1
The Islamic University-Gaza
Deanery of Postgraduate Studies
Faculty of Science
Department of Biological Sciences
Medical Technology
Evaluation of Insulin-Like Growth Factor Binding
Protein-1 among Infertile Women with Poly Cystic Ovary
Syndrome in Gaza strip .
Prepared by
Hana Zuheer Zumarra
B.Sc. Medical Technology
The Islamic University of Gaza
Supervisor
Prof. Dr. Baker M. Zabut
Faculty of Science
The Islamic University of Gaza
A thesis submitted in partial fulfillment of the requirements for the
degree of Master of Biological Sciences-Medical Technology
March,2015
II
Declaration
I hereby declare that this submission is my own work and that to the best of my
knowledge and belief, it contains no material previously published or written by
another person or material which to a substantial extent has been accepted for
qualification of any other degree of the university or other institute.
Hana Zumarra Signature
Hana Zumarra Name
March,2015 Date
III
Dedication
I would like to dedicate my work to:
All those who taught me a letter .
My father who is a good example to be followed.
My mother who has sacrificed every thing in her life for me.
My be loved husband for his encouragement and support.
My brothers and sisters for their support.
All my friends and colleagues in the work.
Every one advise me and share with.
Every one who adore the education.
The souls of martyrs who sacrificed their lives for Palestine.
Palestinian people who have suffered and struggled with the persistence to
have a free Palestine.
IV
Acknowledgments
First of all, the real infinite and grateful praises and thanks to ALLAH for all his
gifts thus this text would not have been possible without the helps of ALLAH.
I would like to express my appreciation to my supervisor Prof. Dr. Baker Zabut,
Professor of Biochemistry, Faculty of Science, The Islamic University of Gaza, who
has cheerfully answered my queries, checked my examples, assisted me in a myriad
ways with the writing and helpfully commented on earlier drafts of this project.
My thanks also to all members in The Islamic University of Gaza and Faculty of
Science for giving me the opportunity to achieve this research.
My great thanks to my dear husband Mohammed El burnia, who have EL arabi
medical laboratory for his support and helpful to achieve this research .
My thanks should be extended to all the doctors and laboratory staff members at
Specialized Medical Centers in Gaza city especially Dr. Fadya Mallhes , Dr. Seeda
Maleha and Dr. Bahaa ALgalaeeni for their helpful.
Also my special thanks to Dr. Akram Elhalak for his helpful to achieve this work.
I am also very grateful to my friends especially Dojana Abu Khater and my family
for their good humor and support throughout the production of this research.
My thanks also to my colleagues in the work Abed Alkader ALottol and Rami
Almasry for their helpful .
At the end, I am very grateful to those who participated and helped me to complete
this study.
V
Evaluation of Insulin-Like Growth Factor Binding Protein-1among
Infertile women with Poly Cystic Ovary Syndrome in Gaza strip .
Abstract Background: Poly cystic ovary syndrome (PCOS) is one of the most common
endocrine disorders among women. It is thought to be one of the leading causes of
female infertility. An elevated circulating concentration of insulin inhibits the
production of insulin-like growth factor binding protein-1 (IGFBP-1), thus increasing
the level of free IGF-I in serum and stimulating ovarian androgen production.
Objective: to evaluate of IGFBP-1 and its relation to other reproductive hormones
among Poly Cystic Ovary infertile women in Gaza strip.
Methodology: The study was a case control and carried out in Specialized Medical
Centers in Gaza strip. A total of 40 women with PCOS infertility were face to face
interview was used for filling questionnaire which is designated for matching the
study need and blood analysis for IGFBP-1, Follicular Stimulating Hormone (FSH),
Luteinizing Hormone (LH), Testosterone, Dehydroepiandrosterone-Sulfate
(DHEA-S), Thyroid Stimulating Hormone (TSH) and Insulin were performed .
Moreover, forty fertile women were served as controls and they have been selected
on the basis of being married, having children and matching the case in age and
residence. Statistical Package for the Social Sciences (SPSS) system was used to
analyze the obtained data.
Results: The mean body mass index (BMI) in patients compare to controls were
(25.8 and 23.7) kg/m2, respectively. The mean of the serum IGFBP-1 levels were
decreased significantly in the patients group compared to controls,(4.57 vs 9.2
ng/ml), (P=0.000). Moreover the serum LH, Testosterone, DHEAS–S, and Insulin
were increased in patients compared to controls (18.77±7.05 vs 7.75± 2.14 mIU/ml,
P=0.00) and (0.54±0.28 vs 0.35±0.18 ng/ml, P=0.00) and (2.06±1.01 v
1.0±0.43μg/ml, P=0.00) and (20.61±5.48 vs 6.86±1.62 mIU/ml), P=0.00),
respectively. But the serum FSH, TSH were no significantly different between
patients and controls (6.11±1.77 vs 6.0±2.09 mIU/ml,P=0.82) and (2.61±1.07 vs
2.84±1.42 mIU/ml, P=0.41), respectively.
IGFBP-1 was negatively correlated with LH, Testosterone, DHEA-S, Insulin and
BMI (r=-0.632,P=0.00),(r=-0.389,P=0.00), (r=-0.546,P=0.01), (r=-0.590,P=0.00) and
(r=-0.411,P=0.00), respectively among the study population. Its correlation with
other parameters FSH and TSH were not observed (P>0.05).
Conclusion: The significance decrease in the serum IGFBP-1 levels between PCOS
infertile women and fertile women suggests that this hormone is involved in
pathophysiology of PCOS infertility. IGFBP-1 was negatively correlated with LH,
Testosterone, DHEA-S and Insulin. In contrast, no significant correlation was
observed between the IGFBP-1 and FSH or TSH hormones which suggests that
physiological concentration of serum IGFBP-1 does not directly influence FSH and
TSH production.
Key words: PCOS, Infertile women, IGFBP-1 status, Gaza strip.
VI
ستخلصم
لدى السيدات العقيمات في باألنسولين النمو الشبيههرمون المرتبط ب 1البروتين الناقل تقييم غزة قطاع
كما ويعتبر أيضا من ˛يعتبر مرض التكيس المبيضى هو أحد االضطرابات الهرمونية األكثر شيوعا بين السيدات: المقدمةالبروتين الناقل رتفاع معدل األنسولين في الدم يعمل على تثبيط إنتاج ا.أهم األسباب التي تؤدى إلى العقم لدى السيدات
والذي 1النمو الشبيه باألنسولين الحر هرمونمما يؤدى إلى ارتفاع نسبة المرتبط بهرمون النمو الشبيه باألنسولين 1 .بدوره يزيد من تحفيز المبايض إلنتاج الهرمونات الذكرية
وبعض الهرمونات المرتبط بهرمون النمو الشبيه باألنسولين 1البروتين الناقل عرفة دور تهدف الدراسة إلى م :الهدف ∙غزة قطاعاألخرى لدى السيدات الالتي يعانين من مرض التكيس المبيضى في
والتي تم اختيارها من المراكز الطبية ˛تتبع الدراسة منهجية اختيار عينة تجريبية وأخرى ضابطة : آليات الدراسة سيدة مصابة بالتكيس المبيضى 04أجريت هذه الدراسة على مجموعة مكونة من ∙غزة قطاعالمتخصصة في العقم في
وتم إجراء مقابلة لتعبئة االستبيان وأخذ ˛(العينة الضابطة ) سيدة لديهن قدرة منتظمة على اإلنجاب 04بالمقارنة مع . IGFBP-1 ,FHS ,HS ,Testosterone, SAEH ,HHS ,nIlusnI ة جراء الفحوصات الهرمونيعينة دم إل
∙لتحليل البيانات والنتائج التي تم الحصول عليها HSHHواستخدمت الحزمة اإلحصائية كما ∙على التوالي 8م/كيلو جرام )8.52و 8.52(أن متوسط مؤشر كتلة الجسم كان أظهرت نتائج الدراسة: النتائج حيث ˛لدى العينة التجريبية المرتبط بهرمون النمو الشبيه باألنسولين 1البروتين الناقل أظهرت النتائج انخفاض معدل و
, على التوالي ls/In ) 2588و 05.2(أعطى دالئل إحصائية واضحة بالمقارنة مع العينة الضابطة وكانت النتائج هيفي العينات التجريبية مقارنة مع العينة HS,HTlsslsTtsIT, SAEH,nIlusnIكما وسجل ارتفاع معدل كل من
في كلتا العينتين التجريبية FHS ˛ HHSفي معدالت كل من ذات داللة احصائيةبينما لم يكن هناك فروقات . الضابطةمع كل النسولينالمرتبط بهرمون النمو الشبيه با 1البروتين الناقل كما بينت الدراسة وجود ارتباط عكسي بين . و الضابطة
.ومؤشر كتلة الجسم, HS ,HTlsslsTtsIT , SAEH ,nIlusnIمن المرتبط بهرمون النمو الشبيه 1البروتين الناقل في مستوي ذو الداللة االحصائية االنخفاض الملحوظ :الخالصة ية كبيرة في الفسيولوجيا له دور ذو أهمانه لدى السيدات الالتي يعانين من التكيس المبيضى يشير إلى باألنسولين
المرتبط بهرمون النمو الشبيه باالنسولين 1البروتين الناقل بين عكسي لوحظ وجود ارتباط. المرضية للتكيس المبيضىالمرتبط بهرمون 1البروتين الناقل ولم يكن هناك ارتباط بين .HS ,HTlsslsTtsIT , SAEH ,nIlusnI وكل من
المرتبط 1للبروتين الناقل وذلك يشير إلي أن التركيز الفسيولوجي FHS,HHS هرمونيوكال من النمو الشبيه باألنسولين . بهرمون النمو الشبيه باألنسولين ال يؤثر على إنتاجهما
, لنمو الشبيه باألنسولينالمرتبط بهرمون ا 1البروتين الناقل , التكيس المبيضى, النساء العقيمات :الكلمات المفتاحية .قطاع غزة
VII
Table of contents
Declaration
II
Dedication
III
Acknowledgment
IV
English abstract
V
Arabic abstract
VI
Table of contents
VII
List of tables
XII
List of figures
XIV
Abbreviation
XVI
Chapter 1: Introduction
1
1.1 Overview………………………………………………………………
1
1.2 General objective………………………….............................................
3
1.3 Specific objectives……………………………………………………
3
1.4 Significance…………………………………………………………
3
Chapter 2: Literature review
4
2.1 Structure of ovaries………………………………………………………
4
2.2 Ovarian Hormone……………………………………………….......
5
2.2.1 Estrogens…………………………………………………………
5
2.2.2 Progesterone………………………………………………………
6
2.3 Oogenesis……………………………………………………………….
7
2.4 Ovarian cycle……………………………………………………………
8
2.4.1 Ovarian cycle and hormonal regulation…………………………… 10
VIII
2.5 Infertility………………………………………………………………….
11
2.5.1 Types of infertility…………………………………………………
11
2.5.2 Risk factor of female infertility……………………………………
12
2.5.3 Symptoms of infertility……………………………………………
12
2.5.4 Causes of female infertility………………………………………
13
2.6 Thyroid hormones and infertility………………………………………..
13
2.7 Ovulatory disorders…………………………………………………….
14
2.7.1 Hormonal problems………………………………………………
14
2.7.2 Malfunction of hypothalamus……………………………………
14
2.7.3 Malfunction of the pituitary gland……………………………
14
2.8 Poly Cystic Ovary Syndrome…………………………………………..
15
2.8.1 Diagnosis of Poly Cystic Ovary Syndrome………………………
15
2.8.2 Clinical feature of Poly Cystic Ovary Syndrome………………
16
2.8.3 Reproductive feature of Poly Cystic Ovary Syndrome……………
17
2.8.3.1 Ovarian dysfunction and infertility…………………………
17
2.8.3.2 Hyper androgenism………………………………………
17
2.8.4 Metabolic feature of Poly Cystic Ovary Syndrome……………
18
2.8.4.1 Dyslipidemia……………………………………………
18
2.8.4.2 Insulin resistance and abnormal glucose metabolism………
18
2.8.4.3 Cardiovascular disease risk…………………………
19
2.9 Extra- ovarian factors…………………………………………………
20
2.9.1 FSH deficiency……………………………………………………
20
2.9.2 Hyper secretion of LH…………………………………………
21
2.10 Intra- ovarian factors……………………………………………… 21
IX
2.10.1 Epidermal growth factors family………………………………
22
2.10.2 Fibroblast growth factors family………………………………
22
2.10.3 Neurotrophin growth factors family……………………………
22
2.10.4 Transforming growth factor-b family………………………
23
2.10.5 Vascular endothelial growth factor family……………………
23
2.10.6 Cytokine family………………………………………………
24
2.11 Other microenviroment factors………………………………………..
24
2.11.1 Homocysteine……………………………………………………
24
2.11.2 Leptin……………………………………………………………
25
2.12 Insulin- like Growth Factor family system………………………
25
2.13 Insulin- like Growth Factor Binding-1………………………………
26
2.13.1 Definition and site of secretion…………………………………
.
26
2.13.2 Action of IGFBP-1 on the ovary………………………………
27
2.13.3 Role of IGFBP-1 during human pregnancy……………………
28
2.13.3.1 Migration………………………………………………..
28
2.13.3.2 Metabolism………………………………………………
30
2.13.4 Physiological condition of IGFBP-1…………………………
31
2.13.5 Regulation of IGFBP-1 action……………………………
31
2.14 Previous studies………………………………………………………..
32
Chapter 3: Material and Methods
35
3.1 Study design…………………………………………………………
35
3.2 Target population……………………………………………………
35
3.3 Sampling and Sample size…………………………………………
35
3.4 Ethical consideration…………………………………………………….. 36
X
3.5 Data collection……………………………………………………………
36
3.5.1 Questionnaire interview………………………………………………
36
3.5.2 Body mass index…………………………………………………
36
3.5.3 Blood sampling and processing……………………………………
36
3.5.4 Determination of serum IGFBP-1………………………………….
37
3.5.5 Hormonal analysis…………………………………………………
39
3.6 Statistical analysis…………………………………………………
49
Chapter 4: Results
50
4.1 Demographic characters of the study population…………………………
50
4.2 Distribution of BMI among the study population……………………
52
4.3 Serum IGFBP-1 of the study population………………………………….
52
4.4 Serum IGFBP-1 of the overweight of the study population……………..
54
4.5 FSH and LH Hormone of the study population…………………………..
54
4.6 Serum Testosterone levels of the study population………………………
57
4.7 Serum DHEA-S Hormone levels of the study population………………..
58
4.8 Serum TSH levels of the study population………………………………
59
4.9 Serum Insulin levels of the study population…………………………….
60
4.10 Correlation of IGFBP-1 with different hormones among the study
population
61
4.10.1 IGFBP-1 levels correlated with FSH and LH among the study
population
61
4.10.2 Correlation of IGFBP-1 levels with Testosterone and DHEA-S
levels of the study population
61
4.10.3 IGFBP-1 levels correlated with TSH and Insulin levels of the
study population
62
4.10.4 IGFBP-1 levels correlated with BMI among the study Population 63
XI
4.11 Correlation of IGFBP-1with different hormones among the cases
63
4.11.1 IGFBP-1 levels correlated with FSH and LH among the cases
63
4.11.2 IGFBP-1 levels correlated with Testosterone and DHEA-S among
the cases
64
4.11.3 IGFBP-1 levels correlated with TSH and Insulin among the cases
65
4.11.4 IGFBP-1 levels correlated with BMI among the cases
65
Chapter 5 : Discussion
66
5.1 Socio demographic of the study population………………………………
67
5.2 BMI of the study population……………………………………………
67
5.3 Assay of IGFBP-1 of the study population………………
68
5.4 Hormonal profile of the study population………………………………
68
5.5 IGFBP-1 correlated with BMI and FSL , LH hormones…………………
70
5.6 IGFBP-1 correlated with TSH,Insulin,Testosterone and DHEA- hormones
70
Chapter 6: Conclusions and Recommendations
72
6.1 Conclusions……………………………………………………………….
72
6.2 Recommendations…………………………………………………………
73
Chapter 7: References
74
Appendices
Annex 1
87
Annex 2
89
Annex 3
90
Annex 4
91
XII
List of Tables
Table Page
Table 2.1: Commonly Used Definitions of Poly cystic Ovary Syndrome
15
Table 4.1: Distribution of Age, Education scale, Family history, Drug
consumption of the study population
51
Table 4.2: Distribution of Body Mass Index among the study population
52
Table 4.3: Distribution of IGFBP-1 of the study population
53
Table 4.4: Distribution of IGFBP-1 of the overweight of the study
population
54
Table 4.5: FSH levels of the study population
55
Table 4.6: LH levels of the study population
56
Table 4.7: Testosterone levels of the study population
57
Table 4.8: DHEA-S Hormone levels of the study population
58
Table 4.9: TSH levels of the study population
59
Table 4.10: Insulin levels of the study population
60
Table 4.11: Correlation of IGFBP-1 levels with FSH and LH of the study
population
61
Table 4.12: Correlation of IGFBP-1 levels with Testosterone and DHEA-S
of the study population
62
Table 4.13: Correlation of IGFBP-1 levels with TSH and Insulin of the
study population
62
Table 4.14: Correlation of IGFBP-1 levels with BMI of the study
population
63
Table 4.15: Correlation of IGFBP-1 levels with LH and FSH among the
cases
64
XIII
Table 4.16: Correlation of IGFBP-1 levels with Testosterone and DHEA-S
among the Cases
64
Table 4.17: Correlation of IGFBP-1 levels with TSH and Insulin among the
cases
65
Table 4.18: Correlation of IGFBP-1 levels with BMI among the cases
65
XIV
List of Figures
Figure
page
Figure 2.1: Cross section of the ovary
5
Figure 2.2:
Stage of oogenesis
7
Figure 2.3: Hormone Levels During Oogenesis
9
Figure 2.4: Regulation of ovarian cycle
11
Figure 2.5: Intra-and extra-ovarian factors associated with the PCOS
pathology that negatively affect oocyte and subsequent
embryo quality
20
Figure 2.6: Insulin-like growth factor family (IGF) system
25
Figure 2.7: Structure of IGFBP-1
26
Figure 2.8: Action of IGFBP-1 on ovary
28
Figure 2.9: Signaling pathways activated by the paracrine factors in
trophoblast cells
29
Figure 2.10: Proposed pathways of IGF-dependent IGFBP action
30
Figure 2.11: Growth hormone - Insulin-like growth factor-1 axis and
modulation by insulin-like growth factor binding proteins-1
and −3
32
Figure 4.1: Bar chart of mean serum IGFBP-1 levels of the study
population
50
Figure 4.2: Bar chart of mean serum FSH levels of the study population
51
Figure 4.3: Bar chart of mean serum LH levels of the study population
52
Figure 4.4: Bar chart of mean serum Testosterone levels of the study
population
53
Figure 4.5: Bar chart of mean serum DHEA-S levels of the study
population
54
XV
Figure 4.6: Bar chart of mean serum TSH levels of the study population
55
Figure 4.7: Bar chart of mean serum Insulin levels of the study population
56
XVI
Abbreviations
ACTH Adreno Cortico Trophic Hormone
AES Androgen Excess Society
AF Amniotic Fluid
AGEs Advanced Glycation End products
AITD Auto Immune Thyroid Disease
AMH Anti-Mullerian Hormone
APA Adrenal Precursor Androgen
ASRM American Society for Reproductive Medicine
BDNF Brain-Derived Neurotrophic Factor
BMI Body Mass Index
BMP Bone Morphogenetic Protein
cAMP Cyclic adenosine monophosphate
CHO Chinese Hamster Ovary
CVD Cardio Vascular Disease
DHEA-S Dehydroepiandrosterone Sulfate
DM2 Diabetes mellitus type 2
DRG Diagnostic Reagent Group
E2 Estradiol
EGF Epidermal Growth Factor family
ELISA Enzyme Linked Immuno Sorbent Assay
ESHRE European Society for Human Reproduction
FF Follicular Fluid
FFFS Follicle Fluid Factors
FGFs Fibroblast Growth Factors family
FSH Follicular Stimulating Hormone
GC Granulose Cell
GDF Growth Differentiation Factor
GDM Gestational Diabetes Mellitus
GnRH gonadotropin releasing hormone
GSK-3 Glycogen Synthase Kinase 3
H2SO4 Sulphuric acid
Hcy Homocysteine
HOXA Homeobox A
hsCRP high-sensitivity C-reactive protein
IGF Insulin-like Growth Factor
IGF1 Insulin-like growth Factor 1
IGF1R Insulin-like Growth Factor1 Receptor
IGF2 Insulin-like growth Factor 2
IGF2R Insulin-like Growth Factor2 Receptor
IGFBP-1 Insulin-like Growth Factor-Binding Protein-1
IGT Impaired Glucose Tolerance
IL Interleukins
IX Christmas factor
LH Luteinizing Hormone
MIS Mullerian Inhibiting Substance
XVII
mIU/ml Milliinternational Unit per milliliters
ml milliliters
mRNA messenger Ribonucleic Acid
NGF Neurotrophin Growth Factor family
NGF Nerve Growth Factor
NIH National Institutes of Health
nm Nanometer
NT Neurotrophin
NT-3 Neurotrophin-3
NT-4 Neurotrophin-4
NT-5 Neurotrophi-5
OD Optical Density
PCOS Polycystic Ovary Syndrome
PI 3-kinase phosphatidylinositol 3- kinase
PID Pelvic Inflammatory Disease
POI Primary Ovarian Insufficiency
PP12 Placental Protein 12
PRL Prolactin
s Fas soluble Fas
sFasL soluble Fas Ligand
SHBG Serum Hormone Binding Globulin
SPSS Statistical Package for the Social Sciences
TGFB Transforming Growth Factor-B family
TIRE Thymine-rich Insulin Response Element
TMB 3,3',5,5'-Tetramethylbenzidine
TNF Tumor Necrosis Factor
TRH Thyroid Releasing Hormone
TSH Thyroid Stimulating Hormone
VEGF Vascular Endothelial Growth Factor family
VII proconvertin
VIII Hemophilia A
XI Thromboplastin
μL Microlitre
1
Chapter 1
Introduction
1.1 Overview
Infertility is the failure of a female to become pregnant after one year of marriage
without birth control [1] and it is defined as If a woman is not able to carry a full
term pregnancy. Pregnancy is the result of a process that has many steps to get
pregnant: a woman's body must release an egg from one of her ovaries (ovulation),
the egg must go through a fallopian tube toward the uterus , a man's sperm must join
(fertilize) the egg along the way and the fertilized egg must attach to the inside of the
uterus (implantation); Infertility can happen if there are problems with any of these
steps, there are many biological and other causes of infertility, including some that
medical intervention can treat. About 40% of the issues involved with infertility are
due to the man, another 40% due to the woman, and 20% result from complications
with both partners. Most cases of female infertility are caused by problems with
ovulation. Without ovulation, there are no eggs to be fertilized. Some signs that a
woman is not ovulating normally include irregular or absent menstrual periods [2].
The regulation of the ovaries function is mediated primary by two hormones;
gonadotropin releasing hormone (GnRH) from hypothalamus and gonadotropin
stimulating hormones FSH and LH released from anterior pituitary. FSH initiates
follicular growth, specifically affecting granulosa cells with the concomitant rise in
inhibin B. FSH levels then decline in the late of follicular phase. This seems to be
critical in selecting only the most advanced follicle to proceed to ovulation. At the
end of the luteal phase, there is a slight rise in FSH that seems to be of importance to
start the next ovulatory cycle [3].
LH is necessary to maintain luteal function for the first two weeks. In case of a
pregnancy, luteal function will be further maintained by the action of human
chorionic gonadotropin (a hormone very similar to LH) from the newly established
2
pregnancy. Luteinizing hormone supports theca cells in the ovary that provide
androgens and hormonal precursors for estradiol production [4].
Ovulation problems are often caused by polycystic ovary syndrome (PCOS). PCOS
is a hormone imbalance problem which can interfere with normal ovulation, and
PCOS is the most common cause of female infertility. Primary ovarian insufficiency
(POI) is another cause of ovulation problems. POI occurs when a woman’s ovaries
stop working normally before she is 40. POI is not the same as early menopause.
PCOS happens when a woman's ovaries or adrenal glands produce more male
hormones than normal leading to cysts (fluid-filled sacs) development in the ovaries.
Women who are obese are more likely to have polycystic ovary syndrome than
normal weight women. Any problem in hypothalamus, pituitary, adrenal glands and
female reproductive system can cause infertility among females [5].
The insulin-like growth factor (IGF)1 axis plays an important role in human fetal
growth and development. Insulin-like growth factor-binding protein-1 (IGFBP-1) is a
major IGF binding protein in amniotic fluid (AF). The physiological role of IGFBP-1
is considered to be highly dependent on its differential phosphorylation.
Phosphorylation of IGFBP-1 increases its affinity for IGF-1 , suggesting that IGFBP-
1 may modulate the action of IGF-1 specifically with respect to fetal and placental
growth. and it is believed to be important in endometrial development during the
menstrual cycle and in the process of implantation [6]. It is a key regulator of fetal
and maternal tissue growth and development during human pregnancy [7].
3
1.2 General objective
The general objective of this study is to evaluate the level of IGFBP-1 and its
relation to the other reproductive hormones among PCOS infertile women in Gaza
Strip.
1.3 Specific objectives
1- To measure IGFBP1 level in PCOS infertile women and compare it with that
of controls.
2- To measure FSH, LH, Testosterone, Insulin, DHEA-S and TSH level in
study population.
3- Study the significance of BMI in patients versus controls.
4- To investigate the possible correlations between IGFBP-1 and the previous
studied parameters.
1.4 Significance
1- This is the first study to assess serum IGFBP1 level in poly cystic ovary
infertile women in Gaza Strip.
2- Understanding the relation between IGFBP-1 and other reproductive
hormones.
4
Chapter 2
Literature review
2.1 Structure of ovaries
The ovaries are almond-shaped structures which located medial to the external iliac
vessels and anterior to the internal iliac vessels and ureter and they are suspended
medially by the ovarian ligaments, originating bilaterally at the cornua of the uterus,
and laterally by the suspensory (infundibulopelvic) ligament, extending from the
infundibulum of the fallopian tube and ovary to the sidewall of the pelvis. The ovary
is also attached to the posterior aspect of the broad ligament via the mesovarium [8].
The ovaries are the only structures within the abdominal pelvic cavity that are not
covered by visceral peritoneum. The germinal epithelium is a single layer of
epithelial cells lining the outer surface of the ovary. The tunica albuginea is a fibrous
connective tissue capsule found beneath the epithelial layer. The ovarian stroma, or
body of the ovary, consists of the peripheral cortex and the central medulla. The
cortex constitutes the bulk of ovarian tissue, and is the site of oogenesis. The medulla
contains the ovarian vasculature, lymphatics, and nerves supported by fibrous
connective tissue (Figure 2.1). Ovarian size varies during the life span depending on
age, menstrual status, pregnancy status, body habitus, and menstrual cycle phase .
Normal measurements during reproductive years range from 2.5 to 5 cm in length,
0.6 to 2.2 cm in anteroposterior thickness or height, and 1.5 to 3 cm in width [9].
The ovary, an ever-changing tissue and dynamic multi compartmental organ, is
unique in the endocrine system in that in every reproductive cycle it develops
entirely new secretory structures, the graafian follicles, from a pool of primordial
follicles [10].The primordial follicles are the major endocrine and reproductive units
5
of the ovary whose numbers determine both reproductive potential and reproductive
life span [11].
Figure 2.1 Cross section of the ovary [ 12].
2.2 Ovarian Hormone
The main source of the female hormones estrogen and progesterone come from the
ovaries. These hormones are responsible for controlling the development of the
female body characteristics including, breast size, and body shape [13].
2.2.1 Estrogens
Estrogens are produced at the level of the ovary and is crucial for the development
of the antrum and maturation of the Graafian follicle, and they promote the
development and maintenance of female reproductive structures, secondary sex
characteristics and the breasts. The secondary sex characteristics include the
distribution of adipose tissue in the breasts, abdomen, and hips; also voice pitch, a
broad pelvis and the pattern of hair growth on the head and body [13].
6
Estrogen is predominant at the end of the follicular phase, directly preceding
ovulation. Estradiol, the most potent and abundant estrogen, is primarily derived
from androgens produced by thecal cells. The androgens migrate from the thecal
cells to the granulose cells, where they are converted into estradiol by aromatase
enzyme.
The actions of estradiol include induction of FSH receptors on granulosa cells,
proliferation and secretion of follicular thecal cells, induction of LH receptors on
granulosa cells, and proliferation of endometrial stromal and epithelial cells [14].
Estrogens increase protein anabolism and lower blood cholesterol level. Moderate
amount of estrogens in the body inhibit both the release of GnRH by the
hypothalamus and secretion of LH and FSH by the anterior pituitary gland. At least
six different estrogens are present in the plasma of human females, but only three are
present in significant quantities: B-estradiol, estrone, and estriol. In non pregnant
females, the principle estrogen is B-estradiol, which is synthesized from cholesterol
in the ovaries [14].
2.2.2 Progesterone
Progesterone is steroid hormone involved in the female menstrual cycle, pregnancy
(supports gestation) and embryogenesis of humans and other species. It is secreted by
the corpus luteum after ovulation and by the placenta and it is responsible for
preparing the body for pregnancy and, if pregnancy occurs, maintaining it until birth.
Progesterone is secreted from corpus luteum to make continues the preparation of
the endometrium for a possible pregnancy, inhibits contraction of the uterus and
inhibits development of a new follicle [14].
7
Figure 2.2 Stage of oogenesis [15].
2.3 Oogenesis
Oogenesis is the creation of an ovum (egg cell). It is the female form of
gametogenesis; the male equivalent is spermatogenesis. It involves the development
of the various stages of the immature ovum.
Female sex cells, or gametes, develop in the ovaries by a form of meiosis called
oogenesis. Early in fetal development, primitive germ cells in the ovaries
differentiate into oogonia. These cells divide rapidly to form thousands of cells, still
called oogonia, which have a full complement of 46 (23 pairs) chromosomes.
Oogonia then enter a growth phase, enlarge, and become primary oocytes ( Fig
2.2).The diploid (46 chromosomes) primary oocytes replicate their DNA and begin
the first meiotic division, but the process stops in prophase and the cells remain in
this suspended state until puberty. Many of the primary oocytes degenerate before
birth, but even with this decline, the two ovaries together contain approximately
700,000 oocytes at birth. This is the lifetime supply, and no more will develop. By
puberty the number of primary oocytes has further declined to about 400,000 [15].
Beginning at puberty, under the influence of FSH , several primary oocytes start to
grow again each month. One of the primary oocytes seems to outgrow the others and
8
it resumes meiosis I. while the other cells degenerate. The large cell undergoes an
unequal division so that nearly all the cytoplasm, organelles, and half the
chromosomes go to one cell, which becomes a secondary oocyte. The remaining half
of the chromosomes go to a smaller cell called the first polar body. The secondary
oocyte begins the second meiotic division, but the process stops in metaphase. At this
point, ovulation occurs. If fertilization occurs, meiosis II continues. Again this is an
unequal division with all of the cytoplasm going to the ovum, which has 23 single-
stranded chromosomes. The smaller cell from this division is a second polar body.
The first polar body also usually divides in meiosis I to produce two even smaller
polar bodies. If fertilization does not occur, the second meiotic division is never
completed and the secondary oocyte degenerates in oogenesis, only one functional
fertilizable cell develops from a primary oocyte. the other three cells are polar bodies
and they degenerate [16].
2.4 Ovarian cycle
The ovarian cycle is a series of events in the ovaries that occur during and after the
maturation of the oocyte (egg or ovum). During their reproductive years, non
pregnant females usually experience a cyclical sequence of changes in their ovaries
and uterus. Each cycle takes about one month and involves both oogenesis, the
process of formation and development of oocyte, and preparation of the uterus to
receive a fertilized ovum (Fig 2.3).
9
Figure 2.3 Hormone Levels During Oogenesis [17].
The ovarian cycle is split into two parts:
1- The follicular phase, also called the preovulatory phase, is the first part of the
ovaria cycle. During this phase, the ovarian follicles mature and get ready to
release an egg. The latter part of this phase overlaps with the proliferative
phase of the uterine cycle.Through the influence of a rise in FSH during the
first days of the cycle, a few ovarian follicles are stimulated. These follicles,
which were present at birth and have been developing for the better part of a
year in a process known as folliculogenesis, compete with each other for
dominance. Under the influence of several hormones, all but one of these
follicles will stop growing, while one dominant follicle in the ovary will
continue to maturity. The follicle that reaches maturity is called a tertiary, or
Graafian, follicle, and it contains the ovum [17].
01
2- The second half, the luteal phase, is also called the postovulatory phase is the
final phase of the ovarian cycle and it corresponds to the secretory phase of
the uterine cycle. During the luteal phase, FSH and LH cause the remaining
parts of the dominant follicle to transform into the corpus luteum, which
produces progesterone. The increased progesterone in the adrenals starts to
induce the production of estrogen. The hormones produced by the corpus
luteum also suppress production of the FSH and LH that the corpus luteum
needs to maintain itself. Consequently, the level of FSH and LH fall quickly
over time, and the corpus luteum subsequently atrophies. Falling levels of
progesterone trigger menstruation and the beginning of the next cycle. From
the time of ovulation until progesterone withdrawal has caused menstruation
to begin, the process typically takes about two weeks, with 14 days
considered normal. For an individual woman, the follicular phase often varies
in length from cycle to cycle; by contrast, the length of her luteal phase will
be fairly consistent from cycle to cycle [18].
.
2.4.1 Ovarian Cycle and Hormonal Regulation
The principle regulator of LH and FSH secretion is GnRH. GnRH is a ten amino
acid peptide that is synthesized and secreted from hypothalamic neurons and binds to
receptors on gonadotrophs. As depicted in the (figure 2.4), GnRH stimulate secretion
of LH, which in turn stimulates gonadal secretion of the sex steroids testosterone,
estrogen and progesterone. In a classical negative feedback loop, sex steroids inhibit
secretion of GnRH and also appear to have direct negative effects on gonadotrophs.
This regulatory loop leads to pulsatile secretion of LH and, to a much lesser extent,
FSH. The number of pulses of GnRH and LH varies from a few per day to one or
more per hour. In females, pulse frequency is clearly related to stage of the cycle.
Numerous hormones influence GnRH secretion, and positive and negative control
over GnRH and gonadotropin secretion is actually considerably more complex than
depicted in the figure. For example, the gonads secrete at least two additional
hormones - inhibin and activin - which selectively inhibit and activate FSH secretion
from the pituitary [19,20].
00
Testosterone is a steroid hormone from the androgen group and is found in
mammals and other vertebrates. In mammals, testosterone is secreted primarily by
the testicles of males and the ovaries of females, although small amounts are also
secreted by the adrenal glands. It is the principal male sex hormone [20].
Figure 2.4 Regulation of ovarian cycle [21]
2.5 Infertility
Infertility is a disease of the reproductive system that impairs the body's ability to
perform the basic function of reproduction [1] and is defined as the inability of
getting pregnant after trying for at least 6 months or one year [2] and is defined as the
lack of conception after an arbitrary period of 12 months without using any
contraception [22].
2.5.1 Types of infertility
There are two types of infertility:
1- Primary infertility refers to couples who have not become pregnant after at
least 1 year having sex without using birth control methods.
2- Secondary infertility refers to couples who have been able to get pregnant at
least once, but now are unable [23,24].
02
2.5.2 Risk factors of female infertility
Risk factors for female infertility include [25]
Age: Fertility begins to decline when a woman reaches her mid-30s, and
rapidlydeclines after her late 30s.
Weight: Extreme weight levels, either high or low, can contribute to
infertility.
Smoking: Cigarette smoking can impair a womans fertility.
Sexually transmitted diseases .
History of Pelvic Inflammatory Disease ( PID ) .
Eating disorders.
Anovulatory menstrual cycles.
Endometriosis .
Defects of the uterus or cervical obstruction .
Long-term (chronic) disease such as diabetes .
Stress :neurotransmitters (chemical messengers) act in the hypothalamus
gland,
which controls both reproductive and stress hormones. Severely elevated levels
of stress hormone can, in fact, shut down menstruation. Whether stress has any
significant effect on fertility or fertility treatments is unclear [25].
2.5.3 Symptoms of infertility
There are two symptoms of infertility
Inability to become pregnant.
A range of emotional reactions by either or both members of the Having couple.
In general, such reactions are greater among childless couples. at least one child
tends to soften these painful emotions [25].
03
2.5.4 Causes of female infertility include
The most common cause of female infertility was problems in the fallopian tubes in
27.4% of the cases, while the second most common cause is typically the
consequence of chronic PID, which can lead to tubal scarring [23]. The third most
common cause was disorders of menstruation in the 20% of the cases, following
infertility due to problems in the uterus in the 9.1% of the cases. Finally, in 2.7% of
the participants infertility was due to age, an additional due to Endometriosis and the
last cause was the ovulatory disorders , which was common for the majority of the
women tested [24].
2.6 Thyroid hormones and infertility
The menstrual pattern is influenced by thyroid hormones directly through impact on
the ovaries and indirectly through impact on Serum Hormone Binding Globulin
(SHBG), Prolactin (PRL) and GnRH secretion and coagulation factors. Treating
thyroid dysfunction can reverse menstrual abnormalities and thus improve fertility
[26]. The prevalence of Auto Immune Thyroid Disease (AITD) is significantly
higher compared to parous age-matched women. This is especially the case in
women with endometriosis and PCOS. During the first trimester, however, pregnant
women with AITD carry a significantly increased risk for miscarriage compared to
women without AITD, Hypothyroidism is associated with a broad spectrum of
reproductive disorders ranging from abnormal sexual development through
menstrual irregularities to infertility.
Severe hypothyroidism is commonly associated with ovulatory dysfunction due to
numerous interactions of thyroid hormones with the female reproductive system
Both hyperprolactinaemia, due to increased Thyroid Releasing Hormone (TRH)
production, and altered GnRH pulsatile secretion, leading to a delay in LH response
and inadequate corpus luteum [27]. Thyroid responsitivity by the ovaries could be
explained by the presence of thyroid hormone receptors in human oocytes.
Progesterone production is another pathway through which hypothyroidism may
impact on fertility is by altering the peripheral metabolism of oestrogen and by
decreasing SHBG production. Both pathways may result in an abnormal feedback at
the pituitary level. Independently of hormonal changes, hypothyroidism can also lead
04
to menorrhagia by altered production of coagulation factors (decreased levels of
factors Proconvertin VII, Hemophilia A VIII, Christmas factor IX and
Thromboplastin XI) [28].
2.7 Ovulatory disorders
Ovulatory disorders are one of the most common reasons why women are unable to
conceive, and account for 30% of women's infertility. Fortunately,approximately
70% of these cases can be successfully treated by the use of drugs such as
Clomiphene and Menogan/Repronex [29].
The causes of failed ovulation can be categorized as follows:
2.7.1 Hormonal Problems
These are the most common causes of anovulation. The process of ovulation
depends upon a complex balance of hormones and their interactions to be
successful, and any disruption in this process can hinder ovulation [30]. In
approximately 50% of the cases of anovulation, the ovaries do not produce normal
follicles in which the eggs can mature. Ovulation is rare if the eggs are immature and
the chance of fertilization becomes almost nonexistent [31].
2.7.2 Malfunction of the hypothalamus
The hypothalamus is the portion of the brain responsible for sending signals to the
pituitary gland, which, in turn, sends hormonal stimuli to the ovaries in the form of
FSH and LH to initiate egg maturation. If the hypothalamus fails to trigger and
control this process, immature eggs will result. This is the cause of ovarian failure in
20% of cases [32].
2.7.3 Malfunction of the pituitary gland
The pituitary's responsibility lies in producing and secreting FSH and LH. The
ovaries will be unable to ovulate properly if either too much or too little of these
substances is produced. This can occur due to physical injury, a tumor or if there is a
chemical imbalance in the pituitary [33,34].
05
2.8 Polycystic Ovary Syndrome (PCOS)
PCOS is the most common endocrine abnormality in reproductive-age women [35]
and is metabolic disorder [36,37].The prevalence of PCOS is traditionally estimated
at 6%-20% of all reproductive-aged women [38].
2.8.1 Diagnosis of poly cystic ovary syndrome ( PCOS)
Table 2.1 Commonly Used Definitions of Polycystic Ovary Syndrome [39]
Definition/year Diagnostic criteria
NIH/1990 Requires Requires the simultaneous presence of:
1. Hyperandrogenism (clinical and/or
biochemical)
2. Ovarian dysfunction
Rotterdam (ESHRE/ASRM)/2003 Requires the presence of at least two
criteria:
1. Hyperandrogenism (clinical and/or
biochemical)
2. Ovulatory dysfunction
3. Polycystic ovarian morphology
AES/2006 Requires the presence of hyper
androgenism
(clinical and/or biochemical) and either:
1. Ovulatory dysfunction
2. Polycystic ovarian morphology
Androgen Excess and PCOS
Society/2009
Requires the simultaneous presence of:
1. Hyperandrogenism (clinical and/or
biochemical)
2. Ovarian dysfunction (ovulatory
dysfunction and/or polycystic ovarian
morphology)
The four most common definitions of the syndrome are presented in Table 2.1.The
1990 National Institutes of Health (NIH) definition requires the simultaneous
presence of hyperandrogenism (clinical and/or biochemical) and menstrual
dysfunction in the absence of other causes [39] , highlighting the importance of
hyperandrogenism in the syndrome’s etiology.
06
In contrast, the 2003 Rotterdam [European Society for Human Reproduction and
Embryology and American Society for Reproductive Medicine (ESHRE/ASRM)]
definition requires only two of the following three criteria: (1) Hyperandrogenism
(clinical and/or biochemical), (2) ovulatory dysfunction (oligo- or anovulation), and
(3) ultrasonographic evidence of polycystic ovaries in the absence of other causes
[40]. Importantly, the Rotterdam criteria broadened the PCOS phenotype to include
women with ovulatory dysfunction and polycystic ovaries but without hyper
androgenism, and eumenorrheic women with hyperandrogenism and polycystic
ovaries (often called ‘‘ovulatory’’ PCOS) [41].
However, the 2006 Androgen Excess Society (AES) definition reemphasized the
importance of hyperandrogenism in the etiology of PCOS, requiring: (1) The absence
of other hyperandrogen-causing disorders, syndromes of severe insulin resistance,
thyroid dysfunction, and hyperprolactinemia, (2) hyperandrogenism (clinical and/or
biochemical), and (3) ovulatory dysfunction (oligo- or anovulation) or polycystic
ovarian morphology.
The 2009 Androgen Excess and PCOS Society’s definition also emphasized the
importance of hyperandrogenism in the syndrome’s etiology, requiring: (1)
Hyperandrogenism (clinical and/or biochemical), (2)ovarian dysfunction (oligo- or
an ovulation and/or polycystic ovaries), and (3) the exclusion of other androgen
excess or related disorders [42].
2.8.2 Clinical features of Poly Cystic Ovary Syndrome (PCOS)
The main clinical features of PCOS are related to hyperandrogenism, such as
hirsutism, acne and menstrual disorders [37,43]. PCOS is also associated with
overweight or obesity mainly abdominal adiposity [44]. It is characterized by a
clustering of hyper secretion of LH, infertility, pregnancy and neonatal
complications [43,45,46]. And metabolic implications ((insulin resistance, metabolic
syndrome, Impaired Glucose Tolerance (IGT), Diabetes Mellitus type 2 (DM2) and
potentially Cardio Vascular Disease (CVD)) [47]. PCOS patients are typically
characterized by producing an increased number of oocytes, they are often of poor
07
quality, leading to lower fertilization, cleavage and implantation rates, and a higher
miscarriage rate [48]. Impaired oocyte maturation and embryonic developmental
competence in PCOS women is possibly linked with abnormal endocrine/ paracrine
factors, metabolic dysfunction and alterations in the intrafollicular
microenvironment during folliculogenesis and follicle maturation [49].
2.8.3 Reproductive features of PCOS
2.8.3.1 Ovarian dysfunction and infertility
Ovarian dysfunction usually manifests as oligomenorrhoea/ amenorrhoea resulting
from chronic oligo-ovulation/ anovulation . However, prolonged anovulation can
lead to dysfunctional uterine bleeding which may mimic more regular menstrual
cycles. The majority of PCOS patients have ovarian dysfunction, with 70% to 80%
of women with PCOS presenting with oligomenorrhoea or amenorrhoea [50].
PCOS is the most common cause of anovulatory infertility. It accounts for 90% to
95% of women attending infertility clinics with anovulation. However 60% of
women with PCOS are fertile (defined as the ability to conceive within 12 months),
although time to conceive is often increased . In those with PCOS and infertility,
90% are overweight [51].
2.8.3.2 Hyperandrogenism The clinical and/or biochemical signs of androgen excess in PCOS result from
increased Synthesis, and release of ovarian androgens. Elevated LH and insulin
synergistically increase androgen production. Insulin resistance leads to hyper
insulinaemia, reduces SHBG and raises free circulating Testosterone and together,
hyperandrogenism and hyperinsulinaemia impairs ovarian follicle development.
Clinical hyper androgenism primarily includes hirsutism, acne and male pattern
alopecia [37,51,52]. Adrenal hyperandrogenism is also common in PCOS patients.
Approximately 20-30% of PCOS women demonstrate excess adrenal precursor
androgen (APA) production, primarily using DHEAS as a marker of APA, The role
of APA excess in determining or causing PCOS is unclear [53].
08
2.8.4 Metabolic features of PCOS
2.8.4.1 Dyslipidemia Dyslipidaemia is common in PCOS compared to weight matched controls with
higher triglycerides and lower high density lipoprotein cholesterol. The
dyslipidaemia occurs independent of BMI, The causes of dyslipidaemia in PCOS are
again multifactorial. Insulin resistance appears to have a pivotal role mediated in part
by stimulation of lipolysis and altered expression of lipoprotein lipase and hepatic
lipase [54].
Life-long apolipoprotein lipid metabolic dysfunction in women with PCOS
exaggerates the risk for CVD with aging , Elevated triglycerides and waist
circumference predict CVD risk and women with PCOS often have these
phenotypes. Diet and exercise interventions followed by selective lipid lowering
medications are encouraged to normalize the dyslipidemia [55].
2.8.4.2 Insulin resistance and abnormal glucose metabolism Insulin resistance occurs in around 50% to 80% of women with PCOS Mechanisms
involved in insulin resistance are likely to be complex with genetic and
environmental contributors. Specific abnormalities of insulin metabolism identified
in PCOS include reductions of insulin sensetivity , reduced hepatic extraction [56],
impaired suppression of hepatic gluconeogenesis and abnormalities in insulin
receptor signalling [57]. Insulin resistance in PCOS results in hyper insulinaemia
with its associated diverse and complex effects on regulating lipid metabolism,
protein synthesis and modulation of androgen production. The cause of insulin
resistance is likewise complex and multifactorial with genetic and environmental
contributors [58]. Hyperinsulinemia may have preferentially impaired oocyte
developmental competence, resulting in reduced rates of fertilization, embryonic
development and implantation in PCOS patients with obesity [59]. Insulin may
induce local androgen production, which results in oocytes of lower quality, post-
maturity . At the molecular level, insulin binds to its receptor, localized on GC and
09
theca cells, and oocytes, to stimulate follicle recruitment, consequently altering
expression of multiple genes involved in meiotic/ mitotic spindle dynamics and
centrosome function in PCOS ooctyes. This indicates that insulin may be an
important mediator of oocyte developmental competence via a ligand-receptor
regulating system [60,61].
Women with PCOS also develop abnormal glucose metabolism at a younger age and
may demonstrate a more rapid conversion from IGT to DM2 , Women with PCOS
also have higher Gestational Diabetes Mellitus (GDM) risk , The risk of GDM
occurs both independent of and is exacerbated by obesity [62].
2.8.4.3 Cardiovascular disease risk
Obesity, particularly of visceral origin, plays a crucial role in both the development
and maintenance of PCOS, and significantly influences the severity of cardiovascular
risk profile. About 50–60% of women with PCOS have central body fat distribution
whereby a disproportionate quantity of adipose tissue is distributed in the visceral
depot. Visceral fat is the main source of free fatty acids and inflammatory cytokines
causing IR and consequent CVD [63]. Mean arterial blood pressure and the risk of
preeclampsia are higher in women with PCOS. Women with PCOS had significantly
elevated high-sensitivity C-reactive protein (hsCRP), homocysteine, plasminogen
activator inhibitor-1 and its activity, vascular endothelial growth factor, asymmetric
dimethylarginine, advanced glycation end products (AGEs), and lipoprotein A [64].
A better understanding of how PCOS is related to abnormalities in extra- and intra-
ovarian factor (Fig. 2.5) and their impact on Granulose Cell (GC)–oocyte
interactions, oocyte maturation and potential embryonic developmental competence,
will be crucial to improving fertility and optimizing clinical stimulation
21
Figure 2.5 Intra-and extra-ovarian factors associated with the PCOS pathology
that negatively affect oocyte and subsequent embryo quality [37].
Ovarian folliculogenesis is regulated by a fine balance between extra and intra-
ovarian factors. Oogenesis is profoundly dependent upon intra-ovarian factors, in
particular follicle fluid factors (FFFs) which are positively related to levels of these
factors in serum. Any imbalance or dysfunction between extra- and intra-ovarian
factors may result in abnormal folliculogenesis and oogenesis disorder [65].
2.9 Extra-ovarian factors
Extra ovarian factor include FSH deficiency, Hyper secretion of LH, Hyper
androgenism and Hyper issulinemia.
2.9.1 FSH deficiency
FSH stimulates follicular growth and recruitment of immature follicles from the
ovary. FSH is the major survival factor during folliculogenesis, when there is a
delicate balance between recruitment and atresia of follicles. Human antral follicles
between 2 and 5 mm become responsive to FSH, whereas slightly larger follicles
20
between 6 and 8 mm acquire aromatase activity and potentially increase the Estradiol
(E2) levels [60]. With the concomitant rise in E2 and inhibin B, FSH levels then
decline in the late follicular phase, and eventually only the most advanced and
mature follicle is selected to proceed to ovulation. At the end of the luteal phase,
there is a slight rise in the FSH level, which is very important in initiating the next
ovulatory cycle [63]. PCOS patients show lower serum FSH levels as compared with
normal cycles Consequently, FSH deficiency results in an increased accumulation of
antral follicles between 2 and 8 mm Clearly, the high number of smaller follicles
indicates many have undergone premature arrest and failed to become the dominant
follicle [65,66].
2.9.2 Hyper secretion of LH
Women with PCOS typically have tonic hypersecretion of LH during the follicular
phase of their cycles High LH levels have been associated with significant decreases
in oocyte maturation, and fertilization rates, and impaired embryo quality,
consequently resulting in impaired pregnancy rates, and higher miscarriage rates
[67]. Hyperseceretion of LH during folliculogenesis may suppress FSH function,
resulting in abnormal GC function by promoting premature GC luteinization and
follicular aresia in small antral follicles from women with PCOS, causing premature
oocyte maturation via inhibition of oocyte maturation inhibitors [64]. LH may also
activate premature meiotic processes by damaging the oocyte nucleus, leading to
apoptosis via a receptor-coupled signal transduction system [68]. Errors in
embryogenesis stemming from abnormal and premature oocyte exposure to increased
LH stimulation may explain the elevated miscarriage rate in PCOS patients [69].
2.10 Intra-ovarian factors
Intra-ovarian factors include Epidermal growth factor family (EGF), Fibroblast
Growth Factors Family (FGFs) , Neurotrophin Growth Factor Family (NGF),
Transforming Growth Factor-b family (TGFB) , Vascular endothelial growth factor
family (VEGF) , Cytokine family and Insulin-like growth factors family (IGFs) [70].
22
2.10.1 Epidermal growth factor family
Epidermal growth factor (EGF) is a soluble growth factor that plays an important
role in the regulation of cell growth, proliferation and differentiation when bound to
its receptor , In the human ovary, EGF is found in the Follicular Fluid (FF),
regulating follicular development and oocyte meiotic maturation [70].
In women with PCOS, FF EGF levels are higher than those of normally ovulating
women which may suggest the involvement of EGF in the maintenance of PCOS ,
EGF inhibits estrogen synthesis in GCs, which may explain why EGF blocks antral
follicle growth and results in follicular arrest in PCOS patients [71]. EGF-like
factors, such as amphiregulin, epiregulin and betacellulin, are reportedly involved in
oocyte maturation through autocrine and paracrine mechanisms [72]. However, the
physiological function of EGF-like factors in PCOS remains unknown [37].
2.10.2 Fibroblast growth factor family (FGF)
Fibroblast growth factors (FGFs) are a group of polypeptides that play a
fundamental role in development, cell growth, tissue repair and transformation. They
are expressed in GC and theca cells of growing follicles, and are considered to be
physiological regulators of FSH action ,this may suggest a role for FGF in oocyte
maturation by affecting surrounding follicular GC and theca cells. FGF contributes to
alterations in the intra-follicle environment, resulting in arrest of follicle
development in patients with PCOS Therefore, FGF alterations in the FF and serum
remain controversial, the impact of EGF on oocyte maturation and embryonic
development requires further elucidation in PCOS patients [71].
2.10.3 Neurotrophin growth factor family
Brain-Derived Neurotrophic Factor (BDNF), Nerve Growth Factor (NGF),
Neurotrophin-3 (NT-3) and Neurotrophin-4/5 NT-4/5 are major members of the
neurotrophin (NT) family of growth factors that are involved in development of the
central and peripheral nervous systems , NTs are not only involved in the nervous
system, but also act on the ovaries of humans and other mammals, NTs play a
fundamental role in folliculogenesis and cytoplasmic competence of the oocyte [73].
23
Data from research using in vitro animal models suggest that co-incubation with
BDNF promotes nuclear and cytoplasmic maturation of the oocyte, which are
essential processes for successful oocyte and preimplantation embryo development
[74]. Evidence from some studies shows that increased FF BDNF and NGF levels
are closely related to the pathology of women with PCOS [75]. Another report found
that FF BDNF and NT-3 levels are increased, but FF NGF is decreased, in women
with PCOS, which may be indicative of the differential status of follicles in PCOS
Patients [73].
2.10.4 Transforming growth factor-b family
Among the many intra-ovarian factors, particular members of the transforming
growth factor (TGF)-b family play an important biological role in follicle growth and
oocyte development. These family members include Anti-Mullerian Hormone
(AMH), Mullerian Inhibiting Substance (MIS), activin, follistatin, inhibins, bone
morphogenetic protein (BMP)-9 and growth differentiation factor (GDF)-9
[58].Under different physiological conditions, TGF-b family members may either
promote or block ovarian follicle growth and/or differentiation of the GC–oocyte
complex, which is also related to the pathogenesis of PCOS [60,76].
2.10.5 Vascular endothelial growth factor family
In the ovary, VEGF is expressed in GCs and theca cells, but rarely in stroma cells
and is also present in the FF [71]. VEGF plays an important role in angiogenesis,
follicular vascularization and intrafollicular oxygenation, consequently impacting
follicular maturation, oocyte quality, fertilization and embryo developmental
competence [77]. In normally ovulating women (NOW), decreased FF and serum
VEGF levels are related to improved ovarian response, consequently increasing the
number of oocytes retrieved, and improving the rates of fertilization and pregnancy;
the reverse has also been shown, as elevated FF VEGF levels are associated with
poor oocyte quality and decreased fertilization and pregnancy rates, especially in
older patients [69]. In women with PCOS, elevated FF VEGF is closely associated
with the development of ovarian hyperstimulation syndrome and is indicative of
immature oocytes and poor fertilization rates [78]. FF VEGF may serve as a dynamic
24
indicator for the evaluation of follicular maturity, subsequently predicting oocyte
maturity, fertilization success and embryo development in PCOS patients [77].
However,further research is required to uncover the true relationship between VEGF
levels and subsequent.
2.10.6 Cytokine family
Cytokines encompass a large family of soluble polypeptide regulators that are
produced widely throughout the body by cells of diverse embryological origin; the
family comprises the interleukins (IL1 _35), leukemia inhibitory factor, tumor
necrosis factor (TNF)α, soluble Fas (sFas) and sFas ligand (sFasL) (TNF subfamily).
Within the ovary, the action of cytokines may be autocrine or paracrine, but not
endocrine; they exist in the FF, suggesting their production by GCs , and have
regulatory functions in follicular maturation and subsequent embryonic development
[70].
Interleukins IL-1, IL-2, IL-6, IL-8, IL-11, IL-12 and other cytokines, play multiple
roles in folliculogenesis, ovulation and corpus luteum function FF IL-12 levels vary
within immature and pre-ovulatory follicles the presence of FF IL-12 has been
associated with fertilization failure, and decreased FF IL-12 level and increased FF
IL-13 level in PCOS patients is correlated with a reduced rate of oocyte maturation,
fertilization and pregnancy, but this reduction did not reach statistical significance
[79].
2.11 Other microenvironment factors
2.11.1 Homocysteine
Homocysteine (Hcy) is a homologue of the amino acid cysteine elevated Hcy
levels in serum and FF are inversely associated with oocyte and embryo quality,
resulting in decreased fertilization and pregnancy rates, and increased miscarriage
rates in PCOS patients undergoing IVF treatment [80 ,81]. elevated levels of Hcy in
FF and serum may suppress E2 synthesis, and consequently interfere with ovarian
follicular developmental competence, oocyte maturation and fertilization in women
with PCOS [80,82].
25
2.11.2 Leptin High leptin levels in the FF and serum are closely associated with decreased oocyte
maturity, poor fertilization and embryo quality, and lower pregnancy rates in PCOS
patients [83,84].
2.12 Insulin-like growth factor family (IGF) system
The insulin-like growth factors (IGFs) are proteins that regulate growth,
diferentiation and survival in a multitude of cells and tissues. with high sequence
similarity to insulin. IGFs are part of a complex system that cells use to
communicate with their physiologic environment. This complex system (often
referred to as the IGF "axis") as at (figure 2.6) consists of two cell-surface receptors
( Insulin-like Growth Factor1 Receptor (IGF1R) and Insulin-like growth factor2
receptor (IGF2R)), two ligands (Insulin-like growth factor 1(IGF-1) and Insulin-like
growth factor 2(IGF-2)), A family of six high-affinity IGF- binding proteins (IGFBP-
1 to IGFBP-6), as well as associated IGFBP degrading enzymes, referred to
collectively as proteases [85].
Figure (2.6) Insulin-like growth factor family (IGF) system [85]
26
2.13 Insulin Like Growth Factor Binding Protein - 1
2.13.1 Definition and site of secretion IGFBP-1 also known as placental protein 12 (PP12), a protein discovered at 1989
that in humans is encoded by the IGFBP1 gene. This gene is a member of (IGFBP)
family and encodes a protein with an IGFBP domain and a type-I thyroglobulin
domain. the protein binds both insulin like growth factors (IGFs) I and II in body
fluids and tissues and circulates in the plasma. Binding of this protein prolongs the
half-life of the IGFs and alters their interaction with cell surface receptors. Alternate
transcriptional splice variants, encoding different isoforms, have been characterized
[86]. IGFBP-1 contains 234 amino acids, with a predicted molecular mass of 25 kDa
(Figure 2.7). The major sites of IGFBP-1 synthesis are the fetal /adult liver, kidneys,
and decidualized endometrium. local synthesis of IGFBP-1 is influenced by feeding
level and exogenous somatotropin. Serum levels of IGFBP-1, which reflect its
synthesis by the liver, exhibit considerable diurnal variation. Circulating IGFBP-1
levels are highest early in the morning and lowest in the evening. The levels are high
in the fetus and newborn, but decline steadily until puberty. The mean level of
IGFBP-1 in healthy adults is 4.4 ng/ml (range 0.6–14.4 ng/ml). After about 65 years
of age, serum IGFBP-1 levels begin to increase [87].
Figure (2.7) Structure of IGFBP-1 [88]
27
The IGFBP-1 protein sequence contains 12 N-terminal and 6 C-terminal cysteine
residues which are conserved in other mammalian, the human IGFBP-1 and IGFBP-
3 genes are contiguous and located in close proximity to the homeobox A (HOXA)
gene cluster on chromosome 7. IGFBP-1 primarily non phosphorylated is found in
follicular fluid from women with normal menstrual cycles and from gonadotrophin –
stimulated luteinizing follicles, it is expressed in granulosa cells of the dominant
follicle following the LH surge, and IGFBP-1 messenger Ribonucleic acid (mRNA)
is expressed in corpus luteum. There may be paracrine feedback cycles operating
because IGFBP-1 inhibits IGF-II and in turn IGF-II inhibits IGFBP-1 production by
luteinized human granulosa cells in vitro. The IGF system is one the various growth
factor systems believed to be important in endometrial cyclic development and
implantation. IGFBP-1, which is by far the most abundantly expressed IGFBP in late
secretory endometrium, continues to be expressed in pregnancy decidua at much
higher concentrations than any of the other IGFBPs [86]. Decidual IGFBP-1 may
regulate trophoblast derived IGF-II auto/paracrine actions. However, in addition to
its regulation of IGF bioavailability, IGFBP-1 has IGF- independent effects, binding
directly to cell membranes and altering cellular motility, IGFBP-1 contains the Arg-
Gly-Asp (RGD) sequence which is known to mediate recognition for several cell
adhesion molecules, including the α 5 β 1 integrin. IGFBP-1 binds, via its RGD
sequence, to the α 5 β 1 integrin of Chinese hamster ovary (CHO) cells and
stimulates their motility in vitro. Therefore, decidual IGFBP-1 may also interact
directly with the invading trophoblast to regulate its activity [89].
2.13.2 Action of IGFBP-1 on ovary
It was proposed that insulin stimulates ovarian androgen synthesis through its
interaction with the insulin-like growth factor (IGF) system and that IGF-I
potentiates LH-stimulated ovarian androgen synthesis as in (figure 2.8). The action
of IGF-I action is modulated by the IGF-binding protein-1, which has been reported
to correlate inversely with the serum level of free IGF-I. Insulin has also been shown
to regulate the serum level of IGFBP-1. It was therefore hypothesized that
hyperinsulinaemia in PCOS inhibits the production of IGFBP-1, causing an increase
28
in the level of free IGF-I in the serum and potentiating LH-stimulated androgen
production [90].
Figure (2.8) Action of IGFBP-1 on ovary [90].
2.13.3 Role of IGFBP-1 during human pregnancy
2.13.3.1 Migration.
Successful implantation, placental development and consequent fetal growth
depend upon adequate migration of IGF-II-producing trophoblasts into a maternal
decidual environment rich in IGFBP-1. the effect of IGFBP-1 that depends upon the
interaction of its RGD site with integrin α5β1 , IGFBP-1 also stimulates human
trophoblast cell migration both in a monolayer wounding assay and a trans-Matrigel
barrier migration assay [89].
29
Downstream signaling pathways activated by the paracrine factors in trophoblast
cells as show in (figure 2.9): Endometrium derived paracrine factors predominantly
activate Jak-STAT, MAPK and TGF-beta mediated signaling pathways to influence
the invasion of trophoblastic cells. Jak-STAT pathway is activated by IL-6 group of
cytokines and inhibition of STAT3 expression by siRNA inhibits the IL-11 and LIF
mediated invasion in trophoblastic cells. Similarly, ERK1/2 dependent MAPK
pathway is activated by EGF, IGF-II, IGFBP etc and to a certain extent by LIF and
IL-11. However, the role of ERK1/2 activation by IL-11 and LIF during invasion of
trophoblast cells has not been elucidated. In contrast to these, TGF-beta dependent
signaling pathway inhibits the invasion of trophoblast cells through activation of
Smad and Rho A dependent signaling mechanism. Red ball with 'Y': phosphorylation
at tyrosine residue while, red ball with 'S': phosphorylation at serine residue. U0126
is the inhibitor for both MEK1 and MEK2 while PD98327 is the inhibitor of MEK1.
Y27632 is a selective inhibitor of the ROCK [91].
Figure (2.9) Signaling pathways activated by the paracrine factors in
trophoblast cells [91].
31
2.13.3.2 Metabolism.
Fuel for normal fetal growth and development depends upon the transfer of
nutrients from mother to fetus via the placenta. Thus, IGFBP-1 may influence fetal
growth by affecting IGF-mediated nutrient transport. IGF-I stimulates transport of
both glucose and amino acids in placental trophoblasts isolated from first trimester.
IGFBP-1 inhibits IGF-I metabolic effects , the effect of IGFBP-1 is dependent upon
its phosphorylation status. IGF-I-stimulated [3H]a-amino isobutyric acid uptake by
human trophoblast cells was inhibited by equimolar concentrations of
phosphorylated IGFBP-1, whereas similar doses of non-phosphorylated IGFBP-1
enhanced IGF-mediated amino acid uptake. In these experiments, IGFBP-1 was
incubated with the trophoblast cells for 24 h before addition of IGF, allowing for the
IGFBP-1 interaction with cell surfaces which, as described above, may be important
for the potentiating effects of IGFBP-1 [92].
The mitogenic activity of IGFs, mediated through IGFRI as in (figure 2.10), is
inhibited by their sequestration by soluble IGFBPs. Proteolysis of IGFBPs leads to
the release of IGFs from the binary complexes and hence a potentiation of IGF
activity. Cell-associated IGFBPs have been reported to eitherpotentiate or inhibit the
IGF effects. Refer to the text for further details of these interactions[93].
Figure (2.10) Proposed pathways of IGF-dependent IGFBP action[93].
30
2.13.4 Physiological conditions of IGFBP-1
Plasma concentrations of IGFBP-1 show marked diurnal variation, In the non
pregnant state, plasma levels show a difference of over 10-fold between midnight
and morning regardless of metabolic and hormonal status. These changes are
obviously associated with nutritional factors, with elevated values during fasting , the
concentration of IGFBP-1 in human fetal serum is 10 times higher than in cord
plasma of term infants. The levels continue to decline after birth until a steady state
is reached at puberty. Serum IGFBP-l concentrations decline in adolescence (Argente
and remain low in adulthood. With advancing age, there may be a slight increase in
serum IGFBP-l concentrations as the inverse correlation with insulin levels becomes
less pronounced [94].
2.13.5 Regulation of IGFBP-1 action
Circulating levels of IGFBP-1 are regulated by a variety of physiological and
pathological stimuli. Metabolic, nutritional and anthropometric factors have been
shown to be associated with plasma concentrations of IGFBP-1. Plasma levels are
subject to diurnal variation reaching their lowest during the afternoon and highest in
the morning coinciding with the increase in IGF-1 levels during the morning hours.
Insulin inhibits hepatic IGFBP-1 transcription, plasma IGFBP-1 levels being
reciprocally related to plasma insulin concentrations. Glucagon acts as a stimulator
of plasma IGFBP-1 independently of insulin levels, thus implying a more complex
metabolic regulation of IGFBP-1[95]. IGFBP-1 levels decline immediately after a
carbohydrate meal, a direct response to increased plasma insulin, however, it has also
been proposed that long-term intake of carbohydrates may increase IGFBP-1 levels
by inducing a state of hepatic insulin insensitivity. Prolonged exercise increases
IGFBP-1 levels, probably an indirect effect of insulin and counter regulatory
hormones. IGFBP-1 expression is also stimulated by glucocorticoids, growth
hormone, thyroid hormones, epidermal growth factor and cytokines. The serum
concentrations of IGFBP-1 increase with age and this could possibly be related either
to decreased suppression by insulin or to other factors such as stimulation by
32
inflammatory cytokines [96]. Factors regulating circulating levels of IGFBP-1 are
shown in (figure 2.11).
Figure (2.11) Growth hormone-Insulin-like growth factor-1 axis and modulation
by insulin-like growth factor binding proteins-1 and −3 [97]
2.14 PREVIOUS STUDIES
At 1992 (Nobles and Dewailly) were Elucidate the relationship and role of insulin-
like growth factor-1(IGF-1), IGF binding protein-1 (IGFBP-1), insulin and
luteinizing hormone (LH) in the pathogenesis of polycystic ovary syndrome (PCOS).
In the main study, serum concentrations of IGF-1, IGFBP-1, insulin and LH in
women with an ovulation associated (n = 23) and not associated (n = 47) with PCOS
were determined. Mean serum IGFBP-1 in PCOS (33.8 ± 21.2 μg/l) was decreased
compared with anovulatory non-PCOS (60.0 ±22 μg/1) (P = 0.0001) [98].
At 2010 (Jakubowicz et al.) hypothesized that serum IGFBP1 concentration would
be reduced in women with PCOS during the first trimester of pregnancy. Fasting
serum insulin and IGFBP-1 were performed in 72 women with PCOS and 62 normal
33
women. Each woman was seen once and assigned to one of three gestational groups:
wk 3-5, 6-8, and 9-11. The insulin sensitivity index during oral glucose tolerance test
was lower in women with PCOS compared with normal women throughout the first
trimester (P < 0.0001). IGFBP-1 was markedly lower in women with PCOS (IGFBP-
1: wk 3-5 and 6-8, P < 0.0001; wk 9-11, P = 0.0003). Comparing women with PCOS
who experienced EPL with those who did not, serum IGFBP-1 concentrations are
markedly decreased in PCOS, implicating endometrial epithelial and stromal
dysfunction during periimplantation and early pregnancy as a possible mechanism
for early pregnancy loss EPL in PCOS [86].
At 2011(Kelly et al.) compared serum IGFBP-1 in two populations: either PCOS
versus controls, or an overweight subgroup versus the normal weight subgroup in
either, the result found the population difference is presented as the Weighted Mean
Difference (95% CI). PCOS subjects had a significantly lower serum concentrations
of IGFBP-1 compared with controls [P< 0.00001; −36.6 (−52.0, −21.2) µg/l].
Overweight PCOS subjects also had lower IGFBP-1 levels compared with normal
weight PCOS subjects [P < 0.006; −30.6 (−52.3, −8.8) µg/l]. No significant
difference was found between overweight PCOS patients and overweight controls [P
= 0.23; −5.1 (−13.5, 3.2) µg/l] or between normal weight PCOS patients and normal
weight controls [P = 0.50; −3.8 (−14.9, 7.3) µg/l]. Overweight controls had
significantly lower IGFBP-1 concentrations than normal weight controls [P = 0.03;
−18.0 (−34.4, − 1.5) µg/l] [99].
At 2011 (Koutsaki et al.) evaluated the expression of hPGH, IGF-I, IGFBP-1
and IGFBP-3 genes in placentas from pregnancies complicated by fetal growth
restriction (FGR).The study group was comprised of term placentas from 47 FGR-
complicated pregnancies of no recognizable cause. Thirty-seven placentas from
normal pregnancies with appropriate for gestational age birth weight were used as
controls. The expression status of the genes was evaluated by quantitative real-time
PCR.hPGH, IGF-I and IGFBP-1 exhibited significantly lower expression compared
to the controls (p=0.003, p=0.049 and p=0.001, respectively). Numerically, lower
34
IGFBP-3 expression was also demonstrated in the FGR-affected group, without
however reaching statistical significance (p=0.129). Significant co-expression [100].
35
Chapter 3
Materials and methods
3.1 Study design
The present study was a case control one .
3.2 Target population
The target population was infertile women with PCOS .
3.3 Sampling and sample size
The sample was collected from women who are visiting the Specializing Medical
Centers in Gaza strip with at least 3 years duration of infertility. The number of
patients ( sample size ) was 40 women with medical reports. A total number of 40
controls were selected at the basis of being married, having children and matching
the case in age and residence.
Inclusion criteria
Infertile women aged 18-40 years .
Having poly cystic ovary syndrome .
Their husbands having normal sperm parameters ( sperm motility equal or
more than 40%, and the count of sperms equal or more than 25 millions).
Exclusion criteria
Fertile women .
Infertile women aged less than 18 and more than 40 years .
Those females whom semen analysis of their husbands gave abnormal results.
Diseased women with Diabetes or women have problem in the reproductive
system.
36
3.4 Ethical consideration
The necessary approval to conduct this study was obtained from local ethical
committee like Palestinian Ministry of Health Annex 2. And from Specialized
Medical Centers in Gaza strip especially Albasma center directed by Dr. Bahaa
Algalaeeni Annex 3. And from gynaecology and obstetrics clinic directed by Dr.
Fadya Malhees Annex 4. and from gynaecology and obstetrics clinic directed by Dr.
Seeda mallha.
3.5 Data collection
3.5.1 Questionnaire interview
A face to face interview was used to fill questionnaire was designed for this study.
The questionnaire (Annex1) was based on female infertility patient questionnaire
with some modification related to medical history and medication intake. During the
study, the interviewer was explained to the patients any of confused question that
was not clear to them. Most questions was yes / no ones, which offer a dichotomous
choice. The questionnaire was piloted with 10 patients, and was modified as
necessary to improve reliability and validity.
3.5.2 Body mass index (BMI)
BMI is a simple index of weight-for-height that is commonly used to classify
underweight, overweight and obesity in adults. It is defined as the weight in
kilograms divided by the square of the height in meter (kg/m2).Women with BMI
<18.50 was considered Underweight. Women with BMI = 18.5- 24.9 was considered
to have normal weight. Women with BMI 25.0 - 29.9 was classified overweight.
Women with BMI ≥30.0 was considered obese [101].
3.5.3 Blood sampling and processing
Venous blood sample (5ml) was drawn from each woman of the study population
by plastic pyrogen-free disposable syringe into a plastic tube. Left the tube for a
37
short time to allow blood to clot. Clear serum samples were obtained by
centrifugation at 3500 rpm for 10 minutes for hormonal analysis.
3.5.4 Determination of serum IGFBP-1
Determination of human serum IGFBP-1 was carried out by sandwich enzyme
immunoassay [102].
Principle of the test
The IGFBP-1 ELISA (Enzyme Linked Immuno Sorbent Assay) is a solid-phase
sandwich enzyme-immunoassay for the quantitative determination of IGFBP1 in
human serum.The ELISA-plate is coated with a monoclonal antibody directed
towards a unique antigenic site of an IGFBP1 molecule. IGFBP1 from samples and
standards bind to the monoclonal antibody and are immobilized on the plate. An
enzyme conjugate containing another monoclonal antibody directed towards a
different region of IGFBP1 molecule and POD binds to the IGFBP1-antibody-
complex during the incubation. Unbound conjugate is washed off with washing
solution. After removal of the conjugate not bound by washing the horseradish
peroxidase oxidizes the substrate TMB (3,3’,5,5’- tetramethylbenzidine) yielding a
color reaction which is stopped with 0.25 M sulphuric acid (H2SO4). The enzymatic
color reaction is stopped after a defined period of time. The concentration of
oxidized TMB correlating proportionally to the concentration of IGFBP1 in the
serum is measured photometrically. The extinction is measured at a wavelength of
450 nm with a microplate reader. The use of a reference measurement with a
wavelength ≥ 550 nm is recommended.
Assay procedure
Bring all reagents and samples to room temperature before use. It is recommended
that all samples, controls, and standards be assayed in duplicate [102].
1- Fix the required number of coated wells or strips in the strip holder.
38
2- Pipette 50 μL of each standard and of each specimen sample into the
respective wells.
3- Add 50 μL of enzyme conjugate to each well.
4- Incubate for 60 minutes at room temperature.
5- Briskly shake out the contents of the wells and then rinse the wells 5 times
with 200 μL distilled or deionized water.
6- Knock the residual water out of the wells by hitting them (in the holder) on
absorbent paper or cloth.
7- Pipette 100 μL of the substrate solution into each well.
8- Incubate for 15 minutes at room temperature.
9- Stop the enzymatic reaction by adding 100 μL stop solution to each well.
10- Measure the extinction of the samples at 450 nm. It is recommended to carry
out the measurement of the extinction within 10 minutes after stopping the
reaction.
Calculation
1- Calculate the average absorbance values for each set of reference standards
and specimen samples.
2- The optical density (OD, absorbance, extinction) of each standard value is
plotted as y value (y-axis), the corresponding IGFBP1 value is drawn in as
the x-value (x-axis). The resulting calibration curve is used to determine the
values of the specimen samples. The OD values of the serum samples are
correlated with the corresponding IGFBP1 concentration values by
interpolation. A four parameter fit (sigmoid) should be used.
3- Using the mean absorbance value for each sample determine the
corresponding concentration of IGBP1 in ng/mL from the standard curve.
4- Any diluted samples must be further converted by the appropriate dilution
factor.
Normal reference value of IGFBP-1 in Follicular and Luteal phase for adult
females are ( 0.6 – 14.4 ) ng/ml [102].
39
3.5.5 Hormonal analysis
3.5.5.1 Determination of serum FSH hormone
FSH hormone level was determined method using ELIZA DRG kit for FSH [103].
Principle of the test
The Diagnostic Reagent Group (DRG) FSH ELISA Kit is a solid phase enzyme-
linked immunosorbent assay (ELISA) based on the sandwich principle. The
microtiter wells are coated with a monoclonal antibody directed towards a unique
antigenic site on a FSH molecule. An aliquot of patient sample containing
endogenous FSH is incubated in the coated well with enzyme conjugate, which is an
anti-FSH monoclonal antibody conjugated with horseradish peroxidase. After
incubation the unbound conjugate is washed off. The amount of bound peroxidase is
proportional to the concentration of FSH in the patient sample.
Assay procedure
All reagents and specimens must be allowed to come to room temperature (25°C)
before use. All reagents must be mixed without foaming. Standards, controls and
samples should be assayed in duplicate.
1- Secure the desired number of Microtiterwells in the holder.
2- Dispense 25 μL of each Standard, controls and samples with new disposable
tips into appropriate wells.
3- Dispense 100 μL Enzyme Conjugate into each well.
Thoroughly mix for 10 seconds. It is important to have a complete mixing in
this step.
4- Incubate for 30 minutes at room temperature.
Briskly shake out the contents of the wells.
5- Rinse the wells 5 times with aqua dest (400 μL per well). Strike the wells
sharply on absorbent paper to remove residual droplets.
6- Add 100 μL of Substrate Solution to each well.
7- Incubate for 10 minutes at room temperature.
41
8- Stop the enzymatic reaction by adding 50 μL of Stop Solution to each well.
9- Determine the absorbance (OD) of each well at 450±10 nm with a microtiter
plate reader. It is recommended that the wells be read within 10 minutes after
adding the stop solution.
Calculation
1- Calculate the average absorbance values for each set of standards, controls
and patient samples.
2- Construct a standard curve by plotting the mean absorbance obtained from
each standard against its concentration with absorbance value on the
vertical(Y) axis and concentration on the horizontal (X) axis.
3- Using the mean absorbance value for each sample determine the
corresponding concentration from the standard curve.
Normal range value of FSH in Follicular and Luteal phase for adult females are
( 2.0 – 10.0 ) mIU/ ml [103].
3.5.5.2 Determination of serum LH hormone
LH hormone level was determined method using ELIZA DRG kit for LH [104].
Principle of the test
The DRG LH ELISA Kit is a solid phase enzyme-linked immunosorbent assay
(ELISA) based on the sandwich principle. The microtiter wells are coated with a
monoclonal [mouse] antibody directed towards a unique antigenic site on a LH
molecule. An aliquot of patient sample containing endogenous LH is incubated in the
coated well with enzyme conjugate, which is an anti-LH monoclonal antibody
conjugated with horseradish peroxidase. After incubation the unbound conjugate is
washed off. The amount of bound peroxidase is proportional to the concentration of
LH in the sample. Having added the substrate solution, the intensity of colour
developed is proportional to the concentration of LH in the patient sample.
40
Assay procedure
All reagents and specimens must be allowed to come to room temperature (25°C)
before use. All reagents must be mixed without foaming. Standards, controls and
samples should be assayed in duplicate.
1- Secure the desired number of Microtiterwells in the holder.
2- Dispense 25 μL of each Standard, controls and samples with new disposable
tips into appropriate wells.
3- Dispense 100 μL Enzyme Conjugate into each well.
Thoroughly mix for 10 seconds. It is important to have a complete mixing in
this step.
4- Incubate for 30 minutes at room temperature.
5- Briskly shake out the contents of the wells. Rinse the wells 5 times with aqua
dest (400 μL per well). Strike the wells sharply on absorbent paper to remove
residual droplets.
6- Add 100 μL of Substrate Solution to each well.
7- Incubate for 10 minutes at room temperature.
8- Stop the enzymatic reaction by adding 50 μL of Stop Solution to each well.
9- Determine the absorbance (OD) of each well at 450 ± 10 nm with a microtiter
plate reader. It is recommended that the wells be read within 10 minutes after
adding the stop solution.
Calculation
1- Calculate the average absorbance values for each set of standards, controls
and patient samples.
2- Using semi-logarithmic graph paper, construct a standard curve by plotting
the mean absorbance obtained from each standard against its concentration
with absorbance value on the vertical(Y) axis and concentration on the
horizontal (X) axis.
3- Using the mean absorbance value for each sample determine the
corresponding concentration from the standard curve.
42
Normal range value of LH in follicular and Luteal phase in adult females are (
1.0 –20.0 ) mIU/ml [104].
3.5.5.3 Determination of serum Testosterone hormone
Testosterone Hormone level determined method using ELIZA DRG kit for
Testosterone [105].
Principle of the test
The DRG Testosterone ELISA Kit is a solid phase enzyme-linked immunosorbent
assay (ELISA), based on the principle of competitive binding. The microtiter wells
are coated with a monoclonal [mouse] antibody directed towards an unique antigenic
site on the Testosterone molecule. Endogenous Testosterone of a patient sample
competes with a Testosterone horseradish peroxidase conjugate for binding to the
coated antibody. After incubation the unbound conjugate is washed off. The amount
of bound peroxidase conjugate is reverse proportional to the concentration of
Testosterone in the sample. After addition of the substrate solution, the intensity of
colour developed is reverse proportional to the concentration of Testosterone in the
patient sample.
Assay procedure
All reagents and specimens must be allowed to come to room temperature (25°C)
before use. All reagents must be mixed without foaming. Standards, controls and
samples should be assayed in duplicate.
1- Secure the desired number of Microtiter wells in the holder.
2- Dispense 25 μL of each Standard, Control and samples with new disposable
tips into appropriate wells.
3- Dispense 200 μL Enzyme Conjugate into each well.
Thoroughly mix for 10 seconds. It is important to have a complete mixing in
this step.
43
4- Incubate for 60 minutes at room temperature (without covering the plate).
5- Briskly shake out the contents of the wells. Rinse the wells 3 times with
diluted WashSolution (400 μL per well). Strike the wells sharply on
absorbent paper to remove residual droplets.
6- Add 200 μL of Substrate Solution to each well.
7- Incubate for 15 minutes at room temperature.
8- Stop the enzymatic reaction by adding 100 μL of Stop Solution to each well.
9- Determine the absorbance (OD) of each well at 450 ± 10 nm with a microtiter
plate reader. It is recommended that the wells be read within 10 minutes after
adding the stop Solution.
Calculation
1- Calculate the average absorbance values for each set of standards, controls
and patient samples.
2- Construct a standard curve by plotting the mean absorbance obtained from
each standard against its concentration with absorbance value on the
vertical(Y) axis and concentration on the horizontal (X) axis.
3- Using the mean absorbance value for each sample determine the
corresponding concentration from the standard curve.
Normal range value of Testosterone in adult females are ( 0.26 - 1.22 ) ng /ml
[105].
3.5.5.4 Determination of serum DHEA- S hormone
DHEA-S Hormone level was determined using ELIZA DRG kit for DHEA-S [106].
Principle of the kit
The DRG DHEA-S ELISA Kit is a solid phase enzyme-linked immunosorbent
assay (ELISA), based on the principle of competitive binding. The microtiter wells
are coated with a polyclonal antibody directed towards an antigenic site on the
DHEA-S molecule. Endogenous DHEA-S of a patient sample competes with a
DHEA-S-horseradish peroxidase conjugate for binding to the coated antibody. After
44
incubation the unbound conjugate is washed off. The amount of bound peroxidase
conjugate is inversely proportional to the concentration of DHEA-S in the sample.
After addition of the substrate solution, the intensity of colour developed is inversely
proportional to the concentration of DHEA-S in the patient sample.
Assay procedure
All reagents and specimens must be allowed to come to room temperature (25°C)
before use. All reagents must be mixed without foaming. Standards, controls and
samples should be assayed in duplicate.
1- Secure the desired number of Microtiter wells in the holder.
2- Dispense 25 μL of each Standard, Control and samples with new disposable
tips into appropriate wells.
3- Dispense 200 μL Enzyme Conjugate into each well.
Thoroughly mix for 10 seconds. It is important to have a complete mixing in
this step.
4- Incubate for 60 minutes at room temperature (without covering the plate).
5- Briskly shake out the contents of the wells. Rinse the wells 3 times with
diluted Wash Solution (400 μL per well). Strike the wells sharply on
absorbent paper to remove residual droplets.
6- Add 100 μL of Substrate Solution to each well.
7- Incubate for 15 minutes at room temperature.
8- Stop the enzymatic reaction by adding 50 μL of Stop Solution to each well
9- Determine the absorbance (OD) of each well at 450±10 nm with a microtiter
plate reader. It is recommended that the wells be read within 10 minutes after
adding the Stop Solution.
Calculation
1- Calculate the average absorbance values for each set of standards, controls
and patient samples.
45
2- Construct a standard curve by plotting the mean absorbance obtained from
each standard against its concentration with absorbance value on the
vertical(Y) axis and concentration on the horizontal (X) axis.
3- Using the mean absorbance value for each sample determine the
corresponding concentration from the standard curve.
Normal range value of DHEA-S in adult females are ( 0.40 – 2.17 ) μg/ml [106].
3.5.5.5 Determination of serum TSH hormone
TSH hormone level was determined method using ELIZA DRG kit for TSH [107].
Principle of the test
The Ultrasensitive-TSH ELISA Test is based on the principle of a solid phase
enzyme-linked immunosorbent assay. The assay system utilizes a unique monoclonal
antibody directed against a distinct antigenic determinant on the intact TSH
molecule. Mouse monoclonal anti-TSH antibody is used fore solid phase (microtiter
wells) immobilization, and goat anti-TSH antibody is used in the antibody-enzyme
(horseradish peroxidase) conjugate solution. The test sample is allowed to react
simultaneously with the antibodies, resulting in the TSH molecule being sandwiched
between the solid phase and enzyme-linked antibodies. After a 2 hour incubation at
room temperature with shaking, the solid phase is washed with distilled water to
remove unbound labeled antibodies. A solution of tetramethylbenzidine (TMB) is
added and incubated for 20 minutes, resulting in the development of a blue color.
The color development is stopped with the addition of 1N HCl, and the resulting
yellow color is measured spectrophotometrically at 450 nm. The concentration of
TSH is directly proportional to the color intensity of the test sample.
Assay procedure
All reagents and specimens must be allowed to come to room temperature (25°C)
before use. All reagents must be mixed without foaming. Standards, controls and
samples should be assayed in duplicate [107].
46
1- Secure the desired number of coated wells in the holder.
2- Dispense 100 μL of standards, specimens, and controls into appropriate wells.
3- Dispense 100 μL of Enzyme Conjugate Reagent into each well.
4- Thoroughly mix for 30 seconds. It is very important to have complete mixing.
5- Incubate at room temperature (18-25°C) with shaking at 175 ± 25 RPM for
120 minutes (2 hours).
6- Remove the incubation mixture by flicking plate contents into a waste
container.
7- Rinse and flick the microtiter wells 5 times with distilled or dionized water.
(Please do not use tap water.)
8- Strike the wells sharply onto absorbent paper or paper towels to remove all
residual water droplets.
9- Dispense 100 μL of TMB Reagent into each well. Gently mix for 5 seconds.
10- Incubate at room temperature, for 20 minutes.
11- Stop the reaction by adding 100 μL of Stop Solution into each well.
12- Gently mix for 30 seconds. Ensure that all of the blue color changes
completely to yellow.
13- Read OD at 450 nm with a microtiter well reader within 15 minutes.
Calculation
1- Calculate the average absorbance value (A450) for each set of reference
standards, controls and samples.
2- Using log-log graph paper, construct a standard curve by plotting the mean
absorbance obtained for each reference standard against its concentration in
μIU/mL, with absorbance on the vertical (y) axis and concentration on the
horizontal (x) axis.
3- Using the mean absorbance value for each sample, determine the
corresponding concentration of TSH in μIU/mL from the standard curve
Depending on experience and/or the availability of computer capability, other
methods of data reduction may be employed.
Normal range value of TSH in adults are ( 0.54 - 4.72 ) μIU/mL [107].
47
3.5.5.6 Determination of serum insulin hormone
Insulin hormone level was determined method using ELIZA DRG kit for Insulin
[108].
Principle of the test
Insulin ELISA is a solid phase two-site enzyme immunoassay. It is based on the
direct sandwich technique in which two monoclonal antibodies are directed against
separate antigenic determinants on the insulin molecule. During incubation insulin in
the sample reacts with peroxidase-conjugated anti-insulin antibodies and anti-insulin
antibodies bound to microplate wells. A simple washing step removes unbound
enzyme labelled antibody. The bound conjugate is detected by reaction with
3,3’,5,5’-tetramethylbenzidine (TMB). The reaction is stopped by adding acid to give
a colorimetric endpoint that is read Spectrophotometrically.
Assay procedure
All reagents and specimens must be allowed to come to room temperature (25°C)
before use. All reagents must be mixed without foaming. Standards, controls and
samples should be assayed in duplicate [107].
1- Prepare enzyme conjugate 1X solution and wash buffer 1X solution.
2- Prepare sufficient microplate wells to accommodate Calibrators, controls and
samples in duplicate.
3- Pipette 25 μL each of Calibrators, controls and samples into appropriate
wells.
4- Add 100 μL of Enzyme Conjugate 1X solution to each well.
5- Incubate on a plate shaker (700-900 rpm) for 1 hour at room temperature
(18°C– 25°C)
6- Discard the reaction volume by inverting the microplate over a sink. Add 350
μL wash buffer 1X solution to each well. Discard the wash solution, tap
48
firmly several times against absorbent paper to remove excess liquid. Repeat
5 times.
7- Add 200 μL Substrate TMB into each well
8- Incubate on the bench for 15 minutes at room temperature (18°C – 25°C)
9- Add 50 μL Stop Solution to each well.
10- Place plate on a shaker for approximately 5 seconds to ensure mixing.
11- Read optical density at 450 nm and calculate results. Read within 30 minutes.
Calculation
1- Plot the absorbance values obtained for the Calibrators, except for Calibrator
0, against the insulin concentration on a log-log paper and construct a
calibration curve.
2- Read the concentration of the samples from the calibration curve.
Normal range value of Insulin in adults are ( 2–25 ) mU/L [108].
49
3.6 Statistical analysis
Data were computer analyzed using Statistical Package for the Social Sciences
(SPSS) version 18.0. Simple distribution of the study variables and the cross
tabulation were applied. Chi-square (X2) was used to identify the significance of the
relations, associations and interactions among various nominal variables. The
independent sample t-test procedure was used to compare means of quantitative
variables by the separated cases into two qualitative groups such as the relationship
between patients and controls hormones. Person's correlation test between IGFBP-1
and other studied variables was applied.
Ranges as minimum and maximum values were used.
The percentage difference was calculated according to the formula:
% difference = (|(V1-V2)| / ((V1+V2)/2)) X 100
Microsoft Excel program version 11.0 for correlation graph plotting.
The results were accepted as statistical significant when the p-value was less than 5%
(p<0.05).
51
Chapter 4
Results
4.1 Personal characters of the study population
Table (4.1) illustrates the personal data of the study population. Age classification
showed that 23 (57.5%) of controls and 26 (65.0%) of cases were ≤ 29 years old.
Age group (30-34) years comprised 11 (27.5%) of controls and 8 (20.0%) of cases.
Both controls and patients aged >34 years old were 6 (15.0%). The difference
between controls and patients in term of age distribution was not significant (χ2
=
0.65 , P = 0.72).
Analysis of educational status of the study population showed that 13 (32.5%)
controls and 20 (50.0%) patients had a university degree, 21 (52.5%) and 23 (32.5%)
finished secondary school and 6 (15.0%), 7 (17.5%) had passed primary school.The
difference between various education levels of controls and patients was not
significant (χ2= 3.44, P= 0.17).
Regarding family history, 0 (0%) controls and 2 (5.0%) patients reported that they
have a family history of female infertility ( χ2= 2.05, P= 0.15 ). Moreover, 28
(70.0%) of patients reported drugs consumption for activation of hormones of
ovulation (P=0.000).
50
Table (4.1) Distribution of Age, Education scale, Family history, Drug
consumption of the study population.
Chi-
Sequare
test
p-value
Age (years)
Control(n=40)
No %
Patients(n=40)
No %
≤29 23 57.5 % 26 65.0 %
0.65 0.72 30-34 11 27.5 % 8 20.0 %
>34 6 15.0 % 6 15.0 %
Mean ± Std.
Dev (years)
28.9±4.38 28.3±4.96 t=-1.6 0.11
Range (years)
(21-38) (19-36)
Education
scale
University 13 32.5 % 20 50.0 % Chi-
Sequare
test
3.44
p-value
0.17
Secondary 21 52.5 % 23 32.5 %
Primary 6 15.0 % 7 17.5 %
Family
history
Yes 0 0.0 % 2 5.0 %
2.05 0.15
No 40 100.0% 38 95.0 %
Drug*
consumption
Yes 0 0.0 % 28 70.0 %
43.07 0.000
No 40 100.0 % 12 30.0 %
*Drug consumption: those patients who have hormonal medication to induce ovulation.
52
4.2 Distribution of Body Mass Index among the study population.
Table (4.2) shows the distribution of various classes of BMI among the study
population. The numbers of normal, overweight and obese patients were 3(7.5%), 32
(80.0%) and 5 (12.5%) respectively whereas in controls the numbers were 24
(60.0%), 15 (37.5%) and 1 (2.5%), respectively (P=0.000).
Table (4.2) Distribution of Body Mass Index among the study population.
Chi-
Square
test
p- value Control(n=40)
NO %
Patients(n=40)
NO %
BMI kg/m2
Normal 24 60.0% 3 7.5%
25.14 0.000 Over weight 15 37.5% 32 80.0%
Obese 1 2.5% 5 12.5%
People with BMI=18.5-24.9 were considered to have normal weight, people with BMI=25.0-
29.9 were classified overweight, people with BMI ≥30.0 were considered obese[95].
4.3 Serum IGFBP-1 of the study population
Mean levels of serum IGFBP-1 among study population is presented in table (4.3)
and figure (4.1). The mean levels of IGFBP-1 were significantly decreased in
patients compared to controls with percentage difference 67.2% (4.57±3.27 ˅
9.2±2.39 ng/ml, P=0.000).
53
Table (4.3) Distribution of IGFBP-1 of the study population
t. test
p-value Control(n=40)
Patients(n=40)
Serum
IGFBP-1
(ng/ml)
Group
statistics
9.2±2.39
Mean±Std.
Dev
4.57±3.27
Mean±Std.
Dev
-4.63 0.000
Range (4.5-15.3)
(min-max)
(0.5-12)
(min-max)
IGFBP-1: Insulin Like Growth Factor Binding Protein -1
Reference range: : 0.4-16.6 ng/ml
All value are expressed as Mean ± SD.
P<0.05 significant
control
patients
S1
9.2
4.57
0
2
4
6
8
10
IGFBP-1
control
patients
Figure (4.1) Bar chart of mean serum IGFBP-1 levels of the study population.
54
4.4 Serum IGFBP-1 among the over weighted study population
Mean levels of serum IGFBP-1 among over weighted study population is presented
in table (4.4). The mean levels of IGFBP-1 showed significant decrease in over
weighted patients compared to over weighted controls with percentage difference
67.7% (4.97±3.43 ˅ 10.03±2.16 ng/ml, P=0.000).
Table (4.4) Distribution of IGFBP-1 among over weighted study population
t. test
p-value Control(n=16)
Patients(n=37)
Serum
IGFBP-1
(ng/ml)
Group
statistics
10.03±2.16
Mean±Std.
Dev
4.97±3.43
Mean±Std.
Dev
-5.05 0.000
Range (5.3-14.0)
(min-max)
(0.5-12)
(min-max)
IGFBP-1: Insulin Like Growth Factor Binding Protein -1
Reference range: : 0.4-16.6 ng/ml
All value are expressed as Mean ± SD.
P<0.05 significant
4.5 FSH and LH hormones of the study population.
Table (4.5 & 4.6) and figures (4.2 & 4.3) illustrates FSH and LH levels of the study
population. The results showed no significant difference of the mean levels of FSH
between patients and controls with percentage difference (1.8%), (6.11±1.77 ˅
6.0±2.09 mIU/ml, P= 0.82 ). In contrast, the results showed highly significant
difference in the mean level of LH which increased in patients compared to controls
with percentage difference (83.1%), (18.77±7.05 ˅ 7.75±2.14 mIU/ml, P= 0.000).
55
Table (4.5) FSH levels of the study population
t. test
p-value Control(n=40)
Patients(n=40)
Serum FSH
(mIU/ml)
Group
statistics
6.0±2.09
Mean±Std.
Dev
6.11±1.77
Mean±Std.
Dev
0.09 0.82
Range (1.2-10.4)
(min-max)
(2.8-9.1)
(min-max)
FSH: Follicle Stimulating Hormone
Reference value: 2.0-10 mIU/ml. All values are expressed as Mean ± SD, P>0.05; not
significant.
control
patients
S1
66.11
0
2
4
6
8
10
FSH
control
patients
Figure (4.2) Bar chart of mean serum FSH levels of the study population
56
Table (4.6) LH levels of the study population
t. test
p-value Control(n=40)
Patients(n=40)
Serum LH
(mIU/ml)
Group
statistics
7.75±2.14
Mean±Std.
Dev
18.77±7.05
Mean±Std.
Dev
11.0 0.000
Range (1.8-11.2)
(min-max)
(7.8-36.2)
(min-max)
LH: Luteinizing Hormone
Reference range: 1.0-20.0 mIU/ml
All values are expressed as Mean ± SD, P<0.05; significant.
control
patients
S1
7.75
18.77
0
5
10
15
20
LH
control
patients
Figure (4.3) Bar chart of mean serum LH levels of the study population
57
4.6 Serum Testosterone levels of the study population
Mean levels of serum Testosterone among study population is presented in table
(4.7) and figure (4.4). The mean levels of Testosterone showed significant increase in
patients compared to controls with percentage difference 42.2% (0.54±0.28 ˅
0.35±0.18 ng/ml, P=0.000).
Table (4.7) Testosterone levels of the study population
t. test
p-value Control(n=40)
Patients(n=40)
Serum
Testosterone
(ng /ml)
Group
statistics
0.35±0.18
Mean±Std.
Dev
0.54±0.28
Mean ± Std.
Dev
0.19 0.000
Range
(0.1-0.9)
(min-max)
(0.1-1.2)
(min-max)
Reference rang: 0.26 - 1.22 ng/ml
All values are expressed as Mean ± SD, P<0.05; significant.
controls
patients
S1
0.35
0.54
0
0.1
0.2
0.3
0.4
0.5
0.6
Testosterone
controls
patients
Figure (4.4) Bar chart of mean serum Testosterone levels of the study
population
58
4.7 Serum DHEA-S hormone levels of the study population
Table (4.8) and figure (4.5) illustrates DHEA-S levels of the study population. The
mean levels of DHEA-S were significantly increased in patients compared to
controls with percentage difference 69.2% (2.06±1.01 ˅ 1.0±0.43 μg/ml, P=0.000).
Table (4.8) DHEA-S hormone levels of the study population
t. test
p-value Control(n=40)
Patients(n=40)
Serum
DHEA-S
(μg/ml)
Group
statistics
1.0±0.43
Mean±Std.
Dev
2.06±1.01
Mean±Std.
Dev
1.06 0.000
Range (0.3-1.9)
(min-max)
(0.6-5.1)
(min-max)
DHEA-S: Dehydroepiandrosterone Sulfate hormone
Reference range: 0.40 – 2.17 μg/ml
All values are expressed as Mean ± SD, P<0.05; significant.
control
paitents
S1
1
2.06
0
0.5
1
1.5
2
2.5
DHEA-S
control
paitents
Figure (4.5) Bar chart of mean serum DHEA-S levels of the study population
59
4.8 Serum TSH levels of the study population
Mean levels of serum TSH among study population is presented in table (4.9) and
figure (4.6). The mean levels of TSH were insignificantly decreased in patients
compared to controls with percentage difference 8.3% (2.61±1.07 ˅ 2.84±1.42
mIU/ml, P= 0.41).
Table (4.9) TSH levels of the study population
t. test
p-value Control(n=40)
Patients(n=40)
Serum TSH
(mIU/ml)
Group
statistics
2.84±1.42
Mean±Std.
Dev
2.61±1.07
Mean±Std.
Dev
-0.23 0.41
Range (0.6-8.2)
(min-max)
(0.8-4.7)
(min-max)
TSH: Thyroid Stimulation Hormone
Reference range: 0.54 - 4.72 mIU/ml
All values are expressed as Mean ± SD, P>0.05; not significant.
control
patients
S1
2.84
2.61
0.5
1
1.5
2
2.5
3
TSH
control
patients
Figure (4.6) Bar chart of mean serum TSH levels of the study population
61
4.9 Serum Insulin levels of the study population
Table (4.10) and figure (4.7) illustrates insulin levels of the study population. The
mean levels of Insulin were significantly increased in patients compared to controls
with percentage difference 96.2% ( 20.61±5.48 ˅ 6.86±1.62 mIU/ml, P= 0.00).
Table (4.10) Insulin levels of the study population
t. test
p-value Control(n=40)
Patients(n=40)
Serum
Insulin
(mIU/ml)
Group
statistics
6.86±1.62
Mean±Std.
Dev
20.61±5.48
Mean±Std.
Dev
13.74 0.00
Range (3.6-12.3)
(min-max)
(4.6-27.1)
(min-max)
Reference range: 2–25 mIU/ml
All values are expressed as Mean ± SD, P<0.05; significant.
control
patients
S1
6.86
20.61
0
5
10
15
20
25
Insuline
control
patients
Figure (4.7) Bar chart of mean serum Insulin levels of the study population.
60
4.10 Correlation of IGFBP-1 with different hormones among the
study population
4.10.1 IGFBP-1 levels correlated with FSH and LH among the study
population
Table (4.11) illustrates the result of person correlation between serum IGFBP-1 and
FSH or LH levels. IGFBP-1 showed no significant negative correlation with FSH (r=
- 0.05,P= 0.626). In contrast, IGFBP-1 showed high significant negative correlation
with LH (r= -0.6, P= 0.000).
Table (4.11) Correlation of IGFBP-1 levels with FSH and LH of the study
population
FSH: Follicle Stimulating Hormone
LH: Luteinizing Hormone
Correlation is significant at the 0.05 level (2-tailed)
4.10.2 Correlation of IGFBP-1 levels with Testosterone and
DHEA-S levels of the study population
Table (4.12) illustrates the results of person correlation between serum IGFBP-1 and
Testosterone or DHEA-S levels. IGFBP-1 levels showed significant negative
correlation with both Testosterone and DHEA-S. (r= -0.38,P= 0.000 and r= -0.54, P=
0.000, respectively).
Result IGFBP-1
Result FSH
Pearson Correlation (r) -0.055
Sig. (2-tailed)* 0.626
N 80
Result LH
Pearson Correlation (r) -0.632
Sig. (2-tailed) 0.000
N 80
62
Table (4.12) Correlation of IGFBP-1 levels with Testosterone and DHEA-S of
the study population
IGFBP-1
Testosterone Pearson Correlation -0.389
Sig. (2-tailed) 0.00
N 80
DHEA-S Pearson Correlation -0.546
Sig. (2-tailed) 0.00
N 80
DHEA-S: Dehydroepiandrosterone Sulfate (DHEA-S) Hormone
Correlation is significant at the 0.05 level (2-tailed)
4.10.3 IGFBP-1 levels correlated with TSH and Insulin levels of the
study population
Table (4.13) illustrates the results of person correlation between serum IGFBP-1
and TSH or insulin levels. IGFBP-1 showed no significant positive correlation with
TSH (r= 0.14, P= 0.21). In contrast IGFBP-1, showed significant negative correlation
with Insulin (r= -0.59, P= 0.000).
Table (4.13) Correlation of IGFBP-1 levels with TSH and Insulin of the study
population
Result IGFBP-1
Result TSH
Pearson Correlation 0.141
Sig. (2-tailed) 0.213
N 80
Result Insulin
Pearson Correlation -0.590
Sig. (2-tailed) 0.000
N 80
TSH: Thyroid Stimulation Hormone
Correlation is significant at the 0.05 level (2-tailed)
63
4.10.4 IGFBP-1 levels correlated with BMI among the study
Population
Table (4.14) illustrates the results of person correlation between IGFBP-1 and BMI
among the study population. The results showed significant negative correlation with
BMI (r= -0.411, P= 0.000).
Table (4.14) Correlation of IGFBP-1 levels with BMI of the study population
Result IGFBP-1
BMI
Pearson Correlation -0.411
Sig. (2-tailed) 0.000
N 80
BMI: Body Mass Index
Correlation is significant at the 0.05 level (2-tailed)
4.11 Correlation of IGFBP-1with different hormones among the
cases
4.11.1 IGFBP-1 levels correlated with FSH and LH among the cases
Table (4.15) illustrates the result of person correlation between serum IGFBP-1 and
FSH or LH levels among the cases. IGFBP-1 showed no significant positive
correlation with FSH (r= 0.018,P= 0.91). In contrast, IGFBP-1 showed significant
negative correlation with LH (r= -0.4, P= 0.007).
64
Table (4.15) Correlation of IGFBP-1 levels with LH and FSH among the cases
Result IGFBP-1
Result FSH
Pearson Correlation 0.018
Sig. (2-tailed) 0.913
N 40
Result LH
Pearson Correlation -0.422
Sig. (2-tailed) 0.007
N 40
FSH: Follicle Stimulating Hormone
LH: Luteinizing Hormone
Correlation is significant at the 0.05 level (2-tailed).
4.11.2 IGFBP-1 levels correlated with Testosterone and DHEA-S
among the cases
Table (4.16) illustrates the results of person correlation between serum IGFBP-1
and Testosterone or DHEA-S levels. IGFBP-1 levels showed negative significant
correlation with both Testosterone and DHEA-S with (r= -0.36, P= 0.013 and r=
-0.382, P= 0.015, respectively).
Table (4.16) Correlation of IGFBP-1 levels with Testosterone and DHEA-S
among the Cases
IGFBP
Testosterone Pearson Correlation -0.36
Sig. (2-tailed) 0.013
N 40
DHEA-S Pearson Correlation -0.382
Sig. (2-tailed) 0.015
N 40
DHEA-S: Dehydroepiandrosterone Sulfate
Correlation is significant at the 0.05 level (2-tailed).
65
4.11.3 IGFBP-1 levels correlated with TSH and Insulin among the
cases
Table (4.17) illustrates the results of person correlation between serum IGFBP-1 and
TSH or insulin levels among the cases. IGFBP-1 showed no significant positive
correlation with TSH (r= 0.17, P= 0.26), in contrast, IGFBP-1 showed significant
negative correlation with insulin (r= -0.577, P= 0.02).
Table (4.17) Correlation of IGFBP-1 levels with TSH and Insulin among the
cases
Result IGFBP-1
Result TSH*
Pearson Correlation 0.179
Sig. (2-tailed) 0.269
N 40
Result Insulin
Pearson Correlation -0.577
Sig. (2-tailed) 0.026
N 40
TSH: Thyroid Stimulation Hormone
Correlation is significant at the 0.05 level (2-tailed)
4.11.4 IGFBP-1 levels correlated with BMI among the cases
Table (4.18) illustrates the result of person correlation between IGFBP-1 and BMI
among the cases. The result showed significant negative correlation with BMI (r= -
0.54, P= 0.000).
Table (4.18) Correlation of IGFBP-1 levels with BMI among the cases
Result IGFBP-1
BMI
Pearson Correlation -0.54
Sig. (2-tailed) 0.000
N 40
BMI: Body Mass Index
Correlation is significant at the 0.05 level (2-tailed).
66
Chapter 5
Discussion
Fertility is the natural capability to produce offspring. As a measure, fertility rate is
the number of offspring born per couple, individual or population. Worldwide,
according to the Centers for Disease Control, about one-third of infertility cases are
caused by women's problems. Another one third of fertility problems are due to the
man. The other cases are caused by a mixture of male and female problems or by
unknown problems [2].
The prevalence of PCOS is traditionally estimated at 20% of all reproductive-aged
women. Women are born with a finite number of eggs. Thus, as the reproductive
years progress, the number and quality of the eggs diminish. The chance of having a
baby decrease by 3% to 5% pre year after the age of 30. The increase of infertility is
noted to a much greater extent after age 40 [109].
The researchers found that the level of female infertility were similar in 2000 and
2012,with only a slight overall decrease in primary infertility and a modest overall
increase in secondary infertility (0.4%). The primary infertility rates among women
wanting children also varied by region, from 1.5% in Latin America and the
Caribbean in 2012, to 2.6% in North Africa and the Middle East. With a few
exception, global and country patterns of secondary infertility were similar to those
of primary infertility [110].
According to results of Family Health survey 2010, the total fertility rate in the
Palestinian Territory had declined to 4.1 births (2008-2009) compared to 6.0 births in
1997. At the regional level, the fertility rate in Gaza Strip is higher than West Bank
during the period between (1997-2009), where it reached 3.8 births during the period
between (2008-2009) in the West Bank compared to 5.6 births in 1997. While it
67
reached 4.9 births in Gaza Strip during the period between (2008-2009) compared to
6.9 births in 1997[111].
However, in Gaza strip there are under- reporting or even no real figure on female
infertility. Therefor, this will be the first preliminary study to assess IGFBP-1 status
and related it to female reproductive hormones among PCOS infertile women in
Gaza strip.
5.1 Sociodemographic of the study population
The presented study was carried out on 40 patients with PCOS infertility. The mean
age of patients (28.3 years) was lower than reported from West Bank (30.1 years)
study[112].
The younger age of our patient samples could be explained on the basis the most
women seeking out to have children at young age immediately after marriage.
Female age is a dependant factor because the fertility clearly declines with advancing
age, especially after the mid-30s, and women who conceive are at greater risk of
pregnancy complications [113].
It was found that only seven patients and six controls were at primary education
stage which do reflect educated community. There was significant difference
between patients and controls regarding drug consumption, which was considered a
risk factor for PCOS infertility. But no significant difference between patients and
controls regarding family history was observed.
5.2 BMI of the study population
Data presented here showed that most body fat patients were overweight compared
to controls. This finding indicated the increase in body fat considered a risk factor for
PCOS infertility. PCOS infertility was reported to be associated with higher
incidence of obesity[114]. The scientists explained the effect of overweight on
infertility that estrogen is found in two sources in the body: the ovary and the adrenal
gland. The ovary makes estrogen in quantities depending on the phase of the
68
menstrual cycle. The adrenal glands make something called androstenedione. These
hormones are connected to cholesterol and in the case of androstenedione ; fat cells
convert it into an estrogen called estrone. If someone is significantly overweight, the
oversupply of estrogen from this conversion interacts with the ovary function. This
will causes abnormal ovulation and leads to infertility [115].
Obesity is a common finding in PCOS in women. However high BMI lead to a poor
pregnancy outcome, such as sudden and unexplained intrauterine death [116].
Moreover increased risk of miscarriage among overweighted women suggested that
is due to impaired folliculogenesis and poor oocyte quality [117].
5.3 Assay of IGFBP-1 of the study population
Assay of IGFBP-1 of the study population showed that the mean level of IGFBP-1
was significantly decreased in patients compared to controls. This finding is in
agreement with that declared by other authors [84, 94]. In contrast, other studies
have failed to demonstrate a significant difference in IGFBP-1 between the two
groups [118,119]. That studies interpreted the relationship between PCOS and
decrease IGFBP-1 hormone suggests that a decreased serum level of IGFBP-1 does
not have a role in the pathogenesis of PCOS but is likely to result from the high
prevalence of obesity in the PCOS population [99] and may also increase the risk of
early pregnancy loss by decreasing IGFBP-1 [120].
In light of these points, a definitive conclusion on the role of IGFBP-1 in PCOS has
not yet to be reached. This highlights the need for further investigation of the serum
level of IGFBP-1 among higher study population.
5.4 Hormonal profile of the study population
Hormonal evaluation is considered one of the most important test of PCOS
diagnosis.
Hormonal profile of the study population of the mean level of FSH obtained in the
present study did not show significant different between patients and controls. In
contrast, LH hormone showed high significant increase in patients compared to
69
controls. These results are in accordance with that documented in other study [121].
Women with PCOS experience an increased frequency of hypothalamic GnRH
pulses, which in turn results in an increase in the LH/FSH ratio 2:1 or 3:1 which
leads to infertility because excessive LH triggers premature ovulation, disrupting the
follicle’s maturation process and leading to an increase in androgen production by
ovarian theca cells [121]. The increase of gonadotrophins may be attributed to a
primary abnormality of the hypothalamic-pituitary axis or may be secondary to
alterations in peripheral signaling. As a result the elevation of circulating LH
concentration may be due to inappropriate estrogen feed-back to neuroendocrine
centers [122].
The present finding showed that the mean level of total Testosterone was
significantly increased in patients compared to controls. This finding result is in
agreement with that declared by another study [123]. Total testosterone refers to the
total amount of all testosterone, including the free testosterone in the body. Women
with PCOS often have an increased level of both total testosterone and free
testosterone. Excessive levels of the androgen testosterone, for example, can cause
unwanted reactions, leading to menstrual irregularity and female infertility [123].
The mean level of DHEA-S hormone in present study was significantly increased in
patients compared to controls. This finding was in accordance with that documented
in another study [124]. The elevated DHEA-S is due to stimulation by ACTH,
produced by the pituitary in response to stress. The excess DHEA-S then converts to
androgens via adrenal metabolism, which in turn contributes to the typical elevated
androgen levels in PCOS. As discussed above increase level of androgen will lead to
infertility[125].
In contrast, the mean level of TSH hormone in present study did not show
significant difference between patients and controls. This finding result is in
agreement with that declared by another report [126] . In contrast, the finding result
is in disagreement with other studies which found an increase the level of TSH
(hypothyroidism) in women with PCOS [127]. TSH is checked to rule out other
71
problems, such as an underactive overactive thyroid, which often cause irregular or
lack of periods and an ovulation which lead to infertility.
The mean level of Insulin hormone in present study showed significant increase in
patients compared to controls. These result are in accordance with that documented
by another recent study [128]. The most common causes of insulin resistance are
obesity, poor diet and stress. Hyperinsulinemia is not a characteristic of
hyperandrogenism in general, but is uniquely associated with PCOS. The scientist
suggests that hyperinsulinemia plays a pathogenic role in causing the
hyperandrogenism of the PCOS by increasing ovarian androgen production.
Presumably, this occurs as a result of insulin-stimulating testosterone production by
the ovarian cell responsible for androgen biosynthesis, namely the thecal cell [128].
5.5 IGFBP-1 correlated with BMI and FSH , LH hormones
Serum IGFBP-1 level was significant negative (inverse) correlated with overweight
as determined by BMI in PCOS infertile women. This finding is consistent with
previous study in predominantly overweight and obese population [99,129]. Thus,
overweight and obese women have small amount of IGFBP-1. This highlights the
need for an investigation of the serum level of IGFBP-1 among PCOS of over
weighted women [99].
Moreover, as indicated in the present data, there was no significant positive
correlation between IGFBP-1 and FSH but showed significant negative correlation
with LH hormones. similar finding was declared by another author [130]. However,
it is suggested that a defect in the cyclic that regulate IGFBP-1 which leads to
increased free IGF1 amplitude, mainly attributed to an increased pituitary stimulation
by the hypothalamic growth hormone releasing hormone (GnRH) has been
demonstrated in normal-weight PCOS women[130].
70
5.6 IGFBP-1 correlated with TSH, insulin, Testosterone and DHEA-
S hormones
As indicated in the present finding there was no correlation between IGFBP-1 and
TSH. This finding result is in agreement with that declared by another author [131].
In contrast,there was inverse correlated between IGFBP-1 with both Insulin and
androgen hormones which determined by Testosterone and DHEA-S. These results
are in accordance with that documented in another study [132]. The suppression of
IGFBP-1 by hyperinsulinemia alone is responsible for the increased bioavailability
of IGF-I in PCOS. We propose that the increased adrenal hyperandrogenism and
peripheral androgen conversion in nonobese PCOS may be mediated by free IGF-I
[21]. An elevated circulating concentration of insulin inhibits the production of
IGFBP-1. Thus increasing the level of free IGF-I in serum and stimulating ovarian
androgen production [99].
72
Chapter 6
Conclusions and Recommendations
6.1 Conclusions
Medical history showed that 70% of patients reported administration drugs
for hormonal activation.
Family history was not associated with PCOS .
The difference of BMI between cases and controls was significant (P= 0.000).
Serum IGFBP-1 was significantly decreased among patients compared to
controls (P= 0.000).
Serum FSH did not show significant difference between patients and controls
(P=0.82).
Serum LH was significantly elevated in patients compared to controls (P=
0.000).
Both serum Testosterone and DHEA-S were significantly increased in
patients compared to controls (P=0.000 for both).
Serum TSH did not show significant difference between patients and controls
(P=0.41).
Serum Insulin was significant increased in patients compared to controls
(P=0.000).
Serum IGFBP-1 was significantly negative correlated with BMI, LH,
Testosterone, DHEA-S and Insulin (P=0.000 for all).
Serum IGFBP-1 did not show significant correlation with FSH and TSH.
Thus direct link between IGFBP-1 and these hormones cannot be ruled out.
In a conclusion, the results of present study indicated that obesity,
hyperinsulinemia, hyperandrogenism, increase level of LH and decreased
level of IGFBP-1 play a role in the process of PCOS among infertile women.
73
6.2 Recommendations
1. The present finding of this preliminary study showed that optimal IGFBP-1
secretion is necessary for normal reproductive function. Therefore, married
women must try to have a normal threshold level of serum IGFBP-1 by
following a specific diet or by consultation a nutritional doctor .
2. Suitable dietary and lifestyle regime for overweight women that affect in
ovulation cycle and the range of IGFBP-1 may improve the fertility.
3. By reducing insulin levels by diet and exercises both hyperandrogenism and
related clinical features tend to be improved.
4. Further studies are recommended to find out clear picture of the role of
serum IGFBP-1 in treatment infertility among large scale of sample of
married women.
74
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Annex
Annex 1
Female infertility Questionnaire
هذه معيمتم أتم إذاسأكون ممتنة لكم أنا الباحثة هنا زهير زمارة طالبة ماجستير من الجامعة اإلسالمية
.حاالت التكيس المبيضى في FPSE 1 1االستبانة لدراسة دور
بيانات شخصية
رقم التليفون
االسم
لعمرا
المرحلة الدراسية
ثانوية عامة جامعة أو دبلوم □ □
إعدادي □ابتدائي □أمي □
:الطول: الوزن مؤشر كتلة الجسم
..................... تاريخ أخذ العينة
..................... تاريخ الزواج
--------------- الدخل الشهرى
(خاص بمرضى العقم)بيانات طبية
ال □نعم □ هل يعاني أحد أفراد عائلتك من العقم؟
ال □نعم □ هل الدورة الشهرية منتظمة؟
ال □نعم □ ؟تعانى من أمراض متعلقة بالجهاز التناسليهل
87
إذا كانت اإلجابة نعم ما هي؟
................................................
هل تم فحص هرمونات سابقا؟
إذا كانت اإلجابة نعم ما هي؟
ال □نعم □
................................................
ال □نعم □ هل تتناولين أحد األدوية المنشطة للهرمون؟
ال □نعم □ حمل لديك قبل ذلك ؟هل حصل ال
ال □نعم □ هل لديك أطفال ؟
ال □نعم □ هل تعانين من أحد األمراض المزمنة؟
ال □نعم □ هل يعاني الزوج من أي مشكله تمنع اإلنجاب؟
النتائج
IGFBP-1*
FSH*
LH*
*Testosterone
DHEA-S*
TSH*
insulin*
.......................................................
88