular morbidity and possibly mortality (Dokras, 2013). The dyslipidemia of PCOS is characterized by elevated plasma levels of cholesterol, low density lipoproteins (LDL), very low density lipoproteins (VLDL), and triglycerides with concomitantly reduced concentrations of 8
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high density lipoproteins (HDL), The causes of dyslipidemia in PCOS are again multifactorial but insulin resistance have a pivotal role by stimulation of lipolysis and altered expression of lipoprotein lipase and hepatic lipase (Hong et al., 2011). Hyperhomocysteinemia represents another independent risk factor for the development of cardiovascular disease; Moderate hyperhomocysteinemia predisposes individuals to endothelial dysfunction via a mechanism involving increased oxidative stress (Brattström and Wilcken, 2000). Both symptomatic and asymptomatic women with PCOS have signs of significant vascular impairment. For example carotid artery vascular compliance is decreased, while arterial stiffness is increased (Lakhani et al., 2002), also increased thickness of arterial intima-media with greater prevalence of subclinical significant occlusion in more arterial segments compared to women with normal appearing ovaries (Meyer et al., 2012), also endothelial dysfunction due to impaired vasodilation in hyper androgenic, insulin-resistant women with PCOS when compared with age- and weight-matched controls (Paradisi et al., 2001). A recent study reported that 33% of young obese women with PCOS had evidence of early coronary atherosclerosis compared to 8% of age and weight matched controls (Shroff et al., 2007).
1.3. Etiology of PCOS PCOS is a complex disorder influenced by both environmental and genetic factors (Franks and McCarthy, 2004). Environmental factors have been shown to play a role in the pathogenesis of PCOS for example socio-economic status (SES) and unhealthy behavior, Individuals with lower SES are more at risk for PCOS including smoking, poor diet, lack of exercise and obesity (Thurston et al., 2005,). Smoking and obesity can exacerbate insulin resistance (Cupisti et al., 2010), which is a condition highly correlated with the pathogenesis of PCOS (Ehrmann et al., 2005). In a recent study, Merkin et al. found a correlation between low childhood SES and PCOS (Merkin et al., 2011). Evidence for genetic contribution includes familial clustering of PCOS, increased prevalence of hyperandrogenemia, T2DM in first-degree relatives of women with PCOS (Ehrmann et al., 2005,), Mode of heritance of PCOS remains unclear, and both dominant and multigene modes of transmission have been proposed ( Vink et al., 2006).
9
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1.4. The Phenotypic Spectrum of Polycystic Ovary Syndrome PCOS can be categorized into four main phenotypes (Table 1.1), These categories are useful in clinical practice because health risks have been defined for at least two subtypes, and this dictates careful evaluation of metabolic disturbances for women with frank or classic PCOS (Norman et al., 2007). Table 1.1: Four major clinical phenotypes of polycystic ovary syndrome (Norman et al., 2007). Frank PCOS
Classic PCOS
✓
✓
Chronic anovulation
✓
✓
Polycystic ovaries
✓
Biochemical /clinical Hyperandrogenism
Prevalence Long-term health risks
Ovulatory PCOS
Mild PCOS
✓ ✓ ✓
✓ 7–16%
46–71%
7–40%
7–18%
known
known
unknown
Unknown
1.5. Pathophysiology of Polycystic Ovarian Syndrome Pathophysiology of PCOS includes:
1.5.1. Abnormal Pituitary Function Disordered regulation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) release has been implicated in the pathogenesis of PCOS. In normal women, the hypothalamic gonadotropin-releasing hormone (GnRH) pulses cause LH and FSH release. LH then stimulates ovarian theca cells to produce androgens (mainly androstenedione) and FSH stimulates granulosa cells to convert the androstenedione to estrone and estradiol, estrogen and progesterone provide negative feedback to GnRH-secreting neurons as well as the pituitary (Tsutsumi and Webster, 2009). In patients with PCOS, LH is secreted at a higher rate in relation to FSH, with resultant increased thecal production of androgens, specifically androstenedione, but in the face of inadequate FSH stimulation of granulosa cell development and aromatase production, these 10
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androgens were not converted to estrogen so more androgen is then available for peripheral tissue conversion to testosterone by 17 β-hydroxysteroid dehydrogenase, the enzyme that converts androstenedione to testosterone. Then androgen excess may counteract the LHsuppressive role of female hormones as well as regulation of GnRH neurons by progesterone (Eagleson et al., 2000).
1.5.2. Abnormal Steroidogenesis Another hypothesis suggests that PCOS is attributable to androgen excess, which arises from primary functional ovarian hyperandrogenism (FOH) and primary functional adrenal hyperandrogenism (FAH). Primary FOH and primary FAH appear to be caused by dysregulation of steroidogenesis (Buggs and Rosenfield, 2005). In normal women androgens are produced equally from both adrenal glands and ovaries But in women with PCOS the ovaries are usually the major source of androgens (especially androstenedione) (Buggs and Rosenfield, 2005). Women with PCOS decrease the liver production of sex hormone-binding globulin (SHBG), the major circulating protein that binds testosterone, thus increasing the free (biologically active) testosterone level. These hormonal abnormalities might be related in part to obesity (Lim et al., 2013). Extra glandular synthesis of androgens particularly in the adipose tissue has been found to be involved in the pathophysiology of PCOS, Adipose tissue contains 17β-hydroxysteroid dehydrogenase type 5 (17HSD5), which can convert the weak androgen (androstenedione) to the more potent androgen (testosterone), Expression of 17HSD5 in subcutaneous adipose tissue proportional to overall adiposity, Thus, excessive adiposity in PCOS women may contribute to increased peripheral production of testosterone (Quinkler et al., 2004), Also in PCOS increased 5α-reductase
activity
which
converts
testosterone
to
the
highly
potent
androgen
dihydrotestosterone (Fassnacht et al., 2003), Its activity appears to correlate with both adiposity and insulin concentrations (Figure 1.3) (Vassiliadi et al., 2009).
11
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Figure 1.3: Abnormal Steroidogenesis in PCOS (Rosenfield, 2001).
12
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1.5.3. Insulin Resistance and Hyperinsulinemia Hyperinsulinemia resulting from insulin resistance has an important role in the pathogenesis of PCOS at several levels (Baillargeon, 2005), Insulin stimulates the production of androgens by ovarian thecal cells because insulin has a direct synergistic effect with LH on the theca cells in enhancing androgen production also high insulin levels increase LH secretion from the pituitary, elevating the LH/FSH ratio, and further contributing to anovulation (Balen, 2004), Hyperinsulinemia further decreases SHBG levels, elevating free testosterone level, Insulin also appears to potentiate basal and adrenocorticotropic hormone (ACTH), stimulated adrenal androgen production (Yildiz and Azziz, 2007).
1.5.4. Oxidative Stress Oxidative stress which is an imbalance between the production of reactive oxygen species and antioxidant evaluate by circulating markers, such as malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx)(Murri et al., 2013). Oxidative stress involve in pathophysiology of PCOS (Orio et al., 2005) and recent evidence suggests that inflammatory cytokines and measures of oxidative stress may play a role in the dysregulation of the thecainterstitial compartment, and both of these factors are elevated in PCOS. In addition to simulating proliferation of theca-interstitial cells, moderate oxidative stress also induces testosterone and progesterone production by enhancing thecal expression of key steroidogenic enzymes, such as CYP17, CYP11A1, Thus the excess androgen production in PCOS is not only due to increased numbers of theca cells, but also to an induction of their steroidogenic capacity (Sabuncu et al., 2001,). Oxidative
stress
also
impairs
insulin
signaling
resulting
in
a
compensatory
hyperinsulinemia, which, Stimulates thecal steroidogenesis (Murri et al., 2013), another significant result of oxidative stress is endothelial dysfunction and the subsequent development of cardiovascular disease (Anderson and Smith, 2005). oxidative stress have a pivotal roles in pathogenesis of some malignant lesions such as endometrial cancer, breast cancer, and ovarian cancer in PCOS patients (Krstić et al., 2015). ROS could cause genetic changes by attacking DNA, leading to DNA damages, such as DNA strand breaks, point mutations, aberrant DNA cross-linking, and DNA-protein cross-linking, As a result, the mutations in protooncogenes and tumor suppressor genes probably hijacked cell 13
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proliferation out of control, when the DNA repair mechanism has been disrupted. On the other hand, OS could cause epigenetic changes as well by DNA methylation, silencing tumor suppressor genes (Bartsch H. and Nair J.).
1.5.5. Sympathetic Nerve Activity involvement of sympathetic nervous system in PCOS pathology is supported by the greater density of catecholaminergic nerve fibers in polycystic ovaries (Heider et al., 2001), Increased ovarian sympathetic nerve activity cause stimulating androgen secretion (Dissen et al., 2000), so the degree of androgen concentration can reflect the severity of PCOS (Sverrisdottir et al., 2008), Nerve growth factor (NGF) is a strong marker for sympathetic nerve activity and recently it was demonstrated that women with PCOS has enhanced ovarian NGF production (Dissen, G. A et al., 2009).
1.6. Diagnosis of Polycystic Ovarian Syndrome PCOS is a syndrome because it is not a specific disease and no single criterion can define its diagnosis. There are three Guidelines for the diagnosis of polycystic ovary syndrome as shown in (Table 1.2) (Dantas et al., 2013). In 1990, the first attempt to consolidate a clinical definition of PCOS by the National Institute of Child Health and Human Development resulted in PCOS being defined as the combined presence of androgen excess and oligo-anovulation with the exclusion of other etiologies of androgen excess and an ovulatory infertility (Carmina, 2004). In 2003, The European Society for Human Reproduction and Embryology and the American Society for Reproductive Medicine amended the consensus criteria for diagnosis of PCOS, The diagnosis can be established when at least two of the three following criteria are present with the exclusion of other etiologies: clinical and/ or biochemical signs of hyperandrogenism, oligo- and anovulation and polycystic ovaries (PCO) observed in an ultrasound examination which is determined by the presence of ≥12 follicles within the ovary with a diameter of 2–9 mm and/or ovarian volume ≥10 cm, Such an ultrasound image in one gonad only is sufficient to define polycystic ovaries (Rotterdam workshop group, 2004). In 2006, Androgen Excess and PCOS Society agreed that androgen excess is crucial in the pathogenesis of PCOS. They accepted this basic criterion as obligatory, with the following signs: 14
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oligo- and anovulation and/or polycystic ovaries observed in a US examination. Also exclusion of other etiologies is necessary (Azziz et al., 2006). Table 1.2: Guidelines for the diagnosis of polycystic ovary syndrome (Dantas, Gualano et al. 2013). NIH 1990 PCOS
diagnosis
Rotterdan 2003 in
present of both criteria:
the PCOS diagnosis in the present of PCOS diagnosis in the present 2 of the 3 criteria:
Menstrual dysfunction and Menstrual hyperandrogenemia exclusion of other causes
AES 2006
of both criteria: dysfunction, Menstrual
after hyperandrogenemia
dysfunction
or
and polycystic ovary morphology
polycystic ovary morphology with hyperandrogenemia after after exclusion of other causes
exclusion of other causes.
1.7. Diagnostic Approach for Polycystic Ovarian Syndrome Approach for diagnosis of PCOS includes:
1.7.1. Screening for polycystic ovary syndrome Polycystic ovary syndrome will be suspected in adolescents with hirsutism, acne, menstrual irregularity, or obesity. The history and physical examination should be taked to rule out the disorders that mimic PCOS which includes: non-classic congenital adrenal hyperplasia (CAH) which suspecte in Premature pubarche or prepubertal acne, hyperprolactinemia indicate by Galactorrhea, Virilizing disorders may be indicated by sudden onset and rapid course and defeminization or clitoromegaly, Cushing’s syndrome should be considered in those with a central fat distribution , especially if easy bruisability are present (Buggs and Rosenfield, 2005). The family history should include information regarding infertility, menstrual disorders and hirsutism in female and features of the metabolic syndrome including obesity, glucose intolerance, diabetes, hypertension, cardiovascular disease and stroke. During the physical examination obesity and fat distribution with calculation of body mass index should be noted also the presence of acne, acanthosis nigricans and male pattern baldness. Severity and
15
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distribution of hirsutism is commonly graded using the Ferriman-Gallwey score also size of the thyroid gland should be evaluated (Cash and Glass, 2014).
1.7.2. Laboratory Evaluations Laboratory evaluation for diagnosis of PCOS includes: Measure plasma total testosterone, free testosterone, and another androgen such as , androstenedione and DHEA sulfate while Plasma free testosterone is the single most sensitive test for the detection of androgen excess ( Ozdemir S et al., 2010), Measure the level of LH and FSH in 2nd-5th day of menstrual cycle and thyroid function tests (TSH) in patients with irregular periods or abnormal thyroid physical exam, Also prolactin levels should be measured to exclude a prolactinoma in girls with irregular menses (Sheehan, 2004). Insulin-like growth factor 1 (IGF-1) levels can be measured if a growth hormone-secreting tumor is suspected, In women with PCOS, the hyperinsulinism may stimulate the IGF-1 receptor with resultant overgrowth and other features mimicking a growth hormone secreting tumor (Harwood et al., 2007). A morning 17-hydroxyprogesterone (17-OHP) level should be obtained to screen for late-onset CAH. 17-OHP of ovarian origin normally rises during the second phase of the menstrual cycle with progesterone; it is a major product of the corpus luteum. It is often slightly elevated in PCOS (generally, a value >50 ng/dl suggests hyperandrogenism); however, if the 17-OHP level is >200 ng/dl, CAH may be the cause (Dessinioti and Katsambas, 2009).
1.7.3. Ultrasound The ultrasonography guidelines, supported by the ESHRE/ASRM consensus group define the polycystic ovary as containing 12 or more follicles measuring 2–9 mm and/or an increased ovarian volume of > 10 cm3 (Lujan et al., 2013).
1.8. Interleukin-18 Interleukin-18 (IL-18) is a proinflammatory cytokine that was first described in 1989 for its ability to induce interferon γ (IFN-γ) production, later cloned and found to have a synergistic effect with IL-12 in the production of IFN-γ from T cells, natural killer (NK) cells and macrophages, This synergism is the result of IL-12 inducing the expression of the IL-18 receptor on T cells (Gerdes et al., 2002), IL-18 functionally related to IL-12, but it is structurally similar 16
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to the IL-1 family of cytokines specifically IL-1β, So IL-18 is part of the IL-1 family (Garlanda et al., 2013).
1.8.1. Structure of Interleukin-18 Interleukin-18 folds into a beta-trefoil structure that comprises 12 β-strands (β1-β12) and 2 α-helices (α1-α2) resembles that of IL-1, Presence of three sites that are important for receptor activation: two binding sites for IL-18 receptor alpha (IL-18Ralpha), one binding site for IL-18 receptor beta (IL-18Rbeta). IL-18-induce heterodimerization of receptor subunits, which is necessary for receptor activation (Figure 1.4) (Kato et al., 2003).
Figure 1.4: Structure of interleukin-18(Kato et al., 2003).
1.8.2. Biological Functions of IL-18 L-18 is a potent proinflammatory cytokine which enhances T cell and natural killer cell maturation, as well as the production of cytokines, chemokines and cell adhesion molecules; Function of IL-18 depends on the surrounding cytokine environment. IL-18 with IL-12 or IL-15 enhances the Th1 responses and induces the expression of the Fas ligand on NK cells and IFN-γ via T cells and NK cells, While IL-18 without IL-12 can stimulate Th2 responses, including 17
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allergic inflammation (Nakanishi et al., 2001). L-18 with IL-2 or IL-4 enhances the production of IL-4, IL-13, and immunoglobulin E (IgE) (Muhl and Pfeilschifter, 2004).
1.8.3. Pro IL-18 and activation by caspase-1 Pro IL-18 an inactive precursor of IL-18, is stored in the intracellular space but after being cleaved and processed by caspase-1 into the biologically active cytokine IL-18 is released into the extracellular (Bellora et al., 2012), Pro IL-18 has been observed in various cells, including keratinocytes, dendritic cells, macrophages, Kupffer cells, astrocytes, microglia, intestinal epithelial cells and osteoblasts, also IL-18 receptor (IL-18R) is found on T cells, natural killer (NK) cells, B cells, macrophages, neutrophils, basophils, endothelial cells, smooth muscle cells, chondrocytes, keratinocytes, fibroblasts, melanocytes, and numerous epithelial cells (Airoldi et al., 2004). IL-18 is activated by caspase-1 following formation of the inflammasome, The inflammasome is a macromolecular structure consisting of: an intracellular NOD-like receptor (NLR) such as NLRP3, an adaptor protein, apoptosis speck-like protein containing a caspase recruitment domain (ASC), and procaspase-1 (Mezzaroma et al , 2011), The activation of inflammasome is initiated by recognition of stimuli such as pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) by Pattern-recognition receptors (PRRs), such as Toll-like receptors (TLRs) or nucleotide oligomerization domain (NOD)-like receptors (NLRs) (Carol M. A, 2012), Oligomerization of the nucleotide-binding domain and leucine-rich repeat pyrin domain containing protein-3 (NLRP3) inflammasome induces the activation of caspase-1 from pro-caspase-1 which subsequently cleaves the inactive precursor pro-IL-18 to active cytokine IL-18 (Figure 1.5)(Boraschi and Dinarello, 2006). L-18 is also activated by mast cell chymase, granzyme B, neutrophil-derived proteinase-3 (Omoto et al., 2006), Which after synthesis of biologically active cytokine IL-18 is released into the extracellular, In extracellular IL-18 may be neutralized by a naturally occurring high-affinity IL18 binding protein (IL-18 BP) which is enhanced as a negative feedback mechanism in response to increased IL-18 production for protection of tissue damage due to uncontrolled proinflammatory activity, or may be interact with the IL-18 receptor (IL-18R) (Garlanda et al., 2013).
18
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IL-18 synthesis are stimulate by: hyperglycemia ( Weiss et al., 2011), nuclear transcription factor-κB (NFκB) and interferon-gamma (IFNγ)(Gracie et al., 2003), cathecholamines (Chandrasekar et al., 2004), angiotensin II and inflammation(Sahar et al., 2005).
Figure 1.5: Interleukin-18 processing stimuli (
: pro IL-18,
IL-18Rβ)(Lee et al., 2015). 19
: active IL-18,
: IL-18Rα,
:
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Introduction and Literature Review
1.8.4. Interleukin-18 Receptors IL-18R is a heterodimer, composed of α and β chains. Active IL-18 attaches to the α chain of the IL-18R then IL-18Rβ recruited to form a signaling complex composed of IL-18, IL-18Rα, and IL-18Rβ, After forming the heterodimer the intracellular Toll-IL-1 receptor (TIR) domain binds to myeloid differentiation factor 88 (MyD88) and IL-1 receptor-associated kinase (IRAK). In the NF-κB signaling pathway, phosphorylated IRAK binds to tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), which degrades inhibitor of κB (IκB). Subsequently, NFκB is released (Boraschi and Dinarello, 2006). Although MyD88-IRAK-TRAF6-NF-κB signaling is the major signaling pathway for IL-18, IL-18 also acts through the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) ( JAE, K. et al., 2004).
1.8.5. Elevate Circulating Level of IL-18 Women with PCOS may have chronic low-level inflammation and induce an increase in serum IL-18 levels, which are also associated with visceral adiposity and insulin resistance in PCOS (Escobar-Morreale et al., 2004). Elevated circulating levels of Il-18 also associated with atherosclerotic lesions, type 2 diabetes mellitus (T2DM), metabolic syndrome (MetS), hypertension (HT), and a worse prognosis in cardiovascular disease (CVD)(Espinola-Klein et al., 2008).
1.8.6. Mechanism for IL-18 in the Development of Cardiovascular Disease Interleukin-18 enhances the maturation of T-cells and natural killer cells and the production
of
cytokines,
chemokines,
cell-adhesion
molecules,
IFNγ
and
matrix
metalloproteinases (MMPs) (Gracie et al., 2003, Nold et al., 2003). High level of MMP-9 have been associated with plaque progression ;disability and rupture these various effects exaggerate the inflammatory process promoting atherosclerosis and increase the risk of atherothrombosis and cardiovascular events,IL-18 have been in focus amongst researchers in cardiovascular disease (Koenig and Khuseyinova, 2007).
20
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1.8.7. Pathology of IL-18 in Some Disease IL-18 was involved in pathogenesis of several autoimmune diseases, myocardial function, emphysema, metabolic syndromes, psoriasis, inflammatory bowel disease, macrophage activation syndrome, hemophagocytic syndromes, acute kidney injury and sepsis (Kang et al., 2012). IL-18 stimulates a subset of macrophages to induce cyclooxygenase (COX)-II and, subsequently, prostaglandins (PGs), which mediate various biological responses, including pain . It has been reported recently that the peritoneal fluid of endometriosis patients contains elevated levels of PGs (Kashiwamura et al., 2002) . Polymorphisms of IL-18 gene involved in the association of recurrent miscarriage(AlKhateeb et al., 2011), which is the occurrence of three or more repeated pregnancies that end in loss of the fetus, usually before 20 weeks of gestation and common in PCOS patients (Branch et al., 2010). IL-18 play a crucial role in the pathogenic network in endometriosis (Oku H. et al., 2004). Endometriosis is one of the common gynecological diseases, which is the presence of tissue with epithelial and stromal characteristics of endometrium outside the uterine cavity. It is a benign disease and cause adhesion, inflammation and pain during menstruation, and even infertility (Kashiwamura et al., 2002). IL-18 also implicated in the Pathogenesis of Epithelial ovarian carcinoma (EOC). which is the most frequent cause of death from gynecologic malignancies and the fifth leading cause of death from all cancers in women (Siegel et al., 2012). The cytokine network in the tumor microenvironment may be involved in many aspects of tumor growth and spread such as proliferation, motility, survival, cell-cell or cell-matrix adhesion, neovascularization, extracellular matrix remodeling, host cell infiltration, and local immune response (Apte et al., 2006), patients with epithelial ovarian cancer have elevated level of IL-18 compared to healthy controls(Medina et al., 2014).
1.9. Homocysteine Homocysteine is a non-protein forming sulfur-containing amino acid (Figure 1.6), formed as a primary intermediate during the metabolism of methionine, And Methionine is an essential amino acid that is derived primarily from the diet Which present naturally in all food high in 21
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Introduction and Literature Review
protein but the daily dietary intake only provides a limited supply of methionine so the ability of a cell for synthesis of new methionine from Hcy is extremely important for optimal cellular function and survival (Škovierová et al., 2016). Homocysteine present in plasma in different forms: around 1% circulates as free thiol, 70–80% remains disulphide-bound to plasma proteins, mainly albumin and 20–30% combines with itself to form the dimer homocysteine or with other thiols (Petras et al., 2014).
Figure 1.6: Structure of homocysteine (Ganguly and Alam, 2015).
1.9.1. Pathways of Methionine and Hcy Metabolism The interconnected pathways of methionine and Hcy metabolism can be split into three essential pathways: methionine cycle, remethylation, and transsulfuration (Figure 1.7). In the methionine cycle methionine is intracellularly converted to S-adenosylmethionine (SAM) in a reaction catalyzed by ATP and the enzyme methionine adenosyltransferase, SAM serves as a primary intracellular methyl donor for reactions catalyzed by methyltransferases (MTs) and is the principal metabolic regulator of the flow of Hcy between the remethylation and transsulfuration metabolic pathways. After a series of MT reactions, SAM is converted to Sadenosylhomocysteine (SAH) then SAH is reversibly hydrolyzed by SAH hydrolase yielding adenosine and Hcy (Obeid and Wolfgang Herrmann, 2009). 22
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Under conditions of negative methionine balance, Hcy is metabolized through remethylation pathway that is widely distributed in all tissues, with greater activity in the human liver and kidney. Remethylation pathway is achieved by the enzyme methionine synthase (MS, 5-methyltetrahydrofolate-homocysteine methyltransferase) that uses cofactor vitamin B12 (cobalamin) and 5-methyltetrahydrofolate (5-MTHF) as the methyl donor. MS transfers a methyl group from the 5-MTHF to Hcy, regenerating methionine, vitamin B12, and THF. The efficient activity of this pathway requires an adequate supply of folic acid and the enzyme MTHF reductase (MTHFR) , After remethylation, the newly recycled methionine can be used in the protein synthesis of taurine or again be reconverted to SAM for another round of methyl reactions (Blom and Smulders, 2011). When methionine synthesis not require, Hcy enters the catabolic transsulfuration pathway. This pathway occurs exclusively in the liver, kidney, small intestine, and pancreas and this is the only pathway capable of ridding the body of excess toxic sulfur-containing amino acids, including increased circulating Hcy. In this pathway homocysteine condenses with serine to form cystathionine in an irreversible reaction catalyzed by the pyridoxal-5’-phosphate (PLP)containing enzyme, cystathionine β-synthase (CBS). Cystathionine is hydrolyzed by a second PLP-containing enzyme, gamma-cystathionase, to form cysteine and alpha-ketobutyrate. Excess cysteine is oxidized to taurine and inorganic sulfates or excreted in the urine (Stipanuk, 2004). Two enzymes and three vitamins play a key role in the regulation of circulating homocysteine levels. The enzyme cystathionine-β-synthase controls the breakdown of homocysteine to cystathionine in the transsulfuration pathway, while methylene tetrahydrofolate reductase (MTHFR) is involved in the remethylation pathway, in which homocysteine is converted back to methionine. Folic acid, vitamin B6 and vitamin B12 are essential cofactors in homocysteine metabolism and a lack of them due to a deficient diet or disease can produce elevated plasma homocysteine. In addition, a genetic defect in one of the enzymes of homocysteine
metabolism
can
lead
to
metabolic
Hyperhomocysteinemia (Sandra et al., 2006).
23
disruption
and
potentially
to
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Introduction and Literature Review
Figure 1.7: A representation of the methionine cycle, remethylation pathway and transsulfuration pathway (Steed and Tyagi., 2011).
24
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1.9.2. Homocysteine in Polycystic Ovarian Syndrom women with PCOS have a clustering of cardiovascular risk factors, such as obesity, dyslipidemia, impaired glucose tolerance ,insulin resistance and elevate serum biomarkers of cardiovascular disease such as high C-reactive protein, homocysteine and interleukin-18 (Boulman et al., 2004), Insulin inhibit the hepatic cystathionine beta synthase, which controls the breakdown of homocysteine to cystathionine in the transsulfuration pathway (McCarty, 2000), So insulin resistance cause to increase the homocysteine levels in women with polycystic ovarian syndrom (Bar-On et al., 2000).
1.9.3. Role of Homocysteine in the Development of Cardiovascular Disease There has been a significant correlation between Hyperhomocysteinemia and cardiovascular disease and its complications such as heart attacks and strokes (Baszczuk and Kopczynski, 2014), The prevalence of Hyperhomocysteinemia may vary between populations, and most likely depend on age, diet, and genetic background (Faeh et al., 2006). Increasing age, smoking, coffee consumption, high blood pressure, unfavorable lipid profile, high creatinine and faulty diet are some of the factors associated with increased homocysteine levels. On the other hand, physical activity, good folate and vitamin B12 status are associated with lower homocysteine levels. Vegetarians may be at a higher risk of Hyperhomocysteinemia due to low plasma B12 levels but the difference is likely to be insignificant ( Vijetha et al., 2014).
1.9.4. Mechanism for Homocysteine in the Development of Cardiovascular Disease Homocysteine produce adverse effects on vascular endothelium and smooth muscle cells which lead to alterations in subclinical arterial structure and function, mechanisms of these effects include an increase in proliferation of vascular smooth muscle cells, endothelial dysfunction, oxidative damage, an increase in synthesis of collagen and deterioration of arterial wall elastic material ( Song et al., 2014). Homocysteine is an independent risk factor for atherosclerosis (Pang et al., 2014). Which defined as a continuous inflammatory damage to the arterial intima with increased permeability to plasma, deposition of plasma lipids in plaques and fibrosis and calcification of plaques
25
Chapter one
Introduction and Literature Review
(Schaffer et al., 2014). This effect may be due to ability of homocysteine for initiating an inflammatory response in vascular smooth muscle cells by stimulating CRP production, which is mediated through NMDAr-ROS-ERK1/2/p38-NF-κB signal pathway (Pang et al., 2014). Hyperhomocysteinemia also increase the risk of venous thrombosis, because increased homocysteine level enhance the adhesion of platelet to endothelial cells and also associated with higher levels of prothrombotic factors for example, β-thromboglobulin, tissue plasminogen activator and factor VII, these lead to the augmentation of thrombus formation (Faeh et al., 2006). Homocysteine may elevate blood pressure through numerous mechanisms such as its effect on vascular endothelial integrity also induced oxidative stress to endothelium and reduced available nitric oxide which is a potent vasodilator so lead to impairment of endotheliumdependent vasodilation in temporary or chronic Hyperhomocysteinemia (Lim and Cassano, 2002). Serum homocysteine concentration also associate with different indices of arterial stiffness such as pulse pressure and aortic stiffness as assessed by carotid-femoral Pulse Wave Velocity (PWV) the carotid-femoral PWV was found to be significantly higher in the high homocysteine group than in the normal homocysteine group ( Song et al., 2014).
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Chapter one
Introduction and Literature Review
Aim of the Study The aim of the present study is to estimate the level of interleukin-18 and homocysteine in the serum of patients with polycystic ovary syndrome and compares these values with a normal individuals.
27
CHAPTER TWO MATERIALS AND METHODS
28
Chapter Two
Materials and Methods
2.1. Materials 2.1.1. Laboratory Equipment /Instruments The equipment and instruments used for the study are listed in table (2.1).
Table 2.1: List of laboratory equipment and instrument
No
Laboratory equipment
Manufacturer
1.
Micro ELISA Reader
Bio Tek , USA
2.
Centrifuge
Heraeus Labofuge 200 , Germany
3.
Incubator
INCD 2 , memmert , Germany
4.
Accent 200 Chemistry Analyzer (for blood sugar, HbA1c and serum uric acid test)
Cormay ,Poland
5.
Cobas e 411 Immunology Analyzer (for LH, FSH, TSH, PRL and testosterone test)
Roche /Germany
6.
Ultra low-temperature freezer(-70 °c )
SANYO , Japan
7.
Water Stills /Distiller
Daihan Labtech , India
8.
Refrigerator ( -20 °c )
Vestel , Turky
9.
Micropipettes (5-50 ,20-200 , 100-1000 µl )
Transferpette , brand , Germany
10.
Gel and clot activator tube
AFCO .Jordan
11.
EDTA K3 tube
AFCO .Jordan
12.
Normal ( no anticoagulant ) tube
Sun. H.K.J
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Chapter Two
Materials and Methods
2.1.2. Reagents and Kits The specific reagents and kits used for this study are listed in table (2.2).
Table 2.2: List of reagents and kits No
Reagents and Kits
Manufacturer
1.
Interleukin-18 ELISA kit
eBioscience/USA
2.
Homocysteine ELISA kit
YH Bio search/ China
3.
Luteinizing hormone kit
Roche /Germany
4.
Follicle stimulating hormone kit
Roche /Germany
5.
Testosterone kit
Roche /Germany
6.
Prolactine kit
Roche /Germany
7.
Thyroid stimulating hormone kit
Roche /Germany
8.
Blood sugar kit
Cormay ,Poland
9.
HbA1c kit
Cormay ,Poland
10.
Serum Uric acid kit
Cormay ,Poland
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Chapter Two
Materials and Methods
2.2. Study Design The study was case-control study and involved 300 females (Figure 2.1). One hundred fifty women participated in this study diagnosed as polycystic ovary syndrome by physician based on the 2003 Rotterdan consensus the diagnostic criteria of PCOS, collected in Central Medical Laboratory one in Sulaimani and also in private clinic. The other one hundred fifty women taked as control group who did not have any symptom of PCOS like irregular menstrual cycle, acne, hirsutism, with a normal laboratory test for LH, FSH, TSH, Prolactin and Total Testosterone. These samples collected in female schools. All of the recruited female gave their informed consent in order to be engaged in the study. Blood samples collected randomly and were started in May 2015 then completed in October 2015, serum samples stored in laboratory of Shar hospital until sample analysis which was done in November 2015. Inclusion criteria include 150 female diagnosed as PCOS who present with two of three criteria (menstrual dysfunction, hyperandrogenemia and polycystic ovary morphology) and 150 apparently healthy control female who did not have any clinical or biochemical symptom of PCOS, while, exclusion criteria for this study include postmenopause women, pregnant women, female subject with thyroid disorder and female with only one symptoms of PCOS.
Figure 2.1: Illustration of the study design and case numbers.
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Chapter Two
Materials and Methods
2.3. Methods 2.3.1. Sample collection Using disposable syringes, five milliliters of venous blood was obtained from participants in the study mainly from arm through cubital vein, one milliliter of blood collected in EDTA tubes for HbA1c test which done immediately, four milliliter of drawn blood had been collected in a serum gel separator tubes and left in room temperature for 5 minutes to clot, Then centrifuged for 20 minutes at 4000 rpm to separate the serum. The serum divided in to two parts one part of serum kept frozen at ( -70 )oC until they were assayed and the other part used directly for testing LH, FSH, TSH, Prolactine, testosterone, blood sugar and serum uric acid .
2.3.2. Human Serum IL-18 2.3.2.1. Principle of assay Enzyme-linked immunosorbent assay (ELISA) based on biotin double antibody sandwich technology was used to assay Human Interleukin-18. An anti-human IL-18 coating antibody is adsorbed onto microwells. Human IL-18 present in the sample or standard binds to antibodies adsorbed to the microwells. A biotin-conjugated anti-human IL-18 antibody is added and binds to human IL-18 captured by the first antibody. Following incubation unbound biotin-conjugated anti-human IL-18 antibody is removed during a wash step. Streptavidin-HRP is added and binds to the biotin-conjugated anti-human IL-18 antibody. Following incubation unbound Streptavidin-HRP is removed during a wash step, and substrate solution reactive with HRP is added to the wells. A coloured product is formed in proportion to the amount of human IL-18 present in the sample or standard. The reaction is terminated by addition of acid and absorbance is measured at 450 nm. A standard curve is prepared from human IL-18 standard dilutions and human IL-18 sample concentration determined (Figure 2.2)(Yoshimoto et al., 2001).
32
Chapter Two
Materials and Methods
Figure 2.2: Principle of human serum IL-18 measurements by ELISA.
2.3.2.2. Reagents Used Reagents for human IL-18 ELISA were: 1. 1 aluminium pouch with a Microwell Plate coated with monoclonal antibody to human IL-18 2. 1 vial (70µl) Biotin-Conjugate anti-human IL-18 monoclonal antibody 3. 1 vial (150 µl) Streptavidin-HRP 4. 2 vials human IL-18 Standard lyophilized, 10 ng/ml upon reconstitution 5. 1 vial (12 ml) Sample Diluent 6. 1vial (5 ml) Assay Buffer Concentrate 20x (PBS with 1% Tween 20, 10% BSA) 7. 1 bottle (50 ml) Wash Buffer Concentrate 20x (PBS with 1% Tween 20) 33
Chapter Two
Materials and Methods
8. 1 vial (15 ml) Substrate Solution (tetramethyl-benzidine) 9. 1 vial (15 ml) Stop Solution (1M Phosphoric acid) 10. 4 Adhesive Films
2.3.2.3. Assay Procedure 2.3.2.3.1. Preparation of Reagents Preparation of reagents includes:
2.3.2.3.1.1. Wash buffer (1X) Added (950 ml) distilled water to (50 ml) wash buffer concentrate (20x) into a clean 1000 ml graduated cylinder then mixed and transferred to clean wash bottle.
2.3.2.3.1.2. Assay Buffer (1x) Added (95 ml) distilled water to (5 ml) assay buffer concentrate (20x) into a clean 100 ml volumetric flask then mixed.
2.3.2.3.1.3. Biotin-Conjugate Concentrated Biotin-Conjugate solution diluted (1:100) with assay buffer (1x) in a clean plastic tube. The solution was stable for 30 minutes.
2.3.2.3.1.4. Streptavidin-HRP Concentrated Streptavidin-HRP solution diluted (1:100) with assay buffer (1x) in a clean plastic tube. The solution was stable for 30 minutes.
2.3.2.3.1.5. Human IL-18 Standards Reconstituted human IL-18 standard by addition of (250 µl) distilled water (Concentration = 10 ng/ml), after that prepared 1:2 serial dilution of standard for standard curve as follows: Labeled 7 tubes one for each standard point. S1, S2, S3, S4, S5, S6, S7, Then Pipetted 225 µl of Sample Diluent into each tube, after that Pipetted 225 µl of reconstituted standard into the first tube, labelled S1, and mix (concentration of standard 1 = 5 ng/ml). Then Pipetted 225 µl of this
34
Chapter Two
Materials and Methods
dilution into the second tube, labelled S2, and mix thoroughly before the next transfer. Repeat serial dilutions 5 more times thus creating the points of the standard curve (Figure 2.3).
Figure 2.3: External standard dilution of IL-18.
2.3.2.3.2. Test Protocol Test protocol for human serum IL-18 measurement mentioned in table (2.3).
Table 2.3: Test protocol for human serum IL-18. Washed the microwell strips twice with Wash Buffer and allowed the Wash Buffer to sit in the wells for about 10 – 15 seconds, After the last wash step emptied wells and tap microwell strips on absorbent pad to remove excess Wash Buffer and use the microwell strips immediately. Reagents Standard Sample diluent
Blank well
Standard well
Sample well
-
100 μl
-
100 μl
-
50 μl
35
Chapter Two
Materials and Methods
Sample
-
50 μl
-
Prepared Biotin-Conjugate anti-human IL-18 monoclonal antibody. Diluted BiotinConjugate
50 μl
50 μl
50 μl
Covered microwell strips with an adhesive film and incubated at room temperature for 2 hours. Then Prepared Streptavidin-HRP, After that emptied and washed microwell strips 6 times with Wash Buffer. Diluted StreptavidinHRP
100 μl
100 μl
100 μl
Covered microwell strips and incubated at room temperature for 1 hour, After that emptied and washed microwell strips 6 times with Wash Buffer. TMB Substrate Solution
100 μl
100 μl
100 μl
Incubated the microwell strips in a dark place at room temperature for about 10 minutes.
Stope solution
100 μl
100 μl
100 μl
The absorbance of each well was measured under 450 nm wavelengths.
2.3.3. Human Serum Homocysteine 2.3.3.1. Principle of Assay Enzyme-linked immunosorbent assay (ELISA) based on biotin double antibody sandwich technology was used to assay human serum homocysteine. Homocysteine (Hcy) is added to each wells that are pre-coated with Homocysteine (Hcy) monoclonal antibody and then incubated. After incubation, added anti Hcy antibodies labeled with biotin to unite with streptavidin-HRP, which forms the immune complex. Removed unbound enzymes after incubation and washing, then added substrate A and B. The solution will
36
Chapter Two
Materials and Methods
turn blue and change to yellow after adding the stop solution. The shades of solution and the concentration of human homocysteine(Hcy) are positively correlated (Miller et al., 2002).
2.3.3.2. Reagents Used Reagents for human homocysteine ELISA were: 1. 1 Coated ELISA plate 12 * 8 wells 2. 1 vial (0.5 ml) Standard solution (64nmol/ml) 3. 1 vial (6 ml) Streptavidin-HRP 4. 1 vial (6 ml) Stop Solution 5. 1 vial (6 ml) Chromogenic reagent A 6. 1 vial (6 ml) Chromogenic reagent B 7. 1 vial (1 ml) Anti Hcy antibodies labeled with biotin 8. 1 vial (3 ml) Standard dilution 9. 1 vial (20ml×30) Washing concentrate 10. 2 seal plate membrane
2.3.3.3. Assay Procedure Procedure for homocysteine ELISA test and standard preparation mentioned in table (2.4) and table (2.5).
Table 2.4: Procedure for Hcy ELISA test.
Reagents
Blank well
Standard well
Assay well
Serum
-
-
40 μl
Standard
-
50 μl
-
Anti IL.18 antibodies
-
10 μl
labeled with biotin
37
Chapter Two
Materials and Methods
Streptavidin-HRP
50 μl
-
50 μl
After reagents were added covered with seal plate membrane, then mixed and incubated for 60 minute at 37℃, Diluted washing concentration (30x) with distilled water Washed with diluted washing solution by carefully removing the seal plate membrane and drained liquid and shaken off the remainder then each well filled with washing solution and let stand for 30 seconds, and then drained. This procedure repeated five times. Chromogenic reagent A Chromogenic reagent B
50 μl
50 μl
50 μl
50 μl
50 μl
50 μl
Reagents were added, covered with seal plate membrane, mixed and incubated for 10 minute in a dark place at 37℃. Stop solution
50 μl
50 μl
50 μl
The absorbance of each well was measured one by one under 450 nm wavelength within 10 minutes after adding stop solution
Table 2.5: Dilution of standard solution of Hcy. 32nmol/ml
Standard No.5
120μl Original Standard + 120μl Standard diluents
16nmol/ml
Standard No.4
120μl Standard No.5 + 120μl Standard diluents
8nmol/ml
Standard No.3
120μl Standard No.4 + 120μl Standard diluent
4nmol/ml
Standard No.2
120μl Standard No.3 + 120μl Standard diluent
2nmol/ml
Standard No.1
120μl Standard No.2 + 120μl Standard diluent
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Chapter Two
Materials and Methods
2.3.4. Blood Sugar Measurements 2.3.4.1. Principle of the test Colorimetric, enzymatic method with glucose oxidase Glucose + H2O + O2
gluconic acid + H2O2 (by glucose oxidase)
2H2O2 +phenol + 4-aminoantipyrine → 4-(p-benzochinonomonoimino)-phenazone + 4 H2O The color intensity is proportional to the glucose concentration (Dungan et al., 2007).
2.3.4.2. Procedure This reagent used in automatic analyzer ACCENT-200, reagent ready to use but for reagent blank deionized water was used and then inserted serum sample.
2.3.4.3. Calculation Automatic analyzer ACCENT-200 automatically calculates the analytic concentration of each sample. Normal range (5.4-9.9 mmole/L)
2.3.5. HbA1c Measurements 2.3.5.1. Principle of the test Method for HbA1c determination has been certified by the National Glycohemoglobin Standardization Program (NGSP). The method utilized the interaction of antigen and antibody to directly determine the HbA1c concentration in whole blood. Total hemoglobin and HbA1c have the same unspecific absorption rate to latex particles, when mouse antihuman HbA1c monoclonal antibody is added; latex- HbA1c-mouse antihuman HbA1c antibody complex is formed. Agglutination is formed when goat anti-mouse IgG polyclonal antibody interact with the monoclonal antibody. The amount of agglutination is proportional to the amount of HbA1c absorbed on to the surface of latex particles. The amount of agglutination is measured as absorbance and the HbA1c value is obtained from a calibration curve ( Alan Wu, 2006).
2.3.5.2. Assay procedure 1. Reagent 1 was ready to use. 39
Chapter Two
Materials and Methods
2. Poured the contents of Reagent 2b vial into Reagent 2a and mixed gently. 3. Sample we used collected from venous blood with EDTA tube, sample pretreated by dispense 500 µl hemolysing reagent into tubes labeled, Then place 10 µl of well mixed whole blood into the labeled lyse reagent tube. It was mixed and allowed to stand for 5 minutes or until complete lysis is evident. 4. For reagent blank 0.9 % NaCl was used. 5. Then these reagents used in automatic analyzer ACCENT-200.
2.3.5.3. Calculation Automatic analyzer ACCENT-200 automatically calculates the analytic concentration of each sample and the value is reported in % of heamoglobin unit in accordance with NGSP standardization. Normal range (4.0-6.0 %)
2.3.6. Uric Acid Measurements 2.3.6.1. Principle of the test Enzymatic, colorimetric method with uricase and peroxidase. Uric acid + 2 H2O + O2→allantoine + CO2 + H2O2 ( catalyse by uricase ) ADPS + 4-aminoantipyrine + 2 H2O2→ quinoneimine dye + 4H2O
(By Peroxidase)
The colour intensity is proportional to the uric acid concentration (Zhao et al., 2009).
2.3.6.2. Assay Procedure These reagents used in automatic analyzers ACCENT-200, Reagent-1 and Reagent-2 were ready to use but for reagent blank deionized water was used.
2.3.6.3. Calculation Automatic analyzer ACCENT-200 automatically calculates the analytic concentration of each sample. Normal range (149-405 μmol/L)
40
Chapter Two
Materials and Methods
2.3.7. Measurement of Follicle Stimulating Hormones Immunoassay (Sandwich method) was used for the in vitro quantitative determination of follicle-stimulating hormone in human serum, by use cobas e411 immunoassay analyzers.
2.3.7.1. Principle of test The method of determination includes two incubations: 1st incubation: 40 μL of sample, a biotinylated monoclonal FSH‑specific Antibody, and a monoclonal FSH‑specific antibody labeled with a ruthenium complex formed a sandwich complex.
2nd incubation: After addition of streptavidin-coated micro particles, the Complex became bound to the solid phase via interaction of biotin and Streptavidin.
The reaction mixture is aspirated into the measuring cell where the Micro particles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with ProCell/ProCell M. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier (Alan H.B. Wu, 2006).
2.3.7.2. Reagents M Streptavidin-coated microparticles, 1 bottle (6.5 mL), Streptavidin-coated microparticles 0.72 mg/mL and preservative. R1 Anti-FSH-Ab~biotin, 1 bottle (10 mL),Biotinylated monoclonal anti‑FSH antibody (mouse) 0.5 mg/L, MES buffer 50 mmol/L, pH 6.0 and preservative. R2 Anti-FSH-Ab~Ru, 1 bottle (10 mL),Monoclonal anti‑FSH antibody (mouse) labeled with ruthenium complex 0.8 mg/L, MES buffer 50 mmol/L, pH 6.0 and preservative.
2.3.7.3. Calculation Results are determined via a calibration curve which is instrument specifically generated by 2-point calibration and a master curve provided via the reagent barcods and automatically calculates the analyze concentration of each sample. 41
Chapter Two
Materials and Methods
Normal range for women Follicular Phase 3.5-12.5 mIU/ml Ovulation Phase 4.7-21.5 mIU/ml Luteal Phase 1.7-7.7 mIU/ml
2.3.8. Measurement of Luteinizing Hormone Immunoassay (Sandwich method) was used for the in vitro quantitative determination of luteinizing hormone in human serum, by use cobas e411 immunoassay analyzers.
2.3.8.1. Test Principle The method of determination includes two incubations: 1st incubation: 20 μL of sample, a biotinylated monoclonal LH‑specific antibody, and a monoclonal LH‑specific antibody labeled with a ruthenium complex form a sandwich complex. 2nd incubation: After addition of streptavidin-coated microparticles, the complex becomes bound to the solid phase via interaction of biotin and streptavidin. The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with ProCell/ProCell M. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier (Kalia et al., 2004).
2.3.8.2. Reagents M Streptavidin-coated microparticles, 1 bottle (6.5 mL), Streptavidin-coated microparticles 0.72 mg/mL and preservative. R1 Anti-LH-Ab~biotin, 1 bottle (10 mL), Biotinylated monoclonal anti‑LH antibody (mouse) 2.0 mg/L, TRIS buffer 50 mmol/L, pH 8.0 and preservative. R2 Anti-LH-Ab~Ru, 1 bottle (10 mL),Monoclonal anti‑LH antibody (mouse) labeled with ruthenium complex 0.3 mg/L,TRIS buffer 50 mmol/L, pH 8.0 and preservative.
42
Chapter Two
Materials and Methods
2.3.8.3. Calculation Results are determined via a calibration curve which is instrument specifically generated by 2-point calibration and a master curve provided via the reagent barcods and automatically calculates the analyze concentration of each sample. Normal range for women Follicular Phase 2.4-12.6 mIU/ml Ovulation Phase 14-95.6 mIU/ml Luteal Phase 1-11.4 mIU/ml
2.3.9. Measurement of Testosterone Immunoassay (Competition method) was used for the in vitro quantitative determination of testosterone hormone in human serum, by use cobas e411 immunoassay analyzers.
2.3.9.1. Test Principle The method of determination includes two incubations: 1st incubation: 20 μL of sample are incubated with a biotinylated monoclonal testosterone‑specific antibody. The binding sites of the labeled antibody become occupied by the sample analyte (depending on its concentration). 2nd incubation: After addition of streptavidin-coated microparticles and a testosterone derivate labeled with a ruthenium complex, the complex becomes bound to the solid phase via interaction of biotin and streptavidin. The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with ProCell/ProCell M. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier (Kane et al., 2007).
2.3.9.2 Reagents M
Streptavidin-coated microparticles, 1 bottle (6.5 mL), Streptavidin-coated microparticles
0.72 mg/mL and preservative. 43
Chapter Two
Materials and Methods
R1 Anti-testosterone-Ab~biotin, 1 bottle (10 mL), Biotinylated monoclonal anti-testosterone antibody (sheep) 40 ng/mL, releasing reagent 2-bromoestradiol; MES buffer 50 mmol/L, pH 6.0 and preservative. R2 Testosterone-peptide~Ru, 1 bottle (9 mL), Testosterone derivative, labeled with ruthenium complex 1.5 ng/mL, MES buffer 50 mmol/L, pH 6.0 and preservative.
2.3.9.3. Calculation Results are determined via a calibration curve which is instrument specifically generated by 2-point calibration and a master curve provided via the reagent barcods and automatically calculates the analyze concentration of each sample. Normal range (0.084-0.481 ng/ml)
2.3.10. Measurement of Prolactin Immunoassay (Sandwich method) was used for the in vitro quantitative determination of prolactin hormone in human serum, by use cobas e411 immunoassay analyzers.
2.3.10.1. Test Principle The method of determination includes two incubations: 1st incubation: 10 μL of sample and a biotinylated monoclonal prolactin specific antibody form a first complex. 2nd incubation: After addition of a monoclonal prolactin‑specific antibody labeled with a ruthenium complex and streptavidin-coated microparticles, a sandwich complex is formed and becomes bound to the solid phase via interaction of biotin and streptavidin. The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with ProCell/ProCell M. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier (Landberg et al., 2007).
44
Chapter Two
Materials and Methods
2.3.10.2. Reagents M Streptavidin-coated microparticles, 1 bottle (6.5 mL), Streptavidin-coated microparticles 0.72 mg/mL and preservative. R1 Anti-prolactin-Ab~biotin, 1 bottle (10 mL), Biotinylated monoclonal anti‑prolactin antibody (mouse) 0.7 mg/L, Phosphate buffer 50 mmol/L, pH 7.0 and preservative. R2
Anti-prolactin-Ab~Ru, 1 bottle (10 mL), Monoclonal anti‑prolactin antibody (mouse)
labeled with ruthenium complex 0.35 mg/L, Phosphate buffer 50 mmol/L, pH 7.0 and preservative.
2.3.10.3. Calculation Results are determined via a calibration curve which is instrument specifically generated by 2-point calibration and a master curve provided via the reagent barcods and automatically calculates the analyze concentration of each sample. Normal range (4.79-23.3 ng/ml)
2.3.11. Measurement of Thyroid Stimulating Hormone Immunoassay (Sandwich method) was used for the in vitro quantitative determination of thyroid stimulating hormone in human serum, by use cobas e411 immunoassay analyzers.
2.3.11.1. Test Principle The method of determination includes two incubations: 1st incubation: 50 μL of sample, a biotinylated monoclonal TSH‑specific antibody and a monoclonal TSH‑specific antibody labeled with a ruthenium complex reacted to form a sandwich complex. 2nd incubation: After addition of streptavidin-coated microparticles, the complex becomes bounded to the solid phase via interaction of biotin and streptavidin. The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed
45
Chapter Two
Materials and Methods
with ProCell/ProCell M. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier (Reixa et al., 2013).
2.3.11.2. Reagents M Streptavidin-coated microparticles, 1 bottle (12 mL), Streptavidin-coated microparticles 0.72 mg/mL and preservative. R1 Anti-TSH-Ab~biotin, 1 bottle (14 mL), Biotinylated monoclonal anti-TSH antibody (mouse) 2.0 mg/L, Phosphate buffer 100 mmol/L, pH 7.2 and preservative. R2 Anti-TSH-Ab~Ru, 1 bottle (12 mL), Monoclonal anti-TSH antibody (mouse/human) labeled with ruthenium complex, 1.2mg/L; phosphate buffer 100 mmol/L, pH 7.2 and preservative.
2.3.11.3. Calculation Results are determined via a calibration curve which is instrument specifically generated by 2-point calibration and a master curve provided via the reagent barcods and automatically calculates the analyze concentration of each sample. Normal range (0.27-4.2 μIU/ml)
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Chapter Two
Materials and Methods
2.4. Statistical Analysis After data collection and prior to data entry and analysis, the questions of study were coded. Data entry performed via using an excel spreadsheet then the statistical analysis was performed by SPSS program, version 21 (IBM SPSS Statistical Package for the Social Sciences). The data presented in tabular forms showing the frequency and relative frequency distribution of different variables among the both groups (polycystic ovarian syndrome cases and healthy controls). Chi-square tests were used to compare the categorical data between these two groups in respect to different variables as age group, and BMI groups. For comparing the quantitative variables between cases and controls their means were compared using independent t-test and analysis of variance (ANOVA). The level of homocysteine and interleukin-18 within PCOS cases compared with the level of FSH, prolactin and other quantitative variables to find the linear relationship between them and the coefficient of this linear relationship (r) with (r2) were calculated. Different types of Bar charts and diagrams as well as arithmetic scale line graphs were used to describe the some variables of the study diagrammatically. P- Values of 0.05 were used as a cut off point for significance of statistical tests.
47
CHAPTER THREE
RESULTS
48
Chapter Three
Results
In this research, the studied samples were collected from Central Medical Laboratory one in Sulaimani and also in private clinic which diagnosed as polycystic ovary syndrome based on the 2003 Rotterdam Guidelines for diagnosis of PCOS.
3.1. Clinical Symptoms of Polycystic Ovarian Syndrome Clinical symptoms of patients with PCOS shown as percentage in table (3.1), the most common symptom was hirsutism with 92.7% and less common symptom was alopecia with 4.0% of PCOS patients.
Table 3.1: Frequency distribution of symptoms among polycystic ovarian syndrome cases.
Symptoms of PCOS
Number of Cases
Percentage
Hirsutism
139
92.7%
Oligo menorrhea
108
72.0%
Acne
72
48.0%
Infertility(in married cases)
30
46.15%
Amenorrhea
39
26.0%
Alopecia
6
4.0%
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Chapter Three
Results
3.2. Hormones Level in PCOS and Control Group The serum hormones measured in this study and their results showed in table (3.2), The mean values of serum LH were (10.87 ± 7.36 mIU/ml) and (5.73 ± 2.25 mIU/ml) in cases and control group respectively, the mean values of serum FSH were (5.84 ± 4.60 mIU/ml) and (6.86 ± 2.43 mIU/ml) in cases and control group respectively, mean value of LH/FSH ratio in PCOS was (2.09 ± 1.57) and in control group was (0.96 ± 0.74), the mean values of serum TSH were (2.40 ± 1.02 μIU/ml) and (2.47 ± 1.18 μIU/ml) in cases and control group respectively, the mean values of serum prolactin were (19.67 ± 10.65 ng/ml) and (16.42 ± 4.52 ng/ml) in cases and control group respectively, and testosterone were (0.90 ± 2.01 ng/ml) and (0.35 ± 0.24 ng/ml) in cases and control group respectively, also notice significant difference for all variables between PCOS and control group except for serum TSH which were not significant difference (p-value 0.60) between PCOS and control. LH and testosterone tests are one of the tests using for diagnosis of PCOS and most of patients have higher concentration than normal range. The results were 43.3 % of patients had high LH level and 44.7% of patients had higher testosterone level than normal range as showed in table (3.3).
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Table 3.2: Levels of LH, FSH, LH/FSH ratio, TSH, PRL and testosterone in serum of PCOS and control group. Mean ± SD Hormones
P- Value PCOS
Control
LH mIU/ml
10.87 ± 7.36
5.73 ± 2.25
< 0.001
FSH mIU/ml
5.84 ± 4.60
6.86 ± 2.43
0.02
LH/FSH ratio
2.09 ± 1.57
0.96 ± 0.74
< 0.001
TSH μIU/ml
2.40 ± 1.02
2.47 ± 1.18
0.60
19.67 ± 10.65
16.42 ± 4.52
0.001
0.90 ± 2.01
0.35 ± 0.24
0.001
Prolactin ng/ml Testosterone ng/ml
Table 3.3: Levels of LH and testosterone in PCOS patients. Type of Tests
LH test
Testosterone test
Reference Range
Number Of Cases
Percentage
Low < 2.4 4 mIU /ml
10
6.7%
Normal 2.4-12.4 mIU /ml
75
50.0 %
High > 12.44 mIU /ml
65
43.3%
Low < 0.084 ng/ml
7
4.7%
Normal 0.084-0.481 ng/ml
76
50.7%
High > 0.481 ng/ml
67
44.7%
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Chapter Three
Results
3.3. BMI and Age Distribution among Cases and Control group The BMI calculated for each participant by this formula, BMI =weight (Kg)/ [high] 2(m), and were classified to four groups by depending on BMI, The results showed in table (3.4). There was highly significant difference (p-value < 0.001) in BMI between cases and controls, and notice that obesity in PCOS higher than controls, In PCOS 38.0% was overweight and 16.7% was obese while in control groups 21.3% overweight and 6.7% obese. Age distribution of PCOS cases and control group shown in table (3.4). All participants were classified in to four age groups include: 10 - 19 years , 20-29 years, 30-39 years, 40-49 years. There was statistically significant difference (p-value < 0.001) in age between both groups as highest percentage of PCOS patients within age interval of 20-29 years while control groups between 10-19 years.
Table 3.4: BMI and Age among cases and control group. BMI and Age
BMI
Number and
Number and
Percentage of PCOS
Percentage of Control
underweight (≤ 18.5)
7 (4.7%)
13 (8.7%)
Normal (18.5 - 24.9)
61 (40.7%)
95 (63.3%)
P- value
< 0.001
2
(kg/m )
Age (years)
Overweight ( 25 29.9)
57 (38.0%)
32 (21.3%)
Obese ( ≥ 30 )
25 (16.7%)
10 (6.7%)
10-19
31 (20.7%)
65 (43.3%)
20-29
70 (46.6%)
42 (28.0%) < 0.001
30-39
43 (28.7%)
38 (25.3%)
40-49
6 (4%)
5 (3.4%)
52
Chapter Three
Results
3.4. Age of Menarche Age of menarche is a woman's first menstruation, for determine the effect of PCOS on age of menarche in female we classified cases and control group according to first time of menstrual cycle in to two groups: 10-14 years and 15-18 years. The result in table (3.5) showed no significant difference in age of menarche between cases and control group (p-value 0.10).
Table 3.5: Age of menarche in PCOS cases and control group. Age of menarche
Number of PCOS
Number of Controls
10 - 14 years
129
138
P-value
0.10 15 - 18 years
21
12
3.5. Family History of PCOS Table (3.6) showed that in 150 PCOS: 61 cases had positive family history, while in 150 control: 49 had positive family history of PCOS. These were no significant difference in family history of PCOS between PCOS cases and control (P-value =0.09).
Table 3.6: Family history of PCOS in first degree relative of control and PCOS cases. Family history PCOS
Positive family history
Number and
Number and
Percentage of PCOS
Percentage of Control
61 (40.7%)
49 (32.7 %)
P-value
0.09 Negative family history
89 (59.3%)
101 (67.3 %)
53
Chapter Three
Results
3.6. Concentration of Blood Sugar, HbA1c and Serum Uric Acid in PCOS Patients and Control Group We measured concentration of Blood Sugar, HbA1c and Serum Uric Acid in 150 PCOS patients and 150 control females, table (3.7) showed: for Blood Sugar, P-value was 0.18 which indicated not significant, while for HbA1c and S.uric acid, P-value were <0.05 and 0.01respectively which indicated significant difference in the HbA1c and S.uric acid level between PCOS patients and control group. Serum uric acid level in PCOS and control group difference with BMI as in PCOS the mean concentration were (250.4 ± 67.5 μmol/L, 262.6 ± 49.1 μmol/L) in underweight and obese cases respectively, and in control group were (195.8 ± 10.2 μmol/L, 256.9 ± 33.8 μmol/L) in underweight and obese female respectively, as shown in table (3.8).
Table 3.7: Shows mean, standard deviation and p-value for blood Sugar, HbA1c and s.uric acid in PCOS and control group. Mean ± SD
P value
Tests (normal range) Cases
Control
Blood sugar (5.4-9.9 mmol/L)
5.67 ± 1.34
5.51 ± 0.75
0.18
S. uric acid (149-405 μmol/L)
252.72 ± 54.96
237.65 ± 47.04
0.01
5.53 ± 0.97
5.33 ± 0.73
< 0.05
HbA1c (4-6 %)
54
Chapter Three
Results
Table 3.8: Serum uric acid concentration in PCOS cases and control group according to BMI Uric acid μmol/L ( Mean ± SD
P- value
2
Nutritional status BMI ( Kg / m ) Cases
Control
Underweight ( BMI ≤ 18.5)
250.4 ± 67.5
195.8 ± 10.2
0.02
Normal ( BMI 18.5 - 24.9)
253.0 ± 45.4
231.9 ± 47.9
0.01
Overweight ( BMI 25 - 29.9)
251.7 ± 44.3
238.0 ± 62.0
0.24
Obese ( BMI ≥ 30)
262.6 ± 49.1
256.9 ± 33.8
0.71
3.7. Concentration of Serum IL-18 in PCOS Patients and Control Group Mean and standard deviation of serum IL-18 was (378.3 ±181.21 pg/ml) in PCOS and (224.98± 131.885 pg/ml) in control group as shown in figure (3.1). According to these data, there was highly significant difference (p-value < 0.001) and high serum concentration of IL-18 in PCOS as compared to control group.
55
Chapter Three
Results
Figure 3.1: Concentration of serum IL-18 in PCOS patients and control group.
3.8. Concentration of Serum Homocysteine in PCOS Patients and Control Group Mean and standard deviation for serum concentration of Hcy was (10.36 ± 5.98 nmol/ml) in PCOS and (5.17± 5.24 nmol/ml) in control group as shown in figure (3.2). These results clearly indicated highly significant differences (P-value < 0.001) in the level of homocysteine in PCOS and control group.
Figure 3.2: Concentration of serum homocysteine in PCOS patients and control group.
56
Chapter Three
Results
3.9. Correlation between Serum IL-18 with age, BMI, LH, FSH, Prolactin and Testosterone Level in PCOS There were a significant positive correlation between serum IL-18 concentration with BMI(r=0.156, p-value=0.03) and FSH(r=0.18, p-value 0.026), also highly significant positive correlation between serum IL-18 and LH level in PCOS patients(r=0.452, p-value=0.001), while no significant positive correlation between serum IL-18 with age(r=0.08, p-value=0.17), prolactin(r=0.12, p-value 0.14), and testosterone level(r=0.124, p-value=0.07) in PCOS as shown in table (3.9).
Table 3.9: Correlation of IL-18 with Age, BMI, LH, LH and FSH, prolactin and testosterone level in PCOS. Variable Age
BMI
LH
FSH
Testosterone
Prolactin
R
R2
P-Value
0.08
0.006
0.17
0.156
0.024
0.03
0.452
0.205
< 0.001
0.18
0.033
0.026
0.124
0.015
0.07
0.12
0.008
0.14
57
Chapter Three
Results
3.10. Correlation between Serum Homocysteine with age, BMI, LH, FSH, Prolactin and Testosterone Level in PCOS There were a significant positive correlation between serum level of homocysteine with age(r=0.177, p-value=0.015), LH(r=0.202, p-value=0.01) and testosterone level(r=0.176, pvalue=0.016), while no significant positive correlation of homocysteine level with BMI (r=0.093, p-value=0.13), FSH(r=0.006, p-value 0.94) and Prolactin level (r=0.038, p-value 0.94) in PCOS cases as shown in table (3.10).
Table 3.10: Correlation of homocysteine with Age, BMI, LH, FSH, prolactin and testosterone level in PCOS. Variable Age
BMI
LH
FSH
Testosterone
Prolactin
R
R2
P-Value
0.177
0.031
0.015
0.093
0.009
0.13
0.202
0.041
0.01
0.006
0.00004
0.94
0.176
0.031
0.016
0.038
0.001
0.94
58
CHAPTER FOUR DISCUSSION AND CONCLUSION
59
Chapter Four
Discussion and Conclusion
4.1. Prevalence Symptoms of PCOS in PCOS Patients In this study, we focused on prevalence of these symptom among cases through questionnaire form was provided to obtain information from cases. According to these data among 150 PCOS cases in sulaimani city 92.7 % have hirsutism, 48.0% acne and 4.0% have alopecia (Table 3.1) which is nearly same with studied in other area (Azziz et al., 2006). Menstrual irregularity and infertility also most common in women with PCOS and appeared in our study as shown in table (3.1), There was 72.0% females had oligo menorrhea, 26.0% had amenorrhea and 46.15% had infertility. This is in agreement with (Teede H. et al., 2010).
4.2. Hormones Level in PCOS and Control Group This study examined several hormonal assays which include: Luteinizing hormone (LH), Follicle stimulating hormone (FSH), LH/FSH ratio, thyroid stimulating hormone (TSH), prolactin (PRL) and testosterone. Women with PCOS due to decreased sensitivity of the GnRH pulse generator to the progesterone suppression lead to the abnormality in the regulation of hypothalamic GnRH secretion, with persistently rapid GnRH pulsatility and pituitary synthesis of LH over that of FSH which cause increased LH concentrations and LH/FSH ratios in PCOS patients (Blank et al., 2009, Blank et al., 2006), also one of the hallmarks of PCOS is the presence of hyperandrogenism, which best indicated by persistent elevation of serum testosterone above normal as determined in a reliable reference laboratory by measurement of total testosterone (TT) and free testosterone (FT) (Azziz et al., 2009), so assays of LH level and testosterone are important in the diagnosis of PCOS (Matsumoto and Bremner, 2004). The results obtained for LH and testosterone was significantly higher in PCOS compare with control group (Table 3.2)( p-value 0.001, p-value < 0.001) respectively, also in PCOS cases 43.3% of cases had higher level of LH than normal, and 44.7% of cases had higher testosterone level (Table 3.3), These results agree with other article (Laven et al., 2002) which observed that concentration of testosterone and luteinizing hormone were elevated in PCOS patients compared to normal control, furthermore, it has been shown that although hyperandrogenism most common in PCOS patients but Testosterone values may be normal in some cases (Sheehan, 2004).
60
Chapter Four
Discussion and Conclusion
Serum FSH level was statistically significant difference (p-value= 0.02) (Table 3.2) between PCOS cases and control. This finding is in agreement with other study (Wiweko et al., 2014). The serum levels of LH/FSH ratio in PCOS was significantly higher in compare with control (Table 3.2) (p-value < 0.001), this result consistent with other article (Banaszewska et al., 2003). The primary function of PRL is the development and maintenance of lactation, release of PRL into the blood stream is thought to be under the control of dopamine which is a prolactininhibitory factor produced by hypothalamus (Grattan, 2015). There was significant difference (pvalue 0.001) in the serum levels of PRL between cases and control (Table 3.2). This finding is in agreement with (Bracero and Zacur, 2001) which mean that women with PCOS may have mildly elevated prolactin levels. On the other hand, disagree with other study ( Szosland et al., 2015). This may be related to several physiological conditions which induce the release of PRL, the most notable being the stimulation of the breast and nipple during nursing, other conditions giving rise to PRL release are sever stress and major surgery involving general anesthesia (BenJonathan et al., 2006) . There was statistically not significant difference (p-value 0.60) in TSH levels between PCOS and control (Table 3.2). This finding is in agreement with other studies (Rashidi et al., 2016, Arora et al., 2016, Kialka et al., 2015).
4.3. Age and BMI in PCOS and Control Group Table (3.4) showed age distribution of PCOS and control group, There was statistically significant difference (p-value < 0.001) as highest percentage of control groups between(10-19) years old, highest PCOS patients within age interval of (20-29) years old and lowest percentage between (40-49) years old, This in agree with two other articles (Tabassum, 2014, Ramanand et al., 2013) who said that PCOS is high in early reproductive age group and gradually decreases as the age advances. Body mass index (BMI) was measured according to the following equation: dividing the weight in kilograms by the height in squared meters (kg/m2) (Flegal et al., 2005). The parameters of Body mass index was used according to the European Society of Human Reproduction and Embryology, 2009; as followed: Underweight ≤ 18.5, Normal 18.5-24.9, 61
Chapter Four
Discussion and Conclusion
Overweight 25-29.9, and Obesity ≥ 30. As shown in table (3.4), percentage of overweight in controls were 21.3% but in PCOS cases 38.0%, percentage of obese in controls groups were 6.7% but in PCOS cases 16.7%. There was statistically significant difference (p-value <0.001) in PCOS and control group this result in agreement with (Sam, 2007) which also found PCOS increased in obese cases.
4.4. Age of Menarche Age of menarche is the medical term for a woman's first menstruation. In the present study, all participants were divided into two classes according to age of menarche: class one (10-14 years) and class two (15-18 years). The results showed that, in 150 PCOS; 129 were class one and 21 were class two, but in 150 control group; 138 were class one and 12 were class two. There was no significant difference in age of menarche between PCOS cases and controls (pvalue =0.1) (Table 3.5) because age of menarche related to other life style and environmental factor for example female with higher MBI age of menarche earlier , This result consistent with other articles (Carroll et al., 2012, Chunyan He et al., 2009, Ibanez et al., 2006).
4.5. Family History of PCOS Among 150 cases of PCOS involved in the study 61 cases (40.0%) had positive family history whereas 89 cases (59.3%) had negative family history of PCOS, while in control groups 49 (32.7%) had positive family history but 101 (67.3%) had negative family history of PCOS (Table 3.6). Although positive family history in PCOS cases higher than control groups but statistically no significant (p-value 0.09) because developing PCOS not only governed by genetic also related to other environmental factors and life style, this result in agreement with previous study ( Moini and Eslami, 2009).
4.6. HbA1c and Blood Sugar Level in PCOS and Control Group The term HbA1c refers to glycated hemoglobin, HbA1c test does not require fasting and provides a time-averaged estimate of blood glucose over the preceding 3 - 12 weeks (Sacks et al., 2002), HbA1c test positively correlated with body weight, body mass index, waist: hip ratio, fat mass, triglyceride, total and free testosterone and fasting insulin (Medeiros et al., 2014). 62
Chapter Four
Discussion and Conclusion
The results of HbA1c were within the normal range in PCOS and control group. there was statistically significant difference (p-value < 0.05) and higher means and standard deviation in PCOS compare to control groups (Table 3.7), this result was agree with ( Lerchbaum et al., 2013, Celik et al., 2013) which found that patients with PCOS increase risk of elevated HbA1c and reported that elevation in 10% of PCOS patients. There was no significant difference in blood glucose level between cases and control group (p-value=0.18) (Table 3.7), this result was agree with (Gareeb et al., 2016, González et al., 2014, Lecke et al., 2013).
4.7. Serum Uric Acid in PCOS and Control group Uric acid is the metabolic end product of purine metabolism in humans. Hyperuricemia which is elevation of serum uric acid level predisposes to disease through the formation of urate crystals that cause gout, but hyperuricemia, independent of crystal formation linked with hypertension, atherosclerosis, insulin resistance, and diabetes (So and Thorens, 2010), also emerged as a cardiovascular risk factors (Luque-Ramirez et al., 2008). We observed that mean and standard deviation of uric acid in PCOS higher than healthy controls and statistically significant (p-value=0.01) (table 3.7). This agreed with ( Rajeswari et al., 2016, Gozukara et al., 2015, N.Swetha et al., 2013) who’s concluded that serum uric acid levels in PCOS were significantly raised when compared to healthy controls, it is due to endothelial dysfunction and chronic inflammation because uric acid exerting pro-oxidant and pro-inflammatory action at the endothelial cell. On the other hand, Serum uric acid level in PCOS and control group difference with BMI as shown in table (3.8) in both groups serum uric acid levels in obese female higher than underweight female and positively correlated which agree with other study (Wang et al., 2014).
63
Chapter Four
Discussion and Conclusion
4.8. Concentration of Serum IL-18 in PCOS and Control Group IL-18 is a proinflammatory cytokine that induces the production of TNF alpha, which in turn promotes the synthesis of IL-6, and IL-6 regulates the synthesis of CRP in the liver (Nakanishi et al., 2001). IL-18 is considered a strong risk marker for cardiovascular disease (Blankenberg et al., 2002). Women with PCOS may have chronic low-level inflammation and induce an increase in serum IL-18 levels, which are also associated with visceral adiposity and insulin resistance in PCOS (Escobar-Morreale et al., 2004). Elevated circulating levels of Il-18 also associated with atherosclerotic lesions, type 2 diabetes mellitus (T2DM), metabolic syndrome (MetS), hypertension (HT), and a worse prognosis in cardiovascular disease (CVD)(Espinola-Klein et al., 2008, , Blankenberg et al., 2003). There was highly significant increase (p-value < 0.001) of serum IL-18 in PCOS patients compared with control group (Figure 3.1). This result agrees with other previous study (EscobarMorreale et al., 2004).
4.9. Concentration of Serum Homocysteine in PCOS and Control Group Homocysteine is an intermediate product formed during the breakdown of the amino acid methionine, and may undergo remethylation to methionine or trans-sulphuration to cystathione and cysteine. Elevated serum levels of Hcy which called Hyperhomocysteinemia have adverse effects on the cardiovascular system. and considered to be an independent risk factor for CVD ((Baszczuk and Kopczynski, 2014). According to our results data in (Figure 3.2) mean and standard deviation of Hcy in PCOS was statistically significant difference (p-value < 0.001) and higher level compare to control group. These results agree with other studies which also notice that serum Hcy concentrations was elevated and risk of CVD have been increased in patients with PCOS compared with healthy controls (Wijeyaratne et al., 2004, Schachter et al., 2003).
64
Chapter Four
Discussion and Conclusion
4.10. Correlation between Serum IL-18 with Age, BMI, LH, FSH, Prolactin and Testosterone Level in PCOS In the present study (Table 3.9) showed correlation of serum IL-18 with age, BMI, LH, FSH, PRL and Testosterone. Regarding correlation of serum IL-18 with age in PCOS, There was not found a significant correlation (r=0.08, p-value 0.17), this result consistent with other study (Zhang et al., 2006), who’s also couldn’t found correlation between IL-18 and age, and said serum IL-18 levels were significantly increased in PCOS firmly associated with insulin resistance (Zhang et al., 2006). There was highly significant and moderate correlation between IL-18 concentration and LH level (r=0.452, p-value < 0.001), significant and weak correlation between IL-18 and BMI (r=0.156, p-value 0.03), and also weak correlation of IL-18 with testosterone (r=0.124, p-value 0.07) in PCOS. These results were in agreement with others previous studies which founded correlation of IL-18 with BMI, LH and testosterone level (Yang et al., 2011, Kowalska et al., 2006). Also there were weak significant correlation of IL-18 with FSH (r=0.18, p-value 0.026), and prolactin which were not significant (r=0.12, p-value 0.14).
4.11. Correlation between Serum Homocysteine with Age, BMI, LH, FSH, Prolactin and Testosterone Level in PCOS In the present study as shown in (Table 3.10) there was no correlation between Hcy concentration and BMI in PCOS (r=0.093, p-value 0.13), this was agree with the results of previous studies which said that Hcy was significantly elevated in both lean and obese PCOS patients, and was related to insulin resistance and not to body weight (Gareeb et al., 2016, Schachter1 et al., 2003,). There was significant correlation of serum Hcy concentration with LH level (r=0.202, pvalue 0.01), and age (r=0.177, p-value 0.015) in PCOS. This finding disagree with the other study (Schachter et al., 2003), this may be due to number of other variables were not focused on our study such as: smoking, renal function, vitamin B status, and enzyme dysfunction state which all of them influence Hcy level (de la Calle et al., 2007).
65
Chapter Four
Discussion and Conclusion
About correlation of Hcy concentration with testosterone level in PCOS patient, There was significant correlation(r=0.176, p-value 0.016) (Table 3.10) between them, this results agree with the results of previous study which also found correlation between Hcy and testosterone level (Gul et al., 2008). There was no correlation of Hcy with FSH (r=0.006, p-value 0.94) and prolactin (r=0.038, p-value 0.94).
4.12. Conclusion In our study the most common symptoms of PCOS in Sulaimani city were hirsutism, while the lest common symptoms were oligo menorrhea, acne, infertility, amenorrhea and alopecia. PCOS was more common in age 20-29 years and family history of PCOS increased the risk of this disease, Most PCOS enrolled in this study have high concentration of LH and testosterone levels which were the tests use for diagnosis of PCOS also PCOS did not effect on age of menarche. In PCOS HbA1c and uric acid level were significantly higher than control group while not significant difference in blood sugar level between both groups. Also in PCOS cases Serum IL18 increased and had correlation with LH, testosterone, FSH, Prolactin levels and BMI while not correlated with age. Furthermore, Serum Hcy increased in PCOS and correlated with age, LH and testosterone levels, while there were no correlations of Hcy with BMI, FSH and prolactin levels.
66
Recommendations
Recommendations 1) Increase number of samples to make more accurate result 2) Further investigations are needed to explain the mechanism of IL-18 and Hcy in developing cardiovascular disease 3) Further investigations are needed to evaluate the risk of elevated level of IL-18 and Hcy in other chronic disease like diabetes mellitus. 4) Further investigations are needed to explain the causes for elevation IL.18 and Hcy in PCOS patients. 5) Studying other hormones like estradiol, progesterone, DHEA and 17-hydroxyprogestrone to find the relation of these hormones with PCOS and serum level of IL-18 and Hcy in PCOS cases. 6) Studying the effects of other factors like diet, exercise, smoking and drugs on serum levels of IL-18 and Hcy in PCOS cases.
67
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68
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ٍ ٗ )μIU/mlدَ٘ػخ اىسٞطشح (ّ ٍِ .)2.47 ± 1.18 μIU/mlبحٞخ أخشّ ،ٙسجخ ٗ HbA1cحَط اى٘ٞسٝل فٜ ٍزالصٍخ رنٞس اىَجبٝط مبّذ ( )5.53 ± 0.97, 252.72 ± 54.96 μmol/Lػي ٚاىز٘اىٗ ٜاىز ٛمبُ أػي ٚثنثٞش ٍِ ٍدَ٘ػخ اىسٞطشح (ٗ ، )5.33 ± 0.73, 237.65 ± 47.04 μmol/Lاخٞشا ً ىٌ ٝنِ ْٕبك فشق مجٞش فٍ ٜسز٘ ٙاىسنش ف ٜاىذً ىَزالصٍخ رنٞس اىَجبٝط (ٍ ٗ)5.67 ± 1.34 mmole/Lدَ٘ػخ اىسٞطشح (.)5.51 ± 0.75mmole/L اظٖشد اىْزبئح صٝبدح ٍؼْ٘ٝخ ) (p-value < 0.001فٍ ٜسز٘ ٙاالّزشى٘م 81-ِٞفٍ ٜزالصٍخ رنٞس اىَجبٝط ( 378.3 ٍ )±181.21 pg/mlقبسّزب ً ٍغ ٍدَ٘ػخ اىسٞطشح (ٗ ، )224.98± 131.885 pg/mlػالٗح ػي ٚرىل ،مبُ ْٕبك اسرجبط ظؼٞف ثٍ 81-IL ِٞغٍ :ؤشش مزيخ اىدسٌ ٗ Testosteroneٗ FSH ،PRL ،BMIاسرجبط ٍؼزذىخ ثٍ 81-IL ِٞغ ٍسز٘ٙ ٗ LHػذً ٗخ٘د ا ٛاسرجبط ثٍ 81-IL ِٞغ اىزقذً ف ٜاىسِ فٍ ٜزالصٍخ رنٞس اىَجبٝط. مَب ٗاظٖشد اىْزبئح صٝبدح ٍؼْ٘ٝخ )(p-value < 0.001فٍ ٜسزٍٕ٘٘٘ ٙسسز ِٞفٍ ٜزالصٍخ رنٞس اىَجبٝط ( 10.36 ٍ )± 5.98 nmol/mlقبسّزب ً ٍغ ٍدَ٘ػخ اىسٞطشح (ٗ ، )5.17± 5.24 nmol/mlػالٗح ػي ٚرىل ،مبُ ْٕبك اسرجبط مجٞش ثٍ ِٞسز٘ ٙاىٍٖ٘٘سسزٍ ِٞغ اىزقذً ف ٜاىسِ ، Testosteroneٗ LH ،ف ٜح ِٞال ػالقخ ث ِٞرشمٞض اىٍٖ٘٘سسزٍ ِٞغ ٍؤشش مزيخ اىدسٌ .PRLٗ FSH ، BMI االضـتٌتــاجــاث اسرفبع فٍ ٜسز٘ ٙاالّزشى٘م ٗ 81-ِٞاىٍٖ٘٘سسز ِٞػْذ ٍشظٍ ٚزالصٍخ رنٞس اىَجبٝط ٍقبسّخ ٍغ ٍدَ٘ػخ اىسٞطشحْٕ .بك اسرجبط ث ِٞاالّزشى٘مٍ 81-ِٞغ ٍؤشش مزيخ اىدسٌٕ ٗ PRL ،FSH ،LH ،شٍُ٘ رسز٘سزٞشُٗٗ ،ىٌ ٝنِ ْٕبك ػالقخ ىالّزشى٘مٍ 81-ِٞغ اىزقذً ف ٜاىسِ فٍ ٜزالصٍخ رنٞس اىَجبٝطْٕ .بك اسرجبط ث ِٞاىٍٖ٘٘سسزٍ ِٞغ اىزقذً ف ٜاىسِLH ، ٍٗسز٘ ٙاىزٞسز٘سزٞشُٗ ،ثَْٞب ال ٘ٝخذ اسرجبط ىيٍٖ٘٘سسزٍ ِٞغ ٍؤشش مزيخ اىدسٌ PRLٗ FSH ،فٍ ٜزالصٍخ رنٞس اىَجبٝط.
الــخــالصـــــــت الـخلفيت: ٍزالصٍخ رنٞس اىَجبٝط ٕٗ ٜاحذح ٍِ أمثش االٍشاض اىٖشٍّ٘ٞخ ش٘ٞػب ً ف ٜاىْسبء خاله سْ٘اد اىخص٘ثخ. ٝزَٞضاىَشض ثـٍ٘ :سف٘ى٘خٞب اىَجٞط اىَزؼذد اىزنٞس ،ػذً اّزظبً اىذٗسح اىشٖشٝخ ،فشط اّزبج اىٖشٍّ٘بد األّذسٗخْٞٞخ سشٝشٝب ً اٗ حٝ٘ٞبً ،صٝبدح خطش اإلصبثخ ثذاء اىسنش ٍِ ٛاىْ٘ع ٗ 2اىجذاّخٍ .زالصٍخ رنٞس اىَجبٝط ٕ٘ اظطشاة ٍؼقذ ٝزأثش ثنو ٍِ اىؼ٘اٍو اىجٞئٞخ ٗاى٘ساثٞخٝ .زٌ رشخٞص ٍزالصٍخ رنٞس اىَجبٝط ثظٖ٘س ػي ٚاألقو اثْبُ اٗ ثالثخ ٍِ االػشاض اىزبىٞخ :فشط اىٖشٍّ٘بد األّذسٗخْٞٞخ سشٝشٝب ً اٗ حٝ٘ٞبً ،ػذً اّزظبً اىذٗسح اىشٖشٝخ ٗرنٞس اىَجبٝط ( )PCOاىَالحع ػْذ اىفحص ثبىَ٘خبد ف٘ق اىص٘رٞخ.
الِـذف هي الـذراضــت: اىٖذف ٍِ ٕزٓ اىذساسخ ٕ٘ رقٍ ٌٞٞسز٘ ٙاّزشى٘مٗ 81-ِٞاىٍٖ٘٘سسز ِٞفٍ ٜصو ٍشظٍ ٚزالصٍخ رنٞس اىَجبٝط ٍٗقبسّخ ٕزٓ اىقٍ ٌٞغ ٍدَ٘ػخ األصحبء.
طـرق الـعوــل: فٕ ٜزٓ اىذساسخ رٌ أخز ػْٞبد اىذً ٍِ 033شخص ( 853ػْٞخ ٍِ اىَشظ ٚاىزٝ ِٝؼبُّ٘ ٍِ ٍزالصٍخ رنٞس اىَجبٝط ٗ 853ػْٞخ ٍِ االصحبء)ٗ .قذ ارخزد خَسخ ٍييٞيزش ٍِ اىذً اى٘سٝذ ٍِ ٛمو فشد ٗرٌ رحيٞو :اّزشى٘مٗ81 - ِٞ ٍٕ٘٘سسزّ ِٞزقْٞخ اىٞالٝضا ) ، (ELISAفحص اىسنش ف ٜاىذًّ ،سجخ ،HbA1cحَط اى٘ٞسٝل ٗفحص اىٖشٍّ٘بد ٗقذ شَيذٕ :شٍُ٘ اىَحفض ىيدسٌ االصفش(ٕ ، )LHشٍُ٘ ٍحفض اىدشٝجبد(ّ ، )FSHسجخ (ٕ ، )LH/FSHشٍُ٘ اىحيٞت()PRL ٗ ٕشٍُ٘ اىشحَُ٘ اىخص.)testosterone(ٛ٘ٞ
الـٌتـائـج: اظٖشد ّزبئح ٍسز٘ ٙاىٖشٍّ٘بد أٍّ :سزٕ٘ ٙشٍُ٘ LHفٍ ٜزالصٍخ رنٞس اىَجبٝط ()10.87 ± 7.36 mIU/m ٗاىز ٜمبّذ أػي ٚثنثٞش ٍِ ٍدَ٘ػخ اىسٞطشح (ٍ ،)5.73 ± 2.25 mIU/mlسز٘ FSH ٙف ٜاىذً فٍ ٜزالصٍخ رنٞس اىَجبٝط ( )5.84 ± 4.60mIU/mlاىز ٛمبُ أقو ثنثٞش ٍِ ٍدَ٘ػخ اىسٞطشح (ّ ،)6.86 ± 2.43mIU/mlسجخ FSH/LHفٜ ٍزالصٍخ رنٞس اىَجبٝط ( ٕ٘ٗ )2.09 ± 1.57أػي ٚثنثٞش ٍِ ٍدَ٘ػخ اىسٞطشح (ٍ ،)0.96 ± 0.74سز٘ PRL ٙفٜ ٍزالصٍخ رنٞس اىَجبٝط ( ٜٕٗ )19.67± 10.65 ng/mlأػي ٚثنثٞش ٍِ ٍدَ٘ػخ اىسٞطشح (،)16.42 ± 4.52 ng/ml ٍسزٕ٘ ٙشٍُ٘ testosteroneفٍ ٜزالصٍخ رنٞس اىَجبٝط (ٗ )0.90 ± 2.01 ng/mlاىز ٜمبّذ أػي ٚثنثٞش ٍِ ٍدَ٘ػخ اىسٞطشح (ٗ ،)0.35 ± 0.24 ng/mlىٌ ٝنِ ْٕبك فشق مجٞش فٍ ٜسز٘ TSH ٙثٍ ِٞزالصٍخ رنٞس اىَجبٝط ( 2.40 ± 1.02
حكْهت اقلين كردضتاى ّزارة التعلين العالي ّ البحث العلوي جاهعت الطليواًيت کليت الصيذلت فرع الكيوياء الحياتيت الطريريت
تـقــيـيـن هـطـــتـْٓ االًـتــرلـْكـيـيّ 81-الِـْهـْضـطــتيـي في هـرضـٔ هتـالزهـت تكـيــص الوـبايــض في هـحـافظـت الطــليـواًـيــت سسبىخ ٍقذٍخ اىٍ ٚديس ميٞخ اىصٞذىخ ف ٚاىدبٍؼخ اىسيَٞبّٞخ مدضء ٍِ ٍزطيجبد ّٞو شٖبدح اىَبخسزٞشف ٜاىنَٞٞبء اىحٞبرٞخ اىسشٝشٝخ/اىزحبىٞو اىسشٝشٝخ
ٍِ قِ َجو ضاكار كرين عبذهللا بكالْريْش في الصيذلت /جاهعت الطليواًيت٠٢٠٢- ثأششاف الوذرش د.باى هْضٔ رشيذ دكتْرا في الكيوياء الحياتيت الطريريت
ٍٞ 2382الدٙ
2182م٘سدٙ
ىٔ الٔٝم ٚرشەٗە ئبسز Homocysteine ٚىٔ فشە مٞسٕ ٚێينٔداّذا ) (10.36 ± 5.98 nmol/mlصٝبرشە ثٔ ثٔساٗسد ىٔمٔڵ ئبفشەر ٚرّٔذسٗسذ)ٗ ،(5.17± 5.24 nmol/mlە پٔ٘ٝەّذٕٔٝٔ ٙىٔگٔڵ رٍُٔٔ ٗئبسزLH, ٚ Testosteroneىٔ ّٔخۆشٔمٔدا ثٔاڵً ثٔ٘ٝەّذ ّٔٞ ٙىٔمٔڵ FSH, PRL, BMIىٔ ّٔخۆشٔمٔدا.
دەرئًَجام: ئبسز )Interleukin-18 and Homocysteine) ٚمٔ ّٞشبّذەسێنِ ثۆّٔخۆشٔٞمبّ ٚدڵ ٗ مۆئّٔذاٍ ٚس٘ڕ ٙخ٘ێِ ىٔ ئبفشەر ٚر٘شج٘ ثٔ فشە مٞسٕ ٚێينٔداُ صٝبرشە ثٔ ثٔساٗسد ثٔ ئبفشەر ٚرّٔذسٗسذ،ئبسز Interleukin-18 ٚپٔ٘ٝحّذٕٔٝٔ ٙ ىٔگٔڵ ئبسز ، (BMI,LH,FSH,PRL and testosterone)ٚثٔاڵً ثٔ٘ٝەّذ ّٔٞ ٙىٔگٔڵ رٍّٔٔذاٗ،ەئبسزHomocysteine ٚ پٔ٘ٝەّذٕٔٝٔ ٙىٔگٔڵ رٍُٔٔ ٗئبسز LH, Testosterone ٚىٔ ّٔخۆشٔمٔدا ثٔاڵً پٔ٘ٝەّذ ّٔٞ ٙىٔگٔڵ FSH, PRL, BMIىٔ ّٔخۆشٔمٔدا.
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پْختَ بٌچيٌَ: فشە مٞسٕ ٚێينٔداُ ٔٝمێنٔ ىٔ ثٔسثاڵٗرشّٔ ِٝخۆش ٚم٘ێشە ڕژێْٔمبُ ىٔ ئبفشەرذا ىٔ سبڵٔمبّ ٚر٘اّبٍْ ٙذاڵجّ٘ٞذا، ّٞشبّٔمبّ ٚثشٝز ِٞىٔ مۆڕاّنبسٔٝمبّٕ ٚێينٔداُّ ،بڕێن ٚس٘ڕٍ ٙبّگبّّٔٞ ،شبّٔمبّٝ ٚبُ ثٔسص ٙشٞنبس ٙڕژێْی ّێشەمٚ ،Androgenصٝبدثٍّ٘ٔ ٚرشس ٚثۆ ر٘شجُ٘ ثٔخۆسی دّٗٗٔ ٙخۆش ٚشٔمشە ٗ صٝبدثّ٘ ٚمێش ٚىٔش ،فشە مٞسٕ ٚێينٔداُ مبسٝزێئٔمشٝذ ثٔ ٕٔسدٕٗٗ٘مبسە ژْٝگٔ ٗ ٚٝثۆٍبٗۀٞٝمبُ .فشە مٞسٕ ٚێينٔداُ دەر٘اّشێذ دەسزْٞشبُ ثنشێذ ىٔ مبر ٚثّ٘ٚ الّ ٚمًٔ دٗاُ ىٔٗ سێ ّٞشبّبّٔ ٙخ٘اسەٗەّٞ:شبّٔمبّٝ ٚبُ ثٔسص ٙشٞنبسٔٝمبّٕ ٚۆسٍۆّّ ،Androgen ٚبڕٝن ٚس٘ڕٙ ٍبّگبّٔ ،فشە مٞسّ ٚبٗ ٕٞينٔداُ مٔ ثجْٞشێذ ىخ سۆّخسدا.
ئاهاًج لَتْێژيٌَّەكَ: ٍٔثٔسزی سٔسەکی ئًٔ ر٘ێژْٗٔٝە ٔٝپشنْ ْٚٞئبسزی ( ٓ)Homocysteine(ٗ )Interleukin-18ىٔ شئ ٙخ٘ێْی ر٘شج٘اُ ثٔ ّٔخۆشی فشە مٞسٕٞ ٚينٔداُ ٗۀٕسٗەٕب ثٔساٗسد مشدّ ٚدەسئّٔدبٍٔمبُ ىٔمٔه مٔسبّ ٚرّٔذسسزذا.
رێگَی ئًَجاهذاًی تْێژيٌَّەکَ: ىًٔ ر٘ێژْٗٔٝۀٝدا( )٠٣٣ئبفشەد ثٔشذاس ٙمشدٗٗٓ مٔ ( )٠٥٣ئبفشەر ٚر٘شج٘ ثٔ ّٔخۆش ٚفشە مٞسٕٞ ٚينٔداُ ثُ٘ٗ ٗە ( )٠٥٣ئبفشەر ٚرّٔذسٗسذ ثُ٘ٗ .پێْح ٍٞييٞيٞزش خ٘ێِ ىٔ خ٘ێِ ٕێْٔسی ٍٕٔ٘ ثٔژداسث٘اُ ٗەسگٞشاثٍٔٔثٔسزی پێ٘أّ کشدّی ئبسزی ) )Interleukin-18 ,Homocysteineثٔثٔکبس ٕێْبّی رٔکْٞکی ( Enzyme Linked Immunosorbent ٗ .)Assayۀٕسٗەٕبپێ٘أّ کشدّی ئبسزی ڕژێْٔکبّی ))LH,FSH, LH/FSH ratio, TSH, PRL, Testosterone ٗپ٘ٞأّ کشدّی ئبسزی ( ) Blood Sugar, HbA1c, Uric acidىٔ شئ ٙخ٘ێْ ٚثٔشذاسث٘اّذا.
ئًَجاهَکاى: ئّٔدبٍ ٚشٞنبسٕ ٙۆسٍۆّٔمبُ ىًٔ ر٘ێژْٗٔٝۀٝدا ثًٔ شێ٘ە :ٔٝٔٝىّٔٔخۆش ٚفشە مٞسٕٞ ٚينٔداّذا ئبسزٕ ٚۆسٍۆّٔمبّٚ )(LH, LH/FSH ratio, PRL and testosteroneصٝبرشەٗ ،ە ئبسزٕ ٚۆسٍۆّFSH ٚ
مٍٔزشە ثٔ ثٔساٗسد ىٔمٔه
ئبفشەر ٚرّٔذسٗسذ ،ثٔاڵً ئبسزٕ ٚۆسٍۆّ TSH ٚخٞبٗاص ّٔٞ ٙىٔ ّ٘ٞاُ ئٔٗ دٗٗ گشٗپٔدإٔ .سٗەٕب ئبسزٗ (uric acid ) ٚ )(HbA1cىّٔٔخۆش ٚفشە مٞسٕٞ ٚينٔداّذاصٝبرشە ثٔ ثٔساٗسد ىٔمٔڵ ئبفشەر ٚرّٔذسٗسزذا ثٔاڵً ئبسز(Blood sugar) ٚ خٞبٗاص ّٔٞ ٙىٔ ّ٘ٞاُ ئٔٗ دٗٗ گشٗپٔدا. ئبسز )Interleukin-18) ٚىٔ ئبفشەر ٚر٘شج٘ ثٔ مٞسٕ ٚێينٔداُ ) (378.3 ±181.21pg/mlصٝبرشە ىٔ ئبفشەرٚ رّٔذسٗسذ)ٗ ،(224.98± 131.885 pg/mlە ئبسز Interleukin-18 ٚپٔ٘ٝەّذ ٕٔٝٔ ٙىٔمٔڵ ئبسزٚ ) (BMI,LH,FSH,PRL and testosteroneثٔاڵً پٔ٘ٝەّذ ّٔٞ ٙىٔگٔڵ رخٍخّذا ىٔ ئبفشەر ٚر٘شج٘ ثٔ فشە مٞسٕ ٚێينٔداُ.
حكْهَتی َُرێوی كْردضتاى ٍّزارٍتی خْێٌذًی بااڵ ّ تْێژيٌٍَّی زاًطتی زاًكۆی ضلێواًی کۆلێژی دەرهاًطازی بَشی کيويای ژياًی کليٌيکی
َُڵطًَگاًذًی ئاضتی Interleukin-18 ّ Homocysteineلَ شلَی خْێٌی تْشبْاى بَ ًَخۆشی كيطٔ ُيلكَداى لَ پارێسگای ضلێواًيذا ّبٍٔٔٝکٔ پێشکٔش کشاٗە ثٔ ئّٔدٍّ٘ٔی کۆىێژی دەسٍبّسبصی ىٔ صاّنۆی سيێَبّی ٗەك ثٔشٞل ىٔ پێذاٗٝسزٔٞکبّی ثٔدەسذ ٕێْبّی ثڕٗاّبٍٔی ٍبسزٔس ىٔ ث٘اسی کَٞٞبی ژٝبّی کيْٞٞکی/ کيْٞٞنی شٞکبسی
ىٔ الُٔٝ ضاکار کرين عبذهللا بَکالۆرۆش لَ دەرهاًطازی /زاًكۆی ضليواًی٠٢٠٢-
ثٔ سٔسپٔسشزی دكتۆرە باى هْضی رشيذ دکتۆرا لَ کيويای ژياًی کليٌيکی
2182ک٘سدی
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