Study size and participants:
In the present cross-sectional case control study, all hypothyroidism patients were selected among referents to the endocrinology clinic of Jahrom city in 2018. In the study, the treatment group included patients with pre-diagnosed hypothyroidism under Levothyroxine therapy or newly diagnosed patients. Written informed consent was acquired from all study subjects. Control group consisted of healthy people, who were similar to other groups in terms of confounding variables. Exclusion criteria were to have collagen vascular and Celiac disease and type-1 diabetes mellitus. Finally, 633 Immune Hypothyroid and 305 non-Immune Hypothyroid and 200 healthy subjects were enrolled (Figure1). The exclusion criteria for this study were type 1 diabetes, lupus, collagen vascular disease, rheumatoid arthritis, celiac disease, and also patients who underwent vitamin D supplementation and vitamin D interacting medications (such as antacids, corticosteroids, orlistat, diabetes medications, antihypertensive drugs, cholestyramine, antiepileptics, calcium supplements). While the participants enrolment were done based on their previous thyroid function workups during referral to thyroid clinic, to investigate the disease and vitamin D interactions, new blood samples were taken to evaluate both vitamin D levels and thyroid function tests in same time at last month of summer with adequate sun exposure. Because, the circulating vitamin D levels ranges from season to season, in our study all blood samples were taken at the same period of august 2018; so, the confounding effect of seasonal variations of vitamin D was eliminated.
Normal thyroid function was considered as 0.3 mIU/L ≤ TSH ≤ 3.6 mIU/L. The diagnosis of overt and subclinical hypothyroidism respectively was done based on TSH levels higher than 10 and 3.6 mIU/L <TSH ≤ 10 mIU/L . Normal T4 levels were considered between 4.5 and 12.0 μg/dL for normal participants. T4 value lower than 4.5 was one of the additional criteria’ for hypothyroidism patients . The values higher than 40 and 100 IU/mL were considered positive for TPOAb and TGAb, respectively. Diagnosis criteria for Hashimoto thyroiditis included decreased T4 value along with an elevated TSH (Overt and subclinical hypothyroidism patients) and the presence of high serum TPOAb or TGAb concentrations. The patients having overt or subclinical hypothyroidism without positive TPOAb or TGAb were considered as having non-autoimmune hypothyroidism disease. Vitamin D levels lower than 8 ng/mL were considered as severe vitamin D deficiency, 9–15 ng/mL concentrations as mild vitamin D deficiency, higher than 16 to 20 ng/mL concentrations as vitamin D insufficiency and higher than 20 ng/mL concentrations as normal vitamin D level .
Blood samples were taken from all participants after at least 8 hours of fasting. T3, Free T4, TSH were measured by Cobas ECLIAs (Roche Diagnostics GmbH, Mannheim, Germany). Thyroid peroxidase antibody (TPOAb) were determined by chemiluminiscenta IMMULITE 2000 XPi (Siemens, Eschborn, Germany). Thyroid globulin antibody (TGAb) levels were analyzed by Enzyme-Linked Immunosorbent Assay (ELISA kit, Diesel). Vitamin D levels were measured by LIAISON vitamin D chemiluminescence immunoassay (DiaSorin, Saluggia, Italy).
In order to compare the quantitative continuous variables, ANOVA for parametric data and Man-u withney and Kruskal Wallis for non-parametric data were used. Chi-square test was used to compare discrete data among different groups. A p-value of less than 0.05 was considered statistically significant. SPSS v.19 was used for statistical analysis.