KWAME NKRUMAH UNIVERSITY OF SCIENCE AND TECHNOLOGY IMMUNOLOGICAL FACTORS IN FEMALE INFERTILITY: ANTITHYROID AND ANTIPHOSPHOLIPID ANTIBODIES A THESIS SUBMITTED TO THE DEPARTMENT OF MOLECULAR MEDICINE, SCHOOL OF MEDICAL SCIENCES IN FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER OF PHILOSOPHY IN IMMUNOLOGY BY ISAAC ACHEAMPONG PG1175613 NOVEMBER, 2016 DECLARATION All the experimental work described in this thesis was done at the Department of Molecular Medicine, KNUST and the University Hospital, KNUST. This thesis has not been submitted for any other degree in this Institution or elsewhere. ………………………….. …………………………… Isaac Acheampong Date (Student) …………………………… …………………………… Prof. (Mrs) M. T Frempong Date (Supervisor) ………………………………. …………………………… Prof. F. A Yeboah Date (Head of Department) i ABSTRACT Background: Mixed findings regarding the association between the presence of autoantibodies (TPO-abs, TG-abs and APL-abs) and female infertility besides spontaneous abortion have been reported in most studies in developed countries. To screen for these autoantibodies still remains divisive. Additionally, very little is known about their relationship with menstrual abnormalities, body mass index (BMI) and haematological parameters in infertile women in developing countries. Aim: The aim of this study was to investigate the relationship between a panel of autoantibodies (TPO-abs, TG-abs and APL-abs) and infertility, spontaneous abortion, menstrual abnormalities, body mass index and haematological parameters. Methods: A case-control study comprising 52 infertile cases and 38 fertile controls in three major hospitals in the Ashanti region of Ghana was undertaken. A standard questionnaire was administered, blood pressure (BP) measured and BMI computed from the weight and height. Blood specimen was collected for haematological (NLR, PLR, #NEUT and #Lymphocyte) and biochemical analysis (TPO-abs, TG-abs and APL-abs). TPO-abs (IgG) >100IU/mL and TG-abs (IgG) >100IU/mL were regarded as positives for the thyroid autoantibodies while APL-abs (IgG/IgM) ≥10U/mL was also regarded as positive for the antiphospholipid antibodies. Results: Infertility was found to be significantly associated with the presence of thyroperoxidase antibody (TPO-abs: χ2 (1) = 7.047, p=0.011) and antiphospholipid antibody positivity (APL-abs: χ2 (1) = 5.55, p=0.02) but not thyroglobulin antibody positivity (χ2 (1) = 0.907, p=0.357). Neither of the three autoantibodies (TPO-abs, TG-abs and APL-abs) was found to be associated with a history of spontaneous abortion (p>0.05). The odds of APL and both (TPO+TG-abs) positivity were found to be 1.71 and 1.45 times greater respectively among infertile women with menstrual abnormalities compared to those without menstrual abnormalities [APL-abs: (OR=1.71, (95% CI): 0.18-15.95; p=0.636) ; TPO + TG-abs: (OR =1.45, (95% CI): 0.27-7.82; p=0.667)], however both associations were not significant. BMI was comparable between infertile cases and fertile controls and did not correlate significantly with any of the autoantibodies. Neutrophil-lymphocyte ratio (NLR), Platelet-lymphocyte ratio (PLR), absolute neutrophils (#NEUT) and absolute lymphocyte count (#Lymphocyte) were significantly lower in infertile women compared to fertile controls (p<0.05). These haematological parameters however did not correlate significantly with any of the autoantibodies in either infertile cases or controls. Conclusion: Infertility among Ghanaian women is associated with TPO-abs and APL-abs positivity therefore these antibodies may be screened as part of routine checks for appropriate intervention. However, these autoantibodies may not be directly associated with a history of spontaneous abortion. BMI, NLR, PLR, #NEUT, and #Lymphocyte do not correlate with the presence of autoantibodies in Ghanaian infertile women. ii DEDICATION This thesis is dedicated firstly to the Almighty God for his abundant supply of grace to finish this project and secondly to my supervisor, Prof. (Mrs) M. T Frempong for her motherly love, patience, mentorship and support throughout this project. iii ACKNOWLEDGEMENT I wish to acknowledge the immense support of the following individuals who helped in diverse ways to make this project a success: Prof. (Mrs) M. T Frempong (my supervisor and Lecturer at the Department of Molecular Medicine) for graciously supervising my work and ensuring the completion of my study. Prof. C. A Turpin (Head, Department Of Obstetrics And Gynaecology, KATH) for allowing me to select some participants from his department for my study. Prof. A. T Odoi (CEO, Trust Care Specialist Clinic) for making his fertility clinic available for me to carry out my study and aiding me to select some participants from his clinic. Dr A. Tawiah (Senior Resident, Department of Obstetrics and Gynaecology, KATH) for his unending support throughout this project. Dr S. S Asamoah (Lecturer at the Department of Molecular Medicine) for availing his laboratory to me to analyse my samples. Mr M. Dompreh (Head, Department of Serology) for helping me store my samples at the requisite temperature in his department prior to the analysis. Mr S. Kwofie (Biochemistry practical instructor, Department of Molecular Medicine), for assisting me with the laboratory analysis of my samples. Mr Enoch Odame (Student, Department of Molecular Medicine) for assisting me with my laboratory work. All my friends and family who helped one way or the other to make this project successful, I say God richly bless you. iv TABLE OF CONTENTS DECLARATION .............................................................................................................. i ABSTRACT ...................................................................................................................... ii DEDICATION ............................................................................................................... iii ACKNOWLEDGEMENT .............................................................................................iv TABLE OF CONTENTS ................................................................................................ v LIST OF TABLES ........................................................................................................ viii LIST OF FIGURES .......................................................................................................... x 1.0 CHAPTER ONE................................................................................................... 1 1.1. INTRODUCTION ................................................................................................ 1 1.2. PROBLEM STATEMENT ................................................................................... 3 1.3. JUSTIFICATION .................................................................................................. 4 1.4. ALTERNATE HYPOTHESIS.............................................................................. 4 1.5. AIM ........................................................................................................................ 4 1.6. SPECIFIC OBJECTIVES ...................................................................................... 5 2.0 CHAPTER TWO .................................................................................................. 6 2.1. INFERTILITY (DEFINITIONS) .......................................................................... 6 2.2. PREVALENCE OF INFERTILITY ..................................................................... 7 2.3. CAUSES OF INFERTILITY................................................................................. 9 2.3.1. Role of immunological factors................................................................................. 9 2.3.2. Thyroid Autoimmunity ........................................................................................ 11 2.3.3. Thyroid Hormone Production and Thyroid Autoantibodies ................................ 11 2.3.4. Thyroid Hormones in Pregnancy and Reproductive Failure ............................... 13 2.3.5. Thyroid Autoimmunity as it Relates With Infertility, Menstrual Abnormalities and Spontaneous Abortion ................................................................................... 14 2.3.6. Antiphospholipid Antibodies Associated Infertility and Spontaneous Abortion . 15 2.4. Male Factor in Infertility ................................................................................... 19 2.5. OBESITY AND INFERTILITY ......................................................................... 19 2.6. HAEMATOLOGICAL PARAMETERS AND INFERTILITY ...................... 21 3.0 CHAPTER THREE ............................................................................................ 22 3.1. METHODOLOGY .............................................................................................. 22 3.1.1. Study Site ............................................................................................................. 22 3.1.2. Study Design ........................................................................................................ 23 3.1.3. Sample Size ........................................................................................................... 24 3.1.4. Sampling Method ................................................................................................. 24 v 3.1.5. Study Population .................................................................................................. 25 3.2. ETHICAL CONSIDERATION ......................................................................... 25 3.3. INCLUSION CRITERIA ................................................................................... 25 3.4. EXCLUSION CRITERIA ................................................................................... 25 3.5. STUDY METHODS ............................................................................................ 26 3.5.1. Data Collection ..................................................................................................... 26 3.5.2. Questionnaires ...................................................................................................... 26 3.5.3. Physical Measurements ........................................................................................ 26 3.5.4. Specimen Collection .............................................................................................. 27 3.6. LABORATORY ANALYSIS ............................................................................. 28 3.6.1. Full Blood Count .................................................................................................. 28 3.6.2. Enzyme linked Immunosorbant Assay (ELISA) .................................................. 29 3.7. STATISTICAL ANALYSIS ............................................................................... 32 4.0 CHAPTER FOUR .............................................................................................. 33 4.1. RESULTS ............................................................................................................. 33 4.1.1. Obstetric Characteristics and BMI of Study Population ..................................... 36 4.1.2. Age, BMI and Diastolic Blood Pressure Distribution of Study Participants ...... 39 4.1.3. Distribution of SBP, Autoantibodies and Haematological Parameters of Study Participants .......................................................................................................... 40 4.1.4. Association between Autoantibodies and Infertility ............................................ 41 4.1.5. Distribution of BMI, DBP and Haematological Parameters of Infertile Women with a History of Spontaneous Abortion .............................................................. 42 4.1.6. Distribution of SBP, Autoantibodies and Haematological Parameters of Infertile Women with a History of Spontaneous Abortion ................................................ 43 4.1.7. Distribution of BMI, DBP and Haematological Parameters of Infertile Women with SA, RA and None ......................................................................................... 44 4.1.8. Distribution of SBP, Autoantibodies and Haematological Parameters of Infertile Women with No Abortion (None), SA and RA ................................................... 45 4.1.9. Association between Autoantibody Positivity and a History of Spontaneous Abortion ................................................................................................................ 47 4.1.10. Odds Ratio of Autoantibody Positive Women’s Association with Spontaneous Abortion ................................................................................................................ 48 4.1.11. Distribution of Autoantibody Levels of Infertile Women with Menstrual Abnormalities ....................................................................................................... 49 4.1.12. Association between Autoantibodies and Menstrual Abnormalities in Infertile Women .................................................................................................................. 50 vi 4.1.13. Odds Ratio of Menstrual abnormality’s association with Autoantibody Positivity among Infertile Women. ....................................................................................... 52 4.1.14. Correlation of Continuous Data Parameters among Infertile Women ................ 53 4.1.15. Correlation of Continuous Data Parameters of Fertile Women ........................... 56 5.0 CHAPTER FIVE ................................................................................................ 59 5.1. DISCUSSION ...................................................................................................... 59 5.1.1. Relationship between Thyroid Autoantibodies, Infertility and a History of Spontaneous abortion. .......................................................................................... 59 5.1.2. Relationship between Antiphospholipid Antibodies (APL-Abs), Infertility and Spontaneous Abortion. ......................................................................................... 60 5.1.3. Relationship between BMI, Infertility and Menstrual Abnormalities ................. 61 5.1.4. Relationship between Haematological Parameters, Systolic and Diastolic Blood Pressure and Infertility......................................................................................... 63 5.1.5. Relationship between Autoantibodies and Menstrual Abnormalities.................. 64 5.1.6. Correlation among Autoantibodies and Haematological Parameters .................. 65 6.0 CHAPTER SIX ................................................................................................... 67 6.1. CONCLUSION ................................................................................................... 67 6.2. RECOMMENDATIONS ................................................................................... 67 7.0 REFERENCES .................................................................................................... 68 8.0 APPENDIX ......................................................................................................... 78 8.1. ETHICAL APPROVAL ..................................................................................... 78 8.2. QUESTIONAIRE ................................................................................................ 79 8.3. PARTICIPANT INFORMATION LEAFLET AND CONSENT FORM ..... 82 vii LIST OF TABLES Table 4.1. Distribution of Socio-demographic Parameters among Study Participants ......................................................................................................... 35 Table 4.2. Distribution of Obstetric Parameters and BMI categories among Study Participants. ........................................................................................................ 38 Table 4.3. Age, BMI, DBP, and Haematological Parameters Compared between Fertile and Infertile Women. ............................................................................ 39 Table 4.4. SBP, Autoantibodies and Haematological Parameters Compared between Infertile and Fertile Women. ............................................................ 40 Table 4.5. Association between Autoantibodies and Infertility .............................. 41 Table 4.6. Distribution of BMI, DBP, and Haematological Parameters of Women with History of Spontaneous Abortion Compared With Those without Any History in the Infertile Group ................................................................. 42 Table 4.7. SBP, Autoantibodies and Haematological Parameters Compared between Women with a History of Spontaneous Abortion and Those without any History in the Infertile Group.................................................... 43 Table 4.8. Distribution of BMI, DBP, and Haematological Parameters of Infertile Women with No History of Spontaneous Abortion Compared With Spontaneous Abortion and Recurrent Abortion ........................................... 44 Table 4.9. SBP, Autoantibodies and Haematological Parameters Compared Between Women with No History of Spontaneous Abortion, Spontaneous Abortion and Recurrent Abortion ................................................................... 46 Table 4.10. Association between Autoantibody Positivity and History of Spontaneous Abortion among Infertile Women ........................................... 47 Table 4.11. Odds of Autoantibody Positive Women’s association with a History of Spontaneous Abortion. ................................................................................. 48 Table 4.12. Autoantibodies Compared Between Infertile Women with One or More Menstrual Abnormalities and Those without Any Menstrual Abnormality........................................................................................................ 49 viii Table 4.13. Association between Autoantibodies and Menstrual Abnormalities among Infertile Women .................................................................................... 51 Table 4.14. Odds Ratio of Autoantibody Positive Infertile Women’s association with Menstrual Abnormality. .......................................................................... 52 Table 4.15. Pearson Correlation among Age, Body Mass Index, Systolic Blood Pressure, Diastolic Blood Pressure, Autoantibodies and Haematological Parameters of the Infertile Women ................................................................. 55 Table 4.16. Pearson Correlation among Age, Body Mass Index, Systolic Blood Pressure, Diastolic Blood Pressure, Autoantibodies and Haematological Parameters of the Fertile Women. ................................................................... 58 ix