UNIVERSITI PUTRA MALAYSIA

INFLUECNE OF ENVIRONMENT AND GENETICS ON

REPRODUCTIVE HEALTH OF TWINS

SHAYESTEH JAHANFAR

FPSK(M) 2009 3

INFLUECNE OF ENVIRONMENT AND GENETICS ON REPRODUCTIVE HEALTH OF TWINS

By

SHAYESTEH JAHANFAR

Thesis submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Master of Science

August 2009

Abstract of thesis presented to the Senate of Uinversiti Putra Malaysia in fulfilment of the requirement for the Degree of Master of Science

INFLUECNE OF ENVIRONMENT AND GENETICS ON REPRODUCTIVE HEALTH OF TWINS

By

SHAYESTEH JAHANFAR

August 2009

Chairman: Prof. Dato’ Dr. Lye Munn Sann, MBBS, DrPH

Faculty: Medicine and Health Sciences

Reproductive health is a dynamic part of our lives with a broad spectrum of events.

Study of twins can assist to investigate the relationship between environmental and

hereditary causes of reproductive diseases, which would otherwise be difficult if not

impossible to study. High concordance between twin pairs suggests a strong influence

for genetic factors, whereas low concordance indicates a weak influence. Preventive

measures can be taken into consideration for those who are at risk of environmental

influences if and only when the role of genetic is minor even though gene therapy may

minimize the occurrence of genetic disease.

This cross-sectional descriptive study of reproductive health of twins investigated the

heritability of qualitative and quantitative measured variables related to reproductive

events or behavior of adult twins. Subjects included 156 identical and 110 non-identical

twins, 15 years and above who were living in urban areas of Iran and Malaysia. Basic

and modern genetic analysis was adopted. Maximum likelihood analysis and model

ii

fitting analysis suggested that birth weight, weight, height, age of menarche,

premenstrual symptoms, acne, hirsutism, baldness and infertility are mostly determined

by genetic factors while characteristics of menstruation were more likely under the

influence of environmental factors. Classical genetic analysis using Falconer’s formula

suggested higher similarity between monozygotic twins than dyzyotics in relation to

reproductive behaviors such as age of first pregnancy and number of pregnancies.

Probandwise concordance rate analysis showed higher similarity between identical twins

in adopting correct reproductive behavior such as undergoing pap smear and using

family planning techniques. Model fitting analysis for the ACE model supported these

findings.

Same-sex twins had a higher risk of congenital abnormality, gynecological problem, and

irregular menstruation than opposite sex twins. Other reproductive events were not

found to be significantly different between the two groups indicating that hormonal

transition from male to female may not be a valid explanation for reproductive ill health.

Birth weight was not found to affect reproductive morbidity during adult life as

frequency of reproductive event was found to be higher among low birth weight twins

compared with normal weight twins. This finding is not consistent with fetal origin

hypothesis or thrifty gene hypothesis.

Reproductive ill-health influenced by environmental factors may be minimized using

careful primary care evaluation, early detection and prevention while genetic

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predisposition can be monitored for those variables under genetic influence. Early

reproductive health consultation for adolescents is recommended to avoid complications

of reproductive ill-health.

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Abstrak tesis yang dikemukakan kepada senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah induk sains

Pengaruh Persekitaran dan Genetik terhadap kesihatan reproduktif

pasangan kembar

Oleh

SHAYESTEH JAHANFAR

August 2009

Pengerusi: Prof. Dato’ Dr. Lye Munn Sann. MBBS, DrPH

Fakulti: Peruabatan dan Sains Kesihatan

Kesihatan reproduktif adalah sebahagian daripada kehidupan kita yang

dinamik yang merangkumi satu spektrum yang luas. Kajian pasangan

kembar boleh membantu mengenal pasti hubungan di antara faktor-faktor

persekitaran dan genetik yang menjadi punca penyakit reproduktif.

Keselarasan yang tinggi antara pasangan-pasangan kembar menandakan

wujudnya pengaruh faktor genetik yang kuat manakala keselarasan yang

rendah pula menandakan pengaruh yang lemah. Walaupun terapi gen

dikatakan dapat mengurangkan kejadian penyakit genetik, langkah-langkah

pencegahan boleh dipertimbangkan bagi mereka yang berada di dalam

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persekitaran yang berisiko hanya jika peranan faktor genetik adalah

minima.

Kajian keratan lintang yang berperihal tentang kesihatan reproduktif

pasangan kembar ini mengukur kebolehan faktor-faktor kualitatif dan

kuantitatif yang berkaitan dengan hal-hal reproduktif ataupun perwatakan

pasangan-pasangan kembar dewasa diwariskan. Subjek termasuk 266

pasangan kembar seiras (monozigotik) dan 110 kembar tak seiras

(dwizigotik) yang berusia 15 tahun ke atas dan tinggal di kawasan bandar

Iran dan Malaysia. Analisa genetik yang asas dan moden telah

dipraktikkan dalam kajian ini. Analisa-analisa ‘maximum likelihood and

model fitting’membuktikan bahawa berat badan kelahiran, berat badan,

ketinggian, usia menarki, simptom-simptom prahaid, jerawat, ‘hirsutism’,

kebotakan dan kemandulan adalah ditentukan oleh faktor-faktor genetik

manakala ciri-ciri kedatangan haid adalah dipengaruhi oleh faktor-faktor

persekitaran. Analisa genetik menggunakan formula Fanconi menunjukkan

persamaan yang tinggi diantara pasangan kembar monozigotik daripada

pasangan kembar dwizigotik berkaitan dengan usia kehamilan yang

pertama, bilangan kehamilan dan usia perkahwinan. Dari segi proban,

analisa kadar keselarasan menunjukkan persamaan yang lebih tinggi bagi vi

pasangan-kembar seiras dalam amalan hal-hal reproduktif yang betul seperti

menjalankan ujian pap smear dan perancangan keluarga. Analisa ‘model

fitting’ menyokong hasil kajian ini.

Pasangan kembar jantina serupa adalah lebih berisiko dalam mengalami

masalah kongenital tidak normal , masalah-masalah ginekologi dan

kedatangan haid yang tidak tetap dibandingkan dengan pasangan kembar

jantina tak serupa. Lain-lain hal reproduktif tidak menunjukkan perbezaan

yang ketara di antara kedua-dua jenis kumpulan pasangan kembar,

seterusnya mencadangkan bahawa transisi hormon lelaki kepada

perempuan tidak boleh dianggap sebagai satu penjelasan terhadap kesihatan

reproduktif yang kurang baik.

Berat badan kelahiran didapati tidak mempengaruhi masalah reproduktif

ketika dewasa dimana frekuensi masalah reproduktif didapati lebih tinggi di

kalangan pasangan kembar yang mempunyai berat badan yang lebih rendah

pada waktu lahir dibandingkan dengan mereka yang mempunyai berat

badan normal. Hasil kajian ini tidak selaras dengan hipotesis ‘fetal origin’

ataupun hipotesis ‘thrifty gene’.

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Pengaruh persekitaran terhadap perihal reproduktif yang tidak sihat boleh

diminimakan dengan menggunakan penilaian penjagaan utama yang teliti,

pengesanan yang awal dan pencegahan manakala bagi yang dibawah

pengaruh genetik dapat diawasi. Perundingan kesihatan reproduktif yang

awal bagi remaja adalah disyorkan untuk mengelak daripada komplikasi

penyakit reproduktif.

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Acknowledgements

I would like to express my deep gratitude to Dato’ Dr. Lye Munn Sann, Professor of

Epidemiology, Dept. of Community Health, Faculty of Medicine and Health Sciences,

Universiti Putra Malaysia for his constant encouragement and support in every aspect of

this study as a supervisor. My heartiest thanks go to Dr. Isthrinayagy A/P S.

Krishnarajah, Senior Lecturer of Statistics, Faculty of Science, Universiti Putra Malaysia

for her professional guidance and support as a co-supervisor.

This study is financially supported by Royal College of Medicine, Perak; Universiti

Kuala Lumpur. Ethical approval has been obtained from the Medical Ethics Research

Committee UPM, Research Committee UniKL, National Malaysian Research Registry,

and Tan Sri Datuk Dr. Haji Mohd Ismail Merican, Director-General, Ministry of Health

Malaysia via a letter (reference: (17)dlm.kkm(AM)09/62/10-23/46 issued on 31st July

2007.)

I would also like to express our heartfelt thanks and gratitude to the following:

• Associate Professor Dr. Mohd Amir Abas, postgraduate and research center, UniKl

for his constant support.

• Professor Hashami Bohari, Dean and CEO of Royal College of Medicine, Perak

(RCMP); Universiti Kuala Lumpur for his guidance and support of this research

project.

• Dr. Sharifah Halimah Binti Jaafar, Senior Lecturer RCMP and Obstetrics and

Gynecologist consultant in Ipoh Specialist Hospital for her devotion and compassion

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in assisting the project, accommodating the National Malaysian Twin Registry

Office in her own office and for her professional and clinical opinions.

• Mrs. Yip Siew Keen for her dedication and hard work as research assistant in

contacting subjects with language barriers during data collection

Most of all I like to thank all the participants in the study who made this work possible.

I salute them and hope the provided services would be beneficial for them and their

families. I like to dedicate this thesis to my husband Dr. Saied Saiedi who was my

backbone and spirit throughout the ups and downs and to my boys Mohammad and

Hamed who are expecting their own Masters’ degrees soon. My sincere love goes to you

both.

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Take me out later

I certify that an examination Committee has met on 27 August 2009 to conduct the final

examination of Shayesteh Jahanfar on her Master of Science degree in Community

Health thesis entitled “Influence of Environment and Genetics on Reproductive Health

of Twins” in accordance with the Universities and University Colleges Act 1971 and the

Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The

committee recommends that the students be awarded the Master of Science.

Members of Thesis Examination Committee were as follows:

Prof. Dr. Syed Tajuddin bin Syed Hassan, PhD Department of Community Health, Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman) Name of Examiner 1, PhD Title: Prof. Madya Dr. Rozita Rosli Name of faculty: Faculty of Medicine and Health Sciences University Putra Malaysia (Internal Examiner) Name of Examiner 2, PhD Title: Dr. Shamarina Shohaimi Name of faculty: Department of Biology, Faculty of Science University Putra Malaysia (Internal Examiner) Name of External Examiner, PhD Title: Prof. Dr. Rama C. Nair Name of faculty: Faculty of Medicine University of Ottawa, Canada Prof. Dr. Bujang Kim Huat

Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia

Date:

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows:

Lye Munn Sann Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman) Isthrinayagy A/P S. Krishnarajah, PhD Faculty of Science Universiti Putra Malaysia (member) HASANAH MOHD. GHAZALI, PhD Professor and Dean

School of Graduate Studies Universiti Putra Malaysia

Date: 16 November 2009

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DECLARATION I declare that the thesis is my original work except for quotations and citations which

have been duly acknowledged. I also declare that it has not been previously, and is not

concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other

institution.

----------------------------- Shayesteh Jahanfar Date: August 2009

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TABLE OF CONTENTS ABSTRACT ii ABSTRAK v ACKNOWLEDGEMENTS ix APPROVAL xi DECLARATION xiii TABLE OF CONTENT xiv LIST OF TABLES xvii LIST OF FIGURES xix LIST OF ABBREVIATIONS

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1. INTRODUCTION 1 2. OBJECTIVE

4

3. LITERATURE REVIEW

5

3.1. Genetic epidemiology 5 3.1.1. Various study designs in genetic epidemiology 7 3.1.2. History of twin studies 9 3.1.3. Analysis, sampling and assumptions of twin studies 14 3.1.4. Benefits of twin studies 16 3.1.5. Limitations of twin studies 18 3.1.6. The assumption of representativeness 19 3.1.7. The assumption of equal environment 21 3.1.8. The assumption of twinning mechanisms 21 3.1.9. The assumption of higher MZ mortality in comparison with DZ in

adulthood 22

3.2. Reproductive Health 24 3.2.1. Why twin study of reproductive health 24

3.2.1.1. Heritability of reproductive ill health 24 3.2.1.2. Biology of twinning and its effect on reproductive health 26

3.2.1.2.1. Birth size 27 3.2.1.2.2. Discordant birth weight 30 3.2.1.2.3. Birth weight and adult life morbidity 31 3.2.1.2.4. Adult morbidity and genetic origin of association with birth

weight 33

3.2.1.2.5. Opposite sex effect

38

4. MATERIALS AND METHODS

42

4.1. Study plan 42 4.2. Questionnaire 46 4.3. Statistical analysis 48

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4.3.1. Data preparation 50 4.3.2. Genetic analysis 51

4.3.2.1. Classical approach 52 4.3.2.2. Maximum likelihood analysis 55 4.3.2.3. Path analysis 55 4.3.2.4. Maximum likelihood estimation 57 4.3.2.5. Univariate twin analysis 62 4.3.2.6. Graphical analysis 64 4.3.2.7. Probandwise Concordance rate 65 4.3.2.8. Genetic modeling for qualitative variables 66 4.3.2.9. Model fitting analysis for qualitative data 69

5. RESULTS

70

5.1. Descriptive analysis 70 5.2. Reproductive events 73

5.2.1. Quantitative variable 73 5.2.1.1. Descriptive analysis 73 5.2.1.2. Genetic analysis 74

5.2.1.2.1. Classical approach using Falconer’s formula 74 5.2.1.2.2. Univariate twin analysis 75 5.2.1.2.3. Graphical analysis 77

5.2.1.2.3.1. Scatter plots 77 5.2.1.2.3.2. CDF plots 83

5.2.2. Qualitative variable 87 5.2.2.1. Probandwise Concordance Rate 87 5.2.2.2. Model Fitting 88

5.3. Reproductive behavior 90 5.3.1. Quantitative variable 90

5.3.1.1. Descriptive analysis 90 5.3.1.2. Genetic analysis 90

5.3.1.2.1. Classical approach using Falconer’s formula 90 5.3.2. Qualitative variable 93

5.3.2.1. Probandwise Concordance rate and Model Fitting 94 5.4. Opposite sex twin 95

5.4.1. Quantitative variables 95 5.4.2. Qualitative variables 96

5.5. Comparison between identical and non-identical twins as a group 98 5.5.1. Descriptive analysis 98 5.5.2. Reproductive Health events and behavior 99

5.6. Comparison between low birth weight and normal birth weight twin 105 5.6.1. Descriptive analysis 105 5.6.2. Reproductive events 105 5.6.3. Analysis of missing data 106

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6. DISCUSSION

108

6.1. Reproductive Events 108 6.1.1. Birth weight 108 6.1.2. Age of menarche 110 6.1.3. Anthropometric measures 113 6.1.4. Menstruation 115 6.1.5. Premenstrual Symptoms 117 6.1.6. Polycystic ovary syndromes 119 6.1.7. Hyperandrogenism 121

6.2. Reproductive behavior 125 6.3. Opposite sex effect 131 6.4. Fetal origin hypothesis: Differences between MZ and DZ twin 135 6.5. Thrifty gene hypothesis: Difference between MZ and DZ twin 140

7. SUMMARY, CONCLUSION AND RECOMMENDATION FOR FUTURE

RESEARCH 145

REFERENCES 151 APPENDICES 173 BIODATA OF STUDENT 208 LIST OF PUBLICATIONS 211

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LIST OF TABLES

Table

Page

1. Frequency of socio-demographic characteristics of twins within the whole population (n=266)

70

2. Test of homogeneity across two nations (Iran and Malaysia) 72

3. Descriptive analysis of quantitative variables (n=193) 73

4: Analysis of genetic and environmental effects for quantitative variables related to reproductive events using basic approach

74

5: Univariate twin analysis for reproductive events inclusive of genetic (A), common environment (C) and shared environment (E) effect and best fitting model estimation

76

6: Probandwise concordance rate of MZ and DZ twins, its ratio, andtetrachoric correlation for qualitative reproductive events

87

7: Modeling results for the concordance data 89

8. Description of quantitative variables of reproductive behavior 90

9. Description of qualitative variables of reproductive behavior 91

10: Analysis of genetic and environment variable for variables related to quantitative reproductive behavior

93

11. Probandwise concordance rate of MZ and DZ twins, its ratio, and tetrachoric correlation for qualitative reproductive behavior

94

12. A comparison between the mean ± SD of quantitative variables related to reproductive health

96

13: A comparison between FM and FF group related to reproductive ill health using Chi-square test

97

14 : A comparison of quantitative variables related to reproductive events between MZ and DZ twins

99

15 : A comparison of qualitative variables related to reproductive events between MZ and DZ twins

101

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16: Differences between reproductive health behavior between MZ and DZ twins

99

17: Difference between low and normal birth weight for quantitative variables 105

18: Difference between low and normal birth weight for qualitative variables

106

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LIST OF FIGURES Page

1. Univariate ACE model for data obtained from MZ and DZ twins 57

2: Twin univariate model fitting 62

3. ACE model and nested sub models, output for BMI 63

4. Contingency table 66

5: Scatter plot of twin 1 and twin 2 between MZ twins for birth weight (gr) 78

6: Scatter plot of twin 1 and twin 2 between DZ twins for birth weight (gr) 78

7: Scatter plot of twin 1 and twin 2 between MZ twins for age of menarche (Y) 79

8: Scatter plot of twin 1 and twin 2 between DZ twins for age of menarche (Y) 79

9: Scatter plot of twin 1 and twin 2 between MZ twins for menstrual duration (day) 80

10: Scatter plot of twin 1 and twin 2 between DZ twins for menstrual duration (day) 80

11: Scatter plot of twin 1 and twin 2 between MZ twins for weight (kg) 81

12: Scatter plot of twin 1 and twin 2 between DZ twins for weight (kg) 81

13: Scatter plot of twin 1 and twin 2 between MZ twins for height (cm) 82

14: Scatter plot of twin 1 and twin 2 between DZ twins for height (cm) 82

15: CDF plot of birth weight (gr) 84

16: CDF plot of age of menarche (year) 84

17: CDF plot of weight (kg) 85

18: CDF plot of height (cm) 85

19: CDF plot of menstrual duration (day) 86

20: CDF plot of menstrual interval (day) 86

21: A comparison between average of weight (kg) for hirsute and non-hirsute subjects within MZ and DZ twins

102

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List of Abbreviations

A Additive Genetic Variation

AIC Akaike's Information Criterion

BMI Body Mass Index

C Common Environment Variation

CANDLES Children of Auschwitz Nazi Deadly Laboratory Experiments Survivors

CDF Cumulative Distribution Function

D Dominant Genetic Variation

DNA Deoxyribonucleic Acid

DSL Diethylstilbestrol

DZ Dizygotic

E Non-shared Environment Variation

FAE Functional Androgen Excess

FF Female-Female

FM Female-Male

GE Genetic Epidemiology

h2 Heritability

HDL High Density Lipoprotein

HPO-Axis Hypothalamic Pituitary Gonadal- axis

ICPD International Conference on Population and Development

IQ Intelligence Quotient

LDL Low density Lipoprotein

MZ Monozygotic

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NMTR National Malaysian Twin Registry

OS Opposite Sex

PCOS Polycystic Ovary Syndrome

PMS Pre Menstrual Syndrome

SD Standard Deviation

SHBG Sex Hormone Binding Globulin

SPSS Statistical Package for the Social Sciences

SS Same Sex

TC Tetrachoric Correlation

UK United Kingdom

UniKL University of Kuala Lumpur

USA United States of America

WC Waist Circumference

WHO World Health Organization

1. INTRODUCTION

Twins play an important role in the study of hereditary risk factors in disease, the on-going quest

for knowledge of twins and multiple births, and the search for answers to the profound questions

posed by genetic research. A wide range of research projects have been undertaken in

conjunction with twins who have registered with twin registries including studies on alcohol and

tobacco use; asthma; cholesterol; diabetes; anxiety, stress, and depression; osteoporosis; heart

disease; epilepsy; dietary salt; Alzheimer's disease; eating disorders; prostate size; male

infertility; premenstrual tension; endometriosis; oral ulcers; attention deficit disorder; and breast

cancer (Hurbed, 1978; Phillips, 2001; Baker, 1998; Christensen, 1995; Vagero, 1994).

History of twin studies goes back to year 1876 where Sir Francia Glaton first suggested that the

examination of twins can be considered as a method of determining the contribution of heredity

and upbringing. His main objective in his research was to find dissimilarities between adult

twins as he constantly followed their growth from childhood looking for difference between

them with increasing age. Siemen (1924) was the first to conduct the classical twin study where

he collected ample representative samples of twins in his study population of school children.

He concluded that a trait was under genetic control if monozygotic (MZ) twins were strongly

concordant for a trait, while dizygotic (DZ) twins were less concordant (Macdonald, 1993).

Twin studies are therefore traditionally used to estimate the genetic contribution to a trait by

comparing MZ twin pairs with DZ twins. Study of twins helps us understand the distinctions

between nature (genes) and nurture (environment) in shaping particular individual traits.

2

The hypothesis behind twin studies is that since MZ twins share 100% of their genes in

common, while DZ twins share only 50% of an excess concordance between MZ twin

pairs may reflect a greater role of genetic factor (Keith, 1994).

Reproductive health, although very broad in spectrum, is an important part of natural

science. It is defined by the World Health Organization (WHO) as a state of mental,

physical and social well being related to the reproductive system (WHO, 1999). It is not

merely the absence of disease, dysfunction or infirmity. Since the International

Conference on Population and Development (ICPD) held in Cairo in 1994 and, more

recently, the ICPD+5 Forum in Hague, many organizations and researchers tried to

define reproductive health and the way it is measured in order to improve different

aspects of reproductive health (Reproductive health strategy, WHO, 2001).

Reproductive health study of twins can assist us to investigate the genetic or

environmental causes of reproductive diseases, which would otherwise be difficult or

impossible to study. For example, by comparing breast cancer concordance in twin

pairs, researchers can estimate the effect of genetics and environment on the

development of this type of cancer. High concordance between twin pairs suggests a

strong influence for genetic factors, whereas low concordance indicates a weak

influence. Preventive measures can be taken in to consideration for those who are at risk

of environmental influences if the role of genetics is minor. Gene-gene interaction and

3

gene-environement interaction for variables are to be studied diligently and future gene

therapies can eliminate some light on the type of interventions possible on genetic

modifications.

The role of shared environment is extremely important. Even though twins are the same

age and usually grow up in the same household, their experiences can differ greatly.

Identical twins share all the same hereditary material (DNA), whereas fraternal twins

share only about half of the same DNA. Greater similarity for a trait among identical

twins than the same trait among fraternal twins implies that the influence of genetic

factors may predominate over that of environmental factors.

Investigating the hereditary or environmental nature of reproductive health variables call

for an interesting research. As far as the author knows, Malaysian and Iranian twins

have never been studied before for reproductive health traits. This study aims at

conducting an investigation on various aspects of reproductive health that has some

indication of possible heredity. The effect of intra-uterine environment, zygosity and

low birth weight on reproductive health was also examined.