Thyroid dysfunction is a prevalent endocrine disorder, with hypothyroidism being the most common manifestation, ranging from overt to subclinical forms. Overt hypothyroidism is characterized by elevated thyroid-stimulating hormone (TSH) and low free thyroxine (fT4), while subclinical hypothyroidism presents with elevated TSH but normal fT4 levels. Both forms are frequently associated with autoimmune thyroiditis, suggesting an underlying immunological etiology ¹.

Vitamin D, beyond its classical role in calcium-phosphate metabolism and bone health, is now recognized for its immunomodulatory properties. A growing body of evidence has shown that Vitamin D deficiency is commonly observed in patients with autoimmune disorders, including autoimmune thyroid diseases (AITD) ^2-3. Several studies have proposed that low serum 25-hydroxyvitamin D [25(OH)D] levels may be implicated in the pathogenesis of hypothyroidism by influencing the immune system and thyroid autoimmunity⁴.

Recent investigations have identified a significant inverse relationship between serum Vitamin D levels and TSH levels in hypothyroid individuals, suggesting that Vitamin D insufficiency could potentially worsen thyroid dysfunction ⁵. Moreover, Vitamin D receptors (VDRs) are expressed in thyroid tissue, and polymorphisms in the VDR gene have been associated with increased susceptibility to AITD ⁶.

In studying Vitamin D status among overt and subclinical hypothyroid patients is crucial to better understand the potential interplay between thyroid function and Vitamin D metabolism. Early identification and correction of Vitamin D deficiency may serve as a supplementary strategy in the management of hypothyroidism, particularly in populations with high prevalence of Vitamin D deficiency.

Aim and Objective:

The aim of this study is to assess and compare serum Vitamin D levels in patients with overt and subclinical hypothyroidism. The objective is to evaluate the correlation between Vitamin D deficiency and thyroid dysfunction, and to determine whether Vitamin D status differs significantly between the two hypothyroid groups.

Study Design and Setting:

A hospital-based case-control study was conducted in the Department of Medicine at Sanjay Gandhi Memorial Hospital, associated with Shyam Shah Medical College, Rewa (M.P.), after obtaining approval from the Institutional Ethics Committee.

Study Population:

The study included adults aged 18–65 years diagnosed with overt or subclinical hypothyroidism based on biochemical parameters. Participants were recruited from outpatient and inpatient departments. Written informed consent was obtained from all subjects.

Inclusion Criteria:

1. Age 18–65 years.
2. Overt hypothyroidism: TSH >10 mIU/L and FT4 below reference range.
3. Subclinical hypothyroidism: Elevated TSH with normal FT4.
4. Willingness to provide informed consent.

Exclusion Criteria:

1. Vitamin D supplementation in past 6 months.
2. Chronic kidney/liver disease or malabsorption syndromes.
3. Pregnant or lactating women.
4. Autoimmune disorders unrelated to thyroid dysfunction.
5. Use of drugs affecting Vitamin D metabolism.

Sample Size:

Based on prior prevalence data and assuming an odds ratio of 3, with 95% confidence interval and 80% power, the required sample size was calculated as 348 (174 cases and 174 controls) using an online statistical formula.

Data Collection:

A structured proforma recorded demographic details (age, sex, BMI, sunlight exposure, diet). Clinical examination and fasting venous blood samples were obtained for analysis.

Biochemical Analysis:

• Thyroid Function Tests: Serum TSH, FT4, and FT3 measured via chemiluminescence immunoassay.
• Vitamin D: Serum 25(OH)D levels assessed using ELISA. Categories: deficiency (<20 ng/mL), insufficiency (20–30 ng/mL), sufficiency (>30 ng/mL).
• Other Parameters: Serum calcium, phosphorus, and alkaline phosphatase to assess bone metabolism.

Statistical Analysis:

Data were analyzed using SPSS v20.0. Results were expressed as mean ± SD or percentages. Independent t-tests compared Vitamin D levels across groups. Pearson’s correlation assessed associations between Vitamin D and thyroid parameters. A p-value <0.05 was considered statistically significant.

Study Design and Setting:

A hospital-based case-control study was conducted in the Department of Medicine at Sanjay Gandhi Memorial Hospital, associated with Shyam Shah Medical College, Rewa (M.P.), after obtaining approval from the Institutional Ethics Committee.

Study Population:

The study included adults aged 18–65 years diagnosed with overt or subclinical hypothyroidism based on biochemical parameters. Participants were recruited from outpatient and inpatient departments. Written informed consent was obtained from all subjects.

Inclusion Criteria:

1. Age 18–65 years.
2. Overt hypothyroidism: TSH >10 mIU/L and FT4 below reference range.
3. Subclinical hypothyroidism: Elevated TSH with normal FT4.
4. Willingness to provide informed consent.

Exclusion Criteria:

1. Vitamin D supplementation in past 6 months.
2. Chronic kidney/liver disease or malabsorption syndromes.
3. Pregnant or lactating women.
4. Autoimmune disorders unrelated to thyroid dysfunction.
5. Use of drugs affecting Vitamin D metabolism.

Sample Size:

Based on prior prevalence data and assuming an odds ratio of 3, with 95% confidence interval and 80% power, the required sample size was calculated as 348 (174 cases and 174 controls) using an online statistical formula.

Data Collection:

A structured proforma recorded demographic details (age, sex, BMI, sunlight exposure, diet). Clinical examination and fasting venous blood samples were obtained for analysis.

Biochemical Analysis:

• Thyroid Function Tests: Serum TSH, FT4, and FT3 measured via chemiluminescence immunoassay.
• Vitamin D: Serum 25(OH)D levels assessed using ELISA. Categories: deficiency (<20 ng/mL), insufficiency (20–30 ng/mL), sufficiency (>30 ng/mL).
• Other Parameters: Serum calcium, phosphorus, and alkaline phosphatase to assess bone metabolism.

Statistical Analysis:

Data were analyzed using SPSS v20.0. Results were expressed as mean ± SD or percentages. Independent t-tests compared Vitamin D levels across groups. Pearson’s correlation assessed associations between Vitamin D and thyroid parameters. A p-value <0.05 was considered statistically significant.

Table 1- Demographic profile of patient’s cases and control

In the study, males comprised a slightly higher proportion in both groups (55.17% cases vs. 52.30% controls). The age distribution differed significantly, with older participants (61–65 years) in the control group (15.52%) compared to cases (3.45%) (χ²=13.53, p=0.0002). Urban residence was more common in both groups, with no significant difference (p=0.4481). Socioeconomic status showed a significant disparity, with more controls from the low-income group (61.49%) compared to cases (33.33%) (p=0.0001). (Table-1)

Table 2- Anthropometry distribution of patients of case and control

In the study, the mean BMI was significantly higher in hypothyroid cases (25.69 ± 2.25) compared to controls (23.62 ± 3.31) with a p value of 0.0001. Serum Vitamin D levels were significantly lower in cases (21.40 ± 4.45 ng/mL) than in controls (30.41 ± 4.54 ng/mL), also statistically significant (p=0.0001). However, mean fasting glucose levels showed no significant difference between cases (104.48 ± 5.86 mg/dL) and controls (103.03 ± 16.44 mg/dL) (p=0.2739).

Table 3- association between different clinical parameters of case and control

The comparative analysis of thyroid profiles between the two groups revealed significant alterations in hypothyroid patients. Cases showed markedly lower mean T3 (1.16 ± 0.38 pg/mL) and T4 (6.54 ± 5.23 µg/dL) levels, alongside significantly elevated TSH levels (49.40 ± 29.54 µIU/mL) compared to controls (T3: 1.72 ± 0.48, T4: 7.91 ± 2.08, TSH: 2.70 ± 2.32), confirming the diagnosis of overt and subclinical hypothyroidism (p<0.001 for all). Renal function tests showed that serum urea (20.79 ± 6.49 mg/dL) and creatinine (0.97 ± 0.20 mg/dL) were significantly elevated in hypothyroid patients as compared to controls (13.59 ± 1.16 and 0.75 ± 0.12, respectively), indicating a potential impact of thyroid dysfunction on renal physiology (p=0.0001). Lipid profile analysis demonstrated significantly higher levels of total cholesterol (200.80 ± 46.74 mg/dL) and triglycerides (198.65 ± 93.91 mg/dL) in hypothyroid cases compared to controls (127.53 ± 15.94 and 148.04 ± 11.96, respectively), supporting the association of hypothyroidism with dyslipidemia. HDL levels were slightly lower in cases but statistically significant (p=0.0001). Serum calcium and phosphorus levels were also found to be significantly elevated in cases, suggesting possible alterations in bone and mineral metabolism associated with thyroid dysfunction. These findings underscore the systemic impact of hypothyroidism on multiple biochemical parameters.

Table 4- Comparison of Vitamin D Status in Overt and Subclinical Hypothyroid Patients (N=348)

Among patients with overt hypothyroidism, the majority (64.37%) were found to have Vitamin D deficiency, while only 7.47% had sufficient levels. In contrast, subclinical hypothyroid patients showed a significantly higher proportion (40.23%) with sufficient Vitamin D levels, and fewer (21.84%) were deficient. The difference in Vitamin D distribution between the two groups was statistically significant (p < 0.0001), suggesting a strong association between lower Vitamin D levels and the severity of hypothyroidism. (Table 4)

Table 5- Pearson Correlation Coefficient between Vitamin D and Thyroid Profile Parameters (N=348)

There was a moderate to strong negative correlation between serum Vitamin D and TSH levels (r = –0.62, p < 0.0001), indicating that as TSH levels increased (worsening hypothyroidism), Vitamin D levels tended to decrease. Conversely, positive correlations were observed between Vitamin D and both T3 (r = +0.44) and T4 (r = +0.36), suggesting that better thyroid hormone levels were associated with higher Vitamin D levels.

These findings support the hypothesis that Vitamin D deficiency is more prevalent in patients with worsening thyroid dysfunction, particularly overt hypothyroidism.

In the present study, males slightly outnumbered females in both overt (55.17%) and subclinical (52.30%) hypothyroid groups, differing from common trends where females predominate. Age-wise, the 61–65 years group showed a significantly higher proportion in subclinical cases (15.52%) compared to overt cases (3.45%) (p=0.0002), indicating age-related increase in subclinical hypothyroidism. No significant difference was found in urban-rural distribution (p=0.4481). Socioeconomic status revealed a higher proportion of low-income individuals among subclinical controls (61.49%) versus overt cases (33.33%) (p=0.0001), suggesting socioeconomic factors influence disease detection and care (Olmos et al., 2015)⁷.

association between different clinical parameters of case and control

The comparative analysis between cases and controls shows significant differences across various clinical parameters. Mean T3 levels were significantly lower in cases (1.16±0.38ng/mL) compared to controls (1.72±0.48ng/mL; p=0.0001), consistent with findings reported by Suma Lingaraju et al. (2021), who noted decreased T3 levels in post-menopausal women with thyroid dysfunction ⁸. Similarly, the mean T4 levels in cases (6.54±5.23µg/dL) were significantly lower than in controls (7.91±2.08µg/dL; p=0.0014), which also aligns with the study by Lingaraju et al. (2021) ⁸. TSH levels were markedly elevated in cases (49.40±29.54µIU/mL) compared to controls (2.70±2.32µIU/mL; p=0.0001), reinforcing the presence of hypothyroidism, as also noted by Lingaraju et al. ⁸.

Renal function parameters showed significant elevations in the case group, with serum urea levels being 20.79±6.49mg/dL in cases versus 13.59±1.16mg/dL in controls (p=0.0001), and serum creatinine levels of 0.97±0.20mg/dL in cases compared to 0.75±0.12mg/dL in controls (p=0.0001). These findings are supported by Pipliwal et al. (2017), who reported elevated urea and creatinine in subjects with impaired renal function ⁹.

Lipid profile alterations were also evident in cases, with total cholesterol being significantly higher (200.80±46.74mg/dL) compared to controls (127.53±15.94mg/dL; p=0.0001), and triglyceride levels similarly elevated (198.65±93.91mg/dL in cases vs 148.04±11.96mg/dL in controls; p=0.0001). These findings mirror the results of Pipliwal et al. (2017), who also reported dyslipidemia in hypothyroid patients ⁹. The HDL levels were slightly lower in cases (47.86±4.72mg/dL) compared to controls (48.34±3.20mg/dL; p=0.0001), a pattern similarly described by Pipliwal et al. ⁹. Additionally, Wu et al. (2023) noted that elevated triglyceride to HDL cholesterol ratios can be an early indicator of metabolic abnormalities ¹⁰.

Mineral metabolism parameters showed that serum calcium levels were slightly higher in cases (9.19±3.74mg/dL) compared to controls (8.54±1.48mg/dL; p=0.033), which aligns with findings by Madeo et al. (2018), who emphasized the relevance of calcium levels in endocrine disorders ¹¹. Serum phosphorus levels were also significantly increased in cases (4.11±1.01mg/dL) compared to controls (3.41±1.21mg/dL; p=0.001), supporting previous research by Pipliwal et al. ⁹.

In the present study, hypothyroid patients showed significantly lower serum vitamin D levels (21.40±4.45ng/mL) compared to controls (30.41±4.54ng/mL; P=0.0001), supporting the association between vitamin D deficiency and hypothyroidism. This finding aligns with Ahi et al. (2020)¹², who observed significantly reduced vitamin D levels in both autoimmune and non-autoimmune hypothyroid patients in Iran. Similarly, Appunni et al. (2021)¹³ reported that vitamin D deficiency increased the risk of hypothyroidism. A study in Bihar, India, also found lower vitamin D levels in hypothyroid patients (23.57±9.77ng/mL) than controls (31.20±10.23ng/mL), along with a negative correlation with TSH levels. Tamang et al. (2023)¹⁴ confirmed significantly lower median vitamin D in hypothyroid individuals. However, Aljohani et al. (2017)¹⁵ in Saudi Arabia reported no significant difference between groups, suggesting population-specific variation. Overall, these findings emphasize the need to assess vitamin D status in hypothyroid patients due to its potential role in thyroid health.

In the present study, vitamin D deficiency (<20ng/mL) was significantly more prevalent in overt hypothyroid patients (64.37%) compared to those with subclinical hypothyroidism (21.84%), while sufficient levels (>30ng/mL) were seen in only 7.47% of overt cases versus 40.23% in subclinical ones. This inverse association between vitamin D levels and hypothyroid severity is consistent with findings by Goswami et al. (2008)¹⁶, who reported widespread vitamin D deficiency among Indian hypothyroid patients. Tamer et al. (2011)¹⁷ also observed high deficiency rates in autoimmune thyroid cases, suggesting a possible immunomodulatory role of vitamin D. Similarly, Chailurkit et al. (2013)¹⁸ found lower vitamin D associated with elevated TSH levels, supporting a physiological link.

However, Aljohani et al. (2017)¹⁵ reported no significant difference in vitamin D levels between hypothyroid patients and controls in a Saudi cohort, indicating that the association may vary by population or other contextual factors. Despite this, most evidence supports routine vitamin D evaluation in hypothyroidism management.

In the present study the Pearson correlation analysis revealed a moderate to strong negative correlation between Vitamin D and TSH levels (r = –0.62, p < 0.0001), indicating that lower Vitamin D levels are associated with higher TSH. Conversely, Vitamin D showed a moderate positive correlation with T3 (r = +0.44) and a weak to moderate positive correlation with T4 (r = +0.36), both statistically significant.

This study highlights a significant association between Vitamin D deficiency and hypothyroidism, particularly in patients with overt and subclinical hypothyroidism. A moderate to strong negative correlation between Vitamin D and TSH levels suggests that declining Vitamin D status may be linked with increased thyroid dysfunction. Additionally, the positive correlations of Vitamin D with T3 and T4 levels support its potential role in maintaining normal thyroid hormone balance. The findings underscore the importance of routine screening for Vitamin D deficiency in hypothyroid patients and suggest that Vitamin D supplementation could be considered as an adjunct in the management of thyroid disorders. Further longitudinal and interventional studies are recommended to establish causality and evaluate the therapeutic impact of correcting Vitamin D deficiency on thyroid function.

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