Hypothyroidism and Pregnancy

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1081 Hypothyroidism and Pregnancy MICHAELA GRANFORS ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2015 ISSN 1651-6206 ISBN 978-91-554-9201-4 urn:nbn:se:uu:diva-247090

Dissertation presented at Uppsala University to be publicly examined in Rosénsalen, Akademiska sjukhuset, ingång 95/96. Barn- och kvinnosjukhuset. NBV (nedre bottenvåning)., Uppsala, Friday, 8 May 2015 at 09:00 for the degree of Doctor of Philosophy. The examination will be conducted in Swedish. Faculty examiner: Professor Ulla Feldt- Rasmussen (Department of Medical Endocrinology PE 2132, Rigshospitalet, Copenhagen University Hospital, Denmark). Abstract Granfors, M. 2015. Hypothyroidism and Pregnancy. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1081. 69 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9201-4. Hypothyroidism is a common endocrine disorder affecting women of reproductive age. On a global level, iodine deficiency is still the most common cause of hypothyroidism. Also genetic variations, in particular SNP rs4704397 in the PDE8B gene, are responsible for a significant proportion of TSH variations. Untreated hypothyroidism has significant adverse effects on pregnancy and fetal outcome. Most international guidelines suggest targeted thyroid testing in pregnant women with risk factors for thyroid disturbances. In a case-control study, an association between homozygous A/A as well as homozygous G/ G carriers of SNP rs 4704397 in PDE8B and recurrent miscarriage was found. The explanation for this association is unknown. In a nationwide survey, all guidelines for thyroid testing and management of hypothyroidism during pregnancy in Sweden were collected and compared with international guidelines. The local guidelines were variable and poorly compliant with the international guidelines. In a follow-up in one district, 5,254 pregnant women were included for subsequent review of their medical reports. We found a targeted thyroid testing rate of 20.1% in clinical practice, with an overall frequency of women with trimester-specific elevated TSH of 18.5%. More disturbingly, half of the women who were on levothyroxine treatment at the time of conception had an elevated TSH level at thyroid testing. In a subsequent cohort study of the 5,254 women, we found the prevalence of trimesterspecific elevated TSH and overt hypothyroidism to be equal in targeted thyroid tested and untested women. In a cross-sectional study, a median urinary iodine concentration (UIC) of 98 μg/l was found in the study population. According to WHO/UNICEF/IGN criteria, the population-based median UIC during pregnancy should be 150-249 μg/l. In conclusion, genetic variations may contribute to adverse pregnancy outcomes. In clinical practice, thyroid testing and the management of hypothyroidism during pregnancy is unsatisfactory, regarding the whole chain from development of local guidelines to their implementation and to targeted thyroid testing. Moreover, our results indicate insufficient iodine status in the pregnant population of Sweden. Keywords: phosphodiesterase 8B, recurrent miscarriage, single nucleotide polymorphism, thyroid, guidelines, hypothyroidism, pregnancy, survey, thyroid testing, screening, iodine, iodine deficiency, median urinary iodine concentration Michaela Granfors, Department of Women's and Children's Health, Akademiska sjukhuset, Uppsala University, SE-75185 Uppsala, Sweden. Michaela Granfors 2015 ISSN 1651-6206 ISBN 978-91-554-9201-4 urn:nbn:se:uu:diva-247090 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-247090)

The more you know, the better you realize how little you actually know. After Aristotle, 384-322 BC To all women who suffer from hypothyroidism during pregnancy

List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I II III IV Granfors M., Karypidis H., Hosseini F., Skjöldebrand-Sparre L., Stavreus-Evers A., Bremme K., Landgren BM., Sundström- Poromaa I., Wikström AK., Åkerud H. (2012) Phosphodiesterase 8B gene polymorphism in women with recurrent miscarriage: A retrospective case control study. BMC Med Genet, 13:121. Granfors M., Åkerud H., Berglund A., Skogö J., Sundström- Poromaa I., Wikström AK. (2013) Thyroid Testing and Management of Hypothyroidism During Pregnancy: A Populationbased Study. J Clin Endocrinol Metab, 98(7):2687-92. Granfors M., Åkerud H., Skogö J., Stridsberg M., Wikström AK., Sundström-Poromaa I. (2014) Targeted Thyroid Testing During Pregnancy in Clinical Practice. Obstet Gynecol, 124(1):10-5. Granfors M., Andersson M., Stinca S., Åkerud H., Skalkidou A., Sundström Poromaa I., Wikström AK., Filipsson Nyström H. (2015) Iodine deficiency in a study population of pregnant women in Sweden. Submitted. Reprints were made with permission from the respective publishers. Cover illustration: Schwangerschaft by Rino Axt, Berlin Reprinted with kind permission from the artist

Contents Preface... 10 Introduction... 11 Historical perspective... 11 Definitions and causes of hypothyroidism... 12 The thyroid gland during fetal life and pregnancy... 12 The thyroid gland during fetal life... 13 The thyroid gland during pregnancy... 13 Prevalence of hypothyroidism during pregnancy... 13 Genetic influences on thyroid hormones... 15 Consequences of hypothyroidism during pregnancy... 15 Recurrent miscarriage... 15 Adverse pregnancy outcomes... 16 Neurocognitive development of the child... 17 Management of hypothyroidism during pregnancy... 19 Guidelines... 19 Treatment of hypothyroidism during pregnancy... 19 Universal screening or targeted thyroid testing?... 20 Iodine... 21 Dietary sources and requirements... 22 Assessment of iodine status... 23 Consequences of iodine deficiency during pregnancy... 24 Global and national iodine status... 25 Aims... 26 Materials and Methods... 27 Overview of the studies... 27 Study populations... 28 Uppsala Biobank of Pregnant Women... 28 Study I... 28 Studies II-III... 29 Study IV... 29 Study procedures... 30 Study I... 30 Study II... 30 Study III... 31 Study IV... 32

Statistics... 33 Ethics... 33 Results... 34 Study I... 34 Study II... 35 Study III... 36 Study IV... 38 Discussion... 40 Methodological considerations... 40 Study designs and representativeness... 40 Significance of the results in a general context... 43 Study I... 43 Studies II and III... 45 Study IV... 48 How to deal with the current situation... 50 Conclusions... 51 Future perspectives... 52 Sammanfattning på svenska... 53 Acknowledgements... 56 References... 59

Abbreviations A AOR BMI CI DNA G hcg IGN IQ LT4 OR PDE8B RCT SNP T3 T4 TBG Tg TPO TPOAb TRH TSH UIC WHO Adenine Adjusted Odds Ratio Body Mass Index Confidence Interval Deoxyribonucleic Acid Guanine Human Chorionic Gonadotropin The Iodine Global Network Intelligence Quotient Levothyroxine Odds Ratio Phosphodiesterase Type 8B Randomized Controlled Trial Single Nucleotide Polymorphism Triiodothyronine Thyroxine Thyroxine-Binding Globulin Thyroglobulin Thyroid Peroxidase Thyroid Peroxidase Antibody Thyrotropin Releasing Hormone Thyroid Stimulating Hormone Urinary Iodine Concentration World Health Organization

Preface After having worked with obstetrics for several years, I was astonished by the various attitudes different doctors had to hypothyroidism during pregnancy. While some advocated testing and treating a large number of women, others seemed to feel that the issue was not important. Most colleagues, however, seemed, as I was, to be confused. Even more confusing, iodine status was never mentioned or discussed. The idea of hopefully being able to contribute knowledge to the subject was an inspiration for me. 10

Introduction Historical perspective In a historical perspective, iodine deficiency has been both an extensive and the most important reason for hypothyroidism worldwide, often visible as a goiter. Until barely a hundred years ago, hypothyroidism and goiter due to iodine deficiency was common in, for instance, parts of central Europe around the Alps and also in parts of Scandinavia. The primary scenario and consequences of severe hypothyroidism was often described as cretinism, which includes impairment of mental and physical growth, development, and has a negative impact on most organ systems. Infertility was common and, in case of pregnancy, the child was often similarly affected. As endemic cretinism was widespread in certain areas, it was often attributed to stagnant air in the valleys or bad water. Marine and Kimball showed in 1917 that, in most cases, goiter was caused by iodine deficiency and could be prevented by iodine supplementation 1. In 1928-9 medical doctor and scientist Axel Höjer conducted an extensive nationwide survey to map the goiter rate in Sweden 2. His group systematically examined 29,000 men and women from all over Sweden, and reported several cases of cretinism and a goiter prevalence of more than 15% in men and more than 33% in women in several parts of the country (Figure 1). Figure 1. Goiter prevalence in Sweden 1929 11

Switzerland and the United States were the first countries to introduce goiter prophylaxis through salt iodization. In Sweden, iodine prophylaxis with iodized salt was established in 1936 3. Definitions and causes of hypothyroidism Definitions used in this thesis: Overt hypothyroidism: a TSH level above the defined upper limit of the reference range in combination with a serum free T4 below the lower limit of the reference range Subclinical hypothyroidism: elevated TSH levels, combined with normal free T4 levels Hypothyroxinemia: normal TSH levels in conjunction with free T4 levels below the lower limit of the reference range Thyroid autoimmunity: presence of thyroid autoantibodies Iodine deficiency is still the most common cause of hypothyroidism worldwide 4, but in areas of iodine sufficiency, chronic autoimmune thyroiditis (Hashimotos thyroiditis) is the main cause of hypothyroidism 5. Other causes of hypothyroidism include prior thyroidectomy or ablative radioiodine therapy, whereas secondary (pituitary) and tertiary (hypothalamic) causes of hypothyroidism are rare. The thyroid gland during fetal life and pregnancy An overview of thyroid homeostasis is shown in Figure 2. Figure 2. The hypothalamic pituitary thyroid axis with positive (+) and negative (-) feedback control. During pregnancy, also hcg stimulates the thyroid gland due to similarities in TSH and hcg glycoprotein. 12

The thyroid gland during fetal life During fetal life, the thyroid gland completes its formation at the end of the first trimester of pregnancy. Before that, maternal free T4 and perhaps T3 in very low concentrations are the only sources of thyroid hormones in the developing fetus 6. From 16-20 weeks of gestation, secretion of thyroid hormones in the fetus reaches significant levels, but the regulatory feedback system including the pituitary and hypothalamus is not fully developed until birth. Besides the thyroid hormones, iodine passes the placenta and accumulates in the fetal thyroid from 12 weeks of gestation, where it is used for thyroid hormone production 7. Thyroid hormones are essential for neural development and myelination of nerve cells in all the stages of fetal life 6. The thyroid gland during pregnancy Pregnancy is a kind of stress test for the thyroid gland. If the thyroid gland is unable to adapt to these physiological changes during pregnancy, hypothyroidism may occur 8. Production of T4 and T3 increases by 50%, along with a 50% increase in the daily iodine requirement from early pregnancy. During normal pregnancy, there are several maternal physiological changes to adapt to the demands of the mother and the fetus. TBG levels increase, which leads to lower levels of free T4, resulting in a feedback stimulation of the thyroid gland. Moreover, fast increasing hcg-levels in maternal plasma in early pregnancy transiently stimulate the thyroid gland due to similarities in TSH and hcg glycoprotein 8. Ten to fifteen percent of pregnant women are estimated to be positive for thyroid autoantibodies, which are indicators of underlying chronic autoimmune thyroiditis such as Hashimotos thyroiditis. TPOAb are the most sensitive and specific autoantibodies for thyroid autoimmunity 9. Even in euthyroid women, thyroid autoimmunity is a risk factor for incurring hypothyroidism later in pregnancy or postpartum thyroiditis 10. Prevalence of hypothyroidism during pregnancy Hypothyroidism is one of the most common endocrine disorders in women of reproductive age 11. The prevalence of overt hypothyroidism during pregnancy has commonly been estimated to be 0.3-0.5% and, in addition, 2-3% of pregnant women may suffer from subclinical hypothyroidism 12-14. Of course, the prevalence of overt or subclinical hypothyroidism depends on the upper TSH cut-off levels used. There is strong evidence that the reference range for TSH is lower throughout pregnancy compared with the nonpregnant state 15-17. The lowest TSH levels are observed during the first tri- 13

mester of pregnancy, and are apparently related to hcg stimulation of the thyroid gland as hcg levels are highest early in gestation 15-17. According to several guidelines, TSH reference intervals should be established from the 95% confidence limits of the log-transformed values of at least 120 apparently healthy individuals without any personal or family history of thyroid disease, goiter, thyroid autoantibodies or medications 18 19. Due to thyroid hormone changes during pregnancy, separate, trimesterspecific TSH reference ranges, as defined in populations with optimal iodine intake, should be established. However, in the absence of local normative data, the following upper cut-off levels for TSH are commonly recommended: 2.5 miu/l in the first and 3.0 miu/l in the second and third trimester of pregnancy 10 20 21. As few laboratories have established trimester-specific reference ranges, the use of these latter TSH reference ranges is widespread. When applying these reference ranges, several studies report a prevalence of hypothyroidism of 12-15% during pregnancy 22-24. Reference intervals for free T4 are important to be able to differentiate overt from subclinical hypothyroidism, and universally accepted free T4 reference ranges would be desirable. In the non-pregnant state, 0.03% of total T4 is unbound to serum proteins and is available for tissue uptake. The remaining 99.97% of total T4 is bound to transport proteins, mainly to TBG (70%) and to a lesser extent to albumin and transthyretin (around 15% each). Pregnancy implies an abnormal binding-protein state with higher levels of TBG and lower levels of albumin. Free T4 levels are usually measured by immunoassays that are known to be sensitive to alterations in binding proteins 25. Due to the abnormal bindingprotein state during pregnancy, these commonly used immunoassays do not accurately reflect free T4 levels during pregnancy 26. Methods of equilibrium dialysis and tandem mass spectrometry have been considered to be the gold standard for the measurement of T4 during pregnancy 27. However, the method is expensive and demanding, and its use is not widespread 10 28. Alternative strategies, for example, the use of free T4 indexes have been suggested 26 and criticized by others 28. In case of using immunoassays, the establishment of method- and trimester-specific reference ranges for free T4 are usually recommended 10. Moreover, it is pointed out that serum TSH is a more accurate indicator of thyroid function during pregnancy than any free T4 measurement apart from equilibrium dialysis and tandem mass spectrometry 10. The Committee for Standardization of Thyroid Function Tests (C-STFT) keeps on working on the development of a reference measurements system for standardization, investigating the status of between-assay comparability, and assessing the key performance attributes of the individual assays for thyroid hormones 27. 14

Genetic influences on thyroid hormones More than 99.9% of human DNA sequences are the same, even in unrelated individuals. Single nucleotide polymorphisms, SNPs, make up about 90% of all human genetic variation. SNPs are DNA sequence variations that occur when a single nucleotide in the genome sequence is altered. Many SNPs have no known effect on cell function, while others are believed to predispose people to disease or influence their response to a drug. Heritability studies have suggested that up to two thirds of circulating thyroid hormone and TSH levels are genetically determined 29 30. However, until now, polymorphisms in only a few genetic loci have been identified to be associated with circulating TSH levels 30 31. One of those genetic loci is the human phosphodiesterase type 8B (PDE8B), which is predominantly expressed in the thyroid gland. PDE8B is located on chromosome 5 and encodes a high affinity camp specific nucleotide phosphodiesterase 32-34. The SNP that to date has been shown to have the strongest association to TSH levels is SNP rs 4704397 in PDE8B, being responsible for approximately 2.3% of TSH variation 30 35. While this association was initially detected in a genome-wide association study 35, it has been confirmed in several subsequent studies 36 37. In addition, the association between SNP rs 4704397 in PDE8B and levels of TSH has been confirmed in pregnant women 38. In SNP rs 4704397 of PDE8B an adenine (A) nucleotide is replaced by a guanine (G). The association between the polymorphism and higher levels of TSH and lower free T4 levels, indicating relative hypothyroidism, is found in homozygous carriers of A/A 37. The SNP rs 4704397 is present in intron 1 of the PDE8B gene, and is thus not directly involved in a coding region 37. Based on previous results it has been proposed that the SNP rs 4704397 in PDE8B and in particular the presence of A alleles might induce increased phosphodiesterase activity in PDE8B, thereby reducing the ability of the thyroid gland to generate free T4 when stimulated by TSH 35. The possible impact of SNP rs4704397 in PDE8B on clinical outcomes has barely been studied. As A/A has been associated with higher TSH levels, we hypothesized that homozygous carriers of A/A might be at increased risk of recurrent miscarriage. Consequences of hypothyroidism during pregnancy Recurrent miscarriage Recurrent miscarriage is commonly defined as three or more consecutive pregnancy losses before 20 weeks of amenorrhoea and affects approximately 1% of all couples trying to conceive 39 40. Predisposing factors include maternal age, parental chromosomal aberrations, uterine abnormalities, antiphos- 15

pholipid syndrome, immunological and thrombophilic disorders, and endocrine diseases such as hypothyroidism and diabetes mellitus 39. Unlike sporadic spontaneous miscarriage, recurrent miscarriage more often occurs despite normal fetal cytogenetic findings, and in 50% of cases the underlying cause remains unexplained 39 41-43. There is a known relation between hypothyroidism and recurrent miscarriage 39. Adverse pregnancy outcomes In relation to overt hypothyroidism Overt hypothyroidism during pregnancy has been clearly associated with adverse pregnancy outcomes such as preeclampsia, gestational hypertension, fetal death, premature delivery, spontaneous abortions, recurrent abortions and cretinism 5 11 13 44 45. Numerous observational studies confirm the benefit of treating overt hypothyroidism during pregnancy 10. A recent review summarized that levothyroxine is effective in reducing the risk of miscarriage and preterm delivery in women with overt hypothyroidism during pregnancy 46. Treatment is generally recommended. In relation to subclinical hypothyroidism Numerous studies have investigated possible associations between subclinical hypothyroidism and adverse pregnancy outcomes such as preeclampsia, gestational hypertension, fetal death, premature delivery, spontaneous abortions or recurrent abortions 21. In comparison to overt hypothyroidism, the data regarding subclinical hypothyroidism are not as consistent. A recent review summarized that subclinical hypothyroidism in early pregnancy, compared with normal thyroid function, was associated with the occurrence of preeclampsia and an increased risk of perinatal mortality 47. Concerning the treatment of subclinical hypothyroidism during pregnancy, current evidence is insufficient, and controlled, randomized trials are lacking 10 46. Pregnancy outcomes related to subclinical hypothyroidism are currently being investigated as secondary outcomes in an ongoing, prospective randomized controlled trial by the National Institute of Child Health and Human Development, USA 48. In relation to hypothyroxinemia Some studies have investigated possible associations between hypothyroxinemia and pregnancy outcomes. Findings are divergent. Korevaar et al found an association between hypothyroxinemia and premature delivery 49, while several other studies did not find hypothyroxinemia to be associated with different pregnancy outcomes 50-52. 16

Studies concerning the treatment of hypothyroxinemia and the effect on pregnancy outcome are lacking. Pregnancy outcomes related to hypothyroxinemia are also being investigated as secondary outcomes in the ongoing, prospective randomized controlled trial by the National Institute of Child Health and Human Development, USA 48. In relation to thyroid autoimmunity In observational studies, the presence of thyroid autoantibodies (mainly TPOAb) in euthyroid pregnant women or women of childbearing age has been associated with an increased risk of unexplained subfertility, miscarriage, recurrent miscarriage, preterm birth and maternal post-partum thyroiditis when compared with the absence of thyroid antibodies 47. One prospective, randomized trial including 984 euthyroid pregnant women showed higher rates of miscarriage and premature deliveries (<37 weeks) in TPOAbpositive women not treated with levothyroxine compared with treated TPOAb-positive or TPOAb-negative women 53. However, it is worth noticing that most cases of miscarriage occurred prior to the initiation of treatment/no treatment. To date, there is insufficient evidence to recommend for or against levothyroxine treatment in euthyroid TPOAb-positive women 1) undergoing assisted reproduction technologies, 2) with sporadic or recurrent abortion and 3) during pregnancy (to prevent miscarriage or preterm delivery) 10 20 46. Two ongoing RCTs assess the effect of levothyroxine treatment on pregnancy outcome in euthyroid, TPOab-positive women with a history of (recurrent) miscarriage or with an ongoing treatment for infertility: The TABLET (Thyroid Antibodies and Levothyroxine) trial in the UK, and the T4 Life trial in the Netherlands. Planned closing dates for these trials are in 2015 and 2016, respectively 54 55. Hopefully, these studies will help to clarify whether to treat euthyroid, TPOab-positive women in the situations described. Neurocognitive development of the child In relation to overt hypothyroidism Overt, untreated hypothyroidism during pregnancy has consistently been shown to be associated with impaired neurodevelopment of the child 56 57. In a sub-analysis within an observational study by Haddow et al, children to women with untreated overt hypothyroidism during pregnancy had lower IQ-scores at the age of seven compared with children of women with levothyroxine treated hypothyroidism or euthyroidism during pregnancy 56. In line with this, several observational studies indicate that child neurocognitive development is normal when overt hypothyroidism is detected and subsequently treated during pregnancy 58-60. 17

There are no randomized controlled trials concerning levothyroxine treatment in overt hypothyroidism during pregnancy. This seems unethical, however, and most likely such a study will never be conducted. It is generally considered that the available data confirm the benefits of treating overt hypothyroidism during pregnancy. In relation to subclinical hypothyroidism or hypothyroxinemia The effect of subclinical hypothyroidism or hypothyroxinemia on neurodevelopment of the child is less clear. Countless observational studies have investigated the association between these mild forms of thyroid dysfunction and outcomes in offspring as, for example, (non)-verbal cognitive function, behavioral problems, motor or IQ scores or vision or hearing development. However, study designs were variable, and the findings in the studies are not consistent. Some studies found an association between subclinical hypothyroidism and neurocognitive delay in offspring 61 62, while others did not 63 64. Others found subclinical hypothyroidism to predict externalizing problems in specific ages 65 or ADHD symptoms in girls 66. Consistently, also studies concerning isolated hypothyroxinemia were inconsistent. While several studies suggested an association between, for example, isolated hypothyroxinemia during pregnancy and neurocognitive delay in offspring 63 67-69, others could not confirm these findings 70 71. To date, only one placebo-controlled, randomized trial has assessed the effect of levothyroxine therapy in mild maternal thyroid failure on offspring IQ 72. The primary outcome was the children s IQ at the age of three years. There were no differences between the groups in mean IQ or the proportion of children with an IQ less than 85. As a secondary outcome, there were no differences in IQ scores between the treatment and control groups when divided into subgroups (overt or subclinical hypothyroidism or hypothyroxinemia). Thus, the study does not support screening for, and treatment of, mild thyroid failure during pregnancy to prevent impaired childhood cognitive function. The children from this study are now aged between 7 and 10 years, and a follow up study is ongoing (CATS II), assessing aspects of the children s cognitive functioning including their IQ 73. Yet another study is also ongoing: a large-scale, prospective randomized controlled trial sponsored by the National Institute of Child Health and Human Development (USA) is screening pregnant women with less than 20 weeks gestation for subclinical hypothyroidism or hypothyroxinemia, and randomizing to treatment with levothyroxine or placebo until delivery. The primary outcome is IQ at five years of age in offspring. The results are expected in 2015 48. Hopefully, these studies will contribute to clarity. 18

In relation to thyroid autoimmunity Studies on the association between thyroid autoimmunity and neurocognitive development of the child are rare 47. One observational study reported that TPOAb positivity in euthyroid women at 16-20 weeks of gestation was associated with lower scores on intellectual and motor development in their children at 25 to 30 months of age 61. Management of hypothyroidism during pregnancy Guidelines In recent years, three evidence-based international and European guidelines have been published: 1) The Endocrine Society Guidelines from 2007 12 with a recent update in August 2012 20, 2) the guidelines from the American Thyroid Association from 2011 10 and 3) the European Thyroid Association Guidelines from 2014 21. The implementation of guidelines into clinical practice is of course essential. Despite existing guidelines, a European survey demonstrated a lack of consensus in thyroid testing and the treatment of hypothyroidism during pregnancy 74. Moreover, only 11.5% of providers in an American survey had read the Endocrine Society Guidelines 75. According to these data, proper implementation of international guidelines into clinical practice is not obvious. Beyond these surveys, few studies have evaluated how international guidelines are implemented into clinical practice. Treatment of hypothyroidism during pregnancy Hypothyroidism during pregnancy is recommended to be treated with levothyroxine. In Figure 3, the recommendations of different international guidelines are illustrated. Isolated hypothyroxinemia or isolated thyroid autoimmunity during pregnancy should not be treated 10 20. According to the European Thyroid Association, however, levothyroxine therapy may be considered when isolated hypothyroxinemia is detected in the first trimester of pregnancy, but not when detected in the second or third trimester 21. In hypothyroid women already treated with levothyroxine before conception, the demand of increase in levothyroxine may vary from 25 to 50% from very early in pregnancy. It has been proposed and widely accepted to increase the dosage of levothyroxine by two additional tablets weekly (nine tablets per week instead of seven tablets per week; 29% increase) at confirmation of pregnancy, with subsequent TSH monitoring every four weeks through midgestation 10 20 76. The goal of treatment is to maintain TSH levels within trimester-specific reference ranges. 19

Figure 3. An algorithm for the interpretation and management of the results of first trimester thyroid testing according to different guidelines Universal screening or targeted thyroid testing? To screen or not to screen that is the question. Whether all pregnant women should be screened in order to identify and treat thyroid dysfunction has been, and still is, extremely controversial 10 77 78. The Endocrine Society Guidelines 2007 recommend targeted case finding in high-risk women for thyroid disease at the first prenatal visit or at diagnosis of pregnancy, suggesting 10 indications for targeted case finding 12. However, according to their updated guidelines from 2012, there is insufficient evidence to recommend universal TSH screening at the first trimester visit, leaving options open for either universal screening or targeted thyroid testing 20. This is in agreement with the guidelines from the American Thyroid Association 10. If applying targeted thyroid testing, verbal screening of all pregnant women at the initial prenatal visit is recommended, and subsequent TSH testing in case of any present risk factors for thyroid dysfunction (Table 1). Finally, the ambivalence on this topic is nicely illustrated by the guidelines of the European Thyroid Association: while concluding that universal screening for subclinical hypothyroidism in early pregnancy is not recommended because of the lack of grade 1 evidence, it is noted that the majority of the authors anyway recommend universal screening 21. 20

It has been shown that targeted case finding in research settings miss about one to two thirds of pregnant women with overt or subclinical hypothyroidism 79 80. The impact of screening method has been ambiguous in the only prospective study concerning this topic: as the main outcome, universal screening compared with case finding did not result in an overall decrease in adverse obstetrical outcomes in the whole study population. As a secondary outcome, when only considering low-risk women, treatment of hypothyroidism or hyperthyroidism identified by screening a low-risk group was associated with a lower rate of adverse outcomes 80. However, these data apply to the research setting, when detailed study protocols are used to identify pregnant women with risk factors for thyroid disease. The efficacy of targeted thyroid testing when implemented in clinical practice is currently unknown. Older data suggest that identifying high risk women for targeted thyroid testing appears to not be very successful outside the research setting. In an audit from 2002, for example, less than 20% of high-risk women for thyroid disease in a district were screened, despite existing local guidelines 81. Table 1. Indications for targeted thyroid testing during pregnancy according to the American Thyroid Association and 2007 and 2012 Endocrine Society Guidelines History of thyroid dysfunction or prior thyroid surgery Family history of thyroid dysfunction Presence of goiter TPOAb positivity (when known) Symptoms or clinical signs of thyroid dysfunction Type 1 diabetes Other autoimmune disorders Infertility (as part of the infertility work-up) History of head or neck radiation History of preterm delivery Age >30 years* History of miscarriage* Residing in an area of known moderate to severe iodine insufficiency* Morbid obesity (BMI > 40 kg/m2) Use of amiodarone or lithium, or recent administration of iodinated radiologic contrast * In American Thyroid Association and 2012 Endocrine Society Guidelines Only in American Thyroid Association Guidelines Iodine Although required only in very small amounts, iodine is an essential nutrient as part of thyroid hormones. This is illustrated in Figure 4. 21

Figure 4. The structural formulas of T4 and T3 Dietary sources and requirements Iodine is unevenly distributed in the earth s environment. While iodinedeficient soils are common in, for example, mountain areas far from the sea, most iodine is found in the oceans. The iodine content of crops and plants is reflective of the iodine content of the soil or water they are growing in. Consequently, the iodine levels of, for example, meat, chicken, eggs, dairy products and fish or seafood are reflective of the iodine content of the animal feed used. The native content of most foods and beverages is low, with the exception of fish, seafood, and especially certain seaweeds. The composition of iodine from dietary sources in a typical food basket can be completely different in different countries, regions, or even within a region. This depends on, for example, the country s iodine prophylaxis program, and the content of iodine in food or personal food habits. In Sweden, iodized salt is the main source of iodine in the diet 82. Iodine fortification of edible table salt is, however, not mandatory in Sweden 83. Dairy products and fish are the second and third contributors to iodine intake in Sweden 84. According to WHO/UNICEF/IGN, the recommended daily nutrient intake for iodine is: 90 μg for pre-school children (0-5 years); 120 μg for schoolchildren (6-12 years); 150 μg for adolescents (above 12 years) and adults; 250 μg for pregnant and lactating women. 22

Assessment of iodine status Currently, there are four different methods generally recommended for assessing iodine nutrition in populations: 1) assessment of thyroid size and goiter rate, 2) urinary iodine concentration (UIC), 3) serum TSH and 4) serum Tg 85. Assessment of thyroid size by inspection and palpation is the historical method used for evaluation of goiter prevalence and iodine nutrition. Today, ultrasonography provides a more precise and objective method. Goiter rates in a population reflect long-term iodine nutrition (months to years). Although the assessment of thyroid size is not as widely used as previously, it remains important in assessing goiter rates previous to the introduction of any intervention to control iodine deficiency. School-aged children are the preferred group for this kind of assessment. Urinary iodine is a good marker of recent iodine intake, as more than 90% of dietary iodine is excreted in the urine within 24-48 hours 86. Epidemiological criteria for the median UIC for different populations are given in Table 2. Table 2. Epidemiological criteria from the WHO for assessing iodine status in a population on the basis of median or range of urinary iodine Median UIC (μg/l) Iodine intake Iodine nutrition status Adults and school-aged children* < 20 Insufficient Severe iodine deficiency 20-49 Insufficient Moderate iodine deficiency 50-99 Insufficient Mild iodine deficiency 100-199 Adequate Optimal 200-299 Above requirements May pose a slight risk of more than adequate intake in the overall population 300 Excessive Risk of adverse health consequences (iodine-induced hyperthyroidism, autoimmune thyroid disease) Pregnant women < 150 Insufficient Iodine deficiency 150-249 Adequate Optimal 250-499 Above requirements - 500 Excessive - Lactating women and children < 2 years of age 100 Adequate Optimal * Applies to adults, but not to pregnant and lactating women For lactating women and children <2 years of age, a median urinary iodine concentration of 100 μg/l can be used to define adequate iodine intake, but no other categories of iodine intake are defined. Although lactating women have the same requirement as pregnant women, the median urinary iodine is lower because iodine is excreted in breast milk. 23

According to WHO/UNICEF/IGN, population-based prevalence surveys of iodine status should be done by measurement of urinary iodine levels from spot urine samples. Surveys should be done separately in school-age children and women of childbearing age/pregnant women. Due to large intraindividual variations of spot UIC, single UIC measurements cannot be used as an individual marker of iodine deficiency 87 88. Ideally, an assessment of iodine status should include a concurrent assessment of household use of iodized salt 85. TSH levels are poor indicators of iodine status. The exceptions are neonates, where TSH is a valuable indicator for iodine deficiency 85. Thyroglobulin (Tg) is a thyroid protein that is a precursor in the synthesis of thyroid hormones. Over a range of iodine intake from severely deficient to excessive, Tg levels show a clear U-shaped curve with the lowest levels in iodine sufficient populations 89. According to WHO, dried whole blood spots Tg can be used for monitoring of iodine status in school-age children after iodine repletion 85. The WHO advises each country to assess the iodine nutrition situation in the population every five years. In case of suboptimal iodine status, supplementation with iodine for vulnerable groups, such as pregnant women, is recommended 85. Recent European and international guidelines have implemented this and recommend supplementation to pregnancy-planning, pregnant and breastfeeding women with formulas containing around 150 μg iodine daily 10 20 21. Consequences of iodine deficiency during pregnancy Iodine deficiency can have multiple adverse effects on fetal and infant growth and neurodevelopment, resulting from inadequate thyroid hormone production. Effects on neurodevelopment of the child Exposure to severe iodine deficiency during fetal life and infancy has a detrimental effect on neurocognitive development and may result in cretinism 91. Several randomized studies have proven that endemic cretinism can be reduced or eliminated with iodine prophylaxis 91. Evidence concerning the effect of mild to moderate iodine deficiency during pregnancy on neurocognitive development of the child is not equally clear. Observational studies have, with few exceptions, reported impaired intellectual function and motor skills in children born to mothers with mild to moderate iodine deficiency during pregnancy compared with those born to iodine sufficient mothers 90 92-95. Results from large-scale controlled trials are lacking, but needed, to clarify whether iodine supplementation will benefit infant and childhood neurodevelopment in countries with mild to moderate iodine deficiency. Results of 24

randomized controlled trials from Thailand, India, Australia and New Zealand on this topic are expected in the near future 96 97. Effects on thyroid function Severe iodine deficiency results in severe maternal hypothyroidism, but the impact of mild to moderate iodine deficiency on maternal thyroid function during pregnancy is not as consistent. In mild to moderate iodine deficient areas, studies among pregnant women have shown that: 1) thyroid volume increased up to 31%, 2) TSH levels either increased slightly or remained stable, 3) free T4 levels either decreased or remained stable and 4) Tg levels increased up to 50% throughout pregnancy 98. Randomized controlled trials have shown that iodine supplementation during pregnancy in mild to moderate iodine deficient areas results in: 1) thyroid volume increasing to a lesser content than in untreated controls, 2) stable TSH levels, 3) either decreasing or stable free T4 levels and 4) in most studies decreasing Tg levels throughout pregnancy 98. Concerning neonatal thyroid function, iodine supplementation during pregnancy resulted in lower Tg levels or smaller thyroid volumes in neonates compared with neonates born after non-supplemented pregnancies. No differences in TSH or free T4 levels were seen between the groups 98. Global and national iodine status On a global level, only a few countries were completely iodine sufficient before 1990. Substantial progress has been made since then, mainly due to effective salt iodization programs 99. However, Europe is lagging behind. Populations in several countries remain mildly to moderately iodine deficient, and some countries have also described re-emerging iodine deficiency after decades of iodine sufficiency 100. The few countries that have performed iodine nutrition surveys in pregnant women suggest iodine deficiency in as many as 2/3 of the examined pregnant populations in Europe 21. Nationwide surveys indicate adequate iodine nutritional status in the general population in Sweden 82 101. Information on iodine nutrition during pregnancy in Sweden is, however, scarce. Two small studies from Uppsala examined the effect of pregnancy on thyroid hormone homeostasis and tested the usefulness of thyroglobulin as an indicator of iodine status during pregnancy 102 103. However, the iodine data were not presented in currently used units, and no conclusions were drawn about the iodine status in Swedish pregnant women. Hence, there are no recent data on iodine status of pregnant women in Sweden and no national recommendations concerning iodine supplementation during pregnancy. 25

Aims The general aim of this thesis was to examine the implementation of international guidelines and a targeted thyroid testing approach for clinical practice, and to map iodine nutritional status in pregnant women. The specific aims of the studies were To investigate whether there is an association between SNP rs 4704397 in the PDE8B gene and recurrent miscarriage. (Study I) To determine the adherence of nationwide local guidelines concerning testing for, and treatment of, hypothyroidism in pregnancy to corresponding international guidelines. (Study II) To analyze the degree of implementation of guidelines into everyday clinical practice. (Study II) To examine the real-life efficacy of a targeted thyroid testing approach in identifying women with elevated TSH and overt hypothyroidism during pregnancy. (Study III) To evaluate iodine nutrition during pregnancy in two regions in Sweden. (Study IV) 26

Materials and Methods All the studies included in this thesis were conducted in Sweden. Antenatal care in Sweden is standardized, decentralized to maternity care centers, and free of charge. In Sweden, all pregnant women are invited to an ultrasound examination at 17-19 weeks of gestation, and approximately 97% of the Swedish pregnant population participates 104. Within each maternity care district, a consultant in obstetrics is responsible for the development and implementation of guidelines. In the guidelines of Uppsala County, a targeted thyroid testing approach was implemented in 2004. Overview of the studies Table 3. Overview of the studies Study and topic Design Inclusion date PDE8B polymorphism and recurrent miscarriage (Study I) Thyroid testing (a) (Study II) Thyroid testing (b) (Study II) Targeted thyroid testing (Study III) Iodine status (Study IV) Case-control April 2009-June 2010 (cases); May 2007-June 2010 (controls) Population / materials n=188 (cases); n=392 (controls) Cross-sectional 2011-2012 Endocrine Society Guidelines 2007; 29 local guidelines Descriptive Jan 2009-Dec 2011 n=5254 Cohort Jan 2009-Dec 2011 n=5254 Cross-sectional Jan 2006-July 2007 Jan 2010-Sept 2012 n=459 27

Study populations Uppsala Biobank of Pregnant Women Data from this biobank have been used for three studies (Studies I III). Starting from May 31 2007, Swedish-speaking women aged 18 years or older from the whole of Uppsala County, without blood-borne disease, have been approached to participate in the Uppsala Biobank of Pregnant Women when attending the second-trimester routine ultrasound screening at 17 to 18 weeks of gestation. In Uppsala County, all routine ultrasound examinations are performed at Uppsala University Hospital, which is also the only available delivery ward within the county. Hence, the biobank participants represent a population-based sample. The biobank is a convenience sample based on the availability of a research nurse. Approximately 30% of the respondents decline participation, mainly due to lack of time or fear of blood or needles. Participation in this biobank includes both clinical approaches, such as, for example, medical record reviews and biobank research. A blood sample is collected before the routine ultrasound examination from all women and is stored at -70 C. Moreover, brief maternal demographic data are collected, including ongoing chronic disorders, medication, smoking, and height and weight. It is estimated that the Biobank covers approximately half of the pregnant population of Uppsala County. Study I Cases Cases (n=188) were recruited from the Department of Obstetrics and Gynecology at Uppsala University Hospital, Karolinska University Hospital, Huddinge University Hospital and Danderyd University Hospital, Sweden. Women with a diagnosis of recurrent miscarriage during 1989-2009, defined as three or more verified consecutive miscarriages in the first or second trimester of pregnancy (5-21 completed weeks of gestation), were identified in the out-patient registers of the participating clinics and invited to participate in the study. Inclusion occurred between April 29, 2009 and June 30, 2010. Women with known risk factors for recurrent miscarriage, such as systemic lupus erythematosus, diabetes mellitus type 1, severe thrombophilia and major chromosomal aberrations were not included. Furthermore, two women with hyperthyroidism were excluded. Controls The control subjects (n=391) were matched for age at first planned pregnancy and were randomly chosen from the Uppsala Biobank of Pregnant Women (May 31, 2007 until June 30, 2010). In the control group, none had a history of miscarriage and 74.9% had at least two spontaneous pregnancies, 28

including the ongoing pregnancy, resulting in a term ( 37 weeks) birth of a live infant. Beyond that, the same inclusion and exclusion criteria were applied for the control group as for the cases. Studies II-III All women from the Uppsala Biobank of Pregnant Women with an ultrasound-estimated date of delivery between January 1, 2009, and December 31, 2011, were included in the second part of Study II and in Study III (n=5,254). Study IV The study population consisted of two cohorts of pregnant women (total n=459). The cohorts were from the counties of Värmland (n=273) and Uppsala (n=186), situated in the western and eastern part of Sweden, respectively. Only Swedish-speaking women older than 18 years of age and with singleton pregnancies were included in the cohorts. For the purpose of the study, women with known thyroid disease before pregnancy, thyroid disease detected during pregnancy, preexisting diabetes mellitus or women who continued smoking during pregnancy had not been included in the cohorts. Spot urine samples were collected during the third trimester of pregnancy (from 28 weeks of gestation or later). The Värmland cohort A cohort of pregnant women without chronic hypertension was enrolled in gestational weeks 8 12 at five participating antenatal maternity care centers in Värmland, Sweden. The cohort was initially gathered for the study of preeclampsia risk factors 105. According to the study protocol, spot urine samples were collected around gestational week 33. The urine samples analyzed in this study were collected between January 2006 and July 2007. We reviewed the medical records of the women in 2014. The Uppsala cohort Participants were recruited within the pregnancy cohort Biology, Affect, Stress, Imaging, and Cognition in Pregnancy and the Puerperium (BASIC), which is a longitudinal study investigating biological correlates of mood and anxiety disorders during pregnancy and in the postpartum period 106. All pregnant women in Uppsala County were invited to participate in BASIC at the time of their routine ultrasound around gestational week 18. According to the study protocol, spot urine samples were collected in a subgroup of 200 randomly invited participants around gestational week 38. The urine samples analyzed in this study were collected between January 2010 and September 29

2012. The demographical data was collected at inclusion, and the medical records were reviewed after childbirth. Study procedures Study I At inclusion, both cases and controls attended a brief health examination and answered standardized questions on reproductive history. Further, we reviewed all medical records. According to routine clinical procedures, TSHlevels were analyzed in all women in the case-group when diagnosed with recurrent miscarriage. In the control-group, only women at risk of thyroid disease were subjected to targeted thyroid testing via TSH according to respective local guidelines. In both groups, hypothyroidism was defined as a TSH above the current defined upper limit of the reference range at the different hospitals. Study II Part one: adherence of local guidelines to international guidelines All existing local guidelines from Sweden concerning thyroid testing or treatment of maternal hypothyroidism during pregnancy were compared with The Endocrine Society Guidelines 2007 12. To identify local guidelines concerning thyroid testing or treatment of maternal hypothyroidism during pregnancy, a nationwide survey by e-mail was conducted in 2011-12. All maternity care consultants (n=41) replied to this inquiry. The guidelines were analyzed with respect to four different aspects: 1) the degree of adherence to the Endocrine Society Guidelines, where 10 high-risk groups as reasons for thyroid testing are described, and the proportion of these reasons that were found in the local guidelines was calculated; 2) recommended thyroid function tests; 3) the trimester-specific TSH upper reference limit for intervention with levothyroxine; and 4) the trimester-specific TSH upper reference limit for monitoring women on treatment with levothyroxine. Part two: degree of implementation of guidelines into everyday clinical practice We reviewed all the women s medical records. During the time-frames for this study, there were about 12,000 deliveries in Uppsala County, and 5,254 pregnant women were included in the biobank. Thus, nearly half of the women from the whole district, pregnant in the second trimester, were included in our study. The included women s medical reports were reviewed between February and May 2012 concerning: 30