Thesis Abstract

<p>                 <b>ABSTARCT&nbsp;</b></p><p> <b>Background</b> Thyroid dysfunction is one of the most common endocrine disorder. Thyroid dysfunction affects the female reproductive system and can be manifested by menstrual irregularities, pregnancy loss and infertility. Unexplained infertility has an incidence of 10 to 15 % worldwide. Aim The general objective of this thesis was to explore the importance of thyroid function for reproduction Material and method Serum levels of thyroid stimulating hormone (TSH) were compared in three groups of women in early pregnancy, one high-risk group (n = 88), one low-risk group (n = 511) and a general screening group (n = 699). Serum levels of TSH, free thyroxine (fT4) and thyroid peroxidases antibodies (TPO Ab) in fertile women (n = 67) were compared to women with unexplained infertility (n = 147). By using immunohistochemistry, the protein staining of thyroid hormone receptors (TRα1 and TRβ1), TSH receptor (TSH R), mono carboxylate transporter-8 (MCT8), and type 2 iodothyronine deiodinases (DIO2)] in endometrial biopsies were compared between fertile women (n = 19) and women with unexplained infertility (n = 28). Thyroid related proteins in different part of Fallopian tube during the menstrual cycle in fertile women (n=13) were analyzed. Additionally, embryo development until day 6, in 38 human embryos cultured in standard media with T4 added were compared to development of 36 embryos cultured in standard media.&nbsp;</p><p><b>Results </b>The incidence of subclinical hypothyroidism and hypothyroidism was almost the same in all three study groups (almost 10 %). Hypothyroid women on levothyroxine (LT4) supplementation had in almost 50 % of cases an inadequate treatment. Women with unexplained infertility had significantly higher serum level of fT4, and lower protein staining of TRα1 and MCT8, in the endometrium. Supplementation of thyroid hormone in vitro culture media improved the blastocyst development. Additionally, we showed thyroid related proteins in the Fallopian tube. Conclusion It can be concluded that a general screening for thyroid dysfunction during early pregnancy, by use of TSH levels, is optimal. Furthermore, the imbalance in the thyroid system in women with unexplained infertility highlights the importance of thyroid hormone for female fertility. The improvement of blastocyst development by adding thyroid hormone in early embryo cultures and the presence of proteins related to thyroid in Fallopian tubes suggest involvement of thyroid hormone in early embryo development.&nbsp;</p><p><b><i>Keywords TSH screening, early pregnancy, thyroid hormone, endometrium, Fallopian tube, embryo culture, unexplained infertility, MCT8, DIO2, thyroid hormone receptor, TSH receptor </i></b> <br></p>

Thesis Overview

<p><b>1.0 INTRODUCTION&nbsp;</b></p><p><b>1.1 THYROID</b>&nbsp;</p><p>1.1.1 Thyroid hormones Thyroid hormones (TH), thyroxine (T4) and 3,3’, 5-trijod-L-tyronin (T3), are secreted from and stored in the thyroid gland. Thyroid hormones regulate energy homeostasis, cell proliferation, and carbohydrate-, fat- and protein metabolism.&nbsp;</p><p>1.1.2 The hypothalamic and the pituitary regulation of thyroid hormone secretion The production of THs is mainly regulated by hypothalamic – pituitary – thyroid axis [1]. Thyroid stimulating hormone (TSH) stimulates the production of TH in response to thyroid releasing hormone, produced by the hypothalamus. Thyroid releasing hormone (TRH) is transported to the pituitary via the hypothalamic hypophyseal portal system. TSH and TRH are regulated by negative feedback by T3 and T4. Furthermore, thyroid hormone levels are under influence of other hormones such as glucocorticoids, somatostatin, dopamine, prolactin, estrogen and growth hormones (Figure 1).</p><p>&nbsp;Figure 1. The hypothalamic-pituitary-thyroid axis. Somatostatin(-) Dopamin(-) Prolactin(-) GH(-) Estrogen(+) Glucocorticoids(-) (-) T3 T4 (-) 20% 80% 8&nbsp;</p><p>1.1.3 Thyroid stimulating hormone, TSH and TSH receptor TSH is a heterodimeric glycoprotein hormone that shares the α-subunit with other glycoprotein hormones, such as human chorionic gonadotrophin (hCG), follicle stimulating hormone (FSH) and luteinizing hormone (LH) but it has an unique β-subunit. TSH exerts its effect by binding to the TSH-receptor (TSH R), which is located in the cell membrane of thyroid follicular cells. TSH R is a member of the G-protein associated receptor family, similar to the hCG and LH receptors [2, 3]. TSH R expression has been shown in thyroidal tissue and also in extra-thyroidal tissues such as adipose tissue, testes, ovaries and endometrium [4, 5].&nbsp;</p><p>1.1.4 Thyroid hormone secretion Follicular epithelial cells located in the thyroid gland produce thyroid hormones, mainly T4. They are hydrophobic hormones that are to more than 99 % bound to proteins, mainly to thyroxine binding globulin (TBG). The free fractions of thyroid hormones (fT4, fT3), which mediate thyroid hormone action in target cells, are estimated to 0.02 % of total T4 and 0.30 % of total T3. The local enzymatic conversion of thyroid hormone in target tissues is regulated by iodothyronine deiodinases [6]. The majority of T3 in the circulation is derived from conversion of T4 by type 2 iodothyronine deiodinases (DIO2) and type 1 iodothyronine deiodinases (DIO1). The inactivation of T4 and T3 to reverse T3 (rT3) is mediated by type 3 iodothyronine deiodinase (DIO3)[7]. Cellular transport of thyroid hormone requires active transport across the plasma membrane. This is mediated through different members of mono carboxylate transporter (MCT) and organic anion transporting polypeptide (OATP) depending on target cells[8] .&nbsp;&nbsp;</p><p>1.1.5 Thyroid hormone receptor Thyroid hormones exert their biologic effect through thyroid hormone receptors (TRs), which act as transcription factors to regulate gene expression [9]. TRs bind to a short DNA sequence of target gene called thyroid response element (TRE), which leads to transcription. By contrast, TRs interaction with the response elements, in absence of T3 leads to suppression of basal transcriptional activity [10] (Figure 2).&nbsp;</p><p>Figure 2. The action of thyroid hormone in the target cells. Thyroid hormone receptors (TRs), Thyroid response element (TRE), mono carboxylate transporter (MCT), organic anion transporting polypeptide (OATP), type 2 iodothyronine deiodinases (D2) and type 1 iodothyronine deiodinases (D1). M C T TRE T R T3 D1,2 O A P T T4,T3 T4 T3 mRNA DNA Cell membrane&nbsp; Thyroid receptors are encoded by two genes, TRα and TRβ, each with three isoforms; TRα1, TRα2 and TRα3 and TRβ1, TRβ2 and TRβ3 [11].&nbsp;</p><p>Thyroid receptors are expressed in most tissues and they have higher affinity to T3 than T4. TRα1 is predominantly expressed in brain, heart and skeletal muscles. [11]. TRβ1 is widely expressed in different organs except testes[12] . TRα1, TRα2 and TRβ1 have been shown in endometrium[4, 5]. 2.2 THYROID DYSFUNCTION Changes in serum concentration levels of TSH are the most commonly used indicator of thyroid dysfunction such as autoimmune thyroid dysfunction, hypothyroidism, subclinical hypothyroidism and hyperthyroidism.&nbsp;</p><p>1.2.1 Hypothyroidism Hypothyroidism is defined as low levels of thyroid hormone combined with elevated levels of TSH. Hypothyroidism can be due to low secretion of hormone from the thyroid gland, primary hypothyroidism, or due to low levels of TSH, that is central hypothyroidism. The worldwide prevalence of hypothyroidism is between 0.6 to 12 per 1000 women and 1.3 to 4 per 1000 men [13]. Iodine deficiency is the most common cause of hypothyroidism worldwide [14, 15]. In iodine sufficient countries like Sweden, the most common thyroid disorder is chronic autoimmune thyroiditis, usually known as Hashimoto’s thyroiditis. The diagnosis of Hashimoto’s thyroiditis is confirmed by the presence of anti-thyroid peroxidase antibodies (TPO-Ab)[16]. Hypothyroidism can also be caused by earlier treatment of Graves´ disease such as antithyroid drugs, thyroidectomy or radioiodine treatment. Symptoms of hypothyroidism are nonspecific and vary due to the severity of the disorder. Dry brittle hair and nails are common in these patients who may also have symptoms of chilliness, fatigue, weight gain and slowing of higher mental function. Treatment of hypothyroidism is thyroid hormone (L-T4) substitution.&nbsp;</p><p>1.2.2 Subclinical hypothyroidism Subclinical hypothyroidism (SCH), defined as elevated serum levels of TSH combined with normal thyroid hormone levels [17]. Studies performed in the United States have shown a prevalence of 3 to 15 % of SCH. Women with SCH may have vague or nonspecific symptoms or have symptoms similar to those with hypothyroidism. Women with TPO-Ab and elevated TSH levels are at higher risk of progressing from SCH to hypothyroidism [18, 19] Women with TPO-Ab are at higher risk to development postpartum thyroiditis [20, 21].&nbsp;</p><p>1.2.3 Hyperthyroidism (thyrotoxicosis) Hyperthyroidism is defined as elevated thyroid hormone levels combined with almost undetectable levels of TSH. It affects approximately 2.0 % of women and 0.2 % of men worldwide. The most common type of hyperthyroidism is Graves´ disease. This condition is due to stimulation of thyroid gland by TRAb on the thyroid follicular cells [18]. Common symptoms of hyperthyroidism are weight loss, palpitations, tremulousness, heat intolerance, and anxiety. Physical findings such as tachycardia, thyroid enlargement and tremor are also seen. Treatment options are: anti-thyroid drugs, surgery and radioiodine treatment.&nbsp;</p><p>1<b>.3 THYROID DYSFUNCTION AND FEMALE REPRODUCTION</b>&nbsp;</p><p>1.3.1 Hypothyroidism&nbsp;</p><p>&nbsp;Women with hypothyroidism have low levels of sex hormone binding globulin (SHBG) and low levels of estrogen and testosterone [22]. Menstrual disturbances such as oligomenorrhea, amenorrhea and menorrhagia are common in hypothyroid women. These 12 disturbances can partly be due to TRH-induced hyperprolactinemia and thus altered pulsatile GnRH secretion and partly due to defect hemostasis with low levels of coagulation factors. [23-25].&nbsp;</p><p>1.3.2 Hyperthyroidism (thyrotoxicosis) Thyrotoxicosis may lead to different symptoms ranging from normal menstrual cycles to menstrual irregularities such as menorrhagia, oligomenorrhea, amenorrhea, anovulation and reduced fertility [26, 27] . Women with Graves´ Disease have 2 to 3 times higher serum levels of estrogen and LH during all phases of the menstrual cycle, probably due to high levels of SHBG [27] . The production of testosterone and androstenedione is also increased in these women [28].&nbsp;</p><p><b>1.4 THYROID AND PREGNANCY&nbsp;</b></p><p>1.4.1 The change in thyroid hormone production during pregnancy In early pregnancy an estrogen derived increase in TBG (2.5-fold higher) occurs which requires an increase in thyroid hormone production and a higher daily intake of iodine (250 µg) [29-32] [33]. While the free fraction of thyroid hormone is slightly increased during the first trimester of pregnancy, the total serum levels of thyroid hormones are 1.5-fold higher in pregnant women than in non-pregnant women. The TSH levels have a transient fall during the first trimester of pregnancy due the thyrotrophic action of hCG [34], the lowest levels are seen around 10-12 weeks of gestation. In iodine sufficient areas the TSH levels will remain stable and similar to pre-gestational levels after the first trimester until the end of pregnancy.&nbsp;</p><p>1.4.2 Thyroid hormones and fetus Despite incorporation of iodine late in the first trimester of pregnancy, the fetus does not start to secrete its own thyroid hormones until 18th to 20th weeks of pregnancy. Thus, the fetus is totally dependent on the trans-placental passage of maternal thyroid hormone during the early stages of pregnancy [35, 36]. During pregnancy, the fetus can also be affected by maternal thyroid receptor antibodies and anti-thyroid drugs due to trans-placental passage (Figure 3).&nbsp;</p><p>Figure 3: The trans-placenta passage of thyroid hormones and thyroid hormone related factors. The fetus normal growth and neurologic development is dependent on optimal levels of thyroid hormones. Thyroid hormones influence neurodevelopmental events, such as neurogenesis, myelination, dendrite proliferation and synaptogenesis [37, 38]. TSH T3, T4 ThyroidAb Antithyroid -Drugs Iodine T4, T3 14&nbsp;</p><p><b>1.5 THYROID DYSFUNCTION DURING PREGNANCY&nbsp;</b></p><p>1.5.1 Hypothyroidism/ subclinical hypothyroidism or isolated hypothyroxinemia Untreated hypothyroidism has been associated with an increased risk of obstetric and fetal complications while this it is not the case in isolated hypothyroxinemia or SCH [39, 40] [41]. Hypothyroidism and SCH have been associated with adverse fetal and obstetric outcome such as miscarriages, preterm labor, before 32 weeks of gestation, postpartum hemorrhage, respiratory fetal distress, intrauterine growth retardation (IUGR) and neurological disorders [39, 40, 42-46]. However, there is less evidence that untreated SCH during pregnancy is associated with neurological disorders in the fetus [47].&nbsp;</p><p>1.5.2 Hyperthyroidism Untreated hyperthyroidism is associated with adverse outcomes of pregnancy such as miscarriage, preeclampsia, and preterm delivery. There is a significantly higher risk of fetal complications including IUGR and congenital heart failure even in euthyroid status in the mother, due to the presence of TRAbs. Both thyroid antibodies and anti-thyroid drugs have the ability to pass through placenta [48]. Pregnancy with positive TRAbs, require careful monitoring of thyroid status and TRAbs and controls of the fetus (ultrasonography) during pregnancy. The change of the immune system during pregnancy leads to remission in Graves´ disease like many other autoimmune diseases with a risk for a postpartum relapsing [49].&nbsp;&nbsp;</p><p>1.5.3 Gestational thyrotoxicosis The production of fT4 increases during pregnancy due to secretion of hCG, with a peak during the 10th to 12th weeks of gestation. hCG is a glycoprotein, which shares the alpha unite with TSH and can act as a TSH agonist. This leads to suppression of TSH and transient hyperthyroxinemia in the first trimester of normal pregnancy as well as multiple pregnancy and may cause hyperemesis gravidarum [50-52].&nbsp;</p><p>1.5.4 Postpartum thyroid dysfunction Postpartum thyroiditis is a destructive autoimmune disease with a prevalence of 5 to 9 % and usually occurs within the first year after delivery. Women with diabetes mellitus type 1 have a threefold higher risk of developing postpartum thyroiditis. Women with positive TPO antibodies during early pregnancy have 50 % risk of developing postpartum thyroiditis and an increased risk of developing a permanent hypothyroidism [17, 53, 54]&nbsp;</p>