RBC PARAMETERS, VITAMIN B12, FOLATE, HOMOCYSTEINE LEVELS AND MTHFR GENE POLYMORPHISM IN POSTMENOPAUSAL MALAY WOMEN AND THEIR RELATION TO HORMONE REPLACEMENT THERAPY
BY
MOHAMED TALAL ABD ALFATTAH AL JABR
Thesis submitted in fulfillment of the requirements for the degree of Master of Science
UNIVERSITI SAINS MALAYSIA
2011
II DEDICATION
I would like to dedicate this dissertation to my wife, whose support has been unwavering and to my beloved parents. Their moral and financial support has been beyond all limits.
III ACKNOWLEDGEMENTS
First of all, I would like to thank Allah (S.W) for giving me the strength and courage to carry out this work.
I would like to thank the Universiti Sains Malaysia for providing the short term grant that funded my research project.
I would also like to express my utmost gratitude and appreciation to my main supervisor, Dr. Suhair Abbas Ahmed, and to my co-supervisors AP Dr. Wan Zaidah Abdullah and AP Dr. Nik Hazlina Nik Hussain, for their invaluable suggestions and expert advice throughout the course of my study.
My sincere and special gratitude to the Head of Department of Haematology, AP Dr.
Rosline Hassan, for her constructive support.
I would like to express my appreciation to Prof. Dr. Fawwaz Al-Joudi, for his kind advice and support throughout my work.
My deepest thanks go to staff and students at Department of Haematology, especially Wan Soriany Bt Wan MD Zain, Puan Suryati Abdullah, Noor Adzha Abd Majid and Mohd Annuar Nordin for their assistance and support during laboratory work.
IV Many thanks are due to Intan Idiana Hassan, for her assistance and support, during sample collection and arrangements with patients.
Special thanks to the staff of the Molecular Haematology Section especially to Ms Selamah Ghazali, Ms Ang Cheng Yong and Mrs Narishah Sharif for their help throughout my study.
I should not forget to thank all the patients who took the time and trouble to participate in my study.
Last, but not the least, I would like to thank all my fellow colleagues and friends who have directly or indirectly participated in making this study possible. While it might be not possible to name them all here, their help is forever unforgettable.
Mohamed Talal Al Jabr
V
TABLE OF CONTENTS
Contents Page TITLE I
DEDICATION II ACKNOWLEDGEMENTS III TABLE OF CONTENTS V LIST OF TABLES XII LIST OF FIGURES XIV LIST OF ABBREVIATIONS XV ABSTRAK XVIII ABSTRACT XXI CHAPTER ONE: INTRODUCTION
1.0. Introduction and literature review 2
1.1. Menopause 3
1.2. Physiological changes 4
1.3. Effects of menopause 5
1.3.1. Short term effects 5
1.3.2. Long term effects 8
1.4. Therapeutic options 14
1.5. Red blood cell parameters 19
1.5.1. Red blood cells/Erythrocytes 20
1.5.2. Haemoglobin 21
1.5.3. Haematocrit or Packed Cell Volume 21
1.5.4. Mean Cell Volume (MCV) 22
VI
1.5.5. Mean Cell Haemoglobin (MCH) 22
1.5.6. Mean Cell Haemoglobin Concentration (MCHC) 22
1.5.7. Red cell distribution width (RDW) 23
1.6. Red blood cell, vitamin B12 and folate metabolism 23
1.6.1. Vitamin B12 23
1.6.1. (A) Biochemistry 25
1.6.1. (B) B12 absorption 26
1.6.2. Folate 30
1.6.2. (A) Biochemistry 31
1.6.2. (B) Folate absorption 32
1.6.3. Megaloblastic anaemia 35
1.6.3. (A) Megaloblastic change in Peripheral blood 35
1.7. Homocysteine and hormones 37
1.7.1. Metabolism of homocysteine 38
1.7.2. Increase Homocysteine levels 40
1.7.2. (A) Life-style 40
1-Smoking 40
2- High alcohol intake 41
3-Increased coffee consumption 42
4-Inadequate nutrition 43
5-Age-related factors 43
6-Hormonal changes 43
1.7.3. Homocysteine and cardiovascular diseases 43
VII
1.7.4. Hyperhomocysteinemia and genetic causes 44
1.8. MTHFR GENE 46
1.9 Rationale of the study 49
2.0 Research Questions 49
CHAPTER TWO: OBJECTIVES 2.0 Objectives of the study 51
CHAPTER THREE: MATERIALS AND METHODS 3.0. Study design 53
3.1. Sample size determination 53
3.2. Subjects selection 54
3.3. Inclusion and exclusion criteria for postmenopausal and premenopausal subjects 54 3.3.1. Inclusion criteria 54
3.3.2. Exclusion criteria 55
3.4. Blood sample and tests 55
3.5. Test methods 57
3.5.1 Full blood count (FBC) 57
3.5.1.1 Assay procedure 58
3.5.1.1 (A). Reagents and samples preparation 58
3.5.1.1 (B). Procedure 58
3.5.2 Measurement of serum B12 level 59
3.5.2.1. Assay procedure 59
3.5.2.1 (A). Reagents and samples preparation 59
3.5.2.1 (B). Principle 59
VIII
3.5.2.1 (C). Procedure 60
3.5.3. Measurement of serum folate level 61
3.5.3.1. Assay procedure 61
3.5.3.1 (A). Reagents and samples preparation 61
3.5.3.1 (B) principle 61
3.5.3.1 (C). Procedure 62
3.5.4. Measurement of RBC folate 62
3.5.4.1. Assay procedure 63
3.5.4.1 (A). Reagents and samples preparation 63
3.5.4.1 (B) principle 63
3.5.4.1 (C). Preparation of haemolysates 63
3.5.4.1 (D). Procedure 64
3.5.5. Measurement of Plasma homocysteine level 65
3.5.5.1 Assay procedure 65
3.5.5.1 (A). Reagents and samples preparation 65
3.5.5.1 (B). Principle of test 65
3.5.5.1 (C). Procedure 66
3.5.6 Detection of methylene tetrahydrofolate reductase (MTHFR) 677C>T gene polymorphisms by Molecular tests (PCR) 66
3.5.6.1 Polymerase Chain Reaction (PCR): Principles and step 66
3.5.6.2. Preparation of glassware and tips 68
3.5.6.3. DNA extraction 69
3.5.6.3 (A) DNA extraction reagents 69
3.5.6.4. Isolation of total cellular DNA from human whole blood 70
IX
3.5.6.5. Quantitative measurement of DNA 72
3.5.6.6 Detection of 677C>T Mutation of the Methylenetetrahydrofolate Reductase (MTHFR) 72
3.5.6.6. (A) Principle 73
3.5.6.6. (B) PCR Method 73
3.6. Analysis of data 79
3.7. Flow chart for Study Design 80
CHAPTER FOUR: RESULTS 4.1. Demographic data 82
4.1.1. Age 82
4.1.2. Race 82
4.1.3. Level of educational background 84
4.1.4. Monthly income 85
4.1.5. Duration of menopause 86
4.2. The comparison between non HRT group and HRT group 86
4.2.1. The comparison on full blood count parameters between the study groups 86 4.2.1.1 Total red blood cell (RBC) count 87
4.2.1.2 Haemoglobin (Hb) 88
4.2.1.3 Haematocrit (HCT) 89
4.2.1.4 Mean Cell Volume (MCV) 90
4.2.1.5 Mean Cell Haemoglobin (MCH) 91
4.2.1.6 Mean Cell Haemoglobin Concentration (MCHC) 92
4.2.1.7 Red Cell Distribution Width (RDW) 92
4.2.1.8 Platelets (PLT) count 93
X
4.2.1.9 Summary table 94
4.2.2. The comparison of mean different on Folate levels between non HRT group and HRT group 95
4.2.2.1 Serum Folate 95
4.2.2.2 Red Blood Cell folate levels 96
4.2.3. The comparison of mean different on Vitamin B12 between non-HRT group and HRT groups 97
4.2.4.The comparison on Homocysteine level (Hcy) between premenopausal women and postmenopausal women 98
4.2.4.1. The comparison on Hcy between premenopausal women and postmenopausal women (HRT group and non-HRT groups) 98
4.2.4.2. The comparison on Homocysteine level between postmenopausal women non-HRT group and HRT group 99
4.2.5. The comparison on MTHFR 677C>T gene polymorphisms between non-HRT group and HRT group with high Hcy level 100
4.2.6. The comparison on MTHFR 677C>T gene polymorphisms between non-HRT group and HRT group with normal Hcy level 102
4.3. Correlation between Hcy and RBC parameters in all menopausal women 105
4.3.1. Correlation between Hcy and vitamin B12 and serum/ RBC folate in all menopausal women 106
4.3.2. Association between Hcy and MTHFR gene polymorphisms 107
4.4. Correlation between vitamin B12 and all variables 108
4.5. Correlation between serum folate and all variables 109
CHAPTER FIVE: DIESCUSION 5.0. Discussion 113
5.1. Limitation and strength of the study 124
XI
5.2. Recommendations 125
CHAPTER SIX: CONCLUSION 6.0. Conclusions 127
REFERENCES 129
APPENDICES 144
PRESENTATIONS 150
Reagent used 159
XII LIST OF TABLES
Page
Table 1.1 Biochemical differentiations of vitamin B12 and Folic acid 34
deficiency Table 3.1 Volume of master mix for MTHFR gene mutation 75
Table 3.2 PCR thermal cycling profile for MTHFR gene mutation 76
Table 3.3 Interpretation of visualized fragments in MTHFR gene mutation 78
Table 4.1 Demographic variables of the study population 83
Table 4.2 The education level results in non-HRT group and HRT group 85
Table 4.3 Income level in non-HRT group and HRT group 85
Table 4.4 The duration of menopause results in non-HRT group and HRT group 86
Table 4.5 The total RBC count results in non-HRT group and HRT group 87
Table 4.6 The total RBC count results in non-HRT group and HRT group 87
Table 4.7 The Hb results in non-HRT group and HRT group 88
Table 4.8 The Hb results in non-HRT group and HRT group 88
Table 4.9 The HCT level results in non-HRT group and HRT group 89
Table 4.10 The HCT level results in non-HRT group and HRT group 89
Table 4.11 The MCV level results in non-HRT group and HRT group 90
Table 4.12 The MCV level results in non-HRT group and HRT group 90
Table 4.13 The MCH level results in non-HRT group and HRT group 91
Table 4.14 The MCH level results in non-HRT group and HRT group 91
Table 4.15 The MCHC level results in non-HRT group and HRT group 92
Table 4.16 The RDW level results in non-HRT group and HRT group 93
Table 4.17 The RDW level results in non-HRT group and HRT group 93
XIII
Table 4.18 The Platelets count results in non-HRT group and HRT group 94
Table 4.19 The Platelets count results in non-HRT group and HRT group 94
Table 4.20 RBC parameter results in non-HRT group and HRT group 94
Table 4.21 The serum folate results in non-HRT group and HRT group 96
Table 4.22 The serum folate results in non-HRT group and HRT group 96
Table 4.23 The RBC folate level results in non-HRT group and HRT group 97
Table 4.24 The RBC folate level results in non-HRT group and HRT group 97
Table 4.25 The Vit.B12 results in non-HRT group and HRT group 98
Table 4.26 The Vit.B12 results in non-HRT group and HRT group 98
Table 4.27 The total Homocysteine results in premenopausal women 99
and postmenopausal women (non-HRT group and HRT group) Table 4.28 The total Homocysteine results in postmenopausal women 100
non-HRT group and HRT group Table 4.29 The Homocysteine results in non-HRT group and HRT group 100
Table 4.30 The MTHFR gene mutation results in non-HRT group and HRT group with high Hcy level 101
Table 4.31 The MTHFR gene mutation results in non-HRT group and HRT group with normal Hcy level 103
Table 4.32 Correlation between Hcy and RBC parameters 105
Table 4.33 Correlation between Hcy and vitamin B12 and serum/ RBC folate 106
Table 4.34 Association between Hcy and MTHFR gene polymorphisms 108
Table 4.35 Correlation between vitamin B12 and all variables 108
Table 4.36 Association between S.B12 and MTHFR gene polymorphisms 109
Table 4.37 Correlation between serum folate and all variables 110
Table 4.38 Association between S. folate and MTHFR gene polymorphisms 110
XIV LIST OF FIGURES
Page
Figure 1.1 Human Red Blood Cells 21
Figure 1.2 Vitamin B12 structure 26
Figure 1.3 Vitamin B12 absorption 27
Figure 1.4 Folate structure 31
Figure 1.5 Folate metabolism 33
Figure 1.6 hypersegmented neutrophil 36
Figure 1.7 Homocysteine metabolism 39
Figure 1.8 Methylenetetrahydrofolate structure 40
Figure 1.9 Effect of alcohol on methyl transfer 42
Figure 4.1 Age of subjects without HRT 83
Figure 4.2 Age of subjects (HRT group) 84
Figure 4.3 Example of MTHFR gene mutation study in subjects with high 102
Hcy level by PCR. Figure 4.4 Example of MTHFR gene mutation study in subjects with normal 104
Hcy level by PCR. Figure 4.5 Example of MTHFR gene mutation study in subjects with normal 105
Hcy level by PCR Figure 4.6 Correlation between Hcy and RBC folate 107
Figure 4.7 Correlation between serum Folate and RBC Folate 111
XV
LIST OF ABBREVIATIONS
a Alanine
Adenosyl-Cbl Adenosylcobalamin ATP Adenosine Triphosfate
BHMT Betaine Homocysteine Methyltransferase BMD Bone Mineral Density
bp Base pair
CAD Coronary Artery Disease CbI Cobalamins
CBS Cystathionine B-Synthase CHD Coronary Heart Disease CN- Cyanide anion
CO2 Carbon dioxide
CVD Cardio Vascular Disease df degree of freedom DNA Deoxyribonucleic acid
E1 Estrone
E2 Estradiol
E3 Estriol
EDTA Ethylenediamine Tetra Acetic Acid
EPT Estrogen Progesterone Treatment
ET Estrogen Therapy
FA Folic Acid FBC Full Blood Count
FH4 Tetrahydrofolate
FMP Final Menstrual Period
FSH Follicle Stimulating Hormone
Hb Haemoglobin
Hct haematocrit
XVI Hcy Homocysteine
HRT Hormone Replacement Therapy
HUSM Hospital Universiti Sains Malaysia ICA Internal Carotid Artery
IF Intrinsic Factor
LH Luteinizing Hormone
LW Low molecular Weight
MAT Methionine Adenosyltransferase
MCH Mean Cell Hemoglobin
MCHC Mean Cell Haemoglobin Concentration
MCV Mean Cell Volume
Methyl-Cbl Methylcobalamin
Methyl-FH4 Methyl-Tetrahydrofolates
MI Myocardial Infarction
MS Methionine Synthase
MTHF Methyltetrahydrofolate
MTHFR Methylenetetrahydrofolate reductase N Number
NC Normal Control NTC No Template Control
O2 Oxygen
PCR Polymerase Chain Reaction
PCV Paced Cell Volume
PGA Pteroylglutamic Acid
PLT Platelets
PML Postmethionine Loading
POF Premature Ovarian Failure POS Positive Control
R Pearson correlation
RBC Red Blood Cell
XVII
RDA Recommended Daily Allowance
SAH S-Adenosylhomocysteine
SAHH S-Adenosyl-L-Homocysteine-Hydrolase
SAM S-adenosylmethionine
SD Standard Deviation
SPSS Statistical Package for Social Sciences TCI Transcobalamin I
TCII Transcobalamin II
V Valine
WHI World Health Initiative
WHO World Health Organization
X2 Pearson Chi-square
XVIII PARAMETER-PARAMETER SEL DARAH MERAH, VITAMIN B12, FOLAT,
ARAS HOMOSISTEINE DAN MUTAS GENE MTHFR DALAM KALANGAN WANITA MELAYU YANG TELAH MENOPAUS DAN HUBUNG KAIT
DENGAN PENGGUNAAN TERAPI PENGGANTIAN HORMON
ABSTRAK
Latarbelakang: Hiperhomosisteinemia terjadi disebabkan oleh penurunan aras kofaktor-kofaktor (vitamin B12, B6 dan folat) dan / atau akibat kekurangan keberkesanan enzim methylene tetrahydrofolate reductase (MTHFR). Keadaan ini telah dikaitkan dengan risiko kejadian ‘atherosclerosis’ yang merupakan salah satu faktor penyumbang kepada penyakit jantung koronari di kalangan wanita menopaus. Adalah tidak di ketahui samada perubahan pada parameter-parameter sel darah merah boleh terjadi tanpa melibatkan ketidak normalan pada aras vitamin B12 dan folat di kalangan wanita menopaus yang berkaitan dengan kehadiran hiperhomosisteinemia.
Objektif: utama kajian ini adalah untuk mengkaji hubungkait parameter-parameter sel darah merah, B12, folat, homosisteina dan polimorfisme gen MTHFR di kalangan wanita Melayu yang mengambil rawatan terapi penggantian hormon selepas menopaus dan yang tidak mengambil rawatan tersebut. Tujuan lain adalah untuk membandingkan paras homosisteina di antara wanita-wanita Melayu sebelum dan selepas menopaus.
XIX Metodologi: Satu kajian keratan silang telah dijalankan di Hospital Universiti Sains Malaysia (HUSM). Seratus wanita Melayu menopaus secara semulajadi telah menjadi subjek kajian ini. Separuh dari mereka mengambil rawatan terapi penggantian hormon dan separuh lagi tidak mengambil rawatan tersebut. Sampel darah diambil dari subjek- subjek ini untuk ujian-ujian aras vitamin B12, serum dan sel darah merah folat dengan menggunakan kaedah ACCESS immunoasai. Aras homosisteina diukur dengan cara latex immunoasai secara ‘automated’ sementara pengesanan polimorfisma gen MTHFR dilakukan ke atas subjek-subjek terpilih dengan menggunakan reagen attomol MTHFR 677C>T. Produk dari ujian ‘polymerase chain reaction’ (PCR) kemudiannya di kesan dengan menjalankan ujian gel elektroforesis untuk menentukan jenis polimorfisma gen ini. Pengiraan darah lengkap dilakukan ke atas semua sampel dengan menggunakan mesin analisa hematologi dari Sysmex.
Keputusan: Umur subjek-subjek dalam kajian ini adalah di antara 50 – 60 tahun.
Terdapat perbezaan yang ketara di antara aras homosisteina di kalangan wanita-wanita ini sebelum dan selepas menopaus. Walaubagaimanapun tiada perbezaan yang ketara secara statistik bagi paras homosisteina di kalangan wanita yang mengambil dan tidak mengambil rawatan terapi penggantian hormon. Purata aras B12 menunjukkan perbezaan yang ketara di kalangan wanita yang mengambil dan tidak mengambil hormon tersebut. Terdapat hubungkait di antara paras sel darah merah folat dan serum folat serta aras homosisteina. Tiada hubungkait di antara homosisteina dan parameter- parameter sel darah merah, B12 dan serum folat serta polimorfisme gen MTHFR di kalangan wanita-wanita ini.
XX Perbincangan/Kesimpulan: Purata aras homosisteina di kalangan wanita Melayu menopaus adalah lebih tinggi berbanding dengan sebelum menopaus. Kajian ini menunjukkan tiada kesan positif pada aras homosisteina di kalangan wanita yang mengambil rawatan terapi penggantian hormon. Hiperhomosisteinemia di kalangan wanita Melayu adalah disebabkan oleh pelbagai faktor dan polimorfisma gen MTHFR tidak menunjukkan hubungkait yang jelas dalam menyumbangkan keadaan ini dalam kajian ini. Kajian serupa yang melibatkan sampel yang lebih besar di masa hadapan dapat mengesahkan faktor-faktor penyumbang kepada hiperhomosisteinemia di kalangan wanita Melayu termasuk gen-gen lain yang ada kaitan dengan metabolisma folat dan B12.
XXI RBC PARAMETERS, VITAMIN B12, FOLATE, HOMOCYSTEINE LEVELS
AND MTHFR GENE POLYMORPHISM IN POSTMENOPAUSAL MALAY WOMEN AND THEIR RELATION TO HORMONE REPLACEMENT
THERAPY
ABSTRACT
Background: Hyperhomocysteinemia occurs as a result of decreased levels of cofactors (vitamin B6, vitamin B12 and folate) and/or reduced efficiency of Methylenetetrahydrofolate reductase (MTHFR) enzyme. Hyperhomocysteinemia is an independent risk factor for atherosclerosis and may be one of the factors predisposing for coronary heart disease (CHD) in postmenopausal women. It is not well known whether changes in red blood cells parameters can occur independently of B12 and folate abnormalities in postmenopausal women in association with hyperhomocysteinemia.
Objectives: The main aim of this study was to investigate the association between red cell parameters, B12, folate, homocysteine level and MTHFR gene polymorphisms in postmenopausal Malay women on hormone replacement therapy (HRT) and those not receiving HRT. Another objective was to compare the homocysteine (Hcy) levels in post and premenopausal women.
Methodology: A comparative cross-sectional study was conducted at Hospital Universiti Sains Malaysia (HUSM). One hundred women were the subjects of this study, half of whom were HRT group and the other half without HRT. Blood samples were taken from all the subjects and tested for B12 and folate levels (serum and red
XXII cell) using ACCESS immunoassay system. The concentration homocysteine was measured using an automated latex-enhanced immunoassay method. The MTHFR gene detection was performed for selected cases with high and normal Hcy levels using attomol® MTHFR 677C>T reagents. PCR products were detected by gel electrophoresis in 3.0% agarose gel. Full blood counts (FBC) were done for all samples using Sysmex haematology analyzer.
Results: The ages of the subjects were 50-60 years and all of them had natural menopause. There was a significant difference in the Hcy levels between premenopausal and postmenopausal women. However, Hcy level did not show any significant difference in postmenopausal women with and without HRT. B12 was significantly lower in subjects in the HRT group compared to non HRT group. There was a significant correlation found between serum folate and RBC folate and a significant correlation between the levels of Hcy and RBC folate. For the RBC parameters, B12 and folate levels and MTHFR gene polymorphisms, no relationship was established with Hcy level.
Conclusions: The Hcy level was higher in Malay postmenopausal women (both HRT group and non HRT group) compared with premenopausal Malay subjects. HRT did not show to have positive effects in on Hcy levels. Hyperhomocysteinaemia in postmenopausal Malay women may be multifactorial and the MTHFR gene polymorphisms were not found to be associated with this condition. In addition, red cell parameters were not shown to have any association with the other variables studied. It
XXIII was further concluded that bigger sample size with other genetic factors known to be involved in B12 and folate metabolism should be included in a similar study to confirm the factors contributing to the hyperhomocysteinaemia in postmenopausal women.
CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
2 INTRODUCTION AND LITERATURE REVIEW
1.0 Introduction and literature review
Menopause is a natural progression of women’s physiology. However it can be a distressing stage for many women and is associated with increased risk of cardiovascular disease and osteoporosis. Hormone replacement therapy (HRT) has been the basis of the treatment of menopausal state. Despite the proven benefit of HRT, only 15% of post menopausal women currently use HRT (Amato et al., 2002) and of those who started HRT, nearly 30% of them subsequently stopped (Ryan et al., 1992). The main reasons for not taking HRT were concerns over its side effects, safety and efficacy of the treatment.
The Women’s Health Initiative (WHI) Study stated an increase risk in cardiovascular disease, breast cancer, stroke and thromboembolic disease with conjugated equine estrogen plus medroxyprogesterone acetate compared with placebo (Prestwood et al., 2003). In view of this problem many women are increasingly turning to alternative medicine in an effort to manage their menopausal symptoms (Amato et al., 2002).
However, there are still questions regarding the effectiveness of HRT and alternative medicines in managing menopausal symptoms and complications.
3 1.1 Menopause:
Menopause is defined as cessation of menses for at least 12 months. The diagnosis is therefore done retrospectively. Although there is a set point for menopause, it involves progressive changes in the hormonal and physiological status in women. Efforts have been made to categorize the stages of change in the menopausal continuum. This includes the menopause transitional period or premenopause which is divided into early and late phase. The early phase is categorized by irregularity in menstrual cycle of 7 days different from the normal cycle. In the late transitional period, there are 2 or more skipped cycles with an interval of amenorrhea of equal to or more than 60 days. The next stage is the actual postmenopausal stage which is divided into two phases, early and late menopause. The early menopause lasts for 5 years after the actual cessation of the menstrual flow and the late menopause begin after 5 years onwards (Soules et al., 2001).
The age of menopause has not changed much over the centuries; it has been fairly consistent between 50 to 52 years of age. There is also not much difference in the age of menopause between regions and across different ethnic groups. There are a variety of factors which have been linked to earlier age of menopause such as genetic, null parity, smoking, low body mass index, a diet high in vegetables fibers and cereals and autoimmune diseases (Lund, 2008).
4 1.2 Physiological changes:
In terms of physiological changes, menopause is generally marked by the decline in the oocytes and ovarian function. At birth the numbers of oocytes reach over a million, at around forty years of age this is reduced to a few thousands and by the time women reach menopause there are few or no oocytes left. It is the depletion of the oocytes that eventually leads to the final menstrual period (FMP) and the decline in the ovarian hormone production appears to reflect the decline in the ovarian follicle numbers.
The major gonadotrophins involved are the follicle stimulating hormone (FSH) and less importantly, the luteinizing hormone (LH). The menopause transition is characterized by a dynamic period whereby there is markedly changing hypothalamic-pituitary feedback between these hormones produced by the aging ovary. This physiological change occurs 10 years prior to the FMP. The hormonal changes in the premenopause transition lead to cycle irregularity and dramatic swings in estradiol from undetectable, to levels that are several times higher than those observed in those still in early reproductive age (Hall, 2004). With the decreasing number of follicles at menopause, estradiol level drops until it is no longer adequate to stimulate the endometrium leading to amenorrhea.
5 The level of inhibin A and inhibin B are also decreased. These hormones along with estradiol are secreted by the developing follicles and their main action is exerting a negative feedback to the hypothalamus, therefore reducing the secretion of FSH by the pituitary (Burger, 1996). At menopause with the reduced negative feedback by the inhibins, FSH tends to be higher to exceed 30 IU/l, a level considered as postmenopausal range.
Other hormones are also affected. Androgens secreted by both the adrenals and ovaries also decline as women age. However plasma testosterone levels fall only slightly after menopause. There is also a difference in the type of estrogen that predominates in the postmenopausal state. The main postmenopausal estrogen is estrone, which is produced by the conversion of adrenal androstenedione at the peripheral adipose tissue.
1.3 Effects of menopause:
1.3.1 Short term effects:
Vasomotor symptoms:
The most common symptoms experienced by menopausal women are vasomotor symptoms. Symptoms include hot flushes and night sweats and it can occur in as many as 88% of postmenopausal women (Feldman et al., 1985). However the prevalence of the symptoms is different across culture and regions. Women in Germany for example experience more hot flushes than those in Papua New Guinea (Kowalcek et al., 2005).
6 A study in Asia showed that only 30% of postmenopausal women in Malaysia complained of hot flushes compared to 70% in Thailand (Ko-En et al., 2010).
The mechanism of hot flushes has yet to be clearly defined. Although, many believe that the mechanism of hot flushes are related to the fall in estrogen levels, this alone does not account for the vasomotor symptoms since the levels of estrogen have not been found to correlate with hot flushes. It is hypothesized that the thermoregulatory zone is narrowed and more sensitive to subtle changes in core body temperature (Gracia and Freeman, 2004).This is thought to be more centrally mediated in the hypothalamus. It is still poorly understood how the changes in gonadal hormones level affect the thermoregulatory set point. Serotonin and norepinephrines are the other two hormones that are pivotal in the mechanism of hot flushes (Rapkin, 2007). Estrogen withdrawal may be correlated with a decline in both of these neuropeptides.
Sexual dysfunction:
Sexual dysfunction is highly prevalent during the menopausal transition. The Melbourne Women’s Midlife Health Project which was a prospective study done in Australia reported the prevalence of sexual dysfunction to be as high as 88% in this group of women (Dennerstein et al., 2002). The reduction in sexual functioning adversely affects women’s quality of life and also their sense of femininity (Ko-En et al., 2010).
7 Estrogen deficiency and androgen deficiency are associated with vaginal atrophy and dryness, and decrease in libido respectively (Van Voorhis, 2005), and these will affect sexual functioning. The presence of vasomotor symptoms such as night sweats, disturbed sleep and depression also lead to diminished libido in postmenopausal women which lead to sexual dysfunction (Reed et al., 2007). Hormonal factors are not the only factors contributing to sexual dysfunction. Other factors such as loss of sexual partner and the aging process itself are also implicated (Nappi and Lachowsky, 2009).
Urogenital atrophy:
The role of estrogen in maintaining normal urogenital structure and function is by stimulating the growth and development of vaginal epithelium, allowing it to remain thick, moist and supple. Vaginal atrophy occurs progressively with the dramatic fall in estrogen that occurs just before the final menstrual period. Clinically it is identified by the appearance of thin, pale and dry vaginal epithelium. It also lacks normal rogation and the introital size is often reduced.
There is also change in the vaginal pH with decrease in estrogen level. In women of reproductive age, vaginal pH is < 4.5. Vaginal pH taken from postmenopausal women was found to increase to between 6.0 and 7.5. This may lead to impairment in the local defense system and lead to vaginal and urinary tract infection (Van Voorhis, 2005).
8 Symptoms of vaginal atrophy include dryness, itching, vaginitis, and dyspareunia. It is a common symptoms reported by postmenopausal women. Vaginal dryness was a complaint in 51% of postmenopausal women in Hong Kong (Ko-En et al., 2010).
1.3.2 Long term effects:
The long term complication of menopause which have the greatest impact on health status both at the individual and public health levels are cardiovascular disease and osteoporosis.
Cardiovascular disease:
Cardiovascular disease is the major cause of death in women worldwide; the third Malaysian National Health Survey showed that the main cause of death of Malaysian women is due to cardiovascular disease (CVD) (Azmi et al., 2009). The figure is almost the same in other parts of developed countries like United State of America. There is a difference between the incidence of cardiovascular disease in men and women. The incidence of death due to cardiovascular disease is almost unheard of before women reach their 50s. However, by the age 70 years the incidence of CVD is equal in men and women, suggesting that menopause with it ensuing estrogen deficiency causes a rapid acceleration in CVD risk (Azmi et al., 2009).
9 The high incidence in cardiovascular disease is due to the increase in the risk factors associated with menopausal state. Menopausal state is associated with the emergence of features of metabolic syndrome which includes increase in central or intra abdominal body fat, a change toward more atherogenic lipid profile, with increased low density lipoprotein particles and increased insulin resistance (Knopp, 2002, Eaten and Anthony 2002). The mechanism for these changes may be a direct result of ovarian failure or, alternatively, an indirect result of the metabolic consequences of central fat redistribution with estrogen deficiency in postmenopausal women (Carr, 2003). There is however controversy regarding the relationship between weights gains and menopause.
Previous data suggest that this is more related to aging rather than failure in ovarian function (Kolasa, 2002).
Another important risk factor for cardiovascular disease is sedentary life style.
Menopausal women have been shown to lead more sedentary lifestyle compared to younger women (Kolasa, 2002). The role of other risk factors like C-reactive protein and homocysteinuria in linking menopause with cardiovascular disease are less well established (Kolasa, 2002). More studies need to be done to establish the associations between these factors.
When vitamin B12 level decreases too low, the methionine cycle breaks down which results in elevated levels of homocysteine. That cycle converts methionine into smaller molecules known as S-adenosylmethionine (SAMe). SAMe then breaks down into thousands of compounds and proteins that are vital for healthy cells, tissue and organs.
10 One of those breakdown products is homocysteine. Recycling of the homocysteine to getting quickly back into methionine needs the assistance of vitamin B12 and folic acid.
If someone is deficient the normal function of the primary pathway of methionine cycle is disrupted and this will result in excess of homocysteine (Nygard et al., 1997).
Excess amount of homocysteine causes blood vessels to lose their elasticity, making it harder for them to dilate and damaging the inner lining. These changes allow cholesterol, collagen and calcium to attach to the inner walls of the blood vessels where they can form sticky deposits called as atherosclerotic plaque. These plaques narrow the arteries and increase the risk of artery disease, myocardial infarction, strokes and thrombosis clot (Nygard et al., 1997).
Coronary artery disease (CAD) or atherosclerotic heart disease is the end result of the accumulation of atheromatous plaques within the walls of the coronary arteries that supply the myocardium (the muscle of the heart) with oxygen and nutrients. It is sometimes also called coronary heart disease (CHD), but it is not the only cause, although CAD is the most common cause of CHD (Tortora & Grabowski 2003).
Osteoporosis:
Osteoporosis is a major health issue since it can lead to fracture with catastrophic consequences. The prevalence of osteoporosis is also rising worldwide. In Western countries for example United States of America, it is estimated that 54%
11 postmenopausal white women are osteopenic and 30% are osteoporotic as quoted by the International Osteoporosis Foundation in 2002. The National Osteoporosis Foundation in the USA reported that by the year 2010, about 12 million people over the age of 50 are expected to have osteoporosis (National Osteoporosis Foundation, 2002). There is an erroneous belief that Asian women suffer less osteoporosis and fall compared to their western counterpart. An audit released by the International Osteoporosis Foundation on the epidemiology of osteoporosis in Asia revealed that the incidence of hip fractures has risen by 2- to 3- fold over the past 30 years (International Osteoporosis Foundation, 2002).
The WHO defines osteoporosis based on bone mineral density, as a value of less than 2.5 standard deviation below the average in young women (T-score ≤ -2.5). This definition initially was more for epidemiological purpose, however in recent times this threshold value has been established as clinical diagnostic tool.
The postmenopausal state is associated with a decrease in the bone mineral density which leads to osteoporosis. There is a widely held belief that the decrease in the estradiol level directly contribute to this. Estradiol directly acts on osteoclast by reducing the rate of bone resorption (Steinweg, 2002). Bone loss in the postmenopausal women, therefore is the result of increase in the rate of bone remodeling and the imbalance in osteoclast.
12 The rate of bone loss differs in relation to period of menopause, with the most rapid loss occurring early in the premenopausal age. The rate of bone resorption then decrease in the late menopause (Sirola et al., 2003). There is also difference regarding the rate of bone loss at different site of the body. The lumbar spine has been shown to have more osteoporotic changes in early postmenopause compared with other sites such as the femur.
Menopausal and cancer risk:
Menopause in itself does not cause cancer, but the risk of developing cancer increases as a woman ages (Barrett-Connor et al., 2009). Women who have been through menopause are more likely to develop cancer because they are older. Among the cancers which are associated with late menopause are breast, ovarian, and endometrial cancers (Barrett-Connor et al., 2009). (Rossouw et al., 2002).
In terms of breast cancer, menopausal women was found in a study to have an additional increase in breast cancer risk if they had high breast density as measured by mammogram (Kerlikowske et al., 2009). This study also showed that advanced-stage breast cancer risk was increased 1.7-fold for postmenopausal HRT users who had very high density (BIRADS-4) compared to those with average density (BIRADS-2). The association between HRT use in postmenopausal women and the increased risk in breast cancer had been shown in various large studies most notably the Women’s Health Initiative (WHI) trial (Rossouw et al., 2002).
13 Endometrial cancer is the commonest gynaecological cancer in the developed countries and it occurs mainly in the postmenopausal women. The main symptom of endometrial cancer is post menopausal bleeding. Postmenopausal women who presented with this symptom have to have their endometrial sampled to exclude the presence of any abnormality. In term of the effect of combined hormone replacement therapy on the cancer incidence, the WHI trial demonstrated the incidence of endometrial cancer during the 5-6 years of follow-up was 56 per 100000 person-years than observed in women taking the therapy or 13 fewer cases per 100000 person-years than observed in women taking placebo (Anderson et al., 2004). This indicated the protective effect of progestin against an increased risk of endometrial cancer associated with unopposed estrogen.
Ovarian cancer is also not associated with menopause but the incidence of the cancer increases as women go into postmenopause. The risk of postmenopausal women having the cancer is increased if they have additional risk factors such as low parity, infertility, early age of menarche, and late age of menopause (Berek and Hacker, 2005).The theory behind the increase risk of ovarian cancer with late age of menopause is thought to be related to the incessant ovulation theory. According to this theory with repeated damage and trauma to the ovarian epithelium during each ovulatory cycle, there is an increased potential for genetic mutation and ovarian neoplasm during the repair process.
Incidence of ovarian cancer has also been shown to be associated with long term combined hormone therapy use (Anderson et al., 2004).
14 Cognitive function:
The effect of estrogen on cognitive function is an intriguing area of research. It is well known that normal aging causes a decline in certain cognitive function, and a decline in the estrogen associated with menopause may contribute to this process. In the past estrogen therapy has been associated with better performance on memory testing in postmenopausal women compared with postmenopausal controls who were not receiving estrogen therapy (Kawas et al., 1997). However data from the WHI do not show improvement in cognitive function in women taking either combined hormone replacement therapy or estrogen only therapy (Rossouw et al., 2002).
1.4 Therapeutic options:
There are three main therapies available in the management of menopausal complications which are hormonal replacement therapy (HRT), bisphosphonate and selective estrogen receptor modulator. The use of these agents is increasingly being challenged by the alternative medicines especially after the release of the result of Women Health Initiative Study. Among the popular alternative medicine used in menopausal symptoms are soy products, black cohosh, dong quai, red clover, ginseng and evening primrose oil (Black et al., 2006).
15 Hormone replacement therapy:
Hormone replacement therapy (HRT) has long been the mainstay of the treatment of menopausal symptoms and its complications especially osteoporosis. The essential component in HRT is estrogen, which is combined with progestogen to prevent endometrial hyperplasia in women with intact uterus (Barrett-Connor et al., 2006).
The oral estrogen commonly used is estradiol valerate 1mg or 2 mg, conjugated equine estrogen (CEE) 0.625 mg or 1.25 mg and estrone 1.25 mg. The minimum effective dose of estrogen has long been questioned. After the WHI, more interest has been shown to the use of low dose estrogen. The usually prescribed dosage of postmenopausal estrogen therapy has declined progressively and in the past 10 years, use of lower dose HRT has grown in popularity. Low-estrogen formulations that are starting to be used recently are 0.3mg CEE, 0.45 mg CEE and 0.5 mg or 1mg micronzed oral 17-estradiol and these are considered low dose HRT. This lower dose of oestrogen is also beneficial in terms of reduced breast tenderness, an effect that should also help to improve compliance and acceptability, particularly in older women (Barrett-Connor et al., 2006).
The progestogens used in HRT are nearly all synthetic. They are added to reduce the risk of endometrial hyperplasia and malignancy. The two classes of progestogens commonly used for in combination with estradiol is C-21 group (medroxyprogesterone acetate, dydrogesterone) derived from native progestogens and a C-19-nor group
16 (norethisterone, norgestrel) derived from testosterone. Dydrogesterone, a retro- progesterone derivative in particular is structurally closest to the body’s own progesterone and fairly neutral effect on the protective properties of estrogen on bone metabolism (Schindler et al., 2003).
Currently progestogens are mainly given orally, however transdermal formulations of levonogestrel and norethisterone are becoming available. Progestogen is only required in women with intact uterus. It can be given either for 10-14 days every four weeks or for 14 days every 13 weeks or even continuously every day. The continuous formulations is suitable for women with early or late postmenopause while the monthly sequential regime is given for those in menopausal transition or premenopause stage (Cieraad et al., 2006).
The controversy with HRT started to appear after the release of the results from Women’s Health Initiative (WHI) trial in 2002. The trial involved 27,347 predominantly healthy postmenopausal women aged 50 to 79 years old from 40 centers across America. The women were randomized to either receive 0.625 mg/d of CEE or 0.625mg/d CEE plus 2.5 mg/d of medroxyprogesterone acetate (MPA) based on their hysterectomy status and they were compared with placebo (Rossouw et al., 2002). The aim of the trial was to see the effect of HRT to a ‘global health index’ which includes coronary heart disease, breast cancer, fractures, stroke, pulmonary embolism, colorectal cancer and endometrial cancer (Rossouw et al., 2002).
17 For every 10,000 women per year who were taking combination HRT, there were eight more invasive breast cancers, seven more coronary heart disease events, eight more strokes and eight more pulmonary embolisms than in the placebo group. Physicians and patients were encouraged to use HRT for the shortest duration possible since the biggest increase in breast cancer was seen after 5 years. Similar result in term of breast cancer risk was seen in the Million Women Study conducted in England (Beral, 2003).The main limitation of WHI trial is that they include older women who were postmenopausal for many years and the result therefore cannot be extrapolated to the younger postmenopausal women.
A variety of drugs are used for prevention and treatment of CHD. Hormone replacement therapy (oestrogen) was reported to give benefit effects to reduce the risk of CHD in postmenopausal women (Smolders et al., 2005).
The main indication for HRT use currently is for the treatment of postmenopausal symptoms and it is no longer recommended for disease prevention (Dull, 2006). A Cochrane review concluded that oral estrogen or combined estrogen and progestogen hormone replacement therapy greatly reduces the frequency and severity of hot flushes and night sweats (MacLennan et al., 2004). The maximum benefit is usually seen within three months of treatment.
18 HRT has long been proven beneficial in the treatment of postmenopausal osteoporosis.
Both observational studies and randomized clinical trial have demonstrated that estrogens or estrogens plus progestin increase bone density and reduce the risk of fractures by preventing bone loss mainly in the vertebral bone (Huot et al., 2008).
Recently, there has been increasing evidence that it is also beneficial in preventing fracture in the non-vertebral bone. A 5 year randomized controlled trial was done to compare the incidence of new non-vertebral bone fractures in non-osteoporotic postmenopausal women taking HRT or low dose vitamin D. This study showed the incidence of non-vertebral bone fractures is significantly reduced compared to the vitamin D group. The fall in fracture rate is also consistent with the increase in bone mass (Komulainen et al., 2008).
Femoston Conti is one of the examples of low dose hormone replacement therapy. It contains 17β-estradiol plus dydrogesterone with a multiple dosing range combination.
The doses of estradiol include 1 and 2 mg, while the doses of dydrogesterone include 5, 10 and 20 mg. There are multiple studies which have shown the effectiveness of this treatment in managing menopausal symptoms and having bone protective effects. A study comparing the 17β-estradiol / dydrogesterone with conjugated equine estrogens(CEE)/norgestrel have shown that it was equally effective in managing climacteric symptoms while showing some advantages in terms of lipid profile and incidence of bleeding (Cieraad et al., 2006). A combination of 1 mg 17β-estradiol with a combination of 5, 10 and 20 mg of dydrogesterone had also been shown to have
