We request permission to apply for funds to perform assays of thyroid hormones- TSH, free thyroxine (fT4) and thyroid-peroxidase antibody (TPO-Ab)- on the serum residuals from maternal samples collected during pregnancy. There is increasing evidence that even mild abnormalities in maternal thyroid hormone levels during pregnancy are associated with both maternal and fetal adverse outcomes. Evidence also exists that elevated TPO autoantibodies can exert these adverse effects even when the mother is euthyroid in early pregnancy. This proposal will examine the impact of maternal thyroid status on pregnancy outcomes and therefore build on the recent work conducted by Debbie Lawlor examining maternal determinants of offspring health in ALSPAC. In particular inspection and abstraction of obstetric data from 13,733 women will soon be completed with complete data on perinatal outcomes exists for ALSPAC.

We would like to address the following objectives in relation to maternal thyroid status:

a. Describe the distribution of maternal thyroid hormones and antibodies in the first and third trimester.

b. Determine the prospective associations of first trimester TSH, fT4 and TPO with maternal weight gain in pregnancy, blood pressure change in pregnancy, smoking status, onset of gestational diabetes/glycosuria and adverse perinatal events including spontaneous and elective preterm birth, preeclampsia, abruption and stillbirth.

c. Determine the prospective associations of first and third trimester maternal thyroid status gestation with offspring birthweight, gestational age, head circumference, offspring fat mass and change in fat mass, growth trajectories from birth to age 15 and offspring cognitive function assessed at age 10 and educational assessment outcomes.

d. Examine the cross-sectional relationships during pregnancy of TSH, fT4 and TPO with vitamin D, calcium and PTH

e. Determine the prospective association of TSH, fT4 and TPO during pregnancy with maternal cardiovascular risk profile in later life (lipids, carotid intima-media thickness (CIMT), blood pressure, height, weight, BMI, fat mass insulin and glucose).

Background

Maternal thyroid status and perinatal outcomes

Maternal thyroid dysfunction has been associated with adverse pregnancy outcomes including spontaneous miscarriage1-3, fetal death4, preterm delivery5, fetal distress6, small head circumference7, low birth weight7 and impaired neuropsychological development8-10. Thyroid autoantibodies have also been independently associated with increased risk of spontaneous miscarriage and preterm delivery5. The results of individual studies on these perinatal outcomes are, however, controversial, reflecting differences in study design, laboratory data, classification of thyroid dysfunction and populations - in particular the inclusion of women with undiagnosed or insufficiently treated thyroid dysfunction. Therefore although clinical hypo and hyperthyroidism have been associated with various adverse perinatal outcomes, at present the role of subclinical thyroid disease is contentious11, 12. The largest study to date suggested that subclinical hypothyroidism -defined as TSH >=97.5th percentile for gestational age at screening and a fT4 greater than 9pmol/l (0.680 ng/dL)- was associated with placental abruption (relative risk 3.0, 95% CI 1.1-8.2) and moderate preterm birth (relative risk, 1.8, 95% CI 1.1-2.9)13. In contrast a recent prospective, population-based cohort study of 5805 women examining maternal thyroid and autoantibody status suggested that subclinical hypothyroidism as defined as TSHgreater than 95th centile and a fT4 between the 5th and 95th percentiles was not associated with adverse perinatal outcomes14. This second study also benefited from assessment of autoantibody status and demonstrated that TPO positivity, defined as greater than 95th centile (n=288), was associated with increased perinatal mortality (adjusted OR 3.2 (1.4-7.1), and a trend towards moderate preterm birth, but no association with birthweight was observed14. Notably subclinical hyperthyroidism has not been associated with adverse pregnancy outcomes15. An association between TPO seropositivity and preterm delivery has recently been demonstrated in a meta-analysis5, and a single intervention study demonstrated that the rate of preterm delivery may be reduced in TPO positive women by levothyroxine treatment16. These findings suggest that the TPO association with preterm delivery may be mediated via impaired thyroid function but obviously requires further study. To our knowledge no study has examined early pregnancy maternal thyroid status on maternal weight gain, an important confounder of adverse perinatal outcomes.

Maternal thyroid status and offspring development

Hypothyroidism caused by iodine deficiency during pregnancy has classically been associated with neurodevelopmental disorders in the offspring, as has late treatment of congenital hypothyroidism. More recently, animal studies have highlighted the importance of the supply of maternal thyroxine to the developing fetal brain17, 18. These studies suggest that even transient periods of hypothyroxinaemia can induce irreversible brain damage during development, specifically abnormal cell migration and cytoarchitecture of the somatosensory cortex and hippocampus17, 18. The role of isolated maternal hypothyroidism or isolated hypothyroxinaemia on offspring neurodevelopment in humans has been more controversial. In 1999 Haddow et al demonstrated an association of maternal hypothyroidism with impaired neurodevelopment at age 8 - with offspring IQ scores 7 points lower than those of mothers on treatment and 19% with IQ scores less than 85 as compared to 5% in controls8. Even more strikingly, isolated maternal hypothyroxinaemia (fT4 less than 10th centile, normal TSH) during the first trimester has also been associated with delayed mental and motor function in the offspring at age 1 and 210. These studies have substantial potential public health importance as the prevalence of subclinical hypothyroidism may be as high as 2-5% of the pregnant population12, 13 but the importance on long term development of subtle changes in maternal thyroid function remain disputed. The long term impact of these milder disruptions of maternal thyroid status on offspring development and educational attainment are unknown, with a NICHD study currently recruiting 5000 women to examine the impact of a fT4 less than 3rd centile on language and motor development at age 2. With respect to TPO autoantibodies, a single study has suggested an independent relationship of positive TPO antibodies and cognitive function independent of concurrently measured fT410. Further an increase in sensorineural loss has been demonstrated in children whose mothers have elevated TPO in the first half of pregnancy in the absence of overt maternal hypothyroidism19. It remains possible that both of these findings are mediated ultimately through changes in maternal or fetal thyroid hormone levels.

Fetal production of thyroid hormones is also essential for normal cardiovascular, respiratory and skeletal development. However, fetal thyroid hormone production does not begin until 10 to 12 weeks gestation, with the fetus dependent on maternal thyroid hormones until then. It is increasingly recognised that adverse perinatal events including growth restriction can be determined as early as 12 weeks gestation; with smaller than expected skeletal growth as determined by crown rump length associated with adverse outcomes including low birth weight20. The significance of this early dependence on maternal thyroid function on offspring skeletal growth is unclear; however, in an extreme example of genetically dependent fetal hypothyroidism21, although head circumference (33.8cm) was normal, bone development was systematically immature and birthweight (2650g) and body length (46cm) were reduced. The potential for a long term impact of maternal hypothyroxinaemia on offspring growth is unknown, although normality due to fetal and neonatal thyroid compensation is likely.

Subclinical hypothyroidism and cardiovascular risk

In adults, subclinical hypothyroidism has been associated with risk factors for cardiovascular disease. Specifically there is a weak association between subclinical hypothyroidism and serum lipids starting at a level of TSHgreater than 5mU/l and assuming significance on approaching a TSH level of 10mU/L22. Treatment by levothyroxine is also associated with improvement in lipid parameters23. C-reactive protein (CRP) levels also increase with progressive thyroid failure24, with replacement reducing CRP in one25 but not another study26. Enhanced central aortic pressure and central arterial stiffness have also been reported in patients with subclinical hypothyroidism27, with again improvement by levothyroxine treatment28. Lastly (CIMT) is greater in conjunction with subclinical hypothyroidism29 and hypothyroxinaemia30, with improvement on normalisation of thyroid function29, 31. The clinical importance of these findings remains disputed but given that consideration of universal screening of thyroid function during pregnancy is currently being considered, if positive associations do exist with later CVD risk this would be an important additional reason for screening and opportunity for early intervention.

Maternal thyroid status and other endocrine axes

There is limited data on the interaction of thyroid hormones and vitamin D, with animal models suggesting that Vitamin D may directly alter thyroid function32, although no association between Vitamin D and thyroid function or TPO status were recently found in a study of 642 Indian adults33.

Methods

All biochemical analyses will be performed at Glasgow Royal Infirmary, which adheres to UK external quality control for all parameters and is Clinical Pathology Accreditation (CPA) accredited.

For objectives a-d relevant datasets will be compiled by DA Lawlor and standard linear/logistic regression models used in analyses. For objective e this will be performed on completion of the BHF funded clinic of the ALSPAC mothers (Lawlor PI).

References

1 Stagnaro-Green, A., Roman, S. H., Cobin, R. H., el-Harazy, E., Alvarez-Marfany, M. and Davies, T. F., Detection of at-risk pregnancy by means of highly sensitive assays for thyroid autoantibodies, Jama, 1990, 264: 1422-1425.

2 Glinoer, D., Riahi, M., Grun, J. P. and Kinthaert, J., Risk of subclinical hypothyroidism in pregnant women with asymptomatic autoimmune thyroid disorders, J Clin Endocrinol Metab, 1994, 79: 197-204.

3 Glinoer, D., Soto, M. F., Bourdoux, P., Lejeune, B., Delange, F., Lemone, M., Kinthaert, J., Robijn, C., Grun, J. P. and de Nayer, P., Pregnancy in patients with mild thyroid abnormalities: maternal and neonatal repercussions, J Clin Endocrinol Metab, 1991, 73: 421-427.

4 Allan, W. C., Haddow, J. E., Palomaki, G. E., Williams, J. R., Mitchell, M. L., Hermos, R. J., Faix, J. D. and Klein, R. Z., Maternal thyroid deficiency and pregnancy complications: implications for population screening, J Med Screen, 2000, 7: 127-130.

5 Stagnaro-Green, A., Maternal Thyroid Disease and Preterm Delivery, J Clin Endocrinol Metab, 2009, 94: 21-25.

6 Wasserstrum, N. and Anania, C. A., Perinatal consequences of maternal hypothyroidism in early pregnancy and inadequate replacement, Clin Endocrinol (Oxf), 1995, 42: 353-358.

7 Blazer, S., Moreh-Waterman, Y., Miller-Lotan, R., Tamir, A. and Hochberg, Z., Maternal hypothyroidism may affect fetal growth and neonatal thyroid function, Obstet Gynecol, 2003, 102: 232-241.

8 Haddow, J. E., Palomaki, G. E., Allan, W. C., Williams, J. R., Knight, G. J., Gagnon, J., O'Heir, C. E., Mitchell, M. L., Hermos, R. J., Waisbren, S. E., Faix, J. D. and Klein, R. Z., Maternal Thyroid Deficiency during Pregnancy and Subsequent Neuropsychological Development of the Child, N Engl J Med, 1999, 341: 549-555.

9 Pop, V. J., Brouwers, E. P., Vader, H. L., Vulsma, T., van Baar, A. L. and de Vijlder, J. J., Maternal hypothyroxinaemia during early pregnancy and subsequent child development: a 3-year follow-up study, Clin Endocrinol (Oxf), 2003, 59: 282-288.

10 Pop, V. J., Kuijpens, J. L., van Baar, A. L., Verkerk, G., van Son, M. M., de Vijlder, J. J., Vulsma, T., Wiersinga, W. M., Drexhage, H. A. and Vader, H. L., Low maternal free thyroxine concentrations during early pregnancy are associated with impaired psychomotor development in infancy, Clin Endocrinol (Oxf), 1999, 50: 149-155.

11 Gyamfi, C., Wapner, R. J. and D'Alton, M. E., Thyroid dysfunction in pregnancy: the basic science and clinical evidence surrounding the controversy in management, Obstet Gynecol, 2009, 113: 702-707.

12 Casey, B. M., Subclinical hypothyroidism and pregnancy, Obstet Gynecol Surv, 2006, 61: 415-420; quiz 423.

13 Casey, B. M., Dashe, J. S., Wells, C. E., McIntire, D. D., Byrd, W., Leveno, K. J. and Cunningham, F. G., Subclinical hypothyroidism and pregnancy outcomes, Obstet Gynecol, 2005, 105: 239-245.

14 Mannisto, T., Vaarasmaki, M., Pouta, A., Hartikainen, A.-L., Ruokonen, A., Surcel, H.-M., Bloigu, A., Jarvelin, M.-R. and 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, 94: 772-779.

15 Casey, B. M., Dashe, J. S., Wells, C. E., McIntire, D. D., Leveno, K. J. and Cunningham, F. G., Subclinical hyperthyroidism and pregnancy outcomes, Obstet Gynecol, 2006, 107: 337-341.

16 Negro, R., Formoso, G., Mangieri, T., Pezzarossa, A., Dazzi, D. and Hassan, H., Levothyroxine Treatment in Euthyroid Pregnant Women with Autoimmune Thyroid Disease: Effects on Obstetrical Complications, J Clin Endocrinol Metab, 2006, 91: 2587-2591.

17 Lavado-Autric, R., Auso, E., Garcia-Velasco, J. V., Arufe Mdel, C., Escobar del Rey, F., Berbel, P. and Morreale de Escobar, G., Early maternal hypothyroxinemia alters histogenesis and cerebral cortex cytoarchitecture of the progeny, J Clin Invest, 2003, 111: 1073-1082.

18 Auso, E., Lavado-Autric, R., Cuevas, E., del Rey, F. E., Morreale de Escobar, G. and Berbel, P., A Moderate and Transient Deficiency of Maternal Thyroid Function at the Beginning of Fetal Neocorticogenesis Alters Neuronal Migration, Endocrinology, 2004, 145: 4037-4047.

19 Wasserman, E. E., Nelson, K., Rose, N. R., Eaton, W., Pillion, J. P., Seaberg, E., Talor, M. V., Burek, L., Duggan, A. and Yolken, R. H., Maternal Thyroid Autoantibodies during the Third Trimester and Hearing Deficits in Children: An Epidemiologic Assessment, Am. J. Epidemiol., 2008, 167: 701-710.

20 Smith, G. C., Stenhouse, E. J., Crossley, J. A., Aitken, D. A., Cameron, A. D. and Connor, J. M., Early-pregnancy origins of low birth weight, Nature, 2002, 417: 916.

21 de Zegher, F., Pernasetti, F., Vanhole, C., Devlieger, H., Van den Berghe, G. and Martial, J. A., The prenatal role of thyroid hormone evidenced by fetomaternal Pit-1 deficiency, J Clin Endocrinol Metab, 1995, 80: 3127-3130.

22 Duntas, L. H. and Wartofsky, L., Cardiovascular risk and subclinical hypothyroidism: focus on lipids and new emerging risk factors. What is the evidence?, Thyroid, 2007, 17: 1075-1084.

23 Danese, M. D., Ladenson, P. W., Meinert, C. L. and Powe, N. R., Effect of Thyroxine Therapy on Serum Lipoproteins in Patients with Mild Thyroid Failure: A Quantitative Review of the Literature, J Clin Endocrinol Metab, 2000, 85: 2993-3001.

24 Christ-Crain, M., Meier, C., Guglielmetti, M., Huber, P. R., Riesen, W., Staub, J. J. and Muller, B., Elevated C-reactive protein and homocysteine values: cardiovascular risk factors in hypothyroidism? A cross-sectional and a double-blind, placebo-controlled trial, Atherosclerosis, 2003, 166: 379-386.

25 Ozcan, O., Cakir, E., Yaman, H., Akgul, E. O., Erturk, K., Beyhan, Z., Bilgi, C. and Erbil, M. K., The effects of thyroxine replacement on the levels of serum asymmetric dimethylarginine (ADMA) and other biochemical cardiovascular risk markers in patients with subclinical hypothyroidism, Clin Endocrinol (Oxf), 2005, 63: 203-206.

26 Perez, A., Cubero, J. M., Sucunza, N., Ortega, E., Arcelus, R., Rodriguez-Espinosa, J., Ordonez-Llanos, J. and Blanco-Vaca, F., Emerging cardiovascular risk factors in subclinical hypothyroidism: lack of change after restoration of euthyroidism, Metabolism, 2004, 53: 1512-1515.

27 Obuobie, K., Smith, J., Evans, L. M., John, R., Davies, J. S. and Lazarus, J. H., Increased Central Arterial Stiffness in Hypothyroidism, J Clin Endocrinol Metab, 2002, 87: 4662-4666.

28 Owen, P. J. D., Rajiv, C., Vinereanu, D., Mathew, T., Fraser, A. G. and Lazarus, J. H., Subclinical Hypothyroidism, Arterial Stiffness, and Myocardial Reserve, J Clin Endocrinol Metab, 2006, 91: 2126-2132.

29 Monzani, F., Caraccio, N., Kozakowa, M., Dardano, A., Vittone, F., Virdis, A., Taddei, S., Palombo, C. and Ferrannini, E., Effect of Levothyroxine Replacement on Lipid Profile and Intima-Media Thickness in Subclinical Hypothyroidism: A Double-Blind, Placebo- Controlled Study, J Clin Endocrinol Metab, 2004, 89: 2099-2106.

30 Dullaart, R. P., de Vries, R., Roozendaal, C., Kobold, A. C. and Sluiter, W. J., Carotid artery intima media thickness is inversely related to serum free thyroxine in euthyroid subjects, Clin Endocrinol (Oxf), 2007, 67: 668-673.

31 Nagasaki, T., Inaba, M., Henmi, Y., Kumeda, Y., Ueda, M., Tahara, H., Sugiguchi, S., Fujiwara, S., Emoto, M., Ishimura, E., Onoda, N., Ishikawa, T. and Nishizawa, Y., Decrease in carotid intima-media thickness in hypothyroid patients after normalization of thyroid function, Clin Endocrinol (Oxf), 2003, 59: 607-612.

32 Misharin, A., Hewison, M., Chen, C.-R., Lagishetty, V., Aliesky, H. A., Mizutori, Y., Rapoport, B. and McLachlan, S. M., Vitamin D Deficiency Modulates Graves' Hyperthyroidism Induced in BALB/c Mice by Thyrotropin Receptor Immunization, Endocrinology, 2009, 150: 1051-1060.

33 Goswami, R., Marwaha, R. K., Gupta, N., Tandon, N., Sreenivas, V., Tomar, N., Ray, D., Kanwar, R. and Agarwal, R., Prevalence of vitamin D deficiency and its relationship with thyroid autoimmunity in Asian Indians: a community-based survey, Br J Nutr, 2009: 1-5.