Early stages of thyroid autoimmunity: follow-up studies in the Amsterdam AITD cohort Effraimidis, G.

Transcription

1 UvA-DARE (Digital Academic Repository) Early stages of thyroid autoimmunity: follow-up studies in the Amsterdam AITD cohort Effraimidis, G. Link to publication Citation for published version (APA): Effraimidis, G. (2012). Early stages of thyroid autoimmunity: follow-up studies in the Amsterdam AITD cohort General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam ( Download date: 04 Oct 2018

2 E arly S tages of T hyroid A utoimmunit y G rigoris E ffr aimidis

3 Early stages of thyroid autoimmunity follow-up studies in the Amsterdam AITD cohort Grigoris Effraimidis

4 Early stages of thyroid autoimmunity, follow-up studies in the Amsterdam AITD cohort Academic thesis, University of Amsterdam, Amsterdam, The Netherlands This study was supported by: - the fellowship program Exchange in Endocrinology Expertise from the Section/Board of Endocrinology of the UEMS and Novo Nordisk - grant NWO from the Netherlands Organization for Scientific Research, The Hague, the Netherlands. Printing of this thesis was financially supported by: - Stichting Amsterdam Thyroid Club - Universiteit van Amsterdam. ISBN: Author Lay-out Cover Design Print Grigoris Effraimidis Grigoris Effraimidis Eugenia Dimopoulou CPI, Zutphen, the Netherlands Grigoris Effraimidis, Amsterdam 2012 All rights reserved. No part of this publication may be reproduced, stored, or transmitted in any form or by any means, without written permission of the author.

5 Early stages of thyroid autoimmunity follow-up studies in the Amsterdam AITD cohort ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus prof. dr. D.C. van den Boom ten overstaan van een door het college voor promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel op dinsdag 19 juni 2012, te 12:00 uur door Grigorios Effraimidis geboren te Thessaloniki, Griekenland

6 PROMOTIECOMMISSIE Promotors: Prof. dr. W.M. Wiersinga Prof. dr. J.G.P Tijssen Overige Leden: Prof. dr. R.J.M. ten Berge Dr. O.M. Dekkers Prof. dr. E. Fliers Prof. dr. J.C.J.M. de Haes Prof. dr. P. Lips Prof. dr. J. A. Romijn Faculteit der Geneeskunde

7 to the memory of my father to my mother to Giorgos and Sofia

8 Στόν πατέρα μοῦ χρωστῶ το ζῆν καί στον δάσκαλό μοῦ το εὖ ζῆν I am indebted to my father for living, but to my teacher for living well Aan mijn vader dank ik mijn bestaan, aan mijn leraar dank ik mijn (spirituele) leven Μέγας Αλέξανδρος/Alexander the Great/ Alexander de Grote

9 CONTENTS Chapter 1 Introduction. 9 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Natural history of the transition from euthyroidism to overt autoimmune hypo-or hyperthyroidism: a prospective study. Effraimidis G, Strieder TGA, Tijssen JGP, Wiersinga WM. European Journal of Endocrinology. 2011;164: Discontinuation of smoking increases the risk for developing thyroid peroxidase antibodies and/or thyroglobulin antibodies: a prospective study. Effraimidis G, Tijssen JGP, Wiersinga WM. Journal of Clinical Endocrinology & Metabolism. 2009;94: Alcohol consumption as a risk factor for autoimmune thyroid disease: a prospective study. Effraimidis G, Tijssen JGP, Wiersinga WM. European Thyroid Journal 2012; 1: issue 2 (in press). Vitamin D deficiency is not associated with thyroid autoimmunity. Effraimidis G, Badenhoop K, Tijssen JGP, Wiersinga WM. European Journal of Endocrinology. 2012; accepted for publication. Involvement of stress in the pathogenesis of autoimmune thyroid disease: a prospective study. Effraimidis G, Tijssen JGP, Brosschot JF, Wiersinga WM. Psychoneuroendocrinology. 2012; Epub ahead of print. Age-dependent changes in recent life events and daily hassles in a cohort of healthy women. Effraimidis G, Brosschot JF, Tijssen JGP, Wiersinga WM. Submitted for publication. No causal relationship between Yersinia enterocolitica infection and autoimmune thyroid disease: evidence from a prospective study. Effraimidis G, Tijssen JGP, Strieder TGA, Wiersinga WM. Clinical & Experimental Immunology. 2011;165: Chapter 9 General discussion. 109 Author s affiliations 135 Summary. 137 Samenvatting. 141 Αcknowledgements. 145

10

11 Chapter 1 Introduction

12 10

13 A short historical overview of Autoimmune Thyroid Disease 11 The first presentation of the classic symptoms of hypothyroidism was in 1883 at the meeting of the Clinical Society of London [1], although there had been already a few other early observations on myxedema in 1850 [2]. It was at the same time that Theodor Kocher observed that patients who had undergone total thyroidectomy developed a clinical picture similar to that of patients with cretinism [3]. In 1888 the Clinical Society of London published a detailed report which linked cretinism and myxedema with the destruction of the thyroid gland [4]. It became clear that the lack of one or more substances secreted by the thyroid was responsible for the disease. At the end of the 19th century hypothyroidism was not curable but very soon thyroid extracts from sheep proved to be effective [5]. In 1912 the Japanese physician Hakaru Hashimoto described a new pathologic entity of the thyroid gland in four women who had goiters with diffuse lymphocytic infiltration, fibrosis and parenchymal atrophy (struma lymphomatosoma) [6]. In 1956, it was reported for the first time by Roitt et al. [7] that serum of patients with lymphadenoid goiter contains antibodies against thyroid tissue (thyroglobulin antibodies) which are associated with Hashimoto s disease. In the same year, Rose and Witebsky provided evidence that immunization of animals with homologous thyroid extract developed thyroiditis lesions with similar features as those seen in the human disease [8]. The production of experimental autoimmune thyroiditis and the detection of antithyroglobulin antibodies laid the foundation stone for the causal relationship between autoimmunization and human disease [9,10]. In 1958, Belyavin and Trotter reported on another type of thyroid autoantibodies which were found to react with crude preparations of thyroid cell membranes, the anti-microsomal autoantibodies [11]. The microsomal antigen remained unidentified until 1985 when it was recognized as the thyroid peroxidase (TPO) [12,13]. The cardinal diagnostic test of hypothyroidism caused by destruction of thyroid gland is an elevated serum TSH. However until the mid 1960s serum TSH assays were not available. Until that time a diagnosis of primary hypothyroidism was confirmed by measuring basal metabolic rate, later replaced subsequently by protein bound iodine, hormonal iodine and T4 assays. By the early 1980s the methods for assay of serum TSH had been improved: TSH assays had become sufficiently sensitive to discriminate between normal and suppressed TSH values. The wide employment of TSH assays in clinical practice led to the realization that besides patients with overt hypothyroidism (elevated TSH in combination with low T4 levels) there were patients with an elevated TSH in the presence of normal T4 serum concentrations, a condition called subclinical hypothyroidism. The first cases of exopthalmos and goiter were published in Ten years later Robert Graves described four women with goiter and palpitations while one of them women had also exophthalmos [14]. The same constellation of symptoms was independently reported by Karl von Basedow in 1840, the historical Merseburger Triad as it is called in textbooks [15]. Paul Julius Moebius first brought up the idea that the disorder is caused by hypersecretion of a ductless gland in 1886 [16]. By the end of 19th century the thyroid was recognized as the causative factor because patients who underwent thyroidectomy were cured from Chapter 1

14 12 thyroid hormone excess. Therefore, thyroidectomy remained the core for the treatment of hyperthyroidism in the first half of the 20th century. In 1956 Adams and Purves while setting up an assay for serum TSH in guinea pigs found a stimulator in the serum of Graves disease patients distinct from TSH [17]. Their findings were confirmed in 1958 by McKenzie who used mice as assay model [18]. The stimulator was initially called long-acting thyroid stimulator (LATS). Kriss et al. in 1964 proved that LATS was an immunoglobulin G, and subsequently the autoimmune nature of the disease became established [19]. During the next 20 years it became obvious that the thyroid stimulating activity is caused by antibodies against the TSH receptor. At present very sensitive assays for the TSH-receptor antibodies are available [20]. Autoimmune Thyroid Disease: a complex multifactorial entit y Hashimoto s thyroiditis (hypothyroidism) and Graves disease (hyperthyroidism) can be considered as two separate diseases. Although clinically the opposite, both conditions share immunological features like a variable degree of lymphocytic infiltration and the presence of autoantibodies to TPO and Tg in serum. Moreover, it is well known that sometimes (although rarely) patients with hypothyroid Hashimoto goiter may convert into Graves hyperthyroid cases; also a number of Graves hyperthyroid patients may spontaneously convert to hypothyroidism in the long term. Furthmore, in the same family there can be patients with Graves hyperthyroidism and Hashimoto s hypothyroidism. Consequently, one may consider the two conditions as representing the opposites sides of the same coin. In this view Graves disease and Hashimoto s thyroiditis belong to one and the same disease entity, namely autoimmune thyroid disease (AITD). AITD is nowadays generally considered to be a complex multifactorial entity in which the interplay between genetic and environmental factors result in the expression of the disease. There is a good evidence that the genetic predisposition plays a major role in the pathogenesis of AITD as siblings and other family members of AITD patients are at increased risk for AITD [21,22]. The sibling recurrence risk ratio (λs) is 11.6 for Graves and 28.0 for Hashimoto s disease [23,24]. In women with at least one first degree relative with AITD, the incidence of Graves hyperthyroidism is times higher and of Hashimoto s hypothyroidism times higher than the general female population [25]. Twin studies suggest that the concordance rate for Graves disease and for autoimmune hypothyroidism is significantly higher in monozygotic twins compared with dizygotic twins (0.35 vs 0.03, p=0.001 and 0.55 vs. 0.0, p=0.01 respectively) [26,27]. A model fitting analysis estimated that 79% and 73% of the likelihood of developing Graves disease and thyroid antibodies respectively is due to genetic factors [27-29] and it can be assumed that the relative impact of genetic factors in the phenotypes of AITD is most likely around 75% [29]. The nature of these genetics factors is gradually being discovered over the last decades. The importance of the HLA gene complex in AITD was early identified. Graves disease has been

15 associated with HLA-DR3 in Caucasians [30] (primary susceptibility allele HLA-DRB1*03 [24]) while Hashimoto s thyroiditis has been reported to be associated with DR3 and DR4 in Caucasians [30] and with a pocket HLA-DR amino acid signature [31]. Later, it has been reported that polymorphisms in the gene that encodes cytotoxic T lymphocyte antigen 4 (CTLA-4) [32] and protein tyrosine phosphatase, non-receptor type 22 (PTPN22) [30,33] are associated with AITD. Polymorphisms in CD40, thyroglobulin and thyrotropin receptor genes have also been identified as contributing to thyroid autoimmunity [22,30]. Two comments can be made in these studies. First, the observed genetic associations are not always specific for AITD as the same genetic factors (HLA, CD40, CTLA-4, PTPN22) are associated with a number of other autoimmune diseases (like type 1 diabetes, rheumatoid arthritis, Addison disease). Second, the contribution of each of these genetic factors to the risk of contracting AITD is very small indeed. It has been estimated that combining the presently known polymorphisms that carry a risk for AITD, ends up to only 5-10% of the genetic susceptibility. Consequently there must be many more genes involved in the pathogenesis of AITD than currently known. Whereas multiple genes contribute to AITD, environmental factors are also important in the pathogenesis of the disease. According to twins studies environmental factors will contribute for about 30% to the development of AITD. Exposure to a multitude environmental factors have been studied and found to be associated with AITD like iodine, smoking, stress, pregnancy, exogenous estrogens and certain infections (e.g. with Yersinia enterocolitica) [34,35]. 13 Chapter 1 Aim of the present thesis So far, the importance of environmental factors in the pathogenesis of AITD has been studied mostly in a retrospective manner in cross-sectional case-control or populationbased studies (exceptions are ambient iodine intake and exposure to particular drugs which have been studied in a prospective manner). For instance the well established association between stress and Graves hyperthyroidism has been studied just in retrospective studies, which are liable to recall bias as Graves disease patients were asked to report on stressful life events either at the time they were still hyperthyroid or even later when euthyroidism was restored. Prospective studies in general provide more solid evidence in establishing associations between environmental factors and autoimmune thyroid disease. Since the first descriptions of hypothyroid and hyperthyroid patients in the 19th century, a large body of literature has provided many pieces of information that help to solve the puzzle of the pathogenesis of AITD. The overall picture that emerges is as follows. AITD starts with a particular genetic background. Next thyroid antibodies (TPO-Ab, Tg-Ab) occur in serum, which can be taken as an early sign of thyroid autoimmunity; thyroid function tests (TSH, ft4) at this stage are still within the normal reference range. Thereafter serum TSH values may become abnormal while serum ft4 concentrations are still normal: the stage of subclinical hypothyroidism or subclinical hyperthyroidism. AITD ends in many cases with

16 14 overt hypo- or hyperthyroidism characterized by both abnormal TSH and ft4 concentrations. However there remain still many unanswered questions. With respect to environmental factors, these have been studied mostly in a retrospective manner and in relation to the occurrence of the final stages of AITD (overt hypo- or hyperthyroidism) and not to the earlier stages of AITD (occurrence of thyroid antibodies). In order to get better insight into environmental factors involved in the pathogenesis of AITD and especially in the early stages of AITD, we evaluated exposure to a number of environmental insults in a prospective manner. This was done by nested case-control studies in the Amsterdam AITD cohort. The Amsterdam AITD cohort consists of healthy euthyroid women at risk for AITD because they were relatives of patients with AITD; subjects were followed prospectively with annual assessments for 5 years. The cohort also allows evaluation of the relative importance of susceptibility genes and assessment of early aberrations in immunological surveillance in AITD, but such studies are outside the scope of the present thesis which is devoted to environmental factors. Amsterdam AITD cohort The cohort has been described previously in detail [36,37]. Subjects willing to participate in our study, were recruited from all over The Netherlands through patients visiting our department, through advertisements in local newspapers, and through thyroid patient self-support associations. The Medical Ethics Committee of the Academic Medical Center in Amsterdam approved the study. All subjects were seen at our Institution and gave their informed written consent. We screened 1003 female subjects with at least one 1st or 2nd degree relative with documented autoimmune hyper- or hypothyroidism, who were in selfproclaimed good health, without a history of thyroid disease, and between 18 and 65 years of age. We checked the medical history of the affected relative(s) regarding the autoimmune nature of their thyroid disease after obtaining their informed consent. Autoimmune thyroid disease for this purpose was defined as documented hyper- or hypothyroidism in the presence of autoantibodies against thyroid peroxidase (TPO), TSH-receptor autoantibodies, histology compatible with autoimmune thyroiditis, or thyroid eye disease. In 200 of the 1003 screened subjects proof of the autoimmune nature of the thyroid disease in at least one family member could not be ascertained, leaving 803 participants to be included in the cohort. After excluding 13 females with overt hyper- or hypothyroidism at baseline, 790 first or second degree female relatives of AITD patients were eligible for follow-up. Endpoints of follow-up were the development of overt hyperthyroidism or hypothyroidism (events) defined by abnormal TSH values in combination with abnormal ft4 concentrations in plasma. Endpoints were assessed every year for 5 years. At each visit blood was sampled for assay of TSH, ft4, TPO-Ab, Tg-Ab, TBII (TSH binding inhibitory immunoglobulins) and Yersinia enterocolitica serology. In addition, annual questionnaires assessed smoking habits, alcohol consumption, use of oral contraceptives or other estrogens, pregnancies, exposure to iodine excess, and stress exposure.

17 At the end of the 5-yr follow-up of the Amsterdam AITD cohort, 38 subjects had developed autoimmune hypothyroidism and 13 hyperthyroidism [25]. The cumulative event rate was 7.5% over 5 years, the mean annual event rate was 1.5%. 15 Outline of the thesis Chapter 1 In chapter 2 we describe the time course of the transition from euthyroidism to either overt hyperthyroidism or overt hypothyroidism during the natural history of AITD. We also evaluated whether exposure to some environmental factors change in the years preceding the development of overt AITD in two nested case-control studies. In chapter 3 we evaluate environmental factors (especially smoking) in relation to de novo occurrence of thyroid antibodies (TPO-Ab, Tg-Ab). Smoking is a well known risk factor for Graves hyperthyroidism, and recent literature data indicate that in contrast smoking seems to protect against overt autoimmune hypothyroidism. In chapter 4 the hypothesis is tested that alcohol consumption reduces the risk of developing AITD. We examined the effect of alcohol intake on both early (development of TPO-Ab) and late stages (overt hypothyroidism) of AITD. Previous studies suggest a protective effect of alcohol on the development of other autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus. In chapter 5 the hypothesis is tested that early stages of thyroid autoimmunity (i.e. genetic susceptibility and development of TPO-Ab) are associated with low vitamin D levels. Vitamin D deficiency has been identified as a risk factor for a number of autoimmune diseases including type 1 diabetes and multiple sclerosis. In chapter 6 the hypothesis is tested that recent life events and daily hassles are associated with the development of TPO-Ab and overt hyper- or hypothyroidism. Exposure to stress has been related to the onset of Graves hyperthyroidism in retrospective studies, but the role of stress in the development of thyroid antibodies and Hashimoto s hypothyroidism has scarcely been studied. In chapter 7 we report age-dependent changes in recent life events and daily hassles in healthy women who remained euthyroid without any serological sign of thyroid autoimmunity during the 5-yr follow-up. In chapter 8 we evaluate the relationship between Yersinia Enterocolitica infection and de novo occurrence of thyroid antibodies and the development of overt autoimmune hypo- or hyperthyroidism. So far, conflicting results are reported in the literature on the putative relationship between Yersinia enterocolitica infection and AITD. In the general discussion of chapter 9, an overview of the above mentioned studies is presented, and the scientific and clinical implications of the obtained results are commented.

18 16 Figure 1. Timeline of the history of autoimmune thyroid disease.

19 References [1] Reports of Societies. Clinical Society of London. Br Med J 1883: [2] Curling TB. Two Cases of absence of the Thyroid body, and symmetrical swellings of fat tissue at the sides of the neck, connected with defective cerebral development. Med Chir Trans 1850;33: [3] Kocher T. Ueber Kropf exstirpation und ihre Folgen. Archiv fur Klinische Chirurgie; 1883 p [4] Longmans G&C, editor. Report of a committee of the Clinical Society of London to investigate the subject of myxoedema. London, UK: Longmans, Green & Co; [5] Murray GR. Note on the Treatment of Myxoedema by Hypodermic Injections of an Extract of the Thyroid Gland of a Sheep. Br Med J 1891;2: [6] Hashimoto H. Zur Kenntnis der lymphomatösen Veränderung der Schilddrüse. Archiv Für Klinische Chirurgie 1912;97: [7] Roitt I, Doniach D, Campbell P, Hudson R. Auto-antibodies in Hashimoto s disease (lymphadenoid goitre). Lancet 1956;271:820. [8] Rose NR, Witebsky E. Studies on organ specificity. V. Changes in the thyroid glands of rabbits following active immunization with rabbit thyroid extracts. J. Immunol. 1956;76: [9] Witebsky E, Rose NR, Terplan K, Paine JR, Egan RW. Chronic thyroiditis and autoimmunization. Jama 1957;164: [10] Mackay IR. Travels and travails of autoimmunity: A historical journey from discovery to rediscovery. Autoimmunity Reviews 2010;9:A251 A258. [11] Belyavin G, Trotter WR. Investigations of thyroid antigens reacting with Hashimoto sera; evidence for an antigen other than thyroglobulin. Lancet 1959;1: [12] Portmann L, Hamada N, Heinrich G, DeGroot LJ. Anti-thyroid peroxidase antibody in patients with autoimmune thyroid disease: possible identity with anti-microsomal antibody. J Clin Endocrinol Metab 1985;61: [13] Sawin CT, Bigos ST, Land S, Bacharach P. The aging thyroid. Relationship between elevated serum thyrotropin level and thyroid antibodies in elderly patients. Am J Med 1985;79: [14] Graves R. Clinical lectures delivered at the Meath Hospital during the session Lecture XII. Newly observed affection of the thyroid. London Med Surg 1835;7: [15] Basedow CA. Exophthalmos durch Hypertrophie des Zellgewebes in der Augenhöhle. Wochenschrift Ges Heilkunde 1840;6: , [16] Moebius PJ. Ueber das Wesen der Basedowschen Krankheit. Arch Psychiatrie; [17] Adams D, Purves H. Abnormal responses in the assay of thyrotropin. Proc. Uniu. Otago Med. Sch 1956;34: [18] McKenzie JM. Delayed thyroid response to serum from thyrotoxic patients. Endocrinology 1958;62: [19] Kriss JP, Pleshakov V, Chien JR. Isolation and identification of the long-acting thyroid stimulator and its relation to hyperthyroidism and circumscribed pretibial myxedema. J Clin Endocrinol Metab 1964;24: [20] Zöphel K, Roggenbuck D, Schott M. Clinical review about TRAb assay s history. Autoimmunity Reviews 2010;9: [21] Chopra IJ, Solomon DH, Chopra U, Yoshihara E, Terasaki PI, Smith F. Abnormalities in thyroid function in relatives of patients with Graves disease and Hashimoto s thyroiditis: lack of correlation with inheritance of HLA-B8. J Clin Endocrinol Metab 1977;45: [22] Jacobson EM, Tomer Y. The CD40, CTLA-4, thyroglobulin, TSH receptor, and PTPN22 gene quintet and its contribution to thyroid autoimmunity: back to the future. Journal of Autoimmunity 2007;28: [23] Villanueva R, Greenberg DA, Davies TF, Tomer Y. Sibling recurrence risk in autoimmune thyroid disease. Thyroid 2003;13: [24] Tomer Y. Searching for the Autoimmune Thyroid Disease Susceptibility Genes: From Gene Mapping to Gene Function. Endocrine Reviews 2003;24: [25] Strieder TGA, Tijssen JGP, Wenzel BE, Endert E, Wiersinga WM. Prediction of progression to overt hypothyroidism or hyperthyroidism in female relatives of patients with autoimmune thyroid disease using the Thyroid Events Amsterdam (THEA) score. Arch Intern Med 2008;168: [26] Brix TH, Kyvik KO, Hegedüs L. A population-based study of chronic autoimmune hypothyroidism in Danish twins. J Clin Endocrinol Metab 2000;85: [27] Brix TH, Kyvik KO, Christensen K, Hegedüs L. Evidence for a major role of heredity in Graves disease: a populationbased study of two Danish twin cohorts. J Clin Endocrinol Metab 2001;86: [28] Hansen PS. The relative importance of genetic and environmental effects for the early stages of thyroid autoimmunity: a study of healthy Danish twins. Eur J Endocrinol 2006;154: [29] Brix TH, Hegedüs L. Twin studies as a model for exploring the aetiology of autoimmune thyroid disease. Clin Endocrinol 2011:Accepted Article. [30] Huber A, Menconi F, Corathers S, Jacobson EM, Tomer Y. Joint Genetic susceptibility to type 1 diabetes and autoimmune thyroiditis: from epidemiology to mechanisms. Endocrine Reviews 2008;29: [31] Menconi F, Monti MC, Greenberg DA, Oashi T, Osman R, Davies TF, Ban Y, Jacobson EM, Concepcion ES, Li CW, Tomer Y. Molecular amino acid signatures in the MHC class II peptide-binding pocket predispose to autoimmune thyroiditis in humans and in mice. Proc. Natl. Acad. Sci. U.S.A. 2008;105: [32] Tomer Y. Genetic Susceptibility to Autoimmune Thyroid Disease: Past, Present, and Future. Thyroid 2010;20: Chapter 1

20 18 [33] Velaga MR. The Codon 620 Tryptophan Allele of the Lymphoid Tyrosine Phosphatase (LYP) Gene Is a Major Determinant of Graves Disease. J Clin Endocrinol Metab 2004;89: [34] Prummel MF, Strieder T, Wiersinga WM. The environment and autoimmune thyroid diseases. Eur J Endocrinol 2004;150: [35] Tomer Y, Huber A. The etiology of autoimmune thyroid disease: a story of genes and environment. Journal of Autoimmunity 2009;32: [36] Strieder TGA, Prummel MF, Tijssen JGP, Endert E, Wiersinga WM. Risk factors for and prevalence of thyroid disorders in a cross-sectional study among healthy female relatives of patients with autoimmune thyroid disease. Clin Endocrinol 2003;59: [37] Strieder TGAF. The Amsterdam Autoimmune Thyroid Disease cohort. Thesis, University of Amsterdam, 2008.

21 19 Chapter 1

22

23 Chapter 2 Natural history of the transition from euthyroidism to overt autoimmune hypo- or hyperthyroidism: a prospective study. Effraimidis G Strieder TGA Tijssen JGP Wiersinga WM Eur J Endocrinol. 2011;164(1):

24 ABSTR ACT 22 Objective: To evaluate the progression in time from euthyroidism to overt autoimmune hypothyroidism or to overt autoimmune hyperthyroidism. Subjects and methods: The design is that of a nested case-control study within the prospective Amsterdam AITD cohort-study, in which 790 healthy euthyroid women with at least one 1st or 2nd degree relative with documented AITD were followed for five years. Thyroid function tests were assessed annually. Contrast between cases (overt hypothyroidism TSH >5.7 mu/l and ft4 <9.3 pmol/l or overt hyperthyroidism TSH <0.4 mu/l and ft4 >20.1 pmol/l ; also referred to as events) and controls (matched for age and duration of follow-up). Results: At baseline, the 38 hypothyroid cases had already higher TSH and lower ft4 concentrations than their 76 controls, and the difference between both groups persisted at one year before occurrence of the event. In contrast, neither TSH nor ft4 values differed between the 13 hyperthyroid cases and their 26 controls at baseline or at one year before the event. The prevalence of TPO-Ab was higher in both hypothyroid and hyperthyroid cases than in controls. At the time of event hypothyroid cases were less often current smokers p=0.083) and more often in the postpartum period (p=0.006) than their controls, whereas hyperthyroid cases had been pregnant more frequently (p=0.063). Conclusions: The data suggest that progression towards overt autoimmune hypothyroidism is a gradual process taking several years, but that in contrast overt autoimmune hyperthyroidism develops faster in terms of months.

25 Introduction In the perception of most clinical endocrinologists, the progression from euthyroidism to overt autoimmune hypothyroidism (Hashimoto s thyroiditis) over time is supposed to be slow and insidious. On the other hand, the progression from euthyroidism to overt autoimmune hyperthyroidism (Graves disease) is believed to occur more rapidly, as symptoms in newly diagnosed Graves hyperthyroid patients have been present for relatively short period of time. Although to date many studies have investigated the progression rate from subclinical to overt autoimmune hypothyroidism or hyperthyroidism [1-13], a careful review of the literature however did not detect studies on the progression from euthyroidism to overt autoimmune hypothyroidism or to overt hyperthyroidism and with these premises we sought to assess the time course from euthyroidism to overt thyroid failure and the supposedly rapid progression from euthyroidism to overt thyroid hyperfunction. 23 Chapter 2 Subjects & Methods Subjects The original Amsterdam AutoImmune Thyroid Disease (AITD) cohort consisted of 803 female subjects between 18 and 65 years of age, without a history of thyroid disease, who were in self-proclaimed good health and had at least one first- or second- degree relative with documented autoimmune hyper- or hypothyroidism. At study entrance thyroid function tests revealed overt hyper- or hypothyroidism in 13 participants, leaving 790 subjects for follow-up [14]. Subjects were seen annually at our institution during a five year follow-up. At each visit blood was withdrawn for thyroid function tests and the participants were asked to fill in questionnaires on smoking habits, pregnancy, estrogen treatment and iodine excess in the previous year. Current smoking was defined as smoking now or having stopped smoking within 1 year before the study visit. Current pregnancy was defined as being pregnant at the time of the study visit, and postpartum as termination of pregnancy in the previous year. Current use of estrogens was defined as using estrogens at the time of the study visit, or having stopped taking estrogens in the previous year. Endpoints of the study were either completion of the 5-yr follow-up period, or the development of overt hypothyroidism ( defined as TSH >5.7 mu/l and ft4 <9.3 pmol/l) or overt hyperthyroidism (defined as TSH <0.4 mu/l and ft4 >20.1 pmol/l) called cases or events. There occurred 38 cases of overt autoimmune hypothyroidism (34x Hashimoto s hypothyroidism, 4x postpartum thyroiditis) and 13 cases of overt autoimmune hyperthyroidism (11 with Graves hyperthyroidism, 1 with postpartum thyroiditis, 1 with silent thyroiditis), as reported elsewhere [15]. In all subjects except one symptoms developed in between two annual visits to our center; these subjects consulted their family physician, and the diagnoisis was confirmed clinically and biochemically. Cases didn t receive further follow-up for this study after the definite diagnosis of overt AITD.

26 24 For each of these 51 cases two controls were selected from the remaining subjects in the cohort. Controls were matched for age at study entrance, and the analysis of their data was limited up to the same visit at which the cases had reached their endpoint, thereby guaranteeing that both groups in this nested case-control study had been exposed to environmental factors for precisely the same follow-up period. The study was approved by the Institutional Review Board of the Academic Medical Center, and all participants gave their informed consent prior to enrollment in the study. Laboratory measurements Serum TSH and ft4 were measured using time-resolved fluoroimmunoassays (Delfia htsh and Delfia ft4 respectively, Wallac Oy, Turku, Finland). Reference values are for TSH mu/l and for ft pmol/l as described previously [14]. Thyroid peroxidase (TPO) antibodies and thyroglobulin (Tg) antibodies were measured by chemiluminescence immunoassays (LUMI-test anti-tpo and LUMI-test anti-tg respectively, Brahms, Berlin, Germany). Improved versions of both assays became available during followup: detection limits of these new assays were for TPO-Ab 30 ku/l and for Tg-Ab 20 ku/l. TPO-Ab concentrations obtained with the old assay were multiplied by a factor 0.72 to obtain comparative values in the new assay. TPO-Ab and Tg-Ab concentrations were considered to be positive at values of >100 ku/l. TSH receptor antibodies were determined as TSH binding inhibitory immunoglobulins (TBII) using the TRAK assay (Brahms, Berlin, Germany); detection limits in the 1st and 2nd generation TRAK assays were 5 and 1 U/L respectively, and values above 12 and 1.5 U/L respectively were considered as positive. Statistical analysis Statistical analysis was carried out with the SPSS18 package. Values are given as mean±sd for age, but as median and interquartile range for all other parameters. Differences between cases and controls were evaluated by Students t test for age, by Mann-Whitney U-test for TSH, ft4, TPO-Ab and Tg-Ab, and by χ2 test or if appropriate Fisher s exact test for the other parameters. A p-value of <0.05 was considered to indicate significant differences between groups. Results The average time period between study entrance and the occurrence of an event was three years. At baseline, the 38 hypothyroid cases had already higher TSH, lower ft4 and higher TPO-Ab and Tg-Ab serum concentrations than their 76 controls, and the difference between both groups persisted at one year before occurrence of the event (Table 1). No differences were observed in TSH, ft4, TPO-Ab and Tg-Ab between study entrance and one year before the occurrence of event, neither among cases nor among controls. At baseline, 7 of the 38 subjects who developed overt hypothyroidism had subclinical hypothyroidism. Exclusion of these 7 subjects from the analysis did not alter the results: the remaining 31 cases had still higher TSH, lower ft4 and higher TPO-Ab and Tg-Ab serum concentrations

27 Table 1. Comparison of characteristics between subjects who developed clinically overt auto-immune thyroid disease (AITD) and their corresponding controls (matched for age and duration of follow-up) at consecutive time points of follow-up, in a nested case-control study of 153 women with 1st or 2nd degree relatives with proven AITD derived from the prospective Amsterdam AITD cohort. Hypothyroidism Hyperthyroidism Cases Controls P-value Cases Controls P-value N At study entrance - Age in years ± ± TSH mu/l 4.2 ( ) 1.6 ( ) < ( ) 1.5 ( ) ft4 pmol/l 10.7 ( ) 12.8 ( ) < ( ) 14.1 ( ) TPO Antibody positive 79 % 15 % < % 35 % TPO Antibody ku/l 1123 ( ) 25 (<30-49) < (< ) <30 (<30-155) Tg Antibody positive 26 % 10 % 0.01* 15 % 8 % 0.46* - Tg Antibody ku/l 75 (40-238) 8 (<20-29) < (23-108) 17 (<20-41) TBII positive 0 % 0 % % 4 % 1.00* One year before occurrence of event - Age in years 40 ± ± ± ± TSH mu/l 4.0 ( ) 1.6 ( ) < ( ) 1.6 ( ) ft4 pmol/l 10.6 ( ) 13.0 ( ) < ( ) 13.5 ( ) TPO Antibody positive 87 % 18 % < % 42 % TPO Antibody ku/l 1076 ( ) 25 (<30-42) < (< ) 36 (<30-91) Tg Antibody positive 45 % 21 % < % 12 % Tg Antibody ku/l 123 (42-235) 10 (<20-32) < (21-133) 15 (<20-57) TBII positive 5 % 0 % 0.11* 0 % 0 % 1.00* Data are given as means ± SD or medians with interquartile range or proportions, P-value of cases versus controls, * = Fisher s exact test. Time of event: visit when diagnosis was made or confirmed. TSH: thyroid stimulating hormone, ft4: free T4, TPO: thyroid peroxidase, Tg: thyroglobuline, TBII: TSH-receptor antibodies. 25 Chapter 2

28 26 than their 62 controls, both at baseline and at one year before the occurrence of the event (data not shown). In contrast, neither TSH nor ft4 values differed between the 13 hyperthyroid cases and their 26 controls at baseline or at one year before the event, but the prevalence of TPO-Ab and Tg-Ab was higher in cases than in controls (Table 1). TBII were present in only one participant at study entrance. No differences were observed in TSH, ft4, TPO-Ab and Tg-Ab between study entrance and one year before the occurrence of event, neither among cases nor among controls. At baseline, 2 of the 13 subjects who developed overt hyperthyroidism had subclinical hyperthyroidism. Exclusion of these 2 subjects from the analysis did not alter the results: TSH, ft4, TPO-Ab and Tg-Ab serum concentrations in the remaining 11 cases and 22 controls were similar at baseline and at one year before occurrence of the event (data not shown). The difference in TPO-Ab and Tg-Ab concentrations was not significant between hypothyroid cases and hyperthyroid cases neither at baseline nor at one year before occurrence of the event. Smoking status didn t differ between cases and controls at study entrance or at one year before the event (Table 2). At the time of the event, however, there were less current smokers in hypothyroid cases than in controls (5 out of 38 (13%) cases vs 21 out of 76 (28%) controls, p=0.083). This was not so in the hyperthyroid subgroup (2 out of 13 (15%) cases vs 7 out of 26 (27%) controls, p=0.7). Cases and controls did not differ with respect to pregnancies at study entrance or at one year before event, but there were more cases in the postpartum period than controls at the time of the event (Table 2). This was exclusively due to more hypothyroid cases in the postpartum period (8 out of 38 (21%) cases vs 3 out of 76 (4%) controls, p=0.006), not to more hyperthyroid cases (2 out of 13 (15%) cases vs 1 out of 26 (4%) controls, p=0.5). The proportion of women who had never been pregnant was lower in hyperthyroid cases than in their controls both one year before the event and at the time of the event (1 out of 13 (8%) cases vs 10 out of 26 (38%) controls, p=0.063); this was not so in the hypothyroid subgroup. The use of oral estrogens didn t differ between cases and controls at any time. (Table 2). Discussion The aim of our case-control study nested in the observational Amsterdam AITD cohort study was to evaluate in a prospective manner the progression from euthyroidism to overt autoimmune hypo- or hyperthyroidism (called events, or cases in the present study) and to examine the involvement of certain environmental factors in the development of the events. In our population, 51 cases were observed during follow-up, consisting of 38 hypothyroid and 13 hyperthyroid events occurring on average 3 years after study entrance, as reported elsewhere [15]. In our nested case-control study, controls had the same sex (all female) and by matching the same age and duration of follow-up as the cases. At study entrance, hypothyroid cases had higher TSH and TPO-Ab and lower ft4 serum levels than controls. This means that a minor degree of thyroid failure already existed at that time and that the transition from euthyroidism to autoimmune hypothyroidism had

29 Table 2. Comparison of exposure to environmental factors between subjects who developed clinically overt auto-immune thyroid disease (AITD) and their corresponding controls (matched for age and duration of follow-up), at consecutive time points of follow-up, in a nested case-control study of 153 women with 1st or 2nd degree relatives with proven AITD derived from the prospective Amsterdam AITD cohort. Exposure Hypothyroidism Hyperthyroidism Cases Controls P-value Cases Controls P-value N % % % % Smoking behaviour At study entrance -never quitters current smokers * One year before occurrence of event -never quitters current smokers * At time of occurrence of event -never quitters current smokers * 27 Chapter 2 Pregnancies At study entrance -never in the past current postpartum * * One year before occurrence of event -never in the past current postpartum * * At time of occurrence of event -never in the past current postpartum * * Oral estrogen use At study entrance -never in the past quitters current users * One year before occurrence of event -never in the past quitters current users * At time of occurrence of event -never in the past quitters current users * Data are given as proportions with P-value of cases versus controls, * = Fisher s exact test. Time of event: visit when diagnosis was made or confirmed. Quitters: used to until previous visit. Current: after previous visit and/or at present visit.

30 28 already started in the past. Although we do not know how long these abnormalities existed, it suggests that progression towards overt autoimmune hypothyroidism is a gradual process taking several years. This is in agreement with population studies focused on the evolution of subclinical hypothyroidism (defined as elevated serum TSH in the presence of normal free T4). In the Whickham survey [1], the annual rate of progression from subclinical to overt hypothyroidism was 3% in women with elevated TSH levels (>6mU/L), 2% in women with positive thyroid autoantibodies and 4.3% when both elevated TSH and thyroid Abs were present. The importance of thyroid antibodies in the progression from subclinical to over hypothyroidism was highlighted in other studies carried out in elderly subjects [2-9,15]. In a prospective study [9] in 107 subjects over the age of 55 with spontaneous subclinical hypothyroidism and no history of thyroid disease it was found that TSH is the most powerful predictor for the outcome of spontaneous subclinical hypothyroidism. At baseline TSH and ft4 concentrations were not different between hyperthyroid cases and controls and this was still true one year before the occurrence of the event. TPO-Ab concentrations were higher in hyperthyroid cases than in controls both at baseline and at one year before the occurrence of the event. Therefore, the biochemical switch from normal TSH and ft4 values to the suppressed TSH and elevated ft4 values must have occurred in the year before hyperthyroidism was diagnosed, suggesting that the transition from euthyroidism to overt autoimmune hyperthyroidism develops fast in terms of months rather than years. This observation is in agreement with a recent cross-sectional multicentre observational study [16] which investigated the duration of thyrotoxic symptoms until Graves hyperthyroidism was diagnosed. The median duration of Graves disease symptoms until diagnosis is four months. Some studies of patients with subclinical hyperthyroidism followed for 1-4 years suggested that subclinical hyperthyroidism may develop into overt hyperthyroidism at a rate of 1-5%/year [2,10-13]. However, these studies did not differentiate autoimmune subclinical hyperthyroidism from other causes of TSH suppression. Our study results further suggest that certain environmental factors provoke the transition from euthyroidism to overt autoimmune hypo/hyperthyroidism. In the present study the proportion of current smokers was lower in hypothyroid cases than in controls but only at the time of the event, not at baseline or one year before the event. In view of the marginal statistical significance the data might be interpreted cautiously that discontinuation of smoking promotes the development of hypothyroidism. This interpretation could be in line with recent observations that smoking protects against the development of TPO-Ab [17] and also against the development of hypothyroidism [18,19]. We could not ascertain the well-known risk of smoking for developing Graves hyperthyroidism most likely to the small sample size [20]. Another potential factor is pregnancy. In the present study, cases were more often in the postpartum period at the time of the event than controls, which was seen only in the hypothyroid cases and due to the occurrence of postpartum thyroiditis. The proportion of women who had never been pregnant was lower in the hyperthyroid cases than in their controls, that is hyperthyroid cases had experienced pregnancies more often. This is in line with the notion that in the postpartum year the risk for developing Graves hyperthyroidism is higher [21,22]. The use of oral estrogens (oral contraceptive pills) was not related in our study to the occurrence of hypo- or hyperthyroidism, although a few studies in the past

31 29 Chapter 2 Figure 1. Proposal model for the natural history of AITD. A particular genetic background consisting of single-nucleotide polymorphisms in AITD susceptible genes allows development of TPO-Ab. Smoking and a low iodine intake favor development toward hyperthyroidism, whereas to refrain from smoking and a high iodine intake favor development toward hypothyroidism. The transition from euthyroidism to overt hyperthyroidism occurs relatively fast in months, provoked, for example, by stress and pregnancy, whereas the transition from euthyroidism to overt hypothyroidism may take years, for example, in the postpartum period and by quitting smoking. GH, Graves hyperthyroidism; HH, Hashimoto s hypothyroidism. Thunderbolt denotes provocative factor. indicate a protective effect of estrogens on the development of hyperthyroidism [23,24]. The limitation of our study is its sample size with a relative low number of cases. The strength of our study is its prospective nature with annual assessments during 5-yr follow-up. We are not aware of studies assessing the transition from euthyroidism to overt autoimmune hypo/ hyperthyroidism in a structured prospective manner. Our observations led us to propose a model as depicted in Figure 1 for the development of autoimmune thyroid disease. It is well accepted that AITD can be viewed as a complex disease arising from the interplay between a particular genetic background involving multiple genes and a variety of environmental factors [25]. The so far identified susceptibility genes can be divided into two major groups: the immune modulating genes (HLA-DR, CD40, CTLA-4, PTPN22) and the thyroid specific genes (Tg and TSHR) [26]. Some polymorphisms confer susceptibility to both Graves and Hashimoto s disease whereas others are specifically related to the risk of Graves or Hashimoto s disease. Environmental factors play also an important role in the development of AITD as the concordance rate for AITD in monozygotic

32 30 twins is not 100% [27,28]. Twin studies suggest that genetic factors account for about 70% leaving about 30% for the environmental factors [27-30]. Identified environmental factors are iodine intake, smoking, stress, pregnancy, estrogens and possibly infections. It has been reported that in areas with sufficient iodine intake, hypothyroidism is more common than in iodine-deficient regions [31], whereas the overall prevalence of thyrotoxicosis is greater in iodine-deficient areas [32]. Smoking is a risk factor for Graves disease [20]. On the other hand, smoking decreases the risk on hypothyroidism [18,19] and has a protective effect on the development of TPO-Ab and Tg-Ab [17]. With regard to stress, several reports [33] showed that Graves patients have a higher number of stressful life events than controls in the months preceding the onset of the disease period but stress exposure has not been linked to the presence of thyroid antibodies [34]. In our view, Hashimoto s hypothyroidism and Graves disease encompass a common entity and they are considered to represent the two ends of the same spectrum: autoimmune thyroid disease. Whether Graves or Hashimoto s disease will develop in a subject, will depend on a number of polymorphisms in susceptibility genes and to a large extent to environmental factors (especially ambient iodine intake and smoking). Our study suggests that the transition from euthyroidism to Graves hyperthyroidism occurs relatively fast within one year in contrast to the transition from euthyroidism to Hashimoto s hypothyroidism which may take years. References [1] Vanderpump MP, Tunbridge WM, French JM, Appleton D, Bates D, Clark F, Grimley Evans J, Hasan DM, Rodgers H, Tunbridge F. The incidence of thyroid disorders in the community: a twenty-year follow-up of the Whickham Survey. Clin Endocrinol 1995;43: [2] Parle JV, Franklyn JA, Cross KW, Jones SC, Sheppard MC. Prevalence and follow-up of abnormal thyrotrophin (TSH) concentrations in the elderly in the United Kingdom. Clin Endocrinol 1991;34: [3] Lazarus JH, Burr ML, McGregor AM, Weetman AP, Ludgate M, Woodhead JS, Hall R. The prevalence and progression of autoimmune thyroid disease in the elderly. Acta Endocrinol 1984;106: [4] Sawin CT, Castelli WP, Hershman JM, McNamara P, Bacharach P. The aging thyroid. Thyroid deficiency in the Framingham Study. Arch Intern Med 1985;145: [5] Sawin CT, Bigos ST, Land S, Bacharach P. The aging thyroid. Relationship between elevated serum thyrotropin level and thyroid antibodies in elderly patients. Am J Med 1985;79: [6] Geul KW, van Sluisveld IL, Grobbee DE, Docter R, de Bruyn AM, Hooykaas H, van der Merwe JP, van Hemert AM, Krenning EP, Hennemann G. The importance of thyroid microsomal antibodies in the development of elevated serum TSH in middle-aged women: associations with serum lipids. Clin Endocrinol 1993;39: [7] Rosenthal MJ, Hunt WC, Garry PJ, Goodwin JS. Thyroid failure in the elderly. Microsomal antibodies as discriminant for therapy. Jama 1987;258: [8] Huber G, Staub J-J, Meier C, Mitrache C, Guglielmetti M, Huber P, Braverman LE. Prospective study of the spontaneous course of subclinical hypothyroidism: prognostic value of thyrotropin, thyroid reserve, and thyroid antibodies. J Clin Endocrinol Metab 2002;87: [9] Díez JJ, Iglesias P. Spontaneous subclinical hypothyroidism in patients older than 55 years: an analysis of natural course and risk factors for the development of overt thyroid failure. J Clin Endocrinol Metab 2004;89: [10] Sawin CT, Geller A, Wolf PA, Belanger AJ, Baker E, Bacharach P, Wilson PW, Benjamin EJ, D Agostino RB. Low serum thyrotropin concentrations as a risk factor for atrial fibrillation in older persons. N Engl J Med 1994;331: [11] Sawin CT, Geller A, Kaplan MM, Bacharach P, Wilson PW, Hershman JM. Low serum thyrotropin (thyroid-stimulating hormone) in older persons without hyperthyroidism. Arch Intern Med 1991;151: [12] Sundbeck G, Edén S, Jagenburg R, Lindstedt G. Thyroid dysfunction in 85-year-old men and women. Influence of nonthyroidal illness and drug treatment. Acta Endocrinol 1991;125: [13] Forfar JC, Miller HC, Toft AD. Occult thyrotoxicosis: a correctable cause of idiopathic atrial fibrillation. Am. J. Cardiol. 1979;44:9 12.

33 [14] Strieder TGA, Prummel MF, Tijssen JGP, Endert E, Wiersinga WM. Risk factors for and prevalence of thyroid disorders in a cross-sectional study among healthy female relatives of patients with autoimmune thyroid disease. Clin Endocrinol 2003;59: [15] Strieder TGA, Tijssen JGP, Wenzel BE, Endert E, Wiersinga WM. Prediction of progression to overt hypothyroidism or hyperthyroidism in female relatives of patients with autoimmune thyroid disease using the Thyroid Events Amsterdam (THEA) score. Arch Intern Med 2008;168: [16] Vos XG, Smit N, Endert E, Tijssen JGP, Wiersinga WM. Variation in phenotypic appearance of Graves disease: effect of genetic anticipation and duration of complaints. Eur J Endocrinol 2009;161: [17] Effraimidis G, Tijssen JGP, Wiersinga WM. Discontinuation of smoking increases the risk for developing thyroid peroxidase antibodies and/or thyroglobulin antibodies: a prospective study. J Clin Endocrinol Metab 2009;94: [18] Asvold BO, Bjøro T, Nilsen TIL, Vatten LJ. Tobacco smoking and thyroid function: a population-based study. Arch Intern Med 2007;167: [19] Belin RM, Astor BC, Powe NR, Ladenson PW. Smoke exposure is associated with a lower prevalence of serum thyroid autoantibodies and thyrotropin concentration elevation and a higher prevalence of mild thyrotropin concentration suppression in the third National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab 2004;89: [20] Prummel MF, Wiersinga WM. Smoking and risk of Graves disease. Jama 1993;269: [21] Amino N, Tada H, Hidaka Y. Postpartum autoimmune thyroid syndrome: a model of aggravation of autoimmune disease. Thyroid 1999;9: [22] Benhaim Rochester D, Davies TF. Increased risk of Graves disease after pregnancy. Thyroid 2005;15: [23] Frank P, Kay CR. Incidence of thyroid disease associated with oral contraceptives. Br Med J 1978;2:1531. [24] Martin-du Pan RC. [Triggering role of emotional stress and childbirth. Unexpected occurrence of Graves disease compared to 96 cases of Hashimoto thyroiditis and 97 cases of thyroid nodules]. Ann Endocrinol (Paris) 1998;59: [25] Tomer Y, Huber A. The etiology of autoimmune thyroid disease: a story of genes and environment. Journal of Autoimmunity 2009;32: [26] Tomer Y, Davies TF. Searching for the autoimmune thyroid disease susceptibility genes: from gene mapping to gene function. Endocrine Reviews 2003;24: [27] Brix TH, Kyvik KO, Hegedüs L. A population-based study of chronic autoimmune hypothyroidism in Danish twins. J Clin Endocrinol Metab 2000;85: [28] Brix TH, Kyvik KO, Christensen K, Hegedüs L. Evidence for a major role of heredity in Graves disease: a populationbased study of two Danish twin cohorts. J Clin Endocrinol Metab 2001;86: [29] Hansen PS, Brix TH, Iachine I, Kyvik KO, Hegedüs L. The relative importance of genetic and environmental effects for the early stages of thyroid autoimmunity: a study of healthy Danish twins. Eur J Endocrinol 2006;154: [30] Ringold DA, Nicoloff JT, Kesler M, Davis H, Hamilton A, Mack T. Further evidence for a strong genetic influence on the development of autoimmune thyroid disease: the California twin study. Thyroid 2002;12: [31] Laurberg P, Pedersen KM, Hreidarsson A, Sigfusson N, Iversen E, Knudsen PR. Iodine intake and the pattern of thyroid disorders: a comparative epidemiological study of thyroid abnormalities in the elderly in Iceland and in Jutland, Denmark. J Clin Endocrinol Metab 1998;83: [32] Laurberg P, Nøhr SB, Pedersen KM, Hreidarsson AB, Andersen S, Bülow Pedersen I, Knudsen N, Perrild H, Jørgensen T, Ovesen L. Thyroid disorders in mild iodine deficiency. Thyroid 2000;10: [33] Tsatsoulis A. The role of stress in the clinical expression of thyroid autoimmunity. Annals of the New York Academy of Sciences 2006;1088: [34] Strieder TGA, Prummel MF, Tijssen JGP, Brosschot JF, Wiersinga WM. Stress is not associated with thyroid peroxidase autoantibodies in euthyroid women. Brain Behav Immun 2005;19: Chapter 2

34

35 Chapter 3 Discontinuation of smoking increases the risk for developing thyroid peroxidase antibodies and/or thyroglobulin antibodies: a prospective study. Effraimidis G Tijssen JGP Wiersinga WM J Clin Endocrinol Metab. 2009;94:

36 ABSTR ACT 34 Context: Autoimmune thyroid disease (AITD) develops in genetic susceptible subjects, provoked by environmental factors. Little is known on the environment in the early stages of autoimmunity. Objective: Evaluation of environmental factors contributing to de novo occurrence of thyroid antibodies. Design: Prospective cohort study of 521 euthyroid women without thyroid antibodies in serum who were relatives of AITD patients. Follow-up was five year. Baseline characteristics were related to the occurrence of thyroid peroxidase (TPO) and/or thyroglobulin (Tg) antibodies. Exposure to environmental factors in the year prior to the occurrence of antibodies was investigated in a nested case-control study. Results: The 5-yr probability for conversion to TPO-Ab and/or Tg-Ab was 20.1%, and for TPO-Ab 14.5%. None of the baseline characteristics except TSH contributed to the risk of seroconversion. Each case (occurrence of antibodies) was matched for age and duration of follow-up with two controls (no seroconversion). Exposure to environmental stimuli was similar between cases and controls except for smoking. At study entrance current smokers among cases and controls were 31.3% and 35.5% respectively (NS). Current smoking decreased in cases during follow-up. Consequently, the odds of smoking for developing TPO-Ab and/or Tg-Ab were 0.62 (95% CI ) one year before seroconversion, and 0.59 (95% CI ) at seroconversion; for conversion to TPO-Ab these figures are 0.58 (95% CI ) and 0.54 (95% CI ) respectively. Conclusion: Discontinuation of smoking is associated with an increased risk for occurrence of TPO-Ab and/or Tg-Ab in serum. The observation is in line with the decreased risk of hypothyroidism in smokers.

37 Introduction Autoimmune thyroid disease (AITD) results from interplay among genetic and environmental factors. The occurrence of TPO-Ab in serum is one of the earliest signs of thyroid autoimmunity that can be detected [1]. In order to get more insight into the early pathogenesis of AITD, we did a prospective 5 years follow up study on the novel occurrence of thyroid antibodies in euthyroid women at risk for AITD. 35 Subjects & Methods Chapter 3 Participants This study was carried out among the 803 subjects from the Amsterdam AITD Cohort [2,3] which consisted of women between 18 and 65 years of age in self-proclaimed good health without a history of thyroid disease, who had at least one 1st or 2nd degree relative with documented autoimmune hyper- or hypothyroidism. Subjects were followed for five years, or shorter when overt hyper- or hypothyroidism had occurred (defined as TSH <0.4mU/L in combination with ft4 >20.1pmol/L, or TSH >5.7mU/L in combination with ft4 <9.3pmol/L respectively). Blood samples were collected annually to measure TSH, ft4, T3, TPO-Ab, Tg-Ab and TBII, and subjects were asked to fill in questionnaires on smoking habits, use of estrogens, pregnancies, iodine excess and stress. Participants were selected as follows: we excluded subjects who at study entrance had overt or subclinical hyper- or hypothyroidism, or thyroid antibodies (i.e. serum concentrations of either TPO-Ab 100kU/L, Tg-Ab 100kU/L, or TBII 12U/L) and those who had no follow-up, 521 participants were finally enrolled. We performed two different analyses. The first was a time-to-event analysis (event was defined as the occurrence of serum TPO-Ab and/or Tg-Ab >100 ku/l); the probability of an event was estimated from characteristics of the 521 participants at study entrance. The second analysis was based on a nested case-control study design. A subject was recruited as a case when she had developed TPO-Ab and/or Tg-Ab. The end-point for a case was the time at which she had become positive for the first time for one of the two antibodies (TPO-Ab or Tg-Ab). Cases and controls (two controls for each case) were matched by age and duration of follow up [4]. Age was calculated as the difference between date of study entry and date of birth in years. Controls should have remained seronegative for thyroid antibodies up to the time at which the case they were matched to, had received her end-point. Subjects who during follow-up developed thyroid antibodies and showed up as cases later, or who developed subclinical or overt thyroid dysfunction were still qualified to act as controls. A subject could only be sampled once as control. Smoking behaviour, pregnancy, and exposure to exogenous estrogens, iodine excess and stress in the year prior to the seroconversion were compared between cases and controls. Current smoking was defined as smoking now, or having stopped smoking in the year prior to the study visit.

38 36 Laboratory measurements Serum TSH and ft4 were measured using time-resolved fluoroimmunoassay (Delphia, Turku, Finland) and total T3 by an in-house radioimmunoassay. Reference values are for TSH mu/l, for ft pmol/l and for T nmol/l. Thyroid peroxidase (TPO) antibodies and thyroglobulin (Tg) antibodies were measured by chemiluminescence immunoassays (LUMI-test anti-tpo and anti-tg respectively, Brahms, Berlin, Germany). Improved versions of both assays became available during follow-up: detection limits of these new assays were for TPO-Ab 30 ku/l and for Tg-Ab 20 ku/l. TPO-Ab concentrations obtained with the old assay were multiplied by a factor 0.72 to obtain comparative values in the new assay. TPO-Ab and Tg-Ab concentrations were considered to be positive at values >100 ku/l. TSH receptor antibodies were determined as TSH binding inhibitory immunoglobulins (TBII) using the TRAK assay (Brahms, Berlin, Germany); detection limits in the 1st and 2nd generation TRAK assays were 5 and 1 IU/L respectively, and values above 12 and 1.5 U/L respectively were considered as positive. Stress assessments have been previously published in detail [5]. Statistical analysis Data processing was done with the statistical software SPSS for windows version 12.0 (SPSS, Inc., Chicago, IL). Normally distributed data are presented as mean±sd and were analyzed by Student s t-test. Data that are not distributed normally are expressed as median and 25th and 75th percentiles and were analyzed by Mann-Whitney U test. Categorical data are expressed as percentages. The significance of differences between groups was analyzed with the χ2 test or with Fisher exact test in the case of small numbers. Prognostic indicators for seroconversion to positive thyroid Abs were identified using Cox regression analysis, univariate and multivariate if necessary. Statistical significance was set at 5 %. Results Cohort study At study entrance mean age was 34.0±11.3 years and mean serum TSH was 1.69±0.77 mu/l. During follow-up 17 subjects (3.3%) developed both TPO-Ab and Tg-Ab, 53 subjects (10.2%) developed only TPO-Ab, and 29 (5.6%) only Tg-Ab. At the time of seroconversion the median TPO-Ab value was 125 ku/l (range ku/l) and the median Tg-Ab value was ku/l, (range ku/l). Kaplan-Meier analysis indicated a 5-yr probability of seroconversion of 20.1% for TPO-Ab and/or Tg-Ab, and of 14.5% for TPO-Ab only. Prognostic indicators at study entrance for conversion during follow-up to TPO-Ab and/or Tg-Ab, and to TPO-Ab independent of Tg-Ab status were identified by Cox regression analysis. Age did not contribute to the risk of seroconversion, nor did any of the environmental factors (smoking, estrogen medication, pregnancy, iodine excess, stress) affect the hazard ratio s significantly (Table 1, data on stress not shown). Only baseline serum TSH was positively related to the risk of seroconversion by univariate analysis.

39 Table 1. Baseline characteristics, event rates and Cox regression analysis in a prospective cohort of 521 euthyroid women with 1st or 2nd degree relatives with proven AITD who were followed for 5 years. TPO-Ab and/or Tg-Ab TPO-Ab N Event rate (%) HR a 95% CI a P-value Event rate (%) HR a 95% CI a P-value All women % 13.4% Age 37 <30 b % - - c 11.8% - - c % c 17.6% c % c 12.1% c % c 11.8% c > % c 10.5% c Chapter 3 TSH in mu/l b % - - d 12.1% - - e % d 15.6% e % d 19.2% e % d 33.3% e Smoking habits Never smoker b % % Ex-smoker % % Current smoker % % Estrogen medication Never use b % % Ex use % % Current use % % Gravida Nullipara b % % Uni- and multipara % % Iodine excess Absent b % % Present % % a HR denotes hazard ratio, CI denotes confidence interval. b This served as the reference group (category) c p-value for trend : non significant d p-value for trend : e p-value for trend : 0.047

40 38 Supplemental Figure 1. Kaplan-Meier curve depicting the probability of de novo occurrence of TPO-Ab and/or Tg-Ab (continuous line) or TPO-Ab (discontinuous line) during the 5 years follow-up of the Amsterdam AITD cohort. Nested case-control study There was a perfect match with respect to age and duration of follow-up between cases and controls (Table 2). The median time to seroconversion in cases was 2.20 years (for developing either TPO-Ab or Tg-Ab) and 2.98 years (for developing TPO-Ab independent of Tg-Ab). Serum TSH increased during follow-up in cases but not in controls; serum ft4 did not change in the follow-up in either group. At study entrance, the frequency of smokers among cases was comparable to that in controls (31.3% vs 35.5%, NS). At one year before seroconversion, there were less current smokers in cases than in controls (28.3% vs 39.0%, p=0.068), and this was still true at the time of seroconversion (27.3% vs 39.0%, p=0.046). The odds of smoking for developing either TPO-Ab and/or Tg-Ab were 0.62 (95% CI ) one year before the seroconversion, and 0.59 (95% CI ) at seroconversion. When only conversion to TPO-Ab is considered, the odds of smoking were 0.58 (95% CI ) one year before seroconversion and 0.54 (95% CI ) at the time of seroconversion. The protective effects of smoking on seroconversion were maintained in a multivariate logistic regression analysis adjusting for pregnancy and estrogen use (data not shown).

41 Discussion The main finding of the present study was that discontinuation of smoking increases the risk of de novo occurrence of serum TPO-Ab and/or Tg-Ab in subjects susceptible for developing AITD. At study entrance smoking behaviour did not differ between those who subsequently developed thyroid antibodies and those who did not: the effect of smoking became obvious only during follow-up. A limitation of our study is that we have not looked after a doseresponse effect between smoking and the occurrence of thyroid antibodies: the relatively small number of subjects who changed their smoking behaviour during the 5-yr follow-up did not allow a meaningful analysis. The still modest sample size of the case-control study on conversion to TPO-Ab may also explain why the upper value of the 95% confidence interval of the odds ratio did just not decrease below the value of 1.00 but remained 1.02 (it decreased below 1.00 when conversion to either TPO-Ab and/or Tg-Ab was studied). In view of the significant fall in odds ratio s during follow-up in both studies we feel however quite confident on the validity of our conclusion. Another finding of the present study is that at study entrance a serum TSH >2mU/L but still within the normal reference range already indicates a small risk for developing thyroid antibodies in the next five years. Thyroid ultrasound may detect signs of thyroid autoimmunity before thyroid antibodies appear in serum [6]. We did not perform thyroid ultrasonography, which is another restriction to our study findings. These limitations are in our view well balanced by the strengths of our study. Its prospective nature guarantees more solid evidence than obtained from cross-sectional studies. Moreover, in the nested case-control study a perfect match existed between both groups with respect to age and duration of follow-up. Higher age and longer exposure time both increase the likelihood of developing thyroid antibodies, constituting possible bias. The matching procedure effectively excluded both bias. The present study is the first to evaluate the relationship between smoking and thyroid antibodies in a prospective manner, but three cross-sectional studies on this topic have been published with essentially similar conclusions. A decreased prevalence of TPO-Ab in smokers compared with nonsmokers was reported among women of the Amsterdam AITD Cohort [2]. The odds ratio (OR) of current smoking for the presence of TPO-Ab was 0.69 (95% CI ). Data from NHANES III [7] indicated that fewer smokers (11%, 95% CI 10-13%) had TPO-Ab and/or Tg-Ab compared to nonsmokers (18%, 95% CI 17-19%). The relationship persisted when analyzing the presence of one antibody independently of the status of the other antibody. Recently, a Danish population study [8] found that smoking was negatively associated with the presence of thyroid antibodies with the strongest association between smoking and Tg-Ab (OR s of ). Taken together, the various studies provide good evidence that smoking decreases the prevalence (cross-sectional studies) and incidence (our present study) of TPO-Ab and Tg-Ab. One may thus ask the question if smoking decreases the risk on hypothroidism. A metaanalysis could not prove such an association [9]. A Danish study found a lower prevalence of mild hypothyroidism (TSH >3.6mU/L) among smokers compared to nonsmokers (2.6% vs 5.3%) with an adjusted OR of 0.47 (95% CI ) [10]. In NHANES III smokers had less frequently an elevated TSH value (>4.5mU/L) compared with nonsmokers 39 Chapter 3

42 40 Table 2. Comparison of characteristics between cases (seroconverters) and controls (non seroconverters matched for age and duration of follow up) at baseline, one year prior to event (seroconversion) and at the time of event. TPO-Ab and/or Tg-Ab BASELINE ONE YEAR BEFORE EVENT AT EVENT P - value a at event one year before event Cases Controls Cases Controls Cases Controls baseline Age (years) 34.7± ± ± ± ± ±10.8 TSH (geom. mean) 1.22 ( ) 1.19 ( ) 1.25 ( ) 1.16 ( ) 1.28 ( ) 1.15 ( ) ft4 (pmol/l) 12.8± ± ± ± ± ± Current smokers 31.3% 35.5% 28.3% 39.0% 27.3% 39.0% Pregnant last year 5.1% 7.0% 8.1% 10.0% 6.1% 9.0% Estrogen use 43.4% 51.5% 31.3% 42.5% 29.3% 40.5% Iodine excess 11.1% 10.2% 7.7% 14.4% 9.5% 19.8% Positive and Negative Affect Schedule Scale (tendency to report) Negative feelings 20.0 ( ) 21.0 ( ) 21.0 ( ) 21.0 ( ) 20.0 ( ) 20.0 ( ) Positive feelings 39.0 ( ) 38.0 ( ) 39.0 ( ) 38.0 ( ) 37.0 ( ) 37.0 ( ) Recent Life Events Total number 10.0 ( ) 10.0 ( ) 9.0 ( ) 9.0 ( ) 8.0 ( ) 8.0 ( ) Amount of total unpleasantness 16.0 ( ) 15.0 ( ) 12.1( ) 13.0 ( ) 12.5 ( ) 11.0 ( ) ( ) 16.0 ( ) 14.0 ( ) 14.0 ( ) 13.0 ( ) 14.0 ( ) Amount of total pleasanteness Daily Hassles Total intensity 30.0 ( ) 27.0 ( ) 31.5( ) 28.0 ( ) 32.0( ) 30.0 ( )

43 TPO-Ab BASELINE ONE YEAR BEFORE EVENT AT EVENT P - value a at event one year before event Cases Controls Cases Controls Cases Controls baseline Age (years) 34.0± ± ± ± ± ±10.8 TSH (geom. mean) 1.21( ) 1.21( ) 1.22( ) 1.17( ) 1.28( ) 1.15( ) ft4 (pmol/l) 13.0± ± ± ± ± ± Current smokers 34.3% 34.8% 27.1% 39.0% 25.7% 39.0% Pregnant last year 1.4% 8.6% 8.8% 8.8% 8.7% 9.4% Estrogen use 44.3% 48.9% 31.4% 39.0% 31.4% 40.4% Iodine excess 12.9% 6.5% 7.3% 12.5% 11.1% 12.7% Positive and Negative Affect Schedule Scale (tendency to report) Negative feelings 21.0 ( ) 21.0 ( ) 21.0 ( ) 20.0 ( ) 19.0 ( ) 21.0 ( ) Positive feelings 39.0 ( ) 39.0 ( ) 39.0 ( ) 38.0 ( ) 37.0 ( ) 37.0 ( ) Recent Life Events Total number 10.0 ( ) 11.0( ) 9.5( ) 9.0 ( ) 8.0 ( ) 8.0 ( ) Amount of total unpleasantness 15.1( ) 16.0( ) 12.0 ( ) 13.0 ( ) 12.0 ( ) 13.0 ( ) ( ) 16.0( ) 14.0 ( ) 14.0 ( ) 13.0 ( ) 14.0 ( ) Amount of total pleasanteness Daily Hassles Total intensity 30.0 ( ) 27.0 ( ) 31.5 ( ) 29.0 ( ) 32.0 ( ) 31.0 ( ) p values indicate differences between cases and controls at each time point. 41 Chapter 3

44 42 Supplemental Figure 2. Frequency of current smoking in cases (seroconverters black bars) and in controls (nonseroconverters, matched for age and duration of follow up white bars) at baseline, one year prior to event (seroconversion) and at the time of event. OR denotes odds ratio and the 95% confidence intervals are in parentheses. (2.6%, 95% CI %, vs 5.5%, 95% CI %) [7]. Recently, a cross-sectional population-based study from Norway reported a lower prevalence of overt and subclinical hypothyroidism among current smokers compared to never smokers (OR s are 0.60, 95% CI for overt and 0.54, 95% CI for subclinical hypothyroidism in women, and 0.51, 95% CI and 0.37, 95% CI respectively in men) [11]. One may therefore hypothesize that the preventive effect of smoking is -at least partlyexplained by the protective effect of smoking on the development of TPO-Ab and Tg-Ab. The findings in hypothyroidism are in sharp contrast with many studies showing that smoking is a risk factor for the development of Graves hyperthyroidism, especially for Graves ophthalmopathy [12]. Apparently, smoking behavior may determine to a certain extent the clinical phenotype of AITD: smokers are less likely to develop hypothyroidism but more likely to get Graves disease. The mechanism behind these divergent effects of smoking is poorly understood.

45 References [1] Vanderpump MP, Tunbridge WM, French JM, Appleton D, Bates D, Clark F, Grimley Evans J, Hasan DM, Rodgers H, Tunbridge F. The incidence of thyroid disorders in the community: a twenty-year follow-up of the Whickham Survey. Clin Endocrinol 1995;43: [2] Strieder TGA, Prummel MF, Tijssen JGP, Endert E, Wiersinga WM. Risk factors for and prevalence of thyroid disorders in a cross-sectional study among healthy female relatives of patients with autoimmune thyroid disease. Clin Endocrinol 2003;59: [3] Strieder TGA, Tijssen JGP, Wenzel BE, Endert E, Wiersinga WM. Prediction of progression to overt hypothyroidism or hyperthyroidism in female relatives of patients with autoimmune thyroid disease using the Thyroid Events Amsterdam (THEA) score. Arch Intern Med 2008;168: [4] Hollowell J, Staehling N, Flanders W. Serum TSH, T4, and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab 2002;87: [5] Strieder TGA, Prummel MF, Tijssen JGP, Brosschot JF, Wiersinga WM. Stress is not associated with thyroid peroxidase autoantibodies in euthyroid women. Brain Behav Immun 2005;19: [6] Marcocci C, Vitti P, Cetani F, Catalano F, Concetti R, Pinchera A. Thyroid ultrasonography helps to identify patients with diffuse lymphocytic thyroiditis who are prone to develop hypothyroidism. J Clin Endocrinol Metab 1991;72: [7] Belin RM. Smoke Exposure is associated with a lower prevalence of serum thyroid autoantibodies and thyrotropin concentration elevation and a higher prevalence of mild thyrotropin concentration suppression in the third National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab 2004;89: [8] Pedersen IB, Laurberg P, Knudsen N, Jørgensen T, Perrild H, Ovesen L, Rasmussen LB. Smoking is negatively associated with the presence of thyroglobulin autoantibody and to a lesser degree with thyroid peroxidase autoantibody in serum: a population study. Eur J Endocrinol 2008;158: [9] Vestergaard P. Smoking and thyroid disorders--a meta-analysis. Eur J Endocrinol 2002;146: [10] Knudsen N, Bülow I, Laurberg P, Perrild H, Ovesen L, Jørgensen T. High occurrence of thyroid multinodularity and low occurrence of subclinical hypothyroidism among tobacco smokers in a large population study. J Endocrinol 2002;175: [11] Asvold BO, Bjøro T, Nilsen TIL, Vatten LJ. Tobacco smoking and thyroid function: a population-based study. Arch Intern Med 2007;167: [12] Prummel MF, Wiersinga WM. Smoking and risk of Graves disease. Jama 1993;269: Chapter 3

46

47 Chapter 4 Alcohol consumption as a risk factor for autoimmune thyroid disease: a prospective study. Effraimidis G Tijssen JGP Wiersinga WM European Thyroid Journal 2012; 1: issue 2 (in press)

48 ABSTR ACT Background: Alcohol consumption has been identified as a protective factor for some autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus. 46 Objective: We hypothesized that alcohol consumption would reduce the risk of developing autoimmune thyroid disease (AITD). Study design: Two nested case-control studies in the prospective Amsterdam AITD cohort. Follow-up was five year, with annual assessments. In study A, we compared alcohol consumption between cases (subjects who during follow up remained euthyroid but developed TPO-Ab, called event) and controls (subjects who remained euthyroid and TPO-Ab negative). In study B, we compared alcohol consumption between cases (subjects who during follow-up developed overt hypothyroidism, called event) and controls (subjects who did not develop overt hypothyroidism). For each case, two controls were selected, matched for age, duration of follow up and smoking behavior at baseline and at the time of event. Results: In study A, alcohol consumption did not differ between cases and controls at any time point. In study B, the number of subjects consuming >10 units of alcohol per week was not different between cases and controls at study entrance (8.3% vs 14.5%, NS), but lower at one year before (5.3% vs 19.7%, p=0.041) and at the time of event (6.7% vs 23.7%, p=0.044); respective odds ratio s are 0.54 ( ), 0.23 ( ) and 0.23 ( ). Conclusion: Alcohol consumption is not associated with de novo development of TPO- Ab, but is lower in subjects who developed overt hypothyroidism. The data suggest alcohol consumption may protect against overt autoimmune hypothyroidism.

49 Introduction Autoimmune thyroid disease (AITD) is a multifactorial condition and genetic factors as well as environmental factors are thought to play a role in its pathogenesis. Twin studies suggest that genetic factors account for about 70% of the risk to get AITD [1-3]. The remaining 30% is likely due to environmental factors which may provoke autoimmune reactions. Iodine intake, stress, smoking, drugs (e.g. estrogens), pregnancy and infections have all been investigated as putative determinants of AITD. Recent studies suggest a protective effect of alcohol on the development of rheumatoid arthritis and systemic lupus erythematosus [4-6]. Available literature data indeed support the possibility that alcohol is a modulator of the immune system [7]. In the present study we hypothesised that alcohol consumption would reduce the risk of developing AITD. We examined the effect of alcohol on both early and late stages of AITD. The occurrence of TPO-Ab in serum represents an early stage of AITD, and the occurrence of overt hypothyroidism may represent the late final stage of AITD [8]. Thus we performed two nested case controls studies in the prospective Amsterdam AITD cohort. In study A, we compared alcohol consumption in euthyroid subjects who did or did not develop TPO-Ab. In study B, we compared alcohol consumption in subjects who did or did not progress to overt hypothyroidism. 47 Chapter 4 Subjects & Methods Participants The present study was carried out among the 803 subjects from the Amsterdam AITD Cohort. The cohort has previously been described in detail [9]. In short, the cohort consisted of women between 18 and 65 years of age in self-proclaimed good health without a history of thyroid disease, who had at least one 1st or 2nd degree relative with documented autoimmune hyper- or hypothyroidism. Subjects were followed for five years, or shorter when overt hyper- or hypothyroidism had occurred (defined as TSH <0.4 mu/l in combination with ft4 >20.1 pmol/l, or TSH >5.7 mu/l in combination with ft4 <9.3 pmol/l respectively). Results of thyroid function tests at study entrance revealed overt hypothyroidism in 10 subjects and overt hyperthyroidism in 3 subjects, leaving 790 subjects to be included in the present study. At each annual visit blood samples were collected to measure TSH, ft4, TPO-Ab, Tg-Ab and TBII, and subjects were asked to fill in questionnaires on alcohol consumption (number of alcoholic drinks per week) and smoking habits (current smoking defined as smoking now or stopped smoking in the last year). All subjects gave informed written consent and the Medical Ethics committee of the Academic Medical Center in Amsterdam approved the study. We performed two nested case-control studies A and B.

50 48 Study A: Alcohol consumption and de novo development of TPO-Ab. In order to evaluate the relationship between alcohol consumption and the de novo occurrence of TPO-Ab we selected participants from the inception cohort of the 790 euthyroid subjects, excluding those women who had thyroid antibodies at baseline (i.e. serum concentrations of either TPO-Ab 100 ku/l, Tg-Ab 100 ku/l or TBII 12U/L), those who had subclinical hyper- or hypothyroidism at baseline, and those who had no follow-up. Consequently, 521 euthyroid participants without any serological sign of AITD at baseline were thus enrolled. A subject was recruited as a case when she had remained euthyroid but had developed TPO-Ab during follow-up. The end-point for a case was the time at which she had become positive for the first time for TPO-Ab without developing abnormal TSH (called event). This happened in 81 subjects. Subjects from the Amsterdam AITD cohort qualified to act as controls if they remained euthyroid and seronegative for TPO-Ab up to the time at which the case they were matched to, had received her end-point. Study B: Alcohol consumption and development of overt hypothyroidism. In order to evaluate the relationship between alcohol consumption and the development of overt hypothyroidism, we designed a nested case-control study in the inception cohort as follows. A subject was recruited as a case when she had developed overt hypothyroidism during follow up (called event). The end-point for a case was the time at which she had developed overt hypothyroidism. Subjects qualified as controls if they had not progressed to overt hypo- or hyperthyroidism up to the time at which the case they were matched to, had received her end-point. In both studies A and B for each case two controls were selected. A subject could only be sampled once as control. Controls for both studies were matched for age, duration of follow up and smoking status at baseline and at the time of event. Alcohol consumption at baseline, one year before the occurrence of the event and at the time of the event was compared between cases and controls. Laboratory measurements Serum TSH and ft4 were measured using time-resolved fluoroimmunoassay (Delphia, Turku, Finland). Reference values are for TSH mu/l and for ft pmol/l. Thyroid peroxidase (TPO) antibodies and thyroglobulin (Tg) antibodies were measured by chemiluminescence immunoassays (LUMI-test anti-tpo and LUMI-test anti-tg respectively, Brahms, Berlin, Germany). Improved versions of both assays became available during follow-up: detection limits of these new assays were for TPO-Ab 30 ku/l and for Tg-Ab 20 ku/l. TPO-Ab concentrations obtained with the old assay were multiplied by a factor 0.72 to obtain comparative values in the new assay. TPO-Ab and Tg-Ab concentrations were considered to be positive at values 100 ku/l. TSH receptor antibodies were determined as TSH binding inhibitory immunoglobulins (TBII) using the TRAK assay (Brahms, Berlin, Germany); detection limits in the 1st and 2nd generation TRAK assays were 5 and 1 IU/L respectively, and values above 12 and 1.5 U/L respectively were considered as positive.

51 Statistical analysis Normally distributed data are presented as mean±sd and group differences were analyzed by Student s t-test. Data that are not distributed normally are expressed as median and 25th and 75th percentiles, and were analyzed by Mann-Whitney U test. Categorical data are expressed as percentages. The significance of differences between groups was analyzed with the χ2 test or with Fisher exact test in the case of small numbers. Statistical significance was set at 5 %. Results Study A: Alcohol consumption and de novo development of TPO-Ab. During the 5-yr follow up period, 81 of the 521 subjects (15.5%) had developed TPO-Ab under maintenance of a normal TSH, and they were considered cases. One hundred sixty two matched controls could be selected. Cases and controls did not differ in age (at baseline 36±12 and 36±12 years respectively), or in duration of follow up (2.8±1.3 and 2.8±1.3 years respectively). The same was true for the proportion of current smokers. Thyroid function did not change during follow up, neither in cases nor in controls. At the time of seroconversion the TPO-Ab concentration had a median value of 140 ku/l (interquartile range ku/l) (Table 1). At study entrance, the frequency of consuming more than 10 units of alcohol per week among cases was comparable to that in controls (14.5% vs 13.4%, NS). The same was true at one year before seroconversion (14.1% vs 15.1%, NS) and at the time of seroconversion (11.3% vs 13.7%, NS) (Figure 1A). 49 Chapter 4 Study B: Alcohol consumption and development of overt hypothyroidism. During the 5-yr follow up period 38 cases of overt autoimmune hypothyroidism occurred, as reported elsewhere [10]. Seventy six matched controls could be selected in whom overt hypo- or hyperthyroidism had not occurred. Cases and controls did not differ with regard to mean age (at baseline 38±12 and 38±12 years respectively), mean follow up (3.2±1.3 and 3.2±1.2 years respectively) and the proportion of current smoking at baseline, one year before the event and at the time of event. Cases had already higher serum TSH and lower serum ft4 concentrations than controls at baseline (Table 1). At study entrance, the frequency of consumers of >10 units of alcohol per week among cases was not significantly different from that in controls (8.3% vs 14.5%, NS). At one year before the occurrence of overt hypothyroidism, there were less consumers of >10 units of alcohol per week in cases than in controls (5.3% vs 19.7%, p=0.041), and this was still true at the time of overt hypothyroidism (6.7% vs 23.7%, p=0.044) (Figure 1B). The odds of consuming >10 units of alcohol per week for developing overt hypothyroidism were 0.54 (95% CI ) at baseline, 0.23 (95% CI ) one year before the event, and 0.23 (95% CI ) at the event (Figure 2).

52 50 Table 1. Comparison of characteristics between cases and controls (matched for age, duration of follow up and smoking habits) at baseline, one year prior to event and at the time of event in study A (de novo occurrence of TPO-Ab) and in study B (development of overt hypothyroidism). STUDY A development of TPO-Ab 81 cases and 162 controls baseline one year before event at event P - value at event 1 yr before event Cases Controls Cases Controls Cases Controls baseline TSH (mu/l) 1.5 ( ) 1.5 ( ) 1.4 ( ) 1.4 ( ) 1.5 ( ) 1.4 ( ) ft4 (pmol/l) 13.1 ( ) 13.4 ( ) 13.0 ( ) 13.6 ( ) 13.1 ( ) 13.3 ( ) TPO-Ab (ku/l) <25 (<25-<25) <25 (<25-<25) <25 (<25-50) <25 (<25-50) 140 ( ) <25 (<25-50) <0.001 Tg-Ab (ku/l) 15 (<10-25) <10 (<10-15) 18 (<10-35) <10 (<10-14) 17 (<10-43) <10 (<10-20) Current smokers 38% 38% 31% 30% 31% 31% NS NS NS Alcohol consumers * 14.5% 13.4% 14.1% 15.1% 11.3% 13.7% STUDY B development of overt hypothyroidism 38 cases and 76 controls baseline one year before event at event P - value at event 1 yr before event Cases Controls Cases Controls Cases Controls baseline TSH (mu/l) 4.2 ( ) 1.5 ( ) 4.0 ( ) 1.5 ( ) 14.8 ( ) 1.5 ( ) ft4 (pmol/l) 10.7 ( ) 13.0 ( ) 10.6 ( ) 13.4 ( ) 7.1 ( ) 13.0 ( ) TPO-Ab (ku/l) 1123 ( ) <25 (<25-150) 1076 ( ) 50 (<25-320) 3000 ( ) <25 (<25-180) <0.001 Tg-Ab (ku/l) 75 (40-238) 15 (<5-40) 123 (42-235) 15 (<10-40) 140 ( ) 10 (<10-50) Current smokers 18% 18% 18% 18% 13% 13% NS NS NS Alcohol consumers * 8.3% 14.5% 5.3% 19.7% 6.7% 23.7% * >10 units/week data given as median (interquartile range)

53 51 Chapter 4 Figure 1. Frequency of alcohol consumption in cases (black bars) and in controls (matched for age, duration of follow up and smoking behavior - grey bars) at baseline, one year prior to event and at the time of event in study A (de novo development of TPO-Ab) and in study B (development of overt hypothyroidism).

54 Discussion 52 The aim of the present case-control studies nested in the observational Amsterdam AITD cohort study was to evaluate in a prospective manner the involvement of alcohol in both the early stages (when thyroid antibodies develop but thyroid function is still normal) and late stages (when overt thyroid dysfunction emerges) of the natural course of AITD. We find that alcohol consumption is not associated with the de novo occurrence of TPO-Ab. However, we observed that alcohol consumption decreases the risk of the development of overt hypothyroidism in subjects susceptible for developing AITD. At study entrance alcohol consumption did not differ between those who subsequently developed overt hypothyroidism and those who did not; the effect of alcohol consumption became obvious during follow-up, although the odds ratio at baseline is already The present study is the first to evaluate the relationship between alcohol consumption and thyroid autoimmunity in a prospective manner. Its prospective nature guarantees solid evidence. Cases and controls were matched for age, duration of follow-up and smoking behaviour as higher age and longer exposure time increase while smoking decrease the likelihood of developing autoimmune hypothyroidism and thyroid antibodies [11-13]. The perfect matching procedure between cases and controls effectively exclude the potential biases and enhance the validity of our results. A weakness of our study is the limited number of subjects who converted from euthyroidism to overt hypothyroidism (N=38), but even with this small sample size we find evidence that alcohol consumption exhibits an inverse association with overt autoimmune hypothyroidism. However, the limited sample size may have precluded to find a dose-response relationship in our study: we did not observe differences between cases and controls when we categorised alcohol consumption into three groups: non drinkers, low consumption (more than zero but below or equal to ten units per week) and high consumption (more than ten units per week). The external validity of the present study is limited because we studied only women who had a family history of AITD. Our findings are in an agreement with a recent case-control study from Denmark [14] in which evidence that alcohol consumption is protective for the development of autoimmune hypothyroidism was also obtained. Cases were 140 patients with incident autoimmune overt hypothyroidism identified in a Danish population-based study and they were matched for age and sex with 560 controls recruited from the same population who had normal thyroid function and no history of thyroid disease. The odds ratio for developing hypothyroidism for alcohol consumers of 1-10 units/week was 0.58 (95% CI ) and for alcohol consumers of >11units/week was 0.40 (95% CI ) while non-drinkers (abstainers) were used as the reference group. The observed odds ratios did not change after multivariate adjustment for smoking habits and family history of hypothyroidism (0.59 (95% CI ) and 0.41 (95% CI ), respectively). Our data on a protective effect of alcohol on autoimmune hypothyroidism are reminiscent to the protective effect of alcohol on the development of SLE as found in a meta-analysis [6]. Alcohol also protects against rheumatoid arthritis: non-drinkers have an odds ratio of 4.17 compared with subjects consuming alcohol on more than 10 days per month [5], and in another study the odds ratio is for subjects in the quartile with the highest alcohol

55 53 Chapter 4 Figure 2. Proportion of subjects consuming >10units/week in cases (black bars) and in controls (matched for age, duration of follow up and smoking behavior - grey bars) at baseline, one year prior to event and at the time of event in study B (development of overt hypothyroidism). OR denotes odds ratio and the 95% confidence intervals are in parentheses. consumption compared with the lowest quartile [4]. The mechanism behind the effect of alcohol on thyroid autoimmunity is not clear. Although experimental and clinical data suggest that alcohol is a potential modulator of the immune system, our findings do not suggest any association between alcohol and the immune response at early stages of thyroid autoimmunity. One may thus ask the question if our findings regarding the development of overt hypothyroidism are due to a direct effect of alcohol on the thyroid gland. Studies in the past have reported decreased serum thyroid hormones, normal TSH and a blunted TSH response to TRH among alcohol-dependent patients [15]. A direct and irreversible toxic effect of alcohol on the thyroid gland has been proposed as explanation for the reduction of serum thyroid hormones. A direct toxic effect could also explain that alcohol protects against the development of goiter, as evident from a significant decrease of thyroid volume observed in alcohol-dependent patients [16,17]. However, in our study alcohol consumption protected against thyroid hormone deficiency, thereby rendering a direct toxic effect of alcohol on the thyroid gland in our study highly unlikely. Recently, we and others [12,13] have reported that smoking has a protective effect on the development of thyroid antibodies and also on the development of Hashimoto s

56 54 hypothyroidism. The mechanisms behind the protective effect of smoking on AITD are also not well understood so far. Because both smoking and alcohol seem to protect against autoimmune hypothyroidism, it may be hypothesized that smoking and alcohol act via the same immune pathways; however, this seems to be less likely if one realises that the association between alcohol consumption and overt hypothyroidism in our study was indepent of smoking. An alternative hypothesis could be based on genetics. Twin studies [18-22] and studies on selected strains of laboratory animals [23] suggest a strong genetic influence on the liability to both alcohol and nicotine dependence. From that perspective it can be hypothesized that genetic factors which predispose to addiction behavior are also involved in the pathogenesis of thyroid autoimmunity. In conclusion, alcohol consumption of >10 units/week may protect against the development of overt hypothyroidism, independent of smoking. The mechanism behind this phenomenon remains to be clarified. References [1] Brix TH, Kyvik KO, Hegedüs L. A population-based study of chronic autoimmune hypothyroidism in Danish twins. J Clin Endocrinol Metab 2000;85: [2] Brix TH, Kyvik KO, Christensen K, Hegedüs L. Evidence for a major role of heredity in Graves disease: a population-based study of two Danish twin cohorts. J Clin Endocrinol Metab 2001;86: [3] Ringold DA, Nicoloff JT, Kesler M, Davis H, Hamilton A, Mack T. Further evidence for a strong genetic influence on the development of autoimmune thyroid disease: the California twin study. Thyroid 2002;12: [4] Kallberg H, Jacobsen S, Bengtsson C, Pedersen M, Padyukov L, Garred P, Frisch M, Karlson EW, Klareskog L, Alfredsson L. Alcohol consumption is associated with decreased risk of rheumatoid arthritis: results from two Scandinavian case-control studies. Annals of the Rheumatic Diseases 2009;68: [5] Maxwell JR, Gowers IR, Moore DJ, Wilson AG. Alcohol consumption is inversely associated with risk and severity of rheumatoid arthritis. Rheumatology 2010;49: [6] Wang J, Pan H-F, Ye D-Q, Su H, Li X-P. Moderate alcohol drinking might be protective for systemic lupus erythematosus: a systematic review and meta-analysis. Clin Rheumatol 2008;27: [7] Romeo J, Wärnberg J, Nova E, Díaz LE, Gómez-Martinez S, Marcos A. Moderate alcohol consumption and the immune system: A review. Bjn 2007;98. [8] Effraimidis G, Strieder TGA, Tijssen JGP, Wiersinga WM. Natural history of the transition from euthyroidism to overt autoimmune hypo- or hyperthyroidism: a prospective study. Eur J Endocrinol 2011;164: [9] Strieder TGA, Prummel MF, Tijssen JGP, Endert E, Wiersinga WM. Risk factors for and prevalence of thyroid disorders in a cross-sectional study among healthy female relatives of patients with autoimmune thyroid disease. Clin Endocrinol 2003;59: [10] Strieder TGA, Tijssen JGP, Wenzel BE, Endert E, Wiersinga WM. Prediction of progression to overt hypothyroidism or hyperthyroidism in female relatives of patients with autoimmune thyroid disease using the Thyroid Events Amsterdam (THEA) score. Arch Intern Med 2008;168: [11] Hollowell J, Staehling N, Flanders W. Serum TSH, T4, and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab 2002;87: [12] Effraimidis G, Tijssen JGP, Wiersinga WM. Discontinuation of smoking increases the risk for developing thyroid peroxidase antibodies and/or thyroglobulin antibodies: a prospective study. J Clin Endocrinol Metab 2009;94: [13] Pedersen IB, Laurberg P, Knudsen N, Jørgensen T, Perrild H, Ovesen L, Rasmussen LB. Smoking is negatively associated with the presence of thyroglobulin autoantibody and to a lesser degree with thyroid peroxidase autoantibody in serum: a population study. Eur J Endocrinol 2008;158: [14] Carle A, Pedersen I, Knudsen N, Perrild H, Ovesen L, Rasmussen L, Jørgensen T, Laurberg P. Alchohol consumption is protective for development of aytoimmune hypothyroidism A population-based study. European Thyroid Journal 2011;-: [15] Hermann D, Heinz A, Mann K. Dysregulation of the hypothalamic-pituitary-thyroid axis in alcoholism. Addiction 2002;97: [16] Hegedüs L. Decreased thyroid gland volume in alcoholic cirrhosis of the liver. J Clin Endocrinol Metab 1984;58: [17] Hegedüs L, Rasmussen N, Ravn V, Kastrup J, Krogsgaard K, Aldershvile J. Independent effects of liver disease and chronic alcoholism on thyroid function and size: the possibility of a toxic effect of alcohol on the thyroid gland. Metab. Clin.

57 Exp. 1988;37: [18] Carmelli D, Swan GE, Robinette D. The relationship between quitting smoking and changes in drinking in World War II veteran twins. J Subst Abuse 1993;5: [19] Enoch MA, Goldman D. The genetics of alcoholism and alcohol abuse. Curr Psychiatry Rep 2001;3: [20] Swan GE, Carmelli D, Cardon LR. Heavy consumption of cigarettes, alcohol and coffee in male twins. J. Stud. Alcohol 1997;58: [21] Goldman D, Oroszi G, Ducci F. The genetics of addictions: uncovering the genes. Nat Rev Genet 2005;6: [22] Bierut LJ, Rice JP, Goate A, Hinrichs AL, Saccone NL, Foroud T, Edenberg HJ, Cloninger CR, Begleiter H, Conneally PM, Crowe RR, Hesselbrock V, Li T-K, Nurnberger JI, Porjesz B, Schuckit MA, Reich T. A genomic scan for habitual smoking in families of alcoholics: Common and specific genetic factors in substance dependence. Am. J. Med. Genet. 2003;124A: [23] Le AD. Increased Vulnerability to Nicotine Self-Administration and Relapse in Alcohol-Naive Offspring of Rats Selectively Bred for High Alcohol Intake. Journal of Neuroscience 2006;26: [18] Carmelli D, Swan GE, Robinette D. The relationship between quitting smoking and changes in drinking in World War II veteran twins. J Subst Abuse 1993;5: [19] Enoch MA, Goldman D. The genetics of alcoholism and alcohol abuse. Curr Psychiatry Rep 2001;3: [20] Swan GE, Carmelli D, Cardon LR. Heavy consumption of cigarettes, alcohol and coffee in male twins. J. Stud. Alcohol 1997;58: [21] Goldman D, Oroszi G, Ducci F. The genetics of addictions: uncovering the genes. Nat Rev Genet 2005;6: [22] Bierut LJ, Rice JP, Goate A, Hinrichs AL, Saccone NL, Foroud T, Edenberg HJ, Cloninger CR, Begleiter H, Conneally PM, Crowe RR, Hesselbrock V, Li T-K, Nurnberger JI, Porjesz B, Schuckit MA, Reich T. A genomic scan for habitual smoking in families of alcoholics: Common and specific genetic factors in substance dependence. Am. J. Med. Genet. 2003;124A: [23] Le AD. Increased vulnerability to nicotine self-administration and relapse in alcohol-naive offspring of rats selectively bred for high alcohol intake. Journal of Neuroscience 2006;26: Chapter 4

58

59 Chapter 5 Vitamin D deficiency is not associated with early stages of thyroid autoimmunity. Effraimidis G Badenhoop K Tijssen JGP Wiersinga WM Eur J Endocrinol. 2012; accepted for publication.

60 ABSTR ACT Context: Vitamin D deficiency has been identified as a risk factor for a number of autoimmune diseases including type 1 diabetes and multiple sclerosis. Objective: We hypothesized that low levels of vitamin D are related to the early stages of autoimmune thyroid disease (AITD). 58 Design: Two case-control studies were performed. In the cross-sectional study A euthyroid subjects with genetic susceptibility for AITD but without thyroid antibodies were compared to controls. Cases were subjects from the Amsterdam AITD cohort (euthyroid women who had 1st or 2nd degree relatives with overt AITD) who at baseline had normal TSH and no thyroid antibodies; controls were healthy women examined at the same time period. In the longitudinal study B subjects who developed de novo TPO-Ab were compared with those who did not. Cases and controls were subjects from the Amsterdam AITD cohort who at baseline had normal TSH and no thyroid antibodies and during follow up developed TPO-Ab (cases) or remained without thyroid antibodies (controls). Controls in both studies were matched for age, BMI, smoking status, estrogen use, month of blood sampling and in study B for the duration of follow up. Results: Serum 25(OH)D levels in ng/ml were as follow: Study A: 21.0±7.9 vs. 18.0±6.4 ng/ml (78 cases vs 78 controls, p=0.01). Study B: baseline, 22.6±10.3 vs. 23.4±9.1; follow-up 21.6±9.2 vs. 21.2±9.3 (67 cases vs. 67 controls, NS). Conclusions: Early stages of thyroid autoimmunity (in study A genetic susceptibility and in study B development of TPO-Ab) are not associated with low vitamin D levels.

61 Introduction The recent description that many tissues and cells in the body express the vitamin D receptor and 1α-hydroxylase resulted in a growing interest in the role of vitamin D in extra-skeletal conditions such as common cancers, cardiovascular disease and autoimmune diseases [1-4]. Lower 25-hydroxy vitamin D levels have been reported in several autoimmune diseases. Low vitamin D intake and vitamin D deficiency have been identified as a risk factor for type 1 diabetes, multiple sclerosis, Crohn s disease and rheumatoid arthritis [5-10] e.g. in a prospective case-control study, the risk of multiple sclerosis significantly decreased with increasing levels of 25(OH)D [8]. Recent studies indicate that 1,25(OH) 2 D, the biologically active form of vitamin D, is a modulator of both the innate and adaptive immune system. Immune cells such monocytes, macrophages, dendritic cells and T-lymphocytes and B-lymphocytes are targets for the active vitamin D. Moreover, 1,25(OH) 2 D seems to play an autocrine and paracrine role in the immune system as immune cells are capable to activate vitamin D [4]. However it is not known which vitamin D levels are sufficient to improve the immune regulatory function and lead to an effective immune response. Very few studies in the past have reported on a putative association between thyroid autoimmunity and vitamin D with inconclusive results [11-13]. In the present study we aimed to assess whether vitamin D deficiency is related to the early stages of autoimmune thyroid disease (AITD) [14]. We hypothesized that vitamin D levels are already low in subjects with genetic susceptibility for AITD, at a time when thyroid antibodies are absent and thyroid function is still normal and also lower in euthyroid subjects who will develop thyroid antibodies de novo. Thus, we compared family members of AITD patients who were euthyroid without thyroid antibodies with healthy controls (study A), allowing to evaluate whether genetic susceptibility for AITD by itself is already associated with lower vitamin D levels. We further evaluated vitamin D levels in euthyroid family members of AITD patients, comparing those who developed TPO-Ab during follow-up (seroconverters) and those who did not (study B), allowing to assess whether the occurrence of thyroid antibodies is associated with lower vitamin D levels. 59 Chapter 5 Subjects & Methods Participants The present study was carried out among the 803 subjects from the Amsterdam AITD Cohort. The cohort has previously been described in detail [15]. In short, the cohort consisted of women between 18 and 65 years of age in self-proclaimed good health without a history of thyroid disease, who had at least one 1st or 2nd degree relative with documented autoimmune hyper- or hypothyroidism. Results of thyroid function tests at study entrance revealed overt hypothyroidism in 10 subjects and overt hyperthyroidism in 3 subjects, leaving 790 subjects to be included in the present study. Subjects were followed for five years, or shorter when overt hyper- or hypothyroidism had occurred (defined as TSH <0.4 mu/l in combination with ft4 >20.1 pmol/l, or TSH >5.7 mu/l

62 in combination with ft4 <9.3 pmol/l respectively). At each annual visit to our institution blood samples were collected to measure TSH, ft4, TPO-Ab, Tg-Ab and TBII. Plasma and serum samples were stored at 20 C until assay. We performed two case-control studies A and B. In both studies, for each case one control was selected. A case-control approach was chosen due to cost-effectiveness. Subjects using drugs affecting vitamin D levels were excluded in both studies. All subjects gave informed written consent and the Medical Ethics committee of the Academic Medical Center in Amsterdam approved the study. 60 Study A: Vitamin D and genetic susceptibility for AITD. Cases were selected from the Amsterdam AITD cohort at inception: euthyroid subjects without serological signs of thyroid autoimmunity at baseline (i.e. normal TSH, normal free T4, negative TPO-Ab, negative Tg-Ab and negative TBII) were included. As a control group, we used female subjects between 20 and 69 years of age, who were recruited through advertisements in local newspapers, to participate in an ongoing program within our institution for delineating reference values of endocrine function tests. They were also in self-proclaimed good health, had no family or personal history of thyroid disease and had normal TSH and no thyroid antibodies. Blood samples were collected over the same period of time as those of the Amsterdam AITD cohort, and processed in the same manner. Because the number of controls satisfying these criteria was limited to 78, we selected also 78 cases matched for known factors to affect 25(OH)D status: age, BMI, smoking status, estrogen use and month of blood sampling accepting a difference of 1 month. We defined seasons as winter (December to February), spring (March to May), summer (June to August), and autumn (September to November). Study B: Vitamin D and de novo development of TPO-Ab. We selected participants from the inception cohort of the 790 euthyroid subjects as follows: first we excluded 241 who had thyroid antibodies at baseline (i.e. serum concentrations of either TPO-Ab of 100 ku/l or greater, Tg-Ab of 100 ku/l or greater, or TBII of 12U/L or greater). From the remaining 549 euthyroid subjects without thyroid antibodies we subsequently excluded those who had subclinical hyper- or hypothyroidism and those who had no follow-up. Consequently, 521 euthyroid participants without any serological sign of AITD at baseline were thus enrolled. A subject was recruited as a case when she remained euthyroid but had developed TPO-Ab during follow-up. The end-point for a case was the time at which she had become positive for the first time for TPO-Ab without developing abnormal TSH. This happened in 67 subjects. Subjects from the Amsterdam AITD cohort qualified to act as controls if they remained euthyroid and seronegative for TPO-Ab up to the time at which the case they were matched to, had received her end-point. Controls were matched by age, BMI, smoking status, estrogen use and month of blood sampling at study entrance and by duration of follow up. 25(OH)D status and 1,25(OH) 2 D status at baseline and at the time of the seroconversion to TPO-Ab were compared between cases and controls.

63 Methods Serum TSH and ft4 were measured using time-resolved fluoroimmunoassay (Delphia, Turku, Finland). Reference values are for TSH mu/l and for ft pmol/l. Thyroid peroxidase (TPO) antibodies and thyroglobulin (Tg) antibodies were measured by chemiluminescence immunoassays (LUMI-test anti-tpo and LUMI-test anti-tg respectively, Brahms, Berlin, Germany). Improved versions of both assays became available during followup: detection limits of these new assays were for TPO-Ab 30 ku/l and for Tg-Ab 20 ku/l. TPO-Ab concentrations obtained with the old assay were multiplied by a factor 0.72 to obtain comparative values in the new assay. TPO-Ab and Tg-Ab concentrations were considered to be positive at values 100 ku/l. TSH receptor antibodies were determined as TSH binding inhibitory immunoglobulins (TBII) using the TRAK assay (Brahms, Berlin, Germany); detection limits in the 1st and 2nd generation TRAK assays were 5 and 1 IU/L respectively, and values above 12 and 1.5 U/L respectively were considered as positive. 25(OH)D and 1,25(OH) 2 D were measured by radioimmunoassay (I125 Radioimmunoassay IA Kit; DiaSorin, Stillwater, MN). Plasma 25(OH)D concentrations of <20 ng/ml was defined as 25(OH)D deficiency and <30 ng/ml as 25(OH)D deficiency and insufficiency [16]. A range of pg/ml of 1,25(OH) 2 D was considered normal according to the manufacturer. 61 Chapter 5 Statistical analysis Differences between cases and controls were evaluated by Student s t-test for age, BMI, duration of follow up and 25(OH)D and 1,25(OH) 2 D status, by Mann-Whitney U-test for TSH, ft4, TPO-Ab and Tg-Ab. Seasonal variation in 25(OH)D levels was assessed by ANOVA. A p-value of <0.05 was considered to indicate significant differences. Results Study A: Vitamin D and genetic susceptibility for AITD. The 78 cases and 78 controls were comparable in age, BMI, proportion of smokers and estrogen users, month of blood sampling, TSH, ft4, TPO-Ab and Tg-Ab levels (Table 1). Seasonal variation in 25(OH)D was observed in cases and controls. Cases had a higher serum 25(OH)D concentration than controls (mean±sd: 21.0±7.9 ng/ml vs. 18.0±6.4 ng/ml, p=0.01). The prevalence of 25(OH)D deficiency (<20 ng/ml) was lower in cases than in controls (48.7% vs. 64.1% respectively, p=0.05); the same was true for the prevalent rates of 25(OH)D deficiency and insufficiency (<30 ng/ml) (83.3% vs. 94.9%, p=0.02). Study B: Vitamin D and de novo development of TPO-Ab. During the 5 year follow up period 67 subjects developed TPO-Ab while their TSH remained normal (Table 2). The mean±sd age at baseline of the converters to TPO-Ab was 35.5±11.5 years and the mean±sd follow up was 2.8±1.3 years. Controls mean age and mean follow up did not differ from cases. The same was true for BMI, proportion of smokers and estrogen users, month of bleed, TSH, ft4, TPO-Ab and Tg-Ab levels at baseline. Seasonal variation in 25(OH)D and 1,25(OH) 2 D was observed in cases and controls.

64 Table 1. Characteristics and 25(OH)D status of healthy euthyroid thyroid antibodies-negative female relatives of AITD patients and healthy controls (Study A). cases controls p-value N age yr 42.1± ±13.1 NS BMI kg/m ( ) 23.8 ( ) NS current smokers % 27% 27% NS 62 current estrogen users % 13% 13% NS blood sampling % (winter/spring/summer/autumn) 23/44/13/20 % 23/50/5/22 % NS TSH mu/l 1.60 ( ) 1.58 ( ) NS ft4 pmol/l 14.0 ( ) 14.1 ( ) NS TPO-Ab ku/l <30 (<30-<30) <30 (<30-<30) NS Tg-Ab ku/l <10 (<10-21) <10 (<10-<10) NS 25(OH)D ng/ml 21.0± ± Data are given as mean±sd or median with interquartile range between parenthesis. BMI: Body mass index, TSH: thyroid stimulating hormone, ft4: free T4, TPO: thyroid peroxidase, Tg: thyroglobulin, 25(OH)D: 25-hydroxyvitamin D3. At the time of seroconversion the TPO-Ab concentration had a median value of 140 ku/l (interquartile range ku/l). Statistical analysis at baseline and at time of seroconversion revealed no significant differences in the 25(OH)D and in the 1,25(OH) 2 D levels between cases and controls. The frequency of 25(OH)D deficiency (<20 ng/ml) at baseline was comparable in cases and in controls (49.2% vs. 34.3% respectively, p=0.05); the same was true for the prevalent rates of 25(OH)D deficiency and insufficiency (<30 ng/ml) (79.1% vs. 86.5%, p=0.25). Similar figures were obtained at the time of seroconversion (52.2% vs 55.2% and 88.1% vs 83.6% respectively, NS). Discussion The main finding of our study is that 25(OH)D levels in the cases were not lower than in the controls, neither in the subjects with genetic susceptibility for AITD in study A, nor in the seroconverters with de novo occurrence of TPO-Ab in study B. We therefore have to reject our hypothesis that the early stages of thyroid autoimmunity are associated with low 25(OH)D levels. The present study is the first to evaluate the relationship between 25(OH)D and thyroid autoimmunity in a prospective manner. All previous studies have been cross-sectional

65 Table 2. Characteristics, 25(OH)D and 1,25(OH) 2 D status of healthy euthyroid thyroid antibodies-negative women who developed TPO-Ab (cases) and who did not develop TPO-Ab (controls) at baseline and at the time of seroconversion, in a nested case-control study of euthyroid women with 1st or 2nd degree relatives with proven AITD derived from the Amsterdam AITD cohort (study B). BASELINE FOLLOW UP cases controls p-value cases controls p-value N age yr 35.5± ±11.2 NS 38.3± ±11.3 NS BMI kg/m ( ) 23.8 ( ) NS 23.0 ( ) 24.2 ( ) NS current smokers % 27% 27% NS 28% 27% NS current estrogen use % 13% 13% NS 28% 37% NS blood sampling % 21/21/28/30 % 21/22/31/26 % NS 21/22/14/43 % 24/21/15/40 % NS (winter/spring/summer/autumn) TSH mu/l 1.50 ( ) 1.50 ( ) NS 1.60 ( ) 1.50 ( ) NS ft4 pmol/l 13.0 ( ) 13.3 ( ) NS 12.9 ( ) 13.3 ( ) NS TPO Antibody ku/l <30 (<30-<30) <30 (<30-<30) NS 140 ( ) <30 (<30-50) <0.001 Tg Antibody ku/l <10 (<10-25) <10 (<10-15) NS 18 (<10-47) <10 (<10-13) NS 25(OH)D ng/ml 22.6± ±9.1 NS 21.6± ±9.3 NS 1,25(OH) 2 D pg/ml 58.5± ±18.1 NS 56.7± ±19.7 NS Data are given as mean ± SD or median with interquartile range between parenthesis. BMI: Body mass index, TSH: thyroid stimulating hormone, ft4: free T4, TPO: thyroid peroxidase, Tg: thyroglobulin, 25(OH)D: 25-hydroxyvitamin D3, 1,25(OH)2D: 1,25 dihydroxyvitamin D3 63 Chapter 5

66 64 in nature. A study from India [11] reported 25(OH)D deficiency (25(OH)D serum levels 10ng/ml) in 87% of 642 students and teachers aged years; blood sampling was done in the winter months. Comparing subjects with 10 ng/ml or >10 ng/ml, there were no differences in the prevalence of TPO-Ab (21.3% vs. 18.1%), the concentration of TPO-Ab (58±4.9 vs. 46±10.4 IU/ml) and the prevalence of subjects with TPO-Ab and decreased TSH (13.2% vs. 12.0%). A study from Hungary [12] (with blood sampling in March) observed a higher prevalence of 25(OH)D deficiency (25(OH)D serum levels 10 ng/ml) in outpatients with thyroid disease than in age-matched healthy controls (63% vs. 30%, p<0.001). However, the prevalence of 25(OH)D deficiency was not different between AITD patients (both Hashimoto s and Graves disease) and non AITD patients (72% vs. 52%, p=0.08); abnormal thyroid function (not specified any further) in these groups occurred in 44% and 15% respectively (p=0.003). This may constitute a bias since 1,25(OH) 2 D levels tend to be low in hyperthyroidism as a result of the increased calcium release from bone; conversely 1,25(OH) 2 D levels are slightly higher in hypothyroidism. The third and last study [13] on this topic comes from Turkey (performed between October 2008 and February 2009). Vitamin D levels were lower in patients with Hashimoto s thyroiditis than in age and sex matched controls (25(OH)D 16.3±10.4 vs 29.6±25.5 ng/ml, p<0.0001), as was the prevalence of 25(OH)D deficiency and insufficiency (25(OH)D <30 ng/ml, 92% vs. 63%, p<0.0001). There were no significant differences in 25(OH)D levels between Hashimoto s patients who were overtly hypothyroid, subclinically hypothyroid or euthyroid (15.4±9.3, 15.7±7.4 and 17.4±12.9 ng/ml respectively, p=0.87). Seasonal variation could still be a confounding factor in this study. The authors concluded there might be an association between vitamin D insufficiency and Hashimoto s thyroiditis but not between vitamin D insufficiency and progress of thyroid damage in Hashimoto s thyroiditis. They recommended further studies to evaluate whether vitamin D insufficiency is a causal factor in the pathogenesis of AITD. Our studies do not support the hypothesis that low 25(OH)D levels in adults are involved in the pathogenesis of AITD neither at the early stages in which there is nothing else except genetic susceptibility, nor in the preclinical stage in which thyroid antibodies develop but thyroid function is still normal. It cannot ruled out that 25(OH)D deficiency at even earlier time points (than those we studied) i.e. in early life, might effect the development of AITD in genetically high risk individuals. This view is supported by the finding that serum 25(OH)D levels of 100 nmol/l before the age of 20 years protect against the development of multiple sclerosis later in life [8]. Studies on type 1 diabetes have shown increased prevalence of type 1 diabetes when vitamin D deficiency was present in the first months of life [17]; also the onset of type 1 diabetes is accelerated in mice with vitamin D deficiency in early life [18]. We observed a high prevalence of 25(OH)D insufficiency in our study, in agreement with previous Dutch population-based studies [19]. One may argue that a high prevalence of 25(OH)D insufficiency may enhance the chance to miss a small difference in 25(OH)D levels between AITD patients and healthy controls. However, the finding of a difference in Turkey with also a high prevalence of 25(OH)D insufficiency, does not support this possibility. However we cannot exclude, that other environmental factors such as infections may interact with vitamin D deficiency and enhance the risk for thyroid autoimmunity by differential immune pathways only operative if these two factors coincide, possibly with additional immunogenetic susceptibility regulating this process. An interaction

67 of vitamin D deficiency with susceptibility to several viral and retroviral infections is documented [20-22]. Serum 25(OH)D and 1,25(OH) 2 D concentrations may also not always accurately reflect the hormonal effect in target tissues, which can be modified in multiple ways, e.g. by paracrine effects of locally synthesized 1,25(OH) 2 D and polymorphisms in the vitamin D receptor [23,24]. The finding that 25(OH)D levels in study A were higher in cases than controls might also be interpreted as to suggest that women with genetic susceptibility to AITD do not have thyroid autoantibodies because they are protected from developing antibodies through higher levels of 25(OH)D. This interpretation, however, does not change our conclusion that low 25(OH)D levels are not associated with early stages of AITD. A weakness of our study is the limited number of subjects and a larger number of cases is required to more accurately outline the role of vitamin D. Another limitation is that we studied adults, as vitamin D levels earlier in life may be critical in conferring protection against autoimmune diseases. The strengths of our studies are their prospective nature and their well executed matching procedure for cases and controls. The large sample size of the Amsterdam AITD cohort allowed to match for a great number of variables known to influence 25(OH)D levels: age, BMI, smoking status, estrogen use and month of blood sampling. We did not inquire about recent holidays in the sun, but most likely variation in sunny holidays between cases and controls is effectively neutralized by our matching for seasonal variation in blood sampling. Thus, we feel rather confident on the validity of our conclusion: vitamin D deficiency in adults is not related with early stages of thyroid autoimmunity. 65 Chapter 5 References [1] Vanoirbeek E, Krishnan A, Eelen G, Verlinden L, Bouillon R, Feldman D, Verstuyf A. The anti-cancer and anti-inflammatory actions of 1,25(OH)2D3. Best Pract Res Clin Endocrinol Metab 2011;25: [2] Woloszynska-Read A, Candace SJ, Trump DL. Vitamin D and cancer: Clinical aspects. Best Pract Res Clin Endocrinol Metab 2011;25: [3] Leu M, Giovannucci E. Vitamin D: epidemiology of cardiovascular risks and events. Best Pract Res Clin Endocrinol Metab 2011;25: [4] Van Belle TL, Gysemans C, Mathieu C. Vitamin D in autoimmune, infectious and allergic diseases: a vital player? Best Pract Res Clin Endocrinol Metab 2011;25: [5] Mohr SB, Garland CF, Gorham ED, Garland FC. The association between ultraviolet B irradiance, vitamin D status and incidence rates of type 1 diabetes in 51 regions worldwide. Diabetologia 2008;51: [6] Zipitis CS, Akobeng AK. Vitamin D supplementation in early childhood and risk of type 1 diabetes: a systematic review and meta-analysis. Archives of Disease in Childhood 2008;93: [7] Mora JR, Iwata M, Andrian von UH. Vitamin effects on the immune system: vitamins A and D take centre stage. Nat Rev Immunol 2008;8: [8] Munger KL, Levin LI, Hollis BW, Howard NS, Ascherio A. Serum 25-hydroxyvitamin D levels and risk of multiple sclerosis. Jama 2006;296: [9] Cutolo M, Otsa K, Laas K, Yprus M, Lehtme R, Secchi ME, Sulli A, Paolino S, Seriolo B. Circannual vitamin d serum levels and disease activity in rheumatoid arthritis: Northern versus Southern Europe. Clin. Exp. Rheumatol. 2006;24: [10] Patel S, Farragher T, Berry J, Bunn D, Silman A, Symmons D. Association between serum vitamin D metabolite levels and disease activity in patients with early inflammatory polyarthritis. Arthritis Rheum 2007;56: [11] Goswami R, Marwaha RK, Gupta N, Tandon N, Sreenivas V, Tomar N, Ray D, Kanwar R, Agarwal R. Prevalence of vitamin D deficiency and its relationship with thyroid autoimmunity in Asian Indians: a community-based survey. Bjn 2009;102: [12] Kivity S, Agmon-Levin N, Zisappl M, Shapira Y, Nagy EV, Danko K, Szekanecz Z, Langevitz P, Shoenfeld Y. Vitamin D and autoimmune thyroid diseases. Cell. Mol. Immunol. 2011;8: [13] Tamer G, Arik S, Tamer I, Coksert D. Relative Vitamin D Insufficiency in Hashimoto s Thyroiditis. Thyroid 2011;21: [14] Effraimidis G, Strieder TGA, Tijssen JGP, Wiersinga WM. Natural history of the transition from euthyroidism to overt

68 66 autoimmune hypo- or hyperthyroidism: a prospective study. Eur J Endocrinol 2011;164: [15] Strieder TGA, Prummel MF, Tijssen JGP, Endert E, Wiersinga WM. Risk factors for and prevalence of thyroid disorders in a cross-sectional study among healthy female relatives of patients with autoimmune thyroid disease. Clin Endocrinol 2003;59: [16] Holick MF. Vitamin D deficiency. N Engl J Med 2007;357: [17] Hyppönen E, Läärä E, Reunanen A, Järvelin MR, Virtanen SM. Intake of vitamin D and risk of type 1 diabetes: a birthcohort study. Lancet 2001;358: [18] Giulietti A, Gysemans C, Stoffels K, van Etten E, Decallonne B, Overbergh L, Bouillon R, Mathieu C. Vitamin D deficiency in early life accelerates Type 1 diabetes in non-obese diabetic mice. Diabetologia 2004;47: [19] van Schoor NM, Visser M, Pluijm SMF, Kuchuk N, Smit JH, Lips P. Vitamin D deficiency as a risk factor for osteoporotic fractures. Bone 2008;42: [20] Bitetto D, Fattovich G, Fabris C, Ceriani E, Falleti E, Fornasiere E, Pasino M, Ieluzzi D, Cussigh A, Cmet S, Pirisi M, Toniutto P. Complementary role of vitamin D deficiency and the interleukin-28b rs C/T polymorphism in predicting antiviral response in chronic hepatitis C. Hepatology 2011;53: [21] Belderbos ME, Houben ML, Wilbrink B, Lentjes E, Bloemen EM, Kimpen JLL, Rovers M, Bont L. Cord blood Vitamin D deficiency is associated with respiratory syncytial virus bronchiolitis. Pediatrics 2011;127:e1513 e1520. [22] Viard J-P, Souberbielle J-C, Kirk O, Reekie J, Knysz B, Losso M, Gatell J, Pedersen C, Bogner JR, Lundgren JD, Mocroft A. Vitamin D and clinical disease progression in HIV infection: results from the EuroSIDA study. Aids 2011;25: [23] Hewison M. An update on vitamin D and human immunity. Clin Endocrinol 2012;76: [24] Ramagopalan SV, Heger A, Berlanga AJ, Maugeri NJ, Lincoln MR, Burrell A, Handunnetthi L, Handel AE, Disanto G, Orton SM, Watson CT, Morahan JM, Giovannoni G, Ponting CP, Ebers GC, Knight JC. A ChIP-seq defined genome-wide map of vitamin D receptor binding: Associations with disease and evolution. Genome Research 2010;20:

69 67 Chapter 5

70

71 Chapter 6 Involvement of stress in the pathogenesis of Autoimmune Thyroid Disease: a prospective study. Effraimidis G Tijssen JGP Brosschot JF Wiersinga WM Psychoneuroendocrinology. 2012; Epub ahead of print

72 ABSTR ACT Background: An association between stress and autoimmune thyroid disease (AITD) (especially Graves hyperthyroidism) has been reported, but all studies so far on this topic have been retrospective. Objective: To evaluate prospectively the relationship between stress and i) de novo occurrence of thyroid antibodies and ii) development of overt autoimmune hyper-/ hypothyroidism. 70 Study design: Two nested case-control studies in a prospective cohort of 790 euthyroid women who were 1st or 2nd degree relatives of AITD patients. Follow-up was five year, with annual assessments including questionnaires on stressful life events, daily hassles, and mood. In study A, cases were subjects who developed TPO-Ab but remained euthyroid during follow-up (called event). In study B, cases were subjects who developed overt hypothyroidism (TSH >5.7mU/L and ft4 <9.3pmol/L) or overt hyperthyroidism (TSH <0.4mU/L and ft4 >20.1pmol/L) during follow-up (called event). For each case, two controls were selected, matched for age and duration of follow up; controls in study A remained TPO-Ab negative, and in study B remained without overt hyper-/hypothyroidism. Outcomes: Contrast in questionnaire responses between cases and controls at baseline, at one year prior to the event and at time of event. Results: Exposure to stress was not different between subjects who developed or did not develop TPO-Ab (study A). No differences were observed in stress questionnaires between hyper-/hypothyroid cases and controls at any time point, but hypothyroid cases had less negative feelings than controls at the time of diagnosis (study B). Conclusion: The data suggest that stress is not involved in the pathogenesis of AITD.

73 Introduction Autoimmune thyroid disease (AITD) results from a complex interplay among genetic and environmental factors. Evidence for a genetic base of this disease is strong as siblings and other family members of AITD patients are at increased risk for AITD [1,2]. The sibling recurrence risk ratio (λs) is 11.6 for Graves and 28.0 for Hashimoto s disease [3]. In women with at least one first degree relative with AITD, the incidence of Graves hyperthyroidism is times higher and of Hashimoto s hypothyroidism times higher than the general female population [4]. Twin studies suggest that genetic factors play a major role, accounting for about seventy percent of the risk to get AITD [5-7]. The remaining thirty percent must be due to environmental factors which alter the immune reaction depending on the duration and intensity of exposure [8]. Among those factors is stress. Stress affects the immune system both directly and indirectly through the activation of neural and endocrine systems [9,10]. The phenotypic expression of AITD is to a large extent dependent on the balance of Th1 versus Th2 immune response [11]. During periods of stress an increase of the secretion of glucocorticoids and catecholamines is observed as a result of the activation of the sympathoadrenal system and the hypothalamic-pituitaryadrenal axis, respectively. Both glucocorticoids and catecholamines cause a selective suppression of Th1 response and a shift toward Th2 mediated humoral immunity [12]. This mechanism may promote the development of Graves disease which is a Th2 predominant disease [13]. On the other hand, a hypoactive hypothalamic-pituitary-adrenal axis may leads to a predominantly Th1-mediated immune activity [14] which may promote thyroid cell destruction and Hashimoto s thyroiditis through apoptotic pathways on thyroid follicular cells. The natural history of AITD probably starts with a particular genetic background and then several stages can be distinguished. The first stage in the natural history of thyroid autoimmunity that can be detected is the occurrence of TPO-Ab. Thereafter, changes in serum TSH at values outside the normal range follow (subclinical hypo- or hyperthyroidism) and the disease ends in many instances with overt hypo- or hyperthyroidism. In order to get more insight in the influence of stress on the natural course of AITD we did two nested case-control studies within the Amsterdam AITD cohort. The aim of the first study was to evaluate the relationship between stress and the de novo occurrence of TPO-Ab and the aim of the second study was to evaluate the relationship between stress and the development of overt hypothyroidism or overt hyperthyroidism. 71 Chapter 6 Subjects & Methods Participants The present study was carried out among the 803 subjects from the Amsterdam AITD Cohort. The original cohort has previously been described in detail [15]. In short, the cohort consisted of women between 18 and 65 years of age in self proclaimed good health without a history of thyroid disease, who had at least one 1st or 2nd degree relative with

74 72 documented autoimmune hyper- or hypothyroidism. Subjects were followed for five years, or shorter when overt hyper- or hypothyroidism had occurred (defined as TSH <0.4 mu/l in combination with ft4 >20.1 pmol/l, or TSH >5.7 mu/l in combination with ft4 <9.3 pmol/l respectively). At each annual visit blood samples were collected to measure TSH, ft4, T3, TPO-Ab, Tg-Ab and TBII, and subjects were asked to fill a number of questionnaires on stressful life events, daily hassles, and mood. The Dutch questionnaire on Recent Experienced Stressful Life Events [16,17] counts the total number of major life events experienced in the last 12 months (checklist of 60 possible events). The respondent scores separately the amount of pleasantness and unpleasantness with each experienced life event, rated on a scale of zero (meaning no (un) pleasantness at all) to four (a huge amount of (un)pleasantness). From this scale, the total amount of pleasantness and unpleasantness is calculated (maximum score 240 for each). When the amount of pleasantness exceeds the amount of unpleasantness the event is categorized as being pleasant and vice versa, yielding the total number of (un)pleasant events (maximum 60). The Dutch Everyday Problem Checklist a validated version of the Daily Hassles Scale [18-20], consists of 114 items concerning daily hassles in the last two months. It also measures the intensity of each hassle on a scale from zero to three, yielding the number of hassles experienced and the total intensity of these hassles (maximum 342). The Positive and Negative Affect Schedule (PANAS, [21]) measures the current mood, in terms of positive and negative affect. It consists of 22 mood states (11 positive, 11 negative) and the respondent is asked to report whether she is affected by each of these states on a scale from 1 (not at all) to 5 (a lot). This yields the tendency to report positive and negative affect states both on a scale from 11 to 55. Results of thyroid function tests at study entrance revealed overt hypothyroidism in 10 subjects and overt hyperthyroidism in 3 subjects, leaving 790 subjects to be included in the present study. Within this cohort we performed two nested case-control studies. In both studies, for each case two controls were selected. Controls were matched by age and duration of follow up. Matching by age was done in view of the increase in prevalence of thyroid antibodies with advancing age [22]. A subject could only be sampled once as control. From all possible subjects who could serve as candidate controls, we selected those that were closest to the corresponding case, first for age and then for the follow up period. Study A. In order to evaluate the relationship between stress and the de novo occurrence of TPO-Ab, we selected participants from the original cohort as follows (Figure 1): From the 790 euthyroid at study entrance subjects, we excluded those who had thyroid antibodies (i.e. serum concentrations of either TPO-Ab of 100 ku/l or greater, Tg-Ab of 100 ku/l or greater, or TBII of 12U/L or greater). From the remaining 549 euthyroid subjects without thyroid antibodies we subsequently excluded those who had subclinical hyper- or hypothyroidism (abnormal TSH in the presence of normal ft4 and T3) and those who had no follow-up. Five hundred twenty one euthyroid participants without any serological sign of AITD at baseline were thus enrolled. In this study, a subject was recruited as a case when she remained euthyroid but had developed TPO-Ab. The end-point for a case was the time at which she had become positive for the first time for TPO-Ab without developing abnormal TSH (called event). Controls should have remained euthyroid and seronegative for TPO-Ab up to the time at which the case they were matched to, had received her end-point.

75 Figure 1. Flowchart of the recruitment of subjects for study A. 73 Chapter 6 Study B. In order to evaluate the relationship between stress and the development of overt hypothyroidism or overt hyperthyroidism, we designed a nested case-control study among the 790 subjects euthyroid at study entrance. A subject was recruited as a case when she had developed over hypothyroidism or overt hyperthyroidism during follow up (called event). The end-point for a case was the time at which she had developed overt hypo- or hyperthyroidism. Controls should have normal TSH up to the time at which the case they were matched to, had received her end-point. Stress measurements at baseline, in the year prior to and at the time of the seroconversion to TPO-Ab for study A and the development of overt hypo- or hyper-thyroidism for study B were compared between cases and controls. Laboratory measurements Serum TSH and ft4 were measured using time-resolved fluoroimmunoassay (Delphia, Turku, Finland). Reference values are for TSH mu/l and for ft pmol/l. Thyroid peroxidase (TPO) antibodies and thyroglobulin (Tg) antibodies were measured by chemiluminescence immunoassays (LUMI-test anti-tpo and LUMI-test anti-tg respectively, Brahms, Berlin, Germany). Improved versions of both assays became available during follow-up: detection limits of these new assays were for TPO-Ab 30 ku/l and for Tg-Ab 20 ku/l. TPO-Ab concentrations obtained with the old assay were multiplied by a factor 0.72 to obtain comparative values in the new assay. TPO-Ab and Tg-Ab concentrations were considered to be positive at values 100 ku/l. TSH receptor antibodies were determined as TSH binding inhibitory immunoglobulins (TBII) using the TRAK assay (Brahms, Berlin, Germany); detection limits in the 1st and 2nd generation TRAK assays were 5 and 1 IU/L

76 respectively, and values above 12 and 1.5 U/L respectively were considered as positive. Statistical analysis Values are given as mean±sd for age and follow up period but as median and interquartile range for TSH and TPO-Ab. Differences between cases and controls were evaluated by Mann-Whitney U-test for stress parameters. A p-value of <0.05 was considered to indicate significant differences between groups. Results 74 Study A. Stress and development of TPO-Ab. During the 5-yr follow up period, 81 of the 521 subjects (15.5%) had developed TPO-Ab while their TSH remained normal (Table 1). The mean age of the converters to TPO-Ab was 36±12 years and the mean follow up was 2.8±1.3 years. Controls mean age and mean follow up did not differ from cases. At the time of seroconversion the TPO-Ab concentration had a median value of 140 ku/l (interquartile range ku/l). Statistical analysis at baseline, at time one year prior the occurrence of event and at time of event revealed no significant differences or trends in reported stress between cases and controls with respect to any of the questionnaires (Table 2). The results for developing either TPO-Ab and/or Tg-Ab were were essentially the same. Study B. Stress and development of overt hypo- or hyperthyroidism. During the 5-yr follow up period 13 cases of overt autoimmune hyperthyroidism and 38 cases of overt autoimmune hypothyroidism occurred after a mean follow-up of 2.7±1.5 and 3.2±1.3 respectively (Table 1). The cause of overt hyperthyroidism was Graves disease in 11 subjects (1 had Graves ophthalmopathy), postpartum thyroiditis in 1 subject, and silent thyroiditis in 1 subject. All subjects with overt hyperthyroidism had positive TPO-Ab and/or Tg-Ab at the time of diagnosis; TSH receptor antibodies were absent in the two patients diagnosed with postpartum thyroiditis and silent thyroiditis, but present in 6 out of 7 patients diagnosed with Graves hyperthyroidism (TBII was not measured in the time of diagnosis in 4 Graves patients). TSH receptor antibodies were undetectable in all controls at each time point, and in all cases at baseline and at the year before the event. Cases and controls did not differ with regard to mean age and mean follow up. We observed no differences in any of the three stress questionnaires between hyperthyroid cases and controls at baseline, at time one year before occurrence of the event and at time of the occurrence of the event (Table 3). At study entrance and at the year prior the event, we did not find any differences between hypothyroid cases and controls in recent life events, daily hassles, or affect scales (Table 3) except a lower amount of total unpleasantness in the hypothyroid cases than in controls at baseline. At the time of event, hypothyroid cases compared to controls reported significantly less frequently negative feelings at the time when diagnosis was made than controls. No other significant differences were found.

77 Table 1. Characteristics of subjects who developed TPO-Ab (study A) or overt autoimmune thyroid disease (study B) and their corresponding controls at time of study entrance and at consecutive time points of follow-up, in a nested case-control study of women with 1st or 2nd degree relatives with proven AITD derived from the prospective Amsterdam AITD cohort. Tg-Ab (ku/l) TPO-Ab (ku/l) ft4 (pmol/l) TSH (mu/l) follow up yr (SD) age yr (SD) N baseline at time of event baseline at time of event baseline at time of event baseline at time of event Study A cases (12) 2.8 (1.3) 1.5 ( ) 1.5 ( ) 13.1 ( ) 13.1 ( ) <25 (<25-<25) 140 ( ) 15 (<5-25) 17 (6-43) controls (12) 2.8 (1.3) 1.5 ( ) 1.4 ( ) 13.1 ( ) 13.1 ( ) <25 (<25-<25) <25 (<25-50) 5 (<5-15) 6 (6-15) p-values * < <0.001 Study B1 (hyperthyroidism) cases (14) 2.7 (1.5) 1.6 ( ) 0.02 ( ) 13.3 ( ) 41.5 ( ) 560 (< ) 1310 ( ) 38(23-108) 175 (44-185) controls (14) 2.7 (1.5) 1.5 ( ) 1.9 ( ) 14.1 ( ) 13.0 ( ) <25 (<25-80) 40 (<25-50) 17 (<5-41) 26 (<5-115) p-values * <0.001 < <0.001 Study B2 (hypothyroidism) cases (12) 3.2 (1.3) 4.2 ( ) 14.8 ( ) 10.7 ( ) 7.1 ( ) 1123 ( ) 3000 ( ) 75 (40-238) 140 ( ) controls (12) 3.2 (1.2) 1.6 ( ) 1.4 ( ) 12.8 ( ) 13.0 ( ) 25 (<25-50) 25 (<25-170) 8 (<5-29) 11 (6-69) p-values * <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 * cases vs. controls Data are given as mean ± SD or median with interquartile range Time of event: first occurrence of TPO-Ab (study A), or diagnosis of hyper- or hypothyroidism (study B). TSH: thyroid stimulating hormone, ft4: free T4, TPO: thyroid peroxidase, Tg: thyroglobulin 75 Chapter 6

78 76 Table 2. Comparison of scores on three questionnaires (Recent Experienced Life events, Daily Hassles, PANAS) between patients who developed TPO-Ab and remained euthyroid (normal TSH) and their corresponding controls matched for age and follow-up, in a nested case-control study of 243 women with 1st or 2nd degree relatives with proven AITD derived from the prospective Amsterdam AITD cohort. BASELINE ONE YEAR BEFORE EVENT AT EVENT P - value at event 1 year before event Cases Controls Cases Controls Cases Controls baseline Positive and Negative Affect Schedule Scale (tendency to report) Positive feelings 39.0 ( ) 38.0 ( ) 38.0 ( ) 38.0 ( ) 37.0 ( ) 37.0 ( ) Negative feelings 22.0 ( ) 21.0 ( ) 21.0 ( ) ( ) 20.0 ( ) ( ) Recent Life Events Total number 10.0 ( ) 11.0 ( ) 9.0 ( ) 9.0 ( ) 8.0 ( ) 9.0 ( ) Number of unpleasant life 5.0 ( ) 5.0 ( ) 4.0 ( ) 3.0 ( ) 3.0 ( ) 3.0 ( ) events Number of pleasant life events 5.0 ( ) 4.0 ( ) 4.0 ( ) 4.0 ( ) 3.0 ( ) 4.0 ( ) Amount of total unpleasantness 15.0 ( ) 17.0 ( ) 12.0 ( ) 12.0 ( ) 12.0 ( ) 13.0 ( ) Amount of total pleasanteness 16.0 ( ) 16.0 ( ) 15.0 ( ) 13.0 ( ) 13.0 ( ) 13.0 ( ) Daily Hassles Total number of daily hassles 22.0 ( ) 23.0 ( ) 25.5 ( ) 22.0 ( ) 25.5 ( ) 23.0 ( ) Intensity per hassle 1.36 ( ) 1.32 ( ) 1.40 ( ) 1.33 ( ) 1.35 ( ) 1.33 ( ) Total intensity of all hasles 30.0 ( ) 29.0 ( ) 33.0 ( ) 30.0 ( ) 33.0 ( ) 29.0 ( ) Data are given as median with interquartile range between parenthesis. P-value of cases versus controls. Time of event: visit when diagnosis of overt hyper- or hypothyroidism was made or confirmed. PANAS: positive and negative affect schedule.

79 We also tested if AITD was more prevalent in subjects who were exposed to severe stress and for that reason we analyzed the data in order to uncover possible differences in the extreme ends of any of the stress measurements for both study A and study B. We found that the proportion of subjects in whom measurements were above the 80th percentile did not differ between cases and controls. When we used the quadratics of the stress measurements, the results did not change either. Discussion The aim of our case-control studies nested in the observational Amsterdam AITD cohort study [23] was to evaluate in a prospective manner the involvement of stress in both the early stages (when thyroid antibodies develop but thyroid function is still normal) and late stages (when overt thyroid dysfunction emerges) of the natural course of AITD. The observed differences between cases and controls in both studies were either nonexistent or of marginal significance at study entrance and during follow-up, with the exception of a lower frequency of negative feelings reported at the time of the occurrence of hypothyroidism. The thyroid autoimmune process takes years [24], resulting in many cases in overt autoimmune hypo- or hyperthyroidism. All studies in the past on the relationship of stress and overt autoimmune hypo- or hyperthyroidism, measured stress by questionnaires at the time of the diagnosis. This approach raises the question of distinguishing between cause and effect, as the diseases themselves might generate stress in a patient. If stress would play a provocative role in the early stages of AITD, one would expect an association of stress and the occurrence of TPO-Ab, as the occurrence of TPO-Ab is one of the earliest events in AITD. Our results do not indicate such an association. These findings are in an agreement with our previous longitudinal cross sectional study [25] in which no association was found between stressful life events, daily hassles and mood and the presence of TPO-Ab in euthyroid women. We observed no increase in stress between hypothyroid cases and controls at baseline, in the year preceding the event or at the time of event in recently life events and daily hassles. Only two papers in the past reported on the association between stress and autoimmune hypothyroidism. Both papers failed to find any association between stress and autoimmune hypothyroidism. In one study [26] the triggering role of stress was assessed in 95 patients with Hashimoto s thyroiditis and 97 patients with benign thyroid nodules used as controls. Stress did not have any triggering role in Hashimoto s thyroiditis. Another study [27] evaluated the association between stress and the occurrence of postpartum thyroiditis. There was no excess of life events in women who developed postpartum thyroiditis with no difference in the number of life events between antibody positive and antibody negative women. In agreement with these studies, our study also didn t observe any differences between hypothyroid cases and controls. We only observed significant difference between hypothyroid cases and their respective controls on the PANAS negative affect scale, indicating a lower tendency to report negative feelings in cases at the time of event. If anything, we observed effects in the direction opposite to our expectation: We found that that hypothyroid cases, compared to their respective controls scored higher at the time of 77 Chapter 6

80 78 Table 3. Comparison of scores on three stress questionnaires (Recent Experienced Life events, Daily Hassles, PANAS) between patients who developed overt auto-immune thyroid disease (AITD) and their corresponding controls matched for age and follow-up. BASELINE ONE YEAR BEFORE EVENT AT EVENT P - value at event 1 year before event Cases Controls Cases Controls Cases Controls baseline HYPERTHYROISM Study B1 Positive and Negative Affect Schedule Scale (tendency to report) Positive feelings 39.0 ( ) 38.0 ( ) 36.0 ( ) 37.0 ( ) 36.0 ( ) 37.0 ( ) Negative feelings 22.0 ( ) 19.0 ( ) 20.0 ( ) 19.0 ( ) 19.0 ( ) 18.0 ( ) Recent Life Events Total number 7.0 ( ) 9.5 ( ) 7.0 ( ) 7.0 ( ) 9.0 ( ) 9.0 ( ) ( ) 5.0 ( ) 4.0 ( ) 5.0 ( ) 2.0 ( ) 5.5 ( ) Number of unpleasant life events 3.0 ( ) 4.5 ( ) 2.0 ( ) 3.0 ( ) 2.0 ( ) 3.0 ( ) Number of pleasant life events 11.0 ( ) 15.6 ( ) 11.0 ( ) 14.0 ( ) 6.0 ( ) 17.0 ( ) Amount of total unpleasantness 14.0 ( ) 15.0 ( ) 7.0 ( ) 9.0 ( ) 8.0 ( ) 12.0 ( ) Amount of total pleasanteness Daily Hassles 24.0 ( ) 22.5 ( ) 21.0 ( ) 20.0 ( ) 16.0 ( ) 24.0 ( ) Total number of daily hassles Intensity per hassle 1.5 ( ) 1.3 ( ) 1.5 ( ) 1.4 ( ) 1.2 ( ) 1.4 ( ) ( ) 29.0 ( ) 31.5 ( ) 29.0 ( ) 19.0 ( ) 32.5 ( ) Total intensity of all hasles

81 BASELINE ONE YEAR BEFORE EVENT AT EVENT P - value at event 1 year before event Cases Controls Cases Controls Cases Controls baseline HYPOTHYROISM Study B2 Positive and Negative Affect Schedule Scale (tendency to report) Positive feelings 39.0 ( ) 38.0 ( ) 35.0 ( ) 39.0 ( ) 37.0 ( ) 38.0 ( ) Negative feelings 21.5 ( ) 23.0 ( ) 20.0 ( ) 22.0 ( ) 17.0 ( ) 23.0 ( ) Recent Life Events Total number 7.5 ( ) 10.5 ( ) 8.5 ( ) 8.0 ( ) 9.0 ( ) 7.5 ( ) ( ) 4.0 ( ) 3.0 ( ) 4.0 ( ) 3.0 ( ) 3.0 ( ) Number of unpleasant life events 3.0 ( ) 4.0 ( ) 3.0 ( ) 3.5 ( ) 5.1 ( ) 3.5 ( ) Number of pleasant life events 8.0 ( ) 13.5 ( ) 10.0 ( ) 12.0 ( ) 9.0 ( ) 10.5 ( ) Amount of total unpleasantness 12.5 ( ) 14.5 ( ) 11.5 ( ) 13.0 ( ) 16.7 ( ) 11.5 ( ) Amount of total pleasanteness Daily Hassles 18.0 ( ) 23.5 ( ) 20.0 ( ) 23.0 ( ) 19.5 ( ) 22.5 ( ) Total number of daily hassles Intensity per hassle 1.2 ( ) 1.3 ( ) 1.4 ( ) 1.3 ( ) 1.2 ( ) 1.4 ( ) ( ) 27.0 ( ) 24.0 ( ) 28.0 ( ) 24.5 ( ) 28.0 ( ) Total intensity of all hasles Data are given as median with interquartile range between parenthesis. P-value of cases versus controls. Time of event: visit when diagnosis of overt hyper- or hypothyroidism was made or confirmed. PANAS: positive and negative affect schedule. 79 Chapter 6

82 80 diagnosis on the PANAS negative affect scale, indicating a lower tendency to report negative feelings in cases. Moreover we found that at the time of diagnosis they also reported to have experienced more pleasant events and a higher total pleasantness of these events in the preceding 12 months. We also did not detect any causal relationship between stressful life events and Graves hyperthyroidism. Our findings are in contrast with several case-control studies in the past which have reported on a positive relationship between stress and the development of Graves hyperthyroidism [28-34]. Αll studies were retrospective in nature and were influenced of recall bias as Graves disease patients were asked to report on stressful life events either at the time they were still hyperthyroid or even later when euthyroidism was restored. Furthermore, it could not be ruled out that negative stressful life events in the year preceding the onset of Graves hyperthyroidism were not the cause but the consequence of thyrotoxicosis. This issue was addressed to a certain extent in a study which reported an increase in negative life events in Graves disease patients compared with non-autoimmune hyperthyroid subjects [35]. A weakness of our study is the limited number of subjects who converted from euthyroidism to overt hypothyroidism (N=38) or hyperthyroidism (N=13). It should be pointed out that cases in study B with thyroid antibodies at baseline, are already at substantial higher risk of developing overt hypo- or hyperthyroidism, independently of stressful situations. A larger number of cases and a longer follow-up period is required to more accurately outline the role of stress. This limitation is in our view well balanced by the strengths of our study. Its prospective nature guarantees more solid evidence than obtained from cross-sectional studies. Moreover, in the nested case-control study a perfect match existed between both groups with respect to age and duration of follow-up. Higher age and longer exposure time both increase the likelihood of developing thyroid antibodies, constituting possible bias. The matching procedure effectively excluded both bias. References [1] Chopra IJ, Solomon DH, Chopra U, Yoshihara E, Terasaki PI, Smith F. Abnormalities in thyroid function in relatives of patients with Graves disease and Hashimoto s thyroiditis: lack of correlation with inheritance of HLA-B8. J Clin Endocrinol Metab 1977;45: [2] Jacobson EM, Tomer Y. The CD40, CTLA-4, thyroglobulin, TSH receptor, and PTPN22 gene quintet and its contribution to thyroid autoimmunity: back to the future. Journal of Autoimmunity 2007;28: [3] Villanueva R, Greenberg DA, Davies TF, Tomer Y. Sibling recurrence risk in autoimmune thyroid disease. Thyroid 2003;13: [4] Strieder, T.G.A., The Amsterdam AutoImmune Thyroid Disease cohort. University of Amsterdam, Amsterdam, p [5] Brix TH, Kyvik KO, Hegedüs L. A population-based study of chronic autoimmune hypothyroidism in Danish twins. J Clin Endocrinol Metab 2000;85: [6] Brix TH, Kyvik KO, Christensen K, Hegedüs L. Evidence for a major role of heredity in Graves disease: a population-based study of two Danish twin cohorts. J Clin Endocrinol Metab 2001;86: [7] Tomer Y, Huber A. The etiology of autoimmune thyroid disease: a story of genes and environment. Journal of Autoimmunity 2009;32: [8] Prummel MF, Strieder T, Wiersinga WM. The environment and autoimmune thyroid diseases. Eur J Endocrinol 2004;150:

83 [9] Chrousos GP. Stressors, stress, and neuroendocrine integration of the adaptive response. The 1997 Hans Selye Memorial Lecture. Annals of the New York Academy of Sciences 1998;851: [10] Elenkov IJ. Glucocorticoids and the Th1/Th2 balance. Annals of the New York Academy of Sciences 2004;1024: [11] Weetman AP. Cellular immune responses in autoimmune thyroid disease. Clin Endocrinol 2004;61: [12] Chrousos, G.P., Elenkov, I.J. Interactions of the endocrine and immune systems. In: DeGroot LJ, Jameson JL, (Ed). Endocrinology. PA:Saunders Elsevier, Philadelphia, 2006;Vol. 1, pp [13] Tsatsoulis A. The role of stress in the clinical expression of thyroid autoimmunity. Annals of the New York Academy of Sciences 2006;1088: [14] Wilder RL. Neuroendocrine-immune system interactions and autoimmunity. Annu Rev Immunol 1995;13: [15] Strieder TGA, Prummel MF, Tijssen JGP, Endert E, Wiersinga WM. Risk factors for and prevalence of thyroid disorders in a cross-sectional study among healthy female relatives of patients with autoimmune thyroid disease. Clin Endocrinol 2003;59: [16] Van de Willige G, Schreurs P, Tellegen B, Zwart F. Het meten van life events : de Vragenlijst Recent Meegemaakte Gebeurtenissen (VRMG). Nederlands Tijdschrift Voor De Psychologie en Haar Grensgebieden [17] Brosschot JF, Benschop RJ, Godaert GL, Olff M, De Smet M, Heijnen CJ, Ballieux RE. Influence of life stress on immunological reactivity to mild psychological stress. Psychosom Med 1994;56: [18] Kanner AD, Coyne JC, Schaefer C, Lazarus RS. Comparison of two modes of stress measurement: Daily hassles and uplifts versus major life events. J Behav Med 1981;4:1 39. [19] Vingerhoets A, Jeninga A, Menges L. Het meten van chronische en alledaagse stressoren: Eerste onderzoekservaringen met de Alledaagse Problemen Lijst (apl) ii. Gedrag en Gezondheid 1989;17: [20] Vingerhoets AJ, Ratliff-Crain J, Jabaaij L, Menges LJ, Baum A. Self-reported stressors, symptom complaints and psychobiological functioning I: Cardiovascular stress reactivity. J Psychosom Res 1996;40: [21] Watson D, Clark LA, Tellegen A. Development and validation of brief measures of positive and negative affect: the PANAS scales. J Pers Soc Psychol 1988;54: [22] Hollowell J, Staehling N, Flanders W. Serum TSH, T4, and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab 2002;87: [23] Strieder TGA, Tijssen JGP, Wenzel BE, Endert E, Wiersinga WM. Prediction of progression to overt hypothyroidism or hyperthyroidism in female relatives of patients with autoimmune thyroid disease using the Thyroid Events Amsterdam (THEA) score. Arch Intern Med 2008;168: [24] Effraimidis G, Strieder TGA, Tijssen JGP, Wiersinga WM. Natural history of the transition from euthyroidism to overt autoimmune hypo- or hyperthyroidism: a prospective study. Eur J Endocrinol 2011;164: [25] Strieder TGA, Prummel MF, Tijssen JGP, Brosschot JF, Wiersinga WM. Stress is not associated with thyroid peroxidase autoantibodies in euthyroid women. Brain Behav Immun 2005;19: [26] Martin-du Pan RC. [Triggering role of emotional stress and childbirth. Unexpected occurrence of Graves disease compared to 96 cases of Hashimoto thyroiditis and 97 cases of thyroid nodules]. Ann Endocrinol (Paris) 1998;59: [27] Oretti RG, Harris B, Lazarus JH, Parkes AB, Crownshaw T. Is There an Association between Life Events, Postnatal Depression and Thyroid Dysfunction in Thyroid Antibody Positive Women? International Journal of Social Psychiatry 2003;49: [28] Winsa B, Adami HO, Bergström R, Gamstedt A, Dahlberg PA, Adamson U, Jansson R, Karlsson A. Stressful life events and Graves disease. Lancet 1991;338: [29] Sonino N, Girelli ME, Boscaro M, Fallo F, Busnardo B, Fava GA. Life events in the pathogenesis of Graves disease. A controlled study. Acta Endocrinol 1993;128: [30] Kung AW. Life events, daily stresses and coping in patients with Graves disease. Clin Endocrinol 1995;42: [31] Chiovato L, Pinchera A. Stressful life events and Graves disease. Eur J Endocrinol 1996;134: [32] Radosavljević VR, Janković SM, Marinković JM. Stressful life events in the pathogenesis of Graves disease. Eur J Endocrinol 1996;134: [33] Yoshiuchi K, Kumano H, Nomura S, Yoshimura H, Ito K, Kanaji Y, Kuboki T, Suematsu H. Psychosocial factors influencing the short-term outcome of antithyroid drug therapy in Graves disease. Psychosom Med 1998;60: [34] Dayan CM. Stressful life events and Graves disease revisited. Clin Endocrinol 2001;55: [35] Matos-Santos A, Nobre EL, Costa JG, Nogueira PJ, Macedo A, Galvão-Teles A, de Castro JJ. Relationship between the number and impact of stressful life events and the onset of Graves disease and toxic nodular goitre. Clin Endocrinol 2001;55: Chapter 6

84

85 Chapter 7 Age-dependent changes in recent life events and daily hassles in a cohort of healthy women. Effraimidis G Brosschot JF Tijssen JGP Wiersinga WM Submitted for publication.

86 ABSTR ACT Introduction. Stress questionnaires are widely used to evaluate the effects of exposure to stress on health and disease, but little is known to which extent the responses are dependent on age. Aim of the study. To describe the effect of age on responses to stress questionnaires in a healthy population. 84 Subjects and methods. 262 healthy adult women participating in a prospective cohort study. Assessment of respondent scores on three questionnaires ( Recent Experienced Stressful Life Events, RLE; daily hassles; and Positive and Negative Affect Schedule, PANAS), completed at baseline and annually during the 5-year follow-up. Results. RLE. Cross-sectional analysis of baseline scores of age groups <30 yr, yr, yr and 50 yr demonstrated a decrease in the number of RLE (p<0.001), a decrease in the number of pleasant events (p<0.001) but not of unpleasant events (P=0.166), and a decrease in the amount of pleasantness (p<0.001) and unpleasantness (p=0.005) with advancing age. Longitudinal analysis of scores during 5-yr follow-up demonstrated a decrease in scores of all of the five RLE items in the group <30 yr, in 3 out of the 5 RLE items in the age group yr, and in none of subjects of 40 yr. Daily hassles. Cross-sectional analysis of baseline scores per age group demonstrated decreases in the number of daily hassles (p=0.024), the intensity per hassle (p=0.052) and the total intensity of daily hassles (p=0.021) with advancing age. Longitudinal analysis of scores during 5-yr follow-up demonstrated that the number and total intensity of daily hassles increased nonsignificantly in subjects 30 yr but significantly in subjects yr, and did not change in the age groups 40 yr. PANAS. Scores did not differ between age groups and did not change during follow-up. Conclusion. In healthy adult women of yr, recent life events and daily hassles decrease with advancing age. Whereas highest scores on stress questionnaires are obtained in the youngest age groups, the effect of age is less pronounced in subjects of 40 yr.

87 Introduction The influence of stress on physical health has been recognized throughout the history of medicine [1]. Negative emotions and psychological distress have been related to depression, post-traumatic stress disorder, coronary heart disease, and immune-related conditions like asthma and Graves hyperthyroidism. Factors which represent a challenge to homoeostasis could be termed stressors, and the complex response that occurs to restore the balance is called the stress response [2]. Experiences of daily life as well as major life events like abuse or trauma may act as stressors [3]. Questionnaires to assess daily hassles and stressful life events in a quantitative manner are well validated, and widely used in studies on health and disease. Relatively little, however, is known on the influence of age on the responses to these questionnaires. Here we report age-dependent changes in stressful life events and daily hassles in a cohort of healthy women who underwent annual assessments during a 5-yr follow-up period. Subjects & Methods Participants For the present study we took advantage of the Amsterdam AITD (autoimmune thyroid disease) cohort, which has previously been described in detail [4,5]. In short, the cohort consisted of women between 18 and 65 years of age in self proclaimed good health without a personal history of thyroid disease, who had at least one 1st or 2nd degree relative with documented autoimmune hyper- or hypothyroidism. Subjects were followed for five years, or shorter when overt hyper- or hypothyroidism had occurred. At each annual visit blood samples were collected to measure thyroid function and thyroid antibodies, and subjects were asked to fill a number of questionnaires on daily hassles, stressful life events and mood. From the original cohort of 803 women we selected for this study those women who had a normal thyroid function (as evident from normal thyrotropin and free thyroxine serum concentrations) and no thyroid antibodies (serum concentrations of antibodies against thyroid peroxidase, thyroglobulin and thyrotropin receptor all below threshold values) at baseline and at each annual visit during the 5-yr follow-up. 262 women met the selection criteria and formed the study population: 113 were <30 yr (mean age±sd 24.0±3.3 yr), 69 were yr (34.4±3.1 yr), 54 were yr (44.1±2.5 yr), and 26 were 50 yr (56.5±4.5 yr). Mean age of all subjects was 34.1±11.1 yr. Questionnaires. The Dutch questionnaire on Recent Experienced Stressful Life Events [6,7] counts the total number of major life events experienced in the last 12 months (checklist of 60 possible events). The respondent separately scores the amount of pleasantness and unpleasantness with each experienced life event, rated on a scale of zero (meaning no (un) pleasantness) to four (a huge amount of (un)pleasantness). From this scale, the total amount of pleasantness and unpleasantness is calculated (maximum score 240 for each). When the amount of pleasantness exceeds the amount of unpleasantness, the event is categorized as being pleasant and vice versa, yielding the total number of (un)pleasant events (maximum 85 Chapter 7

88 60). The Dutch Everyday Problem Checklist is a validated version of the Daily Hassles Scale [6,8-10], and consists of 114 items concerning daily hassles in the last two months. It also measures the intensity of each hassle on a scale from zero to three, yielding the number of hassles experienced and the total intensity of these hassles (maximum 342). Positive and Negative Affect Schedule (PANAS) [11] measures the current mood, in terms of positive and negative affect. It consists of 22 mood states (11 positive, 11 negative) and the respondent is asked to report whether she is affected by each of these states on a scale from 1 (not at all) to 5 (a lot). This yields the tendency to report positive and negative affect states both on a scale from 11 to Statistical analysis Values of scores on questionnaires are given as mean±sd. Age-related differences in questionnaire scores at baseline were evaluated with analysis of variance (ANOVA) for trend. Questionnaire scores during the 5-yr follow-up within each age group were analysed by within-subject ANOVA for trend. Differences in questionnaire scores during follow-up between subjects <30 yr and 30 yr were analysed by mixed between-within subjects ANOVA for trend. A p value of <0.05 was considered to indicate significant differences. Results Age differences in stressors at baseline Table 1 lists the questionnaire scores obtained at baseline, grouped according to age of participants at study entrance. ANOVA for trend demonstrated a decrease in the number of recent life events with advancing age (p<0.001). It was caused by a reduction in the number of pleasant events (p<0.001) rather than a reduction in the number of unpleasant events (p=0.166). The amount of unpleasantness and the amount of pleasantness both decreased significantly with higher age (Figure 1). The number of daily hassles also decreased with advancing age (p=0.024), as did the intensity per hassle (p=0.052) and the total intensity of daily hassles (p=0.021) (Figure 1). Positive and negative affect scores did not differ between the various age groups. Age differences in stressors during 5-yr follow-up Within-subject ANOVA for trend demonstrated in the youngest age group (<30 yr at study entrance) a significant reduction during the 5-yr follow-up in the number of recent life events (p<0.001), the number of unpleasant events (p=0.006) and pleasant events (p=0.001), and the amount of unpleasantness (p=0.005) and pleasantness (p<0.001) (Table 2). In the age group yr, the number of recent life events (p=0.019), the number of pleasant events (p=0.015) and the amount of pleasantness (p=0.001) all decreased significantly; similar trends for the number of unpleasant events and the amount of unpleasantness were visible but not significant. In the age groups yr and 50 yr no significant trends in scores were observed any longer. As the largest changes in scores occurred in

89 Table 1. Mean scores (±SD) of the three questionnaires obtained at baseline as a function of age in a cohort of 262 healthy women. p-value* 50 yr n= yr n= yr n=69 <30 yr n=113 RECENT LIFE EVENTS Number of RLE ± ± ± ± 5.32 <0.001 No of unpleasant events 5.10 ± ± ± ± No of pleasant events 6.59 ± ± ± ± 2.89 <0.001 Amount of unpleasantness ± ± ± ± Amount of pleasantness ± ± ± ± 8.84 <0.001 DAILY HASSLES Number of daily hassles ± ± ± ± Intensity per hassle 1.37 ± ± ± ± Total intensity ± ± ± ± POSITIVE AND NEGATIVE AFFECT SCALE Positive affect ± ± ± ± Negative affect ± ± ± ± * p-value by ANOVA for trend 87 Chapter 7

90 88 Figure 1. Mean scores ± SD of questionnaires on recent life events and daily hassles, obtained at baseline in a cohort of 262 healthy women and presented as a function of age (p values by ANOVA for trend). the youngest age group, we contrasted scores during follow-up between participants <30 yr and 30 yr at study entrance (Figure 2). Mixed between-within subjects ANOVA clearly shows higher scores during the 5-yr follow-up in subjects <30 yr compared to subjects 30 yr. With regard to scores for daily hassles during follow-up, in the age group <30 yr there was a non-significant trend for higher scores of the number and total intensity of daily hassles (p=0.07), but not of the intensity per hassle (p=0.368) (Table 3). In the age group yr the number and total intensity of daily hassles increased during the 5-yr follow-up (p=0.016 and p=0.039 respectively). In the older age groups of yr and 50 yr no significant changes in scores were observed any longer, with

91 Table 2. Mean scores (±SD) of the recently experienced stressful life events (RLE) as a function of age obtained at baseline and at each annual visit during the 5yr follow up in a cohort of 262 healthy women. <30 yr yr yr 50 yr baseline ± ± ± ± yr ± ± ± ± 5.88 Number of RLE 2 yr ± ± ± ± yr ± ± ± ± yr ± ± ± ± 4.65 No of unpleasant events 5 yr ± ± ± ± 5.54 p-value* < baseline 5.10 ± ± ± ± yr 4.78 ± ± ± ± yr 4.18 ± ± ± ± yr 3.82 ± ± ± ± yr 4.34 ± ± ± ± yr 4.17 ± ± ± ± No of pleasant events p-value* baseline 6.59 ± ± ± ± yr 6.67 ± ± ± ± yr 5.92 ± ± ± ± yr 5.47 ± ± ± ± yr 5.60 ± ± ± ± yr 5.47 ± ± ± ± 2.92 Chapter 7 p-value* baseline ± ± ± ± yr ± ± ± ± 9.24 Amount of unpleasantness 2 yr ± ± ± ± yr ± ± ± ± yr ± ± ± ± yr ± ± ± ± 9.77 p-value* baseline ± ± ± ± 8.84 Amount of pleasantness 1 yr ± ± ± ± yr ± ± ± ± yr ± ± ± ± yr ± ± ± ± yr ± ± ± ± 9.56 p-value* < * p-value by within subjects ANOVA for trend

92 90 Figure 2. Mean scores of questionnaires on recent life events (RLE), obtained in a cohort of 262 healthy women who were followed for 5 years (p values indicate differences between subjects <30 yr and 30 yr). the exception of an increase in intensity per hassle (p=0.034) in subjects yr. A comparison of daily hassles scores during follow-up between participants <30 yr and 30 yr at study entrance, demonstrated higher scores in the youngest age group (Figure 3). PANAS scores did not change during the follow-up period in any of the various age groups (data not shown). Discussion Recent life events Cross-sectional analysis of baseline scores on the recent life events (RLE) questionnaire demonstrates lower scores with increasing age: the effect seems large below the age of 40 yr, but small above the age of 40 yr. The longitudinal analysis of follow-up scores confirms this picture. In subjects <30 yr, scores decrease over time in all 5 RLE items. In the age group

93 Table 3. Mean scores (±SD) of the daily hassles as a function of age obtained at baseline and at each annual visit during the 5yr follow up in a cohort of 262 healthy women. <30 yr yr yr 50 yr baseline ± ± ± ± Number of daily hassles 1 yr ± ± ± ± yr ± ± ± ± yr ± ± ± ± yr ± ± ± ± yr ± ± ± ± Intensity per hassle p-value* baseline 1.37 ± ± ± ± yr 1.43 ± ± ± ± yr 1.45 ± ± ± ± yr 1.41 ± ± ± ± yr 1.42 ± ± ± ± yr 1.42 ± ± ± ± 0.39 p-value* baseline ± ± ± ± yr ± ± ± ± Chapter 7 Total intensity 2 yr ± ± ± ± yr ± ± ± ± yr ± ± ± ± yr ± ± ± ± p-value* * p-value by within subjects ANOVA for trend yr a reduction in scores is observed in 3 of the 5 RLE items, and in none of the 5 RLE items in subjects older than 40 yr. Our scores are in reasonable agreement with the few available literature data. The number of recent life events experienced in the last year as reported in cross-sectional studies is 7.21 (44 women, mean age 42.5 yr, Dutch Railways employees) [7], 7.0±3.9 (86 male teachers, mean age 40.5 yr) [6], 11.4±14.2 (98 women, all 27 yr, originally secondary school pupils) [12], and 11.0±5.9 (262 women, mean age 34 yr, present study). One of the previous cross-sectional studies also reports age-related changes. The numbers of RLE were 8.51, 5.54, 5.65 and 4.66 in the age groups <33 yr, yr, yr and >53 yr respectively) [7]. The decline in RLE number with age (p<0.001) was associated with equally significant reductions in total amount of unpleasantness (from in the youngest to in the oldest age group) and pleasantness (from to

94 Figure 3. Mean scores of questionnaires on daily hassles, obtained in a cohort of 262 healthy women who were followed for 5 years ( p values indicate differences between subjects <30 yr and 30 yr) ). Our results are largely the same. There is just one other study with longitudinal data for comparison. That study reports a decrease in the number of RLE over a 2-yr time interval between the age of 27 and 29 yr (in women from 11.4 to 5.6, in men from 10.3 to 6.9) [12]. Our cross-sectional and longitudinal studies fully justifies the conclusion that stressful life events are reported more often by younger age groups in subjects between 18 and 65 yr. Daily hassles As evident from the cross-sectional analysis of our baseline data, daily hassles are decreasing with advancing age: this is true not only for the number of daily hassles but also for the intensity per hassle and the total intensity of hassles. During the 5-yr follow-up, intensity per hassle did not change (except in the yr age group). With respect to the number and total intensity of daily hassles during follow-up, the scores increased just non-significantly in the age group <30 yr, increased significantly in subjects yr, and did not change in subjects 40 yr. The changes during follow-up, if any, are however small. It appears that the youngest age groups below 40 yr have the highest scores of daily hassles associated with relatively large changes during follow-up, and that above the age of 40 yr the changes are smaller. Our results are in partial agreement with literature data. Figures of the mean

95 number of daily hassles experienced in the last two months, as reported in cross-sectional studies, are: 18.9±13.3 (48 women, age 45-64, population based) [8], 15.5±7.3 (36 men and 37 women, mean age 34 yr, employees) [13], 22.6±14.1 (86 male teachers, mean age 40.5 yr) [6] and 18.9±13.2 (98 women, all 27 yr, originally secondary school pupils) [12], in reasonable agreement with our own baseline mean number of 25.1±13.8 (262 women, mean age 34 yr). One of the cross-sectional studies reports on the effect of age: in the age groups 45-49, 50-54, and yr the number of daily hassles were 17.3, 21.1, 20.8, and 23.5 respectively, increasing with age [8]. Another study reports also interaction between age and daily hassles, but in the opposite direction: women of yr had more hassles than older women of yr [14]. Longitudinal studies with monthly assessments for 4, 6, 10 or 24 months did not reveal significant changes in the number of daily hassles in these time periods, except a decrease in the 10-month study (which according to the authors might represent a methodological issue rather than being of substantive interest) [8,12-14]. There is one population-based longitudinal study in 361 premenopausal women between 45 and 55 yr, who were followed for 9 yr with annual assessments [15]. There was a significant decrease in the number of daily hassles and an improvement in mood in the year after children leave home. We conclude that daily hassles overall decrease with advancing age in subjects between 18 and 66 yr. The largest changes occur in the youngest age groups; daily hassles even increased in subjects <40 yr during the 5-yr follow-up period. Due to the small sample size in the age group 50 yr, it cannot be excluded that daily hassles might increase again in the elderly. 93 Chapter 7 Strengths and limitations Our observations on self-reported recently experienced stressful life events and daily hassles are not confounded by the current mood state of participants as scores on the positive and negative affect scales (PANAS) did not differ between age groups and did not change during follow-up. This is relevant because current mood state may influence the perception and thereby the scores of recent life events and daily hassles [8,13,15]. Our PANAS scores at baseline are in good agreement with those reported by Watson et al. [11] in university students and employees: positive affect scale 37.9±5.5 and 35.0±6.4 respectively, negative affect scale 22.3±7.3 and 18.1±5.9. One of the strengths of our study is thus the avoidance of confounding by differences in PANAS scores. Other strengths are the complete data set and the longitudinal nature of the study. Indeed a recent review in this area emphasizes the need for studies with representative cohorts followed over long periods of time [16]. It would help with problems of attrition and recall bias. We think our cohort is likely representative for the adult Dutch female population. Although all participants had in common a family history of autoimmune thyroid disease, they had no evidence of thyroid disease during the whole study period. Participating women came from all regions in The Netherlands. Limitations of our study are the restriction to female gender excluding males, and the underrepresentation of the age group 50 yr. In fact, only seven of the study participants were older than 60 yr at study entrance.

96 94 Concluding remarks Our results clearly indicate age-dependent changes in the responses to stress questionnaires by healthy adult women. Highest scores of RLE and daily hassles were observed in the youngest age groups. Cross-sectional analysis of baseline scores indicated a decrease in recent life events and daily hassles with advancing age. Longitudinal analysis of scores obtained during the 5-yr follow-up period reached a similar conclusion, except a temporary increase in daily hassles in subjects below 40 yr. The question thus arises which factors determine these age-dependent dynamics in responses to stress questionnaires. Although answering this question is outside the scope of the present study, reports in the literature suggest age differences in sources of stress and how people cope with them. Agerelated differences in coping behaviour might be explained by changes in what people must cope with (contextual interpretation), or by changes in the ways people cope as they age (developmental interpretation) [14]. Stressful experiences and learning across lifespan indeed seem to affect coping strategies [12,17-20]. Besides age, other factors like gender, ethnicity and socioeconomic status may affect responses to stress questionnaires [7,8,12,14,16]. Stress questionnaires are often used in predicting subsequent psychological symptoms, in which daily hassles scores perform better than RLE scores [8,13,21]. Recently there has been much interest in exposure to stressful life events as a provocative factor in the development of disease. Stressful life events have been related to the onset of chronic daily headache [22], metabolic syndrome [23], poor metabolic control in type 1 diabetes [24] and asthma [25], but not to multiple sclerosis [26]. The latter paper concludes that repeated and more focussed measures of stress are needed to firmly exclude stress as a potential risk factor for multiple sclerosis [26]. The data in the present paper might be helpful in this respect, providing longitudinal responses to stress questionnaires in a healthy population.

97 References [1] Henley, D., Kaye, J., & Lightman, S. The endocrine response to stress. In: Wass JAH, Stewart PM (eds). Oxford Textbook of Endocrinology and Diabetes, 2nd edition, Oxford University Press 2011, pg [2] Chrousos GP. Stressors, stress, and neuroendocrine integration of the adaptive response. The 1997 Hans Selye Memorial Lecture. Annals of the New York Academy of Sciences 1998;851: [3] McEwen BS. Central effects of stress hormones in health and disease: Understanding the protective and damaging effects of stress and stress mediators. European Journal of Pharmacology 2008;583: [4] Strieder TGA, Prummel MF, Tijssen JGP, Endert E, Wiersinga WM. Risk factors for and prevalence of thyroid disorders in a cross-sectional study among healthy female relatives of patients with autoimmune thyroid disease. Clin Endocrinol 2003;59: [5] Strieder TGA, Tijssen JGP, Wenzel BE, Endert E, Wiersinga WM. Prediction of progression to overt hypothyroidism or hyperthyroidism in female relatives of patients with autoimmune thyroid disease using the Thyroid Events Amsterdam (THEA) score. Arch Intern Med 2008;168: [6] Brosschot JF, Benschop RJ, Godaert GL, Olff M, De Smet M, Heijnen CJ, Ballieux RE. Influence of life stress on immunological reactivity to mild psychological stress. Psychosom Med 1994;56: [7] Van de Willige G, Schreurs P, Tellegen B, Zwart F. Het meten van life events : de Vragenlijst Recent Meegemaakte Gebeurtenissen (VRMG). Nederlands Tijdschrift Voor De Psychologie en Haar Grensgebieden [8] Kanner AD, Coyne JC, Schaefer C, Lazarus RS. Comparison of two modes of stress measurement: Daily hassles and uplifts versus major life events. J Behav Med 1981;4:1 39. [9] Vingerhoets AJ, Ratliff-Crain J, Jabaaij L, Menges LJ, Baum A. Self-reported stressors, symptom complaints and psychobiological functioning I: Cardiovascular stress reactivity. J Psychosom Res 1996;40: [10] Vingerhoets A, Jeninga A, Menges L. Het meten van chronische en alledaagse stressoren: Eerste onderzoekservaringen met de Alledaagse Problemen Lijst (apl) ii. Gedrag en Gezondheid 1989;17: [11] Watson D, Clark LA, Tellegen A. Development and validation of brief measures of positive and negative affect: the PANAS scales. J Pers Soc Psychol 1988;54: [12] Twisk JW, Snel J, Kemper HC, van Mechelen W. Changes in daily hassles and life events and the relationship with coronary heart disease risk factors: a 2-year longitudinal study in year-old males and females. J Psychosom Res 1999;46: [13] Monroe SM. Major and minor life events as predictors of psychological distress: further issues and findings. J Behav Med 1983;6: [14] Folkman S, Lazarus RS, Pimley S, Novacek J. Age differences in stress and coping processes. Psychol Aging 1987;2: [15] Dennerstein EDJG. Empty nest or revolving door? A prospective study of women s quality of life in midlife during the phase of children leaving and re-entering the home 2002:1 6. [16] Hatch SL, Dohrenwend BP. Distribution of traumatic and other stressful life events by race/ethnicity, gender, SES and age: A review of the research. Am J Community Psychol 2007;40: [17] Graham JE, Christian LM, Kiecolt-Glaser JK. Stress, age, and immune function: toward a lifespan approach. J Behav Med 2006;29: [18] Lu L. Daily hassles and mental health: a longitudinal study. Br J Psychol 1991;82 ( Pt 4): [19] Richaud de Minzi MC, Sacchi C. Stressful situations and coping strategies in relation to age. Psychol Rep 2005;97: [20] Shors TJ. Stressful experience and learning across the lifespan. Annu. Rev. Psychol. 2006;57: [21] Chamberlain K, Zika S. The minor events approach to stress: support for the use of daily hassles. Br J Psychol 1990;81 (Pt 4): [22] Scher A, Stewart W, Buse D, Krantz D, Lipton R. Major life changes before and after the onset of chronic daily headache: a population-based study. Cephalalgia 2008;28: [23] Pyykkonen AJ, Raikkonen K, Tuomi T, Eriksson JG, Groop L, Isomaa B. Stressful life events and the metabolic syndrome: the prevalence, prediction and prevention of diabetes (PPP)-Botnia Study. Diabetes Care 2010;33: [24] Helgeson VS, Escobar O, Siminerio L, Becker D. Relation of stressful life events to metabolic control among adolescents with diabetes: 5-year longitudinal study. Health Psychology 2010;29: [25] Lietzen R, Virtanen P, Kivimaki M, Sillanmaki L, Vahtera J, Koskenvuo M. Stressful life events and the onset of asthma. European Respiratory Journal 2011;37: [26] Riise T, Mohr DC, Munger KL, Rich-Edwards JW, Kawachi I, Ascherio A. Stress and the risk of multiple sclerosis. Neurology 2011;76: Chapter 7

98

99 Chapter 8 No causal relationship between Yersinia enterocolitica infection and autoimmune thyroid disease: evidence from a prospective study. Effraimidis G Tijssen JGP Strieder TGA Wiersinga WM Clin Exp Immunol 2011;165:38 43.

100 ABSTR ACT Objective: To evaluate prospectively the relationship between Yersinia Enterocolitica (YE) infection and the development of overt autoimmune hypo- or hyperthyroidism (study A) and the de novo occurrence of thyroid antibodies (study B). 98 Subjects & Methods: Prospective cohort study of 790 euthyroid women who were 1st or 2nd degree relatives of AITD patients. Follow-up was five years, with annual assessments. Study A: Nested case-control study in which YE serological status was measured between cases (subjects who developed overt hypothyroidism (TSH >5.7mU/L and ft4 <9.3 pmol/l) or overt hyperthyroidism (TSH <0.4mU/L and ft4 >20.1 pmol/l) and matched controls. Study B: 388 euthyroid women without thyroid antibodies at baseline were enrolled. The YE serological status was compared between subjects who developed TPO-Ab and/or Tg-Ab at 4-yr follow up and those who remained negative. Results: Study A. The proportion of subjects positive for YOP-IgG or YOP-IgA did not differ between cases and controls at baseline. One year before the development of overt hypo- or hyperthyroidism, the proportion of subjects with YOP-IgG was not different between cases and controls, but YOP-IgA were less prevalent in cases. Study B. De novo occurrence of TPO-Ab (or TPO-Ab and/or Tg-Ab) did not differ between subjects in whom at baseline YOP-IgG were positive or negative. Neither persistence nor emergence of YOP-IgG at 4-yr follow up was associated with the occurrence of TPO-Ab or Tg-Ab. Similar results were observed with respect to YOP-IgA. Conclusions: Yersinia enterocolitica infection does not contribute to an increased risk of thyroid autoimmunity.

101 Introduction Infectious diseases may provoke several autoimmune diseases, as e.g. observed for Coxsackievirus P2-C and type 1 diabetes, Proteus mirabilis and rheumatoid arthritis [1,2]. In the mid 1970s for the first time an association between Yersinia enterocolitica (YE) infection and autoimmune thyroid disease (AITD) was reported [3,4]. Since then, several groups investigated the potential precipitation of AITD development by YE infection. It has been suggested that molecular mimicry of YE membrane antigens to TSH may induce autoimmunity to the TSH receptor [5]. However, reported studies in the literature on the frequency of YE infection in AITD have shown conflicting results [6-15]. They are all cross-sectional in nature, examining patients who had already developed Graves hyperthyroidism or Hashimoto s hypothyroidism and who had been treated for months or years. It is obvious that prospective studies are mandatory to support the hypothesis that YE infection might have an aetiologic role in AITD. To this end, we designed a long-term prospective follow up study in euthyroid women at risk for AITD because they were relatives of AITD patients. The aim of the present study was (a) to evaluate the relationship between YE infection and the development of overt autoimmune hypo- or hyperthyroidism and (b) to evaluate the association between YE infection and the de novo occurrence of thyroid antibodies. Serological evidence of YE infection should precede the occurrence of thyroid antibodies or overt AITD if YE infection is involved in the pathogenesis of AITD. Subjects & Methods 99 Chapter 8 Subjects The present study was carried out among the 803 subjects from the Amsterdam AITD Cohort. The cohort has previously been described in detail [6]. In short, the cohort consisted of women between 18 and 65 years of age in self-proclaimed good health without a history of thyroid disease, who had at least one 1st or 2nd degree relative with documented autoimmune hypo- or hyperthyroidism. Results of thyroid function tests revealed overt hypothyroidism in 10 subjects and overt hyperthyroidism in 3 subjects, leaving 790 subjects to be included in the present study. Subjects were followed for five years, or shorter when overt hypo- or hyperthyroidism had occurred (defined as TSH >5.7 mu/l in combination with ft4 <9.3 pmol/l or TSH <0.4 mu/l in combination with ft4 >20.1 pmol/l, respectively). At each annual visit to our institution blood samples were collected to measure TSH, ft4, T3, TPO-Ab, Tg-Ab and TBII. Antibodies against Yersinia enterocolitica antibodies were measured in subjects who developed overt hypo- or hyperthyroidism, in the last blood sample taken before this event happened; Yersinia antibodies were also measured in serum at baseline and at 4 years follow-up. Plasma and serum samples were stored at 20 C until assay. Within this cohort we performed two studies.

102 Study A. In order to evaluate the relationship between YE infection and the development of overt hypothyroidism or overt hyperthyroidism (called events), we designed a nested case-control study among the 790 subjects euthyroid at study entrance. A subject was considered as a case when she had developed overt hypo- or overt hyperthyroidism during follow up. For each case we selected two controls, matched for age at study entrance and duration of follow up. We compared the serological YE status between cases and controls at baseline and at one year before the occurrence of the event. 100 Study B. In order to evaluate the relationship between YE infection and de novo occurrence of thyroid antibodies, we selected participants from the cohort in the following manner as depicted in Figure 1. First, we excluded the 56 subjects without measurements of YE antibodies at 4 years follow up. Second, from the remaining 734 euthyroid subjects we excluded those who had any serological sign of autoimmune thyroid disease (i.e. serum concentrations of either TPO-Ab 100 ku/l, Tg-Ab 100 ku/l, or TBII 12 U/L) or subclinical hypo- or hyperthyroidism at study entrance. Third, we excluded 98 subjects who had no follow-up. The remaining 388 subjects were thus included in this analysis. The YE serological status was compared between subjects who developed TPO-Ab and/or Tg-Ab at 4 years follow up and those who remained negative for thyroid antibodies. Laboratory measurements Serum TSH and ft4 were measured using time-resolved fluoroimmunoassay (Delphia, Turku, Finland). Reference values are for TSH mu/l and for ft pmol/l. Thyroid peroxidase (TPO) antibodies and thyroglobulin (Tg) antibodies were measured by Figure 1. Flowchart of the recruitment of subjects on the Amsterdam autoimmune thyroid disease (AITD) cohort for study B.

103 chemiluminescence immunoassays (LUMI-test anti-tpo and LUMI-test anti-tg respectively, Brahms, Berlin, Germany). Improved versions of both assays became available during follow-up: detection limits of these new assays were for TPO-Ab 30 ku/l and for Tg-Ab 20 ku/l. TPO-Ab concentrations obtained with the old assay were multiplied by a factor 0.72 to obtain comparative values in the new assay. TPO-Ab and Tg-Ab concentrations were considered to be positive at values >100 ku/l. TSH receptor antibodies were determined as TSH binding inhibitory immunoglobulins (TBII) using the TRAK assay (Brahms, Berlin, Germany); detection limits in the 1st and 2nd generation TRAK assays were 5 and 1 IU/L respectively, and values above 12 and 1.5 U/L respectively were considered as positive. Specific IgG and IgA antibodies against purified plasmid-encoded virulence associated YOPs of YE serotype O9 (LCR) in sera were demonstrated by immunoblotting with a YOP- Ab assay (AID, Strassberg, Germany). In short, antigens (25,34,36,37,39,40,46,48 kda) are blotted onto nitrocellulose. Sera are diluted 1:51 in PBS-Tween and incubated with the antigen-coated nitrocellulose strips overnight at 22 C. The IgG and IgA antibody-antigen complexes formed are quantified after immunostaining with the AID-Scan-System. Controls are included in each assay run, using human acute sera (culture-positive YE infection) containing antibodies to the YOPs. Test sera are judged positive if at least three bands (IgG) or two bands (IgA) are seen in immunoblotting at a level greater than 10% (IgG) or 5% (IgA) of reference standards. The inter-assay variation of the YOP-Ab assay is <3% according to the manufacturer. The YOP-Ab assay was performed without prior knowledge of thyroid function tests or the presence of TPO-Ab in the serum samples. Statistical analysis Differences between cases and controls were evaluated by Students t test for age, by Mann- Whitney U-test for TSH, ft4, TPO-Ab and Tg-Ab, and by χ2 test or if appropriate Fisher s exact test for the other parameters. Values are given as mean±sd for age and TSH, but as median and interquartile range for all other parameters. A p-value of <0.05 was considered to indicate significant differences between groups. 101 Chapter 8 Results YE infection and development of overt hypo- or hyperthyroidism (Study A). During the 5 years follow up period 38 cases of overt autoimmune hypothyroidism and 13 cases of overt autoimmune hyperthyroidism occurred after a mean follow-up of 2 years, as reported previously [6]. The proportion of subjects positive for YOP-IgG was not different between cases and controls, neither at baseline nor at one year before the event (Table 1). The proportion of subjects with Yersinia YOP-IgA antibodies was not different between cases and controls at study entrance. However, the frequency of YOP-IgA antibodies at one year before the event was lower in cases than in controls (p=0.02). No significant differences in YOP IgG and IgA frequencies were observed when hypothyroid and hyperthyroid cases with their respective controls were analyzed separately.

104 102 Table 1. Comparison of characteristics and YE serological status between patients who developed overt hypo- ot hyper-thyroidism and their corresponding controls matched for age and follow up, in a nested case-control study of 153 women with 1st or 2nd degree relatives with proven AITD. At study entrance One year before the event Cases Controls OR (95% CI) P-value Cases Controls OR (95% CI) P-value N = - Age in years ± ± TSH mu/l 2.9 ( ) 1.6 ( ) < ( ) 1.6 ( ) < ft4 pmol/l 11.1( ) 13.0( ) < ( ) 13.1 ( ) < TPO Antibody positive 78 % 20 % < % 25 % < Tg Antibody positive 23 % 10 % % 19 % YOP IgG Antibody positive 43% 47 % 0.85(0.43,1.68) % 33 % 0.91(0.44,1.88) YOP IgA Antibody positive 23% 31% 0.67(0.31,1.45) % 30 % 0.36(0.15,0.90) 0.02 Data are given as means ± SD or medians with interquartile range or proportions, OR: odds ratio, CI: confidence intervals, P-value of cases versus controls. Time of event: visit when diagnosis was made or confirmed. TSH: thyroid stimulating hormone, ft4: free T4, TPO: thyroid peroxidase, Tg: thyroglobuline, YOP: Yersinia enterocolitica outer membrane proteins.

105 YE infection and development of TPO-Ab and/or Tg-Ab (Study B). At study entrance the mean age of the 388 participants was 34.3±11.2 years and the mean serum TSH concentration was 1.67±0.73 mu/l. At 4 years follow up, 36 subjects (9.3%) had developed only TPO-Ab while 51 subjects (13.1%) had developed TPO-Ab and/or Tg-Ab. At the time of seroconversion the TPO-Ab concentration had a median value of 160 ku/l (range ku/l) and the Tg-Ab concentration had a median value of 130 ku/l (range ). The rate of TPO-Ab conversion was not different between subjects with positive or negative YOP IgG status at baseline (6.3% vs 10.7% p=0.16) (Table 2) as was the rate of TPO-Ab and/or Tg-Ab conversion (8.7% vs 15.3% p=0.06). The difference in TPO-Ab conversion rate between YOP IgG positive and YOP IgG negative subjects at baseline was -4.4% (95 CI: -10.6%, 1.7%); the difference for TPO-Ab and/or Tg-Ab conversion rate was -6.7% (95 CI: -13.8%, 0.5%). In subjects positive for YOP IgG at study entrance, the rate of TPO-Ab conversion (and of TPO-Ab and/or Tg-Ab conversion) was not different between participants in whom YOP IgG persisted and in whom YOP IgG disappeared. Likewise, in the group who tested negative for YOP IgG at baseline, no difference in the rate of TPO-Ab conversion (and of TPO-Ab and/or Tg-Ab conversion) was observed in subjects who remained YOP IgG negative and those who became YOP IgG positive. Similar results were observed for YOP IgA status (Table 3). De novo occurrence of TPO-Ab (or TPO-Ab and/or Tg-Ab) was not related to the YOP IgA status at baseline. Neither persistence nor emergence of YOP IgA at 4 years follow up was associated with the TPO-Ab or Tg-Ab seroconversion. 103 Chapter 8 Discussion In the present prospective study we evaluated any association between seroreactivity against YE and both early stages (when thyroid antibodies emerge but thyroid function is still normal) and late stages (when overt thyroid dysfunction develops) of the natural course of AITD. In our nested case-control study, the prevalence of YOP-IgG was similar in cases and controls, both at baseline and at one year before the development of overt hypo- or hyperthyroidism. Whereas the prevalence of YOP-IgA was also not different between cases and controls at baseline, YOP-IgA was less prevalent at follow-up just before the development of overt thyroid dysfunction. If YE would play a causative role in this development, one would expect a higher no lower frequency of YOP-IgA. Our results thus indicate that YE infection is not associated with the development of overt autoimmune hypo- or hyperthyroidism. Studies in the past have shown conflicting results, some reporting a higher rate of seropositivity against YE in patients with Hashimoto s or Graves disease than in controls [7-10] whereas other didn t find any relationship [11,12]. Brix et al. reported in a classical case-control study that the prevalence of YOP IgG-Ab and YOP IgA-Ab in Graves disease was higher than in controls (51% vs 35% and 49 vs 34%, respectively) [13]. Similar results were found in twin pairs discordant for Graves disease. In contrast, we didn t observe differences in YOP IgG

106 Table 2. Yersinia enterocolitica YOP IgG status at baseline and at 4-yr follow up in relation to de novo development of thyroid antibodies. YOP IgG status TPO-Ab converters TPO-Ab and/or Tg-Ab converters 36/388=9.3% 51/388=13.1% N % p-value Δ (95% CI) N % p-value Δ (95% CI) } baseline positive 8/ % 11/ % -4.4% 0.16 (-10.6%,1.7%) baseline negative 28/ % 40/ % } % (-13.8%,0.5%) baseline pos 4yr pos 6/91 6.6% 7/91 7.7% 0.28 baseline pos 4yr neg 2/36 5.6% } 4/ % baseline neg 4yr pos 2/ % 3/ % 0.97 baseline neg 4yr neg 26/ % } 37/ % } 0.57 } 0.95 TPO: thyroid peroxidase, Tg: thyroglobulin, Ab: antibodies, YOP: Yersinia enterocolitica outer membrane proteins. Δ: difference in conversion rate, CI: confidence interval 104 Table 3. Yersinia enterocolitica YOP IgA status at baseline and at 4-yr follow up in relation to de novo development of thyroid antibodies. YOP IgA status TPO-Ab converters TPO-Ab and/or Tg-Ab converters 36/388=9.3% 51/388=13.1% N % p-value Δ (95% CI) N % p-value Δ (95% CI) } baseline positive 3/64 4.7% 4/64 6.2% -5.5% 0.34 (-13.3%,2.3%) baseline negative 33/ % 47/ % } % (-17.3%,0.8%) baseline pos 4yr pos 1/35 2.9% 2/35 5.7% 0.44 baseline pos 4yr neg 2/29 6.9% } 2/29 6.9% baseline neg 4yr pos 5/ % 6/ % 0.57 baseline neg 4yr neg 28/ % } 41/ % } 0.85 } 0.52 TPO: thyroid peroxidase, Tg: thyroglobulin, Ab: antibodies, YOP: Yersinia enterocolitica outer membrane proteins. Δ: difference in conversion rate, CI: confidence interval and IgA frequency when we limited our analysis to the 11 Graves hyperthyroid cases. One of the earliest detectable events in the natural history of AITD is the occurrence of thyroid antibodies in serum. The hypothesis that Yersinia infection is an aetiological factor for AITD presupposes that YE infection precedes the occurrence of thyroid antibodies. In our study, neither chronic Yersinia infection (as reflected by YOP IgG status) nor the more recent stages of Yersinia infection (as reflected by the presence of YOP IgA) had any association with thyroid antibody status. Our study is the first to evaluate the relationship between YE infection and thyroid antibodies in a prospective manner. All studies in the past were cross-sectional studies and concerned patients who had been already treated for Hashimotos s thyroiditis or Graves disease. Three recent cross-sectional studies on this topic reached essentially similar conclusions. Strieder et al reported that the presence of antibodies against YE was unrelated to the presence of TPO-Ab in euthyroid female subjects who were relatives of patients with AITD [14]. A Danish twin study also failed to find an association between thyroid antibodies and YOP antibodies in a case-control study in which controls were recruited either from an externaltwin population or from the co-twins [15]. A recent study from China [16] reported that thyroid microsomal antibodies and Tg-Ab were not correlated with the antibodies to YE.

107 A higher prevalence of YOP IgG and IgA in female relatives of patients with AITD than in controls derived from the general population has been reported previously [14]. The higher rate of persistent YE infection in AITD relatives might be due to susceptibility genes for AITD contributing to the risk for YE infection. The Danish twin study indicated that the genetic contribution in the association with YE is modest, and that it is more likely that environmental exposures to confer to the reported association between and YE and AITD [13]. Our study has several limitations. Sample size was dependent on the size of the Amsterdam AITD cohort: all cohort participants eligible for the present studies were included. Nevertheless, the confidence intervals of the odds ratios (study A) and of the differences in the thyroid antibodies conversion rates (study B) clearly do not support the hypothesis that YE infection is causally related to AITD. We measured YOP-Abs not at the time of the occurrence of overt hyper- or hypothyroidism, but in the year prior to the event. One could argue that we have missed acute YE infection in these cases. This limitation does not apply to study B, in which YE serology was assessed precisely when for the first time thyroid antibodies were detected in serum. Another limitation is that outcome of YE infection may differ in a population with different genetic background. Indeed, some rat strains infected with YE are able to clear the infection and YOP-Ab disappear, whereas other rat strain develop chronic YE infection with persistent YOP-Ab [17]. These limitations are in our view well balanced by the strengths of our study. Its prospective nature provides more solid evidence then obtained for cross-sectional studies. Also the nested case-control study design allowed for equal exposure times to environmental insults in cases and controls. In conclusion, the present prospective study demonstrates that YE infection is not a risk factor neither for the occurrence of thyroid antibodies nor for the development of overt autoimmune hypo- or hyperthyroidism. Thus, Yersinia infection is not a causal factor contributing to the pathogenesis of autoimmune thyroid disease. 105 Chapter 8 References [1] Ebringer A, Wilson C. HLA molecules, bacteria and autoimmunity. J. Med. Microbiol. 2000;49: [2] Pordeus V, Szyper-Kravitz M, Levy RA, Vaz NM, Shoenfeld Y. Infections and Autoimmunity: A Panorama. Clinic Rev Allerg Immunol 2008;34: [3] Bech K, Larsen JH, Hansen JM, Nerup J. Letter: Yersinia enterocolitica infection and thyroid disorders. Lancet 1974;2: [4] Shenkman L, Bottone EJ. Antibodies to Yersinia enterocolitica in thyroid disease. Ann Intern Med 1976;85: [5] Prummel MF, Strieder T, Wiersinga WM. The environment and autoimmune thyroid diseases. Eur J Endocrinol 2004;150: [6] Strieder TGA, Tijssen JGP, Wenzel BE, Endert E, Wiersinga WM. Prediction of progression to overt hypothyroidism or hyperthyroidism in female relatives of patients with autoimmune thyroid disease using the Thyroid Events Amsterdam (THEA) score. Arch Intern Med 2008;168: [7] Wenzel BE, Heesemann J, Wenzel KW, Scriba PC. Antibodies to plasmid-encoded proteins of enteropathogenic Yersinia in patients with autoimmune thyroid disease. Lancet 1988;1:56. [8] Chatzipanagiotou S, Legakis JN, Boufidou F, Petroyianni V, Nicolaou C. Prevalence of Yersinia plasmid-encoded outer protein (Yop) class-specific antibodies in patients with Hashimoto s thyroiditis. Clin Microbiol Infect 2001;7: [9] Asari S, Amino N, Horikawa M, Miyai K. Incidences of antibodies to Yersinia enterocolitica: high incidence of serotype O5 in autoimmune thyroid diseases in Japan. Endocrinol. Jpn. 1989;36: [10] Corapçioğlu D, Tonyukuk V, Kiyan M, Yilmaz AE, Emral R, Kamel N, Erdoğan G. Relationship between thyroid autoimmunity and Yersinia enterocolitica antibodies. Thyroid 2002;12: [11] Arscott P, Rosen ED, Koenig RJ, Kaplan MM, Ellis T, Thompson N, Baker JR. Immunoreactivity to Yersinia enterocolitica antigens in patients with autoimmune thyroid disease. J Clin Endocrinol Metab 1992;75:

108 106 [12] Resetkova E, Notenboom R, Arreaza G, Mukuta T, Yoshikawa N, Volpé R. Seroreactivity to bacterial antigens is not a unique phenomenon in patients with autoimmune thyroid diseases in Canada. Thyroid 1994;4: [13] Brix TH, Hansen PS, Hegedüs L, Wenzel BE. Too early to dismiss Yersinia enterocolitica infection in the aetiology of Graves disease: evidence from a twin case-control study. Clin Endocrinol 2008;69: [14] Strieder TGA, Wenzel BE, Prummel MF, Tijssen JGP, Wiersinga WM. Increased prevalence of antibodies to enteropathogenic Yersinia enterocolitica virulence proteins in relatives of patients with autoimmune thyroid disease. Clin Exp Immunol 2003;132: [15] Hansen PS, Wenzel BE, Brix TH, Hegedüs L. Yersinia enterocolitica infection does not confer an increased risk of thyroid antibodies: evidence from a Danish twin study. Clin Exp Immunol 2006;146: [16] Wang Z, Zhang Q, Lu J, Jiang F, Zhang H, Gao L, Zhao J. Identification of outer membrane porin f protein of Yersinia enterocolitica recognized by antithyrotopin receptor antibodies in Graves disease and determination of its epitope using mass spectrometry and bioinformatics tools. J Clin Endocrinol Metab 2010;95: [17] Curfs JH, Meis JF, Van der Lee HA, Mulder JA, Kraak WA, Hoogkamp-Korstanje JA. Persistent Yersinia enterocolitica infection in three rat strains. Microb. Pathog. 1995;19:57 63.

109 107 Chapter 8

110

111 Chapter 9 General discussion