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1 is committed to open peer review. As part of this commitment we make the peer review history of every article we publish publicly available. When an article is published we post the peer reviewers comments and the authors responses online. We also post the versions of the paper that were used during peer review. These are the versions that the peer review comments apply to. The versions of the paper that follow are the versions that were submitted during the peer review process. They are not the versions of record or the final published versions. They should not be cited or distributed as the published version of this manuscript. is an open access journal and the full, final, typeset and author-corrected version of record of the manuscript is available on our site with no access controls, subscription charges or pay-per-view fees ( If you have any questions on s open peer review process please info.bmjopen@bmj.com : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

2 No Impact of Levothyroxine Therapy on Obstetric, Neonatal and Childhood Outcomes in Women with Subclinical Hypothyroidism Diagnosed in Pregnancy: A systematic review and meta-analysis of randomised controlled trials Journal: Manuscript ID bmjopen-0-0 Article Type: Research Date Submitted by the Author: 0-Mar-0 Complete List of Authors: Yamamoto, Jennifer; University of Calgary, Medicine Benham, Jamie; University of Calgary, Medicine Nerenberg, Kara; University of Calgary, Medicine Donovan, Lois; University of Calgary, Medicine Keywords: Pregnancy, Thyroid disease < DIABETES & ENDOCRINOLOGY, subclinical hypothyroidism, meta-analysis, clinical practice : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

3 Page of No Impact of Levothyroxine Therapy on Obstetric, Neonatal and Childhood Outcomes in Women with Subclinical Hypothyroidism Diagnosed in Pregnancy: A systematic review and meta-analysis of randomised controlled trials Jennifer M. Yamamoto, Jamie L. Benham, Kara A. Nerenberg, Lois E. Donovan Endocrinologist and Clinical Lecturer, Division of Endocrinology and Metabolism, Department of Medicine, Cumming School of Medicine, University of Calgary, Alberta, Canada, Endocrinology Fellow, Division of Endocrinology and Metabolism, Department of Medicine, Cumming School of Medicine, University of Calgary, Alberta, Canada. Assistant Professor, Division of General Internal Medicine, Departments of Medicine and Obstetrics and Gynaecology, Cumming School of Medicine, University of Calgary, Alberta, Canada. Clinical Associate Professor Division of Endocrinology and Metabolism, Departments of Medicine and Obstetrics and Gynaecology, Cumming School of Medicine, University of Calgary, Alberta, Canada. Correspondence to: Lois E Donovan Richmond Road Diagnostic and Treatment Centre, Room 0, 0 Richmond Road SW, Calgary, Alberta, Canada TT C Phone 0 -, Fax 0 -, lois.donovan@albertahealthservices.ca Funding: None Conflict of interest: All authors report no conflicts. - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

4 Page of Abstract Objective To determine in women with subclinical hypothyroidism diagnosed in pregnancy whether levothyroxine treatment compared to control, impacts important obstetrical or childhood outcomes (specifically intelligence quotient [IQ]) in randomised controlled trials. Design Systematic review and meta-analysis. Study eligibility criteria Randomised trials which met all the following were included: ) reported original data of women with subclinical hypothyroidism diagnosed in pregnancy (by any prespecified study definition); ) randomised to either levothyroxine or control (placebo or no treatment); ) reported obstetrical outcomes and/or childhood neurodevelopmental outcomes; and ) published from 0 to January 0 in either English or French language. Data sources Medline, EMBASE, CINAHL, Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials, and Clinicaltrials.gov. Outcome measures Obstetrical, neonatal and childhood outcomes including: miscarriage, gestational hypertension, preeclampsia, preterm delivery, mode of delivery, neonatal intensive care unit admission, birth weight, gestational age at delivery, childhood IQ and neurodevelopmental scores. Results Three trials with, participants were included. Two studies were metaanalysed for maternal and neonatal outcomes and two studies for childhood IQ. No statistically significant differences were found for any clinical outcomes with levothyroxine therapy compared with control. Conclusions Treatment of subclinical hypothyroidism in pregnancy with levothyroxine does not improve obstetrical, neonatal, childhood IQ or neurodevelopmental outcomes. Current trial evidence does not support the treatment of subclinical hypothyroidism diagnosed in pregnancy. Prospero registration 0 CRD : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

5 Page of Strength and limitations of this review This systematic review provides an up to date summary of recently published randomised control trials and unpublished data not included in other systematic reviews and metaanalysis A broad range of outcomes that clinicians consider during medical decision making are analysed. This systematic review is rigorous in its design, methodology and analyses with a prepublished protocol. The inclusion of only randomised control trials minimises the bias associated with observational studies in order to clearly evaluate the effects of levothyroxine treatment in pregnant women with subclinical hypothyroidism. Only three trials were identified for inclusion. - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

6 Page of Introduction Subclinical hypothyroidism, generally defined as a thyroid stimulating hormone (TSH) value greater than the upper limit of the reference range with a normal free thyroxine (free T) and no symptoms of hypothyroidism, is a common biochemical finding in pregnancy, occurring in more than % of pregnant women (depending on the assay and reference ranges used). It must be noted that the diagnostic criteria for subclinical hypothyroidism in pregnancy has changed over the years and varies between countries. TSH cut-offs for subclinical hypothyroidism in pregnancy are heterogeneous, ranging from. mu/l, to above the %ile confidence interval for the population examined and assay used, or mu/l. To date, observational studies of variable methodologic quality have found inconsistent associations between subclinical hypothyroidism in pregnancy and adverse obstetrical outcomes including miscarriage, fetal death, preterm delivery, gestational diabetes, gestational hypertension, eclampsia, placental abruption, low birthweight and lower childhood intelligence quotient (IQ). As a result, despite a paucity of high quality randomised controlled trials (RCT) of levothyroxine replacement therapy, many health organizations have advocated for levothyroxine replacement therapy for women with subclinical hypothyroidism diagnosed in pregnancy. These recommendations are further supported by an underlying belief that levothyroxine therapy is physiologic and, thus is benign. Emerging data challenge this belief due to the demonstration of increased risks of harms associated with levothyroxine replacement and/or elevated free T (even in the subclinical hyperthyroid range) in pregnancy including: - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

7 Page of increased risks of preeclampsia, small for gestational age neonates, preterm delivery, gestational diabetes, and, in fact, lower IQ 0. Observational studies of subclinical hypothyroidism in pregnancy have suggested two main underlying hypotheses for the association of subclinical hypothyroidism diagnosed in pregnancy and adverse pregnancy outcomes. It is important to emphasize that observational studies cannot prove causation, but rather associations. The first hypothesis is that subclinical hypothyroidism directly causes pregnancy complications through altered physiologic mechanisms. Conversely, the second hypothesis suggests that an impaired utero-placental unit early in pregnancy directly leads to both aberrations in maternal thyroid function tests as well as pregnancy complications. In the first setting, levothyroxine therapy for subclinical hypothyroidism in pregnancy is thought to improve clinical outcomes. However, in the latter setting, levothyroxine therapy for treatment of subclinical hypothyroidism would not improve pregnancy outcomes due to a different underlying pathophysiology. Given the lack of information about the rate of spontaneous resolution and natural history of untreated subclinical hypothyroidism in pregnancy, as well as the limitations of observational studies which do not establish causation, the best available evidence to guide clinical decision making is RCTs of levothyroxine therapy in women with subclinical hypothyroidism diagnosed in pregnancy. As such, the objective of this review is to systematically evaluate all RCTs of levothyroxine therapy in pregnancy (compared with placebo or no treatment) to determine whether pregnant women with subclinical hypothyroidism receive benefit from levothyroxine on important obstetrical, neonatal or childhood neurodevelopmental outcomes (specifically IQ). - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

8 Page of Methods We performed a systematic review and meta-analysis as outlined in the PROSPERO published protocol (CRD000). Results are reported in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA). Study Question This review examined the following question: In women with subclinical hypothyroidism diagnosed in pregnancy, does treatment with levothyroxine versus control (either placebo or no treatment) influence obstetrical, neonatal or childhood outcomes? Study Eligibility Criteria Studies that met all the following criteria were included in this review: ) pregnant women diagnosed with subclinical hypothyroidism (by any pre-specified study definition); ) randomised to levothyroxine versus control (i.e., placebo or no treatment); ) reported obstetrical, neonatal and/or childhood neurodevelopmental outcomes; ) reported results in either English or French; and, ) published from 0 to, January, 0. The publication date of 0 was specifically chosen to coincide with the availability of more sensitive TSH assays to minimize classification bias for subclinical hypothyroidism. Study Outcomes - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

9 Page of The following clinically relevant obstetrical, neonatal and childhood outcomes were prespecified. Obstetrical outcomes included: miscarriage, gestational hypertension, preeclampsia, preterm delivery, and mode of delivery. Neonatal outcomes included: admission to neonatal intensive care unit (NICU), birth weight, and gestational age at delivery (weeks). Childhood outcomes included: IQ and any other validated measures of childhood neurodevelopment. Search Strategy The search strategy was developed in consultation with a trained medical librarian. The search strategy was composed of the following terms: Hypothyroidism/ or hypothyroidism.mp, Pregnancy/ or pregnanc* or pregnant, Thyroxine/ or thyroxine.mp or synthroid.mp or lt.mp, and was modified according to search headings for each database. Each term was separated by the Boolean term AND. The search was conducted using the Scottish Intercollegiate Guidelines Network (SIGN) filters for randomised controlled trials where possible. The search was limited to human studies. Data Sources and Searches The following databases were searched in duplicate on, January, 0: Medline, EMBASE, CINAHL, Cochrane Database of Systematic Reviews, and Cochrane Central Register of Controlled Trials. In addition, references of included articles were hand-searched in duplicate to identify additional articles for inclusion. Experts in the field of endocrinology in pregnancy were consulted for potential additional articles. Clinicaltrials.gov was searched for active studies. - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

10 Page of Study Selection Study selection was completed in two separate stages by two independent reviewers following PRISMA guidelines. In the first step, titles and abstracts of all citations retrieved by the search were reviewed for eligibility. In the second step, all citations deemed eligible at the title and abstracts stage were reviewed for eligibility in a full text format. Reasons for exclusion were recorded. Agreement was recorded at each stage and reported as a kappa statistic. Disagreements between reviewers were resolved by consensus and by consultation with a third independent reviewer when necessary. Data Collection Process Two reviewers independently extracted relevant study information in duplicate using standardized data extraction forms. Extracted data elements included: study characteristics; study definition of subclinical hypothyroidism; baseline participant characteristics; and obstetrical, neonatal and childhood neurodevelopmental outcomes (i.e., IQ, or other validated neurodevelopmental outcomes). Data management was performed using Microsoft Excel. Risk of Bias Assessment Each reviewer assessed study quality independently using the Cochrane Risk of Bias Tool (Modified) for Quality Assessment of Randomized Controlled Trials. This tool assesses seven methodologic domains and provides a summary measure of bias for each study categorized as low risk of bias, high risk of bias or unclear risk of bias. Data Synthesis and Analysis - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

11 Page of Meta-analyses were performed using random effect models for continuous outcomes relating to harms and benefits of treatment where there was sufficient data. Dichotomous outcomes were meta-analysed using a Mantel-Haenszel method. Risk ratios and % confidence intervals (% CI) were calculated. A pre-specified meta-analysis of summary measures of the effect size for childhood IQ was planned. Pre-specified sensitivity analyses were planned to evaluate the effects of important clinical predictors on clinical outcomes (e.g., timing of initiation of levothyroxine therapy in pregnancy, dose of levothyroxine, duration of treatment, duration of follow-up, definition of subclinical hypothyroidism, treatment strategy [i.e., treat-to-target versus fixed dose], race or ethnicity, iodine status, body mass index, method of IQ measurement, study quality, and date of publication [i.e. to reflect local practice]). Statistical heterogeneity was quantified using I statistics where appropriate, where I >0% represents moderate and I >% represents substantial heterogeneity across studies. Statistical analyses were performed using Review Manager (Version., The Cochrane Collaboration, Copenhagen, Denmark). Results As outlined in Figure, of the citations identified by the sensitive search strategy, nine citations were identified for full text review -. Three trials (n= pregnant women) met all eligibility criteria and thus were included in the qualitative and quantitative analyses -. An additional study by Hales et al. reported a longer nine year follow-up analysis of one of the included trials. The original trial data were included in the full analysis because the reporting and follow up in the original trial were much more complete. However, we report and discuss the nine year follow up data because it is the longest duration of follow up of offspring of - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

12 Page 0 of women with subclinical hypothyroidism in pregnancy ever reported. The kappa statistic for inter-rater agreement for the title and abstract review was 0.0 (% CI 0., 0.) and for the full text review was 0. (% CI 0.0,.00). Study characteristics Included study characteristics are summarised in Table. One trial compared levothyroxine to placebo and two compared to no treatment. One trial randomised women to thyroid function screening during pregnancy (followed by levothyroxine treatment if a diagnosis of subclinical hypothyroidism was found) versus thyroid function screening of stored frozen maternal blood after delivery. The study definitions of subclinical hypothyroidism differed in each trial (table ). One study included women with thyroid peroxidase (TPO) antibodies, one excluded women with TPO antibodies and the last study did not specify TPO-antibody status. Participant characteristics Baseline participant characteristics are summarized in Table. Gestational age at randomisation was highest for Casey et al., with a mean time of randomisation of approximately weeks gestation. Both Nazarpour et al. and Lazarus et al. randomised participants at an average gestational age closer to weeks. Specifically, in the Nazarpour et al. trial, % of women in the levothyroxine arm were recruited prior to weeks gestation and in Lazarus et al. % of women in the treatment arm were started on levothyroxine prior to weeks gestation. Risk of bias assessment : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

13 Page of Study quality and risk of bias assessments are presented in Figures and. Only one trial was considered low risk in all seven domains of the Cochrane Collaboration s tool for risk of bias assessment. The Lazarus 0 trial randomised participants to either immediate screening and treatment for subclinical hypothyroidism, or delayed screening. Specifically, women randomised to immediate screening had their thyroid function assessed at approximately weeks gestation. If the participants meet criteria for subclinical hypothyroidism they were started on levothyroxine. Women randomised to delayed screening had a blood sample take at the same gestational age, but stored for analysis after delivery. Given that the results were not available until after pregnancy, no placebo was used. Thus, the Lazarus trial scored high risk for performance bias, unclear risk of detection bias, since they not reported if outcome assessment was blinded, and low risk of bias in the remaining five domains. Maternal, obstetrical and neonatal outcomes Data from two trials including a total of 0 women allowed for meta-analysis of the following five outcomes: preterm delivery < weeks gestation, gestational age at delivery, placental abruption, NICU admission and head circumference (Figure ). There were no significant differences between levothyroxine versus control (i.e., placebo or no treatment) for any of these outcomes. Meta-analysis for all outcomes had little heterogeneity (I = 0%) except for placental abruption (I =%), which demonstrated moderate heterogeneity between studies. Additional comparisons reported in a single trial that could not be meta-analysed included stillbirth (0 for both arms; p = not reported); stillbirth or miscarriage (% versus % for levothyroxine and placebo respectively; p=0.); gestational diabetes (% both arms; p=0.); gestational hypertension (0% versus % for levothyroxine and placebo respectively; - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

14 Page of p=0.); preeclampsia (% both groups; p=0.); and neonatal death (0 versus <% for levothyroxine and placebo respectively; p = 0.). Childhood outcomes Two trials reported childhood IQ and other commonly used and validated measures of childhood neurodevelopment. The authors of the Casey et al trial provided additional unpublished data on IQ (mean and standard deviation) allowing for inclusion in the metaanalysis (Figure ). The meta-analysis for childhood IQ showed no significant difference between levothyroxine versus control (figure ) (mean difference -0. [-., 0.]; p = 0., I = 0%). One trial initiated levothyroxine at a mean gestational age of. gestational weeks and reported median IQ at years. The other trial initiated levothyroxine at median of weeks and days and reported mean IQ at years. Both studies showed no impact of levothyroxine on Child Behaviour Checklist T-Scores (Table ). Furthermore, both trials found that the childhood IQ was within the normal range in both levothyroxine and control arms of their trials (Table ). There were no differences in IQ in the Lazarus et al. trial based upon the components of their study definition of subclinical hypothyroidism that included women with either a TSH >. th centile or those with hypothyroxinaemia (free T <. th centile). Specifically, women with TSH >. th centile alone (n = ) had similar IQ s in their children (00.±. versus 00.±.0 for intervention and control arms respectively; p=0.) and women with a free T <. th also had similar IQ s between study arms (.±. versus.±. for intervention and control arms respectively; p=0.). - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

15 Page of Lazarus et al. reported no significant differences in the Brief-P scale T-scores at years (median 0 [IQR, ] and 0 [IQR, ] p=0. for levothyroxine and control respectively). Casey et al. found no significant differences between levothyroxine and placebo arms across several additional neurodevelopmental assessment tools including: Bayley-III scores, Differential Ability Scales-II scores, Conners Rating Scales-Revised ADHD and WPPSI-III. Finally, the follow-up study of the Lazarus et al. trial demonstrated no impact of subclinical hypothyroidism in pregnancy (using the original trial definition [i.e. TSH >. th centile and/or a free T <. th centile]) on childhood IQ at nine years of age; 0.±.0 (n=) versus 0.±. (n=) for the children of mothers treated with levothyroxine and untreated respectively. There was also no IQ difference after nine years of follow up among children of mothers who only had TSH >. th centile (0.0±.0 [n=] versus 0.0±. [n=] levothyroxine and untreated, respectively). When a sensitivity analysis was done by substituting the nine year follow up data for the three year follow data from this same cohort the result remained unchanged and showed no impact of levothyroxine on childhood IQ (mean difference -0. [-.,.]; p = 0., I = 0%) (Figure b). Potential harms associated with treatment of subclinical hypothyroidism Lazarus et al. reported that 0% of women in their levothyroxine arm had their dose of levothyroxine lowered due to either biochemical hyperthyroidism (i.e., a very low TSH or a high free T level) or clinical symptoms of hyperthyroidism (i.e., palpitations) associated with levothyroxine overtreatment. The other two trials did not report similar data. The Casey et al. trial reported similar rates of anticipated adverse events between treatment arms;.% vs - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

16 Page of % in the levothyroxine and placebo arms respectively. Unanticipated adverse events in the levothyroxine arm were: maternal chest/abdominal pain (n=), irregular fetal heart (n=), autism spectrum (n=) and severe hearing loss (n=) whereas the placebo arm reported Hashimoto s thyroiditis (n=) and intrauterine growth restriction (n=). Nazarpour et al. did not report adverse events. Sensitivity analysis Our pre-specified sensitivity analysis could not be performed because of the small number of studies reporting the specific data. One trial evaluated the TSH cut off of mu/l versus < mu/l in a post-hoc analysis. Among women with a TSH mu/l, there was a lower proportion of preterm delivery in the levothyroxine treated group compared to the untreated group (.% vs % respectively; p-value not reported). Whereas in women with a TSH < mu/l, levothyroxine treatment was associated with a higher preterm delivery rate compared to untreated women (.% vs.% respectively; p-value not reported). We could not perform a sensitivity analysis as the number of women that had a TSH mu/l was not reported in this post-hoc analysis. Because only three studies were identified as meeting our inclusion criteria, which all had negative results relating to the primary outcome, we did not do a formal assessment of publication bias. Discussion This systematic review and meta-analysis of RCTs of levothyroxine treatment in women with subclinical hypothyroidism diagnosed in pregnancy demonstrated no benefit of levothyroxine - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

17 Page of treatment across a wide range of pre-specified maternal, obstetrical and neonatal outcomes and childhood IQ. Importantly, this review found no benefit of levothyroxine treatment during pregnancy on childhood IQ and neurodevelopment measures at, or years of follow-up. These findings challenge the current clinical practice guidelines which recommend the use of levothyroxine therapy for treatment of women with subclinical hypothyroidism diagnosed in pregnancy. As outlined below, these results also question the recommendations of levothyroxine therapy earlier in pregnancy than occurred in the available RCTs and at lower TSH thresholds amongst women who are TPO antibody positive This review found that the timing of initiation of levothyroxine therapy was not significantly associated with childhood IQ. First, in the Lazarus et al. trial, % of women were started on levothyroxine at less than weeks gestation. There was no difference in childhood IQ amongst women who were started on levothyroxine prior to weeks gestation compared with no treatment (.±. versus 00.00±. levothyroxine versus control respectively; p=0.), although this study was underpowered to examine this difference. Second, a pre-specified subgroup analysis in the Casey et al. trial, found no significant interaction for childhood IQ by gestational weeks at randomisation but was likely was also underpowered to answer this question. Third, the Nazarpour et al. trial entry visit was at the earliest gestation, a mean of. (SD ±.) weeks. This trial also showed no impact of early timing of initiation of levothyroxine therapy. Given the above findings, it appears implausible that treatment at an earlier gestational age may improve childhood IQ, particularly given the finding of normal childhood IQ up to nine years later. Furthermore, childhood IQ was not different between children of mothers with - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

18 Page of normal thyroid function and those with subclinical hypothyroidism after nine years of follow up. Some have hypothesized that levothyroxine treatment pre-pregnancy might be more appropriate since that might affect placentation and very early fetal neurodevelopment. The T life and TABLET trials, result of which are yet to be reported, are assessing the impact of prepregnancy levothyroxine on pregnancy outcomes in TPO antibody positive women. However, these trials include euthyroid women only. Interestingly a recent large randomised control trial of preconception levothyroxine in TPO antibody positive women with TSH levels between 0. and.miu/l undergoing IVF showed no benefits of levothyroxine on pregnancy, miscarriage, live birth or preterm delivery rates suggesting no benefit of levothyroxine replacement on placental development. Based on their observational studies, some investigators 0 have raised the possibility of a synergistic association of subclinical hypothyroidism and TPO-antibody positivity to adversely influence obstetrical and childhood outcomes. The trials in our review that included thyroid antibody positive women were not specifically powered to analyse the results by maternal antibody status. The Casey study, however, had a large proportion of participants with TPO antibody positivity (% of women with subclinical hypothyroidism) and found no association between TPO antibodies and childhood IQ in either study arm. Furthermore, in addition to their published trial results, Casey et al. s published abstract of their trial demonstrated no impact of TPO status on pregnancy loss or stillbirth, preterm birth, preeclampsia, or placental abruption. The Lazarus et al trial did not specifically report thyroid antibody status, nor was - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

19 Page of it part of their trial exclusion criteria. Based on other studies of reproductive age women it is reasonable to assume that at least % of women in this trial would have had positive antithyroid antibodies. Thus, trial evidence to date shows no benefit of treatment with levothyroxine for women with subclinical hypothyroidism who are TPO antibody positive during pregnancy. The included RCTs did not consistently report harms of levothyroxine treatment and were also of inadequate sample size to fully examine this association. This is important as large observational studies of levothyroxine therapy in pregnancy as well as high free T values in pregnancy have found increased risks of preterm delivery, gestational diabetes, pre-eclampsia, small for gestational age and childhood difficulties in math. Only the Casey trial (n=) reported these outcomes and found no increase in gestational diabetes, pre-eclampsia, and small for gestational age in the levothyroxine arm. No included studies reported on math difficulties in children. The only direct harm of levothyroxine found by this review was iatrogenic hyperthyroidism in 0% of the treated women (either biochemical or symptomatic) in the Lazarus trial, which used higher dosing of levothyroxine compared to the other trials. Reassuringly, the nine-year follow-up analysis suggests that this over-supplementation with levothyroxine did not have a detrimental effect on childhood IQ. None of the included studies examined the cost-effectiveness of treatment with levothyroxine for subclinical hypothyroidism in pregnancy. Given the lack of benefit of levothyroxine on obstetrical, neonatal and neurodevelopment outcomes, the costs of healthcare resources associated with screening, therapy and monitoring of subclinical hypothyroidism in pregnancy - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

20 Page of and postpartum must be rigorously evaluated to ensure appropriate healthcare resource allocation. This systematic review has several strengths. It provides an up to date summary of recently published RCTs not included in other systematic reviews and meta-analyses and it addresses a broad range of clinical outcomes that clinicians consider during medical decision making. This systematic review is rigorous in its design, methodology and analyses. Furthermore, the inclusion of only RCTs minimises the bias associated with observational studies in order to clearly evaluate the effects of levothyroxine treatment in pregnant women with subclinical hypothyroidism. Our review s findings must be interpreted in the context of the study s limitations. First, this review limited the search to trials published in English or French, however, this is unlikely to have biased the results as the included trials were conducted across several countries (North America, Europe and the Middle East) and included mixed racial populations with differing maternal iodine status which did also not impact results (Table ). Thus, our study s results are likely generalizable to a broad range of populations. Second, our review limited study selection to published trials (a priori). These eligibility criteria were specifically chosen to ensure high quality results, though limits the ability to use data from the Casey and Lazarus trials that are currently published only in abstract form. Of note, the Lazarus abstract also reports no significant differences in obstetrical outcomes among women with subclinical hypothyroidism randomised to levothyroxine compared to untreated women. Similarly, analysis of maternal and neonatal outcomes by maternal TPO antibody status in the Casey et al trial is currently - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

21 Page of published in abstract form and also showed no association of TPO antibody status on maternal outcomes. Once these results are published in full text format, an updated systematic review will be important. Lastly, given the median and interquartile or % confidence interval of baseline TSH measurements for women enrolled in these trials it is remains difficult to extrapolate these trial findings to women with subclinical hypothyroidism in pregnancy with TSH measurement between to 0 mu/l. Therefore, a trial of women with subclinical hypothyroidism with TSH >.0 to <0.0 mu/l may be warranted. In summary, evidence from high quality RCTs fails to demonstrate any benefit of treating subclinical hypothyroidism in pregnancy on important obstetrical, neonatal, childhood IQ and neurobehavioral outcomes. Given the lack of benefit of treatment of subclinical hypothyroidism in pregnancy, treatment and ultimately the practice of screening for subclinical hypothyroidism in pregnancy needs careful re-examination by policy makers to prevent over detection and overtreatment of a relatively benign, yet common entity. Acknowledgements We thank Helen Lee Robertson (University of Calgary, Calgary, Canada) for her expert advice on our search strategy. We also thank Dr. Lonnie Zwaigenbaum, Professor and Director of Autism Research in the Department of Pediatrics at the University of Alberta for sharing his expertise on the interpretation of childhood IQ and neurodevelopmental measures. Contributors and Sources Drs Yamamoto and Donovan are Endocrinologists and clinical researchers who conceived the idea for this systematic review and meta-anlysis. Dr Nerenberg, a specialist in Obstetric Medicine, contributed content and methodologic expertise to this review. - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

22 Page 0 of Dr Jamie Benham contributed her expertise in systematic review methodology to the design and conduct of the systematic review. All authors participated in the study design. Drs Yamamoto and Benham conducted the literature searches, study selection and data extraction. Drs Donovan and Yamamoto contributed to writing the first draft of the manuscript. All authors contributed to critical review and approval of the final manuscript. Dr Donovan is the guarantor of this article. Conflicts of Interest All of the authors report no disclosures References. Li C, Shan Z, Mao J, et al. Assessment of thyroid function during first-trimester pregnancy: what is the rational upper limit of serum TSH during the first trimester in Chinese pregnant women? The Journal of clinical endocrinology and metabolism 0;():-. doi: 0.0/jc.0-. De Groot L, Abalovich M, Alexander EK, et al. Management of thyroid dysfunction during pregnancy and postpartum: an Endocrine Society clinical practice guideline. The Journal of clinical endocrinology and metabolism 0;():-. doi: 0.0/jc.0-0 [published Online First: 0/0/0]. Lazarus J, Brown RS, Daumerie C, et al. 0 European thyroid association guidelines for the management of subclinical hypothyroidism in pregnancy and in children. Eur Thyroid J 0;():-. doi: 0./000 [published Online First: 0/0/]. Alexander EK, Pearce EN, Brent GA, et al. 0 Guidelines of the American Thyroid Association for the Diagnosis and Management of Thyroid Disease during Pregnancy and the Postpartum. Thyroid : official journal of the American Thyroid Association 0 doi: 0.0/thy.0.0. Negro R, Stagnaro-Green A. Diagnosis and management of subclinical hypothyroidism in pregnancy. BMJ 0;:g. doi: 0./bmj.g. Haddow JE, Palomaki GE, Allan WC, et al. Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child. The New England journal of medicine ;():-. doi: 0.0/NEJM000. Medici M, Korevaar TI, Schalekamp-Timmermans S, et al. Maternal early-pregnancy thyroid function is associated with subsequent hypertensive disorders of pregnancy: the generation R study. The Journal of clinical endocrinology and metabolism 0;():E-. doi: 0.0/jc.0-0. Leon G, Murcia M, Rebagliato M, et al. Maternal thyroid dysfunction during gestation, preterm delivery, and birthweight. The Infancia y Medio Ambiente Cohort, Spain. Paediatr Perinat Epidemiol 0;():-. doi: 0./ppe : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

23 Page of Maraka S, Mwangi R, McCoy RG, et al. Thyroid hormone treatment among pregnant women with subclinical hypothyroidism: US national assessment. BMJ 0;:i. doi: 0./bmj.i [published Online First: 0/0/] 0. Korevaar TI, Muetzel R, Medici M, et al. Association of maternal thyroid function during early pregnancy with offspring IQ and brain morphology in childhood: a populationbased prospective cohort study. The lancet Diabetes & endocrinology 0;():-. doi: 0.0/S-()00-. Moher D, Liberati A, Tetzlaff J, et al. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. Ann Intern Med 00;():-, W. [published Online First: 00/0/]. Weeks I, Sturgess M, Siddle K, et al. A high sensitivity immunochemiluminometric assay for human thyrotrophin. Clin Endocrinol (Oxf) ;0():-. [published Online First: /0/0]. Nicoloff JT, Spencer CA. Clinical review : The use and misuse of the sensitive thyrotropin assays. The Journal of clinical endocrinology and metabolism 0;():-. doi: 0.0/jcem--- [published Online First: 0/0/0]. Scottish Intercollegiate Guidelines Network. Randomised Controlled Trials [Available from: accessed Oct0 0.. Higgins JPT, Green S. Cochrane Handbook for Systematic Reviews of Interventions Version..0 [updated March 0]. The Cochrane Collaboration, 0. Available from Casey BM, Thom EA, Peaceman AM, et al. Treatment of Subclinical Hypothyroidism or Hypothyroxinemia in Pregnancy. The New England journal of medicine 0;():-. doi: 0.0/NEJMoa00. Lazarus JH, Bestwick JP, Channon S, et al. Antenatal thyroid screening and childhood cognitive function. The New England journal of medicine 0;():-0. doi: 0.0/NEJMoa00. Nazarpour S, Ramezani Tehrani F, Simbar M, et al. Effects of Levothyroxine on pregnant women with subclinical hypothyroidism, negative for thyroid peroxidase antibodies. Journal of Clinical Endocrinology & Metabolism 0;0:0.. Ma L, Qi H, Chai X, et al. The effects of screening and intervention of subclinical hypothyroidism on pregnancy outcomes: a prospective multicenter single-blind, randomized, controlled study of thyroid function screening test during pregnancy. Journal of Maternal-Fetal & Neonatal Medicine 0;():-. 0. Nazarpour S, Ramezani Tehrani F, Simbar M, et al. Effects of levothyroxine treatment on pregnancy outcomes in pregnant women with autoimmune thyroid disease. Eur J Endocrinol 0;():-. doi: 0.0/EJE--0. Negro R, Schwartz A, Gismondi R, et al. Universal screening versus case finding for detection and treatment of thyroid hormonal dysfunction during pregnancy. The Journal of clinical endocrinology and metabolism 00;():-0. doi: 0.0/jc Taylor P. Controlled antenatal thyroid screening (CATS) study: Obstetric outcomes. European Thyroid Journal;Conference:th Annual Meeting of the European Thyroid Association, ETA. 0. Denmark. Conference Start: 000. Conference End: 0 Vol., pp : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

24 Page of Varner M. Thyroid function in neonates of women with subclinical hypothyroidism and hypothyroxinemia. American journal of obstetrics and gynecology Conference: th annual meeting of the society for maternal-fetal medicine: the pregnancy meeting United states Conference start: 00;Conference end: 00 ( Supplement ):S- S0.. Hales C, Taylor PN, Channon S, et al. Controlled Antenatal Thyroid Screening II: effect of treating maternal sub-optimal thyroid function on child cognition. The Journal of clinical endocrinology and metabolism 0 doi: 0.0/jc.0-0 [published Online First: 0/0/]. Liu H, Shan Z, Li C, et al. Maternal subclinical hypothyroidism, thyroid autoimmunity, and the risk of miscarriage: a prospective cohort study. Thyroid : official journal of the American Thyroid Association 0;():-. doi: 0.0/thy.0.00 [published Online First: 0/0/0]. Stagnaro-Green A. Second trimester levothyroxine treatment for subclinical hypothyroidism or hypothyroxinaemia of pregnancy does not improve cognitive outcomes of children. Evid Based Med 0;():. doi: 0./ebmed-0-0 [published Online First: 0/0/]. Vissenberg R, van Dijk MM, Fliers E, et al. Effect of levothyroxine on live birth rate in euthyroid women with recurrent miscarriage and TPO antibodies (T-LIFE study). Contemp Clin Trials 0;:-. doi: 0.0/j.cct [published Online First: 0/0/0]. Coomarasamy A. Thyroid AntiBodies and LEvoThyroxine study (TABLET). 0 doi: Wang H, Gao H, Chi H, et al. Effect of Levothyroxine on Miscarriage Among Women With Normal Thyroid Function and Thyroid Autoimmunity Undergoing In Vitro Fertilization and Embryo Transfer: A Randomized Clinical Trial. JAMA 0;():0-. doi: 0.00/jama.0. [published Online First: 0//] 0. Korevaar TIM, Pop VJ, Chaker L, et al. Dose-dependency and a functional cut-off for TPOantibody positivity during pregnancy. The Journal of clinical endocrinology and metabolism 0 doi: 0.0/jc.0-00 [published Online First: 0//]. Casey B. Thyroid peroxidase antibodies in women with subclinical hypothyroidism or hypothyroxinemia. American journal of obstetrics and gynecology Conference: th annual meeting of the society for maternal-fetal medicine: the pregnancy meeting United states Conference start: 00;Conference end: 00 ( Supplement ):S.. Plowden TC, Schisterman EF, Sjaarda LA, et al. Subclinical Hypothyroidism and Thyroid Autoimmunity Are Not Associated With Fecundity, Pregnancy Loss, or Live Birth. The Journal of clinical endocrinology and metabolism 0;0():-. doi: 0.0/jc.0-0. Thompson W, Russell G, Baragwanath G, et al. Maternal thyroid hormone insufficiency during pregnancy and risk of neurodevelopmental disorders in offspring: A systematic review and meta-analysis. Clin Endocrinol (Oxf) 0 doi: 0./cen.0 [published Online First: 0/0/]. Li J, Shen J, Qin L. Effects of Levothyroxine on Pregnancy Outcomes in Women With Thyroid Dysfunction: A Meta-analysis of Randomized Controlled Trials. Alternative Therapies in Health & Medicine 0;(): : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

25 Page of Brodersen J, Schwartz LM, Heneghan C, et al. Overdiagnosis: what it is and what it isn't. Evid Based Med 0;():-. doi: 0./ebmed-0-0 [published Online First: 0/0/] - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

26 Page of Identification Screening Included Eligibility Records identified through database search n= Figure : PRISMA Flow Diagram Records after duplicates removed n= records screened full-text articles assessed for eligibility articles included in qualitative review articles included in quantitative review Additional records identified through other sources n= records excluded based on title and abstract review full-text articles excluded Not a randomised controlled trial (n=) Did not report outcomes of interest (n=) Outcomes not analysed by specified definition of subclinical hypothyroidism (n=) Repeat cohort (n=) - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

27 Page of Figure Risk of bias assessment - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

28 Page of Figure Trial Quality Assessment Green = good, yellow = not reported, red = not done - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

29 Page of a b c d e Figure Results of meta-analysis of effects of treatment with levothyroxine on clinical outcomes a. Preterm delivery < gestational weeks, b. Gestational age at delivery, c. Neonatal intensive care unit admission, d. Placental abruption e. Head circumference - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

30 Page of a b Figure : Results of meta-analysis of effects of treatment with levothyroxine on childhood IQ a. childhood IQ at and years, b sensitivity analysis of childhood IQ at and years (note all studies were not combined because the Lazarus 0 and Hales 0 were the from the same cohort) - : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.

31 Page of Table Characteristics of Included Randomised Controlled Trials Study Author (Year) Casey et al. (0) Nazarpour et al. (0) Lazarus et al. (0) Country N Intervention Levothyroxine USA Treatment of subclinical hypothyroidism or hypothyroxinaemia Iran Treatment of subclinical hypothyroidism UK and Italy on April 0 by guest. Protected by copyright. Comparator Definition of Subclinical Hypothyroidism Placebo TSH >.00 then >.00 mu/l and a normal free T No treatment TSH.-0 miu/ml and a normal free thyroxine index No treatment TSH >. th centile and/or free T <. th centile Thyroid screening plus treatment if criteria met Percentage of trial subjects with TPO antibody 0IU/mL Clinical Outcomes % Childhood IQ & Pregnancy outcomes 0 Pregnancy outcomes Not reported - Childhood IQ : first published as 0./bmjopen-0-0 on September 0. Downloaded from

32 Page 0 of Table Baseline Participant Characteristics in Included Studies Study Author (Year) Casey et al. (0) Nazarpour et al. (0) Lazarus et al. (0) Maternal age (years) Body mass index (kg/m ) Intervention arm (LT) Control arm Intervention arm (LT) Control arm Intervention arm (LT).±..±..±..±.. (% CI.,.).0±..±..±..0±.. (IQR.,.) 0±. ±. Not reported UK:. (IQR,.,.) Turin:. (IQR.,.0) Baseline TSH (mu/l) Urinary iodine (µg/l) Gestational age at trial entry (weeks) Control arm. (% CI.,.). (IQR.,.) UK:. (IQR.,. Turin:. (IQR.,.) Intervention arm (LT) Control arm Intervention arm (LT) Control (% CI (% CI.±.0 µ.±.0 µ, ), ) 0 (IQR, (IQR,.±.α.±.α ) 0) Not reported + + ( +, + ) # ( +, + ) # Results presented as either means±sd or as median (with interquartile range or % confidence interval [CI]) as described, LT: Levothyroxine Control: Placebo (Casey et al.), no treatment (Nazarpour, Lazarus), µ = at randomization, α = at first visit, # at screening on April 0 by guest. Protected by copyright. - : first published as 0./bmjopen-0-0 on September 0. Downloaded from

33 Page of Table Neurodevelopmental Outcomes of Studies Study (year) Childhood age [years] Casey et al. (0) [] Lazarus et al. (0) [] Hales et al (0) [] Childhood IQ Child Behaviour Checklist T-Score at years Child Behaviour Checklist At years Levothyroxine Control Levothyroxine Control Levothyroxine Control n= n= n=0 n=0 n= n=. ±. (). ±. (% CI, ) (% CI, ) (% CI, ) (% CI, ) n=0.±. n= 0.±.0 n=0 00.0±. n= 0.±. n=0 ±. n=0.±. Results presented as either means±sd or as median (with interquartile range or % confidence interval [CI]) as described on April 0 by guest. Protected by copyright. - : first published as 0./bmjopen-0-0 on September 0. Downloaded from

34 Page of Section/topic TITLE PRISMA 00 Checklist # Checklist item Title Identify the report as a systematic review, meta-analysis, or both. ABSTRACT Structured summary INTRODUCTION Provide a structured summary including, as applicable: background; objectives; data sources; study eligibility criteria, participants, and interventions; study appraisal and synthesis methods; results; limitations; conclusions and implications of key findings; systematic review registration number. Rationale Describe the rationale for the review in the context of what is already known., Objectives METHODS Protocol and registration Eligibility criteria Information sources Search Study selection Data collection process Data items Risk of bias in individual studies Provide an explicit statement of questions being addressed with reference to participants, interventions, comparisons, outcomes, and study design (PICOS). Indicate if a review protocol exists, if and where it can be accessed (e.g., Web address), and, if available, provide registration information including registration number. Specify study characteristics (e.g., PICOS, length of followup) and report characteristics (e.g., years considered, language, publication status) used as criteria for eligibility, giving rationale. Describe all information sources (e.g., databases with dates of coverage, contact with study authors to identify additional studies) in the search and date last searched. Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated. State the process for selecting studies (i.e., screening, eligibility, included in systematic review, and, if applicable, included in the metaanalysis). 0 Describe method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators. List and define all variables for which data were sought (e.g., PICOS, funding sources) and any assumptions and simplifications made. Describe methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis. Summary measures State the principal summary measures (e.g., risk ratio, difference in means). - on April 0 by guest. Protected by copyright. Reported on page # Prospero registration 0 CRD000 : first published as 0./bmjopen-0-0 on September 0. Downloaded from

35 Page of Synthesis of results Section/topic PRISMA 00 Checklist Risk of bias across studies Additional analyses RESULTS Study selection Study characteristics Describe the methods of handling data and combining results of studies, if done, including measures of consistency (e.g., I ) for each metaanalysis. # Checklist item Page of Specify any assessment of risk of bias that may affect the cumulative evidence (e.g., publication bias, selective reporting within studies). Describe methods of additional analyses (e.g., sensitivity or subgroup analyses, meta-regression), if done, indicating which were prespecified. Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram. For each study, present characteristics for which data were extracted (e.g., study size, PICOS, follow-up period) and provide the citations., Reported on page #, and Figure Table Risk of bias within studies Present data on risk of bias of each study and, if available, any outcome level assessment (see item ). 0, and Figures, Results of individual studies 0 For all outcomes considered (benefits or harms), present, for each study: (a) simple summary data for each intervention group (b) effect estimates and confidence intervals, ideally with a forest plot.,,, Figure, Synthesis of results Present results of each meta-analysis done, including confidence intervals and measures of consistency. Figures, Risk of bias across studies Present results of any assessment of risk of bias across studies (see Item ). 0, and Figures, Additional analysis Give results of additional analyses, if done (e.g., sensitivity or subgroup analyses, meta-regression [see Item ]). Figure b, DISCUSSION on April 0 by guest. Protected by copyright. - : first published as 0./bmjopen-0-0 on September 0. Downloaded from

36 Page of Summary of evidence Limitations PRISMA 00 Checklist Summarize the main findings including the strength of evidence for each main outcome; consider their relevance to key groups (e.g., healthcare providers, users, and policy makers). Discuss limitations at study and outcome level (e.g., risk of bias), and at review-level (e.g., incomplete retrieval of identified research, reporting bias). Conclusions Provide a general interpretation of the results in the context of other evidence, and implications for future research.,,,, FUNDING Funding Describe sources of funding for the systematic review and other support (e.g., supply of data); role of funders for the systematic review.,, No funding From: Moher D, Liberati A, Tetzlaff J, Altman DG, The PRISMA Group (00). Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Med (): e0000. doi:0./journal.pmed0000 For more information, visit: on April 0 by guest. Protected by copyright. Page of - : first published as 0./bmjopen-0-0 on September 0. Downloaded from

37 The Impact of Levothyroxine Therapy on Obstetric, Neonatal and Childhood Outcomes in Women with Subclinical Hypothyroidism Diagnosed in Pregnancy: A systematic review and meta-analysis of randomised controlled trials Journal: Manuscript ID bmjopen-0-0.r Article Type: Research Date Submitted by the Author: 0-Jun-0 Complete List of Authors: Yamamoto, Jennifer; University of Calgary, Medicine Benham, Jamie; University of Calgary, Medicine Nerenberg, Kara; University of Calgary, Medicine Donovan, Lois; University of Calgary, Medicine <b>primary Subject Heading</b>: Secondary Subject Heading: Keywords: Evidence based practice Diabetes and endocrinology, Evidence based practice, Obstetrics and gynaecology Pregnancy, Thyroid disease < DIABETES & ENDOCRINOLOGY, subclinical hypothyroidism, meta-analysis, clinical practice, Randomised Control Trials : first published as 0./bmjopen-0-0 on September 0. Downloaded from on April 0 by guest. Protected by copyright.