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The influence of thyroid disorders on adverse pregnancy outcomes

Rosa Vissenberg

Th

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diso

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n ad

verse preg

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cy ou

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Ro

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UITNODIGING

voor het bijwonen van de openbare verdediging

van het proefschrift

The influence of thyroid disorders

on adverse pregnancy outcomes

door

Rosa Vissenberg

PromotiedatumVrijdag 29 april

om 12.00uur

LocatieAgnietenkapel

Oudezijdsvoorburgwal 231 te Amsterdam

Rosa VissenbergValckenierstraat 35-21018 XD Amsterdam

[email protected]

Paranimfen

Josien van [email protected]

06-41854822

Paulien de [email protected]

06-24287558


Rosa Vissenberg

Th

e infl

uen

ce of th

yroid

diso

rders o

n ad

verse preg

nan

cy ou

tcom

es

Ro

sa Vissen

berg

UITNODIGING





door

Rosa Vissenberg


om 12.00uur




[email protected]

Paranimfen


06-41854822


06-24287558

13407_Vissenberg_OM.indd 1 10-02-16 13:17

THE INFLUENCE OF THYROID DISORDERS ON ADVERSE PREGNANCY OUTCOMES

Rosa Vissenberg

Financial support for printing of this thesis was kindly provided by Stichting Gynaecologische Endocrinologie en Kunstmatige Humane Voortplanting, Schildklier Organisatie Nederland, Stichting Fertiliteitsfonds (www.fertiliteitsfonds.nl), Toshiba Medical Systems Nederland, Nutricia Early life Nutrition and by SBOH.

Met dank aan: Saskia’s huiskamerrestaurant

ISBN: 978-94-6299-306-8

Printed by: Ridderprint BV – www.ridderprint.nlLayout: Ridderprint BV – www.ridderprint.nlCover design: Lyanne Tonk, www.persoonlijkproefschrift.nl

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THE INFLUENCE OF THYROID DISORDERS ON ADVERSE PREGNANCY OUTCOMES

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 vrijdag 29 april 2016, te 12.00 uur

door Rosa Vissenberg

geboren te Eindhoven

PROMOTIECOMMISSIE

Promotores: Prof. dr. J.A.M. van der Post Universiteit van Amsterdam Prof. dr. E. Fliers Universiteit van Amsterdam

Co-promotores: Dr. M. Goddijn Universiteit van Amsterdam Dr. P.H.L.T. Bisschop Universiteit van Amsterdam

Overige leden: Prof. dr. J.H. Kok Universiteit van Amsterdam Prof. dr. F. van der Veen Universiteit van Amsterdam Prof. dr. C.B. Lambalk Vrije Universiteit Amsterdam Prof. dr. E.A.P. Steegers Erasmus Universiteit Rotterdam Dr. R.P. Peeters Erasmus Universiteit Rotterdam Dr. C. Ris-Stalpers Universiteit van Amsterdam

Faculteit der Geneeskunde

CONTENTS

Chapter 1 General introduction and outline of the thesis

Chapter 2 Significance of (sub)clinical thyroid dysfunction and thyroid autoimmunity

before conception and in early pregnancy: a systematic review

Human Reproduction Update 2011;17:605-19

Chapter 3 Increased Thyroid Stimulating Hormone in early pregnancy is associated with

breech presentation at term: a nested cohort study

Accepted in adapted form in European Journal of Obstetrics and Gynecology and

Reproductive Biology

Chapter 4 Is subclinical hypothyroidism associated with lower live birth rates in women

with unexplained recurrent miscarriage?

Submitted

Chapter 5 Pathophysiological aspects of thyroid hormone disorders/ thyroid peroxidase

autoantibodies and reproduction


Chapter 6 Treatment of thyroid disorders before conception and in early pregnancy:

a systematic review


Chapter 7 Live-birth rate in euthyroid women with recurrent miscarriage and thyroid

peroxidase antibodies

Gynecological Endocrinology 2015;2:1-4

Chapter 8 Effect of levothyroxine on live birth rate in euthyroid women with recurrent

miscarriage and TPO antibodies (T4-LIFE study)

Contemporary Clinical Trials 2015;44:134–138

Chapter 9 General discussion

Chapter 10 Summary

Nederlandse samenvatting

Addendum

List of co-authors and their contribution

List of publications

Portfolio

Dankwoord

Curriculum Vitae

7

17

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184

186

188

1 |General introduction and outline of the thesis

Chapter 18 |

THYROID DISORDERS AND PREGNANCY

Thyroid hormone physiology

Circulating thyroid hormone concentrations are regulated via a negative feedback system at the level of the hypothalamus and the pituitary. The production of thyroid hormone by the thyroid gland is regulated by thyroid-stimulating hormone (TSH) produced by the anterior pituitary, which itself is regulated by thyrotropin-releasing hormone (TRH) produced by the hypothalamus. Iodide is a rate-limiting element that is needed for the production of thyroid hormone. TSH stimulates expression of the enzyme thyroid peroxidase (TPO) in the thyroid gland, which oxidises iodide to iodine. Subsequently iodine is incorporated in the glycoprotein thyroglobulin (Tg) to form thyroid hormones, the majority in the form of the prohormone thyroxine (T4) and also limited amounts of the biologically active triiodothyronine (T3). Thyroxine-binding globulin (TBG) is a protein that binds T4 and T3 in the circulation. Only the free, unbound T4 and T3 can enter target cells by virtue of specific thyroid hormone transporters (MCT8, MCT10 and Oatp1c1). In target cells, thyroid hormone can be activated (T4 to T3) or inactivated (T4 to rT3 or T3 to T2) depending on the local activity of specific selenium-containing enzymes, known as deiodinases (D1, D2 and D3). Subsequently, T3 can bind to the nuclear thyroid hormone receptors (TR-alpha and TR-beta) and modulate transcription(1).

Thyroid physiology in pregnancy

Thyroid hormone is critical for the development of the foetal and neonatal brain, as well as for many other aspects of pregnancy including placentation and foetal growth. As soon as pregnancy is established, various physiological changes occur to ensure optimal thyroid function to maintain a normal pregnancy and foetal development. During the first trimester, human chorionic gonadotropin (hCG) stimulates the thyroid gland because of its structural resemblance to thyroid stimulating hormone (TSH) (Figure 1). This may temporarily (typically in the first trimester of pregnancy) result in higher free thyroxine (FT4) concentrations and lower TSH concentrations(2;3) than outside pregnancy. Following this period, serum FT4 concentrations decrease by approximately 10 to 15%, and serum TSH values steadily return to normal. Also starting in early gestation, oestrogen concentrations increase, which stimulates production of thyroxine-binding globulin (TBG) by the liver. During pregnancy, serum TBG concentrations increase, peaking around mid-gestation to be maintained thereafter(4). This results in a significant rise in total T4 and T3. There is an increase in renal blood flow and glomerular filtration rate, which leads to increased iodide clearance from plasma and loss of iodine(5). The net effect is an increased demand for the production of thyroid hormones by the thyroid gland in pregnancy. Foetuses are completely dependent on maternal thyroid hormone production in the first trimester. T4 can be detected in foetal serum from approximately 10-12

General introduction | 9

weeks of gestation onwards, although the majority of foetal hormone synthesis occurs after the 18th to 20th week of gestation(6). Maternal thyroid hormones can be transferred across the placenta. The placenta contains deiodinase 3 that can convert T4 to rT3.

Figure 1. Changes in maternal and foetal thyroid function from conception until delivery(7).

Clinical significance of thyroid disorders: pregnancy complications

Thyroid disorders are common in women of reproductive age. The prevalence of thyroid disorders during pregnancy is estimated to be 2-3%(8). Thyroid disorders include (subclinical) hyperthyroidism and (subclinical) hypothyroidism. Overt hyperthyroidism is found in 0.1-0.4% of pregnant women and has been associated with pregnancy complications such as miscarriage, pre-eclampsia, placental abruption, intrauterine growth restriction or preterm birth(9-11). The prevalence of overt hypothyroidism is 0.3-0.5% in pregnant women(8). Hashimoto’s disease is the most common cause of hypothyroidism and is commonly associated with the presence of TPO-Ab. Hypothyroidism has been associated with miscarriage, preterm birth, preeclampsia and impaired psychomotor development(11;12).

The most prevalent thyroid associated disorders in women of reproductive age are subclinical hypothyroidism and thyroid autoimmunity, which is defined as the presence of thyroid peroxidase antibodies (TPO-Ab) in combination with normal thyroid hormone levels. The prevalence of subclinical hypothyroidism in pregnancy is 2.5%(8;13). In 1999 a study was published showing that untreated subclinical hypothyroidism during pregnancy may negatively impact a child’s psychological development, resulting in a significantly lower I.Q.

Chapter

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Chapter 110 |

score and a decrease in motor skills, attention, language and reading abilities. Associations with pregnancy complications including preterm birth or pre-eclampsia were also suggested but the evidence has remained conflicting. Thyroid autoimmunity has a prevalence of 8-14% in women of reproductive age(8) and has also been associated with adverse pregnancy outcomes such as miscarriage, but was only studied in small cohort studies(14-16).

Pathophysiological mechanisms of thyroid hormones and thyroid peroxidase antibodies during conception and pregnancy

The causal mechanism of TPO-Ab, with or without thyroid dysfunction, leading to pregnancy complications is unknown although several mechanisms have been proposed. Three main hypotheses have been reported (17;18). The first is that TPO-Abs are markers of more generalized autoimmunity and that other autoimmune processes cause pregnancy complications. The second hypothesis is that the association is confounded by age, as the prevalence of TPO-Ab increases with age and older women face a higher risk of pregnancy complications. The third is that thyroid autoantibody-mediated disease alters thyroid reserve to such an extent that the thyroid gland is unable to respond adequately to the demands of pregnancy(19). To date, no convincing evidence is present to support any of these hypotheses.

Treatment interventions for thyroid disorders in pregnancy

Women with overt hyperthyroidism or hypothyroidism present with clinical symptoms like weight gain or weight loss, fatigue, palpitations or changes in menstrual patterns. Hyperthyroidism during pregnancy requires treatment with propylthiouracil or thiamazol(20;21) while hypothyroidism during pregnancy should be treated with levothyroxine(11;20;21).

For women with subclinical hypothyroidism, most guidelines advise treatment with levothyroxine, even in case of only marginally increased TSH levels. This advice is based on the negative association with lower I.Q. score of the offspring and on the fact that levothyroxine treatment during pregnancy is regarded safe. Scientific evidence on a reduction of adverse pregnancy complications by levothyroxine administration is, however, limited. The fact that ethnic and trimester specific cut off levels for thyroid hormones exist and different cut off levels are used in different guidelines complicates the diagnosis and treatment evaluation of subclinical hypothyroidism(20-22). Current guidelines state that women with thyroid autoimmunity are at risk of developing hypothyroidism(20;21) and should be monitored during pregnancy, but evidence for effective treatment interventions is lacking(23-25).


Background of the research described in this thesis

Our research project started with a collaboration between gynaecologists and endocrinologists aimed to improve knowledge on the effect of thyroid disorders on pregnancy outcome. Knowledge gaps existed on the clinical significance of thyroid disorders, the pathophysiological mechanisms and treatment possibilities. Especially for thyroid autoimmunity, a wide clinical practice variation existed in Europe relating to screening and treatment of thyroid disorders during pregnancy(26). At that time the available literature on the association between thyroid disorders and adverse pregnancy outcomes was inconsistent for the pregnant population but also for women with a history of recurrent miscarriage. The underlying mechanisms for thyroid disorders causing pregnancy complications were unknown. Evidence on the effect of treatment of subclinical thyroid disorders in pregnancy was insufficient, because of a lack of randomized interventions studies.

The high prevalence of subclinical hypothyroidism and thyroid autoimmunity and the associated pregnancy complications such as miscarriage and preterm birth represent a potentially important health problem. These thyroid disorders remain undiagnosed without an active screening strategy because they are not associated with specific clinical symptoms. Universal screening of thyroid function in pregnancy was under debate at the time when our collaboration started(21;27). A European survey demonstrated that almost 80% of endocrinologists prescribe levothyroxine during pregnancy for women with TPO-Ab in combination with a TSH level within reference range in spite of limited evidence in terms of pregnancy outcomes(26). This may result in unnecessary screening and treatment with increased health care costs.

Study aims

Because of the knowledge gaps concerning the association of thyroid disorders with pregnancy complications, the pathophysiological mechanism and potential effect of treatment interventions we decided to investigate these topics in this thesis.

The objectives of this thesis can be summarized as follows:1 to determine the association between thyroid disorders and pregnancy complications.2 to gain more knowledge on the pathophysiology of thyroid hormones and thyroid

peroxidase antibodies during conception and pregnancy.3 to assess which interventions for women with thyroid disorders are available and effective

in reducing pregnancy complications.

Chapter

1

Chapter 112 |

OUTLINE OF THE THESIS

Chapter 2

We present a systematic review and meta-analysis aimed to assess the clinical significance and associations of (sub)clinical thyroid disorders and thyroid autoimmunity on adverse pregnancy outcomes. A total number of 43 studies were included and 38 could be used for meta-analysis. The association of clinical hypothyroidism, subclinical hypothyroidism and thyroid autoimmunity with early pregnancy complications, late pregnancy complications and adverse neonatal outcomes were studied.

Chapter 3

In chapter 3 we report the results of our retrospective cohort study which investigated the correlation between maternal TSH and FT4 concentrations in early pregnancy and breech presentation at term. 3347 pregnant women were included, 86 women with TSH levels > 97.5th percentile were compared to 3261 women with TSH levels < 97.5th percentile. Also women with FT4 levels < 2.5th percentile were compared to women with FT4> 2.5th percentile.

Chapter 4

We report a retrospective cohort study in which we studied the chance of a live birth in women with subclinical hypothyroidism and recurrent miscarriage. Live birth rates of 20 women with recurrent miscarriage and subclinical hypothyroidism were compared to 818 women with unexplained recurrent miscarriage. Miscarriage rates and ongoing pregnancy rates were also studied.

Chapter 5

In chapter 5 we present the a systematic review investigating pathophysiological aspects of thyroid hormone disorders including the presence of thyroid peroxidase antibodies increasing the risk for unexplained subfertility and early pregnancy loss. Possible interactions of thyroid hormone or thyroid peroxidase antibodies with folliculogenesis, spermatogenesis, fertilization and embryogenesis, the endometrium, implantation or placentation were discussed.

Chapter 6

We conducted a systematic review and meta-analysis to investigate the effect of treatment interventions for (subclinical) thyroid disorders and thyroid autoimmunity during pregnancy on pregnancy outcome. A total of 22 articles were included and 11 were used for meta-


analysis. The treatment interventions of clinical hypothyroidism, subclinical hypothyroidism and thyroid autoimmunity and their treatment effects on early pregnancy complications, late pregnancy complications and adverse neonatal outcomes were studied.

Chapter 7

We describe a retrospective cohort study in which we present the live birth rates and pregnancy rates of 28 women with unexplained recurrent miscarriage with thyroid peroxidase antibodies compared to 174 women with unexplained recurrent miscarriage without thyroid peroxidase antibodies. It was also studied whether women with thyroid peroxidase antibodies who were treated with levothyroxine had higher live birth rates or pregnancy rates compared to women with unexplained recurrent miscarriage and thyroid peroxidase antibodies that did not receive any treatment.

Chapter 8

In chapter 8 we present the study protocol of a multicentre, international, randomised placebo-controlled trial (the T4-LIFE study, NTR 3364). With this study we aim to investigate whether levothyroxine, as compared with placebo, improves the live birth rate among 240 women with recurrent miscarriage and presence of thyroid peroxidase antibodies.

Chapter 9

Finally, the findings from our studies and their implications for clinical practice are discussed and we give recommendations for future research.

Chapter

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Chapter 114 |

REFERENCES

1 Miot F, Dupuy C, Dumont J, Rousset B. Thyroid Hormone Synthesis and secretion. 2012. Ref Type: Online Source

2 Glinoer D, De NP, Robyn C, Lejeune B, Kinthaert J, Meuris S. Serum levels of intact human chorionic gonadotropin (HCG) and its free alpha and beta subunits, in relation to maternal thyroid stimulation during normal pregnancy. J Endocrinol Invest 1993 Dec;16(11):881-8.

3 Guillaume J, Schussler GC, Goldman J. Components of the total serum thyroid hormone concentrations during pregnancy: high free thyroxine and blunted thyrotropin (TSH) response to TSH-releasing hormone in the first trimester. J Clin Endocrinol Metab 1985 Apr;60(4):678-84.

4 Glinoer D. Regulation of thyroid function in pregnancy: maternal and neonatal repercussions. Adv Exp Med Biol 1991;299:197-201.

5 Dworkin HJ, Jacquez JA, Beierwaltes WH. Relationship of iodine ingestion to iodine excretion in pregnancy. J Clin Endocrinol Metab 1966 Dec;26(12):1329-42.

6 Burrow GN, Fisher DA, Larsen PR. Mechanisms of disease: Maternal and fetal thyroid function. New England Journal of Medicine 1994;331:1072-8.

7 Patel J, Landers K, Li H, Mortimer RH, Richard K. Delivery of maternal thyroid hormones to the fetus. Trends Endocrinol Metab 2011 May;22(5):164-70.

8 Krassas GE, Poppe K, Glinoer D. Thyroid function and human reproductive health. Endocr Rev 2010 Oct;31(5):702-55.

9 Casey BM, Dashe JS, Spong CY, McIntire DD, Leveno KJ, Cunningham GF. Perinatal significance of isolated maternal hypothyroxinemia identified in the first half of pregnancy. Obstet Gynecol 2007 May;109(5):1129-35.

10 Earl R, Crowther CA, Middleton P. Interventions for preventing and treating hyperthyroidism in pregnancy. Cochrane Database Syst Rev 2010;(9):CD008633.

11 Reid SM, Middleton P, Cossich MC, Crowther CA. Interventions for clinical and subclinical hypothyroidism in pregnancy. Cochrane Database Syst Rev 2010;(7):CD007752.

12 Haddow JE, Palomaki GE, Allan WC, Williams JR, Knight GJ, Gagnon J, O’Heir CE, Mitchell ML, Hermos RJ, Waisbren SE, Faix JD, Klein RZ. Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child. N Engl J Med 1999 Aug 19;341(8):549-55.

13 Klein RZ, Haddow JE, Faix JD, Brown RS, Hermos RJ, Pulkkinen A, Mitchell ML. Prevalence of thyroid deficiency in pregnant women. Clin Endocrinol (Oxf) 1991 Jul;35(1):41-6.

14 Bussen SS, Steck T. Thyroid antibodies and their relation to antithrombin antibodies, anticardiolipin antibodies and lupus anticoagulant in women with recurrent spontaneous abortions (antithyroid, anticardiolipin and antithrombin autoantibodies and lupus anticoagulant in habitual aborters). Eur J Obstet Gynecol Reprod Biol 1997 Aug;74(2):139-43.

15 Roberts J, Jenkins C, Wilson R, Pearson C, Franklin IA, MacLean MA, McKillop JH, Walker JJ. Recurrent miscarriage is associated with increased numbers of CD5/20 positive lymphocytes and an increased incidence of thyroid antibodies. Eur J Endocrinol 1996 Jan;134(1):84-6.

16 Kutteh WH, Yetman DL, Carr AC, Beck LA, Scott RT, Jr. Increased prevalence of antithyroid antibodies identified in women with recurrent pregnancy loss but not in women undergoing assisted reproduction. Fertil Steril 1999 May;71(5):843-8.


17 Matalon ST, Blank M, Ornoy A, Shoenfeld Y. The association between anti-thyroid antibodies and pregnancy loss. Am J Reprod Immunol 2001 Feb;45(2):72-7.

18 Kaprara A, Krassas GE. Thyroid autoimmunity and miscarriage. Hormones (Athens ) 2008 Oct;7(4):294-302.

19 Prummel MF, Wiersinga WM. Thyroid autoimmunity and miscarriage. Eur J Endocrinol 2004 Jun;150(6):751-5.

20 Abalovich M, Amino N, Barbour LA, Cobin RH, De Groot LJ, Glinoer D, Mandel SJ, Stagnaro-Green A. Management of thyroid dysfunction during pregnancy and postpartum: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab 2007 Aug;92(8 Suppl):S1-47.

21 Stagnaro-Green A, Abalovich M, Alexander E, Azizi F, Mestman J, Negro R, Nixon A, Pearce EN, Soldin OP, Sullivan S, Wiersinga W. Guidelines of the American Thyroid Association for the diagnosis and management of thyroid disease during pregnancy and postpartum. Thyroid 2011 Oct;21(10):1081-125.

22 NIV (Nederlandse Internisten Vereniging), Richtlijn Schildklierfunctiestoornissen. 2007.

23 Negro R, Formoso G, Mangieri T, Pezzarossa A, Dazzi D, Hassan H. Levothyroxine treatment in euthyroid pregnant women with autoimmune thyroid disease: effects on obstetrical complications. J Clin Endocrinol Metab 2006 Jul;91(7):2587-91.

24 Negro R, Mangieri T, Coppola L, Presicce G, Casavola EC, Gismondi R, Locorotondo G, Caroli P, Pezzarossa A, Dazzi D, Hassan H. Levothyroxine treatment in thyroid peroxidase antibody-positive women undergoing assisted reproduction technologies: a prospective study. Hum Reprod 2005 Jun;20(6):1529-33.

25 Thangaratinam S, Tan A, Knox E, Kilby MD, Franklyn J, Coomarasamy A. Association between thyroid autoantibodies and miscarriage and preterm birth: meta-analysis of evidence. BMJ 2011;342:d2616.

26 Vaidya B, Hubalewska-Dydejczyk A, Laurberg P, Negro R, Vermiglio F, Poppe K. Treatment and screening of hypothyroidism in pregnancy: results of a European survey. Eur J Endocrinol 2012 Jan;166(1):49-54.

27 Stagnaro-Green A, Schwartz A. Is universal screening for thyroid disease in pregnancy a cost-effective strategy? Nature Clinical Practice Endocrinology and Metabolism 2008;4:598-9.

Chapter

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2 |Significance of (sub)clinical thyroid dysfunction

and thyroid autoimmunity before conception and

in early pregnancy: a systematic review

E van den BoogaardR VissenbergJA LandM van WelyJAM van der PostM GoddijnPH Bisschop

Human Reproduction Update 2011;17: 605–619

Chapter 218 |

ABSTRACT

Background

Thyroid dysfunction and thyroid autoimmunity are prevalent among women of reproductive age and are associated with adverse pregnancy outcomes. Preconception or early pregnancy screening for thyroid dysfunction has been proposed but is not widely accepted. We conducted a systematic review of the literature on the clinical significance of thyroid dysfunction and thyroid autoimmunity before conception and in early pregnancy.

Methods

Relevant studies were identified by searching Medline, EMBASE and the Cochrane Controlled Trials Register.

Results

From a total of 14 208 primary selected titles, 43 articles were included for the systematic review and 38 were appropriate for meta-analyses. No articles about hyperthyroidism were selected. Subclinical hypothyroidism in early pregnancy, compared with normal thyroid function, was associated with the occurrence of pre-eclampsia [odds ratio (OR) 1.7, 95% confidence interval (CI) 1.1–2.6] and an increased risk of perinatal mortality (OR 2.7, 95% CI 1.6–4.7). In the meta-analyses, the presence of thyroid antibodies was associated with an increased risk of unexplained subfertility (OR 1.5, 95% CI 1.1–2.0), miscarriage (OR 3.73, 95% CI 1.8–7.6), recurrent miscarriage (OR 2.3, 95% CI 1.5–3.5), preterm birth (OR 1.9, 95% CI 1.1–3.5) and maternal post-partum thyroiditis (OR 11.5, 95% CI 5.6–24) when compared with the absence of thyroid antibodies.

Conclusions

Pregnant women with subclinical hypothyroidism or thyroid antibodies have an increased risk of complications, especially pre-eclampsia, perinatal mortality and (recurrent) miscarriage. Future research, within the setting of clinical trials, should focus on the potential health gain of identification, and effect of treatment, of thyroid disease on pregnancy outcome.

Clinical impact of thyroid disorders around conception | 19

INTRODUCTION

Thyroid dysfunction and autoimmunity are not uncommon among women of reproductive age. The prevalence of thyroid dysfunction during pregnancy is estimated to be 2–3% and is mainly caused by chronic autoimmune thyroiditis. Thyroid auto-antibodies are found in 5–15% of women of reproductive age, but are not necessarily accompanied by thyroid dysfunction. Nevertheless, both thyroid dysfunction and thyroid autoimmunity have independently been associated with adverse pregnancy outcomes during all trimesters of pregnancy(1).

In the general population, miscarriage occurs in ~15% of all clinically recognized pregnancies and recurrent miscarriage in 1–3% of all couples trying to conceive(2). Complications later in pregnancy that have been associated with thyroid disorders are pre-eclampsia (incidence 5–10%), preterm delivery (incidence 10–15%) and placental abruption (incidence ~1%)(3;4).

In order to achieve an optimal pregnancy outcome, namely a healthy full-term live birth, all circumstances should be optimal in early pregnancy. Adequate functioning of the maternal thyroid is especially important during the first trimester, when development of the fetal brain starts and the fetus does not yet produce its own thyroid hormones. The exact prevalence of thyroid dysfunction and thyroid autoimmunity among pregnant women as well as the clinical consequences is still unclear: the same applies to the treatment possibilities and their effects on pregnancy outcome.

Guidelines on treatment of hypo- and hyperthyroidism in non-pregnant women and men are generally well defined(5;6) but only a few guidelines are specifically related to obstetric care(7). Endocrinologists agree upon the need for hormone replacement therapy in pregnant women with subclinical hypothyroidism, even in case of only marginally increased thyroid-stimulating hormone (TSH) levels(8;9). Therapy has also been recommended in euthyroid women with circulating antibodies against thyroperoxidase (TPO-Ab) and/or thyroglobulin (Tg-Ab)(10).

General screening for thyroid dysfunction either preconception or in (early) pregnancy has been proposed but is not widely accepted (American College of Obstetricians and Gynecologists, (1;11). It remains to be established whether screening and subsequent treatment will improve clinical outcome and which risk factors contribute to the complications resulting from thyroid abnormalities. The potential benefit of any screening strategy critically depends on the relative contribution of thyroid dysfunction to adverse pregnancy outcomes and on the impact of treatment.

Studies on treatment interventions in patients with thyroid disorders can only be justified if an association between the thyroid condition and obstetric outcome has been demonstrated. Therefore, in order to gain insight into the clinical significance of thyroid dysfunction and thyroid autoimmunity before conception and in early pregnancy, we conducted a systematic review and meta-analyses of the literature.

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Chapter 220 |

METHODS

Relevant studies were identified by searching Medline, EMBASE and the Cochrane Controlled Trials Register, published until May 2010. Date limit for inclusion was based upon the availability of reliable free thyroxine (fT4) assays, which excluded articles published before 1975(12). Search criteria used were related to thyroid function, thyroid autoimmunity and pregnancy outcome. Specifically the following search terms were used: thyroid*, hyperthyr*, hypothyr*, tpo*, tsh, thyrotropin receptor antibod*, thyroid stimulating immunoglobulin*, thyrotropin-binding inhibit*, thyroxine, thyrotropin, thyroid microsomal antibodies, fertility, infertility, abortion*, miscarriag*, pregnan*, obstetric*, gestation* preterm deliver*, premature deliver*, intrauterine growth retardation*, fetal growth restriction*, intrauterine growth restriction* and child development*. Mesh terms used were: thyroid gland, thyroid diseases immunoglobulins, thyroid-stimulating, thyrotropin, thyroxine, fertility, infertility, pregnancy pregnancy outcome, pregnancy complications, fetal growth retardation and child development. There were no language limitations for the initial search. Randomised Controlled Trials (RCTs), cohort studies and case–control studies were included. Data on the effect of T4 replacement therapy were excluded.

Titles and subsequently abstracts of the articles were screened by two reviewers independently (E.v.d.B., R.V.). Included articles for full text screening were compared during a consensus meeting. In case of disagreement, a third reviewer (M.G. or P.B.) was consulted for the decision on inclusion or exclusion for full-text evaluation. Articles that did not contribute to the answer of our research questions after full text evaluation were excluded. Only articles that described at least 10 patients were eligible. Hypothyroidism was defined as low free T4 and TSH concentrations(13) and subclinical hypothyroidism as a high TSH and normal free T4(14). Hyperthyroidism was defined as low TSH with high free T4 or normal free T4 in case of subclinical hyperthyroidism(15). Articles that did not report concentrations of TSH and/or free T4, and articles on thyroid antibodies in non-euthyroid populations were excluded. After consensus the remaining articles were included for critical appraisal and assessed by two reviewers independently (E.v.d.B., R.V.). Articles were judged on scientific quality according to the CONSORT and STROBE statement(16;17). Levels of evidence were attributed according to the Oxford Centre for Evidence-Based Medicine(18). Articles in foreign languages were translated and included if eligible, except for articles in Chinese, Japanese, Russian and Bulgarian.

In case of adequate clinical and statistical homogeneity, summarized odds ratios (ORs) were calculated using random effect models. Software of Review Manager 5 was used to perform the meta-analyses (available from Cochrane). Meta-analysis on thyroid autoimmunity was performed on the presence of antibodies, i.e. TPO-Ab and/or Tg-Ab. In studies that reported both TPO-Ab and Tg-Ab, TPO-Ab was used for meta-analysis, since this is the most commonly and most frequently tested type of antibody. When applicable, i.e. enough data were reported,


a subgroup meta-analysis on TPO-Ab and Tg-Ab was performed separately. This was carried out to approximate clinical practice more precisely and to achieve applicability of the results in all clinical settings.

RESULTS

Figure 1 shows the selection process after the search: 435 articles were selected for critical appraisal, all dealing with fertility, pregnancy outcome and/or the post-natal period. Of the 43 included articles in this systematic review, 4 reported on hypothyroidism(19-22), 5 on subclinical hypothyroidism(23-27) and 36 on thyroid antibodies(10;25;27-60). Articles on subclinical hypothyroidism and antibodies were, in case of the same outcome measures, included in the meta-analysis. Patients in the included studies were pregnant women or non-pregnant women with unexplained subfertility or recurrent miscarriage. Definitions of (unexplained) subfertility and recurrent miscarriage used in the included articles are described in Table I. Controls were all women, either euthyroid or without the adverse pregnancy outcome.

Quality of the studies

The characteristics of the included articles and quality assessment are reported in Table I. Two RCTs were included(22;52). All other studies were evidence-level II studies, i.e. cohort and case–control studies.

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Chapter 222 |

Figure 1. Flowchart of literature search and article selection.


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upTg

-Ab

and

mic

roso

mal

A

b: p

ositi

ve >

+2S

D in

co

ntro

l gr

oup

100

wom

en w

ith

Tg-A

b /

mic

roso

mal

A

bD

etec

tabl

e,

euth

yroi

d

120

wom

en w

ithou

t A

b de

tect

able

, eu

thyr

oid

PPTD

Mat

chin

g: y

es

Feld

t-Ra

smus

sen

et a

l.

1990

Coho

rt73

6 he

alth

y eu

thyr

oid

preg

nant

wom

enTS

H (0

.3-5

mU

/l)T4

(56-

129

nmol

/l)T3

(1.6

-2.8

nm

ol/l)

TPO

-Ab

and/

or T

gAb

(>10

0 U

/ml)

36 w

omen

with

TP

O-A

b an

d/or

Tg-

Ab

in fi

rst t

rimes

ter

20 w

omen

with

out

TPO

-Ab

and/

or

Tg-A

b in

fir

st tr

imes

ter

PPTD

(tra

nsie

nt o

r pe

rsis

tent

thyr

oid

dysf

unct

ion

with

in 1

year

aft

er d

eliv

ery,

thyr

eoto

xico

sis

orhy

poth

yroi

dism

)

Mat

chin

g: n

o

Stag

naro

-G

reen

et a

l.

1990

Coho

rt55

2 pr

egna

nt e

uthy

roid

w

omen

Thyr

otro

pin

(TSH

) (0.

2-5

U/L

)T4

(58-

161

nmol

/L)

TPO

-Ab

and/

or T

g-A

b (<

0.20

arb

itrar

y un

its

by

ELIS

A )

100

wom

en

posi

tive

for T

PO-A

b an

d/or

Tg-

Ab

392

nega

tive

for

TPO

-Ab

and/

or

TgA

b

MC

(in

first

or s

econ

dtr

imes

ter)

Mat

chin

g: n

o

Leje

une

et a

l. 19

93Pr

ospe

ctiv

e co

hort

363

preg

nant

wom

en,

euth

yroi

d, <

14 w

eeks

ge

stat

iona

l age

TSH

not

defi

ned

TPO

-Ab

(>15

0 U

/mL)

Tg-A

b (>

100U

/ml)

23 w

omen

pos

itive

fo

r TPO

-Ab

and/

or

Tg-A

b

340

wom

en

nega

tive

for T

PO-A

b an

d/or

Tg-

Ab

MC

in th

e ne

xt

preg

nanc

yM

atch

ing:

yes

Prat

t et a

l. 19

93Pr

ospe

ctiv

e co

hort

42 n

on-p

regn

ant

euth

yroi

d w

omen

with

a

hist

ory

of R

M

TSH

(0.3

5-7.

0 μI

U/m

l)fT

4 (0

.9-2

.1ng

/dL)

TPO

-Ab,

Tg-

Ab

(> 5

U/

mL)

13 w

omen

pos

itive

fo

r TPO

-Ab

and/

or

Tg-A

b

29 w

omen

neg

ativ

e fo

r TPO

-Ab

and/

or

Tg-A

b

MC

in th

e ne

xt

preg

nanc

yM

atch

ing:

yes

Chapter

2

Chapter 224 |

Sing

h et

al.

1995

Coho

rt48

7 in

fert

ile p

atie

nts

conc

eivi

ng a

fter

ART

(a

rtifi

cial

repr

oduc

tive

tech

niqu

es) (

IVF)

TSH

not

defi

ned

TPO

-Ab

and

Tg-A

b (s

ampl

e an

tibod

y in

dex

0-3.

8)

106

wom

en

posi

tive

for T

PO-

Ab

and/

or T

g-A

b,

euth

yroi

d

381

wom

en

nega

tive

for T

PO-

Ab

and/

or T

g-A

b,

euth

yroi

d

MC

(not

defi

ned)

Mat

chin

g: n

o

Gev

a et

al.

1996

Pros

pect

ive

coho

rt78

pat

ient

s w

ith

mec

hani

cal (

tuba

l ob

stru

ctio

n) o

r un

expl

aine

d in

fert

ility

in

IVF

prog

ram

TgA

b (>

1:40

0)an

timic

roso

mal

Ab

(>1:

1600

)

16 w

omen

pos

itive

fo

r Tg-

Ab

and/

or

antim

icro

som

al A

b,

euth

yroi

d

55 w

omen

neg

ativ

e fo

r Tg

-Ab

and/

or

antim

icro

som

al A

b,

euth

yroi

d

Preg

nanc

y af

ter I

VF,

MC

aft

er IV

FM

atch

ing:

no

Robe

rts

et a

l. 19

96Ca

se-

cont

rol

33 p

regn

ant w

omen

TSH

(0-5

mU

/L)

T4 (5

5-14

4nm

ol/L

)TP

O-A

b (0

-1 U

/ml)

Tg-A

b (0

-8U

/ml)

11 p

regn

ant

wom

en w

ithRM

(≥3

MC

)11

pre

gnan

t w

omen

with

1 M

C

11 h

ealth

y w

omen

in

the

first

trim

este

r of a

non

goin

g pr

egna

ncy

TPO

-Ab,

Tg-

Ab

Mat

chin

g: n

o

Buss

en a

nd

Stec

k19

97Ca

se-

cont

rol

56 n

on-p

regn

ant w

omen

of

repr

oduc

tive

age,

eu

thyr

oid

TPO

-Ab

( > 1

00 IU

/ml)

Tg-A

b (>

100

IU/m

l)28

non

-pre

gnan

t w

omen

with

RM

(≥

3 M

C)

28 m

ultig

ravi

dae

with

out p

revi

ous

MC

or e

ndoc

rine

dysf

unct

ion

TPO

-Ab,

Tg-

Ab

(com

bine

d)M

atch

ing:

no

Iijim

a et

al.

1997

Coho

rt11

79 h

ealth

y eu

thyr

oid

preg

nant

wom

en w

ith

sing

leto

n ge

stat

ions

Tg-A

b,

antim

icro

som

al A

b (t

itre:

>1:

100)

125

antim

icro

som

al

Ab

posi

tive,

32

Tg-

Ab

posi

tive

951

wom

en

nega

tive

for

antim

icor

som

al

Ab

or

Tg-A

b

MC

(pre

gnan

cy

loss

aft

er e

xist

ence

of

ges

tatio

nal s

ac

or fe

tus)

, PTD

(<37

w

eeks

), st

illbi

rth,

PIH

(>

140/

90 m

mH

g),

birt

h w

eigh

t, m

alfo

rmat

ions

, SG

A

(<1.

2 SD

), LG

A (>

1.5

SD)

Mat

chin

g: n

o

Kim

et a

l.19

98Co

hort

79 e

uthy

roid

wom

enw

ith tu

bal f

acto

r or

unex

plai

ned

infe

rtili

ty

who

und

erw

ent I

VF

TPO

-Ab

and

Tg-A

b (>

10

0 U

/ml)

28 e

uthy

roid

po

sitiv

e fo

r TPO

-Ab

and/

or T

g-A

b

51 e

uthy

roid

w

ithou

t TP

O-A

b an

d/or

Tg

MC

Mat

chin

g: n

o

Firs

t aut

hor

Year

Stud

y Ty

pe

Part

icip

ants

Hor

mon

e le

vels

Pa

tien

tsC

ontr

ols

Out

com

e m

easu

re(s

)Q

ualit

y fe

atur

es


Had

dow

et a

l. 19

99Co

hort

2521

6 pr

egna

nt w

omen

Thyr

otro

pin

(>99

.7

‰ o

f th

e m

ean

valu

es o

f all

wom

en o

r bet

wee

n 98

-99.

6 ‰

)

47 p

regn

ant

wom

en>

99.7

‰

15 w

omen

bet

wee

n 98 an

d 99

.6 ‰

of t

he

mea

n va

lue

of a

ll w

omen

124

mat

ched

pr

egna

nt w

omen

w

ith n

orm

al v

alue

s

Neu

rops

ycho

logi

cal

deve

lopm

ent t

ests

in

thei

r chi

ldre

n

Mat

chin

g: y

es

Kutt

eh e

t al.

1999

aCa

se-c

ontr

ol

/ Co

hort

1073

Non

-pre

gnan

t eu

thyr

oid

heal

thy

wom

en

and

wom

en u

nder

goin

g IV

F

TSH

(0.4

5-4.

5 μI

U/m

L)

TPO

-Ab

( > 4

0 IU

/mL)

Tg-A

b ( >

67

IU/m

L)

873

infe

rtile

wom

en

unde

rgoi

ng A

RT

143

TPO

/Tg-

Ab

posi

tive

wom

en

unde

rgoi

ng A

RT

200

heal

thy

repr

oduc

tive-

aged

pa

rous

con

trol

s 14

3 TP

O/T

g-A

b ne

gativ

e w

omen

un

derg

oing

ART

TPO

-Ab,

Tg-

Ab

Preg

nanc

y ra

te,

deliv

ery

rate

Mat

chin

g: n

o

Mat

chin

g: y

es

Kutt

eh e

t al.

1999

bCa

se-c

ontr

ol15

88 w

omen

of

repr

oduc

tive

age

TSH

0.4

5-4.

5 5

μIU

/mL

TPO

-Ab

(0-6

5 IU

/ml)

and

Tg-A

b (0

-120

IU/m

L)

700

wom

en w

ith

RM

(≥2

MC

))68

8 w

omen

with

a

hist

ory

of in

fert

ility

who

w

ere

unde

rgoi

ng

ART

(d

escr

ibed

abo

ve)

200

heal

thy

fem

ales

TPO

-Ab,

Tg-

Ab

Mat

chin

g: n

o

Mav

raga

ni

et a

l. 19

99Ca

se-c

ontr

ol80

wom

en R

o/SS

A p

ositi

ve o

r with

au

toim

mun

e di

sord

er R

o/SS

A n

egat

ive

TPO

-Ab

(> 6

0 IU

/mL)

Tg-A

b (>

50

IU/m

L)40

ant

i Ro-

SSA

po

sitiv

e w

omen

40 a

ge-m

atch

ed

wom

en w

ith a

n au

toim

mun

e di

sord

er a

ge

mat

ched

ant

i Ro/

SSA

neg

ativ

e

TPO

-Ab,

Tg-

Ab

Mat

chin

g: y

es

Mul

ler e

t al.

1999

Coho

rt

173

Non

-pre

gnan

t wom

en

elig

ible

for I

VF

TSH

(0.2

-4.5

μIU

/mL)

TPO

-Ab

(> 8

0 U

/ml)

25 w

omen

TPO

-Ab

posi

tive,

eut

hyro

id14

8 w

omen

TPO

-Ab

nega

tive,

eut

hyro

idPr

egna

ncy

afte

r IVF

Out

com

e of

pr

egna

ncy

afte

r IVF

Mat

chin

g: y

es

Alla

n et

al.

2000

Coho

rt94

03 P

regn

ant w

omen

\at

ges

tatio

nal a

ge o

f 15-

18

wee

ks

TSH

(<6

mU

/l)91

94 p

regn

ant

wom

en

with

nor

mal

TSH

172

preg

nanc

ies

in w

omen

with

in

crea

sed

TSH

PA, P

IH, C

S, F

etal

de

ath,

PN

DM

atch

ing:

yes

Chapter

2

Chapter 226 |

Den

drin

os

et a

l. 20

00Ca

se-c

ontr

ol45

Non

-pre

gnan

tw

omen

, at l

east

6m

onth

s af

ter l

ast

preg

nanc

y

TSH

(0.5

-4.6

µIU

/mL)

TPO

/Tg-

Ab

(<2

IU/m

L)30

RM

pat

ient

s (≥

3 co

nsec

utiv

e lo

sses

)15

hea

lthy

paro

us

cont

rols

TPO

-Ab,

Tg-

Ab

Mat

chin

g: y

es

Mec

acci

et a

l.20

00Ca

se

-con

trol

138

non-

preg

nant

w

omen

with

RM

, PN

Dor

PE

TSH

(0.2

-4.0

μU

/l)fT

4 (7

.8-1

8.4

pg/m

l)TP

O-A

b (>

10 IU

/ml)

Tg-A

b (>

50 IU

/ml)

29 R

M p

atie

nts

(≥2

loss

es

< 1

2 w

eeks

, un

expl

aine

d)

69 h

ealth

y no

n-pr

egna

nt w

omen

TP

O-A

b an

d/or

Tg-

Ab

Mat

chin

g: y

es

Rush

wor

th

et a

l.20

00Co

hort

870

non-

preg

nant

wom

en w

ith R

M (≥

3 co

nsec

utiv

e lo

sses

)

TSH

(0.5

-5.0

mIU

/L)

TgA

b (t

iter >

1:10

0)

antim

icro

som

alA

b (t

iter

1:40

0)

24 w

omen

, eu

thyr

oid

posi

tive

for T

gAb

and/

or

antim

icro

som

al A

b,

euth

yroi

d

81 w

omen

neg

ativ

e fo

r Tg-

Ab

and/

or

antim

icro

som

al A

b,

euth

yroi

d

MC

(firs

t trim

este

r)M

atch

ing:

no

Saka

ihar

a et

al.

2000

Coho

rt40

22 p

regn

ant w

omen

, eu

thyr

oid

TSH

(0.2

-6.0

mU

/L),

fT4

(7.7

-29.

0 pm

ol/L

)Tg

-Ab,

Ant

imic

roso

mal

A

b (1

00-fo

ld d

ilutio

ns)

131

wom

en

posi

tive

for T

g-A

b an

d/or

an

timic

roso

mal

Ab

1030

wom

en

nega

tive

for

Tg0-

Ab

and/

or

antim

icro

som

al A

b

PPTD

(h

yper

thyr

oidi

sm,

hypo

thyr

oidi

sm 1

an

d 3

mon

ths

post

part

um)

Mat

chin

g: n

o

Klei

n et

al.

2001

Case

-con

trol

Offs

prin

g of

164

m

othe

rs w

ho w

ere

test

ed fo

r thy

roid

fu

nctio

n du

ring

preg

nanc

y

TSH

at 1

7 w

eeks

of

gest

atio

n8-

year

-old

offs

prin

g of

20

untr

eate

d hy

poth

yroi

d m

othe

rs (T

SH 8

8-99

.85th

‰) a

nd 2

0 (T

SH >

99.

85th

‰)

8-ye

ar- o

ld o

ffspr

ing

of 1

24 c

ontr

ol

mot

hers

(TSH

< 9

8th ‰

)

IQM

atch

ing:

yes

Popp

e et

al.

2002

Case

-con

trol

538

non-

preg

nant

w

omen

TSH

( 0.

27-4

.2 m

IU/L

)fT

4 ( 9

.3-1

8.0

ng/L

)TP

O- A

b (>

100

kU/l)

438

infe

rtili

ty

patie

nts,

197

fem

ale

(end

omet

riosi

s, tu

bal d

isea

se

and

ovar

ian

dysf

unct

ion)

, 168

m

ale

fact

or, 7

3 id

iopa

thic

)

100

paro

us c

ontr

ols

TPO

-Ab

Mat

chin

g: y

es

Siei

ro e

t al.

2004

Coho

rt53

4 pr

egna

nt w

omen

TS

H (0

.4-3

.8 m

U/L

)fT

4 (0

.8-2

.0 n

g/dL

)TP

O-A

b (0

-40

U/L

)

29 T

PO-A

b po

sitiv

e w

omen

, eut

hyro

id50

5 TP

O-A

b ne

gativ

e w

omen

, eu

thyr

oid

MC

(spo

ntan

eous

en

ding

of p

regn

ancy

be

fore

20

wee

ks)

Mat

chin

g: n

o

Firs

t aut

hor

Year

Stud

y Ty

pe

Part

icip

ants

Hor

mon

e le

vels

Pa

tien

tsC

ontr

ols

Out

com

e m

easu

re(s

)Q

ualit

y fe

atur

es


Stag

naro

-G

reen

et a

l.

2005

Case

-con

trol

953

wom

en w

ho h

ad

deliv

ered

TS

H (0

.35-

2.99

mIU

/L)

TPO

-Ab,

Tg-

Ab

(sen

sivi

ty a

ssay

0.3

U

/mL)

124

wom

en w

ith

pret

erm

del

iver

y12

4 w

omen

who

de

liver

ed

at te

rm

TPO

-Ab,

Tg-

Ab

Mat

chin

g: y

es

Gha

foor

et a

l. 20

06Pr

ospe

ctiv

e co

hort

1500

eut

hyro

id p

regn

ant

wom

enTP

O-A

b (>

100U

/ml)

168

TPO

-Ab

posi

tive

wom

en13

32 T

PO-A

b ne

gativ

e w

omen

MC

, pre

mat

urity

M

atch

ing:

yes

Neg

ro e

t al.

2006

Case

-con

trol

1074

Pre

gnan

t wom

en,

euth

yroi

dTS

H (

0.27

-4.2

mIU

/L)

fT4

(9.3

-18.

0 ng

/L)

TPO

-Ab

(> 1

00kI

U/l

)

58 p

atie

nts T

PO-A

bpo

sitiv

e86

9 pa

tient

s TPO

-A

b ne

gativ

eM

C, P

IH, P

E, P

TD, P

AM

atch

ing:

yes

Shoe

nfel

d et

al.

2006

Case

-con

trol

269

patie

nts

with

au

toim

mun

e di

seas

e an

d/or

repr

oduc

tive

failu

re

(recu

rren

t pre

gnan

cy lo

ss,

infe

rtili

ty)

TPO

-Ab,

Tg-

Ab

(> 2

SD

than

the

mea

n le

vel i

n co

ntro

l gro

up)

109

RM ((

≥3

MC

in

firs

tan

d se

cond

tr

imes

ter)

120

heal

thy

fem

ales

, eu

thyr

oid

TPO

-Ab,

Tg-

Ab

Mat

chin

g: y

es

Aba

lovi

ch

et a

l. 20

07Ca

se-c

ontr

ol39

9 w

omen

of

repr

oduc

tive

age

TSH

(0.5

-5 m

IU/l)

T4 (4

.5-1

2 μg

/dl)

TPO

- Ab

(> 3

5 IU

/ml)

244

wom

en

cons

ultin

g on

in

fert

ility

(>1

year

, 94

% k

now

n ca

uses

)

155

heal

thy

wom

en w

ith

confi

rmed

fert

ility

TPO

-Ab,

sub

clin

ical

hy

poth

yroi

dism

Mat

chin

g: n

o

Case

y et

al.

2007

Coho

rt17

,298

sin

glet

on p

regn

ant

wom

en

TSH

(0.0

8-3.

0 m

U/l)

fT4

(low

er li

mit

0.86

ng

/dL)

598

with

su

bclin

ical

hy

poth

yroi

dism

(n

orm

al T

SH, f

T4 <

0,

86 n

g/dL

)

16 0

11 n

orm

al T

SH,

fT4

euth

yroi

dPI

H, P

E, G

DM

, PA

, PTD

(3

6 w

eeks

or l

ess)

, C

S,

feta

l mal

form

atio

n,

low

Apg

ar

scor

es (<

3 af

ter 5

min

), ad

mis

sion

NIC

U, R

DS,

PN

D, b

irth

wei

ght

Mat

chin

g: y

es

Mam

ede

da

et a

l.20

07Co

hort

98 p

regn

ant w

omen

TSH

(0.4

-3.8

μm

/L),

fT4

( 0.8

-2.0

ng/

dL)

TPO

-Ab

(> 4

0 U

/L)

10 T

PO-A

b po

sitiv

e w

omen

, eut

hyro

id88

TPO

-Ab

nega

tive

wom

en, e

uthy

roid

PPTD

(hyp

o/hy

pert

hyro

idis

m)

Mat

chin

g: y

es

Neg

ro e

t al.

2007

aCo

hort

423

wom

en u

nder

goin

g IV

FTS

H (0

.27-

4.2

mU

/l)fT

4 (1

2- 3

3.5

pmol

/L)

TPO

-Ab

( > 1

00 k

U/l)

49 T

PO-A

b po

sitiv

e,

euth

yroi

d37

4 TP

O-A

b ne

gativ

e, e

uthy

roid

Preg

nanc

y af

ter I

VFO

utco

me

of

preg

nanc

y af

ter I

VF

Mat

chin

g: y

es

Chapter

2

Chapter 228 |

Neg

ro e

t al.

2007

bRC

T21

43 e

uthy

roid

pre

gnan

t w

omen

TSH

(0.2

7-4.

2 m

IU/l)

fT4

(9.3

-18.

0 ng

/L, 1

2-33

.5 p

mol

/L)

TPO

-Ab

(0-1

00 k

IU/l)

84 e

uthy

roid

pr

egna

nt w

omen

TP

O-A

b po

sitiv

e

85 e

uthy

roid

pr

egna

nt w

omen

TP

O-A

b ne

gativ

e

PPTD

( hy

pert

hyro

idis

m,

hypo

thyr

oidi

sm)

perm

anen

t hy

poth

yroi

dism

(12

mon

ths

post

-par

tum

), M

C

Rand

omiz

atio

n:

com

pute

r ge

nera

ted

Conc

eale

d: y

esBl

indi

ng: y

esIT

T: y

es

Bellv

er e

t al.

2008

Case

-con

trol

119

wom

en u

nder

goin

g A

RTTS

H (0

.25-

5 μU

I/ml)

fT4

(0.7

3-2.

2 ng

/dl)

TPO

-Ab

( >25

IU/m

l)Tg

-Ab

(>10

0IU

/ml)

30 R

M p

atie

nts

26 Im

plan

tatio

n fa

ilure

(IF)

26 IF

+31

U

nexp

lain

ed

infe

rtili

ty (U

I) (5

7 su

bfer

tile

coup

les)

32 O

ocyt

e do

nors

31 U

I32

ooc

yte

TPO

-Ab,

Tg-

Ab

Mat

chin

g: y

es

Cle

ary-

Gol

dman

et a

l.

2008

Coho

rt10

990

wom

en w

ith

sing

leto

n pr

egna

ncie

s TS

H a

nd T

4 (b

etw

een

2,5

and

97,5

th ‰

)TP

O-A

b (>

35 IU

/ml)

Tg-A

b (>

40 IU

/ml)

240

subc

linic

al

hypo

thyr

oidi

sm

(TSH

>

97,5

th a

nd fT

4 be

twee

n 2,

5 an

d 97

,5th

‰)

1051

8 eu

thyr

oid

stat

e(T

SH a

nd T

4 be

twee

n 2,

5th a

nd 9

7,5

th ‰

)

MC

(<24

wks

), PI

H

(>14

0/90

mm

Hg)

, PE,

G

DM

, pla

cent

a pr

evia

, PA

, pet

erm

ons

et o

n la

bor (

<37

wee

ks),

PPRO

M (<

37w

eeks

), PT

D (<

37 w

eeks

), LB

W

(<25

00gr

), m

acro

som

ia

(>40

00gr

), PN

D

Mat

chin

g: y

es

Irava

ni e

t al.

2008

Case

-con

trol

910

euth

yroi

d,,

non-

preg

nant

wom

enTS

H (0

.4-4

.4 m

IU/L

)fT

4 (4

.5-1

0.9

μg/d

L)TP

O-A

b ( >

40

IU/m

L)Tg

-Ab

(>12

5 IU

/mL)

641

wom

en w

ith

RM (≥

3)26

9 no

n-pr

egna

nt

heal

thy

euth

yroi

d co

ntro

ls, a

ge-

mat

ched

TPO

-Ab,

Tg-

Ab

Mat

chin

g: y

es

Kilic

et a

l. 20

08Pr

ospe

ctiv

e Co

hort

69 (5

4 el

igib

le) p

atie

nts

with

une

xpla

ined

infe

rtili

ty u

nder

goin

g IV

F

TSH

(0.0

05-1

00.0

μgI

U/

Ml)

fT4

(0.0

23-7

.77

ng/d

L)TP

O-A

b ( >

34I

U/m

l)Tg

-Ab

( > 1

15 IU

/mL)

23 T

PO-A

b or

Tg-

Ab

posi

tive

patie

nts,

euth

yroi

d

31 T

PO-A

b or

Tg-

Ab

nega

tive

patie

nts,

euth

yroi

d

IVF

outc

ome

Mat

chin

g: y

es

Firs

t aut

hor

Year

Stud

y Ty

pe

Part

icip

ants

Hor

mon

e le

vels

Pa

tien

tsC

ontr

ols

Out

com

e m

easu

re(s

)Q

ualit

y fe

atur

es


Mon

tane

r et

al.

2008

Coho

rt61

9 pr

egna

nt w

omen

w

ithou

t for

mer

DM

TP

O-A

b (>

12

IU/m

l)62

TPO

-Ab

posi

tive,

eu

thyr

oid

557

TPO

-Ab

nega

tive,

eut

hyro

idG

DM

Mat

chin

g: y

es

Rao

et a

l. 20

08Ca

se-c

ontr

ol33

3 no

n-pr

egna

nt

wom

enTS

H (0

.3-5

.0 μ

IU/m

l)T4

(5.0

-12.

5 µg

/dl)

163

RM p

atie

nts

170

heal

th c

ontr

ols,

age-

mat

ched

Hyp

othy

roid

ism

Mat

chin

g: y

es

Benh

adi e

t al.

2009

Coho

rt24

97 W

omen

with

si

ngle

ton

preg

nanc

y w

ithou

t ove

rt

hypo

-hyp

erth

yroi

dism

TSH

(0.3

4-5.

60 m

U/l)

fT4

(7.5

-21.

2 pm

ol/l)

TPO

-Ab

(0-8

0 kU

/l)

146

TPO

-Ab

posi

tive

2351

TPO

-Ab

nega

tive

MC

(<22

wee

ks),

feta

l de

ath

(22

wee

ks-d

eliv

ery)

or

neon

atal

dea

th (0

-7

days

aft

er d

eliv

ery)

Mat

chin

g: y

es

Seze

r et a

l. 20

09Co

hort

128

euth

yroi

d he

alth

y pr

egna

nt w

omen

with

1

MC

TSH

(0.3

-4.5

mIU

/l)fT

4 (1

0-22

pm

ol/L

)TP

O-A

b ( <

34IU

/ml)

Tg-A

b (<

115

IU/m

l)

28 T

PO-A

b or

Tg-

Ab

posi

tive

100

TPO

-Ab

or T

g-A

b ne

gativ

eM

C

Mat

chin

g: y

es

Li e

t al.

2010

Coho

rt12

68 h

ealth

y pr

egna

nt

wom

en w

ithou

t ove

rt

thyr

oid

dise

ase

TSH

(0.1

2-4.

21 m

IU/L

)fT

4 (1

1.9-

24.6

pm

ol/L

)TP

O-A

b ( 0

-50

IU/m

L)

18 w

omen

w

ith s

ubcl

inic

al

hypo

thyr

oidi

sm34

TPO

-Ab

posi

tive

euth

yroi

d

36 e

uthy

roid

co

ntro

ls

TPO

-Ab

nega

tive

68 e

uthy

roid

co

ntro

ls T

PO-A

b ne

gativ

e

CS,

Mea

n in

telli

genc

e sc

ores

Mat

chin

g: y

es

Neg

ro e

t al.

2010

RCT

4562

Pre

gnan

t wom

enTS

H (>

2,5

mIU

/ltr)

TPO

Ab

(> 1

00 k

IU/lt

r)34

hyp

othy

roid

fro

m th

e ca

se

findi

ng lo

w ri

sk

for t

hyro

id d

isea

se

grou

p (n

ot u

nive

rsal

sc

reen

ing

grou

p)

1769

eut

hyro

id

patie

nts

with

or w

ithou

t Ab

MC

, PIH

, PE,

GD

M,

PA, C

S, R

D N

ICU

ad

mis

sion

, LBW

(<

2500

gr),

PTD

(<37

wee

ks),

Low

Apg

ar

Scor

e (<

3 af

ter 5

min

), PN

D

Rand

omiz

atio

n:Co

mpu

ter

gene

rate

d Co

ncea

led:

yes

Blin

ding

: yes

ITT:

no

Ab,

ant

ibod

y; A

RT, a

rtifi

cial

repr

oduc

tive

tech

niqu

es; C

S, c

aesa

rean

sec

tion;

GD

M, g

esta

tiona

l dia

bete

s m

ellit

us; I

F, in

fert

ility

; LG

A, l

arge

for g

esta

tiona

l age

; MC

, mis

carr

iage

; NIC

U,

neon

atal

Inte

nsiv

e ca

re u

nit,

PA, p

lace

ntal

abr

uptio

n; P

E, p

re-e

clam

psia

; PIH

, pre

gnan

cy in

duce

d hy

pert

ensi

on; P

ND

, per

inat

al d

eath

; PPT

D, p

ostp

artu

m t

hyro

id d

isea

se; P

TD,

pret

erm

del

iver

y; R

DS,

resp

irato

ry d

istr

ess

synd

rom

e; R

M, r

ecur

rent

mis

carr

iage

; SG

A, s

mal

l for

ges

tatio

nal a

ge; I

TT, i

nten

tion

to tr

eat.

Not

es:

All

stud

ies

have

an

adeq

uate

sam

ple

size

(n>

10).

Two

RCT’

s w

ere

incl

uded

(Neg

ro e

t al.,

2007

; Neg

ro e

t al.,

2010

) All

othe

r stu

dies

wer

e le

vel I

I stu

dies

: coh

ort a

nd c

ase-

cont

rol s

tudi

es.

Mic

roso

mal

ant

ibod

ies

is th

e pr

evio

us n

omen

clat

ure

for T

PO a

ntib

odie

s.

Chapter

2

Chapter 230 |

The effect of thyroid dysfunction and autoimmunity on fertility

One study reported on the relation between subclinical hypothyroidism and unexplained subfertility in 40 women with subclinical hypothyroidism and 359 controls(25). Subclinical hypothyroidism was associated with an increased risk of unexplained subfertility [one study, OR 4.0, 95% confidence interval (CI) 1.7– 9.8]. Four studies reported on the relation between thyroid antibodies and unexplained subfertility and could be included in a meta-analysis (Fig. 2) (25;41;48;55). Summarized data included 334 patients with anti-thyroid antibodies and 1679 controls. In antibody-positive women subfertility occurred more frequently (four studies, OR 1.5, 95% CI 1.1–2.0).

Seven studies reported on thyroid antibodies in relation to IVF outcome. A total of 1760 women undergoing IVF for different reasons could be included in the meta-analysis, 330 with thyroid antibodies and 1430 controls (Supplementary data, Fig. S1a) (28;36;40;41;43;55;57). No association was found between the presence of thyroid antibodies and the clinical pregnancy rates after IVF (seven studies, OR 0.71, 95% CI 0.36–1.4).

Figure 2. Forest plot of Odds Ratio’s and 95% Confidence Interval of pooled studies comparing euthyroid

antibody positive patients with euthyroid negative controls according to the risk of unexplained subfertility.

The effect of thyroid dysfunction and autoimmunity on early pregnancy

One study reported on the relation between untreated hypothyroidism (determined retrospectively using frozen serum) and miscarriages, showing an increased risk for miscarriage in women with untreated hypothyroidism compared with euthyroid controls (OR 5.78, 95% CI 2.4–14)(22). Another study, with 240 patients with subclinical hypothyroidism and 10 518 controls did not show any difference in miscarriage rate (OR 0.69, 95% CI 0.10–5.0)(26). Data from 13 studies were included to determine the risk for miscarriage rate in relation to thyroid antibodies (Fig. 3) (10;28;32-35;37;39;46;49;51;59;60). Data from 13 studies reporting on 966 thyroid antibody positive patients and 7331 controls without thyroid antibodies could be included in the meta-analysis and showed an increased risk of miscarriage in patients with thyroid antibodies (13 studies, OR 3.7, 95% CI 1.8–7.6). Five studies reported on pregnancy outcome after IVF (Supplementary data, Fig. S1b)(28;36;40;41;43). In contrast to spontaneous pregnancy, there was no evidence for an increased risk of miscarriage in IVF pregnancies in


women with antibodies, compared with women without antibodies (five studies, OR 1.6, 95% CI 0.76–3.5).

Thyroid function and recurrent miscarriage was studied in one study, with 8 hypothyroid patients and 325 euthyroid controls(21). There was no evidence for a difference in risk for recurrent miscarriage between the two groups (one study, OR 7.6, 95% CI 0.92–62). Antibodies in women with recurrent miscarriage were investigated in eight of the included studies, reporting on 460 patients with thyroid antibodies and 1923 antibody-negative controls (Fig. 4) (29;37;38;44;45;53;55;56). Patients with recurrent miscarriage more often had thyroid antibodies (eight studies, OR 2.3, 95% CI 1.5–3.5). One study could not be included in the meta-analysis, since only the OR was documented and not the exact number of patients in both groups(42): this study reported an OR for recurrent miscarriage in women with thyroid antibodies of 2.6, with an OR of 2.6 for TPO-Ab and 4.1 for Tg-Ab.


thyroid antibody positive patients with euthyroid antibody negative controls according to the risk of

miscarriage.


antibody positive patients with euthyroid antibody negative controls according to the risk of recurrent

miscarriage.

Chapter

2

Chapter 232 |

The effect of thyroid dysfunction and autoimmunity on late pregnancy complications

The relation between hypothyroidism and gestational diabetes mellitus (GDM) was addressed in one study, reporting no difference between patients and controls (one study, OR 2.3, 95% CI 0.67–7.5)(22). Meta-analysis of two studies on subclinical hypothyroidism and GDM resulted in a pooled OR of 1.4, 95% CI 0.64–2.8 (Supplementary data, Fig. S2)(24;26). The study on antibodies did not report any relationship with GDM (one study, OR 1.2, 95% CI 0.45–3.17) (58).

Pregnancy-induced hypertension was investigated in six studies; one study on hypothyroidism, three studies on subclinical hypothyroidism and two studies on thyroid antibodies. The study among women with hypothyroidism showed no association with pregnancy-induced hypertension (one study, OR 1.8, 95% CI 0.54–6.0)(22). Meta-analysis did not show any association between subclinical hypothyroidism and pregnancy-induced hypertension (three studies, OR 1.00, 95% CI 0.79–1.29) (Supplementary data, Fig. S3a) (23;24;26). The pooled OR for thyroid antibodies versus no antibodies and pregnancy-induced hypertension was 1.2 (two studies, 95% CI 0.59–2.6), indicating no difference (Supplementary data, Fig. S3b) (10;39).

Hypothyroidism and pre-eclampsia, reported in one study, showed no association (one study, OR 1.52, 95% CI 0.36–6.5)(22). Subclinical hypothyroidism compared with normal thyroid function in the studies included in the meta-analysis was significantly related to the occurrence of pre-eclampsia (two studies, OR 1.7, 95% CI 1.1–2.6) (Supplementary data, Fig. S4) (24;26). Data from the included study on antibodies and pre-eclampsia did not indicate any relation (one study, OR 1.4, 95% CI 0.42–4.8)(10).

In one study reporting on placenta praevia the risk in patients with subclinical hypothyroidism when compared with euthyroid patients appeared to be comparable (one study, OR 0.98, 95% CI 0.13–7.1)(26).

One study showed an increased risk for placental abruption in hypothyroid patients (one study, OR 10.7, 95% CI 1.2–94)(22). In a meta-analysis of three studies reporting on placental abruption, the pooled risk was not significantly increased in subclinical hypothyroid patients (three studies, OR 1.9, 95% CI 0.96–3.7) (Supplementary data, Fig. S5) (23;24;26). In 58 euthyroid patients with thyroid antibodies and 869 euthyroid controls without antibodies, no difference in incidence of placental abruption was described (one study, OR 3.8, 95% CI 0.42–35)(10).

The relationship between clinical hypothyroidism and preterm onset of labor was reported in one study, not showing a significant difference (one study, OR 2.6, 95% CI 0.91–7.7)(22). The study reporting on subclinical hypothyroidism also did not show any difference (one study, OR 0.99, 95% CI 0.57–1.7)(26). This latter study also looked at preterm premature rupture of membranes, for which no increased risk was observed (one study, OR 1.6, 95% CI 0.66–4.0). Six studies reported on preterm delivery before 37 weeks of gestational age. The study on hypothyroidism found the risk of preterm birth to be comparable in hypothyroid and in


euthyroid patients (one study, OR 2.6, 95% CI 0.99–6.9)(22). The meta-analysis on subclinical hypothyroidism and preterm delivery, describing 838 patients and 26 529 controls, showed no difference between the two groups (two studies OR 1.0, 95% CI 0.59–1.8) (Fig. 5a) (24;26). Thyroid antibodies in the meta-analysis were associated with an increased risk of preterm delivery (three studies OR 1.9, 95% CI 1.1–3.5) (Fig. 5b) (10;32;39).

Cesarean delivery rate was not increased in patients with hypothyroidism (one study, OR 1.5, 95% CI 0.68–3.2)(22). The meta-analysis on 788 patients with subclinical hypothyroidism and 25 241 healthy euthyroid controls showed a comparable risk for cesarean section (three studies, OR 1.1, 95% CI 0.91–1.3) (Supplementary data, Fig. S6) (23;24;27). Thyroid antibodies were not related to caesarean section(27) (one study, OR 1.2, 95% CI 0.51–2.9).

Figure 5. Forest plot off Odds Ratio’s and 95% Confidence Interval of pooled studies comparing (a) patients

with subclinical hypothyroidism with euthyroid controls and (b) euthyroid thyroid antibody positive patients

with euthyroid antibody negative controls according to the risk of preterm delivery < 37 weeks gestation.

The effect of thyroid dysfunction and autoimmunity on neonatal outcome

Perinatal mortality was reported in one study, and it was not significantly different in hypothyroid and euthyroid patients (one study, OR 2.4, 95% CI 0.14–42)(22). Meta-analysis on three studies, reporting on 1010 subclinical hypothyroid patients and 35 723 euthyroid controls, revealed an increased risk of perinatal mortality in subclinical hypothyroid patients (three studies, OR 2.7, 95% CI 1.6–4.7) (Supplementary data, Fig. S7) (23;24;26). The presence of thyroid antibodies did not increase the risk of perinatal mortality but was reported in only one study (one study, OR 0.49, 95% CI 0.03–8.6)(59).

Low birthweight defined as a weight of < 2500 g at term and high birthweight defined as a weight of > 4000 g were reported in three studies(22;24;26). No evidence was found for a relationship between hypothyroidism and low or high birthweight (one study, OR 2.6, 95% CI

Chapter

2

Chapter 234 |

0.90–7.6 and OR 2.4, 95% CI 0.81–6.8, respectively)(22). In a meta-analysis of 838 patients and 26 259 controls, subclinical hypothyroidism appeared not to be associated with low or high birthweight (two studies, pooled OR 0.93, CI 0.46–1.9 and OR 0.63, CI 0.37–1.1, respectively) (Supplementary data, Fig. S8a and b) (24;26).

The neonatal outcome was significantly worse in hypothyroid patients than in euthyroid patients as was the risk of admission to the Neonatal Intensive Care Unit (NICU) (one study, OR 4.7, 95% CI 1.9–12)(22). This risk was also increased in subclinical hypothyroid patients (one study, OR 1.8, 95% CI 1.2–1.8)(24). There was no evidence for an increase in respiratory distress syndrome (RDS) in children born to hypothyroid patients (one study, OR 2.4, 95% CI 0.31–18)(22). The same was reported for subclinical hypothyroidism, addressed in one study (one study, OR 1.7, 95% CI 0.98–2.8)(24). The risk of an Apgar score <3 after 5 min was comparable in hypothyroid and euthyroid patients (one study, OR 4.8, 95% CI 0.61–39)(22). The study on subclinical hypothyroidism and low Apgar score, reporting on 598 patients and 16 011 controls, indicated an increased risk for low Apgar score in patients (one study, OR 2.2, CI 1.1–4.3) (24).

Congenital malformations were addressed in two studies, reporting no increased risk in children of patients with subclinical hypothyroidism (one study, OR 0.89, 95% CI 0.39–2.0), nor in children of patients with thyroid autoimmunity (1 study, OR 0.54, 95% CI 0.13–2.3)(24;39).

Three studies reported on intelligence score in the offspring of mothers with thyroid dysfunction or autoimmunity(19;20;27). A meta-analysis could not be performed, since outcome measures were reported as intelligence and development scores (continuous variables) and definitions differed between the studies. The study on children of 62 hypothyroid—sometimes treated—women compared with 1245 control children showed an association of hypothyroidism with lower scores on attention and word discrimination (P = 0.01 and P = 0.04, respectively) but no difference in intelligence score(19). The study on subclinical hypothyroidism and TPO-Ab in association with intelligence and motor scores showed decreased intelligence and motor scores in children of women with subclinical hypothyroidism (one study, OR 16, 95% CI 4.7–52 and OR 9.2, 95% CI 2.9–29, respectively, in multivariable analyses)(27). TPO-Ab were also associated with lower scores on intellectual and motor development (one study,OR6.7, 95% CI 2.3–19 and OR 8.3, 95% CI 3.3–21, respectively, in multivariable analyses)(27). The third study showed an inverse correlation between severity of maternal hypothyroidism and intelligence score in the offspring(20). TSH > 99.85th percentile was associated with lower intelligence scores in the offspring (>1 SD below control mean) compared with women with TSH in the normal range (one study, OR 4.7, 95% CI 1.5–14 in multilevel analyses).

The effect of thyroid autoimmunity on post-natal maternal complications

A relation between thyroid autoimmunity and post-partum thyroid disease in the mother was reported in five studies, which were all included in the meta-analysis(30;31;47;52;54). The


meta-analysis, including 305 antibody-positive euthyroid patients and 1342 healthy controls, showed an increased risk of post-partum maternal thyroid disease (five studies, OR 12, 95% CI 5.6–24) (Supplementary data, Fig. S9).

Subgroup analyses of thyroid antibodies

The relationship between the presence of thyroid antibodies and adverse pregnancy outcomes was not different for TPO-Ab compared with Tg-Ab, with the exception of unexplained subfertility. The presence of TPO-Ab was related to unexplained subfertility, while this relationship could not be found for Tg-Ab (four studies, OR 1.5, 95% CI 1.1–2.1 for TPO-Ab, OR 1.1, 95% CI 0.68–1.7 for Tg-Ab) (Supplementary data, Fig. S10) (25;41;48;55). This difference is most likely explained by the fact that Tg-Ab is present less often than TPO-Ab in cases of autoimmune hypothyroidism and is thus a less sensitive marker for detecting of thyroid autoimmunity.

DISCUSSION

The results of this review provide clear evidence for a relationship between the presence of thyroid antibodies or subclinical hypothyroidism on several pregnancy outcome parameters. Subclinical hypothyroidism, compared with normal thyroid function, was associated with the occurrence of pre-eclampsia and showed an increased risk of perinatal mortality. Meta-analyses on the presence of thyroid antibodies showed an increased risk of unexplained subfertility, miscarriage, recurrent miscarriage, preterm birth and post-partum thyroid disease. In contrast to spontaneous pregnancy, miscarriage after IVF was not associated with the presence of thyroid antibodies.

In the current review, by performing meta-analyses we have found associations that have been unclear or underreported so far. Subclinical hypothyroidism in early pregnancy, compared with normal thyroid function, is associated with the occurrence of pre-eclampsia (OR 1.7, 95% CI 1.1–2.6). We also showed a significantly increased risk of perinatal mortality in women with subclinical hypothyroidism in early pregnancy (OR 2.6, 95% CI 1.6–4.7), a relationship which needs attention, especially in respect of therapeutic options. If, for example, thyroxin supplementation early in pregnancy can reduce perinatal mortality, an important clinical health gain may be achieved. A causal relationship cannot be found between subclinical hypothyroidism and a higher incidence of RDS but the increase in mortality may be related to the increased risk of low Apgar scores and NICU admission in the offspring of these patients. Reasons for mortality are not systematically described in the included studies. Our findings emphasize the importance of normal thyroid function in early pregnancy and even before pregnancy. This review is the first to show the association between thyroid antibodies and unexplained subfertility (OR 1.5, 95% CI 1.1–2.0), while individual studies had

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only demonstrated a trend so far. This review showed an association between the presence of thyroid antibodies and recurrent miscarriage (OR 2.3, 95% CI 1.5–3.5). Not all individual studies reported showed this association but the meta-analysis was conclusive on this point, showing the additional value of pooled studies compared with individual studies.

Several hypotheses exist on the causality between thyroid autoimmunity and obstetric complications. The first hypothesis is that the autoimmunity increases the risk for hypothyroidism, owing to the chronic lymphocytic thyroiditis that is associated with the presence of TPO-Ab. The thyroid then may fail to respond adequately to the increased demand for thyroid hormone during pregnancy. The second hypothesis is that thyroid antibodies can be considered an expression of autoimmunity in general and adverse obstetric outcome may be caused by other underlying autoimmune diseases e.g. anticardiolipin antibodies. The third hypothesis assumes that age is more important than the presence of antibodies, since the amount of antibodies increases with aging(61) and age in itself is a risk factor for obstetric complications(62).The third hypothesis seems the least plausible hypothesis for a number of reasons. The majority of the studies included in this review used age-matched control-groups as a reference to their patients. After exclusion of studies not using age-matched control groups, patients with thyroid antibodies still had an increased risk of miscarriage compared with euthyroid patients without antibodies (OR 5.4, 95% CI 1,8–16; Supplementary data, Fig. S11).

Some limitations of this systematic review should be considered. As mentioned, the included articles used different cut-off levels for TSH, T4 and antibodies, and different inclusion criteria for the patients. This should be considered when using the results for clinical application. For instance, antibody positivity was based on the threshold reported in the individual studies and is shown for each study in Table I. TPO-Ab thresholds vary substantially among the studies, but most studies used a more or less generally accepted cutoff value between 50 and 100 kU/l for TPO-Ab. Nevertheless, some degree of population heterogeneity cannot be excluded. Since we used random effect models to perform the meta-analyses of pooled data in case of heterogeneity, and since the majority of data showed a very similar trend, we consider the results to be generally applicable. Individual patient data meta-analysis regarding subclinical hypothyroidism and the different antibodies could be considered in order to calculate a more specific pooled OR for some outcomes and to address the issue of different reference values(63). For some thyroid abnormalities, a limited number of studies on associations with obstetric outcomes were available.

This systematic review does not provide information on the treatment outcome of thyroid dysfunction, as it was not the aim of our study. Nevertheless, the findings in this review are a logical first step prior to any study on the effect of treatment in early pregnancy. Several studies have been performed on the treatment options in thyroid dysfunction and thyroid autoimmunity. The Cochrane review on treatment of (sub)clinical hypothyroidism


in pregnancy was limited to women with a single miscarriage and only included three trials studying different treatment options and a meta-analysis could not be performed(64). The study on the treatment with levothyroxin in TPO-Ab positive women showed a reduction in preterm birth and a non-significant trend towards reduction in miscarriages(10). A reduction in pre-eclampsia was not seen after treatment with levothyroxin(10). This is not surprising, since our meta-analysis did not demonstrate an association between thyroid autoimmunity and pregnancy-induced hypertension, and the single article selected about pre-eclampsia did not show a significant relationship between thyroid autoimmunity and pre-eclampsia. In other words, if an association cannot be demonstrated, treatment options can never be expected to work and, even if causality is suspected, treatment remains to be proven. Not all relationships described in our diagnostic review were addressed in the Cochrane review. This remains an important topic for future research.

We conclude that patients with subclinical hypothyroidism are facing an increased risk of pre-eclampsia and the hitherto underreported risk for perinatal mortality. The presence of thyroid antibodies in euthyroid patients is associated with unexplained subfertility (which was so far unknown), miscarriage, recurrent miscarriage, preterm birth < 37 weeks and post-partum thyroid disease. Special attention in pregnant women at risk for, or diagnosed with, thyroid abnormalities and in non-pregnant patients with a history of recurrent miscarriage is desirable. Therapeutic options and thereby the viability of a standardized screening program remain to be established in the near future.

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32 Stagnaro-Green A, Roman SH, Cobin RH, el-Harazy E, Alvarez-Marfany M, Davies TF. Detection of at-risk pregnancy by means of highly sensitive assays for thyroid autoantibodies. JAMA 1990 Sep 19;264(11):1422-5.

33 Lejeune B, Grun JP, de NP, Servais G, Glinoer D. Antithyroid antibodies underlying thyroid abnormalities and miscarriage or pregnancy induced hypertension. Br J Obstet Gynaecol 1993 Jul;100(7):669-72.

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38 Bussen SS, Steck T. Thyroid antibodies and their relation to antithrombin antibodies, anticardiolipin antibodies and lupus anticoagulant in women with recurrent spontaneous abortions (antithyroid, anticardiolipin and antithrombin autoantibodies and lupus anticoagulant in habitual aborters). Eur J Obstet Gynecol Reprod Biol 1997 Aug;74(2):139-43.

39 Iijima T, Tada H, Hidaka Y, Mitsuda N, Murata Y, Amino N. Effects of autoantibodies on the course of pregnancy and fetal growth. Obstet Gynecol 1997 Sep;90(3):364-9.

40 Kim CH, Chae HD, Kang BM, Chang YS. Influence of antithyroid antibodies in euthyroid women on in vitro fertilization-embryo transfer outcome. Am J Reprod Immunol 1998 Jul;40(1):2-8.

41 Kutteh WH, Schoolcraft WB, Scott RT, Jr. Antithyroid antibodies do not affect pregnancy outcome in women undergoing assisted reproduction. Hum Reprod 1999 Nov;14(11):2886-90.

42 Mavragani CP, Ioannidis JP, Tzioufas AG, Hantoumi IE, Moutsopoulos HM. Recurrent pregnancy loss and autoantibody profile in autoimmune diseases. Rheumatology (Oxford) 1999 Dec;38(12):1228-33.

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44 Dendrinos S, Papasteriades C, Tarassi K, Christodoulakos G, Prasinos G, Creatsas G. Thyroid autoimmunity in patients with recurrent spontaneous miscarriages. Gynecol Endocrinol 2000 Aug;14(4):270-4.

45 Mecacci F, Parretti E, Cioni R, Lucchetti R, Magrini A, La TP, Mignosa M, Acanfora L, Mello G. Thyroid autoimmunity and its association with non-organ-specific antibodies and subclinical alterations of thyroid function in women with a history of pregnancy loss or preeclampsia. J Reprod Immunol 2000 Feb;46(1):39-50.

46 Rushworth FH, Backos M, Rai R, Chilcott IT, Baxter N, Regan L. Prospective pregnancy outcome in untreated recurrent miscarriers with thyroid autoantibodies. Hum Reprod 2000 Jul;15(7):1637-9.

47 Sakaihara M, Yamada H, Kato EH, Ebina Y, Shimada S, Kobashi G, Fukushi M, Fujimoto S. Postpartum thyroid dysfunction in women with normal thyroid function during pregnancy. Clin Endocrinol (Oxf) 2000 Oct;53(4):487-92.

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49 Sieiro NL, Medina CC, Micmacher E, Mamede da CS, Nazar L, Galvao D, Buescu A, Vaisman M. Influence of thyroid autoimmunity and maternal age on the risk of miscarriage. Am J Reprod Immunol 2004 Nov;52(5):312-6.

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51 Ghafoor F, Mansoor M, Malik T, Malik MS, Khan AU, Edwards R, Akhtar W. Role of thyroid peroxidase antibodies in the outcome of pregnancy. J Coll Physicians Surg Pak 2006 Jul;16(7):468-71.

52 Negro R, Greco G, Mangieri T, Pezzarossa A, Dazzi D, Hassan H. The influence of selenium supplementation on postpartum thyroid status in pregnant women with thyroid peroxidase autoantibodies. J Clin Endocrinol Metab 2007 Apr;92(4):1263-8.

53 Shoenfeld Y, Carp HJ, Molina V, Blank M, Cervera R, Balasch J, Tincani A, Faden D, Lojacono A, Doria A, Konova E, Meroni PL. Autoantibodies and prediction of reproductive failure. Am J Reprod Immunol 2006 Nov;56(5-6):337-44.

54 Mamede da CS, Sieiro NL, Coeli CM, Buescu A, Vaisman M. Value of combined clinical information and thyroid peroxidase antibodies in pregnancy for the prediction of postpartum thyroid dysfunction. Am J Reprod Immunol 2007 Oct;58(4):344-9.

55 Bellver J, Soares SR, Alvarez C, Munoz E, Ramirez A, Rubio C, Serra V, Remohi J, Pellicer A. The role of thrombophilia and thyroid autoimmunity in unexplained infertility, implantation failure and recurrent spontaneous abortion. Hum Reprod 2008 Feb;23(2):278-84.

56 Iravani AT, Saeedi MM, Pakravesh J, Hamidi S, Abbasi M. Thyroid autoimmunity and recurrent spontaneous abortion in Iran: a case-control study. Endocr Pract 2008 May;14(4):458-64.

57 Kilic S, Tasdemir N, Yilmaz N, Yuksel B, Gul A, Batioglu S. The effect of anti-thyroid antibodies on endometrial volume, embryo grade and IVF outcome. Gynecol Endocrinol 2008 Nov;24(11):649-55.

58 Montaner P, Juan L, Campos R, Gil L, Corcoy R. Is thyroid autoimmunity associated with gestational diabetes mellitus? Metabolism 2008 Apr;57(4):522-5.

59 Benhadi N, Wiersinga WM, Reitsma JB, Vrijkotte TG, Bonsel GJ. Higher maternal TSH levels in pregnancy are associated with increased risk for miscarriage, fetal or neonatal death. Eur J Endocrinol 2009 Jun;160(6):985-91.

60 Sezer K, Kamel N, Unlu C, Celik HK. Impact of first trimester and postpartum period thyroid autoantibodies on abortus incidence in Turkish pregnant women. Gynecol Endocrinol 2009 Jun;25(6):387-91.

61 Sinclair D. Clinical and laboratory aspects of thyroid autoantibodies. Ann Clin Biochem 2006 May;43(Pt 3):173-83.

62 Dulitzki M, Soriano D, Schiff E, Chetrit A, Mashiach S, Seidman DS. Effect of very advanced maternal age on pregnancy outcome and rate of cesarean delivery. Obstet Gynecol 1998 Dec;92(6):935-9.

63 Broeze KA, Opmeer BC, Bachmann LM, Broekmans FJ, Bossuyt PM, Coppus SF, Johnson NP, Khan KS, ter RG, van d, V, van WM, Mol BW. Individual patient data meta-analysis of diagnostic and prognostic studies in obstetrics, gynaecology and reproductive medicine. BMC Med Res Methodol 2009;9:22.


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SUPPLEMENTARY DATA

Supplementary figure S1a and b. Forest plot off Odds Ratio’s and 95% Confidence Interval of pooled

studies comparing euthyroid thyroid antibody positive patients with euthyroid antibody negative controls

according to (a) the chance of pregnancy in IVF and (b) the risk of miscarriage after IVF.

Supplementary figure S2. Forest plot off Odds Ratio’s and 95% Confidence Interval of pooled studies

comparing patients with subclinical hypothyroidism with euthyroid controls according to the risk of

gestational diabetes mellitus.


Supplementary figure S3a and b. Forest plot off Odds Ratio’s and 95% Confidence Interval of pooled studies

comparing (a) patients with subclinical hypothyroidism with euthyroid controls and (b) euthyroid thyroid

antibody positive patients with euthyroid antibody negative controls according to the risk of pregnancy

induced hypertension.


comparing patients with subclinical hypothyroidism with euthyroid controls according to the risk of

preeclampsia.

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comparing patients with subclinical hypothyroidism with euthyroid controls according to the risk of placental

abruption.


comparing patients with subclinical hypothyroidism with euthyroid controls according to the risk of caesarean

section.



comparing patients with subclinical hypothyroidism with euthyroid controls according to the risk of perinatal

mortality.

Supplementary figure S8a and Sb. Forest plot off Odds Ratio’s and 95% Confidence Interval of pooled

studies comparing patients with subclinical hypothyroidism with euthyroid controls according to the risk of

(a) birth weight <2500 grams and (b) birth weight >4000 grams.

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comparing patients euthyroid thyroid antibody positive patients with euthyroid antibody negative controls

according to the risk of maternal postpartum thyroid disease.


comparing (a) euthyroid TPO antibody positive patients with euthyroid TPO antibody negative controls

and (b) euthyroid Tg antibody positive patients with Tg antibody negative controls according to the risk of

unexplained subfertility.



comparing euthyroid TPO antibody positive patients with euthyroid TPO antibody negative controls according

to the risk of miscarriage, studies included using age-matched controls.

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3 |Increased Thyroid Stimulating Hormone in early

pregnancy is associated with breech presentation

at term: a nested cohort study

R VissenbergTGM VrijkotteJAM van der PostE FliersM GoddijnPH Bisschop

Accepted in adapted form in European Journal of Obstetrics and Gynecology and Reproductive

Biology

Chapter 3 Thyroid hormone levels and breech presentation50 |

ABSTRACT

Objective

Evidence on the relationship between thyroid function disorders and breech presentation is conflicting at present. In this study we aim to determine the association between thyroid function in early pregnancy and breech presentation at term.

Study Design

We used data from the Amsterdam Born Children and their Development (ABCD) cohort. 3347 pregnant women were included between January 2003 and March 2004 in Amsterdam, the Netherlands. Thyroid function tests were performed between 5 and 37 weeks gestational age (median 12.9 weeks). The main outcome measure was the association between thyroid function in early pregnancy and breech presentation at term. Univariate and multivariate analysis were performed to determine the association between thyroid function and breech presentation.

Results

Increased TSH in early pregnancy (mean 13.5 weeks of gestation), defined as thyroid stimulating hormone (TSH) >97.5th percentile (>3.53 mIU/L), was associated with a higher risk for breech presentation at term (aOR 2.32, CI 1.1-4.8, p = 0.02) compared to euthyroidism (TSH between 2.5th and 97.5th percentile). After exclusion of overt hypothyroidism and hyperthyroidism the aOR was 2.34 (CI 1.1-5.0, p = 0.03). Low free thyroxine (FT4) < 2.5th percentile ( < 6.3 pmol/L) was not associated with breech presentation (aOR 1.06, CI 0.4-3.0, p = 0.91).

Conclusions

Increased TSH in early pregnancy is associated with an increased risk for breech presentation at term.

Thyroid hormone levels and breech presentation | 51

INTRODUCTION

Breech presentation has a prevalence of 3-5% in term women and is associated with neonatal and maternal morbidity and mortality(1). Many etiological factors and risk factors for breech presentation have been described, including prematurity, maternal age, BMI, ethnicity, primiparity, pelvic or uterine abnormalities and smoking during pregnancy(2-5). However they only explain 15% of breech presentations(6). Pregnant women who present with breech presentation at birth often undergo a caesarean section, which itself is associated with an increased risk for maternal morbidity and mortality and a greater risk for complications, like uterine rupture in subsequent pregnancies(7;8). Thyroid dysfunction has been described as a possible risk factor for having an abnormal foetal position at birth(9-14). To date five studies have reported on a potential association between thyroid disorders and breech presentation at birth. There were two studies that did not find an association between increased Thyroid Stimulating Hormone (TSH) and/or low free thyroxine (FT4) levels in the first trimester and breech presentation(11;13). One study found an association between low FT4 levels in the first trimester and breech presentation(12). Two studies reported an association between increased TSH levels in the third trimester and increased risk for breech presentation(9;10). In some of the studies the low number of breech deliveries caused difficulties in a precise estimation of the risk because of insufficient statistical power. Moreover, different cut-off levels for plasma TSH and FT4 used in the previous studies hampers direct comparison of the studies. Detection of a possible risk factor for breech presentation is important as breech presentation is associated with maternal and neonatal morbidity and mortality. The other way around, thyroid disorders are also associated with other pregnancy complications(15). Therefore, targeted screening for thyroid disease is advised in pregnant women who are at risk for having thyroid disease, e.g. a previous miscarriage or preterm birth(16;17). If an association with breech presentation exists, targeted screening might be done in women with a previous breech presentation to detect possible thyroid disease and reduce the risk for associated complications in a subsequent pregnancy. Because thyroid disorders are prevalent in pregnancy (2-3%)(18)and the observations from earlier studies are inconsistent, we therefore aim to investigate the association of abnormal TSH and FT4 levels in early pregnancy and breech presentation. This was done in a large Dutch cohort study of more than 3000 pregnant women using population specific reference intervals as spin-off of a large epidemiological study.

MATERIAL AND METHODS

Subjects

Our study was nested within a prospective cohort study of pregnant women from the Amsterdam Born Children and their Development (ABCD) study(19). The main objective of

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the ABCD study is to determine the role of ethnic background, maternal lifestyle factors and psychosocial conditions on pregnancy outcome and health of the offspring. The ABCD study is a collaborative effort of the Municipal Health Services (GGD) and all hospitals and midwife practices in Amsterdam, the Netherlands. All pregnant women living in the city of Amsterdam were invited to participate at their first visit to an obstetric caregiver between January 2003 and March 2004. The study protocol was approved by the Institutional Review Boards of all Amsterdam hospitals and the Registration Committee of Amsterdam. All participants gave their written informed consent.

Physiological changes in TSH and FT4 occur during pregnancy. Trimester specific reference intervals have been shown to vary substantially between different cohorts(20). Therefore we chose to define abnormal thyroid function based 2.5th and 97.5th percentiles. Subjects with normal TSH levels (between 2.5th and 97.5th percentile of the study population) were compared to subjects with high TSH levels > 97.5th percentile and low TSH < 2.5th percentile. In addition, we compared subjects with normal FT4 levels (between 2.5th and 97.5th percentile of the study population) with subjects with low FT4 levels < 2.5th percentile or high FT4 levels > 97.5th percentile. Women that already used thyroid hormone therapy or thyreostatic drugs were excluded. Blood samples were analysed after delivery and therefore no intervention in case of an abnormal TSH or FT4 level was started during pregnancy.

Baseline characteristics

All pregnant women received a questionnaire at their home address within two weeks after their first antenatal visit. The questionnaire contained questions on demographics, health history, medication and lifestyle(21;22). Ethnicity, smoking status during pregnancy, parity, female age, BMI (before pregnancy) and the use of thyroid medication were determined from the self-reported information and completed by information from the national obstetric registry (Perinatal Registration centre of the Netherlands). Information on birth weight, gestational age and foetal sex was based on data from the Youth Health Care Registration of Amsterdam’s Municipal Health Services. Gestational age was based on ultrasound data or, and if ultrasound data were unavailable on the first day of last menstrual period. Information about the foetal position at birth was obtained from the national obstetric registry (Perinatal Registration centre of the Netherlands) after additional informed consent.

Assays

TSH (reference range, 0.34- 5.60 mIU/L) and free thyroxine (FT4) concentration (reference range, 7.5- 21.1 pmol/L) were measured in serum by means of Access immunoanalyzer of Beckman Coultier, Inc. The inter-assay variation for TSH was 5.0% and for free T4 3.1- 5.0%. Antibodies against Thyroid Peroxidase (TPO-Ab) were determined by Elisa ELIZEN TG Ab (E-CK-


96), Zentech, Luik, Belgium. The inter-assay variation was 13.4%. A TPO-ab titre above 80 kIU/L was considered as positive.

Outcomes

The primary outcome was breech presentation at term.

Statistical Analysis

The blood samples were not taken at the same gestational age for all women. Because thyroid function changes physiologically during pregnancy, we corrected TSH and FT4 levels for the gestational age at time of the blood sample (range 34-262 days of gestational age). The data showed a linear association between TSH and gestational age(23). TSH levels were corrected with linear regression ( (Formula: TSHcorrected = TSH-(timebloodsampling-94)*0.002). FT4 levels were quadratic associated with gestational age and corrected according to the formula: FT4corrected = FT4-(timebloodsampling-94)*0.055+ (Squareoftimebloodsampling-94*94/1000)*0.131) where time blood sampling is expressed in days of gestational age). Baseline measurements are presented as means with standard deviations or as numbers with percentages as appropriate. Categorical variables were compared using Pearson’s X2 test or Fisher’s exact test, as appropriate. Student’s t test for independent samples was used to compare continuous variables between two groups. The group with TSH or free T4 levels between the 2.5th and 97.5th percentile served as the reference category. Univariate logistic regression was performed. Maternal age, parity, congenital abnormalities, ethnicity, BMI (before pregnancy), presence of thyroid peroxidase antibodies (TPO-Ab), smoking during pregnancy, gestational age and years of education were considered as potential confounders. Only covariates that were significantly associated with the outcome measure were added to the multivariate model to calculate adjusted odds ratios with 95% confidence intervals. A p value of < 0.05 was considered statistically significant for the univariate analysis. Normal TSH levels ( between 2.5th and 97.5 percentile) were compared with increased TSH levels > 97.5th percentile or with decreased TSH levels < 2.5th percentile. A sensitivity analysis was performed to study the association of subclinical hypothyroidism with breech presentation, after exclusion of women with overt hypothyroidism (TSH >97.5th and FT4 levels <2.5th percentile) and overt hyperthyroidism (TSH < 2.5th percentile and FT4 levels > 97.5th percentile). We also performed univariate and multivariate analysis to determine the association between FT4 and breech presentation. Subjects with normal FT4 levels (between 2.5th and 97.5th percentile) were compared with subject with low FT4 levels < 2.5th percentile and high FT4 levels > 97.5th percentile. P-values of less than 0.05 were considered statistically significant. All statistical analysis was performed using the Statistical package of Social Sciences and Problem Solutions (SPSS version 21.0).

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RESULTS

Of 12,377 pregnant women invited, 8,266 women agreed to participate (response rate 67%). 4,267 women gave additional informed consent for blood collection during their first visit.

We excluded women who gave birth to twins or with missing TSH values. From the remaining 4,183 women, we excluded women who used medication for known thyroid disease: 24 women who used thyroid hormone supplementation, three women who used thyroid hormone supplementation in combination with thyrostatic drugs and three women who only used thyrostatic drugs. Women where the gestational age at the time of blood sampling was unknown were excluded (n=22). Pregnancies that resulted in a miscarriage < 20 weeks of gestation (n = 28) and pregnancies with antenatal mortality or stillbirth (n = 26) or mortality during labour (n = 1) were excluded. Almost 18% (n=729) of the data were missing because women did not provide informed consent to extract this information from the National obstetric registry (Perinatal Registration centre of the Netherlands) or records from respondents could not be linked to a PRN record(24). Eventually 3347 women remained in the study for analysis (Figure 1).

The median TSH level was 1.16 mIU/L (range 0.01 - 50.81 mIU/L). The 2.5th and 97.5th percentiles for TSH were 0.13 en 3.53 mIU/L, respectively. The median FT4 level was 9.56 pmol/L (range 4.1 - 55.5 pmol/L). The 2.5th and 97.5th percentiles for FT4 were 6.26 and 13.42 pmol/L, respectively. Nine women had overt hypothyroidism and 32 had overt hyperthyroidism.

Median time of blood collection was 12.9 weeks of gestation [11.8-14.4]. In 25.0% ( n= 838) blood was assessed in the first trimester ( < 12 weeks of pregnancy). In 70.3 % ( n= 2354) blood analysis took place between 12 and 20 weeks of pregnancy and in 4.6% (n=155) thyroid hormone levels were assessed after 20 weeks of pregnancy.


Figure 1. Flowchart of the selection process.


Baseline characteristics are shown in table 1. Apart from the expected significant lower FT4 levels and a higher percentage of TPO-

Ab positivity in the TSH >97.5th percentile group, baseline characteristics were not different compared with the group women with normal TSH levels. Women with TSH <2.5th percentile were less often nulliparous, of non-western ethnicity, had less years of education, had a higher percentage of offspring of female sex and had higher FT4 concentrations compared to women with TSH levels between 2.5th and 97.5th.

In women with FT4 <2.5th percentile TSH concentrations were higher, there was a lower percentage of nulliparous women parity, a lower percentage of women with a western ethnicity, a significantly higher BMI and less years of education compared with women with FT4 between reference range. Women with increased FT4 levels had a lower TSH level and were less often nulliparous.

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Table 1. Baseline characteristics of 3347 pregnant women.

Characteristic TSH > 97.5th TSH 2.5 - 97.5th TSH < 2.5th FT4 < 2.5th FT4 2.5 – 97th FT4 > 97.5th

(n=86)§ (n=3173)** (n= 88) §§ (n=83) § (n = 3183) ** (n= 81) §§

Maternal age (years) 31.5 ± 4.0 31.1 ± 4.8 30.6 ± 5.1 30.8 ± 6.2 31.1 ± 4.8 30.6 ± 4.8

Gestational age (weeks)

39.3 ± 1.8 39.4 ± 1.9 39.6 ± 1.4 39.2 ± 2.0 39.4 ± 1.9 39.1 ± 2.0

Birth weight 3508 ± 637 3454 ± 570 3478 ± 618 3384 ± 546 3460 ± 572 3382 ± 635

Sex, girl (%) 46 (54) 1608 (51) 56 (64)* 38 (46) 1630 (51) 42 (52)

Thyroid parameters

TSH mU/L 6.1 ± 6.1* 1.3 ± 0.7 0.06 ± 0.06 * 2.7 ±6.5* 1.4 ± 1.0 0.6 ± 0.6*

FT4 pmol/L 8.2 ± 1.6* 9.6 ± 1.7 13.3 ± 6.2* 5.6 ± 0.5* 9.6 ± 1.5 15.7 ± 5.5*

TPO-Ab positivity (%) 42 (49)* 150 (5) 6 (7) 6 (7) 186 (6) 6 (7)

Parity, (% nulliparity) 47 (55) 1846(58) 38 (44)* 38 (46)* 1854 (58) 39 (48)*

Ethnicity

Western (%) 67 (78) 2493 (79) 45 (52)* 43 (52)* 2503 (79) 59 (73)

Nonwestern (%) 19 (22) 680 (21) 43 (48) 40 (48) 680 (21) 22 (27)

BMI (kg/m2) 23.0 ± 3.9 22.9 ± 3.8 23.0 ± 3.3 23.8 ± 4.1* 22.9 ± 3.8 22.7 ± 4.1

Smoking while pregnant , n (% yes)

5 (6) 308 (10) 5 (6) 13 (16) 299 (9) 6 (7)

Years of education 9.5 ± 3.5 9.3 ± 3.8 8.3 ± 4.9* 7.0 ± 4.2* 9.3 ± 3.8 8.8 ± 3.5

Continuous characteristics are expresses as mean SD* Significant difference p < 0.05** Reference group§ n=9 overt hypothyroid

§§ n=32 overt hyperthyroid

Breech presentation

Data on presentation at birth were available for 3347 women. Maternal age (p= 0.03), parity (p=< 0.01), gestational age at delivery (p < 0.01) and years of

education (p=0.03) were significantly associated with breech position in the univariate model and therefore added to the multivariate model. With univariate analysis, presence of TPO-Ab was not significantly related to the risk of breech presentation.


Results from the multivariate logistic regression are shown in table 2. Maternal age (p = 0.006), parity (< 0.01) and gestational age at delivery ( P < 0.01) were still significantly associated with breech presentation in the multivariate model. A maternal TSH level above the 97.5th percentile was associated with an increased risk for breech presentation (aOR 2.32, CI 1.1-4.8, p = 0.02). A maternal TSH level below the 2.5th percentile was not associated with breech presentation (aOR 0.56, CI 0.1-2.3, p 0.42). Low FT4 levels were not associated with an increased risk for breech presentation (aOR 1.06, CI 0.4-3.0, p = 0.91). A high fT4 > 97.5th percentile was also not associated with breech presentation (aOR 0.68, CI 0.2-2.3, p = 0.54).

Table 2. Univariate and multivariate logistic regression analysis of the association between thyroid function and breech presentation.

Breech, n (%) OR 95% CI P-Value aOR 95% CI p-Value

TSH 2.5-97.5th percentile, n= 3173* 154 (5%)

TSH < 2.5th percentile, n = 88 2 (2%) 0.46 0.1-1.9 0.28 0.56 0.1-2.3 0.42

TSH > 97.5th percentile, n = 86 9 (10%) 2.29 1.1-4.7 0.02 2.32 1.1-4.8 0.02

FT4 2.5-97.5 percentile, n= 3183 * 158 (5%)

FT4 < 2.5th percentile, n = 83 4 (5%) 0.97 0.4-2.7 0.95 1.06 0.4-3.0 0.91

FT4 > 97.5th percentile, n = 81 3 (4%) 0.74 0.2-2.4 0.61 0.68 0.2-2.3 0.54

Corrected for the covariates, maternal age, gestational age at delivery, parity and years of education

*Reference group

Sensitivity analysis

Sensitivity analysis was done to correct for the women with overt hypothyroidism (n = 9) and overt hyperthyroidism (n= 32), those were excluded for analysis. The odds for breech position in women with subclinical hypothyroidism was 2.34 (CI 1.1-5.0, p = 0.03) (supplementary table 1).

Forest plot with earlier results from the literature

Data were pooled with data from one other cohort study investigating the association between abnormal TSH levels (> 4 mIU/L) in early pregnancy(11) (supplementary figure 1). Hypothyroidism is significantly related to an increased risk for breech presentation (two studies, OR 2.47, 95% CI 1.40-4.35, p = 0.002).

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COMMENT

This study shows that TSH levels > 3.53 mIU/L in early pregnancy are associated with an increased risk for breech presentation. After exclusion of women with overt hypothyroidism and hyperthyroidism an increased risk for breech presentation remained. This suggests that the observed effect is not explained only by women with overt thyroid disease. The strength of this study is the large and unselected study population in which testing for thyroid disorders was done, but the study also has some limitations. The participation rate was 67% of which 50% agreed to additional blood sampling. There was a relatively high percentage of missing data on breach position (18%). This might have introduced selection bias which is further investigated in supplementary table 2. Baseline characteristics from the group of women with missing data were compared with those of women with available data on the outcome breech. Only a significant difference of 0.7 was found in mean maternal age. It seems unlikely that this small difference would have resulted in different rates of breech presentation in the non-responders.

This is the first study to show a relationship between increased TSH in early pregnancy and breech presentation. Previous studies found no association between TSH levels in the first trimester of pregnancy and breech position(9;11). This can be explained by the fact that they have compared differences in mean TSH levels or quintiles and that the studies were underpowered.

There are two hypotheses on a possible causal relationship between TSH levels and the increased risk for breech position. The first is a possible negative effect of maternal thyroid dysfunction on foetal movements and mobility as motor development of children born to hypothyroid mothers is delayed(12). Foetal movements are necessary to establish a cephalic presentation(25). The second hypothesis is that thyroid dysfunction has a negative effect on the uterine contractions(9). Hypothyroid rats had a lower amplitude and frequency of spontaneous rhythmic contractions of the myometrium probably caused by a reduction of the uterine myometrial Ca 2+ channel function(26;27). Uterine contractions are important for final cephalic presentation at term(3).

Known risk factors of breech presentation only explains 15% all together. This study shows that increased TSH levels is another risk factor for breech presentation at term. However, given the fact that the aOR was 2.32 and the p-value of high TSH in the multiple logistic regression model is only 0.02, this suggests a low effect size and contribution of high TSH to the prediction of breech presentation. The fact that only data on the foetal position at time of birth were available could have resulted in an underestimation of the effect of TSH on breech presentation, as in the Netherlands women are offered an external version when a foetus presents in breech position. This has a success rate of almost 40% (28). No information on external cephalic version attempts was available.


Breech delivery is associated with adverse perinatal outcome. This could be related more to thyroid dysfunction of women than breech position in itself though this needs more research. Targeted screening for thyroid disease is advised in pregnant women who are at risk for having thyroid disease or increased TSH, e.g. a positive family history for thyroid disease, obesity, previous miscarriage or preterm birth etc(16;17). The association found in this study between increased TSH levels and breech presentation at term is not strong enough to have direct clinical utility and to recommend screening of women with a previous breech delivery as well. More research, eg a larger prospective cohort study, is recommended to further investigate this association (preferably within different subgroups) and the exact clinical relevance. Chapter

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REFERENCES

1 Hill LM. Prevalence of breech presentation by gestational age. Am J Perinatol 1990 Jan;7(1):92-3.

2 Rayl J, Gibson PJ, Hickok DE. A population-based case-control study of risk factors for breech presentation. Am J Obstet Gynecol 1996 Jan;174(1 Pt 1):28-32.

3 Albrechtsen S, Rasmussen S, Dalaker K, Irgens LM. The occurrence of breech presentation in Norway 1967-1994. Acta Obstet Gynecol Scand 1998 Apr;77(4):410-5.

4 Roberts CL, Algert CS, Peat B, Henderson-Smart D. Small fetal size: a risk factor for breech birth at term. Int J Gynaecol Obstet 1999 Oct;67(1):1-8.

5 Witkop CT, Zhang J, Sun W, Troendle J. Natural history of fetal position during pregnancy and risk of nonvertex delivery. Obstet Gynecol 2008 Apr;111(4):875-80.

6 Nordtveit TI, Melve KK, Albrechtsen S, Skjaerven R. Maternal and paternal contribution to intergenerational recurrence of breech delivery: population based cohort study. BMJ 2008 Apr 19;336(7649):872-6.

7 Rietberg CC, Elferink-Stinkens PM, Visser GH. The effect of the Term Breech Trial on medical intervention behaviour and neonatal outcome in The Netherlands: an analysis of 35,453 term breech infants. BJOG 2005 Feb;112(2):205-9.

8 Ecker JL, Frigoletto FD, Jr. Cesarean delivery and the risk-benefit calculus. N Engl J Med 2007 Mar 1;356(9):885-8.

9 Kuppens SM, Kooistra L, Wijnen HA, Crawford S, Vader HL, Hasaart TH, Oei SG, Pop VJ. Maternal thyroid function during gestation is related to breech presentation at term. Clin Endocrinol (Oxf) 2010 Jun;72(6):820-4.

10 Kooistra L, Kuppens SM, Hasaart TH, Vader HL, Wijnen HA, Oei SG, Pop VJ. High thyrotrophin levels at end term increase the risk of breech presentation. Clin Endocrinol (Oxf) 2010 Nov;73(5):661-5.

11 Salehidobakhshari M, Bamforth F, Burstyn I. Maternal thyroid hormones in early pregnancy and risk of breech presentation. J Obstet Gynaecol Can 2010 Oct;32(10):948-55.

12 Pop VJ, Brouwers EP, Wijnen H, Oei G, Essed GG, Vader HL. Low concentrations of maternal thyroxin during early gestation: a risk factor of breech presentation? BJOG 2004 Sep;111(9):925-30.

13 Mannisto T, Vaarasmaki M, Pouta A, Hartikainen AL, Ruokonen A, Surcel HM, Bloigu A, Jarvelin MR, Suvanto-Luukkonen E. Perinatal outcome of children born to mothers with thyroid dysfunction or antibodies: a prospective population-based cohort study. J Clin Endocrinol Metab 2009 Mar;94(3):772-9.

14 Wijnen HA, Kooistra L, Vader HL, Essed GG, Mol BW, Pop VJ. Maternal thyroid hormone concentration during late gestation is associated with foetal position at birth. Clin Endocrinol (Oxf) 2009 Nov;71(5):746-51.

15 van den Boogaard E, Vissenberg R, Land JA, van WM, van der Post JA, Goddijn M, Bisschop PH. Significance of (sub)clinical thyroid dysfunction and thyroid autoimmunity before conception and in early pregnancy: a systematic review. Hum Reprod Update 2011 Sep;17(5):605-19.


17 NIV (Nederlandse Internisten Vereniging), Richtlijn Schildklierfunctiestoornissen. 2012.



19 van EM, Vrijkotte TG, Gemke RJ, van der Wal MF. Cohort profile: the Amsterdam Born Children and their Development (ABCD) study. Int J Epidemiol 2011 Oct;40(5):1176-86.

20 Medici M, Korevaar TI, Visser WE, Visser TJ, Peeters RP. Thyroid Function in Pregnancy: What Is Normal? Clin Chem 2015 May;61(5):704-13.

21 Radloff LS. The CES-D scale: a self-report depression scale for research in the general population. Applied Psychological Measurement. 1, 385-401. 1977.

22 Karasek R, Brisson C, Kawakami N, Houtman I, Bongers P, Amick B. The Job Content Questionnaire (JCQ): an instrument for internationally comparative assessments of psychosocial job characteristics. J Occup Health Psychol 1998 Oct;3(4):322-55.

23 Benhadi N, Wiersinga WM, Reitsma JB, Vrijkotte TG, van der Wal MF, Bonsel GJ. Ethnic differences in TSH but not in free T4 concentrations or TPO antibodies during pregnancy. Clin Endocrinol (Oxf) 2007 Jun;66(6):765-70.

24 Tromp M, van EM, Ravelli AC, Bonsel GJ. Anonymous non-response analysis in the ABCD cohort study enabled by probabilistic record linkage. Paediatr Perinat Epidemiol 2009 May;23(3):264-72.

25 Suzuki S, Yamamuro T. Fetal movement and fetal presentation. Early Hum Dev 1985 Sep;11(3-4):255-63.

26 Elmehdawi RR, Lashika EI. Prolonged pregnancy: A possible complication of hypothyroidism due to myometriopathy. Med Hypotheses 2008;70(1):209-10.

27 Parija SC, Raviprakash V, Telang AG, Varshney VP, Mishra SK. Influence of hypothyroid state on 45Ca(2+) influx and sensitivity of rat uterus to nifedipine and diltiazem. Eur J Pharmacol 2001 Jun 15;421(3):207-13.

28 De HM, Vlemmix F, Kok M, Van Der Steeg JW, Bais JM, Mol BW, van der Post JA. External validation of a prediction model for successful external cephalic version. Am J Perinatol 2012 Mar;29(3):231-6.

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SUPPLEMENTARY DATA

Supplementary table 1. Sensitivity analysis with exclusion of women with overt hypothyroidism and hyperthyroidism.

n (%) OR 95% CI P-Value aOR 95% CI p-Value

Breech presentation

TSH 2.5-97.5th percentile, n =3173* 154 (5%)

TSH < 2.5th percentile. n= 88 2 (2%) 0.36 0.04-2.6 0.31 0.42 0.06-3.1 0.40

TSH > 97.5th percentile, n = 77 8 (10%) 2.27 1.1-4.8 0.03 2.34 1.1-5.0 0.03

Corrected for the covariates, maternal age, gestational age at delivery, parity and years of education.* Reference group.

Supplementary table 2. Comparison of baseline characteristics of 3347 study subjects versus 729 women with missing outcome.

Characteristic Data on outcome breech available

Data missing on outcome breech

P-Value

(n=3347) (n=729)

Maternal age (years)* 31.1 ± 4.8 30.4 ±5.0 0.002

Gestational age (weeks) 39.4 ± 1.9 39.4 ± 1.7 0.96

Birth weight (grams) 3456 ± 573 3434 ± 547 0.36

Thyroid parameters

TSH mU/L 1.37 ± 1.42 1.28 ± 0.91 0.09

FT4 pmol/L 9.64 ± 2.03 9.47 ± 1.75 0.24

TPO-Ab positivity (%) 198 (5.9) 40 (5.5) 0.73

Parity, (% nulliparity) 1931 (58) 407 (56) 0.36

Ethnicity

Western (%) 2605 (78) 548 (75%) 0.13

Nonwestern (%) 742 (22) 181 (25)

BMI (kg/m2) 22.9 ±3.8 22.6 ± 3.5 0.12

Smoking while pregnant , n (% yes) 318 (9.5) 69 (9.5) 1.0

Years of education 9.2 ± 3.8 9.1 ± 3.9 0.26

Continuous characteristics are expresses as mean SD* Significant difference p < 0.05


Supplementary figure 1. Forest plot of Odds Ratio’s and 95% Confidence Interval of pooled studies

comparing hypothyroid patients with euthyroid controls in early pregnancy according to the risk of breech

presentation at term.

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4 |Is subclinical hypothyroidism associated with

lower live birth rates in women with unexplained

recurrent miscarriage?

R Vissenberg*MM van Dijk*PH BisschopF DawoodM van WelyM GoddijnRG Farquharson

*These authors contributed equally to the manuscript

Submitted

Chapter 466 |

ABSTRACT

Thyroid disorders, especially hypothyroidism, have been associated with (recurrent) miscarriage. Little evidence is available on the influence of subclinical hypothyroidism on live birth rates. In this study the influence of subclinical hypothyroidism on live birth rates in women with recurrent miscarriage (RM) was investigated. The study was performed in a tertiary recurrent miscarriage clinic from 2000 - 2011 (Liverpool Women’s Hospital, UK). The study group consisted of women with unexplained RM and subclinical hypothyroidism (defined as thyroid-stimulating hormone [TSH] > 97.5th percentile mU/L with a normal free thyroxine) and the control group were women with unexplained RM and a normal thyroid function (defined as TSH level between 2.5th and 97.5th percentile). To investigate the association of subclinical hypothyroidism on live birth rates, ongoing pregnancy rates and miscarriage rates multivariable logistic regression was performed. Data were available for 848 women. Twenty women (2.4%) had subclinical hypothyroidism, 818 women (96%) were euthyroid and 10 women (1.2%) had overt hypothyroidism. The live birth rate was 45% in women with subclinical hypothyroidism and 52% in euthyroid women (OR 0.69, 95% CI 0.28 – 1.71). The ongoing pregnancy rate was 65% in women with subclinical hypothyroidism and 69% in euthyroid women (OR 0.82, 95% CI 0.32 – 2.10). The miscarriage rate was 35% in women with subclinical hypothyroidism and 28% in euthyroid women (OR 1.42, 95% CI 0.55 – 3.67). No differences in live birth rates, ongoing pregnancy rates and miscarriages rates were found when TSH 2.5 mU/l was used as cut-off level to define subclinical hypothyroidism. In our study no significant difference was found in live birth rates, ongoing pregnancy rates and miscarriage rates in women with unexplained RM and subclinical hypothyroidism compared to euthyroid women with unexplained RM.

Recurrent miscarriage and subclinical hypothyroidism | 67

INTRODUCTION

A spontaneous miscarriage occurs in ~15% of all clinically recognized pregnancies in the general population. Recurrent miscarriage (RM) has a prevalence of 1 – 3% of all couples trying to conceive(1). Couples with parental chromosome abnormalities and women with uterine anomalies, endocrine disturbances, hyperhomocysteinemia and antiphospholipid syndrome have a higher risk for RM. Despite comprehensive investigations, an underlying risk factor for RM is identified in less than 50% of couples(2). At present, no effective treatment has been established to improve the live birth rates for women with unexplained RM. For couples with RM a reliable prognosis for the chance of a live birth is of utmost importance in their decision whether or not to conceive again as RM has often distressing physical and emotional consequences(3).

Thyroid disorders, especially hypothyroidism, have been associated with miscarriage. Overt hypothyroidism is associated with an increased risk for miscarriage (OR 5.78, 95% CI 2.4 – 14), but also with other pregnancy complications like low birth weight, premature delivery, placental abruption and pregnancy-induced hypertension(1;4-7). It often presents with clinical symptoms and therefore most women already receive treatment before conception.

Subclinical hypothyroidism is a more common thyroid disorder among women of fertile age. It is defined as a raised serum thyroid-stimulating hormone (TSH) level above the upper limit of normal with a normal level of total thyroxine (TT4). The prevalence of subclinical hypothyroidism has been estimated between 4.0% and 8.5% in the normal population and between 1.5% and 4% in pregnancy(4;8;9). Thus far, the studies on a relation between subclinical hypothyroidism and RM are conflicting. Also, the effect of subclinical hypothyroidism on live birth rates in women with recurrent miscarriage is unclear and limited to one published study. In this observational cohort study that compared 55 patients with RM and subclinical hypothyroidism (TSH ≥ 2.5 mU/L) (19% of the total cohort) to euthyroid women with RM, no significant difference in the subsequent live birth rates was found(10). The evidence on the association of subclinical hypothyroidism with a single miscarriage is conflicting. In a recent prospective cohort study that screened 3147 women with a singleton pregnancy an elevated risk of miscarriage was found in women with subclinical hypothyroidism compared to euthyroid women (OR 3.40, 95% CI 1.62 – 7.15)(11). Another prospective cohort study of 2479 women reported an increased risk of miscarriage, fetal death and neonatal death for every doubling in TSH concentration (OR 1.60, 95% CI 1.04–2.47)(12). A large cohort study with 240 patients with subclinical hypothyroidism and 10.518 euthyroid women did not find a difference in miscarriage rates between both groups (OR 0.69, 95% CI 0.10 – 5.0)(13).

Interpreting the data on studies about subclinical hypothyroidism is complicated since different cut off levels for TSH are being applied and there is also inter-laboratory differences using various different analysers. There is no consensus on the optimal TSH cut-off level to define

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Chapter 468 |

subclinical hypothyroidism. The American Thyroid Association recommends TSH levels to be trimester specific, with 2.5 mU/l as upper limit and the Endocrine Society advices a TSH level of 0.1 – 2.5 mU/L in the first trimester. The British Thyroid association recommends a TSH at the lower end of the reference range or a little below (TSH 0.4 – 2.0mU/L)(14). Recommendations on cut-off levels for diagnosing subclinical hypothyroidism at pre-conception are not available. TSH levels are furthermore population specific with intrinsic ethnic variation and data cannot automatically be extrapolated to all ethnicities(15).

Subclinical hypothyroidism is associated with pregnancy complications and evidence on its association with RM is conflicting. Therefore, we found it important to evaluate the effect of subclinical hypothyroidism on live birth rates in women with RM. We conducted this cohort study to investigate live birth rates in women with unexplained RM and subclinical hypothyroidism.

MATERIAL AND METHODS

Study population

The study population consisted of female patients (18 - 40 years of age) with RM who presented to the recurrent miscarriage clinic of the Liverpool Women’s Hospital, in Liverpool United Kingdom in the period 2000 – 2011.

Recurrent miscarriage was defined according to the Special Interest Group for Early Pregnancy (European Society of Human Reproduction and Endocrinology) consensus statement as two or more, not necessarily consecutive miscarriages before 20 weeks of gestation, verified by a pregnancy test and/or ultrasonography(16). Unexplained RM was defined when an underlying risk factor for RM was not present. Diagnostic work up for RM included testing for antiphospholipid syndrome (lupus anticoagulant, IgG and IgM anti-cardiolipin antibodies), uterine abnormalities, thrombophilia (Factor V Leiden mutation, prothrombin gene mutation, protein C deficiency, protein S deficiency, antithrombin deficiency) and/or hyperhomocysteinemia.

Women with pre-existent thyroid disease or women who were using thyroid drugs were excluded. Women were not included for analysis if their evaluation did not included thyroid function tests (TSH and/or T4) and when no data for the outcome measure was available. The diagnostic work up of RM did not include testing for the presence of TPO-Ab according to recent guidelines, where TPO-Ab screening is not advised in routine RM workup(17-19).

The study group was defined as women with unexplained RM and subclinical hypothyroidism (defined as a serum TSH level above the 97.5th percentile with a normal serum thyroxine (T4) level between the 2.5 - 97.5th percentile). The control group consisted of euthyroid women (TSH between 2.5 and 97.5th percentile) with unexplained RM. Data on RM diagnostic


work up and subsequent pregnancy outcomes were collected. Data were anonymized before analysis. The present study was defined retrospectively.

Assays

Thyroid function tests were measured by an immunometric assay performed on the e602 analyzer (Roche Diagnostics) with a detection limit of 0.01 mU/L and total assay variation of 2-4%. The reference range was 0.3 - 6.0 mU/L for TSH, and 60 – 150 nmol/L for total T4.

Outcomes

The primary outcome was live birth rates (LBR), defined as a live birth after 24 weeks of gestation. Secondary outcome measures were ongoing pregnancy rates, defined as a pregnancy of more than 12 weeks gestational age and miscarriage rates, defined as pregnancy loss before 20 week of gestational age.

Statistical analysis

For descriptive statistics, the mean with standard deviation (SD) was used. To calculate differences in of the outcome measures, independent sample t-tests (two tailed) were used for continuous variables and a normal distribution. For continuous variables without a normal distribution Mann-Whitney U test was applied. To investigate the association of subclinical hypothyroidism on live birth rates, ongoing pregnancy rates and miscarriage rates multivariable logistic regression was performed. The covariates maternal age and number of previous miscarriages were selected a priori and added in the multivariate model. A 2-tailed p < 0.05 was judged statistically significant. Statistical analyses were performed using SPSS 20.0 (IBM). In a sensitivity analysis the above analyses were repeated with a serum TSH above 2.5 mU/L with a normal serum T4 level.

Ethical approval

Approval for this study was obtained from the Medical Regional Ethics Committee of the Liverpool Women’s Hospital (Number LWH0914).

RESULTS

The flowchart of the selection process is presented in figure 1. Between 2000 and 2011, a total of 1956 women visited the recurrent miscarriage clinic of the Liverpool Women’s Hospital. One hundred and fifty-six women did not undergo full diagnostic work-up for recurrent miscarriage. A total of 1800 women (92%) underwent investigations to assess associated factors for RM. In 384 women (21%) thyroid function tests were not assessed. Thyroid function

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test included assessment of TSH and T4. According to the current RM guidelines, the presence of thyroid peroxidise antibodies was not examined. Women with associated factors for RM were excluded: 79 women (4.4%) with antiphospholipid syndrome (APS), 27 women (1.5%) with pre-existing thyroid disease, 25 women (1.4%) with thrombophilia and 11 women (0.6%) with uterine abnormalities. The remainder of the patients were 1274 women with unexplained recurrent miscarriage and thyroid function tests. Data on the primary outcome was available for 848 patients (67%).

Median TSH was 1.7 mU/L (range 0.05 – 13.9 mU/L). The 2.5th and 97.5th percentiles for TSH were 0.5 and 4.6 mU/L, respectively. The median TT4 value was 98 nmol/L, the 2.5th and 97.5th percentiles for TT4 were 71 and 140 nmol/L, respectively. Subclinical hypothyroidism was defined as a serum TSH above 4.6 mU/L, calculated from the 97.5th percentile with normal TT4 levels. According to this definition 20 (2.4%) women met the criteria of subclinical hypothyroidism; 818 (96%) were euthyroid. Ten (1.2%) patients had overt hypothyroidism and were excluded from further analysis.




Baseline characteristics are presented in table 1. There were no differences in baseline characteristics between the two groups. Maternal age in women with RM and subclinical hypothyroidism compared to euthyroid women with RM were comparable (33.8 vs 32.9 years, p = 0.5). No significant differences in previous first trimester pregnancy losses and number of previous live births were found in both groups. The median TSH was 5.4 mU/l (4.6 – 13.9 mU/l) in the group of women with subclinical hypothyroidism and 1.6 mU/l (0.5 – 4.5 mU/l) in the euthyroid group.

Table 1. Baseline clinical characteristics.

Subclinical hypothyroidism(n = 20)

Euthyroidism(n = 818) p-value

Female age (years) (mean (SD)) 33.8 (4.5) 32.9 (5.8) 0.50

Number of previous miscarriages - 1st trimester miscarriages- 2nd trimester miscarriages

Number of previous live births

TSH (mU/l)

3 (2 – 4)2 (0 – 4) 0 (0 – 2)

0 (0 - 1)

5.4 (4.6 – 13.9)

3 (2 - 11)3 (0 – 10) 0 (0 - 4)

0 (0 - 6)

1.6 (0.05 – 4.5)

0.070.030.31

0.16

0.00

Data are expressed as median (range).

Live birth rates

The live birth rates were 45% (9/20) in women with subclinical hypothyroidism and 52% (428/818) in euthyroid women. With multivariate logistic regression, adjusted for maternal age and previous number of pregnancy losses, no evidence of a difference was found in live birth rates between the two groups (OR 0.69, 95% CI 0.28 – 1.71) (Table 2).

Pregnancy rates

The ongoing pregnancy rates were 65% (13/20) in women with subclinical hypothyroidism and 69% (562/818) in euthyroid women. The odds ratio adjusted for maternal age and previous number of pregnancy losses suggested no significance difference in ongoing pregnancy chance between the two groups (OR 0.82 95% CI 0.32 – 2.10) (Table 2).

Miscarriage rates

The miscarriage rates were 35.0% (7/20) in women with subclinical hypothyroidism and 28.1% (230/818) in euthyroid women. The odds ratio adjusted for maternal age and previous number of pregnancy losses suggested no significance difference in miscarriage rate between the two groups (OR 1.42, 95% CI 0.55 – 3.67) (Table 2).

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Table 2. Ongoing pregnancies, live births, miscarriages in women with subclinical hypothyroidism (TSH > 4.5 mU/l) compared to euthyroid women.


Euthyroidism(n = 818) OR 95% CI

Ongoing pregnancies Resulting in live births

Miscarriages

Other

13 (65.0%)

9 (45.0%)

7 (35.0%)

0

562 (68.7%)

428 (52.3%)

230 (28.1%)

26 (3.2%)

0.82

0.69

1.42

0.32 – 2.10

0.28 – 1.71

0.55 – 3.67

Data are expressed as numbers (percentages).Other includes ectopic pregnancies, termination of pregnancy and biochemical pregnancy losses.

Sensitivity analysis

When subclinical hypothyroidism was defined as a serum TSH above 2.5 mU/L with a normal serum TT4 level, 176 (21%) women met the criteria for subclinical hypothyroidism; 660 (78%) were euthyroid; 21 (2.5%) patients had overt hypothyroidism and were excluded from analysis. The live birth rates according to this definition were 55% (97/176) in women with subclinical hypothyroidism and 51% (339/660) in euthyroid women (OR 1.12, 95% CI 0.79 – 1.58). The ongoing pregnancy rates were 70% (123/176) in women with subclinical hypothyroidism and 68% (451/660) in euthyroid women (OR 1.06, 95% CI 0.73 – 1.53).The miscarriage rates were 29% (51/176) in women with subclinical hypothyroidism and 28% (186/660) in euthyroid women (OR 1.06, 95% CI 0.73 – 1.55) (Table 3).

Table 3. Ongoing pregnancies, live births, miscarriages in women with subclinical hypothyroidism (TSH > 2.5 mU/l) compared to euthyroid women.


Euthyroidism(n = 660) OR 95% CI

Ongoing pregnancies Resulting in live births

Miscarriages

Other

123 (69.9%)

97 (55.1%)

51 (29.0%)

2 (1.1%)

451 (68.3%)

339 (51.4%)

186 (28.2%)

23 (3.5%)

1.06

1.12

1.06

0.94

0.73 – 1.53

0.79 – 1.58

0.73 – 1.55

0.83 – 1.65

Data are expressed as numbers (percentages).Other includes ectopic pregnancies, termination of pregnancy and biochemical pregnancy losses.


DISCUSSION

Main findings

We found a prevalence of 2.4% of subclinical hypothyroidism in a cohort of 848 of women with unexplained recurrent miscarriage. No statistically difference was found in the subsequent live birth rates (OR 0.69, 95% CI 0.28 – 1.71), ongoing pregnancy rates (OR 0.82, 95% CI 0.32 – 2.10) and miscarriage rates (OR 1.42, 95% CI 0.55 – 3.67) in women with RM and subclinical hypothyroidism compared to euthyroid women.

Comparison of results with existing literature

The prevalence of subclinical hypothyroidism in this cohort (2.4%) corresponds with the prevalence of 1.5% - 4% in pregnant women and is slightly lower in the general non pregnant population (4% to 8.5%)(4;8). The incidence of subclinical hypothyroidism is affected by age, sex, race, geographic location and varies according to the TSH level used to define subclinical hypothyroidism. When a TSH cut off level of 2.5 mU/L was chosen, a prevalence of 21% of subclinical hypothyroidism was found. This is higher compared to recent publications where a TSH >2.5 mU/L as a threshold is applied, with prevalence’s varying between 15.0 – 15.6%(7;20). When TSH >2.5mU/L was used as a cut-off level to define subclinical hypothyroidism, this could have resulted in an overestimation of the prevalence of women with subclinical hypothyroidism. Moreover, since the diagnosis of subclinical hypothyroidism was made before conception and women tend to have higher levels of TSH compared to during pregnancy. On the other hand, women with recurrent miscarriage could possibly have a higher average serum TSH and could suffer more frequently from subclinical hypothyroidism than stated in this study. Since reference ranges are determined with the 2.5th and 97.5th percentiles, we choose to define subclinical hypothyroidism with TSH with an upper range distracted from the 97.5th percentile. When using a lower cut-off for serum TSH levels, no difference in live birth rates was seen either.

The results of our study are in line with the additional study on live birth rates in women with subclinical hypothyroidism and RM(10). When the results of the two studies are pooled in a meta-analysis no difference in live birth rates were found (OR 1.10, 95% CI 0.81 – 1.49) (supplementary figure 1). We found a high miscarriage rate in our study population (28.3%). This might be explained by the finding that the most important risk factors for another miscarriage after RM are increasing maternal age and number of prior pregnancy losses(21).

Strengths and limitations

This is the second cohort study investigating live birth rates in women with RM and subclinical hypothyroidism. With our study more understanding is generated in this, so far unsolved,

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subject. Since recurrent miscarriage is multifactorial, research becomes challenging in assessing the impact of a single factor in isolation. We accounted for this by excluding women with antiphospholipid syndrome, thrombophilia and uterus anomalies which are all well-known risk factors for recurrent miscarriage(2;22).

Potential limitations of the present study are the retrospective design and the risk for selection bias. The live birth rate in our study was lower than expected. This might be explained by the fact that in 33% of our study population data on the primary outcome measure was missing and therefore excluded from analysis. This might have introduced a selection bias as it can be assumed that women with a normal subsequent pregnancy not always visit the tertiary RM again. Despite the large database, a relatively low number of study subjects with subclinical hypothyroidism remained, due to a low prevalence. Sample size calculation showed that approximately 24.000 women would be required to detect a 5% absolute difference in live birth rate (80% power at a two-sided alpha level of 0.05), assuming a 75% live-birth rate in the euthyroid group and 2.5% prevalence of subclinical hypothyroidism. When a TSH cut-off level of 2.5 mU/l would be used 3600 women would be required. Therefore this study was underpowered. In our study with 848 women, with a prevalence of 2.5% of subclinical hypothyroidism and a live birth rate of 52%, we could prove a difference in live birth rate of 28%. When a TSH cut-off level of 2.5mU/l was applied and a live birth rate of 52%, we could prove a difference of 13% in live birth rates.

The diagnostic work up of RM did not include testing for the presence of thyroid peroxidise antibodies (TPO-Ab) according to recent guidelines, where TPO-Ab screening is not advised in routine RM workup(17-19). More interest is showed in thyroid auto-immunity and the association with RM, therefore this might be a limitation. More knowledge on pregnancy chances and prognostic factors for success in women with recurrent miscarriages is required. Although our study did not find lower birth rates in women with RM and subclinical hypothyroidism such an association could not be ruled out and further prospective studies are mandatory. It would be interesting to investigate TPO-Ab in this relationship simultaneously. Especially, since the American Thyroid Association guideline advises to define subclinical hypothyroidism according to the TPO-Ab status(19).

Current guidelines do not recommend screening and treatment of subclinical hypothyroidism in women with RM(17;18). The results of this study support this advice. It remains to be established whether screening and subsequent treatment will improve pregnancy outcomes in women with subclinical hypothyroidism and RM. The potential benefit of any screening strategy critically depends on the relative contribution of thyroid dysfunction to adverse pregnancy outcomes and on the impact of treatment.


REFERENCES


2 Alijotas-Reig J, Garrido-Gimenez C. Current concepts and new trends in the diagnosis and management of recurrent miscarriage. Obstet Gynecol Surv 2013 Jun;68(6):445-66.

3 Kolte AM, Olsen LR, Mikkelsen EM, Christiansen OB, Nielsen HS. Depression and emotional stress is highly prevalent among women with recurrent pregnancy loss. Hum Reprod 2015 Apr;30(4):777-82.

4 Abalovich M, Gutierrez S, Alcaraz G, Maccallini G, Garcia A, Levalle O. Overt and subclinical hypothyroidism complicating pregnancy. Thyroid 2002 Jan;12(1):63-8.

5 Kabadi UM. ‘Subclinical hypothyroidism’. Natural course of the syndrome during a prolonged follow-up study. Arch Intern Med 1993 Apr 26;153(8):957-61.


7 Negro R, Schwartz A, Gismondi R, Tinelli A, Mangieri T, Stagnaro-Green A. Increased pregnancy loss rate in thyroid antibody negative women with TSH levels between 2.5 and 5.0 in the first trimester of pregnancy. J Clin Endocrinol Metab 2010 Sep;95(9):E44-E48.

8 Negro R, Mestman JH. Thyroid disease in pregnancy. Best Pract Res Clin Endocrinol Metab 2011 Dec;25(6):927-43.

9 Fatourechi V. Subclinical hypothyroidism: an update for primary care physicians. Mayo Clin Proc 2009;84(1):65-71.

10 Bernardi LA, Cohen RN, Stephenson MD. Impact of subclinical hypothyroidism in women with recurrent early pregnancy loss. Fertil Steril 2013 Nov;100(5):1326-31.

11 Liu H, Shan Z, Li C, Mao J, Xie X, Wang W, Fan C, Wang H, Zhang H, Han C, Wang X, Liu X, Fan Y, Bao S, Teng W. Maternal subclinical hypothyroidism, thyroid autoimmunity, and the risk of miscarriage: a prospective cohort study. Thyroid 2014 Nov;24(11):1642-9.

12 Benhadi N, Wiersinga WM, Reitsma JB, Vrijkotte TG, Bonsel GJ. Higher maternal TSH levels in pregnancy are associated with increased risk for miscarriage, fetal or neonatal death. Eur J Endocrinol 2009 Jun;160(6):985-91.

13 Cleary-Goldman J, Malone FD, Lambert-Messerlian G, Sullivan L, Canick J, Porter TF, Luthy D, Gross S, Bianchi DW, D’Alton ME. Maternal thyroid hypofunction and pregnancy outcome. Obstet Gynecol 2008 Jul;112(1):85-92.

14 UK Guidelines for the Use of Thyroid Function Tests. 2006

15 Korevaar TI, Medici M, de Rijke YB, Visser W, de Muinck Keizer-Schrama SM, Jaddoe VW, Hofman A, Ross HA, Visser WE, Hooijkaas H, Steegers EA, Tiemeier H, Bongers-Schokking JJ, Visser TJ, Peeters RP. Ethnic differences in maternal thyroid parameters during pregnancy: the Generation R study. J Clin Endocrinol Metab 2013 Sep;98(9):3678-86.

16 Kolte AM, Bernardi LA, Christiansen OB, Quenby S, Farquharson RG, Goddijn M, Stephenson MD. Terminology for pregnancy loss prior to viability: a consensus statement from the ESHRE early pregnancy special interest group. Hum Reprod 2015 Mar;30(3):495-8.

17 NVOG Dutch Society of Obstetrics and Gynaecology. Guideline: recurrent miscarriage (NVOG). 2007.

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18 Jauniaux E, Farquharson RG, Christiansen OB, Exalto N. Evidence-based guidelines for the investigation and medical treatment of recurrent miscarriage. Hum Reprod 2006 Sep;21(9):2216-22.

19 De GL, Abalovich M, Alexander EK, Amino N, Barbour L, Cobin RH, Eastman CJ, Lazarus JH, Luton D, Mandel SJ, Mestman J, Rovet J, Sullivan S. Management of thyroid dysfunction during pregnancy and postpartum: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2012 Aug;97(8):2543-65.

20 Blatt AJ, Nakamoto JM, Kaufman HW. National status of testing for hypothyroidism during pregnancy and postpartum. J Clin Endocrinol Metab 2012 Mar;97(3):777-84.

21 Lund M, Kamper-Jorgensen M, Nielsen HS, Lidegaard O, Andersen AM, Christiansen OB. Prognosis for live birth in women with recurrent miscarriage: what is the best measure of success? Obstet Gynecol 2012 Jan;119(1):37-43.

22 Palomo I, Segovia F, Ortega C, Pierangeli S. Antiphospholipid syndrome: a comprehensive review of a complex and multisystemic disease. Clin Exp Rheumatol 2009 Jul;27(4):668-77.


SUPPLEMENTARY DATA

Supplementary Figure 1. Forest plot for the comparison of live birth rates between women with RM and

subclinical hypothyroidism versus euthyroid women. Chapter

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5 |Pathophysiological aspects of thyroid hormone

disorder/thyroid peroxidase autoantibodies and

reproduction

R Vissenberg*VD Manders*S MastenbroekE FliersGB AfinkC Ris-StalpersM GoddijnPH Bisschop

*These authors contributed equally to the manuscript

Human Reproduction Update. 2015;21:378-387

Chapter 580 |

ABSTRACT

Background

Thyroid hormone disorders and thyroid peroxidase autoantibodies (TPO-Ab) in women are associated with subfertility and early pregnancy loss. Here, we aim to provide a comprehensive overview of the literature on the pathophysiology of these associations.

Methods

A review of the literature in the English language was carried out. Relevant studies were identified by searching Medline, EMBASE and the Cochrane Controlled Trials Register from 1975 until March 2014.

Results

From a total of 6108 primary selected articles from the literature search, 105 articles were selected for critical appraisal. Observational data indicate that altered thyroid hormone levels are associated with disturbed folliculogenesis, spermatogenesis, lower fertilization rates and lower embryo quality. Triiodothyronine (T3) in combination with FSH enhances granulosa cell proliferation and inhibits granulosa cell apoptosis by the PI3K/Akt pathway. T3 is considered a biological amplifier of the stimulatory action of gonadotrophins on granulosa cell function. T3 increases the expression of matrix metalloproteinases (MMP), MMP-2, MMP-3, fetal fibronectin and integrin a5b1T3 in early placental extravillous trophoblasts. Thyroid hormone transporters and receptors are expressed in the ovary, early embryo, endometrium, uterus and placenta. No other data explaining the associations could be retrieved from the literature. The presence of TPO-Ab is negatively associated with spermatogenesis, fertilization and embryo quality, but no data are available on the potential pathophysiological mechanisms.

Conclusions

Thyroid hormone disorders and TPO-Ab are associated with disturbed folliculogenesis, spermatogenesis, fertilization and embryogenesis. The pathophysiology of these associations remains largely unknown, as evidence is limited and includes studies using small sample sizes, and often restricted to animal models. There are no studies on the pathophysiology underlying the association between TPO-Ab and reproduction. The available evidence, although limited, supports a role of thyroid hormone in fertility and early pregnancy. This justifies clinical intervention studies on the effects of thyroid hormone supplementation in women with subclinical hypothyroidism and in women prone to develop hypothyroidism due to the presence of TPO-Ab. In addition, more research is needed to identify the underlying mechanisms. This would be of particular interest in women undergoing IVF to pinpoint the effects of thyroid hormone on different parameters of reproduction.

Thyroid hormones, autoantibodies and reproduction | 81

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INTRODUCTION

General introduction

Thyroid dysfunction is a common endocrine disorder. In the US National Health and Nutrition Examination Survey (NHANES III), the prevalence of hypothyroidism was 4.6% (0.3 overt and 4.3% subclinical) and the prevalence of hyperthyroidism 1.3% (0.5 overt and 0.7% subclinical) in people without known thyroid disease or a family history of thyroid disease(1). Thyroid dysfunction is usually acquired and may occur any time in life. In women of reproductive age, the most prevalent cause of thyroid dysfunction is thyroid autoimmunity. Thyroid autoantibodies (Ab) that react with key proteins in the thyroid, such as thyroid peroxidase (TPO) or thyroglobulin (Tg), can induce a chronic lymphocytic thyroiditis that ultimately results in destruction and loss of thyroid function. In Graves’ disease, circulating thyroidstimulating hormone (TSH) receptor autoantibodies can activate the TSH receptor resulting in hyperthyroidism. Both hypothyroidism and hyperthyroidism have been associated with altered ovarian function,menstrual irregularities, subfertility and higher (recurrent) miscarriage rates(2;3), suggesting that thyroid hormone affects female reproductive organs. The prevalence of TPO-Ab is 8–14% in women of reproductive age(2). Although the presence of TPO-Ab predisposes to hypothyroidism, the majority of women with TPO-Ab is euthyroid. Importantly, the presence of TPO-Ab combined with normal thyroid function is associated with subfertility, recurrent embryo implantation failure, early pregnancy loss and adverse pregnancy outcomes(3;4). For women with TPO-Ab, no effective treatment intervention is available probably due to lack of knowledge about the underlying pathophysiological mechanisms. Several mechanisms have been proposed for the association between TPO-Ab and subfertility and pregnancy loss. The first hypothesis is that TPO-Ab merely reflects a different level of autoimmunity and that other autoimmune processes cause subfertility or pregnancy loss. The second hypothesis is that the association with subfertility or pregnancy loss is secondary to a subtle deficiency in thyroid hormone. As mentioned above, TPO-Ab can induce a chronic, lymphocytic thyroiditis that results in a lower capacity of the thyroid to adequately adapt to increased demands during pregnancy(5). The third hypothesis is that the association is confounded by age, because the prevalence of TPO-Ab increases with age and older women face a higher risk of subfertility and miscarriage. The last hypothesis has been rejected by two recent meta-analyses showing that the association between TPO-Ab and subfertility and pregnancy loss is independent of age(3;6). In this review,we provide a comprehensive overview on the described pathways by which thyroid hormone, and possibly TPO-Ab, influence reproductive biology with special focus on the elements essential to fertilization and early pregnancy.

Chapter 582 |

Regulation of thyroid hormone action

Both thyroid hormone synthesis and thyroid hormone release to the circulation are driven by the pituitary-gland-derived TSH in a classical negative feedback loop. This explains why hypothyroidism in the presence of a functional hypothalamic–pituitary axis results in increased TSH levels while the reverse occurs in hyperthyroidism(7). The human thyroid predominantly produces the biologically inactive prohormone thyroxine (tetraiodotyrosine, T4) and only a small amount of the bioactive hormone triiodothyronine (T3). Less than 0.1% of the total amount of circulating thyroid hormone (T4 and T3) is in the free or unbound form that can be transferred across the plasma membrane into a target cell. It was long thought that thyroid hormones diffuse passively across plasma membranes(8-11). Currently, we know that thyroid hormone enters the cell by virtue of thyroid hormone transporters, including the monocarboxylate transporters (MCT) 8 and 10 and the solute carrier organic anion transporter family member 1C1 or OATP1C1(12). The intracellular availability of the biologically active thyroid hormone T3 is the net result of a finely tuned system of three distinct iodothyronine deiodinases (DIO1, DIO2,DIO3) with tissue-specific expression that are responsible for thyroid hormone outer ring (type I and II) and inner ring (type I and III) deiodination(13). DIO1 and DIO2 can convert inactive T4 to biologically active T3, whereas both DIO1 and DIO3 are able to inactivate T3. Biologically active T3 finally enters the nucleus and exerts its function through the nuclear thyroid hormone receptors (THR), thyroid hormone receptor alpha (THRA) and beta (THRB) that are expressed in a tissue-specific manner. Since their initial identification, THRs have evolved into central players in a complex system of co-activators and co-repressors(14-16).

TPO and TPO-Ab

TPO

TPO is essential for thyroid hormone synthesis in vivo. TPO is a glycosylated membrane-bound protein that belongs to the family of mammalian haem peroxidases that have as common denominator a haem prosthetic group acting as an electron acceptor. Other members of this family are dual oxidase 1 (DUOX1), dual oxidase 2 (DUOX2), eosinophil peroxidase (EPX), lactoperoxidase, myeloperoxidase (MPO), peroxidasin homolog (PXDN), peroxidasin-like protein (PXDNL), prostaglandin G/H synthase 1 (PTGS1) and prostaglandin G/H synthase 2 (PTGS2)(17;18). Mammalian haem peroxidases are involved in the catalysis of oxidative reactions, antibacterial processes and inflammation.TPOis highly expressed in the thyroid gland where the protein is located on the apical membrane of thyrocytes. TPO oxidizes iodide to active iodine and links iodinated tyrosine residues to form thyroid hormones, in majority the prohormone T4, but also limited amounts of the biologically active T3(19). Human TPO-Ab recognizes an immunodominant region comprising overlapping A and B domains on conformationally intact TPO. The amino acids recognized by TPO-Ab are located


Chapter

5

in the regions with homology to MPO and the complement control protein(20). The apical membrane, where TPO is functional, is located on the inner side of the thyroid follicle and is generally not exposed to the human immune system. The generation of TPO-Ab is most likely due to TPO protein or peptides expressed on the basal membrane or a previous phase of destructive thyroid gland damage, exposing the TPO protein localized on the apical membrane to the host-immune system. It is tempting to speculate that although these autoantibodies recognize the TPO protein, they have in fact been raised against one of the other highly homologous members of the MPO family. Indeed, for some patients, cross-reaction of serum TPO-Ab with MPO has been demonstrated. Amino acid sequences recognized by TPO-Ab are also located in regions with homology to MPO(20;21). This implies that TPO-Ab, as measured with the conventional assay, could have intrinsic affinity to other members of the MPO family that are being expressed in cells involved in reproduction and pregnancy.

METHODS

In order to present an up-to-date overviewof the effects of thyroid hormone disorders and TPO-Ab on fertility, embryogenesis, implantation and placentation, we searched in Medline, EMBASE and the Cochrane Controlled Trials Register, for relevant studies published from 1975 until March 2014. Relevant research articles published in the English language were obtained and reviewed. Medical subject heading terms used were thyroid hormone, liothyronine, thyroxine, Tg, TPO, thyroid antibody, TPO antibody, Tg antibody, endometrium, placenta, embryo, infertility, fertility, menstrual cycle and spontaneous abortion in relation to thyroid hormones and TPO-Ab. A data limit was specified for the availability of reliable free T4 assays, which precluded articles published before 1975(22). The complete literature search is shown in Supplementary data. From the publically available RNA microarray studies within the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus, we performed an in silico analysis. Data from the in silico analysis were used to determine expression of genes related to thyroid hormone metabolism in tissue/cells involved in reproduction(23-25).

RESULTS

From a total of 6108 primary selected articles from the literature search, 105 articles were selected for critical appraisal. The results were extracted from studies and were divided in the different target tissues involved in reproduction, namely oocytes, sperm, embryo, endometrium and the placenta. These results are described below. The results are also summarized in Tables I and II and illustrated in Figs 1 and 2.

Chapter 584 |

Figure 1. Thyroid hormone physiology. Circulating thyroid hormone concentrations are regulated via a

negative feedback system at the level of the hypothalamus and the pituitary. The production of thyroid

hormone by the thyroid is regulated by thyroid-stimulating hormone (TSH) produced by the anterior pituitary,

which itself is regulated by thyrotropin-releasing hormone (TRH) produced by the hypothalamus. Thyroid

hormone circulates as the inactive prohormone thyroxine (T4) and as the active hormone triiodothyronine

(T3). Thyroid hormone can only enter target cells by virtue of specific transporters (MCT8, MCT10 and Oatp1c1).

In target cells, thyroid hormone can be activated (T4 to T3) or inactivated (T4 to rT3 or T3 to T2) depending

on the local activity of specific deiodinases (D1, D2 and D3). Subsequently, active T3 can bind to the nuclear

thyroid hormone receptors (TR-alpha and TR-beta) and induce transcription.


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Oocytes and ovulation

Thyroid hormone disorders

Ovarian follicles either continue to grow from pre-antral to antral follicles due to survival signals, such as gonadotrophins and growth factors, or undergo atresia. The destiny of the ovarian follicle depends on a subtle balance of expression of hormones and growth factors. In humans, disturbances in thyroid hormone production are responsible for a dysregulation of the hypothalamus–pituitary–gonadal axis, and hypothyroidism is associated with oligomenorrhea(2). In rats, hypothyroidism does not influence the classical pre-ovulatory patterns of LH and FSH secretion(26), suggesting that in contrast to humans, hypothyroidism in rats does not have an effect on pituitary gonadotrophin secretion. In domestic cats, no beneficial effect of T4 on in vitro antral follicle growth, diameter gain, morphologic development or the amount of viable follicles was found(27). The composition of follicular fluid might be important for developing oocytes and may play a substantial role in oocyte quality. Both T3 and T4 are present in follicular fluid of humans. Both isoforms of THR mRNA are expressed in the human oocyte, and hence thyroid hormone may directly affect the oocyte(28). Conflicting results have been reported on the correlation between serum thyroid hormone levels and follicular fluid levels. One study showed a positive correlation between serum T4 and follicular fluid T4 values(29). In addition, an animal study showed that in follicular fluid T4 levels are generally lower than in blood serum, whereas T3 concentration in follicular fluid is comparable with blood serum levels. In vitro studies have shown that the growth of rat pre-antral follicles and the levels of ovulated oocytes is stimulated by thyroid hormone. T3 alone is ineffective, but in combination with FSH, it enhances granulosa cell proliferation and inhibits granulosa cell apoptosis by the PI3K/Akt pathway(30-32). Hypothyroid rats showed similar amounts of corpora lutea, and slightly (although not statistically significant) lower ovulation rates compared with control rats. Hypothyroid rats have higher levels of estrogens, estrogen receptor B (ERb) and cyp19A1 aromatase expression after ovulation compared with control rats, favouring survival of the corpus luteum(26). T3 is considered a biological amplifier of the stimulatory action of gonadotrophins on granulosa cell function(33) and all data indicate that thyroid hormone levels seem to play a positive role in follicle development in vitro and are important during folliculogenesis and ovulation in vivo. Therefore, altered thyroid hormone levels may lead to cyclic irregularities and ovulation disturbances lowering the chance of a successful pregnancy. In humans, increased expression of THRs was found during follicular growth(28;34). Fromthe publically available RNA microarray studies within the NCBI Gene Expression Omnibus, it appears that thyroid hormone transporters and receptors are expressed – at least to some level – in the ovary (Supplementary data, Fig. S1).

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TPO-Ab were measurable in all samples of follicular fluid obtained from women with thyroid autoimmunity, while they were absent in women without thyroid autoimmunity(35). The relevance of this observation remains to be elucidated, because it only demonstrates that plasma TPO-Ab can enter follicular fluid. It can however be speculated that thyroid autoantibodies cause a cytotoxic reaction in the follicle fluid leading to damage to the oocyte, which may decrease its quality and development potential. In women with unexplained subfertility and thyroid autoantibodies (TPO-Ab and/or Tg-Ab), the number of oocytes retrieved after ovarian hyperstimulation was not statistically different compared with women without thyroid autoantibodies(36;37). In conclusion, thyroid hormone disorders are associated with disturbed folliculogenesis. This is supported by the fact that thyroid hormone transporters and receptors are expressed in the ovary. T3 in combination with FSH enhances granulosa cell proliferation and inhibits granulosa cell apoptosis by the PI3K/Akt pathway. Therefore, altered thyroid hormone levels may lead to cyclic irregularities and ovulation disturbances, thereby lowering the chance of a successful pregnancy. Although TPO-Ab can be found in follicular fluid, there is no evidence available that it disturbs folliculogenesis.

Sperm


In thyroidectomized prepubertal rats, testosterone levels, number of spermatozoa and sperm motility are decreased(38). In congenitally hypothyroid mice, seminiferous tubules are smaller and contain fewer spermatogonia, spermatocytes, spermatids and spermatozoa compared with controls(39). Together, these studies indicate that physiological thyroid hormone concentrations are required for normal spermatogenesis in rodents. Male Pax8 null mice are hypothyroid due to thyroid agenesis and have complete azoospermia. However, the azoospermia is the result of a direct morphogenic role of Pax8 in the development of the epididymides and the efferent ducts and not due to congenital hypothyroidism(40).

Hypothyroidism has an adverse effect on human spermatogenesis and negatively affects sperm count and motility(41) as well as morphology(42). Hyperthyroidism is associated with abnormalities in sperm motility(43) and DNA damage(44).

TPO-Ab

The presence of thyroid autoantibodies was higher in subfertile men compared with a control group(45).

Altogether, hypothyroidism, hyperthyroidism and presence of TPO-Ab are associated with an adverse effect on sperm parameters. No studies are available showing a causal effect of thyroid hormone and TPO-Abon sperm parameters. No intervention studies are available on


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treating thyroid disorders and the effect on sperm parameters. The clinical significance remains therefore unclear, especially since some of the reported abnormalities in sperm parameters do not affect fertility.

Figure 2. Mechanisms of action of thyroid hormones in the reproductive system. Schematic summary of

known effects and/or associations of thyroid hormone and the reproductive system. Solid lines indicate an

effect of T4 administration. Dotted lines indicate associations without evidence for causality. For each tissue/

cell-type expression of TR, deiodinases (DIO) and thyroid hormone transporters is indicated. Thyroid peroxidase

autoantibody (TPO-Ab) is not shown because a lack of evidence for a causal relationship between TPO-Ab and

function of the reproductive system. MMP, metalloproteinases.

Fertilization and embryogenesis


We identified only one study that reports on the effect of thyroid status on fertilization. This study showed that fertilization rates were significantly lower in cows treated with propylthiouracil and who were hypothyroid, compared with control euthyroid cows(46).

In humans, the number of embryos of higher quality was significantly higher in women with subclinical hypothyroidism who were treated with T4 supplementation compared with those who were not. In addition, women that were treated with T4 had a higher live birth rate per initiated cycle, with no difference in the live birth rate in TPO-Ab positive patients(47).

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The thyroid hormone transporters and receptors, as well as deiodinases, are expressed in the human pre-implantation embryo (Supplementary data, Fig. S1).

TPO-Ab

Evidence for the influence of TPO-Ab on embryo quality is limited. One study showed a decreased percentage of 3–4 cell stage mouse embryos cultured in serum with TPO-Ab compared with mouse embryos cultured in normal mouse serum (74 versus 90%, P < 0.05) but the number of expanded blastocysts (66 versus 73%) and hatching blastocysts (36 versus 37%) did not significantly differ between the two groups(48).

In a study of 14 women with TPO-Ab (also in follicular fluid), oocyte fertilization, gradeA embryos and pregnancy rates were lower compared with 17 women without TPO-Ab and this effect was independent of thyroid hormone status(35). Another study found no statistical differences in the number of grade 1 and grade 2 embryos comparing women with unexplained subfertility and positive thyroid autoantibodies (TPO-Ab and/or Tg-Ab) with unexplained subfertility without thyroid autoantibodies(37). This discrepancy might be due to the fact that this study also included women positive for Tg-Ab.

In conclusion, both hypothyroidism and the presence of TPO-Ab seem to negatively affect fertilization rates and embryo quality, but to date, only associations have been reported and studies exploring the pathophysiology are lacking.

Endometrium


There is ample evidence that DIO2and DIO3 are present in human endometrium throughout the menstrual cycle(49-51). The expression in the mid-secretory phase is lower and the cyclic changes of deiodinase activities show an inverse relationship with progesterone levels(51;52). THRA and THRB are expressed in the glandular endometrium with a peak during the mid-secretory phase. The expression of deiodinases, THRAand THRB in the endometrium indicates a dynamic local regulation of bioavailable thyroid hormone metabolites.

Evaluation of publically available expression data in the NCBI Gene Expression Omnibus supports these data and also demonstrates the presence of thyroid hormone transporters and even relatively high expression of co-activators and repressors of the THRs in the endometrium and uterus (Supplementary data, Fig. S1).

TPO-Ab

Endometrial volume is an important parameter to evaluate endometrial receptivity and therefore a possible predictor for successful implantation(53;54). In euthyroid women with unexplained infertility, there was no difference in endometrial volume between subjects


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positive or negative for TPO-Ab, whereas the pregnancy rate after IVF was lower in the TPO-Ab positive group(37).

Analysis of mRNA expression data from the non-pregnant human uterus(55) (Supplementary data, Table SI) shows that the MPO domain-encoding genes PXDN and PTGS2 have relatively high expression levels compared with TPO and the other MPO domain-encoding genes in this tissue. PTGS2-mediated prostaglandin synthesis in mouse is known to be essential for ovulation, fertilization, implantation and decidualization(56). The function of PXDN homologue in the endometrium is unknown.

In conclusion, deiodinases, THRA and THRB are expressed in the endometrium suggesting a functional role for thyroid hormone, but there are no studies available that demonstrate a direct effect on endometrial receptivity or endometrial function. TPOmRNA is expressed in the endometrium at a relatively low level and protein expression has never been demonstrated, which makes a direct pathophysiological effect of TPO-Ab on TPO in the endometrium unlikely.

Implantation


In this review, implantation is defined as the direct contact between the maternal and fetal interface prior to the invasion of extravillous trophoblasts into the maternal spiral arteries. One study showed that T4 increases progesterone production in human granulosa cells in vitro when administered in combination with insulin and gonadotrophins(34). As progesterone is responsible for building up the endometrial lining for an optimal implantation and for decreasing the maternal immune response to allow for the acceptance of the pregnancy, T4 bioavailability may have a mediating role in this process.

Leukaemia inhibitory factor (LIF) is involved in the embryo implantation process and expressed in the mid-secretory endometrium(57). TSH significantly up-regulated LIF expression in endometrial cell cultures, suggesting a potential role of TSH in the implantation process(51).

TPO-Ab

Analysis of mRNA expression data from non-pregnant human uterus(55) (Supplementary data, Table SI) shows that the MPO domain-encoding genes PXDN and PTGS2 have relatively high expression levels in this tissue. In humans, the effect of PTGS2 is less clear than in mice, but PTGS2 is known to play a role in female fertility(58). Mice where PGTS2 expression was limited were infertile or produced small litters or no litters. A cohort study of 34 women showed that prostaglandin synthesis appears to be disrupted in patients with repeated IVF failure compared with fertile controls(59). This suggests that reduced prostaglandin synthesis in the human endometrium may lead to poor endometrial receptivity. The function of PXDN

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homologue in the endometrium is unknown. The possibility that TPO-Ab might be able to recognize PTGS2 and that PTGS2 is important for an ongoing pregnancy in human and mice and makes it tempting to speculate that there might be an effect of TPO-Ab on PTGS2. No data supporting this speculation are available.

In conclusion, thyroid hormone stimulates the production of progesterone in granulosa cells and up-regulates LIF expression. Both are important for the implantation process. No evidence is available on a direct pathophysiological effect of thyroid hormones or TPO-Ab on implantation.

Placentation


The placenta is responsible for the exchange of oxygen, nutrients, hormones and growth factors and their waste products between mother and fetus. The migration of extravillous trophoblasts into the maternal uterine spiral arteries allows increased blood flow to the placenta. Cell adhesion molecules(60), metalloproteinases (MMP-2 and MMP-3), tissue inhibitors of metalloproteinases, fibronectin and integrina5b1 are important for the invasion process(61;62). T3 is known to increase the expression of MMP-2, MMP-3, fetal fibronectin and integrin a5b1T3 in cultured early (8–12 weeks) placental extravillous trophoblasts, suggesting that thyroid hormone plays a vital role regulating the invasive potential of extravillous trophoblasts(63).

One of the effects of thyroid hormone is the stimulation of the placental secretion of progesterone and human placental lactogen. Progesterone is essential for the endometrial lining and an optimal nidation, aswell as inducing the local immune tolerance that decreases the maternal immune response and prevents rejection of the fetal allograft(33). Human placental lactogen increases the fetal glucose supply by decreasing maternal fatty acids stores through altering maternal insulin secretion. T3 through the THRB stimulates the expression and release of placental lactogen in cultured human trophoblasts(64). T4 both increases vascular endothelial growth factor in trophoblasts as well as the height of the trophoblast epithelium in gilts(65).

Thyroid hormone metabolism in the placenta seems tightly regulated. All three types of deiodinase are expressed in placenta(66), and the relatively high levels of DIO3 expression limit the transfer of maternal circulating thyroid hormones to the fetus(67). The placenta is responsive to T3 and contains thyroid receptors not only at term, but also during early gestation(68-70). High affinity-specific T3-binding proteins are present in the trophoblast membrane and are responsible for uptake of T3 by trophoblast cells(71-73). All these in vitro data were confirmed by our in silico analysis. There is a relatively high placental expression of all factors involved in thyroid hormone action (Supplementary data, Fig. S1).


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TPO-Ab

Although TPO-Ab diffuse through the placental barrier during the third trimester of pregnancy(74), there is no evidence that this is also true in early pregnancy. Since the characteristics of the placental barrier change only slightly after the first trimester, transfer of TPO-Ab at all stages of pregnancy seems likely. A recent cohort study supported that the increased risk of TPO-Ab positive women on adverse pregnancy outcomes was independent of thyroid function(75). Possibly, there are direct targets for TPO-Ab, other thanTPO, at the maternal–fetal interface that affect placentation and ongoing pregnancy. In particular, expression of the MPO-domain-containing protein peroxidasin homologue is very abundant in both trophoblasts and decidua (www.proteinatlas.org and Supplementary data, Table SI).

It is important to bear in mind that TPO-Ab are also associated with the presence of other autoantibodies, such as zona pellucida autoantibodies. Zona pellucida and thyroid tissue seem to share some antigens and might cross react. It has been postulated that the zona pellucida may be the target of TPO-Ab(76). MPO is involved in the catalysis of oxidative reactions, antibacterial processes and inflammation, which hypothetically may lead to an increased immune response. It is also hypothesized that TPO-Ab reflect a general immune response, resulting in subfertility and complications during early pregnancy(77). Kim et al. showed that tumour necrosis factor alpha and interleukin-10-expressing CD3/CD4 cell ratios and non-organ-specific antibodies were significantly increased in women with thyroid autoantibodies(77). They additionally concluded that women suffering from recurrent miscarriage with thyroid autoantibodies have significantly elevated serum levels of natural killer cells(77). No correlation could be established between the presence of TPO-Ab and uterine-natural killer cells in women suffering for recurrent pregnancy loss after IVF and their levels of TPO-Ab(78).

In conclusion, T3 increases the expression of MMP-2, MMP-3, fetal fibronectin and integrin a5b1 in early placental extravillous trophoblasts, suggesting that thyroid hormone plays a vital role in the regulation of the invasive potential of extravillous trophoblasts. TPO-Ab diffuse through the placental barrier but there is no evidence published that TPO-Ab directly affect placentation.

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Table 1. Summary of the available evidence on thyroid hormones and the effect on reproduction.

Thyroid hormones

Oocytes and ovulation Thyroid hormone disorders are associated with disturbed folliculogenesis. T3 in combination with FSH enhances granulosa cell proliferation and inhibits granulosa cell apoptosis by the PI3K/Akt pathway.Thyroid hormone transporters and receptors are expressed in the ovary.

Sperm Hypothyroidism has an adverse effect on human spermatogenesis and negatively affects sperm count and motility as well as morphology. Hyperthyroidism is associated with abnormalities in sperm motility and DNA damage. No studies are available on the mechanisms by which thyroid hormone affects spermatogenesis.


Hypothyroidism is associated with lower fertilization rates and disturbed embryogenesis.No studies on the pathophysiology have been reported .

Endometrium Deiodinases, THRA and THRB are expressed in the endometrium .Evidence for a direct effect of thyroid hormone on endometrial receptivity or function is lacking.

Implantation Thyroid hormone stimulates the production of progesterone in granulosa cells and up-regulates LIF.There are no studies on the effect of thyroid hormone on implantation.

Placentation T3 increases the expression of MMP-2, MMP-3, fetal fibronectin and integrin a5ß1T3 in early placental extravillous trophoblasts.

T3, triiodothyronine; THRA, thyroid hormone receptor alpha; THRB, thyroid hormone receptor beta; LIF, leukemia inhibiting factor; MMP-2,3, matrix metalloproteinase 2,3.

Table 2. Summary of the available evidence on thyroid peroxidase autoantibodies (TPO-Ab) and the effect on reproduction.

TPO-Antibodies

Oocytes and ovulation TPO-Ab are present in follicular fluid.TPO-Ab do not influence the number of retrieved oocytes during controlled ovarian stimulation.There are no studies on a direct effect of TPO-Ab on folliculogenesis.

Sperm TPO-Ab are more often found in subfertile men compared with a control group.No studies are available that showing a direct effect of TPO-Ab on spermatogenesis.


TPO-Ab are associated with lower fertilization rates and disturbed embryogenesis.No literature is available on the pathophysiology.

Endometrium TPO-Ab do not influence endometrial volume.No studies have been published on direct effects of TPO-Ab on endometrial receptivity or endometrial function.

Implantation There are no studies on direct effects of TPO-Ab on implantation.

Placentation TPO-Ab diffuse through the placental barrier.There is no evidence for a direct effect of TPO-Ab on placentation.


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DISCUSSION

An association exists between thyroid hormone disturbances and/or TPO-Ab and subfertility and early pregnancy loss, but the exact pathophysiology is unknown(3).

This review shows that altered thyroid hormone levels are associated with disturbed folliculogenesis and spermatogenesis, lower fertilization rates and lower embryo quality.

Thyroid hormone levels seem to play a positive role for ovulation and folliculogenesis. T3 in combination with FSH enhances granulosa cell proliferation and inhibits granulosa cell apoptosis by the PI3K/Akt pathway(30;31;79). Hypothyroid rats have higher levels of estrogens, ERb and cyp19A1 aromatase expression after ovulation compared with control rats, favouring survival of the corpus luteum(26).

T3 is considered a biological amplifier of the stimulatory action of gonadotrophins on granulosa cell function(33). Thyroid hormone levels seem to play a positive role for embryo quality, because treatment with T4 in women with subclinical hypothyroidism resulted in a higher embryo quality(46).

T3 is known to increase the expression of MMP-2, MMP-3, fetal fibronectin and integrin a5b1 in cultured early (8–12 weeks) placental extravillous trophoblasts, suggesting that thyroid hormone plays a vital role regulating the invasive potential of extravillous trophoblasts(63).

Thyroid hormone transporters, receptors and their associated proteins are expressed in the ovary, the early embryo(25), endometrium(24), uterus and placenta(23) (Supplementary data, Fig. S1). No other data explaining the associations could be retrieved from the literature and the underlying mechanism for these clinical parameters remains unclear.

The available evidence, although limited, supports a role for thyroid hormone in reproduction and early pregnancy. The fact that almost all factors essential for thyroid hormone action, such as THRA and THRB, thyroid hormone transporters and deiodinases, are expressed in several tissues involved in reproduction, namely ovary, endometrium, uterus and placenta, indicates a dynamic local regulation of bioavailable thyroid hormone metabolites. It is important to realize that thyroid hormone and associated proteins are expressed in multiple tissues, other than the reproductive organs, but that the expression levels seems not that high. It is unknown if the relatively low expression levels also mean that there is a functional effect of these factors. Expression of these factors per se does not explain a possible direct pathway.

The presence of TPO-Ab negatively influences folliculogenesis, spermatogenesis, fertilization rates, embryo quality and pregnancy rates, but no data are available on the potential mechanisms. Low-to-absent expression of TPO mRNA expression in the endometrium, uterus and placenta makes a direct effect of TPO-Ab unlikely (Supplementary data, Table SI).

TPO expression is low or absent in the endometrium and placenta, especially compared with other peroxidases. It is still very interesting to speculate that other peroxidases, such as PXDN or PTGS, are the target for TPO-Ab and cause an increased immunological response. There is however no evidence showing that TPO-Ab have binding affinity for these other

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peroxidases. Studies on a possible increased immunological response in women with TPO-Ab are very limited.

Summary and clinical relevance

The available evidence, although limited, supports a role of thyroid hormone in fertility and early pregnancy. The hypothesis that the associated subfertility or pregnancy loss is secondary to a subtle deficiency in thyroid hormone concentrations is therefore more likely than a direct pathogenic effect of TPO-Ab. Future research should be focusing on thyroid hormone disturbances and their clinical and pathophysiological effects. Although understanding the molecular signalling of thyroid hormone is very interesting, a clinical intervention study is more pragmatic to investigate whether thyroid hormone supplementation improves fertility or early pregnancy outcomes in women with subclinical hypothyroidism and in women prone to develop hypothyroidism due to the presence of TPO-Ab. There is a need for clinical studies given the worldwide discussion on treating pregnant women with subclinical thyroid dysfunction. There is a very broad variation in the treatment and screening of pregnant women for thyroid disorders in pregnancy(80). Guidelines provide different advice regarding when to screen or treat pregnant women with thyroid hormone supplementation(81). Currently, two studies are recruiting women with TPO-Ab and a history of (recurrent) miscarriage to investigate if treatment with levothyroxine improves live birth rates, the T4-LIFE study (NTR 3364) and the TABLET Study (ISRCTN15948785). Randomized studies are needed to study the effect of treating subclinical hypothyroidism in pregnancy. This would also be of particular interest in women with subclinical hypothyroidism and/or TPO-Ab undergoing IVF to pinpoint specific effects of thyroid hormone on reproduction. Valuable data on parameters such as number of follicles, number of oocytes, fertilization rates, embryo quality, implantation rates, and pregnancy outcome could be obtained and may lead to approaches to improve the fertility and pregnancy outcomes, and at the same time provide clues on where to start more fundamental studies on the underlying pathophysiological mechanisms.


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56 Lim H, Paria BC, Das SK, Dinchuk JE, Langenbach R, Trzaskos JM, Dey SK. Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 1997 Oct 17;91(2):197-208.

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58 Cha J, Sun X, Dey SK. Mechanisms of implantation: strategies for successful pregnancy. Nat Med 2012 Dec;18(12):1754-67.

59 Achache H, Tsafrir A, Prus D, Reich R, Revel A. Defective endometrial prostaglandin synthesis identified in patients with repeated implantation failure undergoing in vitro fertilization. Fertil Steril 2010 Sep;94(4):1271-8.

60 Damsky C, Sutherland A, Fisher S. Extracellular matrix 5: adhesive interactions in early mammalian embryogenesis, implantation, and placentation. FASEB J 1993 Nov;7(14):1320-9.

61 Aplin JD, Haigh T, Jones CJ, Church HJ, Vicovac L. Development of cytotrophoblast columns from explanted first-trimester human placental villi: role of fibronectin and integrin alpha5beta1. Biol Reprod 1999 Apr;60(4):828-38.

62 Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E, Seiki M. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 1994 Jul 7;370(6484):61-5.

63 Oki N, Matsuo H, Nakago S, Murakoshi H, Laoag-Fernandez JB, Maruo T. Effects of 3,5,3’-triiodothyronine on the invasive potential and the expression of integrins and matrix metalloproteinases in cultured early placental extravillous trophoblasts. J Clin Endocrinol Metab 2004 Oct;89(10):5213-21.

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67 Mortimer RH, Galligan JP, Cannell GR, Addison RS, Roberts MS. Maternal to fetal thyroxine transmission in the human term placenta is limited by inner ring deiodination. J Clin Endocrinol Metab 1996 Jun;81(6):2247-9.

68 Laoag-Fernandez JB, Matsuo H, Murakoshi H, Hamada AL, Tsang BK, Maruo T. 3,5,3’-Triiodothyronine down-regulates Fas and Fas ligand expression and suppresses caspase-3 and poly (adenosine 5’-diphosphate-ribose) polymerase cleavage and apoptosis in early placental extravillous trophoblasts in vitro. J Clin Endocrinol Metab 2004 Aug;89(8):4069-77.

69 Mukku VR, Kirkland JL, Hardy M, Stancel GM. Evidence for thyroid hormone receptors in uterine nuclei. Metabolism 1983 Feb;32(2):142-5.

70 Maruo T, Hiramatsu S, Otani T, Hayashi M, Mochizuki M. Increase in the expression of thyroid hormone receptors in porcine granulosa cells early in follicular maturation. Acta Endocrinol (Copenh) 1992 Aug;127(2):152-60.

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72 Nishii H, Ashitaka Y, Maruo M, Mochizuki M. Studies on the nuclear 3, 5, 3’-triiodo-L-thyronine binding sites in cytotrophoblast. Endocrinol Jpn 1989 Dec;36(6):891-8.


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73 Pontecorvi A, Robbins J. The plasma membrane and thyroid hormone entry into cells. Trends Endocrinol Metab 1989 Nov;1(2):90-4.

74 Seror J, Amand G, Guibourdenche J, Ceccaldi PF, Luton D. Anti-TPO Antibodies Diffusion through the Placental Barrier during Pregnancy. PLoS One 2014;9(1):e84647.

75 Korevaar TI, Schalekamp-Timmermans S, de Rijke YB, Visser WE, Visser W, de Muinck Keizer-Schrama SM, Hofman A, Ross HA, Hooijkaas H, Tiemeier H, Bongers-Schokking JJ, Jaddoe VW, Visser TJ, Steegers EA, Medici M, Peeters RP. Hypothyroxinemia and TPO-antibody positivity are risk factors for premature delivery: the generation R study. J Clin Endocrinol Metab 2013 Nov;98(11):4382-90.

76 Kelkar RL, Meherji PK, Kadam SS, Gupta SK, Nandedkar TD. Circulating auto-antibodies against the zona pellucida and thyroid microsomal antigen in women with premature ovarian failure. J Reprod Immunol 2005 Jun;66(1):53-67.

77 Kim NY, Cho HJ, Kim HY, Yang KM, Ahn HK, Thornton S, Park JC, Beaman K, Gilman-Sachs A, Kwak-Kim J. Thyroid autoimmunity and its association with cellular and humoral immunity in women with reproductive failures. Am J Reprod Immunol 2011 Jan;65(1):78-87.

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79 Jiang JY, Umezu M, Sato E. Improvement of follicular development rather than gonadotrophin secretion by thyroxine treatment in infertile immature hypothyroid rdw rats. Journal of reproduction and fertility 2000;119:193-9.



82 Uhlen M, Oksvold P, Fagerberg L, Lundberg E, Jonasson K, Forsberg M, Zwahlen M, Kampf C, Wester K, Hober S, Wernerus H, Bjorling L, Ponten F. Towards a knowledge-based Human Protein Atlas. Nat Biotechnol 2010 Dec;28(12):1248-50.

83 Su AI, Wiltshire T, Batalov S, Lapp H, Ching KA, Block D, Zhang J, Soden R, Hayakawa M, Kreiman G, Cooke MP, Walker JR, Hogenesch JB. A gene atlas of the mouse and human protein-encoding transcriptomes. Proc Natl Acad Sci U S A 2004 Apr 20;101(16):6062-7.

84 Cheng SY, Leonard JL, Davis PJ. Molecular aspects of thyroid hormone actions. Endocr Rev 2010 Apr;31(2):139-70.

85 McCall MN, Jaffee HA, Zelisko SJ, Sinha N, Hooiveld G, Irizarry RA, Zilliox MJ. The Gene Expression Barcode 3.0: improved data processing and mining tools. Nucleic Acids Res 2014 Jan;42(Database issue):D938-D943.

Chapter 5100 |

SUPPLEMENTARY DATA

Supplementary figure S1. Gene expression of thyroid hormone receptors , thyroid hormone transporters

and deiodinases in different tissues RNA microarray data on genes/proteins related to thyroid hormone


Chapter

5

signaling was obtained from the NCBI Gene Expression Omnibus (12;24;25;83;84). Samples were pre-processed

and barcoded as described by The Gene expression Barcode 3.0(85). The resulting z-scores are shown and

colored according to the scale bar on the right. The z-scores is the number of standard deviations from the

mean expression signal of the presumably non-expressing tissues.

The genes included in the table are: solute carrier family 16, member 2 (thyroid hormone transporter)

(SLC16A2), solute carrier family 16 (aromatic amino acid transporter), member 10 (SLC16A10), solute carrier

organic anion transporter family, member 1C1 (SLC01C1), deiodinase, iodothyronine, type I (DIO1), deiodinase,

iodothyronine, type II(DIO2), deiodinase, iodothyronine, type III(DIO3), thyroid hormone receptor, alpha (THRA),

thyroid hormone receptor, beta (THRB), nuclear receptor coactivator 1 (NCOA1), nuclear receptor coactivator

2 (NCOA2), nuclear receptor coactivator 3 (NCOA3), nuclear receptor corepressor 1 (NCOR1), nuclear receptor

corepressor 2 (NCOR2), nuclear receptor interacting protein 1 (NRIP1), retinoid X receptor, alpha (RXRA), retinoid

X receptor, beta (RXRB), proteasome (prosome, macropain) 26S subunit, ATPase, 5 (PSMC5), mediator complex

subunit 1 (MED1)

Supplementary table S1. mRNA expression levels of MPO domain-containing proteins in human uterus

and placenta Human uterus (from women age 34 and 40) and placenta (women age N/A, 28, 39, 31)

RNA-seq data were obtained from the human protein atlas (www.proteinatlas.org)(82) Values are

expressed as average Fragments Per Kilobase of transcript per Million mapped reads (FPKM).

protein gene symbolHuman non-pregnant

uterus FPKM Human placenta FPKM

Thyroid Peroxidase TPO 1.3 0.0

Peroxidasin homolog PXDN 58.3 30.7

Myeloperoxidase MPO 0.1 2.2

Peroxidasin-like protein PXDNL 0.2 0.1

Eosinophil peroxidase EPX 0.0 0.3

Lactoperoxidase LPO 0.0 0.3

Dual oxidase 1 DUOX1 2.1 2.1

Dual oxidase 2 DUOX2 0.7 0.1

Prostaglandin G/H synthase 2 PTGS2 21.1 5.1

Prostaglandin G/H synthase 1 PTGS1 5.1 3.2

Chapter 5102 |

Appendix 1: Literature search

Mesh terms used were thyroid hormone, liothyronine, thyroxine, thyroglobulin, thyroid peroxidase, thyroid antibody, thyroid peroxidise antibody, thyroglobulin antibody, endometrium, placenta, embryo, infertility exp therapy, fertility, infertility, menstrual cycle and spontaneous abortion. The following search terms were used: thyro*, hormone*, thyronin*, iodothyronin*, diiodothyronin*, triiodothyronin*, tetraiodothyronin*, thyroxin*, thyroglobulin*, T3, T4, iodide peroxidase*, thyroid peroxidase*, TPO, antithyro*, antibody*, autoantibody*, auto-antibod*, TAA*, TPOA*, anti-thyroglobulin*, antithyroglobulin*, TGA*, TGAb*, thyroid hormone receptor*, THRA, THRB, deiodinase, iodothyronine, DIO1, DIO2, DIO3, menstrual cycle*, reproductive cycle*, ovarian cycle*, ovary, follicle, zona pellucida, granulosa cell, corpus luteum, fallopian tube, antral follicle, preantral follicle fertil*, fertile period*, fecund*, infertile*, subfert*, subfecun*, reproduct* adj failure*, Conception, fertilization, oocyte, ovum, zygote, follicular phase*, preovulat*, ovulat*, postovulat*, luteal phase*, menstruat*, spermatozoa, spermatozoon, endometri*, decidu*, blastocyst*, embryo* (adj development*, interaction, attachment*, transfer*), embryogenesis*, embryo grad*, embryo stag*, trophoblast*, trophectoderm*, implantation*, preimplantation*, nidation*, receptiv*, placenta*, placentation , abortion* and miscarr*.


Chapter

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6 |Treatment of thyroid disorders before conception

and in early pregnancy: a systematic review

R VissenbergE van den BoogaardM van WelyJAM van der PostE FliersPH BisschopM Goddijn


Chapter 6106 |

ABSTRACT

Background

Thyroid disorders are associated with pregnancy complications. Universal screening is currently not recommended because of a lack of evidence on the effectiveness of treatment. Women with hyperthyroidism and hypothyroidism evidently require treatment but this is less clear for women with subclinical hypothyroidism and thyroid autoimmunity. Therefore, we conducted a systematic review to provide a comprehensive overview on the available treatment interventions.

Methods

Relevant studies were identified by searching Medline, EMBASE and Cochrane Controlled Trials Register, published until December 2011.

Results

From a total of 7334 primary selected titles, 22 articles were included for the systematic review and 11 were appropriate for meta-analyses. Eight studies reported on hyperthyroidism. Propylthiouracil (PTU) and methimazole reduce the risk for preterm delivery [risk ratio (RR): 0.23, confidence interval (CI): 0.1 –0.52], pre-eclampsia (RR: 0.23, CI: 0.06 –0.89) and low birthweight (RR: 0.38, CI: 0.22 –0.66). The nine studies that reported on clinical hypothyroidism showed that levothyroxine is effective in reducing the risk for miscarriage (RR: 0.19, CI: 0.08 –0.39) and preterm delivery (RR: 0.41, CI: 0.24 –0.68). For treatment of subclinical hypothyroidism, current evidence is insufficient. The five studies available on thyroid autoimmunity showed a not significant reduction in miscarriage (RR: 0.58, CI: 0.32 –1.06), but significant reduction in preterm birth by treatment with levothyoxine (RR: 0.31, CI: 0.11 –0.90).

Conclusion

For hyperthyroidism, methimazole and PTU are effective in preventing pregnancy complications. For clinical hypothyroidism, treatment with levothyroxine is recommended. For subclinical hypothyroidism and thyroid autoimmunity, evidence is insufficient to recommend treatment with levothyroxine. The overall lack of evidence precludes a recommendation for universal screening and is only justified in a research setting.

Review of treatments for thyroid disorders in pregnancy | 107

INTRODUCTION

Thyroid disease affects 2–3% of pregnant women and is associated with adverse pregnancy outcomes(1-3).Thyroid disorders can be divided into (sub)clinical hyperthyroidism, (sub)clinical hypothyroidism and/or thyroid autoimmunity. Hyperthyroidism is found in 0.1 –0.4% of pregnant women and is most commonly caused by Graves’ disease(4). Graves’ disease in pregnancy is associated with miscarriage, preeclampsia, preterm birth, placental abruption and fetal hyperthyroidism(5;6). According to the Endocrine Society Clinical Practice Guideline (ESCPG) and the American Thyroid Association (ATA), the treatment of choice is with propylthiouracil (PTU)(1;7). Treatment with methimazole (MMI) has been associated with a higher risk of congenital disorders, such as aplasia cutis and choanal atresia(8;9). It is advised by ATA to switch to MMI treatment after the first 12 weeks because of reports of hepatotoxicity in children of mothers treated with PTU(10;11). A recent Cochrane review could not identify any randomized controlled trial (RCT) comparing treatment interventions in pregnant women with hyperthyroidism(6). The prevalence of clinical hypothyroidism is 0.3 –0.5% in pregnant women(1). Hypothyroidism in women of reproductive age is most commonly caused by an autoimmune thyroiditis and Hashimoto’s disease(1). Hypothyroidism in pregnancy is associated with miscarriage, placental abruption, neonatal intensive care unit (NICU) admission and lower intelligence scores(3;12;13). Treatment with levothyroxine is therefore recommended and considered safe in pregnancy(1). The prevalence of subclinical hypothyroidism, defined biochemically by the combination of elevated serum thyroid-stimulating hormone (TSH) level and a free thyroxine level within the reference range, is 3–5%(1). There is a strong association with pre-eclampsia and perinatal mortality and lower intelligence scores in the offspring(3;12-14). The ESCPG ‘Management of Thyroid dysfunction during Pregnancy and post-partum’ advises hormone replacement therapy in pregnant women with subclinical hypothyroidism and reports the evidence as fair for pregnancy outcomes but poor for neurological outcome(1). A recent Cochrane review could not find an RCT on levothyroxine treatment for women with clinical or subclinical hypothyroidism on the effect of pregnancy outcomes(15). Given this poor evidence, it is advised in the guidelines from the ATA to treat subclinically hypothyroid women only when thyroid antibodies are detected as well(7). Thyroid autoimmunity is defined as the presence of thyroid antibodies against thyroperoxidase (TPO-Ab) and/or thyroglobulin (Tg-Ab) in combination with a normal thyroid function or euthyroid state. This has an incidence of 8 – 14% among women of fertile age(16). The presence of thyroid autoantibodies in euthyroid women is associated with a significant risk for unexplained subfertility, miscarriage, recurrent miscarriage, preterm birth and maternal postpartum thyroiditis(3;17). Women with thyroid autoimmunity who are euthyroid in the early stage of pregnancy are at risk of developing hypothyroidism in the course of pregnancy and should be monitored(1;7). A systematic review and meta-analysis restricted to thyroid

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Chapter 6108 |

autoimmunity showed that levothyroxine lowers the risk for miscarriage and preterm birth but this was based only on two very small studies(17-19). The effects of treatment with levothyroxine on other pregnancy complications or subfertility, or the effect of other treatment interventions on pregnancy outcomes, were not studied in this review. The high prevalence of thyroid autoimmunity and subclinical hypothyroidism makes it an important health problem. These conditions are not diagnosed without an active screening strategy because they present without any symptoms. The ESCPG guideline supports selective screening in patients who are at increased risk for thyroid disease(1). Universal screening of thyroid function in pregnancy is under debate and is currently not recommended because of lack of evidence on the effect of treatment interventions, especially for subclinical hypothyroidism and thyroid autoimmunity. We therefore conducted a systematic review of the literature to present an overview on treatment interventions and their effects on pregnancy complications in women with thyroid dysfunction and thyroid autoimmunity before conception and in early pregnancy.

METHODS

Relevant studies were identified by searching Medline, EMBASE and the Cochrane Controlled Trials Register, published until December 2011. Search criteria used were related to thyroid function, thyroid autoimmunity, pregnancy outcome and any form of pharmacological intervention used to treat (sub)clinical hypothyroidism, (sub)clinical hyperthyroidism or thyroid autoimmunity. The diagnosis of clinical or subclinical hypothyroidism was based on high TSH concentrations and a decreased free thyroxine or free thyroxine within the reference range in case of subclinical hypothyroidism. The diagnosis of hyperthyroidism was based on a decreased TSH with an increased free thyroxine or free thyroxine within the reference range in case of subclinical hyperthyroidism(20). Worldwide accepted reference intervals for thyroid hormones or thyroid antibodies in pregnant women are not available. We therefore included all cut-off levels for TSH, free thyroxine and/or TPO-Ab as described for the diagnosis of (sub)clinical hypothyroidism, clinical hyperthyroidism and thyroid autoimmunity. The data limit was specified for the availability of reliable free thyroxine assays, which excluded articles published before 1975(21). Search criteria used were relevant to thyroid function, thyroid autoimmunity, pregnancy outcomes and treatment interventions. Specifically, the following search terms were used: thyroid*, hyperthyr*, hypothyr*, tpo*, tsh, thyrotropin receptor antibod*, thyroid stimulating immunoglobulin*, thyrotropin-binding inhibit*, thyroxine, thyrotropin, thyroid microsomal antibodies, fertility, infertility, abortion*, miscarriag*, pregnan*, obstetric*, gestation* preterm deliver*, premature deliver*, intrauterine growth retardation*, fetal growth restriction*, intrauterine growth restriction* and child


development*. Mesh terms used were: thyroid gland, thyroid diseases, immunoglobulins, thyroid-stimulating, thyrotropin, thyroxine, fertility, infertility, pregnancy, pregnancy outcome, pregnancy complications, fetal growth retardation, drug therapy, placebos, antithyroid agents, iodine, MMI, selenium, PTU, triiodothyronine, thioamides, adrenergic beta-antagonists and child development. There were no language limitations for the initial search. RCTs, cohort studies and case–control studies were included. Titles and subsequently abstracts of the articles were screened independently by two reviewers (R.V., E.B.). Included articles for full text screening were compared during a consensus meeting. In case of disagreement, a third reviewer (P.B., M.G.) was consulted for the decision on inclusion or exclusion for full text evaluation. Articles that did not contribute to the answer of our research questions after full text evaluation were excluded. Only articles that described at least 10 patients were eligible. Articles that reported treatment of thyroid disorders after 20 weeks of gestation were excluded. After consensus, the remaining articles were included for critical appraisal and assessed by two reviewers independently (R.V., E.B.). Articles were judged on scientific quality according to the CONSORT and STROBE statement(22;23). Levels of evidence were attributed according to the Oxford centre for evidence-based medicine(24). Articles in foreign languages were translated and included if eligible, except for articles in Chinese, Japanese, Russian and Serbian. In order to reach a consistent presentation of the data, all individual study results were translated into a risk ratio (RR) and 95% confidence interval (CI). In the case of adequate clinical and statistical homogeneity, with the same outcome measure, treatment intervention and control group were described and articles were included in the meta-analysis. Summarized relative RRs were calculated using random effect models. Software of Review Manager 5 (available from Cochrane) was used to perform the meta-analyses.

RESULTS

In Fig. 1, the selection process after the search is represented. Two hundred and fifty-four articles were selected for critical appraisal, all dealing with pregnancy outcome, post-partum period and/or neonatal outcome. Of the 22 included articles in this systematic review, 8 reported on clinical hyperthyroidism(8;25-31), 9 reported on (sub)clinical hypothyroidism(32-40) and 5 on thyroid autoimmunity(18;19;41-43). All patients in the included studies were women with a thyroid disorder who received treatment during pregnancy. Treatment concerned PTU or MMI for hyperthyroidism, levothyroxine for (sub)clinical hypothyroidism and levothyroxine or selenium for thyroid autoimmunity. Controls were women with the same thyroid disorder who did not receive any treatment or euthyroid without any thyroid disorder and without treatment.

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Chapter 6110 |

Figure 1. Flowchart of literature search and article selection.


Quality of the studies

The characteristics of the included articles and quality assessment are reported in Table I. Six RCT’s were included, four of which were about treating thyroid autoimmunity and two RCTs studied treatment of (sub)clinical hypothyroidism (18;19;32;39;41;42). All other studies were evidence-level II studies, i.e. cohort and case – control studies. Eleven studies presented appropriate data, and could be included in meta-analyses on seven different pregnancy outcomes.

Treatment interventions for clinical hyperthyroidism

Eight studies on treatment of clinical hyperthyroidism in pregnancy were included(8;25-31). All these studies evaluated the effectiveness of PTU and/or MMI.

Patients treated with PTU

One cohort study reported on pregnancy outcomes in 115 hyperthyroid women treated with PTU compared with 1141 euthyroid controls without treatment(30). No significant differences were seen for the prevalence of miscarriage (RR: 1.24, CI: 0.64– 2.41: P = 0.53), preterm delivery (RR: 1.71, CI: 0.9 –3.25: P = 0.10), congenital malformations (RR: 0.39, CI: 0.05– 2.83: P = 0.35) or live birth rate (RR: 0.99, CI: 0.94 –1.05: P = 0.86) for PTU treated hyperthyroid women versus euthyroid women without treatment.

Patients treated with MMI

One cohort study reported on pregnancy outcomes in 241 hyperthyroid women treated with MMI compared with 1089 euthyroid controls without hyperthyroidism. The prevalence of miscarriage was not significantly different for MMI-treated hyperthyroid women compared with euthyroid women without treatment (RR: 0.94, CI: 0.55 –1.61: P = 0.83)(8). Congenital malformations occurred in 8 (4%) of the treated cases versus 23 (2%) of the euthyroid controls (RR: 0.88, CI: 0.86 –4.15: P = 0.12). Another cohort study compared 126 infants whose mothers had been treated with MMI for hyperthyroidism with 50 infants whose mothers were hyperthyroid and did not receive any treatment(25). The prevalence of malformed children was 6% in the non-treated group versus 0% in the treated group. This difference was not significant (RR: 0.06, CI: 0.00 –1.09: P = 0.06).

PTU versus MMI

Two cohort studies reported on the prevalence of neonatal hypothyroidism in 133 hyperthyroid women treated with PTU compared with 79 hyperthyroid women treated with MMI. Meta-analysis showed no difference between the two groups (two studies, RR: 1.50, CI: 0.58 –3.88:

Chapter

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30 p

regn

ant

wom

en

Not

defi

ned

241

hype

rthy

roid

pr

egna

nt w

omen

MM

I/ Ca

rbim

azol

e in

divi

dual

dos

e10

89 e

uthy

roid

pr

egna

nt w

omen

Mis

carr

iage

rate

, m

ajor

con

geni

tal

Mal

form

atio

ns

IIM

atch

ing:

no

Tab

le I.

Cha

ract

erist

ics a

nd q

ualit

y fe

atur

es o

f the

22

stud

ies i

nclu

ded

in sy

stem

atic

revi

ew o

f tre

atm

ent o

f thy

roid

diso

rder

s bef

ore

conc

eptio

n an

d in

ear

ly p

regn

ancy

.


Rose

nfel

d et

al.

2009

Pros

pect

ive

coho

rt12

56 p

regn

ant

wom

en

Not

defi

ned

115

hype

rthy

roid

w

omen

rece

ivin

g tr

eatm

ent

PTU

indi

vidu

al d

oses

1141

eut

hyro

id

preg

nant

wom

en

rece

ivin

g no

tr

eatm

ent

Cong

enita

l an

omal

ies,

pret

erm

del

iver

y (<

37w

eeks

), liv

e bi

rth

rate

, mis

carr

iage

, st

illbi

rth

IIM

atch

ing:

no

Che

n et

al.

2011

Case

-con

trol

2830

wom

en w

ith

hype

rthy

roid

ism

Not

defi

ned

516

hype

rthy

roid

pr

egna

nt w

omen

re

ceiv

ing

PTU

PTU

indi

vidu

al d

ose

MM

I ind

ivid

ual d

ose

65 h

yper

thyr

oid

preg

nant

wom

en

rece

ivin

g M

MI

Low

birt

h w

eigh

t (<

2500

g)

IIM

atch

ing:

no

Hyp

othy

roid

ism

Leun

g et

al.

1993

Pros

pect

ive

coho

rt23

wom

en

with

prim

ary

hypo

thyr

oidi

sm45

wom

en

with

sub

clin

ical

hy

poth

yroi

dism

TSH

0.5

-5.0

m

U/m

LfT

4 4.

5-13

.2

mU

/mL

9 hy

poth

yroi

d w

omen

, eu

thyr

oid

with

trea

tmen

t29

sub

clin

ical

hy

poth

yroi

d w

omen

, eu

thyr

oid

with

tr

eatm

ent

Levo

thyr

oxin

e in

divi

dual

dos

e14

hyp

othy

roid

w

omen

, hy

poth

yroi

d de

spite

trea

tmen

t16

sub

clin

ical

hy

poth

yroi

d w

omen

, hy

poth

yroi

d de

spite

trea

tmen

t

Ges

tatio

nal

Hyp

erte

nsio

nII

Mat

chin

g: n

o

Aba

lovi

ch

et a

l. 20

02Re

tros

pect

ive

coho

rt15

0 hy

poth

yroi

d pr

egna

nt w

omen

TSH

0,5

-4,0

m

IU/L

fT4

0,8-

2,0

ng/d

L

99 h

ypot

hyro

id

preg

nant

wom

en,

euth

yroi

d w

ithtr

eatm

ent

Levo

thyr

oxin

e in

divi

dual

dos

e16

hyp

othy

roid

w

omen

at fi

rst

visi

t, no

trea

tmen

t 35

sub

clin

ical

hy

poth

yroi

d at

firs

t vis

it, n

o tr

eatm

ent

Preg

nanc

y ra

te,

mis

carr

iage

rate

,pr

eter

m d

eliv

ery

IIM

atch

ing:

no

Blaz

er e

t al.

2003

Case

-con

trol

27.3

86 fu

ll-te

rm

new

born

sN

ot d

efine

d24

6 in

fant

s bo

rn to

trea

ted

hypo

thyr

oid

mot

hers

Levo

thyr

oxin

e in

divi

dual

dos

e13

9 in

fant

s bo

rn

to

heal

thy

euth

yroi

d m

othe

rs

Ges

tatio

nal a

ge,

birt

h w

eigh

t, A

pgar

sc

ores

, neo

nata

lth

yroi

d fu

nctio

n

IIM

atch

ing:

yes

Idris

et a

l. 20

05Re

tros

pect

ive

coho

rt16

7 w

omen

w

ith s

ubcl

inic

al

or c

linic

al

hypo

thyr

oidi

sm

TSH

0.1

-5.5

m

U/l

127

hypo

thyr

oid

wom

en w

ith

trea

tmen

t

Levo

thyr

oxin

e in

divi

dual

dos

e40

hyp

othy

roid

w

omen

des

pite

tr

eatm

ent

Caes

area

n se

ctio

n ra

te, N

ICU

ad

mis

sion

, LBW

IIM

atch

ing:

no

Chapter

6

Chapter 6114 |

Mat

alon

et

al.

2006

Pros

pect

ive

coho

rt13

9.16

8 w

omen

w

ith s

ingl

eton

pr

egna

ncie

s

Not

defi

ned

1102

wom

en

with

trea

ted

hypo

thyr

oidi

sm

Levo

thyr

oxin

e13

8, 0

66

euth

yroi

d co

ntro

ls

with

out t

hyro

id

dise

ase

Caes

area

n se

ctio

n ra

te, p

erin

atal

m

orta

lity,

co

ngen

ital

mal

form

atio

ns

IIM

atch

ing:

no

Rasm

usse

net

al.

2007

Case

- con

trol

Not

defi

ned

431

infa

nts

with

cr

anio

syno

stos

is40

94 in

fant

s w

ithou

t co

ngen

ital

mal

form

atio

ns

Mat

erna

l le

voth

yrox

ine

use

IIM

atch

ing:

no

Neg

ro e

t al.

2010

RCT

4500

pre

gnan

t w

omen

: 22

59 u

nive

rsal

sc

reen

ing

grou

p,22

57 w

omen

ca

se fi

ndin

g gr

oup

TSH

0.2

7-2,

5 m

IU/lt

r fT

4 9.

3-18

.0

ng/li

ter (

12.-

33.5

pm

ol/

liter

)TP

O-A

b 0-

100

kU/L

82 h

ypot

hyro

idpr

egna

nt

wom

en re

ceiv

ing

trea

tmen

t

Levo

thyr

oxin

e in

divi

dual

dos

e34

hyp

othy

roid

pr

egna

nt w

omen

, re

ceiv

ing

no

trea

tmen

t43

84 e

uthy

roid

w

omen

, rec

eivi

ng

no tr

eatm

ent

MC

, PIH

, PE,

GD

M,

PA, C

S, R

D, N

ICU

ad

mis

sion

, LBW

(<

2500

g), P

TD

(<37

wee

ks),

low

Apg

ar

scor

e (<

3 af

ter 5

m

in),

PND

IRa

ndom

izat

ion:

com

pute

r ge

nera

ted

Conc

eale

d: y

esBl

indi

ng: y

esIT

T: n

o

Kim

et a

l. 20

11RC

T64

infe

rtile

w

omen

with

su

bclin

ical

hy

poth

yroi

dism

un

derg

oing

IVF/

IC

SI

TSH

< 4

.5

mIU

/LfT

4 no

t de

fined

32 w

omen

with

tr

eatm

ent

Levo

thyr

oxin

e 50

-12

5 μg

32 w

omen

w

ithou

t tr

eatm

ent

Num

ber h

igh

qual

ity e

mbr

yo’s,

em

bryo

im

plan

tatio

n ra

te,

clin

ical

pre

gnan

cy

rate

/ cy

cle,

m

isca

rria

ge ra

te,

live

birt

h ra

te/

cycl

e

IRa

ndom

izat

ion:

com

pute

r ge

nera

ted

Conc

eale

d: y

esBl

indi

ng: n

oIT

T: y

es

Behr

ooz

et a

l.20

11Ca

se-c

ontr

ol38

wom

en w

ith

hypo

thyr

oidi

sm

TSH

< 3

m

U/L

fT4

> 7

.0

μg/d

L

19 h

ypot

hyro

id

wom

en w

ith

trea

tmen

t,eu

thyr

oid

durin

g pr

egna

ncy

Levo

thyr

oxin

e in

divi

dual

dos

e19

hyp

othy

roid

w

ith tr

eatm

ent,

subc

linic

ally

hy

poth

yroi

d du

ring

preg

nanc

y

IQ le

vel,

cogn

itive

pe

rfor

man

ce,

verb

al

perf

orm

ance

IIM

atch

ing:

no

Firs

t aut

hor

Year

Stud

y ty

pe

Pop

ulat

ion

Refe

ren

ce

valu

esIn

terv

enti

on

grou

pIn

terv

enti

onC

ontr

ols

Out

com

e m

easu

re(s

)Le

vel o

fev

iden

ceQ

ualit

y fe

atur

es


Thyr

oid

aut

oim

mun

ity

Noh

r et a

l. 20

00RC

T46

pre

gnan

t w

omen

eu

thyr

oid

with

TP

O-A

b

TSH

0.4

0-4.

0 m

U/L

fT4

10-2

5 pm

ol/L

TPO

-Ab

0-10

0 U

/mL

22 w

omen

with

tr

eatm

ent

150µ

g io

dine

/day

an

d50

µg

sele

nium

fir

st tr

imes

ter t

ill

post

part

um

24 w

omen

w

ithou

t tr

eatm

ent

PPTD

IM

atch

ing:

yes

Rand

omiz

atio

n:no

t des

crib

edCo

ncea

led:

not

de

scrib

edBl

indi

ng: y

esIT

T: n

o

Neg

ro e

t al.

2005

RCT

490

euth

yroi

d w

omen

, un

derg

oing

IVF/

ICSI

TSH

0.2

7-4.

2 m

IU/L

fT4

9.3-

18.0

ng

/L o

r 12-

33.5

pm

ol/L

TPO

-Ab

0-10

0 kI

U/L

36 e

uthy

roid

w

omen

, TP

O-A

b po

sitiv

e re

ceiv

ing

trea

tmen

t

Levo

thyr

oxin

e 1

mg/

kg/d

36 e

uthy

roid

w

omen

, TP

O-A

b po

sitiv

e re

ceiv

ing

plac

ebo

412

euth

yroi

d w

omen

, TPO

-A

b ne

gativ

e un

derg

oing

IVF/

ICSI

Preg

nanc

y ra

te,

mis

carr

iage

rate

IRa

ndom

izat

ion:

com

pute

r ge

nera

ted

Conc

eale

d: y

esBl

indi

ng: y

esIT

T: y

es

Neg

ro e

t al.

2006

RCT

984

euth

yroi

d pr

egna

nt

wom

en

TSH

0,2

7-4,

2 m

IU/lt

rfT

4 9,

3-18

,0

ng o

r12

-33,

5 pm

ol/lt

r.TP

O-A

b 0-

100

kIU

/L

57 e

uthy

roid

w

omen

, TPO

-Ab

posi

tive

rece

ivin

g tr

eatm

ent

Levo

thyr

oxin

e 0,

5.

0,75

or 1

,0 µ

/kg

58 e

uthy

roid

w

omen

, TPO

-Ab

posi

tive

rece

ivin

g no

trea

tmen

t 86

9 pr

egna

nt

wom

en e

uthy

roid

w

ithou

t TP

O-A

b re

ceiv

ing

no tr

eatm

ent

Mis

carr

iage

, ge

stat

iona

l hy

pert

ensi

on

(>14

0/90

), pr

e-ec

lam

psia

, pr

eter

m b

irth

(<37

w

eeks

), pl

acen

tal

abru

ptio

n

IRa

ndom

isat

ion:

com

pute

r ge

nera

ted

Conc

eale

d: y

esBl

indi

ng: y

esIT

T: y

es

Neg

ro e

t al.

2007

RCT

232

euth

yroi

d pr

egna

nt

wom

en

TSH

0,2

7-4,

2 m

IU/L

fT4

9,3-

18,0

ng

/L o

r 12-

33.5

pm

ol/L

TPO

-Ab

0-10

0 kI

U/L

77 e

uthy

roid

w

omen

, TPO

-Ab

posi

tive

rece

ivin

g tr

eatm

ent

Sele

nom

ethi

onin

e 20

0µg/

day

star

ted

in th

e fir

st tr

imes

ter t

ill 1

2 m

onth

s po

st-p

artu

m

74 e

uthy

roid

w

omen

, TP

O-A

b po

sitiv

e re

ceiv

ing

plac

ebo

81 C

auca

sian

pr

egna

nt w

omen

, eu

thyr

oid

TPO

-Ab

nega

tive

PPTD

, per

man

ent

hypo

thyr

oidi

sm

12 m

onth

s af

ter

deliv

ery

IRa

ndom

izat

ion:

com

pute

r ge

nera

ted

Conc

eale

d: y

esBl

indi

ng: y

esIT

T: y

es

Chapter

6

Chapter 6116 |

Reve

lli e

t al.

2009

Retr

ospe

ctiv

e co

hort

93 e

uthy

roid

w

omen

un

derg

oing

IVF

TPO

-Ab

0-35

UI/m

l Tg

-Ab

0-40

U

I/mL

55 e

uthy

roid

w

omen

, TPO

-Ab

and/

or T

g-A

b po

sitiv

e, re

ceiv

ing

trea

tmen

t

Levo

thyr

oxin

e 50

µ/

day

Firs

t day

of

stim

ulat

ion

at

leas

t 10

wee

ks o

f pr

egna

ncy

38 e

uthy

roid

w

omen

, TP

O-A

b an

d/ o

r Tg

-Ab

posi

tive,

rece

ivin

g no

trea

tmen

t20

0 in

fert

ile

wom

en e

uthy

roid

, w

ithou

t TP

O-A

b or

Tg-

Ab

rece

ivin

g no

tr

eatm

ent

Mis

carr

iage

rate

, Pr

egna

ncy

rate

/ IVF

cy

cle

IIM

atch

ing:

yes

Firs

t aut

hor

Year

Stud

y ty

pe

Pop

ulat

ion

Refe

ren

ce

valu

esIn

terv

enti

on

grou

p I

nter

vent

ion

Con

trol

sO

utco

me

mea

sure

(s)

Leve

l of

evid

ence

Qua

lity

feat

ures

Not

e: M

icro

som

al a

ntib

odie

s is

the

prev

ious

nom

encl

atur

e fo

r TPO

ant

ibod

ies

All

stud

ies

have

an

adeq

uate

sam

ple

size

n >

10

Ab,

ant

ibod

y; C

S, c

aesa

rean

sec

tion;

ET,

em

bryo

tra

nsfe

r; G

DM

, ges

tatio

nal d

iabe

tes

mel

litus

; IF,

infe

rtili

ty; L

GA

, lar

ge fo

r ge

stat

iona

l ag

e; L

T4, l

evot

hyro

xine

; MC

, mis

carr

iage

; MM

I, m

ethi

maz

ole;

NIC

U, N

eona

tal I

nten

sive

Car

e U

nit,

PA, p

lace

ntal

abr

uptio

n; P

E, p

re-e

clam

psia

; PIH

, pre

gnan

cy in

duce

d hy

pert

ensi

on; P

ND

, pe

rinat

al d

eath

; PPT

D, p

ost-

part

um th

yroi

d di

seas

e; P

TD, p

rete

rm d

eliv

ery;

PTU

, pro

pylth

iour

acil;

RD

S, re

spira

tory

dis

tres

s sy

ndro

me;

RM

, rec

urre

nt m

isca

rria

ge; S

GA

, sm

all f

or g

esta

tiona

l ag

e; T

g, th

yrog

lobu

lin; T

PO. t

hyro

id p

erox

idas

e.


P = 0.40)(Fig.2)(28;29). One of these two studies also reported on congenital malformations but did not find any significant difference between PTU and MMI (RR: 1.09, 0.12– 10.15: P = 0.94)(28). One case–control study compared low birthweight in babies of 581 mothers with hyperthyroidism treated with PTU or MMI(31). No significant difference was found (RR: 0.55, 0.28– 1.07; P = 0.08).

Adequate versus not adequately treated women

Two cohort studies reported on differences in pregnancy complications between women with hyperthyroidism who were adequately, and women who were not adequately, treated(26;27). Not adequately treated hyperthyroidism was defined as a TSH lower and a thyroxine higher than the reference interval, despite treatment. The first study found no significant differences in miscarriage rate, number of preterm deliveries and neonatal hypothyroidism between 90 women who had been adequately treated for hyperthyroidism with PTU compared with 22 women who were still hyperthyroid despite treatment with PTU (RR: 0.24, CI: 0.02 –3.76: P = 0.31, RR: 1.34, CI: 0.32 –5.63: P =0.69, RR: 0.90, CI: 0.27 –2.94: P = 0.86)(26). The other study found evidence of a significantly lower risk for low birthweight (RR: 0.38, CI: 0.22 –0.66: P = 0.0005), preterm delivery (RR: 0.23, 0.1 –0.52: P = 0.0004) and severe pre-eclampsia (RR: 0.23, 0.06 –0.89: P = 0.03) in 90 women who had been adequately treated with MMI or PTU for hyperthyroidism compared with 57 inadequately treated women. No significant differences were seen in neonates being small for gestational age (RR: 0.81, CI: 0.32 –2.06: P = 0.67) between the two groups(27).

Figure 2. Forest plot of risk ratio for neonatal hypothyroidism of hyperthyroid patients treated with

methimazole or PTU.

Treatment interventions for (sub)clinical hypothyroidism

Nine studies on treatment of (sub)clinical hypothyroidism in pregnancy were included. All these studies used levothyroxine as treatment. Five studies reported on the effect of treatment interventions for clinical and/or subclinical hypothyroidism(33;35;37-39).

Chapter

6

Chapter 6118 |

One randomized study reported a significantly lower miscarriage rate in 82 women receiving levothyroxine treatment for hypothyroidism compared with 34 women with hypothyroidism without any treatment (RR: 0.24, CI: 0.07 –0.76: P = 0.02)(39). These were patients randomized for universal screening or selective screening in potentially high-risk patients for thyroid disorders in early pregnancy. No differences were found in the prevalence of gestational hypertension, pre-eclampsia, gestational diabetes, placental abruption, caesarean delivery, respiratory distress syndrome, birthweight, preterm birth, Apgar scores or perinatal death. The treated group was also compared with 4384 euthyroid controls without any known thyroid disorder. For all pregnancy complications, including miscarriage, no significant differences were found. A meta-analysis could be performed for the outcome Caesarean section rate, for which no significant differences could be detected between hypothyroid-treated women compared with healthy euthyroid controls (two studies, RR: 1.21, CI: 0.54 –2.7: P = 0.65 ( Fig. 3a)(37;39). There was significant statistical heterogeneity (I2 of 90%) between the studies; therefore, these finding should be considered with care. The second cohort study reported no differences in perinatal mortality (RR: 1.02, CI: 0.6 –1.68: P = 0.79) or congenital malformations (RR: 0.83, 0.21 –3.32: P = 0.075)(37). A meta-analysis could be performed, including the third study, for the outcome miscarriage and preterm birth: a significant decrease was seen in women treated with levothyroxine compared with women without treatment (two studies, RR: 0.18, CI: 0.08 –0.39, P < 0.01) (Fig. 3b)(33;39). A significant decrease was also shown for preterm delivery (two studies, RR: 0.41, CI: 0.24 – 0.68, P < 0.01) ( Fig. 3c)(33;39). One case–control study found a significantly lower birthweight in 246 neonates born to treated hypothyroid mothers compared with 139 neonates born to healthy euthyroid mothers. No difference was found in Apgar scores at 1 and 5 min. Overall, both TSH and free thyroxine serum levels were significantly higher in the study group compared with TSH and free thyroxine levels of the control group(35). The fifth study, a case–control study, described a higher risk for an infant with craniosynostosis if the mother was on levothyroxine substitution (RR: 3.05, CI: 1.8–5.14, P < 0.001)(38). Meta-analysis could be performed on congenital malformations. No significant differences were found in treated hypothyroid women compared with healthy euthyroid controls (two studies, RR: 1.86, 0.52 – 6.64: P = 0.34)(37;38) (Fig. 3d). There was large heterogeneity between the studies (I2 of 68%), although no statistical significance. The sixth study was a randomized, not placebo controlled, trial that studied the effect of treatment with levothyroxine on IVF/ICSI in women with subclinical hypothyroidism(32). In 32 women receiving treatment, a significantly higher number of Grade I or II embryos (data presented as mean ±SD; P = 0.007), embryo implantation rate (RR: 1.8, 1.00– 3.25; P = 0.05) and live birth rate (RR: 2.13, 1.07 –4.21; P = 0.03) were found compared with 32 untreated women. No significant differences were found for clinical pregnancy rate (RR: 1.42, 0.81 –2.45; P = 0.22) or miscarriage rate (RR: 0.8, 0.00 –1.36; P = 0.08).


Figure 3. (a) Forest plot of risk ratio for Caesarean section for treated hypothyroid patients versus healthy

euthyroid controls. (b) Forest plot of risk ratio for miscarriage for hypothyroid patients treated with or without

levothyroxine. (c) Forest plot of risk ratio for preterm delivery for hypothyroid patients treated with or without

levothyroxine. (d) Forest plot of risk ratio for congenital malformations for treated hypothyroid patients versus

healthy euthyroid controls.

Adequate versus not adequately treated women

Two cohort studies reported on pregnancy complications for women with clinical or subclinical hypothyroidism who were adequately, and women who were not adequately, treated(34;36). Not adequately treated hypothyroidism was defined as a TSH higher and a thyroxine lower than the reference interval, despite treatment. In the case of subclinical hypothyroidism, a TSH higher than the reference interval despite treatment was defined as not adequately treated. The first study showed no significant difference in the prevalence of gestational hypertension in 68 women not adequately treated for subclinical or clinical hypothyroidism compared with 38 women who were still hypothyroid despite treatment (RR: 0.14, CI: 0.01

a

b

c

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–2.20: P = 0.16 for clinical hypothyroidism; RR: 0.41, CI: 0.11 –1.62: P = 0.21 for subclinical hypothyroidism)(34). The second study reported no significant difference in NICU admissions (RR: 0.31, CI: 0.08 –1.2: P = 0.09). A significant difference was found in low birthweight (RR: 0.31, CI: 0.11 –0.92: P = 0.04) for 127 women with (sub)clinical hypothyroidism with normal TSH level with levothyroxine treatment compared with 40 women with abnormal TSH levels in the first trimester despite levothyroxine treatment, while Caesarean section rates were equal in the two groups, respectively 27.5 and 29.1%(36). One case –control study reported on 38 women with hypothyroidism treated with levothyroxine during pregnancy(40). From the 19 children of mothers who were subclinically hypothyroid despite treatment, no significant difference was found in the IQ level, verbal performance or cognitive performance compared with 19 children of mothers who were euthyroid with treatment (data were continuous variables and presented as mean and SD).

Treatment interventions for thyroid autoimmunity

Five studies reported on the effect of treatment interventions for thyroid autoimmunity. Treatment with levothyroxine was reported in three studies(18;19;43) and treatment with selenomethionine in two studies(41;42).

Levothyroxine in thyroid autoimmunity

The effect of levothyroxine treatment on pregnancy outcomes was evaluated in three studies, of which two were RCTs. One randomized study was in unselected pregnant women(19) and the other two studies (one cohort and one randomized) were in women scheduled to have IVF(18;43). Levothyroxine was used at a dose of 1 μ/kg/day(18), a fixed dose of 50 μ/day(43) or a titrated dose(19). Controls were euthyroid women with thyroid autoimmunity receiving placebo or no treatment. When the results on miscarriage rates were pooled, a near-significant relative risk reduction of 52% in miscarriages was found (total 175 women, three studies, RR: 0.58, CI: 0.32 – 0.1.06; P = 0.07 (Fig. 4a). One randomized study reported on preterm birth, gestational hypertension, pre-eclampsia and placental abruption. One hundred and fifteen women were studied; a significant reduction in preterm births with levothyroxine was shown (RR: 0.31, CI: 0.11–0.90; P = 0.03). No significant differences were shown for hypertension (RR: 0.73, CI: 0.24 –2.16; P = 0.57), pre-eclampsia (RR: 2.04, CI: 0.19 –21.82; P = 0.56) or placental abruption (RR: 0.34, CI: 0.01 –8.15; P = 0.51)(19). One study also reported the effect of treatment with levothyroxine combined with acetylsalicylic acid and prednisolone in 36 women with thyroid autoimmunity who underwent an IVF treatment, and found significantly higher pregnancy rates compared with 38 controls receiving no treatment (RR: 4.14, CI: 1.47 –11.66: P = 0.007). No difference was found in miscarriage rates (RR: 2.27, CI: 0.27 –19.23: P = 0.45)(43).


If only the two randomized studies were included for meta-analyses using a random effect model, no significant risk reduction could be demonstrated (total 160 women, two studies, RR: 0.51, CI: 0.22 – 1.15; P = 0.10) (Fig. 4b)(18;19).

Figure 4. (a) Forest plot of risk ratio for miscarriage for patients with thyroid autoimmunity, treated with

or without levothyroxine. (b) Forest plot of risk ratio for miscarriage for patients with thyroid autoimmunity,

treated with or without levothyroxine (randomized trials only). (c) Forest plot of risk ratio for post-partum

thyroid disease for patients with thyroid autoimmunity treated with or without selenium.

Selenomethionine in thyroid autoimmunity

In one randomized study, 111 euthyroid women with thyroid autoimmunity were given selenomethionine 200 μ/day or placebo started in the first trimester until 12 months post-partum(42). The prevalence of post-partum thyroiditis in the 77 treated patients was compared with those of 74 controls. A significant decrease in post-partum thyroiditis was shown in the treatment group (RR: 0.59, CI: 0.38 –0.9: P = 0.01). At the end of the post-partum period, 11.7% of the women treated with selenomethionine had become permanently hypothyroid and 20.3% of the women who received placebo. This was not a significant difference (RR: 0.58, CI: 0.27 –1.24: P = 0.16). One other randomized study evaluated postpartum thyroiditis in 46 women with thyroid autoimmunity(41). Pooling the post-partum thyroiditis data of these two studies

a

b

c

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did not result in evidence of a difference in post-partum thyroiditis for selenium versus no treatment (RR: 0.85, 0.39 –1.85: P = 0.69) (Fig. 4c)(41;42). As there was significant heterogeneity between the two studies (I2 of 80%), this finding should be considered with care.

DISCUSSION

This review presents all available evidence on the effectiveness of treatment interventions for thyroid disorders in early pregnancy. Overall, we found that both MMI and PTU were effective in preventing pregnancy complications in hyperthyroid women. Furthermore, treatment with levothyroxine prevented miscarriage and preterm birth in women with clinical hypothyroidism. In thyroid autoimmunity, however, there was insufficient evidence for the effectiveness of levothyroxine. The quantity and quality of the evidence on the effectiveness of any treatment intervention for thyroid disorders on pregnancy complications were low. For none of the thyroid disorders was an RCT with enough statistical power available.

Hyperthyroidism

The ESCPG and the ATA guidelines advise to treat hyperthyroidism(1;7). The European Society of Human Reproduction and Embryology (ESHRE) and the Royal College of Obstetricians and Gynaecologists (RCOG) do not have any guidelines on hyperthyroidism in pregnancy. The evidence of the ESCPG on treatment is classified as good according to the GRADE system (Grade 1: ⊕⊕⊕⊕). The US Preventive Services Task Force (USPSTF) recommendation level is A(44). Treatment of choice is PTU because MMI is associated with typical malformations, such as aplasia cutis and choanal atresia. Our review shows that the PTU and MMI treatment options have the same risks for developing neonatal hypothyroidism and congenital malformations and that the risk is the same compared with the normal euthyroid population. Because the prevalence of congenital malformations is very low, very large cohort studies are necessary to detect a possible teratogenic effect. The available studies did not have enough statistical power to reach a final conclusion. Case reports or case series point at a possible association of MMI use with aplasia cutis and choanal atresia but the low prevalence hampers to establish a causal teratogenic relation(45;46). We did not include case reports in our systematic review. However, 35 case reports on MMI treatment and congenital scalp defects were found in our literature search(47-49). One study found that two of the eight observed cases were malformations, typically associated with MMI use i.e. choanal atresia and oesophageal atresia(8). It cannot be excluded that hyperthyroidism is teratogenic by itself. The findings from Momotomi suggest that maternal uncontrolled hyperthyroidism may cause congenital malformations and the beneficial role of MMI treatment outweighs its eventual teratogenic effect(25). There are no data to support an association between congenital abnormalities and PTU. Only small cohort studies show a reduction in pregnancy complications by PTU


treatment in hyperthyroidism. Low birthweight, preterm delivery and pre-eclampsia were reduced by treatment with both PTU and MMI. The risk for miscarriage and preterm delivery in women with treated hyperthyroidism was equal to a healthy population. With the available evidence this review supports the ESCPG and ATA guidelines to use PTU as the treatment of first choice. A preconception surgical intervention, such as subtotal thyroidectomy, or treatment with radioactive iodine should also be considered for hyperthyroidism. This might prevent any necessary treatment with antithyroid drugs during pregnancy. Women should be well informed before pregnancy on these possible treatment interventions and on the fact that it is only safe to become pregnant 6 months after treatment with radioactive iodine(1).

Clinical hypothyroidism

The ESCPG and the ATA guidelines advise to treat clinical hypothyroidism with levothyroxine. The evidence is classified as good according to the Grade system (Grade 1: ⊕⊕⊕⊕). The USPSTF recommendation level is A. The ESHRE and the RCOG do not have any guidelines on clinical hypothyroidism in pregnancy. Only two small cohort studies compared untreated women with treated women and hence evidence of a direct treatment effect is poor. Withholding treatment from these women is not considered to be ethical, therefore large comparative studies or RCTs will not be performed. The other studies compared treated women with euthyroid controls or with women who were not adequately treated. Treatment of (sub)clinical hypothyroidism seems to lower the risk for miscarriage and preterm delivery. No studies were available on the effect of treatment on neonatal intelligence scores. But even with treatment there is a higher risk for pregnancy complications, such as low birthweight and neonatal thyroid disorder. One study showed that neonates from treated hypothyroid mothers had a higher incidence of thyroid dysgenesis compared with the normal population(35). This might reflect an insufficient level of hormone replacement therapy, despite an assumed adequate management. Or, this might reflect that hypothyroidism itself, or levothyroxine use, is a risk factor for pregnancy complications. This needs further attention. Based on the seven studies, our review supports the guidelines in their advice to treat clinical hypothyroidism with levothyroxine.

Subclinical hypothyroidism

The ESCP guideline recommends levothyroxine replacement in women with subclinical hypothyroidism, given the fact that the potential benefits outweigh the potential risks. For obstetrical outcome, USPSTF recommendation level is B; evidence is fair (Grade 1: ⊕⊕OO). For neurological outcome, USPSTF recommendation level is I; evidence is poor (Grade: 0000). The ESHRE and the RCOG do not employ guidelines on subclinical hypothyroidism in pregnancy. From the seven studies on (sub)clinical hypothyroidism, only one study reported separate data on subclinical hypothyroidism. This study showed that gestational hypertension was more

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often found in not adequately treated women than in adequately treated women, though the difference was not significant. The recommendation in the current guidelines to treat subclinical hypothyroidism is based on minimal evidence and thought that the potential benefits outweigh the potential risks. For subclinical hypothyroidism, our review shows that there is currently insufficient evidence to recommend for or against universal treatment with levothyroxine. Thyroid autoimmunity The ESCPG and the ATA guidelines advise to monitor women with thyroid autoimmunity during pregnancy because these women are at risk for developing hypothyroidism. The ESCPG evidence is classified as good according to the Grade system (Grade 1: ⊕⊕⊕O) and here the USPSTF recommendation level is A. In the ATA guidelines, USPSTF recommendation level is B. The ESHRE and the RCOG do not have any guidelines on thyroid autoimmunity in pregnancy. Following our review, only three studies were available on the effect of levothyroxine on miscarriage rate in euthyroid women with thyroid autoimmunity. One of these studies was a retrospective study that showed a non-significant risk reduction of 49%(43). The other two studies were prospective randomized trials and were included for meta-analysis. One study showed a significant reduction in preterm birth(19). The findings from a recently published systematic review on thyroid autoimmunity showed a significant difference at the meta-analysis of the two randomized studies using a fixed effect model(17). In view of the large clinical heterogeneity between included studies, pooling using the random effect model is preferable to the fixed method. The random-effect method provides identical results to fixed effects when there is no heterogeneity among the studies but more conservative claims of statistical significance in the presence of heterogeneity(50). Using a random model effect we were unable to demonstrate a significant difference. These results confirm that thyroid function tests during pregnancy in women with thyroid autoimmunity are necessary but there is insufficient evidence to support treatment with levothyroxine in a euthyroid state.

Intelligence scores in the offspring

Associations have been reported between (sub)clinical hypothyroidism and lower intelligence scores in the offspring(12;14). Also thyroid autoimmunity has been associated with lower scores on intellectual and motor development(3;13). For many clinicians this is reason to treat subclinical hypothyroidism, especially in the presence of TPO-Ab, despite the current lack of evidence on the effectiveness of treatment(51). In this systematic review, only one case–control study of limited sample size (n = 38) could be included for this outcome(40). This study showed that IQ level and cognitive performance in children born of mothers treated with levothyroxine who had subclinical hypothyroidism during their pregnancy were similar to those who remained euthyroid(40). The definite results of the ‘Controlled Antenatal Thyroid Screening’ study (ISRCTN 46178175) should reveal whether screening for and treatment of subclinical hypothyroidism and/or hypothyroxinaemia in pregnancy is of benefit for the


intellectual development: preliminary results presented at the International Thyroid Conference in 2010 did not show a significant difference in the intention to treat analysis(7). The ‘TSH trial’ (NCT 00388297) is a study on the effect of levothyroxine treatment on intellectual scores in the offspring at age 5 years. The study population consists of pregnant women with first trimester subclinical hypothyroidism or hypothyroxinaemia diagnosed during pregnancy .The recruitment has been completed. The follow-up is until 2014 and final analyses are planned in 2015.

Heterogeneity and quality of the included studies

Cut-off levels

Different cut-off levels and assays have been used for the diagnosis of thyroid disorders. For now, standardized or trimester-specific reference intervals are unavailable. These intervals are needed to improve treatment of thyroid disorders in pregnancy and to compare study results. Geographical differences in iodine intake or ethnicities can complicate standardization of reference intervals(52). Aiming for national reference intervals seems therefore better.

Sample size

Many studies used small sample size and did not use any power analysis. This makes the results less solid for the studies that were not appropriate for meta-analysis.

Treatment

Two treatment interventions, PTU or MMI, were used in the studies, with varying dosages. Some studies included both PTU and MMI treatment in the same study group. This makes it hard to interpret study results and conclude on treatment effect comparing PTU and MMI.

Control groups

It is difficult to draw final conclusions from the studies that employ a euthyroid population without any thyroid disease as a control group. The same holds for studies where treated women with or without normalization of their thyroid function are compared.

Matching of the study subjects

In only nine of the 22 included studies, study subjects and controls were matched. This can also have distorted the results. Because of the heterogeneity the outcome of meta-analysis comparing hypothyroid patients with euthyroid controls for the outcome Caesarean section rate and congenital malformations should be considered with care. The same holds for the meta-analysis

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comparing treatment with selenium in women with thyroid auto-immunity for the outcome post-partum thyroid disease. The little evidence makes it difficult to make a clear statement about screening for thyroid disease in early pregnancy. The ESCPG recommend selective screening at the first prenatal visit or at diagnosis of pregnancy for women who are at risk for thyroid disease. USPSTF recommendation level is B; evidence is fair (Grade 2: ⊕⊕OO). ATA guidelines state that there is insufficient evidence to recommend for or against screening, Level I USPSTF. Only one RCT was available on universal screening versus case finding(39). This study showed that in both groups the total number of adverse pregnancy outcomes was the same but that treatment of hypothyroidism or hyperthyroidism in a low-risk group was associated with a lower rate of adverse outcomes. This study did not include screening for thyroid autoimmunity and also a power analysis was not performed to determine sample size. Cost-effectiveness analysis was not performed. Also the results will be influenced, because in both groups the patients classified as being at high risk received the same intervention. There are conflicting data on whether case finding is sufficient to identify women with thyroid disorder. One study describes that about one-third of thyroid disorders will be missed with case finding(53). It should be realized that universal screening will be difficult to introduce as most women have their first visit at 8–10 weeks of pregnancy. This is late to start treatment, especially for preventing early miscarriages. Preconception screening seems therefore better, but thyroid function often starts changing in the first trimester because of an increased need for thyroid hormone(54).

CONCLUSION

For the treatment of hyperthyroidism, we conclude that both MMI and PTU are effective in preventing pregnancy complications. Since PTU is equally effective and has not been associated with typical malformations, such as aplasia cutis or choanal atresia, reported for MMI, it is the preferred thioamide during pregnancy. Treatment with levothyroxine is recommended for women with clinical hypothyroidism because it lowers the risk for miscarriage and preterm delivery. For subclinical hypothyroidism, there is insufficient evidence to recommend for or against universal treatment with levothyroxine. Levothyroxine seems to lower the risk for miscarriage and preterm birth in women with thyroid autoimmunity but this is based on only three small studies. Randomized, placebo controlled trials are highly warranted to study the effects of treatment with levothyroxine, especially for thyroid autoimmunity, on pregnancy outcomes in view of its high prevalence. This overall lack of evidence precludes a recommendation for universal screening. Screening of thyroid dysfunction in pregnancy can only be justified within a setting of an RCT. Cost-effective analysis is required to resolve the debate of universal screening.


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53 Vaidya B, Anthony S, Bilous M, Shields B, Drury J, Hutchison S, Bilous R. Detection of thyroid dysfunction in early pregnancy: Universal screening or targeted high-risk case finding? J Clin Endocrinol Metab 2007 Jan;92(1):203-7.

54 Panesar NS, Li CY, Rogers MS. Reference intervals for thyroid hormones in pregnant Chinese women. Ann Clin Biochem 2001 Jul;38(Pt 4):329-32.

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7 |Live-birth rate in euthyroid women with recurrent

miscarriage and thyroid peroxidase antibodies

R VissenbergE FliersJAM van der PostM van WelyPH BisschopM Goddijn

Gynecological Endocrinology 2015;2:1-4

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ABSTRACT

Thyroid autoimmunity with normal thyroid function is associated with recurrent miscarriage (RM), but the association with live birth is less clear. Therefore, we determined the association between thyroid peroxidase antibodies (TPO-Ab) and live-birth rate (LBR) in a retrospective cohort of euthyroid women with unexplained RM. We included 202 women of which 28 were TPO-Ab positive (13.9%) and 174 were TPO-Ab negative. TPO-Ab positive women (n=10) without levothyroxine treatment had a lower LBR (29%) compared to TPO-Ab negative women (51%) (HR 0.23, 0.07–0.72, p = 0.012). The LBR in women with TPO-Ab receiving levothyroxine was not different compared women without TPO-Ab (60% versus 51%, p =0.50). In conclusion, TPO-Ab are associated with a lower LBR in euthyroid women with unexplained RM and these women may benefit from treatment with levothyroxine.

Recurrent miscarriage and TPO-Ab | 133

INTRODUCTION

Recurrent miscarriage (RM), defined as two or more miscarriages, presents a significant health problem for couples with a desire to have children(1). RM occurs in approximately 5% of couples trying to conceive. In most cases, the etiology is unknown(1). Thyroid disorders have frequently been linked to pregnancy loss(2-4). The most common thyroid-related condition in pregnant women is thyroid autoimmunity reflected by the presence of circulating antibodies against thyroid peroxidase (TPO-Ab)(2). The prevalence of TPO-Ab ranges for 8–14% in women of reproductive age(2). Although TPO-Ab positivity predisposes to hypothyroidism, the majority of women with TPO-Ab have a normal thyroid function. Several studies, including a recent systematic review and meta-analysis, provide convincing evidence that women with TPO-Ab and normal thyroid function have a 2.5 fold risk for RM(4;5). In addition, TPO-Ab have been associated with other adverse pregnancy outcomes including unexplained subfertility, single miscarriage, preterm birth, respiratory distress and postpartum thyroiditis(4;5). To date, only two cohort studies have been published on the association between TPO-Ab and live-birth rate (LBR) in euthyroid women with unexplained RM (6;7). In both studies, the LBR was not different between women with and without TPO-Ab, but only pregnant women were included. The aim of the current study was to determine the association between TPO-Ab and LBR in euthyroid women with unexplained RM. The effect of treatment with levothyroxine was also studied.

METHODS

The study was designed as a retrospective cohort study. The study was carried out using data from medical files from women who consulted the RM clinic at the Academic Medical Centre of the University of Amsterdam between 2005 and 2011. All women with unexplained RM received a written questionnaire at their home address and were asked to return the questionnaires by prepaid mail. A written reminder was sent 2 months after the initial mailing. Women who did not fill out the questionnaire were contacted by telephone and asked permission to be interviewed. Information was collected on the medical history, parity, age,

body mass index (BMI), use of medication, smoking status during pregnancy and the outcome of subsequent pregnancies after the consultation for RM. Execution of this study was approved by the Institutional Review Board under the national legal requirements for clinical research in the Netherlands.

Study subjects

All participants were women with unexplained RM that consulted the RM clinic. All women were screened for the presence of TPO-Ab. RM was defined according to the Special Interest

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Group for Early Pregnancy (European Society of Human Reproduction and Endocrinology) consensus statement as two or more, not necessarily consecutive, miscarriages before 20 weeks of gestation, verified by a pregnancy test and/or ultrasonography(8). Unexplained RM was defined when an underlying risk factor for RM was not present. Known causes of RM (uterine abnormalities, anti-phospholipid syndrome, thrombophilia, hyperhomocysteinemia, abnormal parental karyotyping) were ruled out. Women with a Thyroid Stimulating Hormone (TSH) level outside the institutional reference range were excluded. The study subjects were divided in three groups: (1) euthyroid women with TPO-Ab in whom levothyroxine was started, (2) euthyroid women with TPO-Ab who did not receive levothyroxine and (3) euthyroid women without TPO-Ab.

Assays

Before 2008, TSH was measured by a time-resolved fluorimmunoassay (Delfia, hTSH Delfia Ulta, Perkin Elmer, Turku, Finland): reference range 0.40–4.0 mU/L, detection limit 0.01 mU/L and total assay variation 4–5%. As of 2008, TSH was measured with an electrochemiluminiscent immunometric assay performed on the cobas e602 analyzer (Roche Diagnostics, Almere, The Netherlands): reference range 0.50–5.0 mU/L and total assay variation of 2–4%. TPO-Ab was measured by a chemiluminescence immunoassay (LUMI-test anti-TPO, BRAHMS, Berlin, Germany) with a detection limit of 30 kU/L and total assay variation of 8–12%. TPO-Ab-positivity was defined as TPO-Ab > 60 kU/L.

Levothyroxine therapy

No standardized treatment exists for women with RM and thyroid autoimmunity(9-12). Women were referred to an endocrinologist, who either started empirical levothyroxine (LT4) therapy preconceptually or recommended thyroid function tests during a subsequent pregnancy. There was no formal randomization process. The choice whether to start treatment was based on the preference of the clinician and the patient.

Outcomes

The primary outcome was LBR. A live birth was defined as the birth of a living fetus after 24 weeks of gestation. The secondary outcome was pregnancy rate (defined as a reported pregnancy in the questionnaire).

Statistical analysis

All statistical analyses were performed using the Statistical package of Social Sciences and Problem Solutions (IBM SPSS version 20.0, Armonk, NY). Baseline measurements are presented as means with standard deviations, as median with ranges or as numbers with percentages


as appropriate. Differences between the three groups were evaluated using Chi-square tests for categorical variables, and ANOVA or Kruskal–Wallis tests for continuous variables. In case of statistical significance, Bonferroni post-hoc analysis was used to evaluate differences between the three groups. We expected subjects to have different follow-up times. As pregnancy chances are highly influences by follow-up time, we accounted for time by creating Kaplan–Meier curves. To account for time to event while controlling for potential confounders, we analyzed the data using Cox proportional hazard. LBRs and pregnancy rates were visualized in Kaplan–Meier curves. Cox proportional hazard analysis was done to correct for maternal age and previous number of miscarriages. Differences in LBR or pregnancy rate were expressed as a hazard ratio with a 95% confidence interval (CI).

RESULTS

Between 2005 and 2011, 408 women visited the RM clinic. Women with known causes for miscarriage (n = 47) and women with abnormal TSH levels (n = 17) were excluded. This left 344 euthyroid women with unexplained RM. The response rate was 71% (244/344) and the participation rate was 59% (202/344) (Figure 1). From 202 women who agreed to participate, 28 were TPO-Ab-positive (13.9%) and 174 were TPO-Ab-negative (86.1%). After collection of the baseline data, levothyroxine treatment was initiated in 10 of 28 euthyroid women with TPO- Ab. The decision to start empirical levothyroxine treatment was based on the reference of the treating physician and patient.


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Baseline characteristics are shown in Table 1. Apart from a slightly higher baseline TSH in women with TPO-Ab, who were not going to use levothyroxine (p =0.023), baseline characteristics were not different between the groups.

Table 1. Patient characteristics of women with unexplained recurrent miscarriage.

TPO-Ab, treatment LT4(n=10)

TPO-Ab, no treatment (n=18)

TPO-Ab negative(n= 174)

p value

Age (years) 33 ± 5.9 36 ± 6.2 34 ± 4.6 0.22

Previous miscarriages (n) 3.4 ± 1.3 3.7 ± 2.3 3.3 ±1.8 0.77

Mean TSH (mU/L) (SD) at baseline* 1.8 ± 1.1 2.2 ± 0.9 1.7 ± 0.8 0.028*

Smoking (%) 9 (10) 2 (11) 25 (14) 0.87

Body mass index (kg/m2) 24 ± 5.0 28 ± 8.4 24 ± 5.2 0.08

Follow up time (months) 49 (17-74) 45 (12-86) 41 (10-96) 0.98

* Post hoc Bonferroni analysis: TPO-Ab treatment versus TPO-Ab negative, p = 1.00.TPO-Ab treatment versus TPO-Ab no treatment, p = 0.46.TPO-Ab no treatment versus TPO-Ab negative, p = 0.023.

Live-birth rate

The LBR at 12 months was 29% in women with TPO-Ab without treatment versus 51% in the group women without TPO-Ab. After correction for maternal age and previous number of miscarriages, LBR remained lower in women with TPO-Ab not receiving LT4 treatment compared to women without TPO-Ab (HR 0.23, CI 0.07–0.72, p =0.012). The LBR at 12 months was 60% in the group women with TPO-Ab who received treatment with LT4 versus 51% in the group women without TPO-Ab. After correction for maternal age and previous number of miscarriages, the group of women with TPO-Ab that received treatment with LT4 had a similar chance of a live-birth compared to women without TPO-Ab (HR 1.28, CI 0.62–2.63, ) (Table 2).

Pregnancy rate

The pregnancy rate at 12 months was 44% in the group women with TPO-Ab without treatment versus 69% in the group women without TPO-Ab. After correction for maternal age and previous number of miscarriages, women with TPO-Ab showed a lower pregnancy rate compared to women without TPO-Ab (HR 0.47, 95% CI 0.24–0.95, p =0.032) (Table 2).


The pregnancy rate at 12 months was 67% in the group women with TPO-Ab who received treatment with LT4 versus 69% in the group women without TPO-Ab. After correction for maternal age and previous number of miscarriages, the group of women with TPO-Ab that received treatment with LT4 had a similar subsequent pregnancy rate compared to women without TPO-Ab (HR 0.86, 95% CI 0.40–1.84, p= 0.695) (Table 2).

Table 2. Cox proportional hazard live-birth rate and pregnancy rate of women with unexplained recurrent miscarriage.

Live birth rate1 HR 95% CI p-value

TPO-Ab negative

TPO-Ab, treatment LT4 1.28 0.62-2.63 0.502

TPO-Ab, no treatment 0.23 0.07-0.72 0.0122

Pregnancy rate1 HR 95% CI p-value

TPO-Ab negative

TPO-Ab, treatment LT4 0.86 0.40-1.84 0.6952

TPO-Ab, no treatment 0.47 0.24-0.95 0.0322

1 Corrected for the covariates maternal age and previous number of miscarriage.2 In comparison with the TPO-Ab negative group.

DISCUSSION

This study reveals a 13.9% prevalence of TPO-Ab-positivity in a Dutch cohort of women with unexplained RM. The presence of TPO-Ab in women with RM not receiving LT4 treatment was associated with a lower subsequent LBR and pregnancy rate compared to women with RM without TPO-Ab. Women with RM and TPO-Ab that received treatment with LT4 had similar LBRs and pregnancy rates compared to women without TPO-Ab.

Strengths of this study are the accurate calculation of the LBR with adjustment for time to pregnancy, age and previous number of miscarriages and the fact that LBR s were calculated per individual patient. This cohort study is of great value because data on this specific topic are scarce hitherto. This study concerns a well-described patient population that has been followed for almost 5 years.

The shortcomings of the present study are the relatively small study population and the retrospective and non-randomized design. This study might be subject to allocation, selection and recall bias. In the statistical analysis, we corrected for maternal age and the number of previous miscarriages. Because of the relatively small study population, it was not possible to correct for additional confounders.

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Current guidelines advise not to screen for TPO-Ab in the RM workup(9;10). Arguments are a lack of evidence on the association between TPO-Ab and LBR and the absence of an effective treatment for women with RM and TPO-Ab(6;7). This study suggests that women with unexplained RM and TPO-Ab who receive empirical treatment with LT4 have higher chance for a live birth compared to the women that did not receive any treatment. This study was not an intervention trial and could at most provide an indication for the effectiveness of LT4. The results of this study therefore justify further research, especially randomized controlled trials on this topic. There remains a clear need for combining follow-up studies like current study and other published studies within a Cochrane collaboration or by means of an individual patient data analysis. The evidence to result from such structural review studies should ideally be followed by further evidence to be collected in a randomized controlled setting. The T4-LIFE study (NTR3364), an international multicenter randomized trial, has just started and randomizes women with RM and TPO-Ab between levothyroxine and placebo (http://www.studies-obsgyn.nl/T4-LIFE). The TABLET study is another RCT focusing on women with a spontaneous miscarriage and/or RM and TPO-Ab (ISRCTN Number: 15948785).


REFERENCES

1 Rai R, Regan L. Recurrent miscarriage. Lancet 2006 Aug 12;368(9535):601-11.






7 Yan J, Sripada S, Saravelos SH, Chen ZJ, Egner W, Li TC. Thyroid peroxidase antibody in women with unexplained recurrent miscarriage: prevalence, prognostic value, and response to empirical thyroxine therapy. Fertil Steril 2012 Aug;98(2):378-82.


9 Goddijn M, van den Boogaard E, Steepers EA, Erwich JJ, Macklon NS, Land JA, Ankum WM. [The guideline ‘Recurrent miscarriage’ (first revision) of the Dutch Society for Obstetrics and Gynaecology]. Ned Tijdschr Geneeskd 2008 Jul 26;152(30):1665-70.


11 Franssen MT, Korevaar JC, van d, V, Boer K, Leschot NJ, Goddijn M. Management of recurrent miscarriage: evaluating the impact of a guideline. Hum Reprod 2007 May;22(5):1298-303.

12 Vissenberg R, van den Boogaard E, van WM, van der Post JA, Fliers E, Bisschop PH, Goddijn M. Treatment of thyroid disorders before conception and in early pregnancy: a systematic review. Hum Reprod Update 2012 Jul;18(4):360-73.

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8 |Effect of levothyroxine on live birth rate in

euthyroid women with recurrent miscarriage and

TPO antibodies (T4-LIFE study)

R VissenbergMM van DijkE FliersJAM van der PostM van WelyKWM BloemenkampA HoekWK KuchenbeckerHR VerhoeveHCJ ScheepersS Rombout- de WeerdC KoksJJ ZwartF BroekmansW VerpoestOB ChristiansenM PostDNM PapatsonisMFG VerbergJ SikkemaBW MolPH BisschopM Goddijn

Contemporary Clinical Trials 2015;44:134–138

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ABSTRACT

Background

Thyroid peroxidase antibodies (TPO-Ab) in euthyroid women are associated with recurrent miscarriage (RM) and other pregnancy complications such as preterm birth. It is unclear if treatment with levothyroxine improves pregnancy outcome.

Aim

The aim of this study is to determine the effect of levothyroxine administration on live birth rate in euthyroid TPO-Ab positive women with recurrent miscarriage.

Methods/design

We will perform a multicenter, placebo controlled randomized trial in euthyroid women with recurrent miscarriage and TPO-Ab. Recurrent miscarriage is defined as two or more miscarriages before the 20th week of gestation. The primary outcome is live birth, defined as the birth of a living fetus beyond 24 weeks of gestation. Secondary outcomes are ongoing pregnancy at 12 weeks, miscarriage, preterm birth, (serious) adverse events, time to pregnancy and survival at 28 days of neonatal life. The analysis will be performed according to the intention to treat principle. We need to randomize 240 women (120 per group) to demonstrate an improvement in live birth rate from 55% in the placebo group to 75% in the levothyroxine treatment group. This trial is a registered trial (NTR 3364, March 2012). Here we discuss the rationale and design of the T4-LIFE study, an international multicenter randomized, double blind placebo controlled, clinical trial aimed to assess the effectiveness of levothyroxine in women with recurrent miscarriage and TPO-Ab.

Protocol of T4-LIFE study | 143

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INTRODUCTION

Recurrent miscarriage represents a significant health problem. Approximately 5% of couples trying to conceive suffer recurrent miscarriage (RM)(1;2). Different definitions for RM have been described. In this article recurrent miscarriage has been defined as two or more – not necessarily consecutive – miscarriages(3-8). Known risk-factors for RM are parental chromosome abnormalities, uterine anomalies and antiphospholipid syndrome(1;9). Even after comprehensive investigations, no underlying risk factor for RM is identified in ≥50% of couples(1).

The presence of thyroid peroxidase antibodies (TPO-Ab) indicates a state of thyroid autoimmunity and is strongly associated with sporadic and recurrent miscarriages(10). Thyroid autoimmunity is present in 8–14% among all women at reproductive age(11). The presence of thyroid peroxidase antibodies is not only associated with miscarriage, but also with other adverse pregnancy outcomes such as unexplained subfertility, preterm birth and postpartum thyroiditis(10). A higher prevalence of TPO-Ab is reported in women with recurrent miscarriage, varying from 19 to 36%(11-16).

Given the high prevalence of TPO-Ab and its association with RM and other pregnancy complications, screening for thyroid dysfunction in the work-up for RM or during pregnancy is proposed, but not generally accepted. The current guidelines for RM of the European Society of Human Reproduction and Embryology (ESHRE 2006), the Royal College of Obstetricians and Gynecologists (RCOG 2011) and the ‘Nederlandse Vereniging voor Obstetrie en Gynaecologie’ (NVOG 2007), advise not to screen for thyroid antibodies because no evidence exists for an effective treatment intervention(4;17-19). The guidelines on thyroid disorders and pregnancy of the Endocrine Society Clinical Practice Guideline (ESCPG 2012) and the American Thyroid Association (ATA 2011) state that screening during pregnancy is not indicated because the treatment possibilities and effects for women with thyroid autoimmunity are thus far unclear(20;21).

Two small, randomized studies, including a total of 160 women with thyroid antibodies evaluated the effect of levothyroxine (T4) treatment on pregnancy outcomes. One trial studied pregnant euthyroid women with thyroid antibodies. The other trial studied women with TPO-Ab undergoing assisted reproduction technologies(22;23). Both studies showed a reduction in miscarriage rates (36% and 75% relative reductions). One of the studies found a 69% relative risk reduction in preterm births. Both studies did not have an adequate sample size(22;23). Meta-analysis of these studies showed a non-significant reduction in miscarriage rate, but the studies were too small to draw robust conclusions(19).

Although current RM guidelines do not support the screening for thyroid disorders, since lack of evidence on effective treatment interventions, endocrinologists are eager to prescribe levothyroxine during pregnancy for euthyroid women with TPO-Ab (21;24). A recent European survey demonstrated that almost 80% of endocrinologists prescribe levothyroxine

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during pregnancy for women with TPO-Ab in combination with a normal Thyroid Stimulating Hormone (TSH) level(25). This can result in unnecessary screening and treatment.

The aim of this study is to determine the effect of levothyroxine treatment on live birth rates and pregnancy complications in women with recurrent miscarriage and TPO-Ab. To achieve this, we designed an international randomized double blinded placebo controlled trial with inclusions in multiple centers.

METHODS

Study sample

Women with unexplained recurrent miscarriage and thyroid autoimmunity are eligible for the study. Women aged 18 years until 42 years at randomization will be included. Recurrent miscarriage is defined as two or more, not necessarily consecutive, pregnancy losses before 20 weeks of gestational age(5;6). The definition of miscarriage included documentation of pregnancy by a positive pregnancy test and clinical manifestations of miscarriage (e.g., abdominal pain, cramps and vaginal bleeding); it does not include the loss of a bio-chemical pregnancy. Women with a history of RM after natural conception or after assisted reproductive technology are both included. All participants receive routine diagnostic work-up for recurrent miscarriage, including testing for antiphospholipid syndrome or thrombophilia if indicated, karyotyping if indicated, testing for uterine abnormalities and TSH and TPO-Ab assessments. Thyroid autoimmunity is defined as euthyroidism (TSH level within the center’s reference range) with the presence of TPO antibodies. Euthyroidism will be defined according to the cut-off levels per participating center, as a result of minimal differences in reference ranges between centers due to different assay methods. Cut-off levels will be registered. Most commonly used cutoff levels for TPO antibodies are 60 kIU/L or 100 kIU/L. For TSH, the most commonly used reference interval is 0.5–5.0 mIU/L.

Exclusion criteria are: antiphospholipid syndrome (lupus anticoagulant and/or anticardiolipin antibodies IgG or IgM and/or B2-glycoprotein IgG or IgM positivity), other auto-immune conditions, e.g. diabetes mellitus or other known thyroid diseases, previous enrolment in the T4-LIFE-trial, participation in other (double blind randomized) drug trials, and contraindications for levothyroxine use (acute cardiac arrest, acute pancreatitis or acute myocarditis).

Setting and design

Participating centers

We will perform an international multicenter randomized, double blind placebo controlled, clinical trial, in the Departments of Obstetrics and Gynecology, in both academic and non-academic hospitals in the Netherlands, Denmark and Belgium. This multicenter study will be


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carried out within the infrastructure of the Dutch Consortium for studies on women’s health. This consortium provides a unique clinical research infrastructure for studies in the field of reproductive gynecology. The trial design is presented in Fig. 1. Inclusion has started in January 2013. Currently, as per July 2015, 64 women have been included in the study

At the start of the study, it was expected to enroll around 7 women per month. The original estimated duration of the recruitment phase of the study was supposed to be 30 months. When the recruitment started only few centers have been participating. Currently, 14 centers do participate in the Netherlands, one center in Belgium and one center in Denmark. The number of participating centers is still increasing. With the current recruitment rate, it is expected that the total recruitment phase will be 45 months.

Randomization

If after the diagnostic work-up for recurrent miscarriage positive TPO-Ab are detected with a normal TSH level and women match the inclusion criteria, they will be asked to participate. After signing informed consent preconceptually, they will be randomized and allocated to levothyroxine or placebo (double-blinded). Randomization will be done on the internet in a 1:1 ratio, once the patient data have been entered in a web-based database. Randomization will be performed using stratified blocks or minimization with a block size of 4 for two groups.

All assays have their own specific reference intervals and therefore we believe that treatment differences between centers will be minimal. To correct for possible treatment differences randomization will be stratified for study center. All patients will start taking their study medication preconceptually, immediately after randomization.

Blinding

The study is double blinded. The TSH levels will be assessed at three time points: preconceptually, at the first trimester (before the 12th week of gestation) and at the second trimester (before the 20th week of gestation). When the TSH level is outside the center’s reference range, women will stop taking the study medication and will be referred to an endocrinologist to receive standard care. They will not be excluded from the trial; their data will be used for analysis as well. If the TSH level is within the reference interval, patients will continue their study medication. In this case both the patient and the doctor will remain blinded to the study medication.

Intervention

The intervention will consist of treatment with levothyroxine or placebo tablets (tablets of 25 μg). An individual dosage for each study subject will be calculated based on their body weight and the initial TSH level at diagnosis (the formula is represented in Fig. 1)(23).

The pregnancy itself will be monitored by ultrasounds in the first trimester and patients

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receive standard obstetrical care. Levothyroxine or placebo tablets will be continued till the end of the pregnancy.

Use of co-intervention (if applicable)

Patients with other auto-immune conditions who might use other medications are excluded from the trial. Co-treatment with aspirin, vitamin and other dietary interventions is permissible. Drugs that will interact with the absorption of levothyroxine will be taken at least 2 h apart from the levothyroxine tablets (which will be taken in the morning).

Assays

Euthyroidism will be defined according to the cut-off levels for TSH per participating center. All assays for TSH and TPO-Ab per recruiting center, and center specific reference ranges are mentioned in Appendix 1.

Figure 1. Flowchart of T4-LIFE trial, including all standard visits (example for Academic Medical Center, The

Netherlands).


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Primary outcome

The primary outcome measure is live birth. Live birth is defined as the birth of a living fetus beyond 24 weeks of gestational age.

Secondary outcomes

Ongoing pregnancy at 12 weeks, miscarriage (defined as pregnancy loss before the 20thweek of gestation), preterm delivery (preterm birth defined as delivery before 37 weeks of gestation), adverse events (defined as any untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical product and which does not necessarily have to have a causal relationship with this treatment), serious adverse events (SAE) (a serious adverse event or reaction which is any untoward medical occurrence that at any dose results in death or is life-threatening (requirement of inpatient hospitalization or prolongation of existing hospitalization, results in persistent or significant disability/incapacity, or is a congenital anomaly/birth defect)), time to pregnancy (defined as the interval between thyroid function test and the month of conception of the next pregnancy) and survival at 28 days of neonatal life.

Estimation of power and sample size

We expect an increase in live birth rate from 55% to 75% (intention to treat analysis), based upon a live birth rate of 55% in a previously published cohort of women with recurrent miscarriage and untreated thyroid autoimmunity(16). A relative risk reduction of 52% for miscarriage was shown in a recent meta-analysis for women with thyroid autoimmunity treated with levothyroxine(19;26). To detect an increase of 20% in live birth beyond 24 weeks (from 55% to 75%), with an alpha error rate of 5% and beta error rate of 20% (i.e. 80% power), 90 women will need to be randomized to the intervention arm, and 90 women to the control arm (180 women in total). Assuming and adjusting for a worst case scenario of a loss to follow-up rate of 5%, the total number of participants required will be 200 (100 in each arm of the trial). As participants are randomized preconceptually, and final outcome assessment is at the end of the pregnancy we expect the number of participants lost to follow-up will be minimal because of frequent standard obstetrical care. This is based on several years of experience in the recruitment and follow-up of patients enrolled in RM studies. We expect 15% of the patients in each group will have to stop the study medication after randomization because of development of (sub)clinical hypothyroidism. In this case women will be referred to an endocrinologist for usual care (in most cases treatment with levothyroxine)(23). To make sure we can account for these drop-outs we will allocate 120 patients to each group.

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Statistical analyses

Baseline data and outcome data will be summarized separately. For continuous variables, we will examine the distribution of the observations, and if normally distributed then we will summarize them as means with standard deviations (SDs). If they are not normally distributed, then medians and inter-quartile ranges (IQRs) will be reported. For dichotomous data, we will provide proportions (or percentages). In addition to the baseline and outcome data, we will also summarize the recruitment numbers, those lost to follow-up, protocol violations and other relevant data. Dichotomous outcomes will be analyzed using either Fisher’s exact test or chi-square as appropriate. For continuous outcomes we will use t-test if the observations in each study arm are normally distributed, and if non-normally distributed, Mann– Whitney-U test will be employed. Although p-values will be reported, the focus will be on providing 95% confidence intervals around point estimates as these are more useful in interpreting the findings of the study.

Efficacy analyses will be done by intention-to-treat (ITT) and will include all randomized women. We will compare the primary outcome, i.e. live birth, in the intervention group and the control group. Differences in live-birth rates will be expressed as absolute differences and relative risks, with associated 95% confidence intervals, with the placebo group as the reference. Relative risks and 95% confidence intervals will be calculated also for the secondary outcomes. We will construct Kaplan–Meier curves, estimating the cumulative probability of conception leading to live birth rate over time. All statistical analysis will be performed using the Statistical Package of Social Sciences and Problem Solutions (SPSS version 21.0).

Endpoints

The trial ends for each patient after the end of the first subsequent clinical pregnancy, whether it is a miscarriage, ectopic pregnancy, molar pregnancy or a live birth, or after a two-year period of preconceptual use of the study medication, not resulting in a pregnancy.

Protection of human participants

Approval for this study was obtained from the Institutional Review Board (IRB) of the Academic Medical Centre and from the Central Committee on Research involving Human Subjects (CCMO), in the Netherlands. IRB approval was also obtained from the participating centers in Denmark and Belgium.

In patients who meet the inclusion criteria, written informed consent is obtained before randomization is carried out. The trial is registered with the Dutch Trial registry (NTR3364), Institutional Review Board (ID: NL24082.018.08 MEC Academic Medical Centre (Amsterdam)).

A centralized and independent Data and Safety Monitoring Board (DSMB) provides oversight and monitoring to ensure the safety of participants and the validity and integrity


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of the trial. All SAE are reported to the Institutional Research Board and DSMB. In consultation with the DSMB an interim analysis will not be performed, because of the small number of patients needed to recruit, the low risk of levothyroxine use in pregnancy and the primary outcome being live birth.

DISCUSSION

Thyroid autoimmunity is associated with miscarriage, recurrent miscarriage, preterm birth and postpartum thyroiditis. Miscarriage and preterm birth have a high prevalence. Miscarriage occurs in 10– 15% of every pregnancy and preterm delivery occurs in 7% of all deliveries (12,000 cases in the Netherlands per year). Both conditions are associated with a high maternal and neonatal morbidity. Miscarriage has a high emotional impact and causes distress in the subsequent pregnancy(27). If a miscarriage needs to be managed surgically, this may increase the risk for preterm birth in the subsequent pregnancy(28). Preterm birth is the most important cause of neonatal mortality and morbidity in the Netherlands and is extremely costly for the health care system(29).

To date testing for TPO-Ab in the diagnostic work-up for recurrent miscarriage is not advised in the current guidelines because of a lack of adequate intervention studies(18). There is no proven treatment for women with recurrent miscarriage and thyroid autoimmunity(4;17-19).Despite or perhaps due to this lack of evidence there is substantial variation among European clinicians in the way they manage thyroid autoimmunity during pregnancy in Europe. Almost 80% of endocrinologists prescribe levothyroxine during pregnancy for women with thyroid autoimmunity, although the effectiveness has not been demonstrated(25). This may result in unnecessary screening, medicalization and costs. Therefore a randomized controlled trial is highly warranted on this topic and for this reason we propose a randomized intervention study.

The T4-LIFE study is the first study to determine the effectiveness of levothyroxine treatment in women with recurrent miscarriage. Currently, another randomized controlled trial, the TABLET study (ISRCTN number: 15948785), is including women with TPO-Ab and one or more spontaneous miscarriages.

If the results of our study show that levothyroxine treatment increases live birth rate, it would result in a major change in diagnostic testing and treatment policy in these women. Major health benefits could be obtained. If treatment of thyroid autoimmunity with levothyroxine increases live birth rates it could result in a clear advice on screening all women with recurrent miscarriage for the presence of TPO-Ab and TSH, and if applicable start treatment with levothyroxine. If levothyroxine is effective in preventing miscarriage, this intervention could be highly cost-effective. Levothyroxine is an intervention of extremely low costs (€0.02/tablet levothyroxine 25 μg) with the potency of achieving a major health gain. An additional cost

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effectiveness study should investigate this.If demonstrated that levothyroxine does not increase live birth rates, the outcome could

be implemented in protocols and could be used to reduce unnecessary screening, treatments and costs since many of these women worldwide use this medication now.

CONCLUSION

The T4-LIFE study is a double-blinded, placebo-controlled, randomized, multicenter, international trial that will generate novel data about the efficacy and safety of levothyroxine treatment in TPO-Ab positive women with recurrent miscarriage. If levothyroxine proves to be effective, it may justify screening for TPO-Ab in women with unexplained recurrent miscarriage followed by levothyroxine treatment to increase the live-birth rate. If levothyroxine is not effective, this could prevent referral to endocrinologists and unnecessary prescription of levothyroxine. Independent of the effectiveness of levothyroxine, the results of this study may contribute to standardization of health care and correct use of levothyroxine.


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REFERENCES

1 Rai R, Regan L. Recurrent miscarriage. Lancet 2006 Aug 12;368(9535):601-11.

2 Kaandorp SP, Goddijn M, van der Post JA, Hutten BA, Verhoeve HR, Hamulyak K, Mol BW, Folkeringa N, Nahuis M, Papatsonis DN, Buller HR, van d, V, Middeldorp S. Aspirin plus heparin or aspirin alone in women with recurrent miscarriage. N Engl J Med 2010 Apr 29;362(17):1586-96.

3 Evaluation and treatment of recurrent pregnancy loss: a committee opinion. Fertil Steril 2012 Nov;98(5):1103-11.

4 Goddijn M, van den Boogaard E, Steepers EA, Erwich JJ, Macklon NS, Land JA, Ankum WM. [The guideline ‘Recurrent miscarriage’ (first revision) of the Dutch Society for Obstetrics and Gynaecology]. Ned Tijdschr Geneeskd 2008 Jul 26;152(30):1665-70.

5 Farquharson RG, Jauniaux E, Exalto N. Updated and revised nomenclature for description of early pregnancy events. Hum Reprod 2005 Nov;20(11):3008-11.

6 Jaslow CR, Carney JL, Kutteh WH. Diagnostic factors identified in 1020 women with two versus three or more recurrent pregnancy losses. Fertil Steril 2010 Mar 1;93(4):1234-43.

7 van den Boogaard E, Kaandorp SP, Franssen MT, Mol BW, Leschot NJ, Wouters CH, van d, V, Korevaar JC, Goddijn M. Consecutive or non-consecutive recurrent miscarriage: is there any difference in carrier status? Hum Reprod 2010 Jun;25(6):1411-4.


9 Li TC, Makris M, Tomsu M, Tuckerman E, Laird S. Recurrent miscarriage: aetiology, management and prognosis. Hum Reprod Update 2002 Sep;8(5):463-81.



12 Iravani AT, Saeedi MM, Pakravesh J, Hamidi S, Abbasi M. Thyroid autoimmunity and recurrent spontaneous abortion in Iran: a case-control study. Endocr Pract 2008 May;14(4):458-64.


14 Glinoer D, Riahi M, Grun JP, Kinthaert J. Risk of subclinical hypothyroidism in pregnant women with asymptomatic autoimmune thyroid disorders. J Clin Endocrinol Metab 1994 Jul;79(1):197-204.

15 Bussen S, Steck T. Thyroid autoantibodies in euthyroid non-pregnant women with recurrent spontaneous abortions. Hum Reprod 1995 Nov;10(11):2938-40.


17 The Investigation and Treatment of Couples with Recurrent First-trimester and Second-trimester Miscarriage. Green-top Guideline No.17; RCOG.

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27 McCarthy F, Moss-Morris R, Khashan A, North R, Baker P, Dekker G, Poston L, McCowan L, Walker J, Kenny L, O’Donoghue K. Previous pregnancy loss has an adverse impact on distress and behaviour in subsequent pregnancy. BJOG 2015 Jan 6.

28 McCarthy FP, Khashan AS, North RA, Rahma MB, Walker JJ, Baker PN, Dekker G, Poston L, McCowan LM, O’Donoghue K, Kenny LC. Pregnancy loss managed by cervical dilatation and curettage increases the risk of spontaneous preterm birth. Hum Reprod 2013 Dec;28(12):3197-206.

29 Iams J. Prevention of preterm birth. N Engl J Med 1998 Jan 1;338(1):54-6.


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SUPPLEMENTARY DATA

Appendix 1. Assays per participating center

Academic Medical Center, Amsterdam, The Netherlands

As of 2008, TSH was measured by an electrochemiluminiscent immunometric assay performed on the cobas e602 analyzer (Roche Diagnostics, Almere, The Netherlands): reference range 0.50 – 5.0 mU/L and total assay variation of 2-4%. TPO-Ab was measured by a chemiluminescence immunoassay (LUMI-test anti-TPO, BRAHMS, Berlin, Germany) with a detection limit of 30 kU/L and total assay variation of 8-12%. TPO-Ab-positivity was defined as TPO-Ab >60 kU/L.

University Medical Center Leiden, Leiden, The Netherlands

TSH was measured by a Modular E170 immunoanalyser (ECLIA) (Roche Diagnostics): reference range 0.30 – 4.80 mU/L. TPO-Ab was measured by a Immulite 2000 Xpi immunoanalyser (ILMA) (Siemens Healthcare Diagnostics): reference range 0 – 35 kU/L.

University Medical Center Groningen, Groningen, The Netherlands

As of 2006, TSH was measured by an electrochemiluminiscentie immunoassay (Roche Diagnostics Almere, The Netherlands): reference range: 0.5-4.0 mU/L.As of 2008, TPO-Ab was measured using a commercially available automated fluorescence enzyme-linked immunoassay (ImmunoCAP anti-TPO on Phadia250 analyzer, ThermoFisherScientific/Phadia, Freiburg, Germany) with a detection limit of 33 IU/ml: reference range 0-100 IU/ml.

Isala Clinics, Zwolle, The Netherlands

TSH was measured by a Cobas 8000 immunoanalyser (Roche Diagnostics): reference range 0.4-4.0 mU/L.TPO-Ab was measured by a fluorescence enzyme-linked immunoassay (Phadia): referenge range.

Chapter 8154 |

Onze Lieve Vrouwe Hospital, Amsterdam, The Netherlands

TSH was measured by an Electrochemiluminescence assay (ECLIA) performed on a Cobas e602 analyser (Roche Diagnostics, Almere, The Netherlands): Reference range 0.3 - 4.6 mIU/l (WHO 2nd IRP 80/558), and total assay variation of 2-4 %.TPO-Ab was measured by a fluorescence enzyme immunoassay (FEIA) ( Phadia 250,Thermo Scientific, Uppsala, Sweden), with a detection limit of 4 IU/ml and a total assay variation of 4.1-4.7%. TPO-Ab-positivity was defined as TPO-Ab > 25 IU/ml.

University Medical Center Maastricht, Maastricht, The Netherlands

TSH was measured with a electrochemiluminiscent immunometric assay performed on the cobas e602 analyzer (Roche Diagnostics, Almere, The Netherlands): reference range 0.4 – 4.3 mU/L and total assay variation of 2%.TPO-Ab was measured by an enzyme-linked immunosorbent assay (ELISA) (Euroimmun. Luebeck, Germany) with a detection limit of 10 kU/L and inter-assay variation of 2-3.5%. TPO-Ab-positivity was defined as TPO-Ab >50 kU/L.

Albert Schweizer Hospital, Dordrecht, The Netherlands

As of 2013, TSH was measured with a electrochemiluminiscent immunometric assay performed on the Vista analyzer (Siemens Diagnostics, The Netherlands): reference range 0.4– 4.0 mU/L and total assay variation of <5%. TPO-Ab was measured by a chemiluminescence immunoassay (anti-TPO, Centaur XP, Siemens Diagnostics) with a detection limit of 28 kIU/L and total assay variation of 10%: Reference range 0-60 kIU/L.

Maxima Medical Center, Veldhoven, The Netherlands

TSH was measured by a Cobas 8000 immunoanalyser (Roche Diagnostics): reference range 0.40-4.0 mU/L.TPO-Ab was measured by an immunoassay, C-module (C502 resp. C501) Cobas 8000 en Cobas 6000 (Roche Diagnostics): reference range 0-35 kU/L.

Deventer Hospital, Deventer, The Netherlands

TSH was measured by an electrochemiluminiscent immunometric assay performed on the Cobas 6000 analyzer (Roche Diagnostics, Almere, The Netherlands): reference range 0.40 – 4.0 mU/L and total assay variation < 2.1%.TPO-Ab was measured by an electrochemiluminiscent immunometric assay on the Cobas 6000 analyzer (Roche Diagnostics, Almere, The Netherlands); reference range 0-35 IU/ml, total assay variation < 7,0%.


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University Medical Center Utrecht, Utrecht, The Netherlands

TSH was measured with a chemoluminometric immunoassay on the Beckman-Coulter Unicel DXi800 (CA, USA). Total assay variation ranged from 3.7 to 4.8% (0.38-35 mU/L). Reference range 0.35-5 mU/L. Lower limit of detection: 0.015 mU/L.TPO-Ab was measured with the Brahms luminescence immunoassay (Henningsdorf, Germany). Total assay variation ranged from 4.6 to 8.1% (85-1113 U/ml); reference range: < 60U/ml. Lower limit of detection is 30 U/ml.

Medical Center Leeuwarden, Leeuwarden, The Netherlands

TSH was measured with a sandwich immunoassay on the MODULAR ANALYTICS E170 (Roche Diagnostics GmbH, Mannheim, Germany): reference range 0.4-4.0 mU/L. TPO-Ab was measured with the 2-phase competitive immunoassay on the MODULAR ANALYTICS E170 (Roche Diagnostics GmbH, Mannheim, Germany): reference range 0-34 kU/L.

Amphia Hospital, Breda, The Netherlands

As of 2009, TSH is measured with a electrochemiluminiscent immunometric assay performed on the cobas e602 analyzer (Roche Diagnostics, Almere, The Netherlands): reference range 0.4 – 4.0 mU/L and total assay variation of 4-5%. TPO-Ab is measured by a chemiluminescence immunoassay performed on the Immulite 2000 analyzer (Siemens Healthcare Diagnostics, Den Haag, the Netherlands) with a detection limit of 10 kU/L and total assay variation of 8-10%: Referenge range 0-35 kU/L.

Medical Spectrum Twente, Enschede, The Netherlands

As of 2003 TSH was measured with a electrochemiluminiscent immunometric assay performed on the Modular analytics E170 (Roche Diagnostics, Almere, The Netherlands): reference range 0.005 µU/L – 100 µU/L. Assay variation of 1.8-3.0 %.

Ziekenhuisgroep Twente, Hengelo, The Netherlands

As of 2003 TSH was measured with a electrochemiluminiscent immunometric assay performed on the Modular analytics E170 (Roche Diagnostics, Almere, The Netherlands): reference range 0.005 µU/L – 100 µU/L. Assay variation of 1.8-3.0 %.

University Hospital Brussels, Brussels, Belgium

TSH was measured by the Elecsys TSH assay on the Cobas6000 immunoanalyzer (Roche Diagnostics); reference range 0.27-4.20 mIU/L. TPO-Ab was measured on the Elecsys anti-TPO assay op Cobas6000; reference range 0 -34 kIU/L.

Chapter 8156 |

Copenhagen University Hospital, Copenhagen, Denmark

TSH is measured by a electrochemiluminiscent “ECLIA” using the Elecsys and cobas 8000, e602 analyzers (Roche Diagnostics GmbH, Mannheim, Germany): reference range 0.65-4.80 mU/L, total assay variation (CVmax) 4-6%.TPO-Ab is measured by an immunoflourometric competitive assay (IFMA) using the B.R.A.M.S. anti-TPO kryptor (B.R.A.M.S. GmbH, Hennigsdorf, Germany). The detection limit is 28 kU/L and total assay variation (CVmax) is 8-13%. Reference range 0-60 kU/L.


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9 |General discussion

Chapter 9160 |

In this thesis we explored the association between thyroid disorders and adverse pregnancy outcomes, the underlying pathophysiology and treatment possibilities. The findings from this exploration emphasize the importance of normal thyroid function during pregnancy. Both subclinical hypothyroidism and the presence of thyroid antibodies in euthyroid women increase the risk of adverse pregnancy outcomes, including unexplained subfertility, (recurrent) miscarriage, pre-eclampsia, preterm birth, breech presentation, perinatal mortality and maternal post-partum thyroiditis. Causality has not yet been demonstrated and the underlying mechanisms remain unclear(1-4). Whether treatment with thyroid hormone of the women involved in these studies might beneficially influence this outcome, remains to be elucidated.

Implications for clinical practice and recommendations for future research

Most guidelines advocate treatment of subclinical hypothyroidism (SCH) in pregnancy (5-7), but two very important factors, namely lack of a clear universal definition of subclinical hypothyroidism and the absence of intervention trials, cast doubt on this recommendation. The definition of SCH, and thereby the cut-off level of TSH, depends on the guidelines to which a physician adheres. Subclinical hypothyroidism is defined by the American Thyroid Association (ATA) as a TSH > 2.5 mU/L with or without TPO-Ab. The Endocrine Society, however, defines SCH as TSH > 2.5mU with presence of TPO-Ab or a TSH > 4.0 mU/L, whereas the Dutch guidelines define SCH as a TSH above the assay specific reference range which is usually about 4.0 mU/L.

A main contributor to the existence of various SCH definitions are differences in local reference intervals for thyroid hormone plasma levels, which are dependent on ethnicity, iodine status, TPO-Ab status and analytical method/platform used for TSH quantification(8;9). Geographical differences in iodine intake can complicate standardization of reference intervals, although iodine deficiency no longer exists in the Netherlands(10). A large Dutch cohort study showed ethnic differences in thyroid hormone levels. Significant diagnostic discrepancies were found depending on whether population or ethnicity-specific reference ranges were used to diagnose thyroid disease. A comparison of disease prevalence between a population-based versus an ethnicity-specific reference range changed the diagnosis to euthyroidism for 18% of women who were initially found to have abnormal thyroid function test results(11). The same study showed that in the Dutch population almost 10% of pregnant women had TSH-levels > 2.5 mU/L. These findings should be considered when using the results for clinical application.

As TSH reference intervals are higher in women with SCH with TPO-Ab compared to women with SCH without TPO-Ab, it might be considered to define SCH according to TPO-Ab status.

There is a clear need for population-based, trimester-specific reference intervals of thyroid hormone levels in pregnancy. This will improve diagnosis and treatment of thyroid disorders

General discussion | 161

in pregnancy and will lead to a more individualized approach. In the Netherlands this has been accomplished by two large cohort studies which have investigated thyroid hormone levels in Dutch women in pregnancy(8;12).

Intervention studies are necessary to see if treatment with levothyroxine is effective in preventing pregnancy complications in women with suspected thyroid dysfunction. Thus far, the CATS-trial is the only published trial on this topic showing that treatment of overt and subclinical hypothyroidism with levothyroxine did not result in improved cognitive function in children at 3 years of age. The results of the cognitive screening tests at 7 years of age are awaited for(13). The National Institute of Child Health and Human Development (NICHD) has set up the Thyroid Therapy for Mild Thyroid Deficiency in Pregnancy (TSH) trial to determine whether treatment of women with SCH affects their children’s intellectual development at the age of 5 years. It is expected that the results will be presented in the first half of 2016. Together with the future publication of the results of the T4LIFE study (NTR 3364), described within this thesis(14), as well as the results of the TABLET study, which is a multi-centre, randomized controlled trial focusing on women with a spontaneous miscarriage and/or recurrent miscarriage and TPO-Ab (ISRCTN Number: 15948785), these upcoming data will hopefully clarify whether levothyroxine supplementation for pregnant women with subclinical hypothyroidism or presence of TPO-Ab improves pregnancy outcomes.

Currently, the guideline ‘Thyroid Function Disorders’ from the Dutch Association of Internal Medicine was updated in 2012(15). Treatment of subclinical hypothyroidism in pregnancy and screening of high risk patients for the presence of TPO-Ab was recommended in the previous guideline (2007). Based on our findings the current guideline states that screening for TPO-Ab in pregnancy is not indicated and that evidence is insufficient to recommend treatment of subclinical hypothyroidism pregnancy, irrespective of the presence or absence of TPO-Ab. The ATA guideline was published in 2011 and the Endocrine Society guideline dates from 2012. Results from future randomised intervention studies will hopefully lead to worldwide standardised evidence based guidelines.

Screening for thyroid disease

Universal screening of pregnant women for thyroid disorders is under debate and not advocated in current guidelines. It is advised to screen pregnant women with an increased risk for thyroid disease, although studies show that with a targeted screening strategy a significant amount of cases will remain undiagnosed (6;7;15;16). This thesis shows that evidence on effective treatment interventions for subclinical hypothyroidism and thyroid antibodies is limited. The results from the T4-LIFE study, the TABLET study and the Thyroid Therapy for Mild Thyroid Deficiency in Pregnancy (TSH) trial will aid in determining if universal screening of thyroid disease in pregnancy should be recommended.

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Chapter 9162 |

Fertility

In this thesis an association between subclinical hypothyroidism, thyroid autoimmunity and unexplained subfertility is reported(2). To date, only randomized trials with small study sizes are available, and these trials suggest that treatment with levothyroxine in women with SCH undergoing IVF has a positive effect on implantation rate, miscarriage rates and live birth rates(17). We need more research to define whether thyroid hormone supplementation improves fertility outcome in these women. This would be of particular interest in women undergoing IVF to pinpoint specific effects of thyroid hormone on reproduction. Valuable data on parameters such as number of follicles, number of oocytes, fertilization rates, embryo quality, implantation rates, and pregnancy outcome could be obtained and may lead to approaches to improve the fertility and pregnancy outcomes. At the same time, this will provide helpful information for more fundamental studies on the underlying pathophysiological mechanisms.

CONCLUSION

This thesis shows that subclinical hypothyroidism and thyroid autoimmunity are associated with unexplained subfertility, (recurrent) miscarriage, pre-eclampsia, preterm birth, breech presentation, perinatal mortality and maternal post-partum thyroiditis. Observational data suggest that thyroid function disorders and thyroid peroxidase antibodies are associated with disturbed folliculogenesis, spermatogenesis, lower fertilisation rates and lower embryo quality. The underlying mechanisms for these associations remain largely unknown. Evidence is still insufficient to advise treatment with levothyroxine for pregnant women with subclinical hypothyroidism or thyroid autoimmunity. Population, ethnic and assay specific reference intervals of thyroid hormone levels in pregnancy are warranted for correct diagnosis of thyroid disorders. We need more research to investigate whether thyroid hormone supplementation improves fertility and pregnancy outcomes in (subfertile) women with subclinical hypothyroidism and in women prone to develop hypothyroidism due to the presence of TPO-Ab. If treatment with levothyroxine is effective, cost effectiveness analyses should be done to investigate whether universal screening for thyroid disease in pregnancy is indicated. The three ongoing randomised trials, the T4-LIFE, the TABLET and TSH trial, will hopefully answer the question whether levothyroxine is an effective treatment intervention for subclinical hypothyroidism and thyroid autoimmunity during pregnancy.


REFERENCES

1 Vissenberg R, Fliers E, van der Post JA, van WM, Bisschop PH, Goddijn M. Live-birth rate in euthyroid women with recurrent miscarriage and thyroid peroxidase antibodies. Gynecol Endocrinol 2015 Oct 2;1-4.



4 Vissenberg R, Manders VD, Mastenbroek S, Fliers E, Afink GB, Ris-Stalpers C, Goddijn M, Bisschop PH. Pathophysiological aspects of thyroid hormone disorders/thyroid peroxidase autoantibodies and reproduction. Hum Reprod Update 2015 May;21(3):378-87.

5 NVOG (Nederlandse Vereniging voor Obstetrie en Gynaecologie). Richtlijn Schildklier en Zwangerschap. 2010.




9 Dashe JS, Casey BM, Wells CE, McIntire DD, Byrd EW, Leveno KJ, Cunningham FG. Thyroid-stimulating hormone in singleton and twin pregnancy: importance of gestational age-specific reference ranges. Obstet Gynecol 2005 Oct;106(4):753-7.

10 Wiersinga WM, Podoba J, Srbecky M, van VM, van Beeren HC, Platvoet-Ter Schiphorst MC. A survey of iodine intake and thyroid volume in Dutch schoolchildren: reference values in an iodine-sufficient area and the effect of puberty. Eur J Endocrinol 2001 Jun;144(6):595-603.

11 Korevaar TI, Medici M, de Rijke YB, Visser W, de Muinck Keizer-Schrama SM, Jaddoe VW, Hofman A, Ross HA, Visser WE, Hooijkaas H, Steegers EA, Tiemeier H, Bongers-Schokking JJ, Visser TJ, Peeters RP. Ethnic differences in maternal thyroid parameters during pregnancy: the Generation R study. J Clin Endocrinol Metab 2013 Sep;98(9):3678-86.

12 Medici M, de Rijke YB, Peeters RP, Visser W, de Muinck Keizer-Schrama SM, Jaddoe VV, Hofman A, Hooijkaas H, Steegers EA, Tiemeier H, Bongers-Schokking JJ, Visser TJ. Maternal early pregnancy and newborn thyroid hormone parameters: the Generation R study. J Clin Endocrinol Metab 2012 Feb;97(2):646-52.

13 Hales C, Channon S, Taylor PN, Draman MS, Muller I, Lazarus J, Paradice R, Rees A, Shillabeer D, Gregory JW, Dayan CM, Ludgate M. The second wave of the Controlled Antenatal Thyroid Screening (CATS II) study: the cognitive assessment protocol. BMC Endocr Disord 2014;14:95.

14 Vissenberg R, van Dijk MM, Fliers E, van der Post JA, van WM, Bloemenkamp KW, Hoek A, Kuchenbecker WK, Verhoeve HR, Scheepers CJ, Rombout-de WS, Koks C, Zwart JJ, Broekmans F, Verpoest W, Christiansen O, Post M, Papatsonis DN, Verberg MF, Sikkema J, Mol BW, Bisschop PH, Goddijn M. Effect of levothyroxine on live birth rate in euthyroid women with recurrent miscarriage and TPO antibodies (T4-LIFE study). Contemp Clin Trials 2015 Aug 5.

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15 NIV (Nederlandse Internisten Vereniging), Richtlijn Schildklierfunctiestoornissen. Mei 2012.

16 Vaidya B, Anthony S, Bilous M, Shields B, Drury J, Hutchison S, Bilous R. Detection of thyroid dysfunction in early pregnancy: Universal screening or targeted high-risk case finding? J Clin Endocrinol Metab 2007 Jan;92(1):203-7.

17 Velkeniers B, Van MA, Poppe K, Unuane D, Tournaye H, Haentjens P. Levothyroxine treatment and pregnancy outcome in women with subclinical hypothyroidism undergoing assisted reproduction technologies: systematic review and meta-analysis of RCTs. Hum Reprod Update 2013 May;19(3):251-8.


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10 |Summary

Nederlandse Samenvatting

Chapter 10168 |

SUMMARY


This thesis explores the association between thyroid disorders and adverse pregnancy outcomes, the underlying pathophysiology and treatment possibilities.

In chapter 1 we provide a general introduction of this thesis and describe the objectives of this thesis.

In chapter 2 we present a systematic review and meta-analysis that provides an overview of the associations of thyroid disorders and adverse pregnancy outcomes. A total of 43 articles were selected. Publications on untreated hyperthyroidism were not available. Subclinical hypothyroidism was associated with the occurrence of pre-eclampsia (odds ratio (OR) 1.7, 95% confidence interval (CI) 1.1–2.6) and an increased risk of perinatal mortality (OR 2.7, 95% CI 1.6–4.7). The presence of thyroid peroxidase (TPO) and/or thyroglobulin (Tg) antibodies was associated with an increased risk of unexplained subfertility (OR 1.5, 95% CI 1.1–2.0), miscarriage (OR 3.73, 95% CI 1.8–7.6), recurrent miscarriage (OR 2.3, 95% CI 1.5–3.5), preterm birth (OR 1.9, 95% CI 1.1–3.5) and maternal post-partum thyroiditis (OR 11.5, 95% CI 5.6–24).

We conclude that women with subclinical hypothyroidism and thyroid autoimmunity have an increased risk for pregnancy complications. Special attention during preconception and early pregnancy is desirable for women at risk for, or diagnosed with these thyroid conditions.

In chapter 3 a large retrospective cohort study is described that investigated the association between abnormal TSH and FT4 levels in early pregnancy with breech presentation at term. Data on 3347 pregnant women were available. Hypothyroidism, defined as TSH levels > 3.53 mIU/L, was associated with breech presentation at term (aOR 2.32, 95% CI 1.1-4.8, p = 0.02). Abnormal FT4 levels in early pregnancy, defined as FT4 < 6.3 pmol/L, were not associated with an increased risk for breech presentation. Given the fact that the aOR was 2.32 and the p-value of high TSH in the multiple logistic regression model is only 0.02, this suggests a low effect size and contribution of high TSH to the prediction of breech presentation.

The association found in this study is not strong enough to have direct clinical consequences. More research, e.g., a larger prospective cohort study, is recommended to further investigate this association and its clinical relevance.

In chapter 4 we focus on women with recurrent miscarriage and subclinical hypothyroidism. In this retrospective cohort study we compared 20 women with subclinical hypothyroidism and recurrent miscarriage with 818 euthyroid women with recurrent miscarriage. No significant

| 169Summary

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differences were found in live birth rates (OR 0.69, 95% CI 0.28 – 1.71), ongoing pregnancy rate (OR 0.82, 95% CI 0.32 – 2.10) or miscarriage rates (OR 1.42, 95% CI 0.55 – 3.67). We conclude that screening women with recurrent miscarriage for subclinical hypothyroidism is not indicated.

In chapter 5 we present a review on potential mechanisms by which thyroid function disorders and thyroid peroxidase antibodies affect fertility and early pregnancy. Observational data showed that thyroid function disorders and thyroid peroxidase antibodies are associated with disturbed folliculogenesis, spermatogenesis, lower fertilisation rates and lower embryo quality. The underlying mechanisms for these associations remain largely unknown. Available evidence showed that thyroid hormone transporters and receptors are expressed in the ovary, the early embryo, endometrium, uterus and placenta suggesting that thyroid hormone has a direct effect on reproduction and pregnancy. Triiodothyronine (T3) in combination with follicle stimulating hormone (FSH) enhances granulosa cell proliferation and inhibits granulosa cell apoptosis by the PI3K/Akt pathway. T3 is considered a biological amplifier of the stimulatory action of gonadotropins on granulosa cell function. T3 increases the expression of matrix metalloproteinases (MMP), MMP-2, MMP-3, fetal fibronectin and integrin α5ß1T3 in early placental extravillous trophoblasts. There was no data on the mechanisms underlying the association between thyroid peroxidase autoantibodies and reproduction.

In chapter 6 we systematically review the treatment interventions for thyroid disorders during pregnancy. 22 articles were included, of which eight studies reported on hyperthyroidism. Treatment of hyperthyroidism with propylthiouracil or methimazole reduced the risk of preterm delivery (risk ratio (RR): 0.23, CI: 0.1–0.52), pre-eclampsia (RR: 0.23, CI: 0.06–0.89) and low birth weight (RR: 0.38, CI: 0.22–0.66). The nine studies that reported on clinical hypothyroidism showed that levothyroxine is effective in reducing the risk of miscarriage (RR: 0.19, CI: 0.08–0.39) and preterm delivery (RR: 0.41, CI: 0.24–0.68). Current evidence is insufficient to advise treatment of subclinical hypothyroidism. The five studies available on thyroid autoimmunity showed a non-significant reduction in miscarriage (RR: 0.58, CI: 0.32–1.06), but a significant reduction in preterm birth after treatment with levothyroxine (RR: 0.31, CI: 0.11–0.90).

We conclude that in case of clinical hyperthyroidism and hypothyroidism treatment improves pregnancy outcomes, but evidence is insufficient to recommend treatment with levothyroxine in case of subclinical hypothyroidism and thyroid autoimmunity.

In chapter 7 a retrospective cohort study is presented in which we compared live birth rates between TPO-Ab positive and TPO-Ab negative women with unexplained recurrent miscarriage. Data from 202 women with normal thyroid stimulating hormone concentrations and unexplained recurrent miscarriage were analysed. 174 women were TPO-Ab negative, whereas 28 women were TPO-Ab positive (13.8%). Of these 28 women, 10 women were being

Chapter 10170 |

treated with levothyroxine. TPO-Ab positive women without levothyroxine treatment had a lower live birth rate (29%) compared to TPO-Ab negative women (51%) (HR 0.23, 0.07–0.72, p = 0.012). The live birth rate in women with TPO-Ab receiving levothyroxine was not different compared to women without TPO-Ab (60% versus 51%, p = 0.50). Women with TPO-Ab that received treatment with LT4 had similar pregnancy rates compared to women without TPO-Ab.

We conclude that TPO-Ab are associated with a lower live birth rate in euthyroid women with unexplained recurrent miscarriage. These women may benefit from treatment with levothyroxine. Because this study was not an intervention trial and the results could at best provide an indication for the effectiveness of LT4 we designed a randomised intervention trial on this topic.

In chapter 8 we describe the study protocol for an intervention study to assess the efficacy of thyroid hormone treatment on live birth rate in women with recurrent miscarriage and thyroid peroxidase antibodies. The T4-LIFE study has been designed as a multicentre, international, randomized, placebo controlled study (Dutch Trial Registry number 3364). The primary outcome is live birth rate, defined as the birth of a living foetus beyond 24 weeks of gestation. Secondary outcomes are ongoing pregnancy at 12 weeks, miscarriage rate, preterm birth, survival at 28 days of neonatal life and time to pregnancy. The analysis will be performed according to the intention to treat principle. We need to randomize 240 women (120 per group) to demonstrate an improvement in live birth rate from 55% in the placebo group to 75% in the group with levothyroxine treatment. The study started including patients in January 2014. Currently 80 women have been included.

In chapter 9 we discuss the findings of this thesis. The clinical implications of our studies are described.

| 171Summary

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172 | Hoofdstuk 10

SAMENVATTING

De invloed van schildklieraandoeningen op ongewenste zwangerschaps-uitkomsten

In dit proefschrift onderzoeken wij de associaties tussen schildklieraandoeningen en zwangerschapscomplicaties, de onderliggende pathofysiologische mechanismen en de behandelmogelijkheden.

In hoofdstuk 1 verschaffen we een algemene inleiding van dit proefschrift en beschrijven we de doelstellingen van dit proefschrift.

In hoofdstuk 2 presenteren we een systematisch literatuuroverzicht en meta-analyse die als doel hadden een overzicht te geven van de associaties tussen onbehandelde schildklieraandoeningen en zwangerschapscomplicaties. Er werden 43 artikelen geselecteerd waarvan 38 artikelen gebruikt konden worden voor de meta-analyses. Studies over onbehandelde hyperthyreoϊdie en zwangerschapsuitkomsten werden niet gevonden. Er waren 5 artikelen die de associatie tussen onbehandelde subklinische hypothyreoϊdie en zwangerschapsuitkomsten beschreven. Subklinische hypothyreoïdie was geassocieerd met pre-eclampsie (odds ratio (OR) 1.7, 95% betrouwbaarheidsinterval (BI) 1.1–2.6) en een verhoogd risico op perinatale mortaliteit (OR 2.7, 95% BI 1.6–4.7). Er waren 36 artikelen die onderzochten of schildklier auto-immuniteit (gedefinieerd als de aanwezigheid van thyroperoxidase antistoffen (TPO) en/of thyroglobulin (Tg) antistoffen met een normale schildklierhormoonconcentratie) geassocieerd was met negatieve zwangerschapsuitkomsten. Er werd bij aanwezigheid van deze antistoffen een verhoogd risico op onverklaarde subfertiliteit (OR 1.5, 95% BI 1.1–2.0), miskraam (OR 3.73, 95% BI 1.8–7.6), herhaalde miskraam (OR 2.3, 95% BI 1.5–3.5), vroeggeboorte (OR 1.9, 95% BI 1.1–3.5) en maternale postpartum thryreoïditis (OR 11.5, 95% CI 5.6–24) gevonden.

Met name vrouwen met subklinische hypothyreoïdie en schildklier auto-immuniteit hebben een verhoogd risico op zwangerschapscomplicaties. Het is daarom wenselijk dat er extra aandacht wordt besteed in de preconceptieperiode of in de vroege zwangerschap aan vrouwen met een risico op, of diagnose van, schildklieraandoeningen.

In hoofdstuk 3 geven we een retrospectieve cohort studie weer waarbij we de associatie tussen afwijkende thyroid stimulating hormone (TSH) en vrij thyroxine (FT4) concentraties en stuitligging onderzochten. Een totaal van 3347 zwangere vrouwen werd geïncludeerd. Hypothyreoïdie, gedefinieerd als TSH > 3.53 mIU/L, in de eerste helft van de zwangerschap was geassocieerd met stuitligging bij de geboorte (aOR 2.32, 95% BI 1.1-4.8, p = 0.02). Afwijkende FT4 concentraties, gedefinieerd als FT4 < 6.3 pmol/L, in de eerste helft van de zwangerschap waren niet geassocieerd met een verhoogd risico op stuitligging. De gevonden aOR 2.32

| 173Nederlandse samenvatting

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10

suggereert een beperkte bijdrage van een verhoogd TSH in de voorspelling van stuitligging. De gevonden associatie in deze studie is niet sterk genoeg om direct aanleiding te geven

tot veranderingen in de klinische praktijk. Meer onderzoek, bijvoorbeeld in een grotere prospectieve studie, wordt aanbevolen om deze associatie en de klinische relevantie hiervan verder te onderzoeken.

In hoofdstuk 4 beschrijven we een retrospectieve cohort studie gericht op vrouwen met herhaalde miskraam en een subklinische hypothyreoïdie. Er werden 20 vrouwen met subklinische hypothyreoïdie en herhaalde miskraam vergeleken met 818 vrouwen met herhaalde miskraam en een normale schildklierfunctie. Er werden geen significante verschillen aangetoond in het percentage levendgeborene (OR 0.69, 95% CI 0.28 – 1.71), het percentage doorgaande zwangerschappen (OR 0.82, 95% CI 0.32 – 2.10) of het percentage miskramen (OR 1.42, 95% CI 0.55 – 3.67).

Wij kunnen concluderen dat het screenen van vrouwen met herhaalde miskraam op subklinische hypothyreoïdie daarom niet geïndiceerd is.

In hoofdstuk 5 presenteren we een literatuuroverzicht van de mechanismen die ten grondslag kunnen liggen aan de negatieve invloed van schildklierfunctiestoornissen en de aanwezigheid van TPO-antistoffen op fertiliteit en zwangerschap. Observationele data toonden aan dat schildklierfunctiestoornissen en TPO-antistoffen geassocieerd zijn met een gestoorde folliculogenese en spermatogenese, met verminderde fertilisatie en verminderde embryo kwaliteit. De pathofysiologie van deze associaties bleef echter onduidelijk. De beschikbare literatuur liet zien dat triiodothyronine (T3) in combinatie met follicle stimulating hormone (FSH) de granulosa cel proliferatie verhoogt en de granulosa cel apoptose remt via de PI3K/Akt-route. T3 lijkt een biologische versterker van het stimulerende effect van gonadotropines op de granulosa cel functie. Ook werd door T3 de expressie van matrix metalloproteinases (MMP), MMP-2, MMP-3, foetaal fibronectine en integrine α5ß1T3 verhoogd in de vroege placentaire extravilleuze trophoblasten. De beschikbare literatuur toonde aan dat schildklierhormoontransporters -en receptoren tot expressie komen in het ovarium, het embryo, het endometrium, de uterus en de placenta. Dit suggereert dat er sprake is van direct regulerend effect van schildklierhormoon op de voortplanting en zwangerschap.

Klinische interventiestudies zijn noodzakelijk om het effect van schildklierhormoon suppletie bij vrouwen met subklinische hypothyreoïdie en bij vrouwen met een verhoogd risico op het ontwikkelen van een subklinische hypothyreoïdie door aanwezigheid van TPO-antistoffen, te onderzoeken. Aanvullend is er meer onderzoek noodzakelijk om onderliggende mechanismen te identificeren. Dit zou bij uitstek interessant zijn bij vrouwen die een IVF behandeling ondergaan om de effecten van schildklierhormoon op verschillende reproductieve uitkomsten te onderzoeken.

174 | Hoofdstuk 10

In hoofdstuk 6 presenteren we een systematisch literatuuronderzoek gericht op behandel interventies voor schildklieraandoeningen in de zwangerschap. Er werden 22 artikelen geïncludeerd. Acht studies onderzochten de uitkomst van behandeling van hyperthyreoïdie. Behandeling met propylthiouracil of methimazole vermindert de kans op vroeggeboorte (risk ratio (RR): 0.23, BI: 0.1–0.52), pre-eclampsie (RR: 0.23, BI: 0.06–0.89) en een laag geboortegewicht (RR: 0.38, BI: 0.22–0.66). De geëxtraheerde data uit negen studies lieten zien dat behandeling van klinische hypothyreoïdie effectief is in het verminderen van de kans op miskraam (RR: 0.19, BI: 0.08–0.39) en vroeggeboorte (RR: 0.41, BI: 0.24–0.68). Er was een gebrek aan wetenschappelijk bewijs om te pleiten voor de behandeling van subklinische hypothyreoïdie in de zwangerschap. Er waren vijf studies die naar de behandeling van schildklier auto-immuniteit hebben gekeken. Behandeling met levothyroxine resulteerde in een niet significante daling van het risico op miskraam (RR: 0.58, CI: 0.32–1.06), maar ook in een significante daling van het risico op vroeggeboorte. (RR: 0.31, BI: 0.11–0.90).

Voor klinische hyperthyreoïdie en hypothyreoïdie verbetert behandeling de zwangerschapsuitkomsten, maar voor subklinische hypothyreoïdie en schildklier auto-immuniteit ontbreekt bewijs om behandeling met levothyroxine aan te bevelen.

In hoofdstuk 7 beschrijven we een retrospectieve cohort studie waarbij zwangerschaps-uitkomsten van vrouwen met onverklaarde herhaalde miskraam en aanwezigheid van TPO-antistoffen werden vergeleken met die van vrouwen met onverklaarde herhaalde miskraam zonder TPO-antistoffen. De gegevens van 202 vrouwen met normale schildklierhormoon concentraties en onverklaarde herhaalde miskraam werden geanalyseerd. 174 vrouwen hadden geen TPO-antistoffen en 28 vrouwen hadden wel TPO-antistoffen (13.8%). Van deze 28 vrouwen werden 10 vrouwen behandeld met levothyroxine. Vrouwen met TPO-antistoffen die geen behandeling kregen hadden een lagere kans op een levend geboren kind (29%) vergeleken met vrouwen zonder TPO-antistoffen (51%) (Hazard Ratio (HR) 0.23, 0.07–0.72, p = 0.012). De vrouwen met TPO-antistoffen die behandeld werden met levothyroxine hadden dezelfde kans op een levend geboren kind vergeleken met vrouwen zonder TPO-antistoffen (60% versus 51%, p = 0.50). Deze studie liet ook zien dat euthyreote vrouwen met onverklaarde herhaalde miskraam en TPO-antistoffen een lagere kans hebben op een levendgeboren kind en mogelijk baat hebben bij behandeling met levothyroxine. Aangezien het artikel geen interventie studie was en dus geen sluitende conclusie oplevert, hebben we een gerandomiseerde interventiestudie opgezet voor patiënten met herhaalde miskraam en TPO-antistoffen.

| 175Nederlandse samenvatting

Chapter

10

In hoofdstuk 8 presenteren we het studieprotocol van de T4-LIFE study: ‘levothyroxine voor euthyreote vrouwen met herhaalde miskraam en positieve TPO-antistoffen; een gerandomiseerde gecontroleerde studie’. Dit is een multi-centre, internationale, gerandomiseerde, placebo-gecontroleerde studie met als doel te effectiviteit te evalueren van levothyroxine behandeling bij vrouwen met herhaalde miskraam en TPO-antistoffen (trial registratienummer NTR 3364). De primaire uitkomst is een levendgeboren kind, gedefinieerd als de geboorte van een levende foetus na 24 weken zwangerschap. Secundaire uitkomstmaten zijn doorgaande zwangerschap (> 12 weken), miskraam, vroeggeboorte, overleving na 28 dagen en tijd tot zwangerschap. Er zal een ‘intention to treat’ analyse worden gedaan. In totaal moeten 240 vrouwen gerandomiseerd worden (120 per groep) om een verbetering in percentage levendgeborenen aan te tonen van 55% naar 75%. De studie is in januari 2014 gestart met het includeren van vrouwen. Op dit moment zijn er 80 vrouwen geïncludeerd in de studie.

In hoofdstuk 9 bediscussiëren wij de bevindingen van dit proefschrift en reflecteren we op de klinische implicaties van onze studies.

AddendumList of co-authors and their contribution

List of publications

Portfolio

Dankwoord

Curriculum Vitae

Addendum178 |

LIST OF CO-AUTHORS AND AFFILIATIONS

GB Afink Academic Medical Center, Reproductive Biology Laboratory, Amsterdam

PH Bisschop Academic Medical Center, Department of Endocrinology and Metabolism, Amsterdam

KWM Bloemenkamp University Medical Center Leiden, Department of Obstetrics and Gynaecology, Leiden

E van den Boogaard Academic Medical Center, Department of Obstetrics and Gynaecology, Amsterdam

F Broekmans University Medical Center Utrecht, Department of Obstetrics and Gynaecology, Utrecht

OB Christiansen Copenhagen University Hospital, Fertility clinic, Copenhagen, Denmark

F Dawood Liverpool Women’s Hospital, Department of Obstetrics and Gynaecology, Liverpool, United Kingdom

MM van Dijk Academic Medical Center, Center for Reproductive Medicine, Department of Obstetrics and Gynaecology, Amsterdam

RG Farquharson Liverpool Women’s Hospital, Department of Obstetrics and Gynaecology, Liverpool, United Kingdom

E Fliers Academic Medical Center, Department of Endocrinology and Metabolism, Amsterdam

M Goddijn Academic Medical Center, Center for Reproductive Medicine, Department of Obstetrics and Gynaecology, Amsterdam

A Hoek University Medical Center Groningen, Department of Reproductive Medicine, Groningen

C Koks Maxima Medical Center, Department of Obstetrics and Gynaecology, Veldhoven

| 179List of co-authors and their contribution

WK Kuchenbecker Isala Clinics, Department of Obstetrics and Gynaecology, Zwolle

JA Land University Medical Center Groningen, Department of Obstetrics and Gynaecology, Groningen

VD Manders Academic Medical Center, Reproductive Biology Laboratory, Amsterdam

S Mastenbroek Academic Medical Center, Center for Reproductive Medicine, Amsterdam

BWJ Mol The Robinson Institute, School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, Australia

DNM Papatsonis Amphia Hospital, Department of Obstetrics and Gynaecology, Breda

JAM van der Post Academic Medical Center, Department of Obstetrics and Gynaecology, Amsterdam

M Post Medical Center Leeuwarden, Department of Obstetrics and Gynaecology, Leeuwarden

C Ris-Stalpers Academic Medical Center, Department of Obstetrics and Gynaecology, Reproductive Biology Laboratory, Amsterdam

S Rombout Albert Schweizer Hospital, Department of Obstetrics and Gynaecology, – de Weerd Dordrecht

HCJ Scheepers University Medical Center Maastricht, Department of Reproductive Medicine, Maastricht

J Sikkema Ziekenhuisgroep Twente, Department of Obstetrics and Gynaecology, Hengelo

MFG Verberg Medical Spectrum Twente, Department of Obstetrics and Gynaecology, Enschede

Addendum180 |

HR Verhoeve Onze Lieve Vrouwe Hospital, Department of Reproductive Medicine, Amsterdam

W Verpoest University Hospital Brussels, Department of Reproductive Medicine, Brussels, Belgium

T Vrijkotte Academic Medical Center, Department of Public Health, Amsterdam

M van Wely Academic Medical Center, Department of Obstetrics and Gynaecology, Amsterdam

JJ Zwart Deventer Hospital, Department of Obstetrics and Gynaecology, Deventer

CONTRIBUTION OF CO-AUTHORS

Chapter 2

JA Land, JAM van der Post, M Goddijn and PH Bisschop all contributed substantially to the conception and design of this review. E van den Boogaard and R Vissenberg screened all titles, abstracts, articles and extracted data for meta-analyses. M Goddijn and PH Bisschop were third reviewer in case consensus could not be reached directly. M van Wely supervised the analysis and interpretation of data. E van den Boogaard drafted the article, all other authors critically revised multiple versions of the manuscript. All authors gave their final approval of the version to be published.

Chapter 3

TGM Vrijkotte, JAM van der Post, E Fliers, M Goddijn and PH Bisschop all contributed substantially to the conception and design of this study. TGM Vrijkotte supervised the analysis and interpretation of data. R Vissenberg drafted the article, all other author critically revised multiple versions of the manuscript. All authors gave their final approval of the version to be published.

Chapter 4

MM van Dijk, R Vissenberg, M Goddijn and PH Bisschop all contributed substantially to the conception and design of this article. RG Farquharson and F Dawood contributed to the

| 181List of co-authors and their contribution

acquisition of the data. M van Wely contributed substantially to the analysis and interpretation of data. MM van Dijk and R Vissenberg drafted the article. All other authors critically revised multiple versions of the manuscript. All authors gave their final approval of the version to be published.

Chapter 5

C Ris-Stalpers, E Fliers, S Mastenbroek, M Goddijn and PH Bisschop all contributed substantially to the conception and design of this review. R Vissenberg and VD Manders performed the literature search, screened all titles, abstracts and articles and extracted data. GB Afink was responsible for the in silico analysis and gene expression data. C Ris-Stalpers and GB Afink drafted the introduction of the article. M Goddijn drafted the subchapter sperm and the subchapter fertilization and embryogenesis. VD Manders drafted the subchapters oocyte and ovulation and the subchapter implantation. R Vissenberg drafted the other sections of the article. All other authors critically revised multiple versions of the manuscript. All authors gave their final approval of the version to be published.

Chapter 6

JAM van der Post, E Fliers, PH Bisschop and M Goddijn all contributed substantially to the design of this review. R Vissenberg and E van den Boogaard screened all titles, abstracts, articles and extracted data for meta-analyses. M Goddijn and PH Bisschop were third reviewer in case consensus could not be reached directly. M van Wely supervised the analysis and interpretation of data. R Vissenberg drafted the article, all other authors critically revised multiple versions of the manuscript. All authors gave their final approval of the version to be published.

Chapter 7

E Fliers, JAM van der Post, M Goddijn and PH Bisschop all contributed substantially to the conception and design of this study. R Vissenberg was responsible for the data collection and analysis. M van Wely supervised the analysis and interpretation of data. R Vissenberg drafted the article. All other authors critically revised multiple versions of the manuscript. All authors gave their final approval of the version to be published.

Chapter 8

E Fliers, JAM van der Post, BW Mol, M Goddijn, PH Bisschop and R Vissenberg all contributed substantially to the conception and design of this study. R Vissenberg drafted the article. E Fliers, JAM van der Post, M Goddijn and PH Bisschop critically revised multiple versions of the manuscript. All other authors gave their final approval of the version to be published.

Addendum182 |

LIST OF PUBLICATIONS

Abnormal thyroid function parameters in the second trimester of pregnancy are associated with breech presentation at term: a nested cohort study.R Vissenberg, TGM Vrijkotte, JAM van der Post, E Fliers, M Goddijn, PH BisschopEuropean Journal of Obstetrics and Gynecology and Reproductive Biology, in press.

Live-birth rate in euthyroid women with recurrent miscarriage and thyroid peroxidase antibodies.R Vissenberg, E Fliers, JAM van der Post, M van Wely, PH Bisschop, M GoddijnGynecological Endocrinology 2015;2:1-4.

Effect of levothyroxine on live birth rate in euthyroid women with recurrent miscarriage and TPO antibodies (T4-LIFE study).R Vissenberg, MM van Dijk, E Fliers, JAM van der Post, M van Wely, KWM Bloemenkamp, A Hoek, WK Kuchenbecker, HR Verhoeve, HCJ Scheepers, S Rombout-de Weerd, C Koks, JJ Zwart, F Broekmans, W Verpoest, O Christiansen, M Post, DN Papatsonis, MFG Verberg, J Sikkema, BW Mol, PH Bisschop, M GoddijnContemporary Clinical Trials 2015;44:134-138.

Pathophysiological aspects of thyroid hormone disorders/ thyroid peroxidase autoantibodies and reproduction.R Vissenberg, VD Manders, S Mastenbroek, E Fliers, GB Afink, C Ris-Stalpers, M Goddijn, PH BisschopHuman Reproduction Update 2015;21:378-387.

Recurrent miscarriage clinics.MM van den Berg, R Vissenberg, M GoddijnObstetrics and Gynecology Clinics of North America 2014;41:145-155.

Recurrent Miscarriage, Chapter: Which investigations are relevant?PG de Jong, E van den Boogaard, CR Kowalik, R Vissenberg, S Middeldorp, M GoddijnEditor dr. OB Christiansen: ISBN: 978-0-470-67294-5

Wiley-Blackwell, 2014.

Evaluatieonderzoek verzuipt in regelgeving.R Vissenberg, W Hehenkamp, M Oudijk, M van GoldeMedisch contact 2013:45:2354-2356.

| 183List of publications

Schildklierdisfunctie bij zwangeren.R Vissenberg, M Goddijn, BW Mol, JAM van der Post, E Fliers, PH BisschopTijdschrift voor Verloskunde 2013;6:17-22.

Number and sequence of preceding miscarriages and maternal age for the prediction of antiphospholipid syndrome in women with recurrent miscarriage.E van den Boogaard, DM Cohn, JC Korevaar, F Dawood, R Vissenberg, S Middeldorp, M Goddijn, RG FarquharsonFertility and Sterility 2013;99:188-92.

Schildklierdisfunctie bij zwangeren.R Vissenberg, M Goddijn, BW Mol, JAM van der Post, E Fliers, PH BisschopNederlands Tijdschrift voor Geneeskunde 2012;156:A5163.

Treatment of thyroid disorders before conception and in early pregnancy: a systematic review.R Vissenberg, E van den Boogaard, M van Wely, JAM van der Post, E Fliers, PH Bisschop, M Goddijn Human Reproduction Update 2012;18:360-73.

Is There a Role for Assisted Reproductive Technology in Recurrent Miscarriage?R Vissenberg, M GoddijnSeminars in Reproductive Medicine 2011;29:548-556.

Treatment of (sub) clinical thyroid dysfunction and thyroid autoimmunity before conception and in early pregnancy: a systematic review, abstract.R Vissenberg, E van den Boogaard, JAM van der Post, M Goddijn, PH BisschopJournal of Reproductive immunology 2011;90:148-149.

Significance of (sub)clinical thyroid dysfunction and thyroid autoimmunity before conception and in early pregnancy: a systematic review.E van den Boogaard, R Vissenberg, JA Land, JAM van der Post, M Goddijn, PH BisschopHuman Reproduction Update 2011;17 605-619.

Addendum184 |

PHD PORTFOLIO

Name PhD student: Rosa VissenbergPhD period: February 2011-October 2014Name PhD supervisors: J.A.M van der Post and E. Fliers

1. PhD Training Year Workload (ECTS)

General courses

Pubmed course 2011 0.1

Basic Course Legislation and Organisation for Clinical Researchers 2011 1.0

Entrepreneurship in Health and Life Sciences 2013 1.5

Specific Courses

Practical biostatistics 2012 1.1

Seminars

Weekly department lunch meetings 2011-2014 3.0

Weekly department seminars 2011-2014 3.0

Weekly department journal club 2011-2014 3.0

Oral Presentations

Treatment of (sub)clinical thyroid dysfunction and thyroid autoimmunity before conception and in early pregnancy: a systematic review. Joint European Society for Reproductive Immunology and European Society of Human Reproduction and Embryology Meeting, Copenhagen

2011 0.5

Treatment of (sub)clinical thyroid dysfunction and thyroid autoimmunity before conception and in early pregnancy: a systematic review. Klinische Endocrinologie Dagen, Noordwijkerhout

2012 0.5

Significance of (sub)clinical thyroid dysfunction and thyroid autoimmunity before conception and in early pregnancy: a systematic review. OLVG Wetenschapsdag, Amsterdam

2012 0.5

Treatment of (sub)clinical thyroid dysfunction and thyroid autoimmunity before conception and in early pregnancy: a systematic review. 28th Annual Meeting of European Society of Human Reproduction and Embryology, Istanbul

2012 0.5

When to screen for thyroid function abnormalities? 29th Annual Meeting of European Society of Human Reproduction and Embryology, London

2013 0.5

TPO antibodies and live birth rate in women with recurrent miscarriage, 37th Annual Meeting European Thyroid Association, Leiden

2013 0.5

TPO antibodies are associated with a lower live birth rate in women with recurrent miscarriage. Wetenschapsdag Zaans Medisch Centrum, Zaandam

2014 0.5

| 185Portfolio

Conferences

Joint European Society for Reproductive Immunology and European Society of Human Reproduction and Embryology Meeting, Copenhagen

2011 1.0

28th annual meeting European Society of Human Reproduction and Embryology. Istanbul

2012 1.0

Klinische Endocrinologie Dagen, Noordwijkerhout 2012 0.5

37th Annual Meeting European Thyroid Association, Leiden 2013 0.5

29th Annual Meeting of European Society of Human Reproduction and Embryology, London

2013 0.5

44e Gynaecongres, Arnhem 2013 0.5

2. Teaching Year Workload (ECTS)

Supervising

Lindsey Smits: thyroid autoimmunity in women with unexplained recurrent miscarriage. Student coaching/mentoring scientific research project bachelor thesis

2013 1.0

3. Parameters of esteem Year

Grants

Fonds NutsOhra: Levothyroxine for euthyroid women with recurrent miscarriage and positive TPO antibodies (T4-LIFE trial)

2011

Jan Dekker en dr. Ludgardine Bouwmanstichting: Levothyroxine for euthyroid women with recurrent miscarriage and positive TPO antibodies (T4-LIFE trial)

2012

Schildklier Organisaties Nederland: Levothyroxine for euthyroid women with recurrent miscarriage and positive TPO antibodies (T4-LIFE trial)

2012

ZonMw Goed Gebruik Geneesmiddelen: Levothyroxine for euthyroid women with recurrent miscarriage and positive TPO antibodies (T4-LIFE trial)

2012

Addendum186 |

DANKWOORD

Dit proefschrift is tot stand gekomen dankzij de inzet van velen. Allereerst gaat mijn dank uit naar alle patiënten die bereid zijn geweest tot deelname aan de studies.

Mijn promotores, Prof. dr. J.A.M van der Post en Prof. dr. E. Fliers. Beste Joris, dank voor het mogelijk maken van dit promotietraject en voor je vertrouwen in de afronding van dit proefschrift. Beste Eric, uit jouw snelle, vriendelijke en kritische reacties bleek je betrokkenheid en wist ik dat ik bij je aan kon kloppen als het nodig was. Dank voor je begeleiding.

Mijn co-promotores dr. M. Goddijn en dr. P.H. Bisschop. Samen vormden jullie een hele sterke combinatie. Mariette, altijd beschikbaar, snel een reactie en punctueel in het nakijken van de manuscripten. Jij had vaak weer nieuwe ideeën of projecten om verder te gaan en bewaakte de voortgang van mijn promotie. Ik vind het bewonderenswaardig dat je dit consequent voor al je promovenda doet. Peter, betrokken, vrolijk, enthousiast en inhoudelijk ontzettend kritisch. Als ik helemaal vastliep of me blind aan het staren was op een manuscript moest ik van jou verplicht even afstand nemen, het geheel overdenken en het in de grotere context plaatsen. Ik vind het knap hoe jij dit continue toepast. Heel veel dank voor jullie begeleiding.

De leden van de leescommissie: prof. dr. J.H. Kok, prof. dr. F. van der Veen, prof. dr. C.B. Lambalk, prof. dr. E.A.P. Steegers, dr. R.P. Peeters en dr. C. Ris-Stalpers ben ik zeer erkentelijk voor de inzet van hun deskundigheid ter beoordeling van dit proefschrift en hun bereidheid zitting te nemen in de commissie.

Graag wil ik alle medeauteurs van de hoofdstukken in dit proefschrift bedanken voor hun bijdrage en de goede samenwerking, in het bijzonder Myrthe, Emmy, Vera en Carrie.

Alle gynaecologen en researchmedewerkers die betrokken zijn bij de T4-LIFE studie wil ik bedanken voor hun inzet bij het opzetten van de studie in hun centra en het includeren van patiënten.

Ik wil alle collega-onderzoekers en CVV medewerkers bedanken voor de samenwerking en alle gezelligheid! In het bijzonder Lotte, Merel en Femke en natuurlijk Kai Mee, Gert Jan en Marjet.

Vrienden en vriendinnen, zonder jullie steun en gezelligheid was het nooit gelukt.

| 187Dankwoord

Mijn paranimfen Josien en Paulien. Drie keer met elkaar in de Agnietenkapel, als dat geen mijlpaal is. Lieve Joos, super vriendin! Weer een nieuw avontuur erbij. Laten we elkaar op nog vele ‘vrijdagen’ trakteren, dan is de rest van de week automatisch geslaagd. Lieve Pau, hoe leuk en fijn was het samen de LIFE-studies te doen. Ik ben blij dat dit tot zo’n mooie vriendschap heeft geleid en dat je nu naast mij staat.

Lieve zussen, jullie onvoorwaardelijke steun is de afgelopen jaren ontzettend belangrijk voor mij geweest. Laura, jouw immer vrolijke en stoere karakter maken mij altijd blij. Charlotte, jouw humor, doorzettingsvermogen en sterke wil zijn een inspiratie voor mij! Gelukkig staan wij altijd voor elkaar klaar.

Lieve papa en mama, het is gezien, het is niet onopgemerkt gebleven. Jullie hebben mij zoveel moois gegeven. Bedankt voor wie jullie zijn, bedankt voor alles.

Mijn lieve Ruud, de momenten samen met jou zijn mijn gelukkigste. Wat fijn dat er nu meer tijd komt voor momenten samen.

Addendum188 |

CURRICULUM VITAE

Op 28 september 1984 werd Rosa Vissenberg, dochter van Ron Vissenberg en Lizan Baudoin, geboren in Eindhoven. Zij groeide hier op samen met haar tweelingzusje Laura en haar oudere zus Charlotte. Later verhuisden zij naar Rotterdam en vanaf haar 15e jaar woonde zij in Amsterdam. In 2002 behaalde ze haar diploma aan het Barlaeus Gymnasium, waarna ze in hetzelfde jaar startte met de studie geneeskunde aan de Universiteit van Amsterdam. De doctoraalfase rondde ze af met een wetenschappelijke stage in Semarang, Indonesië. Na haar artsexamen in 2009 ging zij aan de slag als arts-assistent

op de afdeling Verloskunde & Gynaecologie van het Onze Lieve Vrouwe Gasthuis (OLVG) in Amsterdam en werd zij in contact gebracht met Mariëtte Goddijn en Peter Bisschop, werkzaam in het Academisch Medisch Centrum (AMC), voor het schrijven van een systematische review. Dit leidde tot de start van haar promotieonderzoek, gericht op schildklierafwijkingen tijdens de zwangerschap, onder begeleiding van prof. dr. J.A.M. van der Post (promotor), prof. dr. E. Fliers (promotor), dr. M. Goddijn (co-promotor) en dr. P.H. Bisschop (co-promotor). Hiernaast werkte zij als arts-assistent bij de polikliniek voortplantingsendocrinologie en vruchtbaarheidsonderzoek van het OLVG en later bij het Centrum voor Voortplantingsgeneeskunde in het AMC. In september 2014 is zij met veel plezier gestart met de huisartsopleiding verbonden aan het AMC-UvA. Rosa woont samen met haar vriend Ruud.

| 189Curriculum Vitae


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