Maternal thyroid function in pregnancy may program offspring blood pressure, but not adiposity at 20 y of age

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1 nture publishing group Popultion Study Mternl thyroid function in pregnncy my progrm offspring blood pressure, but not diposity t 20 y of ge Dorte Rytter 1, Stine L. Andersen 2,3,4, Bodil H. Bech 1, Thorhllur I. Hlldorsson 5,6, Tine B. Henriksen 7, Peter Lurberg 2,3 nd Sjurdur F. Olsen 6 Bckground: Experimentl evidence exists indicting tht mternl thyroid hormones during pregnncy my ffect the metbolic set point nd crdio-vsculr function in the offspring. The objective of this study ws to investigte the ssocition between mternl thyroid function in week 30 of gesttion nd offspring diposity nd blood pressure t 20 y. Methods: The study ws bsed on the follow up of Dnish birth cohort from 1988 to 1989 (n = 965). A blood smple ws drwn from the pregnnt women in week 30 of gesttion (N = 877). In , the offspring were followed up with selfreported nthropometrics (N = 645) nd cliniclly mesured blood pressure (N = 425). Multiple liner regressions were used to estimte the ssocition between mternl thyroid function nd offspring BMI, wist circumference, nd blood pressure. Results: Offspring of subclinicl hypothyroid women hd higher systolic blood pressure (djusted difference = 3.6, 95% confidence intervl: 0.2, 7.0 mmhg) nd tendency towrd higher distolic blood pressure (djusted difference = 2.3, 95% confidence intervl: 0.2, 4.9 mmhg) compred to offspring of euthyroid women. No ssocition ws found with offspring BMI nd wist circumference. Conclusion: Mternl thyroid function during third trimester of pregnncy my ffect long-term blood pressure in the offspring. Thyroid disese is reltively common disorder in women of reproductive ge (1 3) nd overt s well s subclinicl disese hs been shown to be ssocited with incresed risk of dverse pregnncy outcomes such s miscrrige, preterm birth, smll for gesttionl ge, nd stillbirth (1,4 11). The development of mny diseses tkes yers nd fctors ffecting disese risk my operte throughout life. Hence, the progrmming theory proposes tht n unsuitble environment t sensitive periods of development might result in long-term chnges in the structure nd function of the orgnism nd thereby increse the risk of developing diseses lter in life (12). The fetl thyroid glnd is not cpble of producing hormones during the first hlf of pregnncy, mking the fetus entirely dependent on mternl supply. Thyroid hormones re importnt for proper regultion of fetl brin development (13,14) nd overt mternl hypothyroidism during pregnncy hs been found to be ssocited with impired neurologicl development in the offspring, (11,15,16) wheres the ssocition with subclinicl hypothyroidism is less cler (17 20). Generlly, little is known bout the possible long-term consequences in offspring exposed to mternl thyroid disese during fetl life. However, recent evidence points towrd progrmming effect on neurodevelopmentl disorders such s seizure disorders, utism, nd ttention deficit hyperctivity disorder (ADHD) (21 23). Also, experimentl studies in mice hve shown tht thyroid hormones my be importnt for the development of specific hypothlmic centres governing crdio-vsculr function (24), indicting tht mternl thyroid function during pregnncy my hve the potentil to permnently progrm offspring crdiovsculr function. Likewise, experimentl studies hve shown tht thyroid hormone signling during fetl life is involved in determining the metbolic set point in the offspring nd thereby potentilly ffecting future diposity (25). Only one study hs previously investigted the ssocition between mternl thyroid dysfunction nd offspring crdiometbolic risk fctors (26). In this study, no ssocition ws found with either subclinicl or overt mternl thyroid disese. However, the results indicted tht both low mternl thyroidstimulting hormone (TSH) nd high free T4 were ssocited with lower BMI in the 6-y-old offspring nd tht low TSH ws ssocited with lower distolic blood pressure. The offspring in this study were, however, reltively young nd more reserch is needed on the subject. Hence, the objective of this study ws to investigte the ssocition between mternl subclinicl hypo- nd hyperthyroidism s well s quintiles of TSH nd free T4 (ft4) in week 30 of gesttion nd offspring diposity nd blood pressure t ge y. 1 Section for Epidemiology, Deprtment of Public Helth, Arhus University, Arhus, Denmrk; 2 Deprtment of Endocrinology, Alborg University Hospitl, Alborg, Denmrk; 3 Deprtment of Clinicl Medicine, Alborg University, Alborg, Denmrk; 4 Deprtment of Clinicl Biochemistry, Alborg University Hospitl, Alborg, Denmrk; 5 The Unit for Nutrition Reserch, Fculty of Food Science nd Nutrition, School of Helth Sciences, University of Icelnd, Reykjvik, Icelnd; 6 Centre for Fetl Progrmming, Sttens Serum Institut, Copenhgen, Denmrk; 7 Peditric Deprtment, Arhus University Hospitl, Arhus, Denmrk. Correspondence: Dorte Rytter (dr@ph.u.dk) Received 6 October 2015; ccepted 8 Jnury 2016; dvnce online publiction 13 April doi: /pr Volume 80 Number 1 July 2016 Peditric Reserch 7

2 Rytter et l. Tble 1. Mternl nd offspring chrcteristics t different levels of prticiption, Arhus, Denmrk, Level of prticiption No exposure informtion (n = 38) No prticiption (n = 210) Questionnire only (n = 242) Clinicl exmintion (n = 425) Mternl chrcteristics Smoking 16 (47) 98 (49) 81 (36) 150 (37) Prity 0 27 (73) 109 (54) 135 (57) 249 (59) 1 7 (19) 68 (34) 80 (34) 129 (31) 2 3 (8) 24 (12) 21 (9) 43 (10) Prepregnncy BMI 21,7 (3.0) 21,8 (3.8) 21,1 (2.5) 21,4 (3.0) Age, yers 29,2 (4.0) 28,8 (4.6) 29,1 (4.0) 29,3 (4.1) Mternl eduction Elementry school 4 (12) 41 (22) 27 (13) 37 (9) High-school or technicl school 7 (21) 66 (35) 53 (25) 89 (23) University 13 (39) 55 (29) 84 (39) 159 (40) Higher cdemic 6 (18) 11 (6) 38 (18) 76 (19) Other 3 (9) 16 (8) 11 (5) 33 (8) TSH (miu/l) 1.66 (0.95) 1.51 (0.77) 1.63 (1.08) Free T4 (pmol)l) (1.76) (1.38) (1.56) Offspring chrcteristics b Sex, mle 13 (34) 143 (68) 150 (62) 170 (40) Birth weight, g 3,350 (568) 3,387 (551) 3,562 (530) 3,524 (505) Gesttionl ge, dys 285 (10) 282 (11) 283 (12) 283 (119) Smoking (%) 48 (20) 72 (17) Prentl overweight c 88 (38) 147 (37) Self-reported BMI 22.6 (2.0) 21.8 (2.8) Exercise, yes 165 (68) 308 (74) Strenous exercise, yes d 134 (58) 255 (64) Dt re mens (SD) or n (%). Informtion collected from self-dministered questionnire nd structured interview of the pregnnt women in week 30 of gesttion. b Informtion collected from self-dministered web-bsed questionnire to the offspring t the ge of y. Gender, birth weight, nd gesttionl ge collected from birth records. c Prticipnts were sked whether they would consider ny of their prents to be overweight. d Defined s exercise of t lest 20 min durtion, resulting in brethlessness. RESULTS Offspring of mothers with no exposure informtion were more often girls nd tended to hve lower birth weight compred to offspring of mothers with exposure informtion (Tble 1). Among offspring of mothers with exposure informtion, nonprticipnts hd lower birth weight nd their mothers tended to hve higher prepregnncy BMI nd were more likely to smoke during pregnncy compred to prticipnts. In ddition, prticipnts who only filled out the questionnire but did not prticipte in the clinicl exmintion were more likely to be mle nd the femles hd higher self-reported BMI compred to those who prticipted in the clinicl exmintion. None of the mothers fulfilled the criteri for overt hypothyroidism, wheres two women could be clssified s hyperthyroid. The distribution of both mternl nd offspring covrites were similr mong euthyroid, subclinicl hypothyroid, nd subclinicl hyperthyroid mothers (Tble 2). However, there ws tendency towrd lower birth weight in offspring of hypothyroid mothers nd in this group offspring lso reported prentl overweight less frequently t follow-up. No ssocition ws found between mternl subclinicl thyroid disese nd offspring diposity mesured s BMI nd wist circumference t 20 y (Tble 3). However, mternl subclinicl hypothyroidsm ws ssocited with higher systolic nd distolic blood pressure in the 20-y-old offspring compred to offspring of euthyroid women in the minimlly djusted model (model 1) (Tble 4). Further djustment for mternl ge, smoking, nd dietry iodine intke during pregnncy, eduction, nd prity tended to slightly ttenute the ssocitions (model 2) which however remined sttisticlly significnt for systolic blood pressure. The sme tendency ws seen in offspring of women with subclinicl hyperthyroidism. However, the very low number of prticipnts in this exposure group (N = 9 13, depending on the outcome) resulted in wide confidence intervls nd high vulnerbility to djustments. 8 Peditric Reserch Volume 80 Number 1 July 2016

3 Thyroid function nd offspring BP Tble 2. Mternl nd offspring chrcteristics dependent on mternl thyroid function in week 30 of gesttion in the DFO88 Cohort, Arhus, Denmrk Subclincl hypothyroidsm (n = 48) Euthyroidism (n = 606) Subclinicl hyperthyroidism (n = 13) Mternl chrcteristics Prity 0 33 (70) 445 (58) 6 (46) 1 11 (23) 194 (33) 4 (31) 2 3 (6) 58 (10) 3 (23) Age 28.2 (3.5) 29.2 (4.1) 30.7 (3.2) Smoking (yes) 19 (43) 208 (36) 4 (33) Prepregnncy BMI 21.5 (4.2) 21.3 (2.8) 22.1 (2.5) TSH, miu/l 3.88 (1.79) 1.44 (0.58) 0.20 (0.12) ft4, pmol/l 13.7 (1.52) 13.9 (1.50) 14.4 (1.22) Eduction Elementry school 2 (4.8) 61 (11.0) 1 (18.33) High-school or technicl school 10 (23.8) 127 (23.0) 5 (41,7) University 18 (42.9) 221 (40.0) 4 (33.3) Higher cdemic 7 (16.7) 105 (19.0) 2(16.7) Other 5 (11.9) 39 (1,7) 0 (0) Birth weight, g 3,389 (560) 3,548 (508) 3,608 (573) Gesttionl ge, dys 281 (10) 283 (11) 284 (16) Offspring chrcteristics b Smoking Yes 7 (15) 113 (19) 0 (0) Ex 2 (4) 25 (4) 0 (0) Occsionl 11 (24) 144 (24) 3 (25) No 26 (57) 309 (52) 9 (75) Exercise 34 (72) 428 (72) 11 (85) Strenous exercise c 24 (51) 356 (62) 9 (69) Prentl overweight d 12 (26) 218 (38) 5 (39) Selfreported BMI, kg/m (3.7) 22.0 (2.8) 22.9 (3.9) Sex, mle (%) 27 (56) 288 (48) 5 (38) Dt re mens (SD) or n (%). Informtion collected from self-dministered questionnire nd structured interview of the pregnnt women in week 30 of gesttion. b Informtion collected from selfdministered web-bsed questionnire to the offspring t the ge of y. Gender, birth weight, nd gesttionl ge collected from birth records. c Defined s exercise of t lest 20 min durtion, resulting in brethlessness. d Prticipnts were sked whether they would consider ny of their prents to be overweight. Exmining mternl levels of TSH nd T4 seprtely, no overll ssocition ws observed (Tbles 5 nd 6). Sensitivity nlyses using multiple imputtions to impute missing vlues tended to ttenute the estimted differences for the nthropometric mesures, prticulrly for the offspring of subclinicl hyperthyroid mothers. With regrd to blood pressure, the sensitivity nlysis further strengthened the ssocition between mternl subclinicl hypothyroidism nd offspring blood pressure (difference in systolic blood pressure: 3.7, 95% confidence intervl: 0.6, 6.8 mmhg nd difference in distolic blood pressure: 2.5, 95% confidence intervl: 0.2, 4.9 mmhg compred to offspring of euthyroid mothers) wheres the ssocition between mternl subclinicl hyperthyroidism nd blood pressure ws completely bolished. In the sensitivity nlyses using cliniclly mesured nthropometric mesures, estimtes chnged to some degree compred to the nlyses using self-reported mesures, but still no sttisticlly significnt ssocition ws found with mternl thyroid function (Supplementry Tbles S1 S3 online). The estimted differences for the nthropometric mesured showed some sensitivity towrd djustments for offspring TSH or T4, but djustments did not chnge the overll conclusion of no ssocitions. Adjustments for offspring TSH nd free T4 did not chnge the estimted ssocitions between mternl thyroid function nd offspring blood pressure. Volume 80 Number 1 July 2016 Peditric Reserch 9

4 Rytter et l. Tble 3. Assocition between mternl thyroid function in week 30 of gesttion nd offspring diposity t 20 y, Arhus, Denmrk, Mternl thyroid function BMI (kg/m 2 ) Men (SD) Model 1 Model 2 b Diff 95% CI Diff 95% CI Subclin hypothyroid c (46) 22.6 (3.7) , , 1.39 Euthyroid (586) 22.0 (2.8) Ref Ref Subclin hyperthyroid d (13) 22.9 (3.9) , , 3.16 Wist (cm) Subclin hypothyroid c (45) 83.0 (11.8) , , 4.2 Euthyroid (593) 81.3 (9.9) Ref Ref Subclin hyperthyroid d (13) 83.7 (11.7) , , 9.8 Model 1: Adjusted for mternl prepregnncy BMI nd offspring sex. b Model 2: Adjusted for mternl prepregnncy BMI, mternl eduction, smoking during pregnncy, mternl ge, prity, mternl dietry iodine intke, nd offspring sex. c Defined s TSH>3 miu/l nd ft4 < 18.8 pmol/l. d Defined s TSH < 0.36 miu/l nd ft4 > 9.8 pmol/l. A totl of two women with offspring prticipting in the follow-up nd norml TSH levels hd free T4 concentrtion bove the reference level nd nother two hd free T4 concentrtion below the reference level. According to the pplied definition, these women were ctegorized s euthyroid. Excluding them from the nlyses did not chnge the results. DISCUSSION Results from the present study indicte n ssocition between mternl subclinicl thyroid disese during pregnncy nd blood pressure in the 20-y-old offspring. Hence, mternl subclinicl hypothyroidism during pregnncy ws ssocited with higher distolic nd systolic blood pressure. The sme tendency ws observed in offspring of women with subclinicl hyperthyroidism, but the number of women in this ctegory ws reltively low. No overll ssocition ws found between mternl thyroid function nd offspring diposity nd between mternl TSH nd free T4 in quintiles nd offspring blood pressure or diposity. A mjor strength of this study is the long-term follow-up into young dulthood. The unique personl identifiction number in Denmrk enbled us to identify nd contct lrge proportion of the offspring from the DFO88 cohort y fter their birth. As is often the problem in these long-term follow-up studies, however, we experienced problems with ttrition. Hence, 76% gve informtion bout self-reported nthropometry nd 48% prticipted in the clinicl exmintion. We cnnot exclude the possibility tht prticiption ws ssocited with diposity nd blood pressure. This is lso indicted by the dt showing tht nonprticiption ws ssocited with lower birth weight, higher mternl prepregnncy BMI, mternl smoking during pregnncy, offspring sex nd for the clinicl exmintion lso higher self-reported BMI nd tendency towrds less enggement in physicl ctivity. However, TSH nd free T4 concentrtions were not different for mothers of offspring prticipting in the follow-up nd those who did not, indicting tht the risk of selection bis ws limited. Tble 4. Assocition between mternl thyroid function in week 30 of gesttion nd offspring blood pressure t 20 y, Arhus, Denmrk, Mternl thyroid Model 1 Model 2 b function Men (SD) Diff 95% CI Diff 95% CI Systolic blood pressure (mmhg) Subclin hypothyroid c (33) (11.9) , , 7.0 Euthyroid (383) (10.5) Ref Ref Subclin hyperthyroid d (9) (9.3) , , 10.4 Distolic blood pressure (mmhg) Subclin hypothyroid c (33) 67.8 (7.7) , , 4.9 Euthyroid (383) 65.4 (6.7) Ref Ref Subclin hyperthyroid d (9) 67.7 (6.7) , , 6.2 Model 1: Adjusted for mternl prepregnncy BMI nd offspring sex. b Model 2: Adjusted for mternl prepregnncy BMI, mternl eduction, smoking during pregnncy, mternl ge, prity, mternl dietry iodine intke nd offspring sex. c Defined s TSH > 3 miu/l nd ft4 < 18.8 pmol/l. d Defined s TSH < 0.36 miu/l nd ft4 > 9.8 pmol/l. The ssocition between mternl thyroid function nd offspring diposity, ws estimted using self-reported informtion, which might hve led to some degree of underreporting. However, wheres vlidtion of the self-reported informtion using cliniclly mesured BMI (n = 423) nd wist circumference (n = 407) did indicte some underreporting, this underreporting did not depend on mternl TSH or free T4. We cnnot exclude tht this hs bised the ssocition between mternl thyroid function nd offspring diposity towrds no ssocition. However, repeting the diposity relted nlyses using cliniclly mesured BMI nd wist circumference did not chnge the overll conclusion of no ssocition. In the study, we djusted for number of potentil confounders. We cn, however, not exclude the possibility of residul nd unmesured confounding. The size of the study nd in prticulr the smll number of women with subclinicl thyroid disese lso limited the possibility of djustments. It ws decided not to include offspring life style hbits in the nlyses, since this informtion ws collected cross-sectionlly with the outcome. Hence, the direction of n ssocition between, e.g., exercise nd BMI ws uncertin nd djustments could led to collider bis. Also, birth weight nd gesttionl ge were not djusted for in the min nlysis since they could be prt of the cusl pthwy. Adjusting for these fctors in secondry nlyses, however, did not chnge estimtes mterilly. We tried to djust for dietry intke of iodine during pregnncy. The intke of iodine ws estimted bsed on self-reported diet, nd considerble mount of residul confounding is ssumed to be present. It is, however, strength tht ll women were living in the sme geogrphicl re indicting tht the iodine intke through the drinking wter ws similr for ll women. The mternl concentrtion of free T4 nd TSH ws mesured from blood smples collected in week 30 of gesttion. At this time of gesttion, the fetl thyroid glnd is functioning nd the fetus is probbly less sensitive to mternl concentrtions. It is however likely, tht women ctegorized s hving 10 Peditric Reserch Volume 80 Number 1 July 2016

5 Thyroid function nd offspring BP Tble 5 Assocition between quintiles of mternl TSH in third trimester of pregnncy nd offspring diposity nd blood pressure t 20 y, Arhus, Denmrk, Quintiles mternl TSH N Men (SD) Adjusted difference b BMI (kg/m 2 ) 95% CI Q (3.2) , 0.87 Q (2.7) , 0.88 Q (2.5) Reference Q (3.0) , 0.97 Q (3.1) , 0.94 Wist circumference (cm) Q (10.4) , 1.6 Q (11.0) , 3.2 Q (9.3) Reference Q (9.4) , 2.4 Q (10.4) , 3.6 Systolic blood pressure (mmhg) Q (11.1) , 2.7 Q (10.1) , 1.4 Q (11.3) Reference Q (10.7) , 0.5 Q (10.3) , 2.6 Distolic blood pressure (mmhg) Q (6.9) , 2.1 Q (7.6) , 1.8 Q (5.9) Reference Q (5.8) , 1.5 Q (7.4) , 1.6 Rnge of TSH concentrtions mong mothers with offspring prticipting in the followup: Q1 ( miu/l); Q2 ( miu/l); Q3 (1.28; 1.62 miu/l); Q4 ( miu/l); Q5 (2.15; 13.5 miu/l). b Adjusted for mternl prepregnncy BMI, mternl eduction, smoking during pregnncy, mternl ge, prity, mternl dietry iodine intke, nd sex. subclinicl thyroid disese in week 30 of gesttion lso were ffected in erly pregnncy. Also, mternl supply of thyroid hormones still could influence the regultion of fetl hormone production in lte gesttion. The ssocition between subclinicl hypothyroidism nd offspring blood pressure is in line with experimentl evidence (24). Hence, studies in mice hve indicted tht mternl hypothyroidism during pregnncy my be importnt in regulting the development of specific fetl hypothlmic centers involved in the utonomic control of blood pressure. It is possible tht devition from norml thyroid function, independently of whether it is hyper- or hypothyroidism, is ffecting the development of these hypothlmic cells. This could potentilly explin why we in this study lso found n dverse ssocition between subclinicl hyperthyroidism nd offspring blood pressure. However, in our study, very few women were ctegorized s hving subclinicl hyperthyroidism nd the sensitivity nlysis using multiple imputtions completely Tble 6. Assocition between quintiles of mternl ft4 in third trimester of pregnncy nd offspring diposity nd blood pressure t 20 y, Arhus, Denmrk, Quintiles mternl ft4 Men (SD) Adjusted difference b N (95% CI) BMI (kg/m 2 ) Q (2.8) , 0.7 Q (2.7) , 0.5 Q (3.0) Reference Q (3.4) , 0.9 Q (2.6) , 1.0 Wist (cm) Q (9.7) , 3.6 Q (8.5) , 2.8 Q (1.1) Reference Q (1.2) , 4.2 Q (9.4) , 3.5 Systolic BP (mmhg) Q (10.3) , -0.2 Q (11.1) , 0.4 Q (11.8) Reference Q (9.5) , 1.7 Q (10.5) , 3.7 Distolic BP (mmhg) Q (7.2) , 1.3 Q (6.7) , 0.5 Q (6.1) Reference Q (6.5) , 1.3 Q (7.2) , 1.9 Rnge of T4 concentrtions in the different qurtiles mong mothers with offspring prticipting in the follow-up: Q1 ( pmol/l); Q2 ( pmol/l); Q3 ( pmol/l); Q4 ( pmol/l); Q5 ( pmol/l). b Adjusted for mternl prepregnncy BMI, mternl eduction, smoking during pregnncy, mternl ge, prity, mternl dietry iodine intke, nd sex. bolished the ssocition with blood pressure. Hence, lrger studies re needed to explore the potentil ssocition between subclinicl hyperthyroidism nd blood pressure. The lck of overll ssocition between mternl TSH nd free T4 when nlyzed seprtely could indicte tht only thyroid hormone levels outside the norml rnge ffect the development of blood pressure in the offspring. Hence, in the highest nd lowest quintiles of TSH, only 38 nd 11% hd levels bove 3 miu/l or below 0.36 miu/l respectively, indicting tht possible ssocition between extreme vlues of, e.g., TSH my hve been diluted by women within the reference rnge. Only one previous study hs investigted the ssocition between mternl thyroid function during pregnncy nd offspring crdio-metbolic helth in humns. In this study, mternl hypothyroidism or hyperthyroidism ws not ssocited with offspring blood pressure or diposity t 6 y. However, the study did see n ssocition between both low mternl TSH nd high free T4 nd lower BMI in the 6-y-old Volume 80 Number 1 July 2016 Peditric Reserch 11

6 Rytter et l. Pregnnt women scheduled to ttend routine ntentl cre in gesttionl week 30 t midwife prctice in the city of Arhus, Denmrk, April 1988-Jnury 1989 n = 1,212 Women enrolled in the DFO88 cohort n = 965 Mother-child pirs in the initil study n = 959 Mother-child pirs t follow-up with mesurement of mternl TSH nd FT4 t inclusion n = 877 Mother-child pirs t follow-up with outcome ssessment Outcome self-reported only n = 242 n = 667 Outcome self-reported & from clinicl exmintion n = 403 Outcome from clinicl exmintion only n = 22 Not willing to prticipte n = 247 Missing mternl identifiction number n = 5 Missing child identifiction number n = 1 Lost to follow-up (ded or emigrtion) n = 42 Multiple pregnncy n = 2 No mternl blood smple n = 38 No follow-up on child n = 210 Figure 1. Flowchrt for the DFO88 Cohort, Arhus, Denmrk offspring. Also, low mternl TSH ws ssocited with lower distolic blood pressure. The discrepncies between the two studies could be relted to the different ge of the offspring. Hence, the consequences of being exposed to subclinicl thyroid disese during fetl life my not hve mnifested in the young offspring yet. In conclusion, our dt support the hypothesis tht mternl subclinicl thyroid dysfunction during pregnncy is ssocited with incresed blood pressure in the offspring. An ssocition tht could be the result of mternl thyroid hormone levels interfering with fetl hypothlmic development. If indeed mternl thyroid disese is ssocited with offspring long-term crdiovsculr function, screening, nd tretment of pregnnt women for modest thyroid dysfunction could be reltively chep nd esily implemented strtegy for the prevention of crdiovsclur disese in the offspring. However, limited evidence still exists nd the present study should be seen s hypothesis generting. Therefore, lrger studies re needed before firm conclusions cn be drwn. METHODS The study followed-up birth cohort estblished in Arhus, Denmrk from April 1988 until Jnury 1989 (DFO88 cohort). A totl of 965 pregnnt women, representing 80% of consecutive smple of 1,212 women ttending routine ntentl cre t specific ntentl cre centre in the city, were included in the study. The women filled out self-dministered dietry questionnire 1 wk prior to the scheduled ntentl visit in week 30 of gesttion, nd provided consent ws given, supplementry fce to fce interview ws undertken fter the visit. Also in connection with the visit, blood smple ws obtined, processed, nd stored t 20 C. The men ge of the prticipting women ws 29 y. A totl of 915 (95%) mother nd child dyds were live, living in Denmrk nd could be identified in the centrl registrtion registry in where the offspring from DFO88 were invited for followup t ge y. The offspring were contcted nd sked to complete self-dministered web-bsed questionnire concerning nthropometric mesures, helth, nd lifestyle. Additionlly, they were invited to tke prt in physicl exmintion. A sufficient mount of serum for thyroid hormone nlyses ws vilble for 877 mothers in Of the 877 offspring of mothers with exposure informtion, totl of 663 (76%) filled out the questionnire nd 425 (48%) prticipted in the clinicl exmintion (Figure 1). Exposure Assessment Mternl concentrtions of TSH nd free T4 were quntified in 2014 from serum collected in gesttionl week 30 of pregnncy on Dimension Vist utomted immunossy (Siemens Helthcre Dignostics, Germny) using luminescent detection nd LOCI technique. For TSH, the mnufcturer reference rnge ws miu/l, functionl sensitivity miu/l, nlyticl mesurement rnge miu/l, nd interssy CV % for five concentrtions in the rnge from miu/l. For free T4, the mnufcturer reference rnge ws pmol/l, nlyticl mesurement rnge pmol/l, nd interssy CV % for five concentrtions in the rnge from pmol/l. For both, TSH nd free T4 internl nd externl controls were included. Offspring TSH nd free T4 ws nlyzed from fsting blood smples collected t the time of follow-up using the sme method. The thyrotropic ctivity of humn gondotropin cuses decrese in serum TSH most pronounced in the first trimester of pregnncy nd it hs been recommended to use trimester-specific reference rnges in pregnncy. Therefore, ccording to guidelines from the Americn Thyroid Assocition (27), mothers were ctegorized s being either hypothyroid/subclinicl hypothyroid (TSH > 3.00 miu/l nd free T4 within or below reference levels), hyperthyroid/subclinicl hyperthyroid (TSH < 0.36 nd free T4 within or bove reference levels) or euthyroid (the remining women). Outcome Assessment The offspring gve self-reported informtion bout height, weight, nd wist circumference in the web-bsed questionnire. Blood pressure ws mesured t the physicl exmintion which ws performed by trined personl. Following 7 min rest, blood pressure ws mesured three times (2 min prt) with n utomtic blood pressure device (OMRON M6 Comfort (HEM-7000-E), Omron Helthcre Europe, the Netherlnds). The men of the lst two mesurements ws used in the nlyses. Sttistics Multiple liner regression nlyses were used to estimte the ssocition between mternl thyroid function nd self-reported BMI nd wist circumference s well s cliniclly mesured systolic nd distolic blood pressure. In the primry nlyses, mternl thyroid function ws ctegorized s subclinicl hypothyroidism, euthyroidism, or subclinicl hyperthyroidism. In dditionl nlyses, mternl thyroid function ws ctegorized ccording to quintiles of either TSH or T4 bsed on the entire study popultion with the third quintile s reference. Potentil confounders were identified priori nd the following covrites were included in the fully djusted model: Mternl prepregnncy BMI (< 5% missing), ge (<2 % missing), prity (<2% missing), eduction (7 9% missing), smoking during pregnncy (< 6% missing), nd dietry iodine intke during pregnncy, estimted from the dietry questionnire in week 30 of gesttion (< 2% missing). In ddition, sex of the child ws considered strong predictor for the 12 Peditric Reserch Volume 80 Number 1 July 2016

7 Thyroid function nd offspring BP outcomes nd ws lso included in the model (0% missing) % of prticipnts hd missing informtion on t lest one covrite. Since the subclinicl hyperthyroid nd hypothyroid exposure groups were reltively smll, overdjustments could cuse unstble estimtes nd n dditionl model only djusting for sex nd mternl prepregnncy BMI ws included. Only prticipnts with complete informtion on covrites were included in the min nlyses. Due to the reltively high loss to follow-up, sensitivity nlyses were performed using multiple imputtion. Assuming the vlues missing were missing t rndom, vlues were imputed bsed on sex, mternl prepregnncy BMI, prity, mternl smoking during pregnncy, mternl eduction, nd offspring birth weight. In ddition, the imputtion of blood pressure ws lso bsed on self-reported height nd BMI. Additionlly, sensitivity nlyses were run using cliniclly mesured nthropometrics. To investigte whether potentil ssocition between mternl thyroid function nd offspring diposity nd blood pressure could be medited through offspring thyroid function, dditionl nlyses were run djusting for offspring TSH or T4. Ethics All procedures involving humn subjects were pproved by the locl ethics committee for Region Midtjyllnd (journl no ) nd the Dnish Dt Protection Agency (journl no ). Written informed consent ws obtined from ll prticipnts. SUPPLEMENTARY MATERIAL Supplementry mteril is linked to the online version of the pper t STATEMENT OF FINANCIAL SUPPORT This work ws supported by The Dnish Council for Strtegic Reserch (grnts no: , nd ). Disclosures: The uthors hve no disclosures. References 1. Alln WC, Hddow JE, Plomki GE, et l. Mternl thyroid deficiency nd pregnncy complictions: implictions for popultion screening. J Med Screen 2000;7: Crlé A, Lurberg P, Pedersen IB, et l. Epidemiology of subtypes of hypothyroidism in Denmrk. Eur J Endocrinol 2006;154: Crlé A, Pedersen IB, Knudsen N, et l. Epidemiology of subtypes of hyperthyroidism in Denmrk: popultion-bsed study. Eur J Endocrinol 2011;164: Andersen SL, Olsen J, Wu CS, Lurberg P. Spontneous bortion, stillbirth nd hyperthyroidism: dnish popultion-bsed study. Eur Thyroid J 2014;3: Andersen SL, Olsen J, Wu CS, Lurberg P. Low birth weight in children born to mothers with hyperthyroidism nd high birth weight in hypothyroidism, wheres preterm birth is common in both conditions: Dnish Ntionl Hospitl Register Study. Eur Thyroid J 2013;2: Csey BM, Dshe JS, Wells CE, et l. Subclinicl hypothyroidism nd pregnncy outcomes. Obstet Gynecol 2005;105: Benhdi N, Wiersing WM, Reitsm JB, Vrijkotte TG, Bonsel GJ. Higher mternl TSH levels in pregnncy re ssocited with incresed risk for miscrrige, fetl or neontl deth. Eur J Endocrinol 2009;160: Ashoor G, Miz N, Rots M, Jwdt F, Nicolides KH. Mternl thyroid function t 11 to 13 weeks of gesttion nd subsequent fetl deth. Thyroid 2010;20: Negro R, Schwrtz A, Gismondi R, Tinelli A, Mngieri T, Stgnro-Green A. Universl screening versus cse finding for detection nd tretment of thyroid hormonl dysfunction during pregnncy. J Clin Endocrinol Metb 2010;95: Negro R, Schwrtz A, Gismondi R, Tinelli A, Mngieri T, Stgnro- Green A. Incresed pregnncy loss rte in thyroid ntibody negtive women with TSH levels between 2.5 nd 5.0 in the first trimester of pregnncy. J Clin Endocrinol Metb 2010;95:E Negro R, Mestmn JH. Thyroid disese in pregnncy. Best Prct Res Clin Endocrinol Metb 2011;25: Olsen J. [Fetl progrmming of chronic diseses]. Ugeskr Leger 2003;165: Ahmed OM, El-Greib AW, El-Bkry AM, Abd El-Twb SM, Ahmed RG. Thyroid hormones sttes nd brin development interctions. Int J Dev Neurosci 2008;26: Bernl J, Nunez J. Thyroid hormones nd brin development. Eur J Endocrinol 1995;133: Hddow JE, Plomki GE, Alln WC, et l. Mternl thyroid deficiency during pregnncy nd subsequent neuropsychologicl development of the child. N Engl J Med 1999;341: Mn EB, Brown JF, Serunin SA. Mternl hypothyroxinemi: psychoneurologicl deficits of progeny. Ann Clin Lb Sci 1991;21: Lzrus JH, Bestwick JP, Chnnon S, et l. Antentl thyroid screening nd childhood cognitive function. N Engl J Med 2012;366: Henrichs J, Bongers-Schokking JJ, Schenk JJ, et l. Mternl thyroid function during erly pregnncy nd cognitive functioning in erly childhood: the genertion R study. J Clin Endocrinol Metb 2010;95: Ghssbin A, Henrichs J, Tiemeier H. Impct of mild thyroid hormone deficiency in pregnncy on cognitive function in children: lessons from the Genertion R Study. Best Prct Res Clin Endocrinol Metb 2014;28: Li Y, Shn Z, Teng W, et l. Abnormlities of mternl thyroid function during pregnncy ffect neuropsychologicl development of their children t months. Clin Endocrinol (Oxf) 2010;72: Andersen SL, Lurberg P, Wu CS, Olsen J. Attention deficit hyperctivity disorder nd utism spectrum disorder in children born to mothers with thyroid dysfunction: Dnish ntionwide cohort study. BJOG 2014;121: Andersen SL, Lurberg P, Wu CS, Olsen J. Mternl thyroid dysfunction nd risk of seizure in the child: Dnish ntionwide cohort study. J Pregnncy 2013;2013: Andersen SL, Olsen J, Lurberg P. Foetl progrmming by mternl thyroid disese. Clin Endocrinol (Oxf) 2015;83: Mittg J, Lyons DJ, Sällström J, et l. Thyroid hormone is required for hypothlmic neurons regulting crdiovsculr functions. J Clin Invest 2013;123: Vujovic M, Nordström K, Guthier K, et l. Interference of mutnt thyroid hormone receptor lph1 with heptic glucose metbolism. Endocrinology 2009;150: Godoy GA, Korevr TI, Peeters RP, et l. Mternl thyroid hormones during pregnncy, childhood diposity nd crdiovsculr risk fctors: the Genertion R Study. Clin Endocrinol (Oxf) 2014;81: Stgnro-Green A, Ablovich M, Alexnder E, et l.; Americn Thyroid Assocition Tskforce on Thyroid Disese During Pregnncy nd Postprtum. Guidelines of the Americn Thyroid Assocition for the dignosis nd mngement of thyroid disese during pregnncy nd postprtum. Thyroid 2011;21: Volume 80 Number 1 July 2016 Peditric Reserch 13