Al-Azhar University – Gaza

Faculty of Pharmacy

Deanship of Postgraduate Studies and Research

A thesis Submitted in Partial Fulfillment of the Requirements for the Master Degree of Clinical Nutrition

By

Asmaa Mhareb Slman Meghari

Supervisors

Dr. Amin T. Hamed Dr. Ihab A. Nasser

Assoc. Prof. of Pharmacology Ass. Prof. of Community Nutrition

Faculty of Pharmacy Faculty of Applied Medical Sciences Al-Azhar University-Gaza Al-Azhar University-Gaza

Gaza, 2019 Al-Azhar University – Gaza

Faculty of Pharmacy

Deanship of Postgraduate Studies and Research

The Impact of Oral Contraceptive Use on Selected Vitamins and Minerals in Women of Reproductive Age

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master in Clinical Nutrition in Gaza Strip

By:

Asmaa Mhareb Slman Meghari

This thesis was defended successfully on / / 2019

Committee of Evaluation Signature

Main Supervisor Dr. Amin T. Hamed ………………………………

Co-Supervisor Dr. Ihab A. Nasser ………………………………

Internal Examiner Dr. Mohammed Q. Shubair ………………………………

External Examiner Dr. Marwan O. Jalambo ………………………………

Gaza, 2019

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D E C L A R A T I O N

I, the undersigned hereby, declare that the thesis titled: The Impact of Oral Contraceptive Use on Selected Vitamins and Minerals in Women of Reproductive Age, is my own research and the work provided in this thesis, unless otherwise referenced, is the researcher own work, and has never been submitted elsewhere for any other degree qualifications nor for any academic titles, nor for any other academic or publishing institutions.

I, here to, affirm that I will be completely responsible in academic and legal terms if this work proves the opposite.

Asmaa Mhareb Slman Meghari

Date: / / 2018

Signature: …………………….

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D E D I C A T I O N

I dedicate my dissertation work to my family and many friends. A special feeling of gratitude to my loving late father and to my mother, whose words of encouragement and push for tenacity ring in my ears.

To my dear husband Sa'ad Abdalwahed for helping me until finishing my thesis writing, and to my kids; Majd, Muhammad, and Lama for their lovely smile.

To my brothers and sisters who have never left my side and are very special.

I also dedicate this dissertation to my many friends who have supported me throughout the thesis writing. I will always appreciate all they have done.

Asmaa Mhareb Slman Meghari

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A C K N O W L E D G M E N T

Firstly, I would like to express my sincere gratitude to my supervisors Dr. Amin Hamed and Dr. Ihab Nasser for the continuous support of my master study and related research, for their patience, motivation, and immense knowledge. Their guidance helped me in all the time of research and writing of this thesis.

Besides my supervisors, I would like to thank the evaluation committee for their insightful comments and encouragement, but also for the hard questions which incented me to widen my research from various perspectives.

My sincere thanks also goes to the director of Mansour Lab Dr. Ramzy Mansour, who provided me an opportunity to join his team as intern.

Finally, thanks for all the participants who participated voluntarily in this study.

Asmaa Mhareb Slman Meghari

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ABSTRACT

Background: Childbearing women on Combined Oral Contraceptive Agents (COCA's) method have been linked with the development of various diseases and possible changes in serum micronutrients.

Objective: To investigate the vitamins and trace mineral changes associated with the use of COCA's among child bearing healthy women in Gaza Strip.

Methods: Forty-five long-term user (>1 years) COCA’s (150 microgram Levonoenorgestrel and 30 microgram ) users aged 18-35 years were recruited and these represent the cases in this case-control study, while the controls are 45 women who are not COCA’s users and using other methods of contraception such as intrauterine device (IUD or coil), condoms, or Mechanical (withdrawal method). After meeting the following criteria: nonsmoking, weight within normal range for height, free of metabolic disorders, and judged to be healthy by extensive medical histories and physical examinations. Informed consent was obtained. Questioners, anthropometrics measurements and blood samples were collected from all the respondents who further were categorized users and nonusers of COCA’s.

Results: The results of this study did not show any statistical difference regarding the anthropometric measurements between users and non-users of COCA's. However, there was significant (p = 0.044) association between the use of COCA's and cholesterol level. Also, a significant statistical difference in homocysteine levels and vitamin B6 was found between the two groups (p = 0.008 and p = 0.010, respectively). No significant difference was reported between the groups with respect to the any of the minerals measured. The results also reported positive association between using of COCA's and homocysteine level (b = 2.65 M CI 95%; 1.01, 4.31, P = 0.002). Negative association was observed between COCA's users and the level of vitamin B6 (b = -2.41, CI 95%; (-4.61, -0.20, P = 0.033).

Conclusion: The present study indicated and confirmed micronutrients changes in women on COCA's. Such changes could contribute to a number of diseases and health conditions in these women. For example; heart attack, stroke, diabetes, and colon and uterine cancer.

Key words: Contraceptives, micronutrients, childbearing age.

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تأثير استخدام حبوب منع الحمل الفموية على بعض الفيتامينات والمعادن على النساء اللواتي سن اإلنجاب في قطاع غزه

ملخص الدراسة

مقدمة: النساء في عمر اإلنجاب واللواتي يستخدمن حبوب منع الحمل المركبة، قد يعانين من أمراض مختلفة ونقص أو تغيرات في مستوى المعادن والفيتامينات.

هدف الدراسة: تهدف هذه الدراسة لمعرفة وتعقب التغيرات التي قد تحصل على الفيتامينات والمعادن في أوساط النساء األصحاء في عمر اإلنجاب في قطاع غزة.

منهجية البحث:خمس وأربعون من النساء )مجموعة الحالة( اللواتي يستخدمن حبوب منع الحمل المركبة من الجيل الثاني لمدة تزيد عن عام، وأعمارهن ما بين 18-35 عام تم ضمهن لهذه الدراسة )دراسة الحاالت والشواهد(، وخمس وأربعون أخريات )المجموعة الضابطة( تم ضمهن لمجموعة المقارنة واللواتي ال يستخدمن حبوب منع الحمل المركبة ويستخدمن نوع آخر من موانع الحمل مثل اللولب أو الواقي الذكري.

بعد استيفاء المعايير التالية: غير مدخنة، والوزن ضمن المعدل الطبيعي بالنسبة للطول، وخالية من االضطرابات األيضية، وأن تكون صحية من خالل التاريخ الطبي الشامل والفحوصات البدنية،تم الحصول على موافقة مستنيرة من السيدات الموافقات للمشاركة في هذا البحث.

تم توزيع وجمع جميع المعلومات المتعلقة بقياسات الجسم وعينات الدم )التحاليل المخبرية( من جميع النساء اللواتي وافقن وشاركن في الدراسة واللواتي تم تقسيمهن إلى مستخدمات لحبوب منع الحمل المركبة والمستخدمات ألدوات منع الحمل األخرى.

النتائج:نتائج الدراسة لم تثبت عن أي فرق إحصائي فيما يتعلق بقياسات الجسم بين النساء اللواتي يستخدمن حبوب منع الحمل المركبة واللواتي ال يستخدمن. وقد أوضحت هذه الدراسة وجود عالقة ذات داللة إحصائية)0.044( بين استخدام حبوب منع الحمل المركبة ومستوى الكولسترول في الدم. باإلضافة إلى وجود فروقات ذات داللة إحصائية بين المجموعات في كل من الهومسيستين وفيتامين ب 6 )0.008 و0.010 بالترتيب(. وأكدت هذه الدراسة عن عدم وجود أي فروقات ذات داللة إحصائية بين المجموعتين في معادن الدم التي تم قياساها.

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باإلضافة إلى ذلك، أكدت نتائج الدراسة إلى وجود عالقة طردية بين استخدام حبوب منع الحمل المركبة ومستوى الهومسيستين في الدم )0.002(، وعن وجود عالقة عكسية بين استخدام حبوب منع الحمل المركبة ومستوى فيتامين ب 6 في الدم )0.033(.

االستنتاجات:أشارت وأكدت الدراسة على وجود تغيرات مهمة في المغذيات الدقيقة لدى النساء اللواتي يستخدمن حبوب منع الحمل المركبة. هذه التغيرات في المغذيات الدقيقة قد تساهم في عدد من األمراض والحاالت المرضية المختلفة مثل النوبات القلبية والجلطات الدماغية والسكري وسرطان القولون والرحم.

كلمات مفتاحيه: موانع الحمل، الفيتامينات والمعادن، سن اإلنجاب.

VIII

TABLE OF CONTENTS

DECLARATION III

DEDICATION IV

ACKNOWLEDGMENT V

ABSTRACT VI

VIII ملخص الدراسة

TABLE OF CONTENTS IX

LIST OF TABLES IX

LIST OF ABBREVIATIONS IX

CHAPTER 1: INTRODUCTION

1.1. Background 1

1.2. Problem Statement 2

1.3. Justification of the Study 3

1.4. Objectives 4

1.5. Hypothesis 4

1.6. Limitations of the Study 5

1.7. Definition of Terms 5

CHAPTER 2: LITERATURE REVIEW

2.1. Background 7

2.2. methods 7

2.2.1. Long Acting Reversible Contraceptive 7

2.2.2. Barrier Methods 9

2.2.3. Sterilization 9

2.2.4. Hormonal Methods 10

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2.3. Pharmacological Classifications of COCA's 12

2.4. Mode of Action of COCA's 13

2.5 Metabolic Effect of COCA's 14

2.5.1. Blood Pressure 14

2.5.2 Risk of Cardiovascular Diseases 15

2.5.3 Obesity and Overweight 16

2.6 Effect of COCA's on Macronutrient and Micronutrient 18 Metabolism

2.6.2. Effect of COCA's on Micronutrients (Vitamin &Mineral) 21

2.6.2.1. Minerals 21

2.6.2.1. Vitamins 27

2.7. Effect of COCA's on Homocysteine 29

2.8. Effect of COCA's on Lipid Profile 30

2.9. Functions, Dietary Sources, and the Recommended Daily 31 Allowance (RDA)

2.9.1. Minerals 31

2.9.2. Vitamins 33

CHAPTER 1: METHODOLOGY

3.1. Study Design 37

3.2. Study Sitting 37

3.3. Study Population 38

3.4. Sample Size Calculation 38

3.5. Eligibility Criteria 38

3.6. Pilot Study 39

3.7. Sampling Frame 39

3.8. Sampling Design 39

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3.9. Ethical Consideration 40

3.10. Data Collection 40

3.11. Study Program 40

3.12. Material And Methods 41

3.12.1. Questionnaire 41

3.12.2. Food Frequency Questionnaire 41

3.12.3. Anthropometric Measurements 42

3.12.4. Blood Pressure 44

3.12.5. Biochemical Analysis 44

3.13. Data Management, Treatment And Statistical Analysis. 46

3.13.1. Data Management And Treatment 46

3.13.2. Statistical Analysis 47

CHAPTER 4: RESULTS

4.1. Socio-Demographic and Socioeconomic 48

4.2. Medical and Family History 50

4.3. Obstetric and Gynecological History 52

4.4. Anthropometric Measurements 54

4.5. Blood Pressure Measurements 55

4.6. Food Frequency Questionnaire 56

4.7. Laboratory Assessment 56

4.7.1 Complete Blood Count 56

4.7.2. Lipid Profile 57

4.7.3. Micronutrients 59

CHAPTER 5: DISCUSSION

5.1. Socio-Demographic and Socio-Economic Characteristics 64

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5.2. Medical and Family History 65

5.3. Anthropometric Measurements 66

5.4. Blood Pressure Measurements 67

5.5. Hematological Profile 68

5.6. Lipid Profile 68

5.7. Vitamins and Minerals 69

5.7.1. Vitamins 69

5.7.2. Minerals 72

CHAPTER 6: CONCLUSION AND RECOMMENDATIONS

6.1. CONCLUSION 76

6.2. RECOMMENDATIONS 76

REFERENCES 78

Appendices

Appendix A Permission and Ethical Approval 96

Appendix A-1 Approval from Helsinki Committee 96

Appendix A-2 Consent Form 97

Appendix B Questionnaires 100

Appendix B-1 Questionnaires 100

Appendix B-2 Food Frequency Questionnaires 106

Appendix C Power and Sample Size Software Result 111

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LIST OF TABLES

Table:2.1 Recommended Daily Allowances for Iron 32

Table:2.2 Recommended Daily Allowances for Zinc 33

Table:2.3 Recommended Daily Allowances for Folate 34

Table:2.4 Recommended Daily Allowances for Vitamin B6 35

Table:2.5 Recommended Daily Allowances for Vitamin B12 36

Table:3.1 Classification of Body Mass Index 43

Table:3.2 The WHO Cut-off Points and Risk of Metabolic 44 Complications

Table:3.3 Measurement Units of Biochemical Tests 46

Table:4.1 Socio-Demographic and Socio-Economic 48

Table:4.2 Socio-Demographic and Socio-Economic and COCA's 49 Use

Table:4.3 Medical and Family History 50

Table:4.4 Medical and Family History and COCA's Use 51

Table:4.5 Obstetric and Gynecological History 52

Table:4.6 Obstetric and Gynecological History and COCA's Use 53

Table:4.7 Anthropometric Characteristics of All Respondents 54

Table:4.8 Anthropometric Measurements Comparison Between 54 Groups

Table:4.9 Anthropometric Category and COCA's Use 55

Table:4.10 Blood Pressure Characteristics 55

Table:4.11 Blood Pressure Measurements Comparison Between 55 Groups

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Table:4.12 Food Items Comparison Between Groups 56

Table:4.13 CBC Comparison Between Groups 57

Table:4.14a Lipid Profile Characteristics 57

Table:4.14b Lipid Profile Comparison Between Groups 58

Table:4.14c Lipid Profile Category and COCA's Use 58

Table:4.15 Micronutrients Characteristics for All Respondents 59

Table:4.16 Micronutrients Comparison between Groups 60

Table:4.17 Biochemical Category and COCA's Use 60

Table:4.18 Predictor Factors of Homocysteine Level –Stepwise 62 Method*

Table:4.19 Predictor Factors of B6 Level –Stepwise Method* 63

LIST OF ABBREVIATIONS

APC Activated Protein C

BMI Body Mass Index

CBC Complete Blood Count

COCA's Combined Oral Contraceptive

CRP C-Reactive Protein

Cu-IUD Copper Intrauterine Device

CVD Cardiovascular Diseases

DMT-1 Divalent Metal Transport-1

FDA Food and Drug Administration

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Fe2+ Ferrous Iron

FFQ Food Frequency Questionnaire

FP Family Planning

GnRH Gonadotropin Releasing Hormone

Hb Hemoglobin

Hct Hematocrit

HDL-c High Density Lipoprotein Cholesterol hs-CRP High-Sensitivity C-reactive Protein

HTN Hypertension

IMP Integrin and Mobilferrin

IUDs Intrauterine Devices

LARC Long Acting Reversible Contraceptive

LDL-c Low Density Lipoprotein Cholesterol

MDA Malondialdehyde

MOH Ministry of Health

OCA's Oral Contraceptive Agents

PCC Pre Conception Care

POCs Progestin Only Contraceptive

POPs Only Pills

PRSC Plasma-Renin-substrate Concentration

RBCs Red Blood Cells

RDA Recommended Dietary Allowance

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STD Sexually Transmitted Disease

TC Total Cholesterol

TG Triglyceride

VLDL-c Very Low Density Lipoprotein Cholesterol

WHO World Health Organization

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CHAPTER 1

INTRODUCTION

1.1.Background

Contraceptives is a method or device used to prevent pregnancy. Contraception use by females in their childbearing years and the choice of having safe, effective, affordable and acceptable contraceptive methods as part of family planning (FP) are important dimensions of reproductive health (WHO, 2010). The most effective methods of birth control are sterilization by means of vasectomy in males and tubal ligation in females, Intrauterine Devices (IUDs), and implantable birth control, this is followed by a number of hormone-based methods including Oral Contraceptive Agents (OCA's), patches, vaginal rings, and injections (WHO, 2007). OCA's are currently the most commonly used contraception method in developed countries. In developing countries, female sterilization and intrauterine devices are the two most commonly used contraceptive methods (Darroch & Singh, 2013). There are two types of OCA's; the Combined Oral Contraceptive (COCA's), which contain both and a and the progestogen-only pills (sometimes called minipills) (Ammer, 2009). The most frequently used agents are a combination of drugs containing both an estrogen and a progestin. This combination is considered to be highly efficacious, with a theoretical effectiveness generally considered 99.9% and a use effectiveness of 97% to 98% (Palmery et al., 2013).

According to the UNRWA annual report, the number of women who used FP methods in their primary care clinics in Gaza was estimated at 66.56 %. The most common use was the IUD (51.8%) followed by OCA's (25.7%) and condom (16.1%), injectable (3.3%) and the least one were spermicide suppositories (0.1%) (UNRWA annual report, 2016).

Recently, there has been growing interest concerning alterations of metabolic processes and trace element profiles associated with the use of OCA's. Such changes are driven by genetic predisposition as well as environmental factors, changes in lifestyle, dietary habits and active ingredients of hormonal compounds (Dante et al., 2016). The interactions of OCA's with vitamins and minerals were studied in a large population of women. In the upper socioeconomic groups, higher incidences of

1 abnormal clinical signs related to micronutrient abnormalities were seen in OCA's users than in the control subjects (Park & Kim, 2016). Some of these micronutrients are co-factors and or coenzymes involved in important metabolic pathways. Changes in their tissue level or bioavailability might play a significant role in health risk and might be involved in the pathogenesis of some disorders (Shere et al., 2015). OCA's may create certain nutrient deficiencies and excesses as well as increase the nutritional needs of the user. Most of the B vitamins, particularly pyridoxine (B6) and folic acid, are needed in higher amounts when OCA's are used (Dante et al., 2016). The copper level usually rises, and zinc levels often fall (Akinloye et al., 2011), and more zinc is needed as well. An increased need for vitamins C, E, and K may also result from the use of OCA's. There have been a number of reports indicating that OCA's use is associated with impaired status of a number of nutrients, including folate, vitamins B6 and B12 (Lussana et al., 2003). The decrease in the serum micronutrients levels were proportional to the duration of contraceptive use, and this reduction may increase the risk of serious illness (Dante, et al., 2016). In this study, we investigated the possible effect of COCA's use on micronutrients among childbearing women.

1.2.Problem Statement

Micronutrient deficiencies are a significant public health problem among reproductive age women, that including vitamins and minerals aid the body in the production of hormones, enzymes, and other substances that are critical to the function of body systems (WHO, 2012). Deficiency of these micronutrients are associated with increased risk of adverse consequences (Ramakrishnan, et al., 2008; WHO, 2012; &Ramakrishnan, et al., 2012).

The primary underlying causes of micronutrient deficiencies are insufficient intake and poor bioavailability of micronutrients. Oral contraceptives are a major class of prescription drug, used by a large proportion of women at reproductive age. Much research has been conducted to investigate the physiological changes that occur in women who take Oral contraceptives. These include changes in general health as well as in nutritional status (Palmery, et al., 2013).

In terms of nutrition, several studies investigated whether women on COCA's need different amounts of some vitamins and minerals (Fallah, et al., 2009 &Lussana,

2 et al., 2003). In particular, a report from the WHO points out that the influence of OCAs on nutrient requirements is a topic of high clinical relevance and should receive great attention (WHO, 2012).

As a research knowledge this study is one of the first to describe micronutrient status among women of reproductive age in Gaza Strip who use COCA's. As a result, our findings will form the bases for further nutritional interventions.

1.3.Justifications of the study

Globally, approximately 60% of those who are married and able to have children use birth control, and this percentage varies widely between countries, and 35% of them control birth via female sterilization, 30% is via IUDs, 12% is via OCA's, 11% is via condoms, and 4% is via male sterilization (Darroch, 2013). Unfortunately, some of the literature data report the administration of various COCA's is able to change micronutrient levels (Park & Kim, 2016). It is not always clear which component of the COCA's is responsible for the observed changes. Even nutritional experts’ opinions differ in the interpretation of the biochemical results and the uncontrolled administration of dietary supplements to COCA's users making comprehension of the effects on the women’s health more difficult (S. M. Wilson et al., 2011). Although, there is growing evidence that women using COCA's show changes in serum trace elements and vitamins, the impact of COCA's on the nutritional micronutrients are still controversial issue in literature. This controversy may result from different sample sizes, used different methodology, and different dietary background.

Despite the reports from the World Health Organization (WHO) pointing out that, the influence of COCA's on nutrient requirements as a topic of high clinical relevance and should therefore receive great attention(Palmery et al.). There is no published information available on the effect of OCA's on the micronutrients level in Palestine. Findings of this study will give further insight into the effects of OCA's on the nutritional status of the users. The current study will not only give accurate information about micronutrient status among COCA's users. Also, the researcher are trying to collect a specific information about COCA's users, in order to apply it in nutritional trials in further studies.

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1.4. Objectives

General Objective

The general objective of this study is to evaluate the vitamins and trace minerals changes associated with the use of COCA's among child bearing healthy women in Gaza Strip.

Specific Objectives

 To compare the anthropometric measurements between COCA's users and non-users.

 To compare the level of selected vitamins namely; Pyridoxine B6, Cyanocobalamin B12, and Folic acid between users and non-users of COCA's.

 To compare the levels of selected trace minerals namely; iron, zinc and copper between COCA's users and non-users.

 To compare the hematological profiles among users and non-users of COCA's.

 To compare the level of homocystien between users and non-users of COCA'S.

 To compare the lipid profile between users and non-users of COCA'S.

1.5.Hypothesis

H0: Null hypothesis

There is no significant differences between the users of COCA's and non-users in anthropometric measurements, hematological profile, lipid profile, vitamins, and minerals.

H1: Hypothesis of difference

There is a significant differences between the users of COCA's and non-users in anthropometric measurements, hematological profile, lipid profile, vitamins, and minerals.

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1.6.Limitations of the study

Although this research was carefully prepared, there are some unavoidable limitations.

 The current study was a case control in nature and assessed nutritional status among the users of the COCA's; It might be better if the study followed by dietary interventional program.

 The sample size is relatively small in comparison with other studies. Forty- five respondents is not enough for the researcher to investigate all of the users of COCA's.

 Because of the biochemical tests cost, and lack of supporting agencies for funding, some important and specific biochemical tests were not done.

 Limited or no open access references like books and journals.

1.7.Definition of Terms

Family planning: The practice of controlling the number of children one has and the intervals between their births, particularly by means of contraception or voluntary sterilization.

Contraceptives: Deliberate prevention of conception or impregnation.

Combined Oral Contraceptives: Microgynon ED Fe coated tablet (combined oral contraceptives) contain two hormones, which are 0.15 mg Levonoenorgestrel and 0.03 mg Ethinyloestreadiol. Each blister strip contains 28 tablet. The tablet should be taken at about the same time each day. After taken all 21 hormone containing tablet, the users take the hormone free tablet (Ferrous Fumerate) for the next 7 days.

Progesterone Only Pills used in this research contains 0.03 mg progestogen Levonoenorgestrel. The dosage of Microlut is one tablet daily without any break, taken at the same time each day with some liquid as needed. Tablet must be taken in the order directed on the package throughout the 35 days without regard to bleeding.

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Micronutrients: Essential elements such as a vitamin or mineral, that is essential in minute amounts for the proper growth and metabolism of human body. The included vitamins and minerals in this study are; Pyridoxine B6, Cyanocobalamin B12, Folic acid, Iron, Zinc and Copper.

Women of reproductive age: All women aged 18-35 years, who can become pregnant, and matched with the eligibility criteria in this study.

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CHAPTER 2

LITERATURE REVIEW

2.

2.1.Background

Since ancient times, humans tried hard to control the timing of pregnancy where pregnancy was the leading cause of death in the middle ages as reported by medical history books concerning the complications of pregnancy and childbirth (Alkema et al., 2015). In addition, it is not surprising that we find the issue of FP in the forefront when scanning any topic related to world population. FP has a direct relationship to population inflation, food security, water crises and the consumption of global energy sources, where the world population is expected to reach 8 Billion in 2025 (Ellis & Abarbanel, 2013). Many contraceptive methods have been discovered, most of them for women and a few for men. Presently, available FP methods have been modified to make them more efficient, effective and safer.

Contraception includes all methods that permit intercourse between fertile partners without producing impregnation. There are different methods to prevent pregnancy and choosing the appropriate one vary from person to person, depending on his / her health, age, sexual activity and desire to have children in the future and family history of disease.

2.2.Birth control methods

These methods can be classified into long acting reversible contraceptive (LARC), hormonal methods, barrier methods, emergency contraceptive and sterilization. The effectiveness is generally expressed as the percentage of women who become pregnant using a given method during the first year (Edlin et al., 2000).

2.2.1. Long acting reversible contraceptive 2.2.1.1.Intra uterine device

Intra Uterine Devices is a small, often T-shaped birth control device that is inserted into a woman's uterus to prevent pregnancy, and considered as one of LARC forms (Winner et al., 2012). It is placed in uterus to prevent pregnancy by damaging or killing sperm. It is allowed for use in situations where the use of pills is

7 contraindicated. It is forbidden to use in women who suffer from infections as in the pelvic inflammatory disease and who have more than one partner. There are two types of IUDs; hormonal IUDs and copper (Cu) IUDs. Among types of birth control, they along with birth control implants result in the greatest satisfaction among users (Obstetricians & Gynecologists, 2012). IUDs are the most widely used form of reversible contraception, with more than 180 million users worldwide (Speroff & Darney, 2010). The IUDs do not affect breastfeeding and can be inserted immediately after delivery (Gabbe et al., 2016) and immediately after an abortion(Steenland et al., 2011), and fertility returns to normal immediately after removal (Falcone et al., 2007).

A. Hormonal IUD

This type of IUDs releases a progesterone hormone (levonorgesterl) into the uterus. This hormone leads to thickening in the mucous membrane of cervix. Thus, preventing sperm from reaching and fertilize the ova. Also, makes the endometrium (lining of uterus) thin to prevent a fertilized ova to implant and grow. It is effective in preventing pregnancy more than the Copper Intrauterine Device (cu-IUD) (Ortiz & Croxatto, 2007). The 5-year cumulative pregnancy rate per 100 users was 0.5 and the 5- year Pearl rate was 0.11(Furlong, 2002). b. Copper IUD

It is the most commonly used type of IUD. The copper ions released from cu-IUD is excreted into the uterine fluid. This leads to an inflammatory reaction in the uterine lumen. As a result, women who use this type of IUD have a high- level of Copper in genital tract fluids.(Tal et al., 2016) This substance is considered toxic and harmful to the sperm and affect their motion so that they cannot sterilized the ova. If fertilization occur, the presence of the IUD prevents the fertilized ova from implantation in the endometrium, cu-IUD may remain in the body for 10 years (Ortiz & Croxatto, 2007). Recommended for emergency contraception if you have had unprotected sex in the past few days and need to avoid pregnancy and your plane to continuous using the IUD for birth control.

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2.2.1.2.Implant

Contraceptive implant is small flexible tube inserted under the skin of women's upper arm. A sub-dermal implant contain a number of synthetic progesterone (). The major side- effect of this method is irregular bleeding (Sivin et al., 1980). The implant can remain for up to five years, and the cumulative pregnancy rate was 0.6 per 100 women-years(Ali et al., 2016).

2.2.2. Barrier methods

It acts as a barrier to prevent sperm from reaching the ova, and may form a protect against sexual transmitted diseases. These inexpensive methods are easy to use and can be used immediately after childbirth and safe while breastfeeding. However, these methods are not effective in preventing pregnancy such as other methods like injection and IUD, for example, about 18 to 28 per 100 women who use it got pregnant(Trussel & Guthrie, 2011), methods like (spermicide, condom, sponge ,diaphragm and cervical cap). One of the most widely used methods in primary care in Gaza male condoms and spermicide. Male condoms are generally made of latex or intestinal membrane of lambs(Nordenberg, 2003). Latex condoms reduce the risk of spreading sexually transmitted disease (STD) while lambskin condoms do not prevent STD(Workowski and Bolan, 2015). spermicides is a chemical that inactivates sperm and the most common spermicidal agent is called nonoxynynol-9 (N-9). It is available in several concentrations as forms (foam, Jelly, cream, suppository, pessaries, capsules and film)(Lech, 2002).

2.2.3. Sterilization

Sterilization is a permanent form of birth control that either prevents a woman from getting pregnant or prevents a man from releasing sperm. These procedures usually are not reversible(Bartz and Greenberg, 2008).

A. Tubal ligation

A surgical contraceptive method can be implemented by laparotomy, mini laparotomy, colpotomy, and laparoscopy (Kondo et al., 2009). The incidence of pregnancy is small after tubal ligation, but increase the risk of an ectopic pregnancy.

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Ectopic pregnancy accounts for 9% of all causes of death in women (Peterson et al., 1997). The expected pregnancy rates per 1000 women at one year are 57for hysteroscopic sterilization, seven forlaparoscopic silicone rubber band applicationand three for laparoscopic bipolar coagulation (Gariepy et al., 2014).

B. Sterilization implant

It is considered a non-surgical procedure done by hysteroscopy called (Essure system). A device is inserted through the vagina and then placed in the fallopian tubes to seal them. This method does not need surgical incision, but need local anesthesia. This method may take up to three months to be effective as the scar tissue form. Pregnancy uncommon after any type of sterilization, but infection, bleeding and ectopic pregnancy may occur (Peterson et al., 1997).

C. Vasectomy

It is a permanent, highly effective birth control method. This surgical procedure cuts, closes or blocks the vas deferens. So that it will close the path between the tests and urethra(Fainberg and Kashanian, 2018). Failure rate less than 1% which are usually related to operation failure, unprotected intercourse before the semen is cleared of sperm and spontaneous early or late recanalization of the vas (Cook et al., 2007).

2.2.4. Hormonal methods

All hormonal contraceptive methods contain a progestin, which is responsible to regulate or stop ovulation. Ovulation is the biological process in which the ovary releases an ova, making it available for fertilization (Adashi, 2013). Progestin only methods such as Progestin Only Contraceptive (POCs), injectable medroxyprogestrone acetate and implanted progestin(Phillips et al., 2016). In addition, the progestin in these methods can thicken cervical mucous membrane, which helps block sperm from reaching the ova or thin the lining of the uterus. In contrast, combined hormonal methods contain a synthetic estrogen (Ethinyl ) and one of the many progestin, which has been approved. In combined (i.e. COCA's, vaginal ring and contraceptive implant), estrogen support the action of progestin in inhibiting ovulation (Ryden & Blumenthal, 2009).

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2.2.4.1.Progesterone only pills (POPs)

Progesterone pillsor called mini pills that contain very low doses of a progestin like the natural hormone progesterone. A woman takes one pill daily, preferably at the same time each day. It is best used in women who have been prevented from using COCA's(Becker, 2001). In addition, it is safe to use during breast feeding, because it doesn't contain estrogen (Truitt et al., 2003). POPs may interfere with ovulation in about 50%of women, but primary mechanism of action is thickening of cervical mucosa, making it difficult to sperm to swim into the uterus or enter fallopian tube. With perfect use of POPs, the incidence of pregnancy is 0.5% within the first year (Sarina, 2002). Mini pills alter the normal cyclical changes in the uterine lining and may result in abnormal uterine bleeding and it is the major cause of its discontinuation.This hormone do not appear to be associated with an increased risk of blood clotting (Lidegaard et al., 2009).

2.2.4.2.Injectable birth control

Depo-Provera (depot medroxyprogestrone acetate DMPA) injection150 mg, given in the arm or buttocks once every three months ±14 day because it inhibits the process of ovulation for 14 weeks. This method of birth control is effective in lactating and non- lactating women (Babre & Phadke, 2016). Moreover, it is characterized as effective, safe and not need to take it daily (Westhoff, 2003). It has a contraceptive efficacy exceeding 99%, no effects of DMPA on weight or blood pressure or permanent effect on fertility,it also decreases the incidence of endometrial cancer(Tal et al., 2016). It can cause a temporary loss of bone density. However, this bone loss generally is reversible after discontinuation. The main reason to stop this method is vaginal spotting, prolonged bleeding and amenorrhea(Kaunitz et al., 2008).

2.2.4.3.Vaginal ring

The ring is thin, flexible and approximately two inches in diameter. It delivers a combination of EthinylEstradiol and a progestin. The ring is inserted into the vagina, where it continually releases hormones for three weeks. Women remove it in the fourth week and reinserts a new ring seven days later (Roumen et al., 2001). This method works by inhibiting ovulation and rapidly absorbed through vaginal epithelium, results in steady serum concentration(Kovacs and Briggs, 2015). A

11 vaginal ring may not be recommended for women with certain health conditions like hypertension and heart disease. Also it may increase the incidence of venous thrombosis similar to that in COCA's(Lete et al., 2007). However, it is considered safe and effective in regulating the menstrual cycle and reduce the symptoms associated with it (Kerns & Darney, 2011).

2.2.4.4.Contraceptive patch

This is a thin, plastic patch that sticks to the skin and release hormones (progestinnorelgestramin and the estrogen ethinyloestreadiol) through the skin into the bloodstream(Wiegratz et al., 2014). The patch is placed on the lower abdomen, buttocks, outer arm. Anew patch is placed once weekly for three consecutive weeks (21 day) and no patch on the fourth week to enable menstruation (Abrams et al., 2002). This transdermal contraceptive patch provides effective contraception and cycle control and is well tolerated (Smallwood et al., 2001). The failure rate is 7 pregnancy per 1000 women (Burkman et al., 2001).

2.2.4.5.Combined oral contraceptives

Pills that contain low doses of two hormones (progestin and an estrogen) like natural hormones, since their introduction and approval by Food and Drug Administration (FDA) in the 1960 and become public. Oral contraceptive have rapidly been accepted as an effective method of contraception, with over 100 million users worldwide (Christin-Maitre, 2013). Lower dose pills have been formulated over the years. So that, the majority of the pills is extremely safe, often with health benefits, currently there are more than 50 FDA approved COCA's(McMillan et al., 2006). COCA's maybe monophasic, where each of the active pills releases constant dose of estrogen and progesterone throughout the women menstrual cycle or multiphasic, where the active pills mimic normal hormonal fluctuation of the natural menstrual cycle by providing different level of estrogen and progesterone at different times of the month. Multiphasic pills reduce total hormonal dose and side-effect (Rengel, 2000).

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2.3.Pharmacological classifications of COCA's

COCA's can be classified to different generations (Kumar &Nayak, 2014); First generation COCA's contain high dose Ethinyl estradiol 50µg or more. Second generation COCA's Ethinyl estradiol 30-35µg and progestin as levonorgestrel or norgestimat, these tend to "oppose" the action of estrogen, resulting in pills that have relatively high progestogenic activity (progestogenic dominant). Third generation COCA's Ethinyl estradiol 20-30 µg include , . Unclassified contain Drospirenon (in combination with EthinylEstradiol) differ from other in COCA's; it has very mild diuretic properties due to antiamineralocorticoid activity. This may help to oppose the salt and fluid retaining effects of Ethanol Estradiol and to reduce symptom of fluid retention (Kumar &Nayak, 2014).

2.4.Mode of Action of COCA's

COCA's inhibitsfollicular development and prevent ovulation by suppressing the release of gonadotropins (Gonadotropin Releasing Hormone (GnRH), LH, FSH) as their primary mechanism(Hatcher and Nelson, 2007). Progesterone negative feedback decrease the pulse frequency of gonadotropin releasing hormone GnRH by the hypothalamus, which decrease the release of FSH and greatly decreases the release of LH by the anterior pituitary gland. Decreased levels of FSH inhibit follicular development, preventing an increase in estradiol levels. Progesterone negative feedback on LH release prevent a mid-cycle LH surge inhibition of follicular development and the absence of LH surge prevent ovulation. Estrogen was also found to inhibit follicular development and help prevent ovulation, thus done by Estrogen negative feedback on the anterior pituitary which greatly decreases the release of FSH(Robert Anthony Hatcher & Nelson, 2007). A secondary mechanism of action is inhibition of sperm penetration through the cervix into the upper genital tract (uterus and fallopian tubes) by altering in tubal peristalsis, thin lining endometrial and increase the viscosity of cervical mucosa and its thickening(Cooper and Adigun, 2017).

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Efficacy

The contraceptive method is very effective when used correctly and consistently. Theoretical failure rate of COCA's is being 1 pregnancy/1000women in the first year of use. However, poor compliance is a major factor in limiting effectiveness, particularly in young users. In addition, late starts is the main problem when the pill free interval is extended beyond the usual seven days. Consequently, the failure rate with typical use is about 80 per 1000 women / year (Kileyet al., 2007).

Advantages

COCA's provide a reversible and conventional method of contraception that is independent of intercourse. They also have several non-contraceptive benefits including reduction in premenstrual symptoms, heavy periods and irregular bleeding and benign breast cancer(Schindler, 2013). Also, decrease risk of the ovary and endometrial cancer. There are some evidence of reduced colorectal cancer and fracture in some women. Additional benefits for current users include reduced symptomatic endometriosis, acne and pelvic inflammatory disease(Huber et al., 2008).

2.5.Metabolic effects of COCA's

There is no question that oral contraceptive have brought an unprecedented level of freedom to women in making choices about their health, pleasure and FP. It is important to know the side effects of birth control pills to stay safe and healthy. COCA's can deplete human body nutrients(Bartlik et al., 2018). Health problems that can develop from these nutrients depletion include depression, sleep disorders, anemia, migraine, heart attacks, strokes, blood clots, diabetes and cancer of uterus, colon and breast(Pelton and Association, 2001).

2.5.1. Blood pressure

The association between COCA's and Hypertension (HTN), noted and confirmed as early as 1961, was explored in multicenter clinical trial carried out by (Aronson, 2009). This is more prevalent with older and higher estrogen dose (not currently available) and may subside three to six months after its discontinuation. Women who

14 take low doses of estrogen (less than 35µg) have very low incidence of elevated blood pressure (Legato & Bilezikian, 2004). The effect being much more marked in some individuals than other. If a patient have a family history of HTN or have renal disease, age parity and obesity; the physician needs to be cautious about giving her COCA's and then monitor her blood pressure frequently. Administration of an estrogen – gestagen increase the concentration of angiotensinogen (renin substrate) in plasma. The generation of angiotensin was enhanced by higher substrate concentration. Plasma renin activity was consistently higher during oral contraceptive administration than after discontinued .Aldosterone secretion and excretion rate were higher during estrogen – gestagen administration than after the medication was withheld. This aldosterone level correlate with plasma renin activity. As a result, sodium and water retention is increased by estrogen and gestagens, as is the plasma volume and cardiac stroke volume (Kang et al., 2001). A controlled prospective survey, concluded that, women who took COCA's pills, showed increased mean systolic and diastolic blood pressure 14.2-8.5 mmHg, respectively, after four years. Only one case showed the largest increase in blood pressure 36 mmHg systolic and 20 mmHg diastolic. However, after stopping COCA's, the BP returned to normal after three months(Weir et al., 1974).

All women using COCA'sshould have their blood pressure checked before initiating use and periodically thereafter. Women with well controlled hypertension who elect to use oral contraceptives should be counseled that it is uncertain whether blood pressure control eliminates the associated increases in the risks of stroke and myocardial infarction. Oral contraceptive use should be discontinued immediately in any woman with symptoms suggestive of stroke, myocardial infarction, or venous thrombosis (Petitti, D. B, 2003).

Lubianca and Faccinin 2003 conducted a prospective cross sectional study design to assess the association between systolic and diastolic blood pressure and the use of COCA's in hypertensive women. They were evaluated 171 women who were referred to the Hypertension Outpatient Clinic; 66 current users of COCA'S, 26 users of other contraceptive methods and 79 women who were not using contraception. Diastolic blood pressure was higher in COCA'S users (100.2 ± 15.9 mmHg) than in patients

15 using other contraceptive methods (93.4 ± 14.7 mmHg) and not using contraceptives (93.3 ± 14.4 mmHg, p = 0.016). Women using COCA's for more than 8 years presented higher age-adjusted blood pressure levels than women using COCA's for shorter periods. These results were independent of antihypertensive drug use. They pointed that, COCA'S use independently and significantly associated with prevalence of uncontrolled hypertension. They concluded that hypertensive women using COCA'S present a significant increase in diastolic blood pressure and poor blood pressure control, independent of age, weight and antihypertensive drug treatment (Lubianca and Faccin, 2003).

2.5.2. Risk for Cardiovascular Diseases (CVD)

Since the introduction of COCA's, their use has been associated with an increased risk of both venous and arterial thrombosis. Pulmonary embolism, myocardial infarction and stroke are serious disorders with considerable risk of mortality(Rosendaal, 2003). The risk is associated with COCA's use and CVD, approximately two to four-fold increase relative risks of arterial and venous thromboembolic events, respectively. The highest risk for venous thromboembolism occur in the first year of use and is reduced to 2.76 over baseline risk after four years of use. However, the risk of myocardial infarction does not correlate to the length of COCA's and disappears after discontinuation of pills use. But, there are some factors that increase the risk of CVD in women who took COCA's, either genetic factors or acquired risk factors for thrombosis. Acquired one like age, so it is better not to use these pills after the age 35(Weill et al., 2016). In addition to smoking and hypertension considered by oral diabetes, and hypercholesterolemia and body weight as risk factors for CVD in females who use COCA's. Therefore, in women with multiple cardiovascular risk factors, the use of POPs should be considered. POCs is associated with substantially less risk of cardiovascular events than COCA's (Kaminski et al., 2013).

It is thought that the hormones contained in oral contraceptive formulas create an environment which affect the clotting cascade by altering clotting factors. Estrogen alter the levels of clotting factors by increasing (factors VII ,VIII, X, fibrinogen ) and plasminogen, lowering antithrombin III and protein S levels, and altering Activated Protein C (APC) resistance. APC induce decreased factor V activity. With increased APC resistance, this inhibition is not in effect and the coagulation cascade proceed.

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The net effect of combination pills is a procoagulant effect. The overall hemostatic effect is partly caused by estrogen (found in all pills, but at different doses) and partly caused by the type of progestogens. Various progestogens induce various changes. Third generation pills contain desogestrel that was found to induce a greater increase in factor II and VII levels (Middeldorp et al., 2000). So that, the risk is twice as high for desogestrel and gestodene (third generation) containing COCA's as well as for levonorgestrel (second-generation) containing COCA's (Rosendaal et al., 2003).

2.5.3. Obesity and overweight

Many women and clinicians believe that combination of estrogen–progestin contraceptive use can lead to weight gain(Gallo et al., 2008). This concern can deter women from starting hormonal contraception or lead to premature quitting. A study examined the effect of COCA's on body weight, fat mass, percent body fat, and lean mass in young female distance runners, the study conducted on 150 female competitive distance runners aged 18-26 years who had participated in a two-year randomized trial of the effect of the OCAs Lo/Ovral (30 microg of Ethinyl Estradiol and 0.3 mg of ) on bone health. Weight and body composition were measured approximately yearly by balance beam scales and dual-energy x-ray absorptiometry, respectively. The same study confirms that COCA's use does not cause weight or fat mass gain, at least among young female runners (Procter-Gray et al., 2008). They found that COCA's was associated with lean mass gain in eumenorrheic runners, but not in those with irregular menses, warrants examination in other studies.

The randomized controlled trials measuring weight change that were at least three treatment cycles in duration and that compared a combination contraceptive to a placebo or to a combination contraceptive that differed in drug, dosage, regimen, or study length were eligible for inclusion, of the 570 reports of randomized controlled trials of eligible interventions that were identified, 42 trials were included in the systematic review (Gallo et al., 2004). Depending on the data available, the weighted mean difference using a fixed effect model with 95% confidence intervals was calculated for the mean change in weight between baseline and post treatment measurements or the Peto odds ratio with 95% confidence intervals was calculated by using the proportion of women who gained or lost more than a specified amount of

17 weight. The three placebo-controlled, randomized trials did not find evidence supporting association between combination COCA's or a combination skin patch and weight gain. Most comparisons from the 40 trials that compared two or more combination contraceptives showed no substantial difference in weight. In addition, discontinuation of combination contraceptives because of weight gain did not differ between groups when this factor was studied. This means that available evidence is insufficient to determine the effect of combination contraceptives on weight, but no large effect is evident.

2.6.Effect of COCA's on macronutrient and micronutrient metabolism

The steroid hormone in oral contraceptive affect not only macronutrient (carbohydrate, lipid and protein) metabolism, but produce alteration in the metabolism and /or absorption of certain vitamins and minerals as well(Dante et al., 2016). The change may not be induced in all women taking OCA's because is much dependent on their prior and current nutritional status. Certain women because of an inadequate, maybe more vulnerable to a vitamin and mineral depletion, but the symptoms of actual deficiency have occurred rarely.

2.6.1. Macronutrient effect of COCA's 2.6.1.1.Effect of COCA's on carbohydrates metabolism

Impairment of glucose tolerance and elevation of insulin level indicating insulin resistance. Insulin resistance will lead to metabolic disturbances associated with development of coronary heart disease. The change observed in carbohydrate metabolism with oral contraceptive use are believed to be attributed almost entirely to progestin component of COCA's and are dose related. Ethinyl estradiol administration alone, even in high doses, does not cause glucose tolerance deterioration or abnormal insulin response(Godsland et al., 1990).

Mastorakos and his colleagues in 2004compared the effects of combined oral contraceptives containing cyproteroneacetate or desogestrel on insulin sensitivity in adolescents with polycystic ovary syndrome. Thirty-six adolescent girls with hyperandrogenism and six or less menses in the preceding 12 months.Patients were separated in two groups: group A (n = 18) received 0.15 mg of desogestrel plus 0.030 mg of Ethinyl E2 daily; and group B (n = 18) received 2 mg of cyproterone acetate

18 plus 0.035 mg of Ethinyl E2 daily, for 21 days followed by a 7-day rest, for 12 months.Hirsutism score, lipid, , and sex hormone-binding globulin levels were evaluated at baseline. An oral glucose tolerance test was performed and metabolism indices, based on previously studied mathematical formulas, were assessed at baseline and at 12 months.After 12 months of treatment, the homeostasis model assessment index of insulin resistance increased significantly in both groups. The fasting glucose-to-insulin ratio and predicted insulin sensitivity index decreased in group B. The delta of the area under the oral glucose tolerance testcurve for insulin and predicted first and second phase insulin secretion indices increased significantly only in group B(Mastorakoset al., 2004).

Another study investigated 296 women who used COCA's and 95 who not use pills to detect if there was a difference between the effect of COCA's on insulin by using intravenous glucose tolerance test, insulin and c-peptide concentration. Four estrogen progestin combination, with similar estrogen but different progestin content, and one POCs were studied. Effect on intravenous glucose test glucose, insulin and C- peptide concentrations varied according to progestin content, with levonorgestrel- containing combination having the greatest effect followed by desogestrel and norethindrone. These formulation increased insulin resistance to a similar extent. The POCs did not affect insulin resistance. Levonorgestrel combinations increased second phase pancreatic insulin secretion by 60-90%, but did not affect the insulin half-life. The desogestrel combination increased the insulin half-life by 28%, but did not affect insulin secretion. The effects of different COCA's on glucose tolerance test glucose, insulin, and C-peptide concentration profiles appears to be due to a combination of estrogen-induced insulin resistance and progestin-associated changes in insulin half- life (Sitruket al., 2011).

2.6.1.2.Effect of COCA's on protein metabolism

Brandle and his colleagues evaluated the effect of COCA's on kidney function as well as protein metabolism, 28 healthy women not taking contraceptives and 46 healthy women (aged 20–28 y) on one of three different types of OCA's (combination preparations) were investigated [Minulet / Femovano, Marvelon, Diane]. In all groups the results were, creatinine clearance was significantly increased, the potassium excretion rate was significantly elevated in the groups taking Marvelon and Diane,

19 and the sodium excretion rate was significantly increased in those on Minulet/Femovan and Diane, the albumin excretion rate was numerically but not significantly elevated. On comparing the groups taking contraceptives with the control group, no significant differences were found in the daily oral protein intake or the nitrogen excretion rate. However, the ratio nitrogen excretion rate/daily protein intake was significantly increased in those on Minulet/ Femovan and Diane. The study has shown that besides their various effects on renal tubular function, COCA's were able to increase the glomerular filtration rate, and certain types have a protein catabolic effect (Brandle et al., 1992).

Another study evaluated the effect of COCA's containing 150 micrograms of levonorgestrel and 30 micrograms of Ethinyl estradiol on protein and amino acid metabolism in a group of 34 healthy non-lactating women. Assessments were made prior to, and during the third week of the 4th, 7th, and the 13th cycles of OCA'S treatment. In women, use of COCA's showed significant increases in the fasting concentrations of ceruloplasmin and retinol binding protein by an average of 155% above baseline, while Fasting plasma concentrations of sex hormone binding globulin showed no changes. At the same time, free amino acid concentrations were depressed in OCA's users by 13-33%, with the largest decreases occurring for tyrosine, glycine, ornithine, and proline. Finally, mean concentrations of alpha-1 and beta-globulins increased in COCA's users, while albumin and total protein decreased significantly. A significant reduction of several plasma amino acids and plasma albumin concentrations was observed, suggesting that the increase in the visceral and other types of protein concentrations previously noted was due to increased hepatic protein synthesis rather than increased breakdown or excretion as result of OCA'S intake. Biochemical alterations and metabolic effects may well be related to altered synthesis and release of proteins and/or protein binders as well as induction of certain metabolic enzymes from the liver (Amatayakul et al., 1994).

Wang and his colleagues investigated 5841 women (age range 24-49 years) divided into 2 population, first one women using COCA's or POCs were compared with those who did not use hormonal contraception. In this longitudinal study, comprehensive molecular profile (75 metabolic measures and 37 cytokines) was measured. Metaboloic profiles were reassessed for 869 women after 6 years to

20 uncover the metabolic effects of starting, stopping and persistently using hormonal contraception. There were multiple new findings on the metabolic associations with the use of COCA's. They were positively associated increased lipoprotein subclasses, including all HDL-c subclasses, also increase TG and with TC. But, the associations with fatty acids and amino acids were strong and variable in direction. COCA's use was negatively associated with albumin and positively associated with creatinine and inflammatory markers, including glycoprotein acetyls and several growth factors and interleukins. POCs were only weakly associated with metabolic and inflammatory markers. In contrast, the changes were maintained in consistent users and normalized in those who stopped using. After these results, suggesting the use of COCA's causes widespread metabolic and inflammatory effects. These changes were not permanent, they arereversed after discontinuation (Wang et al., 2016).

2.6.2. Effect of COCA's on micronutrients (vitamin &mineral) 2.6.2.1.Minerals

Iron

Iron is a mineral found in every cell of the body. A Total average of body iron about 4 gram in men and a little more than 2 grams in women, the difference relates to women ̍s smaller body size and decreased stores iron because of iron loss due to menses(Simon et al., 2016). Body iron can be considered as two main component namely functional (70% of total body iron) and stored iron. More than two thirds of functional body iron is found in Red Blood Cells (RBCs) called Hemoglobin (Hb) and small quantity in body tissue, in muscle cells (myoglobin) and various heme and non- heme enzyme like cytochrome. Stored iron occur in two forms namely ferritin and hemosiderin, both of them do not have any physiological function other than to serve as reservoir from which loses from functional component can be replaced. Ferritin is a protein binds to iron stored mainly in liver, spleen and bone marrow. There is a small amount of ferritin found in the blood, this amount will reflect how much iron is stored in the body. The body regulate its iron status by balancing absorption, transport, storage and excretion (Insel et al., 2010).

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Zinc

Zinc is an essential mineral that is required for the activity of > 300 enzymes (McCall et al., 2000). Zinc is involved in numerous aspects of cellular metabolism. It plays role in immune function, protein synthesis, wound healing, DNA synthesis, cell division (structural function). It is important for health of our skin, teeth, bones, hair, nails, muscle, nerve, and brain function(Deshpande et al., 2013). Zinc also supports normal growth and development during pregnancy, childhood, adolescences and decrease mortality in developing countries. In spite of the proven benefits of adequate Zinc nutrition, approximately two billion people still remain at risk of Zinc deficiency (Bhowmik & Chiranjib, 2010).

Zinc homeostasis is primarily maintained via the gastrointestinal system by the processes of absorption of exogenous Zinc and gastrointestinal secretion and excretion of endogenous Zinc. The body has no Zinc stores so that absorption depend on person's Zinc status, if more is needed more is absorbed. In the intestinal cell, Zinc has two directions, involved in the metabolic function in these cells or retained within the cell by metallothionein which are a special binding protein similar to iron storage protein, mucosal ferritin (Whitney & Rolfes, 2007). Zinc is lost from the body through kidney, menstrual flow, semen, and sloughed skin, nails, and hair, although quantitatively these other routes of Zinc loss are relatively small compared with gastrointestinal excretion (Prasad, 2013). There are many factors that influence Zinc absorption, diet composition like Zinc in liquid will be more absorbed than Zinc in solid diet. Although presence of dietary inhibitors, for example phytates bind Zinc, thus limiting its absorption and possibly Iron.Large amounts of supplemental Iron (greater than 30 mg) might decrease Zinc absorption and calcium when consumed as supplements (Mangels et al., 2011).

Also, there are some factors that affect Zinc status mainly Zinc plasma levels like infection, stress, fever, food intake and pregnancy which decrease its level (all of which increase the demand for absorbed Zinc) but other factors like long term fasting increase it (Maret, 2013). Zinc is a natural element essential to life found in all plants and animals and plays a crucial part in the health mainly in developing countries, for maintaining healthy growth of the human body especially for infants and young children's growth and development. In addition, therapeutic Zinc supplementation

22 during diarrheal episodes reduces the duration and severity of the illness (Wessells & Brown, 2012). The human body contain about 1.5-3 g of Zinc and is found in all organs, tissues (especially muscle and bone) and body fluids. It is almost universally found as the divalent zinc. Lean red meat, whole-grain cereals, pulses, and legumes provide the highest concentrations of Zinc. Processed cereals with low extraction rates, polished rice, and lean meat or meat with high fat content have a moderate Zinc content. Fish, roots and tubers, green leafy vegetables, and fruits are only modest sources of Zinc. Separated fats and oils, sugar, and alcohol have a very low Zinc content (Gropper et al., 2016). Clinical consequences of Zinc deficiency are growth delay, low birth weight, delayed sexual development, Impaired learning, loss of smell and taste sensation, loss of appetite, diarrhea, pneumonia, disturbed,neuropsychological performance and abnormalities of fetal, development, impaired wound healing, immune deficiency, some hair and joint conditions, cataract, dermatitis and others.

Copper

Copper is an essential trace mineral present in all body tissue required by human beings to support the biological processes of life. The adult human body contains approximately 100-120 mg of Copper, which is distributed in muscle tissue, internal organs, blood, bones and hair and nail(Kaneko et al., 2008). The absorption efficiency ranges from 10%-50% and is variable depending on both requirement and bioavailability of the diet. The majority of absorption occur in the duodenum via carrier-mediated mechanism. It is fairly rapidly absorbed usually within 15 minutes. Copper is transferred by albumin across the gut wall and carried to the liver, where it is formed into ceruloplasmin, a copper-protein complex. About 90 percent of the average 100 mcg of Copper in the blood is in the form of ceruloplasmin. As a balancing mechanism to minimize Copper toxicities, absorption of Copper is decreased when ceruloplasmin levels are adequate. Most Copper in the tissue is attached to intracellular metallothionein which are proteins bind metals especially Zinc, Copper, cadmium and mercury (Broadley et al., 2012).Aging probably decreases the efficiency of Copper homeostasis. There are some factors that affect Copper absorption like supplementation with minerals that have similar chemical characteristics will reduce Copper absorption. Fiber content of diet may have an

23 indirect effect on Copper bioavailability by altering the bioavailability of mineral antagonists. Protein, soluble carbohydrate and ascorbic acid are likely to have a positive effect on Copper absorption (Broadley et al., 2012). The major function of Copper is a catalytic center in numerous enzymes involved in redox reactions. Copper deficiency is mostly associated with prolonged diarrhea (Copper like Zinc is depleted by acute diarrhea) and or malnutrition particularly in infants. Menke's disease (kinky hair syndrome) is a very rare congenital disorder of Copper absorption that is present before birth. It occurs in male infants. The increased Copper in these tissues leads to hepatitis, kidney problems, brain disorders, and other problems. Oysters and other shellfish whole grains, beans, nuts, potatoes, and organ meats (kidneys, liver) are good sources of Copper. Dark leafy greens, dried fruits such as prunes, cocoa, black pepper, and yeast are also sources of Copper in the diet (Geissler & Powers, 2017).

Copper is found in many enzymes; most important is the cytoplasmic superoxide dismutase, an enzyme that is involved in antioxidant reaction. Copper enzymes play a role in oxygen-free radical metabolism, and in this way have a mild anti-inflammatory effect. Copper also functions in certain amino acid conversions. Being essential in the synthesis of phospholipids(Adlard and Chung, 2016), Copper contributes to the integrity of the myelin sheaths covering nerves(Rao, 2010). It also aids the conversion of tyrosine to the pigment melanin, which gives hair and skin their coloring. Copper is important as a catalyst in the formation of Hb, our oxygen- carrying molecule. Copper in the RBCs is bound to erythrocuprein, a substance thought to have superoxide dismutase activity, which is energy enhancing (Insel et al., 2004).

Iron, Copper, and Zinc fulfill various essential biological functions and are vital for all living organisms. They play important roles in oxygen transport, cell growth and differentiation, neurotransmitter synthesis, myelination, and synaptic transmission. The interaction between these minerals absorption may be explained by competitive binding to the transporter protein DMT-1, which participates in divalent metal transport (Iron, Copper, and Zinc) (Broadley et al., 2012). DMT-1 is the main Fe2+ transporter, that it participated actively in copper transport (Arredondo et al., 2003), and this transport could be regulated by both Iron and Copper (Arredondo et al., 2004). Long exposure to Iron leads to a down-regulation of DMT-1 expression

24 that subsequently will produce a decrease in Copper and Zinc absorption (Troost et al., 2003). Furthermore, recent findings suggest that a shared absorption pathway for Iron and Zinc is distinct from DMT-1, although the actual absorption mechanism remains to be elucidated (Yamaji et al., 2001). These results confirm that DMT-1 is involved in active transport of Iron, Copper, and Zinc although Zinc showed a different relative capacity (Espinoza et al., 2012). The dietary proportions of Zinc, Copper, and Iron appear to influence Zinc, Copper, and Iron metabolism at the intestinal and cellular transport levels over a given period of time (Broadley et al., 2012).

There has been a growing awareness of possible alterations in the trace element profiles of hormonal contraceptive users and their consequences. Kamp and his colleagues was evaluate the effect of COCA'S use on several zinc, iron and copper biochemical indices, and on their response to zinc supplementation. Serum and urinary zinc, erythrocyte zinc and metallothionein, plasma alkaline phosphatase, serum iron, total iron-binding capacity (TIBC), transferrin saturation, serum ferritin, serum copper, plasma ceruloplasmin, erythrocyte copper and Cu–Zn superoxide dismutase (SOD), and serum Fe/Zn and Cu/Zn and erythrocyte Cu/Zn were compared between women users (+OC, n = 19) and non-users (−OC, n = 17) of OC, before and after 8 weeks of zinc supplementation (22 mg/d). Results presented lower serum and urinary zinc (P < 0.005) but higher serum iron, TIBC, ferritin (P ≤ 0.005), copper and ceruloplasmin (P < 0.001), SOD (P < 0.05), and serum mineral molar ratios (P < 0.0001). Zinc supplementation did not affect iron indices but it increased serum and urinary zinc (P < 0.05) only in −OC. In +OC, zinc supplementation decreased erythrocyte zinc (P = 0.05) but increased serum copper, ceruloplasmin, and serum Cu/Zn ratio (P < 0.05).They concluded that, OC use influenced zinc, iron and copper homeostasis. Zinc supplementation further altered copper utilization in OC users, possibly favoring oxidative stress (Kamp et al., 2011).

Increased iron stores are associated with greater cardiovascular risk in postmenopausal women. Oral contraceptive pill (OCP) use decreases the volume of menstrual blood loss and increases iron stores, but the link between OCP use, iron stores and cardiovascular risk in premenopausal women has not been characterized.Friedman and his colleaguesconducted a cross-sectional study of 23

25 healthy COCA's users to determine the association between type and duration of COCA's exposure, iron stores, and vascular endothelial function [flow-mediated dilation in the brachial artery].Median duration of COCA's use was 45 months. Flow- mediated dilation in the brachial artery was significantly associated with progestin type used (estranes/gonanes vs. ) and duration of COCA's use (both p<.05) but not iron stores. In multivariate analysis, progestin type was the only independent predictor of flow-mediated dilation. The use of COCA's containing drospirenone was independently associated with greater Flow-mediated dilation in the brachial artery and, thus, a potentially more favorable cardiovascular risk profile, when compared with use of COCA's containing estranes/gonanes(Friedman et al., 2011).

Fallah and her colleague studied the influence of OCA's uptake on serum Zinc and selenium in OCA's users. The concentration of Zinc and selenium was determined by atomic absorption spectrophotometer in 50 healthy women with normal menstrual cycles as a control group and 50 women taking low-dose OCA's. The control reference values were 81.61±9.44 and 70.35±25.57 mcg/dL, which were obtained for Zinc and selenium, respectively. Use of COCA's resulted in a significant decrease in serum Zinc levels (p≤.009, t=−3.666) and alteration of selenium levels but not significant (p=.08, t=0.935). The duration of use beyond 3 months had no effect on the magnitude of the decrease in serum Zinc levels. These findings may be important because selenium is currently believed to offer protective benefits against carcinogenesis. It has been thought that the decrease in serum Zinc could be reflected in a reduction of tissue Zinc status due to changes in Zinc absorption, excretion or tissue turnover. If these changes occur, the dietary Zinc requirement would be greater in women using COCA's (Fallah et al., 2009).

Another study was conducted on Babylon maternity and pediatric hospital in Hilla city where sixty three healthy women obtained as sample, Thirty women were using Microgynon R (low dose COCA's, each pill containing levonorgestrel 0.15 mg and Ethinyl estradiol 0.03 mg) and thirty three women used Copper T 380A IUDs . Thirty apparently healthy women were taken as control group. Blood samples obtained from women used contraceptives and control group were used to determine the effect of OCA's and IUDs on of Malondialdehyde (MDA), TC, HDL-c, TG,

26

VLDL-c, LDL-c and some trace elements (Copper, Zinc and Iron) concentration. The results of the study showed (significant increase in MDA, Copper, Iron, TC, TGs, VLDL-c and LDL-c concentration), significant decrease in HDL-c and non- significant decrease in Zinc concentration in sera of women who used COCA's when compared to both women who used IUDs and the control group. In addition, this study showed non-significant difference in MDA, Copper, Zinc, TC, HDL-c, TGs, VLDL-c and LDL-c concentration and significant decrease in Iron concentration in sera of women who used IUDs when compared to those of the control group (Al- Gazally et al., 2010).

2.6.2.2.Vitamins

Widespread use of COCA's by a large proportion of women led to much research conducted to investigate the biochemical and metabolic alterations that occur in women who took these agents mainly change in blood levels of some vitamins namely B12, B6 and folic acid. The alterations reported in blood levels of these vitamins are generally believed to be related in large measure to alterations in levels of homocysteine. The significance of these changes have a greater incidence of atherosclerosis and CVD in COCA's users in comparison to women COCA's non- users of the same age(Fallah et al., 2012). In terms of nutrition, several studies investigated whether women need different amounts of some vitamins during COCA's usage.

Vitamin B12

Several studies have found low mean serum vitamin B12 levels in women using COCA's, as compared to non-users. Wilson and his colleaguesrevealed that there was no significant effect on serum vitamin B12 observed as a result of COCA's use (Wilson et al., 2011). Another study was designed to evaluate the influence COCA's of on serum B12 level (Shojania& Wylie, 1979). This study investigated serum vitamin B12 levels in 199 women who were on a regimen of OCA's of either the combination or sequential type and in a control group of 196 women. Serum levels of vitamin B12 in the group using COCA's was significantly lower as compared to those of the control group. A total of 19 women using COCA's had serum vitamin B12 levels that were lower than normal values. However, the Schilling test and

27 urinary methylmalonate excretion in this group were normal. The COCA's group had a significantly lower total serum vitamin B12 binding capacity, a lower total transcobalamin I level, and a higher transcobalamin III level. The study suggests that the fall of serum vitamin B12 in COCA's users is due to the changes in vitamin B12 binders of serum and does not represent vitamin B12 deficiency. So that, there is no justification for vitamin B12 supplementation in users of COCA's.Lussana and her colleague indicated that vitamin B12 was significantly lower in COCA's users than in non-users (Lussana et al., 2003).

Recent studies showed a controversy on their subjects. One study indicated that, functional indicators of vitamin B12 status were not significantly impacted by COCA's use (Wilson et al., 2011). While another one concluded that, the use of COCA's among young women resulted in a significantly lower serum vitamin B12 concentrations (McArthur et al., 2013).

Folic acid

There is a growing evidence that oral contraceptives interfere with and impair the body's metabolism of folic acid, or folate. Case control study was conducted to investigate folate status among 71 healthy female nulligravidae using OCAs (low dose) for ≥3 months and 170 controls. In this study, factors that interfere with vitamin metabolism were thoroughly controlled. Results of this study indicated that folate levels did not differ between the groups (Sutterlin et al., 2003).

Another cross-sectional study investigated the impact of COCA's vs. not users on serum levels of folate, 219 healthy women were included in the study, 159 of them had not been using COCA's for at least 12 months prior to the study, while 60 were on regular COCA's. The result of this study indicated that, the median levels of folate was significantly lower in COCA's users than in non-users (Lussana et al., 2003). Wilson and his colleague studied the potential impact of using COCA's on folate status when the estrogen content of COCA's was much higher. Results did not support a conclusion that used COCA's negatively impact folate status (Wilson et al., 2011).

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Vitamin B6

Vitamin B6 status across studies were different among users of COCA's. Lussana and her colleague, 2003 studied the impact of using COCA's on vitamin B6. Results revealed that, vitamin B6 level was significantly lowered about 24.2 vs. 32.9 nmol/l in the user's group compared to non-users. Since low vitamin B6 levels are independently associated with heightened risks for arterial and venous thromboembolism, they could partly account for the increased thromboembolism risk of COCA's users(Lussana et al., 2003).

Wilson and his colleague also investigated the effect of COCA's on vitamin B6, when the estrogen content of COCA's was much higher. In regard to vitamin B6. However, existing population-based data do provide evidence that current low-dose

OCA's may negatively impact vitamin B6 status (Wilson et al., 2011). Serum level of pyridoxal 5′-phosphate concentrations in COCA's users was depressed which is reflected as decreased body reserves of the vitamin, so that women who discontinue COCA's and become pregnant at risk for vitamin B6 inadequacy.

2.7.Effect of COCA's on homocysteine

Homocysteine is a non-protein α-amino acid. A high level of homocysteine in the blood makes a person more prone to endothelial cell injury, which leads to inflammation in blood vessels, which in turn may lead to atherogenesis that can result in ischemic injury (Gallino et al., 2014). We aimed to assess the effect of COCA's on homocysteine level through literature.

One study evaluated the effect of third-generation COCA's on homocysteine level in a population of young, fertile, non-obese women, 277 healthy white women were included in the study, 77 users of COCA's were compared with 200 non-users. The results indicated that, the use of COCA's did not affect homocysteine levels (Cauci et al., 2008).

Another study investigated the effect of COCA's on homocysteine level among young healthy women, this observational cross-sectional analysis conducted on 90 healthy, non-obese women. Forty-five healthy women on COCA's and 45 healthy controls were studied for homocysteine level. The results showed that the

29 homocysteine (13.268±3.475 vs. 7.288±2.621 μmol/L) level were significantly higher in women receiving COCA's in comparison with the control group (Norouzi et al., 2011). This study confirmedthat the alteration in homocysteine level could be attributed to the COCA's, suggesting that use of these agents should be reviewed in women with increased risk of atherosclerosis and other cardiovascular risk factors. Tanis and his colleagues tried in their study to assess the association between myocardial infarction and elevated level of homocysteine, low folate or methylenetetrahydrofolatereductasegenotypes (Tanis et al., 2004).

2.8.Effect of COCA's on lipid profile

Many researchers have suggested that the use of contraceptives is beneficial but also have some side effects too. The researchers believe that the widespread use of OCAs provides an opportunity for assessing the influence of and progesterone on lipid profile among users.

A cross sectional study was conducted from January 2011 to December 2011 in FP Department to find out the effect of COCA's on lipid profile in females of reproductive age, A total of 200 married fertile women of child bearing age (14-49 years) participated in the study. They were divided in two groups: Group 1 (COCA's users at least for six months) and group 2 (age matched controls not using COCA's). Fasting levels of serum TC, TG, HDL-c, LDL-c and Very Low Density Lipoprotein Cholesterol (VLDL-c) were analyzed. Results; comparing females of group-1 vs. group-2, there was significant increase of TC (185+3.27mg/dl vs. 158.26+2.81mg/dL; p=0.0001), TG (207.33+4.92mg/dl vs. 135.63+4.49mg/dL; p=0.0001), LDL-c (98.20+ 3.11mg/dl vs. 85.19+2.65mg/dl; p= 0.002) (Mohammad et al., 2013).

Emokpae and his colleguestudied the effect of COCA's duration use on lipid and lipoprotein in Nigerian women. The study group consisted of 120 women (mean age 24±5years) who were on biphasic lofeminal tablets for a period ranging from 1-48 months. Fifty age of women have regular menstruation with no history of hormonal use within the last 6 months before the investigation were used as controls. Statistical significant increase were observed in TG, LDL-c, VLDL-c with the duration of oral contraceptive use, this study suggested that, hormonal contraceptive induced dyslipidemia may not be regarded as proatherogenic, COCA's may not necessarily

30 lead to pathologic concentration of lipid within four years duration of use in Nigerian women (Emokpae et al., 2010).

2.9.Functions, Dietary sources, and the Recommended Daily Allowance (RDA).

2.9.1. Minerals

Iron

Iron is a trace mineral that is essential for making Hemoglobin (Hb), a protein in red blood cells which carries oxygen to other cells, it is an important component of myoglobin; it helps supply oxygen to the muscles, collagen; proteins in bone, cartilage, and other connective tissues, because of that iron bioavailability is a critical point in pregnancy (Abbaspour et al., 2014). Iron also plays an important role in specific processes within the cell that produce the energy for the body (Abbaspour et al., 2014). It is for this reason that one of the first symptoms of low body iron stores is tiredness and fatigue (Abbaspour et al., 2014). Iron is required for the production of red blood cells known as haematopoiesis, but it is also part of hemoglobin that is the pigment of the red blood cells binding to the oxygen and thus facilitating its transport from the lungs via the arteries to all cells throughout the body (Nagababu et al., 2008). Iron is also involved in the conversion of blood sugar to energy (Lasocki et al., 2014). The immune system is dependent on iron for its normal functioning (Cherayil, 2011). The production of enzymes which plays a vital role in the production of new cells, amino acids, hormones and neurotransmitters also depends on iron (Abbaspour et al., 2014).

The best sources of Iron are Chicken liver, Oysters, Beef, Kidney Beans, Sardines, Turkey, Lentils, Spinach, White Rice, Tomatoes, Chickpeas and Potatoes. Table 2.1 shows the RDA of Iron needs according to American institute of medicine (Food &Nutritional Board, 2001).

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Age Female Pregnancy Lactation

Birth to 6 0.27 mg months

7–12 months 11 mg

1–3 years 7 mg

4–8 years 10 mg

9–13 years 8 mg

14–18 years 15 mg 27 mg 10 mg

19–50 years 18 mg 27 mg 9 mg

51+ years 8 mg

Food & Nutritional Board ( FNB,2001)

Copper

Copper is an essential trace element that is vital to the health of all living things. In humans, copper is essential to the proper functioning of organs and metabolic processes (Collins et al., 2010). Copper plays a vital role in the formation of red blood cells with iron (Collins et al., 2010; Jain & Williams, 1988), production of collagen which is responsible for the health of bones, cartilage and skin (Harris et al., 1980). Furthermore, it is one of the antioxidant minerals, which protects against free radical damage (Lobo et al., 2010). Moreover, Copper is necessary for the production of adrenal hormones (Gaetke et al., 2014) helps in the absorption of iron (Collins et al., 2010), and maintains nerve fibers and it is essential for the utilization of vitamin C (Telang, 2013).

Copper is highly concentrated in Beef liver, Sunflower seeds, Lentils, Almonds, Dried apricots, Dark chocolate, Asparagus, Mushrooms and Turnip greens. The RDA of copper according to American Institute of Medicine is 900 mcg/day. The Daily Value is 2 mg(Micronutrients et al., 2002).

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Zinc

Zinc is an essential mineral that is taken as a natural over-the-counter remedy for fighting colds and symptoms of illnesses and increases immunity (Maggini et al., 2012). Zinc is an effective anti-inflammatory and antioxidant agent, helping fight oxidative stress and decreases the chance for disease development, including a natural cancer treatment(Prasad, 2014). Moreover, Zinc have beneficial role in hormonal health and fertility because it plays a vital role in hormone production, including increasing testosterone naturally, which has very widespread roles in both men and women (Dissanayake et al., 2009; Favier, 1992). Furthermore, Zinc is needed to balance most hormones, including insulin, the main hormone involved in the regulation of blood sugar and as a diabetes natural cure(Wilcox, 2005). Also, Zinc is needed to maintain the health of cells within the cardiovascular system (Plum et al., 2010), in addition to reducing inflammation and oxidative stress (Marreiro et al., 2017).

Zinc is highly concentrated in Lamb, Pumpkin Seeds, Grass-Fed Beef, Chickpeas, Cocoa Powder, Cashews, Yogurt, Mushrooms, Spinach and Chicken. Table 2.2 shows the RDA of Zinc needs according to American Institute of Medicine (Food &Nutrtional Board, 2001).

Age Female Pregnancy Lactation

0–6 months 2 mg

7–12 months 3 mg

1–3 years 3 mg

4–8 years 5 mg

9–13 years 8 mg

14–18 years 9 mg 12 mg 13 mg

19+ years 8 mg 11 mg 12 g

Food & Nutritional Board ( FNB,2001)

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2.9.2.Vitamins

Folic acid

Folate, forms of folic acid and vitamin B9, is one of the B vitamins(Czeizel et al., 2013). Vitamin B9 plays a vital role for several bodily functions as synthesis and repair of DNA and RNA (Cantarella et al., 2017), aiding rapid cell division and growth, to produce healthy red blood cells (Greenberg et al., 2017), enhances brain health, improve memory, slow the onset of age-related hearing loss (Lasisi et al., 2010), and promote heart health(Bazzano, 2009). It is particularly important for pregnant women to have enough folic acid to prevent major birth defects of her baby's brain or spine (neural tube defects, including spina bifida and anencephaly) (Green, 2002). Women planning to get pregnant should take a folic acid supplement for a full year before conception (Bixenstine et al., 2015).

Vitamin B9 is highly concentrated in Dark Leafy Greens, Asparagus, Broccoli, Citrus Fruits, Beans, Peas, and Lentils, Avocado, Okra, Brussels Sprout, Seeds and Nuts and Carrots. Table 2.3 shows the RDA of folate needs according to American Institute of Medicine (Food &Nutrtional Board, 2001).

Age Female Pregnant Lactating

Birth to 6 months 65 mcg DFE

7–12 months 80 mcg DFE

1–3 years 150 mcg DFE

4–8 years 200 mcg DFE

9–13 years 300 mcg DFE

14–18 years 400 mcg DFE 600 mcg DFE 500 mcg DFE

19+ years 400 mcg DFE 600 mcg DFE 500 mcg DFE

Food & Nutritional Board ( FNB,2001)

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Vitamin B6

Vitamin B6, also called pyridoxine, is a water-soluble nutrient that is part of vitamin B group of essential nutrients. Its active form, pyridoxal 5′-phosphate, serves as a coenzyme in some 100 enzyme reactions in amino acid, glucose, and lipid metabolism (Jabeen et al., 2017). Vitamin B6 helps in the production of neurotransmitters, the chemicals that allow brain and nerve cells to communicate with one another (Kennedy, 2016), ensuring that metabolic processes such as fat and protein metabolism run smoothly (Pregnolato et al., 1993), and is important for immune system function in older individuals (Dai & Koh, 2015). Furthermore, it can help to address a number of conditions, including nerve compression injuries like carpal tunnel syndrome (Ryan-Harshman & Aldoori, 2007), premenstrual syndrome (Ebrahimi et al., 2012), and is often used to treat high homocysteine levels along with folic acid and vitamin B12 (Coppen & Bolander-Gouaille, 2005).

Vitamin B6 is concerted in Turkey Breast, Grass-Fed Beef, Pistachio Nuts, Tuna, Avocado, Chicken Breast, Sunflower Seeds, Sesame Seeds and Chickpeas. Table 2.4 shows the RDA of Vitamin B6 needs according to American Institute of Medicine.

Table 2.4: Recommended Daily Allowances for Vitamin B6

Age Male Female Pregnancy Lactation

Birth to 6 months 0.1 mg 0.1 mg

7–12 months 0.3 mg 0.3 mg

1–3 years 0.5 mg 0.5 mg

4–8 years 0.6 mg 0.6 mg

9–13 years 1.0 mg 1.0 mg

14–18 years 1.3 mg 1.2 mg 1.9 mg 2.0 mg

19–50 years 1.3 mg 1.3 mg 1.9 mg 2.0 mg

51+ years 1.7 mg 1.5 mg

Food & Nutritional Board ( FNB,2001)

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Vitamin B12

Vitamin B12, also called cobalamin, is a water-soluble vitamin that can dissolve in water and travel through the bloodstream (Brescoll & Daveluy, 2015). Any excess or unwanted vitamin B12 is excreted in the urine. Vitamin B12 plays a key role in converting carbohydrates into glucose, which is used to produce energy and helps the body use fats and protein (Kennedy, 2016). Also, heart and entire cardiovascular system needs B12 to remove a dangerous protein called homocysteine from the blood that can damage arteries leading to inflammation and heart disease (Lars et al., 1988; OLeary & Samman, 2010). Moreover, it has a vital role in the normal functioning of the brain and nervous system via the synthesis of myelin(Black, 2008). Vitamin B12 works closely with vitamin B9, also called folate or folic acid, to help make red blood cells and to help iron work better in the body (Remacha et al., 2015).

The best sources of Vitamin B12 include eggs, milk, cheese, milk products, meat, fish, shellfish and poultry. Some soy and rice beverages as well as soy based meat substitutes are fortified with vitamin B12. Table 2.5 shows the RDA of Vitamin B12 needs according to American Institute of Medicine.

Age Female Pregnancy Lactation

0–6 months 0.4 mcg

7–12 months 0.5 mcg

1–3 years 0.9 mcg

4–8 years 1.2 mcg

9–13 years 1.8 mcg

14+ years 2.4 mcg 2.6 mcg 2.8 mcg

Food & Nutritional Board ( FNB,2001)

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CHAPTER 3

RESEARCH METHODOLOGY

3. Hkjjh 3.1.Study Design

This is a case control study designed to investigate the association between use of COCA's and adverse nutritional outcomes. Ninety apparently healthy married women, visiting and receiving care from Family Planning Departments and user of at least one method of contraception were recruited in the study and later they were categorized into users of combined oral contraceptive agents (case group) and other-users (control group) which include other contraceptive methods.

This design has its own advantages that we can prove and/or disprove assumptions, many findings and outcomes can be analyzed to create new theories/studies or in- depth research. It can answer questions and will generate hypothesis for future research, and data useful to many different researchers.

3.2.Study Sitting

The current study was designed to investigate the effect of using COCA's on selected vitamins and minerals in women of reproductive age. The study was conducted at the primary health care clinicsof the Ministry of Health in Rafah Governorate, Gaza Strip, Palestine after obtaining the approvals from the ethical and scientific committees.

The Gaza strip goes along with the midterrain sea between occupied land in year 1948 and Egypt. It is about 360 square kilometers. Its length from Rafah in the south to Beit- Hanoon in the north measures 50 kilometer long and 5-12 kilometer wide. The Gaza Strip administratively divided into five governorates, north, Gaza, Middle zone, Khanyounis and Rafah. It has four towns, fourteen villages and eight refugee camps (Cerone, 2012).

The estimated midyear population size of Palestinian in the year 2016 is about 4.884,336. In the Gaza Strip the population size is estimated at, 1,912,267 and constitutes about 39.15 % of the total population in Palestine in Gaza Strip and west bank. In the Gaza Strip, 42.7% of the total population are under age of 15 years (PCBS, 2016). Regarding population density in Gaza strip, it is about 3,808

37 inhabitants per one square kilometer.Palestine is classified as lower-middle income country. The economy of it is mainly dependent upon agriculture, small industries and employment with the local national authority. The instability of the Palestinian economy is mainly due to the unstable political situations that are related to Israeli acts. Demographic changes have led to increased demand for housing and resulted in high priced housing (Saleh 2018).

3.3.Study Population

All women at childbearing age, who were visiting and receiving care from the Family Planning Department at primary health clinic in Rafah Governorate

3.4.Sample Size calculation

The sample size was calculated by using power and sample size software (Appendix C). Where α is the Type I error probability for a two sided test based on 95 % confidence interval. Power = 80%. P1 is the possibilities of having B12 deficiency among oral contraceptive users = 40% (Wertalik., etal 1972). P0 is the possibility of exposure in the control group = 14%. The calculated sample size according to PS software is 45 cases in each group.

The total number of cases in each group = 45+10% (Non response rate).

n= 50 cases in each group.

Due to the limited resources, the sample size was reduced to 45 cases in each group.

3.5.Eligibility Criteria

The selection of the cases was done in accordance with eligibility criteria after applying the inclusion and exclusion criteria.

Inclusion criteria

 Visitors and recipients of care at Family Planning Departments(Ministry of Health in Rafah Governorate).  Healthy women at reproductive age 18-35 years.

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 Users of at least one method of family planning (OCA's, intrauterine device (IUD or coil), condoms, or Mechanical (withdrawal method) for at least 12 - 24 months.  Agree to sign the consent form.

Exclusion criteria

 Pregnant or lactating mothers.  Having serious medical illness (Renal, heart failure), digestive tract disorders (celiac disease, Crohn's disease, ulcerative colitis, chronic pancreatitis, or cystic fibrosis) and chronic disease (diabetes, hypertension, etc…).  Recipients of any nutritional supplements mainly multivitamins and minerals supplementations.  Taking hormonal , other than OCA's.  Smoking women (cigarettes or Shisha).

3.6.Pilot study

The pilot study was conducted before the intended study. The purpose of the pilot study was to test the research process and protocol, to develop or test the efficacy of research instruments, to maintain maximum objectivity and reduce observer drift, to check reliability and validity of the questionnaire and the other tools as well as to evaluate the possible outcomes. The piloting represented more than 10% of the main study sample (10 cases). We modified the final version according to the feedbacks from respondents. The cases participated in the pilot study were not included in the major study.

3.7.Sampling Frame

The records of the Family Planning Department at the primary Health Care Center of Ministry of Health at Rafah Governorate were our sampling frame. Refuse to sign the consent form.

3.8.Sampling design

After applying the eligibility criteria, systematic sampling design was employed. The number of visitors and recipients of care at Family Planning Department at MOH

39 primary health center in Rafah Governorate from October, 2014 to October, 2015 = 1500 cases, and to calculate the interval number = 1500 /90 = 16.6, This would make the interval number = 17. Meaning that every 17th women after the 34th person (the beginning women) would be selected until we had a total of 90 respondents.

3.9. Ethical consideration

All relevant authorities have agreed and approved the study;

1. Approval from Dean of postgraduate studies.

2. Faculty of pharmacy.

3. Approval from general directorate for Human Resource Development at MOH.

4. Approval from Helsinki Committee (Appendix A-1).

5. The interviewed respondents received a complete explanation of the study and signed consent form (Appendix A-2).

3.10. Data collection.

Data collection was conducted from November 2016 to October 2017. The respondents were interviewed using a structured questionnaire to obtain their demographic and socioeconomic information. Then we arranged meetings with the respondents. The interviews lasted 15 to 20 minutes. We used ABCD method for Data collection and nutritional assessment of the subjects through specific and well- designed tools and materials.

3.11. Study program.

This research was carried out on a total of 90 healthy women at reproductive age, who were visiting and receive care from the Family Planning Department and general practitioners at primary health clinic in Rafah Governorate. The subjects were distributed into two groups; the First group consists of 45 women, who used COCA's as a family planning method (case group). The Second group was a control group 45 women, who do not use COCA's and used another family planning method namely condom, mini-pills, intra-uterine device IUD and Depo-Provera injection (control

40 group). Women were selected randomly according to the inclusion and exclusion criteria adopted. This research studied the effect of third generation monophasic COCA's (28 days’ pack), which contains Ethinyl estradiol 0.03mg and 0.15mg levonorgestrel in 21 white tablets, and seven brown tablets contain 75mg ferrous fumarate. After obtained the written informed consent, all parameters and measurements were compared between the groups.

3.12. Materials and methods

The recruited participants who matched the eligibility criteria of the study were randomly assigned into two different groups of the same size (users and non-users group of COCA's). Random assignment was done by using a computer-generated, random-number sequence. All participants were given a written informed consent (Appendix A-2) to participate in the study after explaining the study program.

3.12.1. Questionnaire

Data collection was carried out through pre tested questionnaire (Appendix B-1) to collect all pertinent research information through face-to-face, individual interviews with subjects. The questionnaire was designed and checked for competence and accuracy to collect the following data (Socio-demographic information and Medical nutritional information).

3.12.2. Food Frequency Questionnaire (FFQ)

FFQ (Appendix B-2) has been developed as a favorite method in large studies because it is easy for respondents to complete, can be analyzed by computers, and is inexpensive. The questionnaire consists of a structure items of individual foods and drinks. The FFQ used in this study was derived from National Institute of Health in America, and translated into the Arabic language, reliable and validated by (Tayyem et al., 2013).

FFQ that included 93 items, it is used to collect the qualitative and descriptive information regarding food habits consumed by participants during the past 12 months. The 93 items of food and drinks did not ask the respondents about the portion sizes that they consume. It is worth mentioning that, the items represented the major food groups; cereals and cereal products, meat and meat products, fish, fruits,

41 vegetables, legumes and nuts, milk and dairy products, soups and beverages. The questionnaire was piloted and face validated with a sample of 10 women to evaluate the clarity and readability.

3.12.3. Anthropometric measurements

Anthropometric measurements are used to assess the size, shape and composition of the human body. Anthropometric measurements also have used in epidemiology and medical anthropology, for example in helping to determine the relationship between various body measurements (height, weight, percentage of body fat, etc.) and medical outcomes. Anthropometric measurements are frequently used in the majority of clinical settings.

Body weight

The term human body weight is used in the medical sciences to refer to a human body mass or weight. Strictly speaking, body weight is the measurement of weight without items located on the person. Practically though, body weight may be measured with light clothes on, but without shoes or heavy accessories such as mobile phones and wallets and using manual or digital weighing scales.

Weight was measured by trusted calibrated weighting device. Weight was calculated in kilograms and grams.The weight was taken twice for all the respondents and the average weight was calculated. The weight was recorded to the nearest (the nearest 0.1 kg).

Height

Human height or stature is the distance from the bottom of the feet to the top of the head in a human body, standing erect. In this study, height was measured after removing the subject's footwear and any head covering. The subject was placed on her back in the middle of the board with her arms at the sides and feet at right angles to the board. The heels, knees, buttocks, back of the head and shoulders were touching the board, and respondents looking straight ahead, and we gently bringing the top of the head to touch the fixed end of the board. The height was recorded in meters and centimeter. The height was taken twice for all the respondents and the average height was calculated.

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Body mass index (BMI)

The BMI is defined as the body mass divided by the square of the body height, and is universally expressed in units of kg/m2, resulting from mass in kilograms and height in meters. WHO and government health departments of the major countries have all adopted BMI as the standard way of diagnosing overweight and obesity. BMI is the indicator used to evaluate nutritional status of adult in both normal situations and emergencies. However, there is some debate about where on the BMI scale the dividing lines between categories should be placed. Commonly accepted BMI ranges are underweight below18.5, normal weight from 18.5 to < 25, overweight from25 to 30, obese over 30 as shown as in table 3.1.

Table:3.1 Classification of BMI according to WHO BMI Classification

Below 18.5 Underweight

From 18.5 – to24.99 Normal

From 25.0 – to29.99 Overweight

From 30 to 34.99 Obese Class I (Moderately obese)

From 35 to 39.99 Obese Class II (Severely obese)

Over 40 Obese Class III (Very severely obese)

(WHO, 2016)

Waist circumference

The waist is that part of the abdomen between the rib cage and hips. The waistline refers to the horizontal line where the waist is narrowest, or to the general appearance of the waist. Visceral and central abdominal fat and waist circumference show a strong association with a lot of health conditions like Type 2 Diabetes (Anjana et al., 2004). Waist circumference was measured at the midpoint between the lower margin of the last palpable rib and the top of the iliac crest, using a stretch resistant tape that provides a constant 100g tension (WHO, 2011). The average of three measurements were recorded.

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Table 3.2: The WHO cut-off points and risk of metabolic complications for women (WHO, 2011) Indicator Cut-off points Risk of metabolic complication

Waist circumference >80 cm (W) Increased

Waist circumference >88 cm (W) Substantially increased

Waist–hip ratio ≥0.85 cm (W) Substantially increased

W, women

3.12.4. Blood pressure

Blood pressure was measured by using Sphygmomanometer, taken twice for all the subjects and the average blood pressure was recorded. Blood pressure was measured in a quiet place after removing any tight-sleeved clothing while the subjects were comfortable and relaxed with a recently emptied bladder, rest for five to ten minutes and Sit up straight with her back against the chair, legs uncrossed. Rest your forearm on the table with the palm of your hand facing up.

3.12.5. Biochemical analysis

Venous blood samples were drawn from all participants by using disposable syringes in the sitting position. About 10 ml of blood was obtained from each subject by vein puncture and pushed slowly into plain disposable tubes. About four ml of the blood were collected in a tube without anti-coagulation. Blood was allowed to clot at 37Cºfor 10-15 minutes, and then centrifuged at 3000 rpm for 20 min then the Sera were obtained and stored at -20Cº Until analysis. The measured biochemicals include (Total cholesterol, T.G, Vitamin B12, Vitamin B6, Folate, homocysteine, copper, iron and zinc). The reminder quantity of the blood was placed into EDTA to perform CBC test.

Kits and devices

The device used for chemistry analysis (total cholesterol and Triglyceride) was (biosystem BTS 350,SN 80/751490), and the kits used in this device were for:

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 Determination of serum cholesterol; Serum cholesterol was determined by cholesterol oxidase (CHOD-POD method) using Bio Systems kit, Spain. Reference value according to the recommendation of the European atherosclerosis society: (less than 200mg/dl recommended, 200-299mg/dl upper limit, more than 300 mg/dl high value). Sensitivity, as detection limit 2 mg/dl.  Determination of serum triacylglycerol; Serum triacylglycerol was determined by Glycerol phosphate oxidase/peroxidase (triglyceride GPO- POD. Liquid) using Bio Systems kit, Spain. Reference value according to the recommendation of the European atherosclerosis society: (less than 200mg/dl recommended, 200-299mg/dl upper limit, more than 300 mg/dl high value). Sensitivity, as detection limit 3 mg/dl.  Determination of serum copper; Serum copper was determined by colorimetric method, using Bio Systems kit, Spain. Reference value for women is 80-155µg/dl. Sensitivity, as detection limit 2 µg/dl.  Determination of serum zinc; Serum zinc was determined by colorimetric method, using Bio Systems kit, Spain. Serum reference value was 60- 110µg/dl. Sensitivity, as detection limit 4 µg/dl.  Determination of serum iron; Serum iron was determined by photometric test using ferene. Serum reference value at age 25 years is 37-165µg/dl(6.6- 29.5µmol), and for women at age 40 years is 23-134 µg/dl(4.1-24 µmol). Sensitivity, as detection limit 5 µg/dl(0.9 µmol).

The device used for vitamin B6,B12 and folic analysis was (Mindry Microplate reader, model MR-96A, S.N.WH-IB 102540), and the kit used for:

 Determination of serum B6; Vitamin B6 was determined by using competitive inhibition method (ELISA Kit for Vitamin B6, product number CEA916Ge). Serum reference value 3.70-300ng/ml. Sensitivity, the minimum detection dose of this kit is typically less than 1.42ng/ml.  Determination of serum B12/Folate; The quantitative determination of vitamin B12 and Folate concentration in serum and plasma by a Microplate Enzyme Immunoassay, Colorimetric (Folate /Vit B12 VAST 2 Analytes 1Kit ELISA Microwells ,product code7825-300). Folate Serum reference value for

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normal adult is >3ng/ml and for serum B12 is 200-835 pg/ml (148- 616pmol/L). Sensitivity, for Folate 0.52ng/ml and for Vitamin B12 70.13 pg/ml.  Determination of serum homocysteine; The Axis Homocysteine Enzyme Immunoassay is intended for the quantitative determination of total homocysteine in plasma or serum (using ELISA Kit AX51301, Germany). Homocysteine Serum reference value is from 2.0 to 50.0µmol/L.

The device used for CBC and hematological parameters were measured by ABX.MICROS 60 S.N.104OT90402.

Table 3.3: illustrates the measurement units of biochemical tests Test Measurement units Test Measurement units

WBC x103/mm3 RDW %

RBC x106/mm3 Cholesterol mg/dl

HGB g/dl Triglyceride mg/dl

HCT % Homocysteine Mmol/ml

PLT x103/mm3 Vita B6 ng/l

LYM x103/mm3 Vita B12 pg/ml

Granulocyte x103/mm3 Folic acid ng/l

MCV Mm3 Iron mg/dl

MCH Pg Zinc mg/dl

MCHC g/dl Copper mg/dl

3.13. Data management, treatment and statistical analysis. 3.13.1. Data management and treatment

Following data collection, the questionnaires, anthropometric measurements and biochemical data were reviewed before being entered into the study database. All data

46 were checked for missing or unclear responses following completion of the interview. The data treatment involved cleaning and organizing the data for analysis, which included numbering the questionnaires, codifying the data, entering the data into the computer, checking the data for accuracy, and transforming the data if necessary. Data were entered into an SPSS (Statistical Package for Social Sciences) version 18.0 database for Windows. The level of significance was set at 0.05.

3.13.2. Statistical analysis

The data analysis was divided into two steps: descriptive statistics and analysis of variance. The data is presented in tables. The quantitative data is represented in the form of proportions (%) and of means with standard deviations or medians. All measurements and indicators of the two groups were compared. Mann Whitney test was used for 2 independent nonparametric data, while Pearson Chi Square will be used for categorical data.

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CHAPTER 4

RESULTS

4. This chapter presents the result of the study following statistical analysis. Data analysis was performed using SPSS (statistical package for social sciences) version 18. Data base for windows. Data analysis was organized into two steps: descriptive statistics and analysis of variance.

4.1.Socio-demographic and Socioeconomic

Table (4.1) presents the socio-demographic and socio-economic characteristics of all respondents. The mean age of the respondents was 29.3 years. More than half of respondents (56.7%) lived in the city. out of ninety,42 and 40 respondents had attained secondary and university level of education, respectively. Almost, 83.3% of respondents did not have any type of job. The average of total income was 1485.3 NIS monthly. The majority of respondents 77.8% were under absolute or deep poverty, and 20% were not considered poor.

Table 4.1: Socio-demographic and Socio-economic characteristics of respondents Profile Variables Mean (SD) Frequency Percentage Age 29.3 (4.087) City 51 56.7 Residence Refugee 39 43.3 CampsTotal 90 100.0 Privet 87 94.6 Accommodation type Rental 5 5.4 Total 92 100.0 Never been to School 0 0.00 Primary 0 0.00 Education level Preparatory 8 8.9 Secondary 42 46.7 Graduate 40 44.4 Total 90 100.0 Yes 15 16.7 Employment status No 75 83.3 Total 90 100.0%

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Table 4.1: Continue Profile Variables Mean (SD) Frequency Percentage Government 6 6.7 sectorsUNRWA 6 6.7 Type of employment Private sector 3 3.3 Un employed 75 83.3 Total 90 100.0 Total Income NIS 1485.3(1546.4) Less than 1783 NISa 70 77.8 Family Income NIS Between 1783- 2236.9 NISb 2 2.2 More than 2237 NISc 18 20.0 Total 90 100.0 a Deep or absolute poverty b Under relative poverty c Not poor

Table (4.2) indicates a significant statistical associations between housing area and educational level and COCA's use; χ2=13.077p<0.001 and χ2=12.495p= 0.002, respectively. About 70% of the non-users group had attained a university level of education.

Table 4.2: Socio-demographic and Socio-economic and COCA's use Test P Profile Non- users Users Value value City 34 (66.7) 17 (33.3) Housing area χ2=13.077 <0.001 Refugee camp 11 (28.2) 28 (71.8)

Preparatory 4(50)% 4(50%)

2 Educational Secondary 13(31%) 29(69%) χ =12.495 0.002 level Graduate 28(70%) 12(30%) Employ 11(73.3) 4(26.7) Employment χ2=3.920 0.440 status Not employ 34(45.3) 41(54.7)

<1783 NIS 32(54.7%) 38(54.3)

2 Monthly 1783-2236 NIS 1(50%) 1(50%) χ =2.514 0.279 income >2236 NIS 12(66.7%) 6(33.3%)

χ2: chi-square value. Level of significance P< 0.05.

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4.2. Medical and family history.

Table 4.3 shows that, the majority (98.9%) of respondents did not take any type of medication. In addition, the majority 83.3 %, 92.2%, 66.7% of respondents have no history of surgery, family history of cancer, and family history of DM, respectively. This indicates that almost all respondents had no diseases and no serious family chronic diseases were noticed from the results.

Table 4.3: Medical and family history of respondents Profile Variables Frequency Percentage

History of chronic Yes 0 00.0 illness No 90 100 Total 90 100.0 Take medications 0 00.0 Not take any medications 90 100 Drug history Total 90 100.0 Cesarean Section 10 11.1 Varicose vein 1 1.1

Hemorrhoid 2 2.2 Past history of Subtotal thyroidectomy 1 1.1 surgery C/S + Hernial repair 1 1.1 No past history of surgery 75 83.3 Total 90 100.0 Other health Yes 0 00.0 Diseases No 90 100 Total 90 100.0 Colon cancer 5 5.6 Ovarian 1 1.1 Family history of Liver 1 1.1 cancer No family history of cancer 83 92.2 Total 90 100.0 Family history of Yes 30 33.3 DM No 60 66.7 Total 90 100.0 HTN 27 30 HTN + Hypothyroidism 1 1.1 Family history of Glaucoma 1 1.1 other diseases Asthma 1 1.1 No family history of disease 60 66.7 Total 90 100.0

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Table 4.4 shows that, there are no statistically significant associations between the use of COCA's and all medical and family history profiles, except in family history of cancer χ2=3.873, p =0.039, the majority (6 out of 7) of all cancer cases were from the non-users group.

Table 4.4: Medical and family history and COCA's use Test P Profile Non- users Users Value value Yes 7(46.7) 8(53.3) Past history of χ2=0.080 0.777 surgery No 38(50.7) 37(49.3)

C/S 6(60%) 4(40%) Varicose vain 1(100%) 0(0.0%) Employment 2 Hemorrhoid 0(0.0%) 2(100%) χ =5.357 0.122 area STT 0(0.0%) 1(100%) C/S + Hernia repair 0(0.0%) 1(100%)

Family Yes 6(85.7%) 1(14.3%) history of χ2=3.873 0.039 39(47.0%) 44(53%) cancer No Colon / Rectal 5(100%) 0(0.0%) Type of 2 Ovarian 1(100%) 0(0.0%) χ =7.000 0.057 Cancer Liver 0(0.0%) 1(100%)

Family Yes 4(66.7%) 2(33.3%) history of χ2=0.714 0.394 41(48.8%) 43(51.2%) heart diseases No 19(63.3%) 11(6.7%) Family Yes χ2=3.200 0.720 history of DM No 26(43.3%) 34(56.7%) HTN 19(70.4%) 8(29.6%)

Family HTN+Hypothyroidism 1(100%) 0(0.0%) history of χ2=3.192 0.280 Glaucoma 1(100%) 0(0.0%) other diseases Asthma 0(0.0%) 1(100%)

χ2: chi-square value. Level of significance; p < 0.05. DM= diabetes mellitus, HTN=hypertension, C/S=caesarian section, STT=subtotal thyroidectomy

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4.3. Obstetric and Gynecological history

Table (4.5) presents the Obstetric and Gynecological history of all respondents. About one third of respondents had a history of abortion. The majority 92.2% of respondents practicing breast-feeding for more than 1 year. About two third 60.2% of subjects had past history of FP, and 50% of them use COCA's.

Table 4.5: Obstetric and Gynecological history of respondents Profile Variable Frequency Percentage

Yes 32 35.6 History of abortion No 58 64.4 Total 90 100 Yes 0 0.0 Complication No 90 100 during labor Total 90 100 Yes 89 98.9 Breast feeding No 1 1.1 Total 90 100 <6month 2 2.2 6month-1 year 4 4.4 > 1 year 83 92.2 Duration of breast No breast feeding 1 1.1 feeding Total 90 100 Yes 56 60.2 Past history of FP No 34 27.8 Total 90 100 Condom 23 25.6 IUD 21 23.3 Current FP COCA's 45 50 method Depo-Provera injection 1 1.1 Total 90 100 Yes 0 0.0 Side effect of FP No 90 100 use Total 90 100 Yes 2 2.2 Weight gain with No 88 97.8 use FP Total 90 100 Yes 0 0.0 Compliance to FP No 90 100 Total 90 100 Yes 0 0.0 Change in mood No 90 100 and sex drive Total 90 100 FP= Family planning

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Table (4.6) shows there was no statistical associations between COCA's use and breast feeding and previous use of FP method χ2=6.580 p = 0.012 and χ2=14.690p = 0.033, respectively.

Table 4.6: Obstetric and Gynecological history and COCA's use by respondents Test P Profile Non- users Users value value Yes 18(56.3%) 14(43.8%) History of χ2=0.776 0.378 abortion No 27(46.6%) 31(53.4%)

Yes 44(49.4%) 45(50.6%) Breast / bottle χ2=1.011 0.237 feeding No 1(100%) 0(0.0%)

<6 month 2(100%) 0(0.0%)

2 Duration of 6 month -1 year 4(100%) 0(0.0%) χ =6.580 0.012 breast feeding >1 year 38(45.8%) 45(54.2%)

Yes 27(48.2%) 29(51.8%) 2 History of FP χ =0.189 0.466 No 18(52.9%) 16(47.1%) Condom 7(50%) 7(50%)

IUD 9(64.3%) 5(35.7%)

OCA's 3(20%) 12(80%)

DPI 0(0.0%) 1(100%)

Previously MINIPILLS 1(100%) 0(0.0%) 2 used FP χ =14.690 0.033 OCA's+ Condom 2(100%) 0(0.0%) method OCA's +Mminipills 2(100%) 0(0.0%)

IUD+OCA's 2(40%) 3(60%)

Minipills 1(100%) 0(0.0%)

DPI + OCA's 0(0.0%) 1(100%)

Depo-Provera injection= DPI, FP= Family planning

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4.4. Anthropometric measurements

Table (4.7) summarizes the anthropometric measurements characteristics of all respondents. The mean BMI of all respondents is 25.27 kg/cm2. About half of respondents 47% lies in normal weight classification. The mean waist circumferences of all respondents is 87.68 cm.

Table 4.7: Anthropometric characteristics of all respondents Variables n (%) Mean (SD) Weight(kg) 65.87 (12.669) Height(cm) 161.22 (5.528) BMI(kg/cm2) 25.27 (4.515) Under weight 3 (3.3%) Normal 43 (47%) Over weight 32 (35.6%) Obese 12 (13.3%) Waist circumference (cm) 87.68 (10.648) People with BMI ≤ 18.5 were considered underweight. People with BMI = 18.5 - 24.9 were considered to have normal weight, people with BMI=25.0-29.9 were classified over weight, people with BMI≥30.0 were considered obese (WHO, 2016).

Table (4.8) shows no statistically significant differences between the groups, but there was some differences as noted by the Mean Differences, for example, mean BMI in the non-users group is greater than the mean in the COCA's users group by about 0.9 kg/m2. Also, the mean waist circumferences in the non-users group is greater than the mean in the COCA's users group about 1.4 cm.

Table 4.8: Anthropometric measurements comparison between groups Variables Non users Users Mean Z P (n = 45) (n =45) Diff statistics Value Median (IQR) Median (IQR) Weight 65(20) 64(21) 3.056 -1.175 0.240 Height 160(9) 160(6) 0.978 -0.595 0.552 BMI 25(5.1) 24(5.9) 0.896 -0.896 0.370 W.C. 90(16) 88(11) 1.444 -0.715 0.475 Mann-Whitney U Test for 2 independent nonparametric data. The level of significance is < 0.05.

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Table (4.9) shows no statistical significant associations between BMI classification and the use of COCA's.

Table 4.9: Anthropometric category and COCA's use

Test P Profile Non- users Users value value Under weight 0(0.0%) 3(100%)

Normal weight 22(51.2%) 21(48.8%) 2 BMI category χ =3.857 0.170 Over weight 18(56.3%) 14(43.8%)

Obese 5(41.7%) 7(58.3%)

4.5. Blood pressure measurements

The blood pressure characteristics is illustrated in the table 4.10. The mean systolic blood pressure is 108.44 mmHg, and the mean diastolic blood pressure is 70 mmHg.

Table 4.10: Blood pressure characteristics

Variables Mean Stander Deviation (SD) Systolic BP 108.44 (9.230) Diastolic BP 71.00 (7.503)

Table (4.11) represent the differences in systolic and diastolic blood pressure between the groups.

Table 4.11: Blood pressure measurements comparison between groups Variables Non users Users Mean Z P (n = 45) (n =45) Diff Statistics Value Median Median (IQR) (IQR) Systolic Bp 110(20) 108(10) 1.333 -1.013 0.311 Diastolic Bp 70(10) 70(10) -1.556 -0.602 0.547

Mann-Whitney U Test for 2 independent nonparametric data. The level of significance is < 0.05.

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4.6. Food Frequency questionnaire

Table (4.12) shows no statistical significant differences between the groups in the score of consumed foods items. Except in nuts, snacks, and soups and sauces. However, if we look at the mean score of nuts and snacks we find that, the non-users group was higher than the COCA's group.

Table 4.12: Food items comparison between groups Variables Non users Users Mean Z P (n = 45) (n =45) Diff statistics Value Median (IQR) Median (IQR) Vegetables 105.00(29) 104(20) -1.511 -0.234 0.815 Fruits 89.00(31) 87.00(19) 3.733 -0.868 0.385 Meat &meat products 19.00(7) 16.00(7) 1.244 -0.874 0.382 Poultry 11.00(7) 10.00(4) 0.533 -0.463 0.958 Fish & sea food 8.00(8) 9.00(6) 0.400 -0.053 0.958 Grain 27.00(9) 27.00(7) 1.089 -0.931 0.352 Dry beans 24.00(8) 24(6) -0.511 -0.365 0.715 Dairy products 20(14) 22(16) -0.356 -0.295 0.768 Egg 7(4) 7(4) 2.067 -0.160 0.873 Nuts 5(2) 3(2) 0.911 -2.080 0.038 Beverages 23(14) 24(11) 0.178 -0.008 0.994 Snacks 27(11) 21(10) 5.778 -2.954 0.003 Soups & sauces 6(6) 9(4) -1.356 -2.169 0.030 Sweet 14(9) 12(8) -42.511 -1.052 0.293

Mann-Whitney U Test for 2 independent nonparametric data. The level of significance is < 0.05.

4.7. Laboratory Assessment: 4.7.1. Complete blood count (CBC):

Table (4.13) shows a statistical significant differences between the groups in the RBC, HGB, LYM, and MCHCp = 0.047, p< 0.001, p = 0.036, p = 0.004, respectively. On the other hand, the results reported no statically significant differences between the groups in the remainder CBC profile.

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Table 4.13: CBC comparison between groups Variables Non users Users Mean Z P (n = 45) (n =45) Diff statistics Value Median (IQR) Median (IQR) WBC 6.400(2.4) 6.600(2.5) 0.4467 -0.448 0.654 RBC 4.400(0.5) 4.500(0.6) -0.1931 -1.985 0.047 HGB 11.600(1.3) 12.700(1.0) 0.7711 -3.546 <0.001 HCT 35.300(3.6) 36.600(4.9) -7.7756 -1.792 0.073 PLT 278.00(61.5) 305.00(100) -20.0667 -1.642 0.101 LYM 37.500(13.2) 30.500(13.1) 3.3956 -2.094 0.036 Granulocyte 54.100(13.5) 59.700(11.7) -1.4622 -1.388 0.165 MCV 81.00(7.5) 82.00(75) -1.6222 -0.461 0.645 MCH 26.800(2.8) 27.900(4.1) -0.3778 -1.614 0.106 MCHC 33.00(1.4) 33.700(2.7) -0.9956 -2.895 0.004 RDW 14.600(119.0) 15.900(2.7) 0.9622 575.000 1.610

Mann-Whitney U Test for 2 independent nonparametric data. The level of significance is < 0.05.

4.7.2. Lipid profile

Table (4.14a) presents thecharacteristics of lipid profile of all respondents. 15 and 20 out of 90 respondents have abnormal T.C and TG, respectively.

Table 4.14a: Lipid profile characteristics Lipid profile Mean (SD) Frequency Percentage

Cholesterol 163.88 (53.4728) Normal 75 83.3% Abnormal 15 16.7% Total 100 100.0% TG 122.60 (67.2849) Normal 70 77.8% Abnormal 20 22.2% Total 100 100.0%

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Table (4.14b) reported that, there is no statistical differences between the groups in the cholesterol and triglyceride levels. With reference to mean differences, the mean cholesterol in the users group is greater than the mean in the non-users group by about 7 mg/dL. While the mean triglyceride in the non-users group is less than the mean in the users group by about 15.4 mg/dL.

Table 4.14b: Lipid profile comparison between groups Variables Non users Users Mean Z P (n = 45) (n =45) Diff Statistics Value Median (IQR) Median (IQR) Cholesterol 155.00(40.0) 142.00(94.5) - 7.000 -0.835 0.404 TG 106.00(93.5) 93.00(60.0) 15.378 -0.569 0.569

Mann-Whitney U Test for 2 independent nonparametric data. The level of significance is < 0.05.

Table (4.14c) indicate a statistical significant association between cholesterol category and use of COCA's (χ2=3.920, p = 0.044). More than two thirds of cases had high blood cholesterol level were from the COCA's users.

Table 4.14c: Lipid profile category and COCA's use Test P Profile Non- users Users Value value Normal 41(54.7%) 34(45.3%) 2 Cholesterol χ =3.920 0.044 High 4(26.7%) 11(73.3%) Normal 34(47.9%) 37(52.1%) 2 Triglycerides χ =0.600 0.438 High 11(57.9%) 8(42.1%)

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4.7.3. Micronutrients

Table (4.15) presents the biochemical characteristics for all respondents. The mean Homocysteine was 11.03. The copper level was below normal in 27 out of 90 cases.

Table 4.15: Micronutrients characteristics for all respondents Profile Mean(SD) Frequency Percentage Homocysteine 11.03 (4.2046) Normal 66 73.3 Below normal 9 10.0 High 15 16.7 Total 90 100.0 Vitamin B6 10.27 (5.4486) Normal 74 82.2 Below normal 16 17.8 Total 90 100.0 Vitamin B12 330.40 (171.235) Normal 68 75.6 Below normal 22 24.4 Total 90 100.0 Folic acid 8.40 (3.2837) Normal 84 93.3 Below normal 6 6.7 Total 90 100.0 Iron 105.69(32.7358) Normal 86 95.6 Below normal 3 3.3 High 1 1.1 Total 90 100.0 Zinc 77.21(20.4453) Normal 67 74.4 Below normal 21 23.3 High 2 2.2 Total 90 100.0 Copper 92.78 (33.4777) Normal 62 68.9 Below normal 27 30.0 High 1 1.1 Total 90 100.0

Table (4.16) indicates that, there is a statistical significant differences between the groups in the Homocysteine and Vit B6 p = 0.008 and p = 0.010, respectively. The mean Vita B6 in the users group is greater than the mean in the non-users group by about 55.6 ng/l.

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Table 4.16: micronutrients comparison between groups Variables Non users Users Mean Z P (n = 45) (n =45) Diff Statistics Value Median (IQR) Median (IQR) Homocysteine 9.300(6.8) 12.00(5.9) -2.2333 - 2.667 0.008 Vita B6 11.200(8.1) 7.500(8.1) 2.6412 - 2.571 0.010 Vita B12 269.00(211.5) 330.00(341.0) -55.600 - 1.445 0.149 Folic acid 8.300(5.1) 7.300(4.6) 0.4467 - 0.848 0.397 Iron 98.00(60.0) 116.00(42.00) -11.556 - 1.384 0.166 Zinc 76.000(28.0) 81.000(30.5) 2.5111 - 0.448 0.654 Copper 98.000(54.5) 95.000(54.5) 5.2378 - 0.771 0.441 Mann-Whitney U Test for 2 independent nonparametric data. The level of significance is < 0.05.

Table (4.17) shows a statistical association between homocysteine and vitamin B6 and the use of COCA's χ2=7.172 p = 0.019 and χ2=7.601p = 0.004, respectively.

Table 4.17: Biochemical category and COCA's use Test P Profile Non- users Users Value value Normal 32(48.5%) 34(51.5%) Homocysteine Below normal 8(88.9%) 1(11.1%) χ2=7.172 0.019 High 5(33.3%) 10(66.7%) Normal 42(56.8%) 32(43.2%) Vitamin B6 χ2=7.601 0.004 Below normal 3(18.8%) 13(81.3%) Normal 34(50%) 34(50%) Vitamin B12 χ2=0.000 >0.999 Below normal 11(50%) 11(50%) Normal 42(50%) 42(55%) Folic acid χ2=0.000 >0.999 Below normal 3(50%) 3(50%) Normal 35(52.2%) 32(47.8%) Zinc Below normal 9(42.9%) 12(57.1%) χ2=0.563 0.754 High 1(50%) 1(50%) Normal 30(48.4%) 32(51.6%) Copper Below normal 14(51.9%) 13(48.1%) χ2=1.102 0.475 High 1(100%) 0(0.0%)

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Predictor Factors of Homocysteine and Vitamin B6 Level

Prior to the stepwise multiple linear regression (MLR) analysis, Simple Linear Regression (SLR) was conducted to investigate the association between Homocysteine and vitamin B6 (dependent variables) and using of COCs well as other predicted variables (independent variables). Simple linear regression (SLR) was conducted to identify possible significant independent variables for the multivariable analysis. SLR was performed on each independent variable. The assumption of normality of the score and the linearity for each domain were checked and found to be approximately normally distributed; the residuals appeared linear and randomly scattered.

Table 4.18 shows the direction of the association between users/none users of oral contraceptives and the Homocysteine level via MLR (stepwise) to obtain the final model. The MLR results for the model cam be summarized as follow:The final model of the MLR analysis implied a significant association between using of oral contraceptives and the Homocysteine level (p = 0.002). MLR showed a significant association between Homocysteine level and the SBP (p = 0.007) as well as intake of nuts (p = 0.029) and the cholesterol level (p = 0.020). An increase of one percent COCA's using increase the Homocysteine level by 2.7 as b = 2.65 units. In addition, a one-point increase in the SBP and cholesterol level increase the homocysteine level by 0.12 and 0.02 unit, respectively. In addition, a one-point decrease in the nuts intake score increase the homocysteine level by 0.43 unit.

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Table 4.18 : Predictor Factors of Homocysteine Level –Stepwise Method* Simple Linear Regression Multiple Linear Regression Variables b(95%CI) p value Adjustedb(95%C R2 p value I) User/Nonuser 2.233(0.53, 3.94) 0.011 2.65(1.01, 4.31) 0.01 0.002 Age -0.05( -0.26, 0.17) 0.685 2

City/ Village 2.17(0.45, 3.90) 0.014 Education Level 0.64( -1.13, 2.42) 0.473 Employment Status 0.02( -2.36, 2.39) 0.982 Monthly Income 0.37( -1.85, 2.58) 0.743 Household Size 0.26( -0.26, 0.79) 0.323 Medical Surgery 0.04( -2.34, 2.42) 0.973 Breast Feeding 1.68( - 6.76, 10.13) 0.693 BMI -0.07(-0.26, 0.13) 0.513

SBP 0.09( -0.00, 0.19) 0.057 0.12(0.03, 0.21) 0.007 DBP 0.14(0.02, 0.25) 0.022 PLT -0.01( -0.02, 0.00) 0.060 Nuts 0.21(-0.20, 0.61) 0.081 0.43(0.04, 0.81) 0.029

Cholesterol 0.02(0.00, 0.03) 0.054 0.02(0.02, 0.03) 0.020 TG 0.01( -0.01, 0.02) 0.281 Note: * Regression p values are included only for variables in the final model.

Table 4.19 implies that there is a significant association between B6 level and using of COCA's (p = 0.033) and breast-feeding (p = 0.047). As the using of oral contraceptive increase by one percent, the level of vitamin B6 decrease by 2.41 units as b = -2.41 and a one percent increase in the breast-feeding increases vitamin B6 level by 10.65 units as b = -10.65.

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Table 4.19 :Predictor factors of B6 level –Stepwise method* Simple Linear Regression Multiple Linear Regression Variables b(95%CI) p value Adjustedb(95%C R2 p value

User/ Nonuser -2.64(-4.87, -0.42) 0.021 -2.41(-4.61I) , -0.20) 0.08 0.033

Age 0.15(-0.13, 0.43) 0.302 ) 1 City/Village 2.30(-0.45, -0.025) 0.048

Education Level -0.79(-3.09, 1.52) 0.498

Employment Status -0.21(-3.29, 2.87) 0.895

Monthly Income -0.70(-3.56, 2.17) 0.743

Household Size -0.12(-0.80, 0.57) 0.735

Medical Surgery 1.03(-2.04, 4.11) 0.505 Breast Feeding 11.86(1.21, 22.52) 0.030 10.65(0.15, 21.15) 0.047 BMI 0.034(-.22, 0.30) 0.760 SBP -0.04-(0.17, 0.09) 0.525 DBP -0.09(-0.24, 0.07) 0.257 PLT 0.00(-0.014, 0.02) 0.787 Cholesterol -0.01(-0.03, 0.010) 0.308 TG -0.01(-0.023, 0.01) 0.487 Note: * Regression p values are included only for variables in the final model.

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CHAPTER 5

DISCUSSION

The present study was designed to investigate whether COCA's impact the micronutrients status in women on COCA's. Ninety healthy childbearing women were enrolled in this study, 45 out of them used COCA's and the other group used another type of FP methods. Results of this study prove that, the use of COCA's impact some important micronutrients like vitamin B6, vitamin B12, zinc , copper and iron.

5. discussion 5.1.Socio-demographic and Socio-economic characteristics

The subjects of this study lived in Rafah governorate, and they were equally distributed either on the city or refugee camps. Although about half of respondents (44.5%) in the study were highly educated (i.e. had post-secondary education), and most of the highly educated mothers (70%) tend to avoid long-acting reversible methods of contraception and prefer to use the conventional method of contraceptives such as condom and IUD. Low uptake of the injections, which was observed in this study and in studies conducted in other regional countries, could be due to the different reasons such as the long acting effect, gain of weight and micronutrients depletion. Also women’s choice of contraception was found to be heavily influenced by their partner’s opinion, we believe that partner education is equally important (Gosavi, et al., 2016).

The economic status is another factor that may affect the micronutrients status especially in women and childhood. The mean total income of the respondents was about 1485 NIS, and 75 out of 90 respondents were not working. While the relative poverty line and the deep poverty line according to consumption patterns (for reference household consisted of 2 adults and 3 children) in the Palestinian Territory in 2010 were 2,237 NIS, and 1,783 NIS respectively (PCBS, 2016). The majority of respondents suffered from absolute or deep poverty. Micronutrient deficiency increases as food prices increase (Iannotti et al., 2012), whether globally or locally. Since foods are the primary micronutrients source, an increase in prices means less money for fruits, vegetables, meat, dairy, and fortified processed foods. As a result, the epidemic of micronutrient deficiency has been with us for as long as there have been people with low income. The iron deficiency anemia that has resulted from low

64 intake of iron; is an example of the link between poverty and micronutrient deficiency (Iannotti et al., 2012).

5.2.Medical and family history

Although the largest proportion of cases who complain of one or more of the diseases was from the COCA's non-users group, the COCA's users group suffered from some of micronutrients deficiency more than the non-users group. As a result, the door opens wide for important questions; to what extent can the COCA's affect micronutrients status and how can these agents affect the micronutrients status. These questions will be discussed below.

In addition, micronutrients deficiencies are important public health issues due to their contributions to the pathogenesis, progression, mortality and morbidity burdens in many chronic diseases, including cardiovascular diseases. High blood homocysteine levels for example, have been linked to an increased chance of developing heart disease. The vitamins B2, B6 and B12, and particularly folate, have been shown to lower homocysteine levels. Thus, people with sub-optimal B vitamin intake may be able to reduce their risk of cardiovascular disease by increasing their consumption of B vitamins (Geissler & Powers, 2017). As an important fat-soluble antioxidant vitamin E has been shown to reduce the oxidation of LDL-c, involved in the build-up of plaque in the arteries (Suzukawa et al., 1995). A lot of studies indicated that vitamin E could be protective against cardiovascular disease (Saremi& Arora, 2010). The pathophysiological mechanisms underlying micronutrients deficiency induced cardiovascular disease include; induction of imbalance in antioxidant defense mechanisms, inflammation and immune system dysfunction.

Moreover, any significant changes in micronutrient status can exacerbate the existence of diabetes, and targeted consumption of micronutrients can help improve metabolic control, optimize treatment and reduce the risk of developing diabetic complications. As coenzymes, the B vitamins play a central role in carbohydrate, protein and lipid metabolism. A diabetic metabolic status is characterized by both higher requirements and increased renal elimination of B vitamins, especially when the diabetes is not well managed. As well as vitamins B6 and B12 supports nervous system functions and helps prevent diabetic neuropathies (Martini et al., 2010). A lack of folic acid and or vitamin B12 leads to

65 impaired metabolism of the amino acid methionine and is frequently accompanied by elevated plasma homocysteine concentrations. Elevated homocysteine levels are regarded as an independent risk factor for stroke, heart attack, dementia and macular degeneration. Patients with type 2 diabetes, elevated homocysteine levels and vitamin B12 deficiency are at substantially greater risk of developing diabetic retinopathy (Satyanarayana et al., 2011). Regular administration of vitamin B12 is often indicated for diabetic patients being treated with metformin to prevent a medication-induced deficiency. In one study with type 2 diabetics, treatment with metformin led to a dip in cognitive performance which improved again when treatment was accompanied by supplementation with vitamin B12 and calcium (Grber et al., 2013).

As a highly effective antioxidant, vitamin E protects enzymes and hormones, as well as the polyunsaturated fatty acids of biological membranes and LDL-c, against oxidation by oxygen radicals. During this process, vitamin E is oxidized and must be regenerated by vitamin C or flavonoids. In this way, the vitamin combats the oxidative degradation of fatty acids (lipid peroxidation) and in particular the oxidative modifications of LDL-c that contribute to the incidence of atherosclerosis. Moreover, vitamin E lowers thrombocyte aggregation and hence the risk for thromboses, and reduces the extent of protein glycosylation HbA1C (Suksomboon et al., 2011). In addition, by inhibiting enzymes the vitamin slows inflammatory processes and the proliferation of connective tissue in the blood vessels and therefore reduces the threat or advance of diabetic complications (Montero et al., 2014). As one of an important element of insulin structure; zinc has a stabilizing effect and protects against oxidative damage. Zinc deficiency can lead to decreased synthesis of insulin receptors and to a decline in glucose tolerance and insulin sensitivity (Chausmer, 1998). Moreover, an insufficient intake of zinc could promote the incidence of atherosclerotic vascular changes and its consequences for diabetics; e.g., coronary heart disease(Soinio et al., 2007).

5.3.Anthropometric measurements.

Results of this study shows small differences between the COCA's users and non- users groups. This result is similar to a clinical trials that showed COCA's use did not affect weight change (Berenson & Rahman, 2009; De Melo et al., 2004; Gallo et al., 2011). While, other studies showed a significant increment in body weight in the

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COCA's users (Edelman et al., 2010; Park & Kim, 2016). With reference to the proportions of BMI category, the obese participants in the COCA's users group were more than the non-users group. As a result, COCA's users had an increased tendency to be obese.

Until this moment, studies carried out on women using COCA'shave not conclusively determined if COCA's have a significant impact on body weight in reproductive aged women. Interestingly, there were a number of studies demonstrating mild to moderate weight loss with COCA's use (Coney et al., 2001; Risser et al., 1999; Rosenberg, 1998), or at least no weight gain (Berenson & Rahman, 2009; Uras et al., 2009). These conflicting results may be due to different methodologies used between studies, and ideal clinical control trials may help.

There are several possible mechanisms onhow weight gain could occur because of OCA's use. The first suggested mechanism is that, estrogen cause fluid-retention weight gain by direct stimulation of the renin–angiotensin system, which can lead to water retention (Neel et al., 1987), which in turn leads to sodium retention. It is noteworthy that, low-dose estrogen COCA's do not cause weight gain and do not cause fluid retention (Reubinoff et al., 1995), but it reduce fluid retention (Khoo et al., 1980; Reubinoff et al., 1995). Also, COCA's containing the progestin drospirenone may assist in helping women who are susceptible to weight gain minimize fluid retention, in particular related to premenstrual syndrome. This progestin has antimineralocorticoid and antiandrogenic properties (Milsom et al., 2006; Rapkin, 2008). The second possible mechanism is increased appetite as a result of suppression of serum cholecystokinin.

5.4.Blood pressure measurements.

Results of this study indicated that, there wasminor fluctuation between COCA's users and non-users. This result was similar with other studies (Weir, 1982; Weir et al., 1974; E. Wilson et al., 1984). While other studies found increased risk of hypertension among users of COCA's that was highest among long-term users and decreased shortly after COCA's cessation, and the risk of hypertension increased with increases in the potency of progestin (Chasan-Taber et al., 1996; Hickson et al., 2011). The mechanisms involved in the production and maintenance of COCA's induced hypertension are not well understood. But the renin-angiotensin system has

67 been implicated; users of high-dose OCA's may have greatly elevated levels of angiotensinogen (Oelkers et al., 2000). Changes in blood pressure related to COCA's are reversible in a short time (Weir et al., 1974).

Hypertension is not a single disease entity with a single identifiable etiology; it is a clinical condition brought on by a number of factors. It is generally recognized that stress, poor diet, lack of exercise and related features of our civilization contribute significantly to the development of hypertension. Some vitamins have been identified as having a role in blood pressure regulation. Vitamin E is the antioxidant vitamin in the aqueous and membrane regions of the cell respectively and, hence, indirectly affect blood pressure regulation (Lonn et al., 2002). Apart from these vitamins, the most consistent and significant effect of a vitamin on blood pressure regulation is that of vitamin B6. Investigations have established that even a moderate deficiency of vitamin B6 could lead to hypertension. Results of this study indicate that there wasreduction in the mean of all antioxidants (vitamins and minerals). Hence, blood pressure may be changed with time.

5.5.Hematological profile.

Results of this study revealed that, COCA's significantly lowered the hemoglobin, RBCs, and MCHC levels, and significantly increased lymphocytes level in the blood. Few studies conducted to investigate the impact of oral contraceptives on hematological profile among humans. One animal study indicated that COCA's negatively impacted hemoglobin, RBCs, lymphocytes, and MCV (Toryila et al., 2014). The changes of Hb, RBCs, and lymphocytes were similar with the finding of (Sajida et al., 2006), and however, result obtained by(Abdalla et al., 2012)showed no changes in full blood count in women on COCA's. The only possible mechanism by which these changes could result is of the haemodilutary effect of estrogen. Thus, the changes of hematological profile depends on the concentration of estrogen and progesterone and the duration of use.

5.6.Lipid profile

Many researchers studied the effects of COCA's on lipid profile, some of these studies indicated negative effect, while others showed no association. Evidence suggests that the composition of different COCA's, in terms of estrogens dose and

68 progestogen type, also influences their respective effects on lipids and lipoproteins (Sitruket al., 2011).

In our study, it was noted that there was a significant association between COCA's use and cholesterol level in the blood. A lot of recent studies confirmed that, the use of COCA'ssignificantly increased the total cholesterol in the blood (Lizarelli et al., 2009; Mohammad et al., 2013; Obisesan et al., 2002). In addition, results of this study showed that no significant association between COCA's use and triglyceride level in the blood. These results were in contrast with the study of Mohammad and his colleges that confirmed the association (Mohammad et al., 2013). It is well known that, the increase in total cholesterol in the blood increases the risk of HTN and CVD.

5.7.Vitamins and minerals

5.7.1. Vitamins

Vitamin B12

Several studies have confirmed that the OCAs negatively impact serum Vitamin B12 levels as for example the study of(McArthur et al., 2013; A. S. Wilson et al., 2011). In contrast, results of this study found that the serum Vitamin B12 was higher in the non-users group than the COCA's group. The functional indicators of vitamin B12 status were not significantly impacted by OCAs use (Wilson et al., 2011). These conflicting results may be due to several causes; for example different dietary background. Also, results of the this study indicated that the non-users of COCAs group have higher scores of the food items that considered the best sources of Vitamin B12 than the users group.

However, the mechanisms by which serum vitamin B12 is reduced in OCAs users are not fully understood. The total vitamin B12 binding capacity of the serum was significantly lower in women using OCAs than in nonusers; the levels of transcobalamin I, a glycoprotein serves to protect vitamin B12 from acid degradation in the stomach, were also lower in OC users (Shojania, 1982; Shojania& Wylie, 1979). In addition, the absorption and the urinary excretion of vitamin B12 in OCAs users were normal and their lower serum levels of vitamin B12 were not associated with evidence of tissue depletion, the lower total vitamin B12 binding capacity and

69 lower transcobalamin I levels in the serum of the users could explain their low serum levels of Vitamin B12 (Shojania, 1982; Shojania& Wylie, 1979).

Vitamin B-6

Results of this study revealed that there was a significant association between the use of COCA's and Vitamin B6 level in the plasma, and as noted by mean differences in the COCA's users group, was lower than in nonusers group by 2.64 ng/l. This result was seen in other studies (McArthur et al., 2013; Var et al., 2014).

Usually, Vitamin B6 deficiency is linked most commonly with neuropsychiatric disorders, including seizures, migraines, chronic pain and mood disorders like depression (Malouf & Grimley Evans, 2003). Also, increased risk of heart disease and rheumatoid arthritis (Lotto et al., 2011; Morris et al., 2010). Other evidence suggested that vitamin B6 deficiency is more common among older people, with the risk of Alzheimer’s disease and other forms of dementia (Malouf&Grimley Evans, 2003; Mecocci et al., 2014; &Morris et al., 2006). The richest sources of vitamin B6 include fish; liver and other organ meats, potatoes, starchy vegetables, legumes, nuts, bananas, avocados, egg yolks, whole grains and vegetables (Stover & Field, 2015).

Folic Acid

In several studies, women taking oral contraceptives developed folic acid deficiency (Stterlin et al., 2003). Although our results indicated that, there was no statistically significant association between groups, however, the mean folate in the COCA's group was less than the non-users group by 0.44 ng/l. Other data did not support a conclusion that the use of OCAs negatively affected folate status (Wilson et al., 2011). Although a number of early studies concluded that OCAs negatively impacted folate status, the majority of these studies were conducted when the estrogen content of OCAs was much higher, the interpretation of findings from many of these studies is problematic since no controls were included for potentially confounding factors. The recent available data do not support a conclusion that currently used OCAs negatively impact folate status (Wilson et al., 2011).

However, there is no conclusive evidence to indicate the negative impact of OCAs on folate status; hence, the reduction in folate may be due to low intake of folic acid or problems with intestinal absorption prior to taking birth control pills. Current data

70 suggest that 45.7% of women of childbearing age become pregnant within three months of stopping oral contraceptive (OC) use (Cronin et al., 2009), and that 28.1% take folic acid before conception. Folic acid supplementation is associated with a reduction in the risk of neural tube defects. Hence, it is critical that women begin supplementing with folic acid in the periconceptional period, at least three months before becoming pregnant (Colapinto, 2014).

A number of studies investigated the association between the duration of OCAs use and the folate status. Some studies shows no significant relationship between duration of therapy with either of two OCAs preparations versus controls on serum folate concentrations (Castren & Rossi, 1970; McLean et al., 1969). While a different study found that serum folate concentrations were significantly reduced with continued OCAs use, and that women who used oral contraceptives for two years or more had significantly lower serum folate levels than those who had been using them for one year or less (Shojania, 1971). A different study found significant reduction inRBC folate concentrations, in comparison to baseline levels, among users of two OCAs preparations (Wilson et al., 2011).Thus, the literature regarding the duration of oral contraceptive therapy shows mixed associations with blood folate concentrations, but in many cases, this could be attributed to differences in the length of the study period or other pre-existing factors that may influence the difference.

In trying to establish a cause-and-effect relationship, many studies further analyzed the composition of oral contraceptives to determine if certain types of combination oral contraceptives had a greater chance of being associated with lowered folate status than others. One study found no relationship between estrogen dose and serum folate levels among the two preparations they used ( with either norethindrone or norgestrel), even though the doses of mestranol varied (McLean et al., 1969). Given that oral contraceptives have evolved in the type of progestins used, as well as in the doses of estrogens, some researchers focused on whether this difference may be responsible for the differences in reported effects. Many of the previous studies involved oral contraceptives containing 35 to 50 µg of ethinyl estradiol (EE), whereas currently used oral contraceptives contain lower EE doses (Stterlin et al., 2003), and it is important to investigate whether this folate- lowering effect could still pose a concern.

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5.7.2. Minerals

COCA's are composed of an estrogens and a progestins. Estrogens involved in combined oral contraceptive pills includes ethinyl estradiol or mestranol. There are nine progestins in combined oral contraceptive pills which are norethindrone, norethindrone acetate, ethynodioldiacetate, levonorgestrel, norgestrel, , desogestrel, and gestodene (Borgelt-Hansen, 2001; Robert A Hatcher & Nelson, 2004).

Results of this study indicated that, there was no significant differences between the users of the COCA's and non-users in the Iron, Zinc, and Copper. With reference to mean differences, iron level in the plasma is higher in the COCA's users group than non-users by 11.5 mg/dl. The zinc and copper levels in the plasma were lower in the COCA's users than non-users by 2.5 and 5.2 mg/dl, respectively.

Copper, zinc and iron are crucial for the functioning of several enzyme systems and are required in a number of metabolic processes in the body. They are involved in gene expression, RNA and DNA metabolism and cellular immune functions. Thus they are of fundamental importance in living organisms (Khanna et al., 2009). The decrease in serum zinc concentration may be an important risk factor in oxidant release and the development of DNA damage and cancer, zinc is co-factor in proteins involved in antioxidant defense, electron transport, DNA repair and protein expression (Ho, 2004).

Results of present study are in agreement with other studies whish reveled that OCAs decrease zinc level in the plasma (De Groote et al., 2009). COCA's can increase the serum iron level by reducing the menstrual period to 3-4 day and reduce the amount of blood that is lost in each menstrual cycle to 25 mL (35 mL of blood are lost in normal menstrual cycle), by reducing the endometrial thickness that shorten the menstrual period. Hence, COCA's can be used in treatment of anemia that results from menorrhagia (Huber et al., 2008). While, the estrogen can increase the hepatic synthesis of transferrin that lead to increases in the serum iron level.

The increased level of serum copper in COCA's users women due to estrogen, may cause an increase in plasma copper level that run parallel to that of ceruloplasmin (Benes et al., 2005). The estrogen component is mainly responsible for the increased

72 level of serum ceruloplasmin while progesterone causes a less drastic rise (Sontakke & More, 2004). Estrogen acts as an inducer for synthesis of ceruloplasmin RNA templates causing subsequent increase in synthesis of the protein. For this reason the increase in ceruloplasmin level cause an increase in serum copper concentration.

Reduction in plasma zinc, increase in plasma iron and, particularly, in plasma copper, have been frequently observed in users of OCAs (Akhter et al., 2006; Fallah et al., 2009). In contrast to zinc, a nutrient with antioxidant proprieties, iron and copper are known to be pro-oxidants (Fang et al., 2002). Therefore, women who use OCAs may be more susceptible than non-users to an imbalance between zinc, iron and copper status, and its physiological consequences. The extent of zinc, iron and copper imbalance with OCAs use is unclear because most studies measured only plasma mineral concentrations. Oxidative stress is defined as an imbalance between prooxidants and antioxidants in the cells, which is manifested by elevated levels of free radicals. The free radical mediated peroxidation of membrane lipids, increase membrane fluidity and permeability with loss of its integrity that lead to cell damage (Abdoljalal et al., 2011). Alterations of these structures are associated with the development of several human pathologies including atherosclerosis, cardiovascular disease, cancer, diabetes complications and arthritis (Cutler, 2005).

Although, COCA's increase iron level in the plasma, women taking oral contraception do not need to reduce the amount of iron-rich foods they eat or avoid multivitamins that contain iron. In addition, COCA's use influenced zinc, iron and copper homeostasis. Zinc supplementation further altered copper utilization in contraceptive users, possibly favoring oxidative stress (Kamp et al., 2011).

5.7.3. Homocysteine

Homocysteine is an amino acid that is derived from demethylation of methionine, and the total serum of homocysteine level varies in the range of 5-15 μmol/L in the normal population (Yan et al., 2010). Homocysteine can be recycled into methionine or converted into cysteine with the aid of certain B-vitamins. Also, folic acid involvement in metabolism of methionine could lead to increased total homocysteine concentration. The following figure illustrate the homocysteine metabolism;

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Homocysteine is metabolized to methionine by remethylation and cystathionine by transsulfuration. Coenzymes are shown in gray. BHMT, betaine-homocysteine S- methyltransferase; DMG, dimethylglycine; MAT, methionine adenosyltransferase; SAM, S- adenosylmethionine; SAH, S-adenosylhomocysteine; SAHH, S-adenosylhomocysteine hydrolase; MS, methionine synthase; THF, tetrahydrofolate; SHMT, serine hydroxymethyltransferase; CH2THF, methylene tetrahydrofolate; CH3THF, methyl tetrahydrofolate; CBS, cystathionine β- synthase; CγL, cystathionine γ-lyase(Zhang et al., 2013).

Results of this study indicated a non-significant reduction in total serum folate. Homocysteine metabolism may be influenced by dietary habits and lifestyle factors. In particular, homocysteine is inversely related to folate and causes oxidative stress, vascular inflammation, damages endothelial cell, inhibits endothelium-dependent relaxation, and enhances thrombogenecity(Libby, 2012; Yan et al., 2010). High levels of homocysteine are related to increased risk of venous thrombosis, cardiovascular diseases, (CVD), thrombotic, neurodegenerative, pregnancy-associated diseases and disorders of the central nervous system (Ganguly & Alam, 2015).

It is proposed that the whole body homocysteine metabolism could be impaired due to OCAs conversion to reactive species, and OCAs may induce the formation of free radicals, which could stimulate homocysteine synthesis (Norouzi et al., 2011). Probably, OCAs has a direct effect on hepatic homocysteine synthesis. Previous studies have shown that OCAs may directly modulate hepatic synthesis of several factors at the transcriptional level and may also have various immunomodulatory effects, therefore predispose thromboembolic events by stimulating inflammatory

74 mechanisms (Krivak & Zorn, 2007). The homocysteine level seems to be sensitive to hormonal changes in OCAs users. It is suggested that even a small amount of steroid compounds in OCAs could be converted to peroxide and is sufficient to induce synthesis of a new molecules of homocysteine. Homocysteine can be degraded by two mechanisms, the remethylation and transsulfuration pathways, which depend on folate and vitamin B12 respectively (Chiantera et al., 2003).

Results of this study indicated that, there was a non-significant reduction in the B vitamins in women who consume OCAs, which might decrease the bioavailability of these vitamins and the possible deficiency of one or more of these vitamins might cause elevated homocysteine levels. Hence, supplementation of B vitamins such as folate and B12 could reduce the risk of cardiovascular events that may resulted from increased homocysteine level (Albert et al., 2008).

It should be noted that if we could determine the homocysteine level before initiation of OCAs, we would be able to compare the levels of homocysteine before and during OCAs use to understand their effects on the aforementioned biomarkers, which would improve the conclusion drawn from this study.

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CHAPTER 6

CONCLUSION AND RECOMMENDATIONS

6.1. Conclusion

Considering the fact that many women begin taking oral contraceptives in early adolescence and continue their use for many years, it is important to look at the possible nutritional effects of oral contraceptives on woman’s body especially when it comes to the depletion of vital nutrients and increase in inflammatory markers.Although, results of this study indicated that there was;

 A significant association between the use of COCA's and vitamin B6 deficiency.

 A significant increase in homocysteine.

 An important reduction in minerals and vitamins in COCA's users group.

6.2. Recommendations

This study has investigated one of the hot clinical topics in nutrition, which is micronutrients status among COCA's users. The following recommendations are suggested depending on our results.

6.2.1. Birth control method users We recommend women who use or plan to use COCA's type of contraception, to be in touch with clinical nutritionist, for assessment and management purposes.

6.2.2. Clinical nutritionist.

There are several different ways that, these agents and the medications in general can affect the amount of a nutrient in the human body. The long-term use of a medicine affects the human body ability to create or maintain a healthy nutrients level. This can cause low levels of nutrients in the body, and this is usually a slow process, occurring over time. Hence, we recommend the clinical nutritionists to assess and to make sure that the micronutrient status among the users of COCA'sare within normal level.

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6.2.3. Special Recommend.

There has been a growing public awareness of the role of nutrition in the etiology of many acute and chronic diseases. Understanding and applying nutrition knowledge and skills to all aspects of health care are extremely important, and all health care professions need basic training to effectively assess dietary intake and provide appropriate guidance, counseling, and treatment to their patients.

The inclusion of nutrition specialists within the health care providers will help in developing plans for solving health problems, and supporting nutritional research, because it may contribute in managing and preventing of different health conditions.

6.2.4. Future studies Based on finding of the current study, the researcher recommend other researchers to conduct clinical trials, to investigate the effects of one or more of these vitamins and minerals on nutritional status in general and on micronutrient in particular.

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Appendices Appendix A Permission and Ethical Approval Appendix A1- Approval from Helsinki Committee

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Appendix A2- Consent form

إقرار موافقة للمشاركة في دراسة بحثية

جامعة األزهر بغزة تأثٌر حبوب منع الحمل على بعض الفٌتامٌنات والمعادن على النساء فً سن اإلنجاب

أن ِت مدعوه للمشاركة فً هذه الدراسة البحثٌة بواسطة الباحثة أسماء محارب مغاري

الرجاء مراجعة هذا النموذج وطرح ومناقشة أي أسئلة ترٌدٌنها مع أخذ الوقت الكافً لذلك، حٌث ٌمكنك مناقشة ومراجعة طبٌبك المختص، عائلتك، وأصدقائك وذلك التخاذ القرار بالمشاركة أو عدم المشاركة فً هذا البحث. قد ٌحتوي هذا النموذج بعض الكلمات التً ال فهمها، لذلك الرجاء طرح األسئلة حولها لغرض تفسٌرها بشكل واضح.

الهدف من الدراسة هً الختبار مدى تأثٌر حبوب منع الحمل ثنائٌه النوع على مستوى بعض الفٌتامٌنات والمعادن فً الدم، وهل هناك تأثٌر أكبر مرتبط بمدة استخدام حبوب منع الحمل.

فً هذه الدراسة سوف ٌتم اختٌارك عشوائٌا فً إحدى مجموعات الدراسة التً سٌتم شرحها فً األسفل. حٌث أن كلمة عشوائٌا تعنً أنه لدٌك نفس الفرصة فً أن تكون فً أحد تلك المجموعتٌن، مثل أن نرمً قطعة نقود.

المجموعة األولى: هً المجموعة المقارنة، وهً التً تأخذ أي نوع من وسائل منع الحمل غٌر حبوب منع الحمل ثنائٌة التركٌب.

المجموعة الثانية: هً المجموعة المستهدفة، وهً التً تأخذ حبوب منع الحمل ثنائٌة التركٌب.

تشمل المشاركة في البحث اإلجراءات التالية:

1.اخذ بعض البٌانات الشخصٌة مثل االسم، العنوان، السكن ورقم الهاتف.

2.اخذ التارٌخ الصحً للشخص المرشح.

3.اخذ الطول الوزن وحساب كتلة الجسم ومحٌط الوسط.

4.سحب عٌنات من الدم على ٌد مختص.

5.كل البٌانات سٌتم إدخالها لبرامج التحلٌل االحصائً، وسٌتم مناقشة ونشر النتائج.

6.سٌتم ابالغ المشتركٌن بنتائجهم أوال بأول.

7.المشاركة فً هذا البحث هً مشاركة تطوعٌة.

المخاطر التي تصحب الدراسة:

ال توجد مخاطر على صحة المشتركٌن فً االنضمام لهذا البحث.

الفوائد المحتملة:

هذه البحث هو امتداد ال بحاث اخرى تهد ف الى التعرف عل بعض الجوانب السٌئة لحبوب منع الحمل ثنائٌه التركٌب، والخروج بتوصٌة تهدف الى استخدام بعض الفٌتامٌنات والمعادن قبل الشروع بالحمل.

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مصروفات الدراسة:

جمٌع مصروفات الدراسة من قٌاسات وتحلٌل عٌنات الدم ومقارنتها وأخذ قٌاسات الجسم وكل إجراءات الدراسة ستكون بشكل مجانً للمشتركات فً الدراسة

حماية سرية البيانات:

سوف ٌتم نشر جمٌع النتائج والتحلٌالت اإلحصائٌة للنتائج التً تم جمعها خالل الدراسة فً المؤتمرات العلمٌة وجمٌع المحافل العلمٌة، مع األخذ بعٌن االعتبار أنه لن ٌتم نشر اسمك أو اي بٌنات تعرٌفٌة عنك.

سٌتم التعامل مع جمٌع البٌانات الشخصٌة والسجالت الطبٌة التً تحتوي على هوٌتك على أنها سرٌة وفقا لقانون جامعة األزهر والمٌثاق األخالقً الخاص بذلك. على الرغم أننا سنبذل جهود قاسٌة لحفظ سرٌة بٌاناتك، إال أن هذه الجهود لٌست مطلقة.

سوف أقوم باستبدال اسمك الشخصً برقم أو كود من جمٌع السجالت الطبٌة التً ستطلب منا لغرض مراجعة إجراءات البحث من قبل الجامعة أو المشرفٌن، حٌث سٌتم حذف أي رقم هاتف أو أي معلومة خاصة بك منها.

سٌتم االحتفاظ بكافة السجالت الطبٌة التً تحتوي على اسمك ومعلومات شخصٌة عنك فً مكان امن، حٌث أن البحاث هو فقط من سٌقوم باالطالع على هذه البٌانات لغرض الدراسة فقط. حٌث سٌتم إخطار طبٌب األسرة أو طبٌبك المختص بمشاركتك فً هذا البحث.

نوع المشاركة:

مشاركتك فً هذا البحث هً مشاركة تطوعٌة، حٌث بإمكانك عدم المشاركة أو االنسحاب فً أي وقت ترٌدٌنه أثناء المشاركة، وسف لن تتحملً أي نتائج أو عقوبات أو غرامة مالٌة أو أي نوع من أنواع العقوبات بسبب االنسحاب من البحث.

أسئلة حول الدراسة:

لدٌك الحق الكامل فً طرح أي سؤال ٌخطر ببالك حول حقوقك أو أي معلومات تردٌنها أو ترٌدٌن أن تستفسري عنها خالل أو قبل أو بعد الدراسة، وذلك بالتواصل مع الباحثة اسماء محارب مغاري، هاتف رقم6511741476 وعلى البرٌد االلكترونً: [email protected]

ولالستفسار حول حقوقك كمشتركه فً بحث سرٌري ٌمكنك مراسلة مجلس جامعة األزهر للدراسات العلٌا على البرٌد االلكترونً التالً: [email protected]

ال توقعً على الموافقة فً هذا البحث فً حال عدم أخذ الفرصة الكافٌة فً الرد على استفساراتك أو أنك غٌر مقتنع باألجوبة المقدمة من الباحثة.

معلومات االتصال:

إذا كان لدٌك أي أسئلة أو استفسارات حول هذه الدراسة أو إذا حدثت أي مشاكل، ٌرجى االتصال مباشرة فً البحاث

)اسماء محارب مغاري( على رقم الهاتف التالً: ،6511741476 البرٌد اإللكترونً التالً:

. [email protected]

بيان الموافقة:

لقد قرأت نموذج الموافقة للمشاركة فً هذا البحث.وقد أتٌحت لً الفرصة الكاملة لمناقشة هذه الدراسة البحثٌة مع الباحثة أسماء محارب مغاري.ولقد أجابت عن جمٌع أسئلتً باللغة التً أفهمها حٌث تم شرح جمٌع المخاطر والمنافع الخاصة بالدراسة. أأكد أنه لم ٌتم التأثٌر على للمشاركة فً هذا البحث من قبل الباحثة اسماء محارب مغاري أو أي شخص اخر بأي شكل من األشكال.

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أنا أفهم أننً سوف أعطى نسخة من هذا االقرار على نموذج الموافقة بعد التوقٌع علٌه، وأفهم أن مشاركتً فً هذا البحث هً مشاركة طوعٌة وتطوعٌة، وأنه لدي الحق الكامل فً االشتراك او رفض االشتراك أو االنسحاب من الدراسة فً اي وقت ارٌد.

إننً أعلم أن جمٌع المعلومات المتعلقة بهوٌتً الشخصٌة ستبقى سرٌة ولكن لٌست بشكل مطلق، كما أننً أفوض جامعة األزهر والمشرفٌن على الباحثة أسماء محارب مغاري بأن ٌقوموا بالتفتٌش على جمٌع السجالت الطبٌة الخاصة بً وذلك لغرض المراقبة والدراسة فقط.

من خالل توقٌعً على هذا نموذج إقرار الموافقة على المشاركة فً هذا البحث أنا لن أتنازل عن أي من حقوقً القانونٌة كمشارك فً هذه الدراسة البحثٌة

الموافق على االشتراك في هذا البحث:نعم ال 

اقرا ر الموافقة:

لقد قرأت هذا النموذج وأعطٌت الفرصة لطرح األسئلة واإلجابة عنها بشكل واضح وبلغة مفهومة جدا. لذلك أعطً موافقتً على المشاركة فً هذه الدراسة.

االسم رباعً: ......

التوقٌع: ...... تارٌخ التوقٌع: ......

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Appendix B Questionnaire Appendix B1- Questionnaire

أختً العزٌزة، السالم علٌكم ورحمة هللا وبركاته...

هذه االستبانة التً بٌن ٌدٌك،هً جزء مهم من دراسة أقوم بها أنا الباحثة/ أسماء محارب سلمان المغاري، والتً تهدف الى معرفة مدى تأثٌر حبوب موانع الحمل المركبة على الفٌتامٌنات و المعادن بشكل أساسً ، وذلك لنٌل درجة الماجستٌر فً برنامج التغذٌة االكلٌنٌكٌة- كلٌة الصٌدلة بجامعة األزهر-غزة، سٌأخذ االستبٌان بضع الدقائق من وقتكم، مع العلم أن المشاركة فً هذه الدراسة هً مشاركة طوعٌة، وأن جمٌع المعلومات سٌتم معالجتها بشكل سري، وهً تستخدم ألغراض البحث العلمً فقط, الرجاء إجابة األسئلة التالٌة بما ٌتناسب مع وجهة نظرك فً الخانة المناسبة.

المعلومات الديموغرافية. م المتغٌر اإلجابة 1. االسم 2. تارٌخ المٌالد 3. العمر بالسنة 4. العنوان 5. رقم الهاتف 6. المواطنة مواطن ألجًء 7. مكان السكن المدٌنة المعسكر القرى 8. المستوى التعلٌمً ابتدائً إعدادي ثانوي توجٌهً دبلوم جامعً 9. هل ٌعمل معٌل األسرة ٌعمل ال ٌعمل 11. نوع العمل حكومً وكالة غوث مجتمع مدنً خاص 11. الدخل اإلجمالً لألسرة بالشٌكل 12. عدد األبناء 13. عدد أفراد االسرة االجمال عدد األبناء الذٌن ٌذهبون .14 للمدرسة 15. الدخل الشهري لألسرة بالشٌكل

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المعلومات الطبية التغذوية. م المتغٌر اإلجابة 1. السكري نشاط الغدة الدرقٌة كسل الغدة الدرقٌة أمراض الكلى إذا كنت قد أصبت بأحد التهابات المفاصل أمراض الكبد األمراض التالٌة الرجاء)/( الصرع أمراض البنكرٌاس إشارة صح أمام المرض األزمة المزمنة المرارة الذي أصبت به لٌن أو هشاشة العظام النقرص أمراض الرنة المزمنة أخرى 2. أذكر أي مرض أو إعاقة صحٌة قد اصبت بها ولم ٌذكر فً البند السابق 3. هل تستخدم االدوٌة بالنظام نعم ال 4. إذا كانت اإلجابة نعم الرجاء ذكر النوع 5. هل تستخدم مكمالت غذائٌة بانتظام نعم ال 6. إذا كانت اإلجابة نعم الرجاء ذكر النوع 7. هل قم بإجراء عملٌات سابقا نعم ال 8. ما هو نوع العملٌة وتارٌخها 9. أمراض القلب جلطات أو أي امراض فً المخ ارتفاع ارتفاع الدهون الثالثٌة الكولٌسترول هل تعانً أحد هذه األمراض أمراض االوعٌة ارتفاع الدهون الثالثٌة الدموٌة أمراض االوعٌة األنٌمٌا الدموٌة السرطان 11. ما هو نوع السرطان 11. هل عانٌت من أحد أمراض الجهاز نعم ال الهضمً 12. إذا كانت اإلجابة نعم الرجاء ذكر نوع العالج المستخدم 13. القولون المعدة الثدي البنكرٌاس هل ٌعانً الوالدٌن أو أحد األقارب الرحم المخ أو الحبل الشوكً الدرجة األولى من أحد أنواع السرطان الفم المثانة التالٌة البلعوم الكبد أخرى 14. هل توفى أحد الوالدٌن أو األقارب الدرجة األولى من الجلطات الدماغٌة أو نعم ال القلبٌة

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.15 هل ٌعانً أحد الوالدٌن من مرض نعم ال السكري 16. هل ٌعانً أحد الوالدٌن أو األقارب الدرجة األولى من مرض اخر لم ٌذكر نعم ال سابقا

Gynecological and Obstetric History

17. عدد األطفال داخل األسرة 18. هل سبق لك وأن حصل عملٌة إجهاض نعم ال 19. إذا كانت اإلجابة نعم الرجاء ذكرها 21. المسافة بٌن الوالدة وسابقاتها بالمسنٌن 21. فً الوالدات السابقة هل حصل تشوه فً الجنٌن نعم ال 22. إذا كانت اإلجابة نعم الرجاء ذكرها 23. هل استخدمت سابقا أدوات تنظٌم حمل نعم ال 24. الرجاء ذكر األداة المستخدمة 25. الرجاء كتابة الفترة المستخدمة من -الى 26. سبب انهاء استخدام أدوات تنظٌم الحمل 27. هل تستخدمٌن حالٌا أدوات تنظٌم حمل نعم ال 28. الرجاء كتابة الفترة المستخدمة )من –الى( 29. سبب اختٌارك لهذه األداة 31. هل أوقفت استخدام مانع الحمل ألي سبب كان نعم ال 31. الرجاء ذكر سبب التوقٌف 32. هل أثرت أداة مانع الحمل على مزاجك نعم ال

33. هل الدورة الشهرٌة منتظمة نعم ال 34. مدة الدورة الشهرٌة 3 – 5 أٌام 5 – 7 أٌام أكثر من 7 أٌام الحالة الفسيولوجية.

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103

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Appendix B2- Food Frequency Questionnaire

استثانح تزدد الغذاء خالل العام

االسن: ...... التاريخ: ......

الزقن: ......

# الغذاء ال هزج واحذج 2-3 هزاخ 1-2 هزج 3 فأكثز 1-2 هزج 3 فأكثز اتناول تالشهز تالشهز تاألسثىع تاألسثىع تاليىم تاليىم

الخضزاواخ الطاسجح/السلطح

A1 اٌشضغاٚاث اٌٛعل١ت اٌّطبٛست ِثً اٌـبأز، اٌٍّٛس١ت، اٌـٍك

A2 اٌشضغاٚاث اٌٛعل١ت اٌطاػجت ِثً، اٌبمضؾٔٚ، اٌجغج١غ

A3 اٌٍّفٛف )ؿٍطت اٚ ِطبٛر(

A4 اٌجؼع )طاػج اٚ ِشًٍ اٚ ِجّض(

A5 اٌمغع-ا١ٌمط١ٓ

A6 اٌطّاطُ )طاػجت أٚ ِعٍبت اٚ عو١غ(

A7 بطاطا دٍٖٛ

A8 اٌفاه١ٌٛا اٌشضغاء )طاػجاً أٚ ِجّضة(

A9 اٌبا٠ػالء اٌشضغاء

A11 اٌٍٛب١اء سضغاء

A11 اٌبا١ِت

A12 اٌظعة )طاػجت أٚ ِعٍبت أٚ ِجّضة(

A13 اؼٌ٘غة )اٌمغٔب١ظ(

A14 بطاطؾ

A15 فٍفً داع

A16 س١اع

A17 بوً

A18 سضاع ِشٍٛطت

A19 اٌف١ٍفٍت أٚ اٌفٍفً اٌذٍٛ )األدّغ أٚ األهفغ أٚ األسضغ(

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A21 اٌباطٔجاْ

A21 وؿٛا

A22 اٞ سضاع اسغٜ ٌُ ٠غص طوغ٘ا

الفىاكه الطاسجح وعصائزها

ػِٛ B1

B2 حفاح

B3 وّثغٜ )االجام(

B4 كّاَ

B5 بط١ز

B6 بغحماي

B7 اّٛ١ٌٍْ

B8 وٍّأخ١ٕا /ِأضع٠ٕا

B9 جغ٠ب فغٚث

B11 اٌعٕب

B11 اٌشٛر/صعاق/اٌـٕخغػٚة

B12 اٌّلّق

B13 اٌخ١ٓ

B14 اٌجٛافت

B15 اٌّأجا

B16 اٌفغاٌٚت

B17 اٌبٍخ اٚ اٌغطب اٚ ِعجْٛ اٌخّغ

B18 اٌعوائغ اٌطب١ع١ت ٌٍفٛاوٗ

B19 اٞ فٛاوٗ اسغٞ ٌُ ٠غص طوغ٘ا

اللحىم وهنتجاخ اللحىم

C1 اٌّغحض٠ال اٚ اٌالٔلْٛ ٌٍذُ اٌبمغٞ

C2 اٌٍذّت اٌّفغِٚت ِثً اٌىفختأٚ اٌىبت

C3 ٌذُ اٌعجً اٚ اٌشاعٚف اٌّطبٛر ِع اٌشضغٚاث أٚ ِعجٕاحاٌٍذّت )عغاؾ٠(

C4 ٌذّت اٌعجً اٚ اٌشاعٚف اٌّلٞٛ أٚ اٌّذّغ

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C5 االدلاء )اٌىبضة اٚ اٌىالٞٚ اٚ اٌطذاي(

C6 ٘اِبٛعجغ اٚ حلؼ١بغغغ

الذواجن

D1 اٌلغائخ اٌّب ّغصة ِٓ اٌضجاج أٚ اٌذبق )ِغحض٠ال اٌضجاجأٚ اٌذبق(

D2 اٌضجاج اٌّـٍٛق أٚ اٌّلٞٛ

D3 اٌضجاج اٌّمٍٟ

االسواك والوأكىالخ الثحزيح

E1 االؿّان ِم١ٍت اٚ ِل٠ٛت

E2 ِعٍباث األؿّان

E3 اٌخٛٔت

E4 اٌجّبغٞ

الحثىب

F1 اٌشبؼ

F2 األعػ

F3 اٌّعىغٚٔت اٚ اٌـباج١خٟ

F4 اٌبغغً اٚ اٌفغ٠ىٗ

F5 إٌٛصؼٌ اٚ االٔضِٟٚ

F6 اٌلعغ٠ت

الثقىلياخ الجافح

G1 فٛي دب اٚ ِجغٚف

G2 دّن

G3 فاه١ٌٛاء أٚ با٠ػالء

G4 عضؽ دب اٚ ِجغٚف

G5 اٌفالفً

الثيض وهشتقاته

H1 اٌب١ض

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# الغذاء ال هزج واحذج 2-3 هزاخ 1-2 هزج 3 فأكثز 1-2 هزج 3 فأكثز اتناول تالشهز تالشهز تاألسثىع تاألسثىع تاليىم تاليىم

الحلية وهشتقاته

I1 د١ٍب

I2 د١ٍب ل١ًٍ اٌضؿُ

I3 ٌبٓ )ك١ّٕج(

I4 ٌبٓ عاصٞ اٚ اٌٍبٕت

I5 جبٕت ب١ضاء بٍض٠ت

I6 األجباْ اٌوفغاء

الوكسزاخ

J1 اٌفـخك اٌذٍبٚ ٟاٚ ػٌٍٛاٌىاكٚ ٛاٌبٕضلٛغ١غ٘ا

الوشزوتاخ

K1 اٌلاٞ

K2 اٌمٛٙة

K3 اٌمٛٙة باٌذ١ٍب اٚ اٌىغ٠ّا

K4 اٌّلغٚباث اٌغا٠ػت

K5 اٌعوائغ اٌوٕاع١ت )إٌىٙاث اٌّشخٍفت(

الىجثاخ الخفيفح )السناكس( والحلىياخ

L1 اٌّعجٕاث – بضْٚ ٌذّٗ

L2 اٌب١خؼا

L3 اٌب١خٟ فٛع

L4 اٌىعه ٚاٌّعّٛي

L5 اٌىٕافت،اٌبمالٚة، إٌّٛعة، اٌٙغ٠ـت(

L6 اٌى١ه

L7 اٌلىٛالحت

الشىرتاخ والصلصاخ

M1 كٛعباث اٌشضاع

M2 كٛعباث اٌبم١ٌٛاث

M3 هٍوت اٌّاؼ١ٔٛ٠

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M4 هٍوت باٌجبٓ

السكاكز

N1 اٌـىغ

N2 اٌعـً

N3 اٌضبؾ، اٌّغبٝ

N4 اٌجٍٟ

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Appendix C Power and Sample Size Software Result

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World Health Organization. (2007). Family Planning: A Global Handbook for Providers: Evidence-based Guidance Developed Through Worldwide Collaboration: Johns Hopkins Ccp-Info.

World Health Organization. Reproductive (2010). Medical eligibility criteria for contraceptive use: World Health Organization.

Yamaji, S., Tennant, J., Tandy, S., Williams, M., Singh Srai, S. K., & Sharp, P. (2001). Zinc regulates the function and expression of the iron transporters DMT1 and IREG1 in human intestinal Cacoa cells. FEBS letters, 507(2), 137- 141.

Yan, J., Liao, J. K., & Wang, D. (2010). Elevated homocysteine and C-reactive protein levels independently predict worsening prognosis after stroke in Chinese patients. Journal of Huazhong University of Science and Technology [Medical Sciences], 30(5), 643-647.

Zhang, Y., Zhu, T., Wang, L., Pan, Y. H., & Zhang, S. (2013). Homocysteine homeostasis and betaine-homocysteine S-methyltransferase expression in the brain of hibernating bats. PloS one, 8(12), e85632.

Wiegratz, I., Bassol, S., Weisberg, E., Mellinger, U. & Merz, M. (2014) Effect Of A Low-Dose Contraceptive Patch On Efficacy, Bleeding Pattern, And Safety, Sage.

Workowski, K. A. & Bolan, G. A. (2015) Sexually Transmitted Diseases Treatment Guidelines, 2015. Mmwr. Recommendations And Reports: Morbidity And Mortality Weekly Report. Recommendations And Reports, 64, 1.

Appendices Appendix A Permission and Ethical Approval Appendix A1- Approval from Helsinki Committee

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Appendix A2- Consent form

إقرار موافقة للمشاركة في دراسة بحثية

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جامعة األزهر بغزة تأثير حبوب منع الحمل على بعض الفيتامينات والمعادن على النساء في سن اإلنجاب

أن ِت مدعوه للمشاركة في هذه الدراسة البحثية بواسطة الباحثة أسماء محارب مغاري

الرجاء مراجعة هذا النموذج وطرح ومناقشة أي أسئلة تريدينها مع أخذ الوقت الكافي لذلك، حيث يمكنك مناقشة ومراجعة طبيبك المختص، عائلتك، وأصدقائك وذلك التخاذ القرار بالمشاركة أو عدم المشاركة في هذا البحث. قد يحتوي هذا النموذج بعض الكلمات التي ال فهمها، لذلك الرجاء طرح األسئلة حولها لغرض تفسيرها بشكل واضح.

الهدف من الدراسة هي الختبار مدى تأثير حبوب منع الحمل ثنائيه النوع على مستوى بعض الفيتامينات والمعادن في الدم، وهل هناك تأثير أكبر مرتبط بمدة استخدام حبوب منع الحمل.

في هذه الدراسة سوف يتم اختيارك عشوائيا في إحدى مجموعات الدراسة التي سيتم شرحها في األسفل. حيث أن كلمة عشوائيا تعني أنه لديك نفس الفرصة في أن تكون في أحد تلك المجموعتين، مثل أن نرمي قطعة نقود.

المجموعة األولى: هي المجموعة المقارنة، وهي التي تأخذ أي نوع من وسائل منع الحمل غير حبوب منع الحمل ثنائية التركيب.

المجموعة الثانية: هي المجموعة المستهدفة، وهي التي تأخذ حبوب منع الحمل ثنائية التركيب.

تشمل المشاركة في البحث اإلجراءات التالية:

1.اخذ بعض البيانات الشخصية مثل االسم، العنوان، السكن ورقم الهاتف.

2.اخذ التاريخ الصحي للشخص المرشح.

3.اخذ الطول الوزن وحساب كتلة الجسم ومحيط الوسط.

4.سحب عينات من الدم على يد مختص.

5.كل البيانات سيتم إدخالها لبرامج التحليل االحصائي، وسيتم مناقشة ونشر النتائج.

6.سيتم ابالغ المشتركين بنتائجهم أوال بأول.

7.المشاركة في هذا البحث هي مشاركة تطوعية.

المخاطر التي تصحب الدراسة:

ال توجد مخاطر على صحة المشتركين في االنضمام لهذا البحث.

الفوائد المحتملة:

هذه البحث هو امتداد ال بحاث اخرى تهد ف الى التعرف عل بعض الجوانب السيئة لحبوب منع الحمل ثنائيه التركيب، والخروج بتوصية تهدف الى استخدام بعض الفيتامينات والمعادن قبل الشروع بالحمل.

مصروفات الدراسة:

جميع مصروفات الدراسة من قياسات وتحليل عينات الدم ومقارنتها وأخذ قياسات الجسم وكل إجراءات الدراسة ستكون بشكل مجاني للمشتركات في الدراسة

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حماية سرية البيانات:

سوف يتم نشر جميع النتائج والتحليالت اإلحصائية للنتائج التي تم جمعها خالل الدراسة في المؤتمرات العلمية وجميع المحافل العلمية، مع األخذ بعين االعتبار أنه لن يتم نشر اسمك أو اي بينات تعريفية عنك.

سيتم التعامل مع جميع البيانات الشخصية والسجالت الطبية التي تحتوي على هويتك على أنها سرية وفقا لقانون جامعة األزهر والميثاق األخالقي الخاص بذلك. على الرغم أننا سنبذل جهود قاسية لحفظ سرية بياناتك، إال أن هذه الجهود ليست مطلقة.

سوف أقوم باستبدال اسمك الشخصي برقم أو كود من جميع السجالت الطبية التي ستطلب منا لغرض مراجعة إجراءات البحث من قبل الجامعة أو المشرفين، حيث سيتم حذف أي رقم هاتف أو أي معلومة خاصة بك منها.

سيتم االحتفاظ بكافة السجالت الطبية التي تحتوي على اسمك ومعلومات شخصية عنك في مكان امن، حيث أن البحاث هو فقط من سيقوم باالطالع على هذه البيانات لغرض الدراسة فقط. حيث سيتم إخطار طبيب األسرة أو طبيبك المختص بمشاركتك في هذا البحث.

نوع المشاركة:

مشاركتك في هذا البحث هي مشاركة تطوعية، حيث بإمكانك عدم المشاركة أو االنسحاب في أي وقت تريدينه أثناء المشاركة، وسف لن تتحملي أي نتائج أو عقوبات أو غرامة مالية أو أي نوع من أنواع العقوبات بسبب االنسحاب من البحث.

أسئلة حول الدراسة:

لديك الحق الكامل في طرح أي سؤال يخطر ببالك حول حقوقك أو أي معلومات تردينها أو تريدين أن تستفسري عنها خالل أو قبل أو بعد الدراسة، وذلك بالتواصل مع الباحثة اسماء محارب مغاري، هاتف رقم0599741476 وعلى البريد االلكتروني: [email protected]

ولالستفسار حول حقوقك كمشتركه في بحث سريري يمكنك مراسلة مجلس جامعة األزهر للدراسات العليا على البريد االلكتروني التالي: [email protected]

ال توقعي على الموافقة في هذا البحث في حال عدم أخذ الفرصة الكافية في الرد على استفساراتك أو أنك غير مقتنع باألجوبة المقدمة من الباحثة.

معلومات االتصال:

إذا كان لديك أي أسئلة أو استفسارات حول هذه الدراسة أو إذا حدثت أي مشاكل، يرجى االتصال مباشرة في البحاث

)اسماء محارب مغاري( على رقم الهاتف التالي:0599741476، البريد اإللكتروني التالي:

. [email protected]

بيان الموافقة:

لقد قرأت نموذج الموافقة للمشاركة في هذا البحث.وقد أتيحت لي الفرصة الكاملة لمناقشة هذه الدراسة البحثية مع الباحثة أسماء محارب مغاري.ولقد أجابت عن جميع أسئلتي باللغة التي أفهمها حيث تم شرح جميع المخاطر والمنافع الخاصة بالدراسة. أأكد أنه لم يتم التأثير على للمشاركة في هذا البحث من قبل الباحثة اسماء محارب مغاري أو أي شخص اخر بأي شكل من األشكال.

أنا أفهم أنني سوف أعطى نسخة من هذا االقرار على نموذج الموافقة بعد التوقيع عليه، وأفهم أن مشاركتي في هذا البحث هي مشاركة طوعية وتطوعية، وأنه لدي الحق الكامل في االشتراك او رفض االشتراك أو االنسحاب من الدراسة في اي وقت اريد.

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إنني أعلم أن جميع المعلومات المتعلقة بهويتي الشخصية ستبقى سرية ولكن ليست بشكل مطلق، كما أنني أفوض جامعة األزهر والمشرفين على الباحثة أسماء محارب مغاري بأن يقوموا بالتفتيش على جميع السجالت الطبية الخاصة بي وذلك لغرض المراقبة والدراسة فقط.

من خالل توقيعي على هذا نموذج إقرار الموافقة على المشاركة في هذا البحث أنا لن أتنازل عن أي من حقوقي القانونية كمشارك في هذه الدراسة البحثية

الموافق على االشتراك في هذا البحث:نعم ال 

اقرا ر الموافقة:

لقد قرأت هذا النموذج وأعطيت الفرصة لطرح األسئلة واإلجابة عنها بشكل واضح وبلغة مفهومة جدا. لذلك أعطي موافقتي على المشاركة في هذه الدراسة.

االسم رباعي: ......

التوقيع: ...... تاريخ التوقيع: ......

Appendix B Questionnaire Appendix B1- Questionnaire

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أختي العزيزة، السالم عليكم ورحمة هللا وبركاته...

هذه االستبانة التي بين يديك،هي جزء مهم من دراسة أقوم بها أنا الباحثة/ أسماء محارب سلمان المغاري، والتي تهدف الى معرفة مدى تأثير حبوب موانع الحمل المركبة على الفيتامينات و المعادن بشكل أساسي ، وذلك لنيل درجة الماجستير في برنامج التغذية االكلينيكية- كلية الصيدلة بجامعة األزهر-غزة، سيأخذ االستبيان بضع الدقائق من وقتكم، مع العلم أن المشاركة في هذه الدراسة هي مشاركة طوعية، وأن جميع المعلومات سيتم معالجتها بشكل سري، وهي تستخدم ألغراض البحث العلمي فقط, الرجاء إجابة األسئلة التالية بما يتناسب مع وجهة نظرك في الخانة المناسبة.

المعلومات الديموغرافية. م المتغير اإلجابة 1. االسم 2. تاريخ الميالد 3. العمر بالسنة 4. العنوان 5. رقم الهاتف 6. المواطنة مواطن ألجيء 7. مكان السكن المدينة المعسكر القرى 8. المستوى التعليمي ابتدائي إعدادي ثانوي توجيهي دبلوم جامعي 9. هل يعمل معيل األسرة يعمل ال يعمل 10. نوع العمل حكومي وكالة غوث مجتمع مدني خاص 11. الدخل اإلجمالي لألسرة بالشيكل 12. عدد األبناء 13. عدد أفراد االسرة االجمال عدد األبناء الذين يذهبون .14 للمدرسة 15. الدخل الشهري لألسرة بالشيكل

المعلومات الطبية التغذوية. م المتغير اإلجابة 1. إذا كنت قد أصبت بأحد السكري نشاط الغدة الدرقية األمراض التالية الرجاء)/( كسل الغدة الدرقية أمراض الكلى إشارة صح أمام المرض التهابات المفاصل أمراض الكبد

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الذي أصبت به الصرع أمراض البنكرياس األزمة المزمنة المرارة لين أو هشاشة العظام النقرص أمراض الرنة المزمنة أخرى 2. أذكر أي مرض أو إعاقة صحية قد اصبت بها ولم يذكر في البند السابق 3. هل تستخدم االدوية بالنظام نعم ال 4. إذا كانت اإلجابة نعم الرجاء ذكر النوع 5. هل تستخدم مكمالت غذائية بانتظام نعم ال 6. إذا كانت اإلجابة نعم الرجاء ذكر النوع 7. هل قم بإجراء عمليات سابقا نعم ال 8. ما هو نوع العملية وتاريخها 9. أمراض القلب جلطات أو أي امراض في المخ ارتفاع ارتفاع الدهون الثالثية الكوليسترول هل تعاني أحد هذه األمراض أمراض االوعية ارتفاع الدهون الثالثية الدموية أمراض االوعية األنيميا الدموية السرطان 10. ما هو نوع السرطان 11. هل عانيت من أحد أمراض الجهاز نعم ال الهضمي 12. إذا كانت اإلجابة نعم الرجاء ذكر نوع العالج المستخدم 13. القولون المعدة الثدي البنكرياس هل يعاني الوالدين أو أحد األقارب الرحم المخ أو الحبل الشوكي الدرجة األولى من أحد أنواع السرطان الفم المثانة التالية البلعوم الكبد أخرى 14. هل توفى أحد الوالدين أو األقارب الدرجة األولى من الجلطات الدماغية أو نعم ال القلبية .15 هل يعاني أحد الوالدين من مرض نعم ال السكري 16. هل يعاني أحد الوالدين أو األقارب الدرجة األولى من مرض اخر لم يذكر نعم ال سابقا

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Gynecological and Obstetric History

17. عدد األطفال داخل األسرة 18. هل سبق لك وأن حصل عملية إجهاض نعم ال 19. إذا كانت اإلجابة نعم الرجاء ذكرها 20. المسافة بين الوالدة وسابقاتها بالمسنين 21. في الوالدات السابقة هل حصل تشوه في الجنين نعم ال 22. إذا كانت اإلجابة نعم الرجاء ذكرها 23. هل استخدمت سابقا أدوات تنظيم حمل نعم ال 24. الرجاء ذكر األداة المستخدمة 25. الرجاء كتابة الفترة المستخدمة من -الى 26. سبب انهاء استخدام أدوات تنظيم الحمل 27. هل تستخدمين حاليا أدوات تنظيم حمل نعم ال 28. الرجاء كتابة الفترة المستخدمة )من –الى( 29. سبب اختيارك لهذه األداة 30. هل أوقفت استخدام مانع الحمل ألي سبب كان نعم ال 31. الرجاء ذكر سبب التوقيف 32. هل أثرت أداة مانع الحمل على مزاجك نعم ال

33. هل الدورة الشهرية منتظمة نعم ال 34. مدة الدورة الشهرية 3 – 5 أيام 5 – 7 أيام أكثر من 7 أيام الحالة الفسيولوجية.

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Appendix B2- Food Frequency Questionnaire

استبانة تردد الغذاء خالل العام

االسم: ...... التاريخ: ......

الرقم: ......

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# الغذاء ال مرة واحدة 2-3 مرات 1-2 مرة 3 فأكثر 1-2 مرة 3 فأكثر اتناول بالشهر بالشهر باألسبوع باألسبوع باليوم باليوم

الخضراوات الطازجة/السلطة

A1 الخضراوات الورقية المطبوخة مثل السبانخ، الملوخية، السلق

A2 الخضراوات الورقية الطازجة مثل، البقدونس، الجرجير

A3 الملفوف )سلطة او مطبوخ(

A4 الجزر )طازج او مخلل او مجمد(

A5 القرع-اليقطين

A6 الطماطم )طازجة أو معلبة او عصير(

A7 بطاطا حلوه

A8 الفاصوليا الخضراء )طازجاً أو مجمدة(

A9 البازيالء الخضراء

A10 اللوبياء خضراء

A11 البامية

A12 الذرة )طازجة أو معلبة أو مجمدة(

A13 الزهرة )القرنبيط(

A14 بطاطس

A15 فلفل حار

A16 خيار

A17 بصل

A18 خضار مخلوطة

A19 الفليفلة أو الفلفل الحلو )األحمر أو األصفر أو األخضر(

A20 الباذنجان

A21 كوسا

A22 اي خضار اخرى لم يرد ذكرها

الفواكه الطازجة وعصائرها

B1 موز

B2 تفاح

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B3 كمثرى )االجاص(

B4 شمام

B5 بطيخ

B6 برتقال

B7 الليمون

B8 كلمانتينا /ماندرينا

B9 جريب فروت

B10 العنب

B11 الخوخ/دراق/السنتروزة

B12 المشمش

B13 التين

B14 الجوافة

B15 المانجا

B16 الفراولة

B17 البلح او الرطب او معجون التمر

B18 العصائر الطبيعية للفواكه

B19 اي فواكه اخري لم يرد ذكرها

اللحوم ومنتجات اللحوم

C1 المرتديال او الالنشون للحم البقري

C2 اللحمة المفرومة مثل الكفتةأو الكبة

C3 لحم العجل او الخاروف المطبوخ مع الخضروات أو معجناتاللحمة )عرايس(

C4 لحمة العجل او الخاروف المشوي أو المحمر

C5 االحشاء )الكبدة او الكالوي او الطحال(

C6 هامبورجر او تشيزبرغر

الدواجن

D1 الشرائح المب ّردة من الدجاج أو الحبش )مرتديال الدجاجأو الحبش(

D2 الدجاج المسلوق أو المشوي

D3 الدجاج المقلي

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االسماك والمأكوالت البحرية

E1 االسماك مقلية او مشوية

E2 معلبات األسماك

E3 التونة

E4 الجمبري

الحبوب

F1 الخبز

F2 األرز

F3 المعكرونة او السباجيتي

F4 البرغل او الفريكه

F5 النودلز او االندومي

F6 الشعرية

البقوليات الجافة

G1 فول حب او مجروش

G2 حمص

G3 فاصولياء أو بازيالء

G4 عدس حب او مجروش

G5 الفالفل

البيض ومشتقاته

H1 البيض

# الغذاء ال مرة واحدة 2-3 مرات 1-2 مرة 3 فأكثر 1-2 مرة 3 فأكثر اتناول بالشهر بالشهر باألسبوع باألسبوع باليوم باليوم

الحليب ومشتقاته

I1 حليب

I2 حليب قليل الدسم

I3 لبن )شمينت(

I4 لبن عادي او اللبنة

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I5 جبنة بيضاء بلدية

I6 األجبان الصفراء

المكسرات

J1 الفستق الحلبي واللوز والكاشو والبندقوغيرها

المشروبات

K1 الشاي

K2 القهوة

K3 القهوة بالحليب او الكريما

K4 المشروبات الغازية

K5 العصائر الصناعية )النكهات المختلفة(

الوجبات الخفيفة )السناكس( والحلويات

L1 المعجنات – بدون لحمه

L2 البيتزا

L3 البيتي فور

L4 الكعك والمعمول

L5 الكنافة،البقالوة، النمورة، الهريسة(

L6 الكيك

L7 الشكوالتة

الشوربات والصلصات

M1 شوربات الخضار

M2 شوربات البقوليات

M3 صلصة المايونيز

M4 صلصة بالجبن

السكاكر

N1 السكر

N2 العسل

N3 الدبس، المربى

N4 الجلي

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Appendix C Power and Sample Size Software Result

109

110