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

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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: 17 Dec 2017

2 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:

3 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.

4 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.

5 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.

6 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

7 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).

8 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

9 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 ( )

10 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

11 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.

12 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