Objective

Researchers have long been captivated by the complex molecular interactions between vitamin D and the thyroid gland. Hypothyroidism affects 2% to 4% of women of reproductive age and can impact fertility through anovulatory cycles, luteal phase defects, hyperprolactinemia, and sex hormone imbalances. This study investigated the relationship between thyroid disease and the severity of vitamin D deficiency across different age groups.

Methods

A retrospective study was conducted of 286 patient samples from individuals aged 18 to 60 years who were processed in the clinical biochemistry laboratory of our hospital. Samples were tested for thyroid-stimulating hormone (TSH) and vitamin D (specifically, vitamin D3) levels. The study samples were categorized into four clinically relevant groups based on TSH levels and into three groups based on serum 25-hydroxyvitamin D (25(OH)D) levels.

Results

Most of the samples were from female patients (n=269), and the most common age group was 18 to 35 years (n=191, 66.78%). Subclinical hypothyroidism was identified in 120 patients, while vitamin D deficiency was present in 237 (82.87%) participants. A significant association was observed between vitamin D deficiency and the presence of thyroid disorders. Additionally, a significant negative correlation was found between TSH and vitamin D levels. Polycystic ovary syndrome was noted in 103 female patients (36.01%).

Conclusion

TSH and 25(OH)D levels should be screened in all women of reproductive age, not just those in high-risk groups, as subclinical and occult hypothyroidism may otherwise go undiagnosed. Furthermore, TSH should be considered the primary screening test.

Keywords: Calcifediol, Hypothyroidism, Infertility, Thyrotropin

Introduction

Vitamin D is known for its favorable effects across various systems, attributable to its pleiotropic properties and ubiquitous receptor expression. The complex molecular interactions between vitamin D and the thyroid gland have long captivated researchers. Consequently, efforts have been made to elucidate the role of vitamin D in thyroid disorders, which stand out from other conditions in their ease of diagnosis and accessibility of treatment.

Thyroid function disorders are prevalent worldwide and represent the most common endocrine diseases in India. The prevalence rates of overt hypothyroidism and subclinical hypothyroidism are approximately 4%–5% [1,2] and 4%–15%, respectively, in the developed world [1,3]. The reported prevalence of thyroid disorders in India varies widely, ranging from 15% to 25% [4-6]. Specifically, the prevalence of hypothyroidism in India is 11%, in contrast to 2% in the United Kingdom and 4.6% in the United States. Additionally, the prevalence rate of vitamin D deficiency is 41.6% [7].

The purpose of this study was to examine the relationship between thyroid-stimulating hormone (TSH) levels and the severity of vitamin D deficiency. Our hospital’s clinical biochemistry laboratory frequently receives samples for thyroid hormone and vitamin D testing, particularly from female patients of reproductive age who are referred by the gynecology outpatient department. Addressing thyroid disorders and vitamin D deficiency in this population could meaningfully contribute to the prevention of congenital hypothyroidism and reduce the rate of recurrent pregnancy loss. Notably, vitamin D and TSH both interact with similar types of receptors, known as steroid hormone receptors, making the exploration of their correlation particularly relevant. This study included participants of both sexes and various types of thyroid disorders; this is because limited data are available on hyperthyroidism, and we sought to investigate any potential sex-based associations. Therefore, we initiated this study with the following objectives: (1) to analyze the association between thyroid disease and the severity of vitamin D deficiency across several age groups; (2) to determine the statistical correlation between thyroid disorders and vitamin D (specifically, vitamin D3) levels, particularly among those of reproductive age; and (3) to explore statistical correlations between vitamin D levels, thyroid disorders, other concomitant diseases, and TSH levels.

Methods

1. Study design

1. Study design

This hospital-based retrospective analytical study was conducted in the clinical laboratory of the Department of Biochemistry at Hindu Rao Hospital, a 900-bed tertiary care facility in New Delhi. The study received approval from the institutional ethical review committee, under reference number F.No: IEC/NDMC/2022/130, dated 17/06/2022. Written informed consent by the patients was waived due to a retrospective nature of our study.

2. Sample selection and sample size

2. Sample selection and sample size

The study group comprised patients who were referred to the laboratory for thyroid hormone and vitamin D measurements. Demographic parameters were extracted from the requisition forms submitted by the patients. A simple random sampling technique was employed to select laboratory records from both male and female outpatients aged 18 to 60 years.

1) Sample size

1) Sample size

Given an estimated prevalence of thyroid disorders of 11% (based on a similar study population), an absolute precision of 5%, and a 95% confidence level, a sample size of 150 was required.

2) Exclusion criteria

2) Exclusion criteria

Pregnant women, those younger than 18 years, and those older than 60 years were excluded from the study.

3. Data collection

3. Data collection

The study included TSH and vitamin D levels from 286 patient samples, comprising 17 male and 269 female patients. Blood samples were collected on the same day for both thyroid hormone and vitamin D measurements. Thyroid hormone levels were determined using an electrochemiluminescence immunoassay analyzer (Cobas 411; Roche Diagnostics), while vitamin D levels were measured with the immunoturbidimetric method on a SYS1300 device (DiaSys Diagnostics India Pvt Ltd.), utilizing specific kits. The study samples were categorized into four groups based on their TSH levels: overt hypothyroidism, with TSH levels greater than 10 IU/mL; hyperthyroidism, with TSH levels less than 0.4 IU/mL; subclinical hypothyroidism, with TSH levels ranging from 4.2 to 10 IU/mL; and normal thyroid function, with TSH levels from 0.4 to 4.2 IU/mL [8]. Circulating vitamin D status was assessed by measuring serum 25-hydroxyvitamin D (25(OH)D) levels. Based on the Endocrine Society Clinical Practice Guidelines [9], participants were classified into three clinically relevant vitamin D categories: optimal (≥30 ng/mL), intermediate (20 to <30 ng/mL), and deficient (<20 ng/mL). For analytical purposes, participants were also grouped by age to examine the prevalence of thyroid disorders across sex and age demographics, with age groups of 18–35, 36–45, and 46–60 years.

4. Statistical analysis

4. Statistical analysis

Categorical variables were presented as counts and percentages (%). Normally distributed quantitative data were expressed as means±standard deviations. Data normality was assessed using the Kolmogorov-Smirnov test, and nonparametric tests were employed for data that did not follow a normal distribution. The associations between variables that were quantitative and normally distributed were analyzed using analysis of variance. The associations between qualitative variables were analyzed using the chi-square test. When any cell had an expected count of less than 5, the Fisher exact test was employed instead. The Spearman rank correlation coefficient was used to evaluate the correlation between vitamin D levels (measured in ng/mL) and TSH concentrations (assessed in mIU/mL). The data were entered into a Microsoft Excel spreadsheet (Microsoft Corp.), and the final analysis was conducted using SPSS version 25.0 (IBM Corp.). A p-value of less than 0.05 was considered to indicate statistical significance.

Results

A total of 286 participants were enrolled in the study based on laboratory data. Of these, 269 (94.06%) were female (Table 1, Figure 1). The mean age of the participants was 32.92 years, ranging from 18 to 60 years. Most patients fell within the 18 to 35 year age group (n=191, 66.78%). Regarding concomitant diseases, hypertension and diabetes mellitus were present in 19 participants (6.64%). Eighteen participants (6.29%) had a history of thyroid dysfunction, including thyroid surgery. Hyperthyroidism was observed in five patients (1.75%), subclinical hypothyroidism in 120 patients (41.96%), and overt hypothyroidism in 36 patients (12.59%). Polycystic ovary syndrome (PCOS) was identified in 103 female patients (36.01%), while vitamin D deficiency was noted in 237 participants (82.87%). The mean TSH level among all participants was 6.05 mIU/L, and the mean vitamin D level was 10.72 ng/mL.

Table 2, Figure 2 presents the association between demographic characteristics and thyroid disorders as determined by the Fisher exact test. The data indicate no statistically significant association between sex and the different types of thyroid disorders (p=0.63). Within the female subgroup, 95% of those with subclinical hypothyroidism and 100% of those with hyperthyroidism s were found to have severe vitamin D deficiency. For male and female participants combined, the mean age difference across various thyroid disorders was not statistically significant (p=0.843). However, a statistically significant difference was observed in the severity of vitamin D deficiency among the various thyroid conditions (p<0.0001).

The associations between vitamin D levels and the presence or absence of thyroid disorders are shown in Table 3, Figure 3. The data are presented as counts and percentages for each category. The p-value indicates a significant association between vitamin D deficiency (levels below 20 ng/mL) and the presence of thyroid disorders. Percentages in each cell of the table reflect the distribution of cases according to the intersection of vitamin D levels and thyroid disorder status.

Table 4, Figure 4 indicates significant differences (p<0.0001) in the distribution of thyroid conditions between patients with and without PCOS. This low p-value suggests a strong association between PCOS and the presence of thyroid dysfunction.

The relationship between PCOS and vitamin D deficiency, focusing on vitamin D levels, is presented in Table 5, Figure 5. The extremely low p-value of 0.0001 indicates a highly significant correlation between PCOS and vitamin D deficiency. A considerable percentage of individuals with PCOS, approximately 40.93%, were found to be vitamin D deficient, in contrast to a mere 12.24% who were not deficient. This suggests that individuals with PCOS have a higher likelihood of experiencing vitamin D deficiency compared to those without the condition.

Table 6, Figure 6 presents a negative correlation coefficient of −0.501, indicating a moderate to strong inverse relationship between vitamin D levels and TSH levels. This suggests that as vitamin D levels rise, TSH levels tend to fall, and vice versa. The extremely small p-value (<0.0001) signifies that the correlation is highly statistically significant.

Discussion

In our study, we discovered that women within the reproductive age range of 18 to 35 years were most strongly affected by thyroid disorders, including subclinical hypothyroidism. Furthermore, we observed that 82.87% of patients with thyroid disorders had concomitant vitamin D deficiency. Additionally, 46.69% of patients with thyroid disorders were diagnosed with PCOS, and among these cases, 40.93% were found to be severely deficient in vitamin D. A unique finding of our study was the correlation between the presence of PCOS and the levels of both TSH and vitamin D.

Many studies have established a connection between serum 25(OH)D levels and the levels of TSH, thyroid hormones, and antithyroid antibodies. Research focusing on the impact of vitamin D (cholecalciferol) supplementation on thyroid function has been primarily conducted in patients with autoimmune thyroid diseases. Significant reductions in thyroid peroxidase antibodies, thyroglobulin antibodies, TSH, thyroid hormone, and thyroglobulin levels were observed in participants after 12 months of vitamin D supplementation [10].

Thyroid diseases are highly prevalent among women of reproductive age, encompassing conditions such as chronic thyroiditis, thyroid dysfunctions, Hashimoto thyroiditis, and Graves’ disease. Investigating the factors that affect thyroid function tests is of considerable interest [11,12]. Therefore, in cases of both immune and nonimmune thyroid diseases, the vitamin D level should be taken into consideration [13,14].

Several studies have indicated that vitamin D deficiency and impaired thyroid function can lead to a range of adverse outcomes during pregnancy. These include gestational hypertension, preeclampsia, preterm delivery, and intellectual and neurological developmental disorders in children, among others [15]. Consequently, maintaining optimal vitamin D levels is crucial for a healthy pregnancy, normal fetal skeletal development, and the prevention of preeclampsia, all of which help ensure the well-being of the fetus [16,17]. Additionally, hypothyroidism is prevalent among pregnant women. If screening is limited to high-risk groups, approximately 25% of pregnant women with subclinical or occult hypothyroidism may go undiagnosed [18].

Despite growing criticism, TSH testing remains the best—and often the only—thyroid function test required for evaluating most patients [19]. Verma et al. [20] presented data suggesting that even slight variations in TSH levels and those on the borderline of clinical hypothyroidism—such as, 4–5, 5–6, and >6.0 μIU/mL—should not be overlooked in infertile women who are otherwise asymptomatic for clinical hypothyroidism. Undiagnosed and untreated thyroid dysfunction is a prevalent cause of infertility, yet it can be readily addressed by normalizing thyroid hormone levels. Among 394 women with infertility, 23.9% were found to be hypothyroid, with TSH levels exceeding 4.2 μIU/mL. Following treatment for hypothyroidism, 76.6% of these women conceived within a period ranging from 6 weeks to 1 year. Additionally, women with infertility who displayed both hypothyroidism and hyperprolactinemia responded well to treatment, resulting in the normalization of prolactin levels [20]. In another study, 20 participants (16%) were observed to have thyroid dysfunction associated with infertility. The most common thyroid dysfunctions were overt hypothyroidism (9.6%) and subclinical hypothyroidism (4.0%), with a particularly high prevalence of dysfunction in cases of secondary infertility (21.8%). Therefore, thyroid function evaluation, particularly serum TSH measurement, should be included as a routine part of the infertility assessment protocol, especially in cases of secondary infertility [21]. Similarly, our study determined the thyroid disorder status in individuals of reproductive age by assessing their TSH levels. Furthermore, the high prevalence of subclinical hypothyroidism observed in our study aligns with findings from prior research.

Arojoki et al. [22] reported that the prevalence of abnormal TSH levels was highest in patients with ovulatory dysfunction (6.3%) and unknown infertility (4.8%) and lowest in those with tubal infertility (2.6%) and male infertility (1.5%), although no statistically significant differences observed. Notably, 67% of women with elevated TSH levels presented with oligo/amenorrhea, underscoring the importance of TSH screening in these patient populations [22].

Several cross-sectional and case-control studies have shown that patients with PCOS have lower levels of vitamin D than controls [23]. Research by Bindayel [24] found that individuals with PCOS not only had significantly lower vitamin D levels (p<0.05) but also a notably higher obesity rate (p<0.05) and significantly increased serum triglyceride levels compared to the control group [25]. Vitamin D deficiency should be considered an additional risk factor for the development of PCOS [26,27]. Our data further support a strong association between PCOS and vitamin D deficiency, as evidenced by a p-value of 0.0001; this indicates a highly significant correlation between PCOS and a deficiency in vitamin D.

In conclusion, primary hypothyroidism is one of the most prevalent endocrine disorders that primary care providers encounter and manage. These providers must depend on biochemical testing to confirm or exclude a diagnosis of hypothyroidism. Despite increasing criticism, TSH remains the best—and frequently the only—thyroid function test required for evaluating most patients. Like severe vitamin D deficiency, undiagnosed and untreated thyroid dysfunction frequently causes infertility, particularly secondary infertility. However, this issue can be readily addressed by correcting the levels of both thyroid hormones and vitamin D. It is essential to screen all patients with thyroid disorders for 25(OH)D deficiency and to initiate appropriate replacement therapy to avert adverse outcomes. Therefore, TSH and vitamin D laboratory assessments should be considered important and mandatory screening tests for women of reproductive age.

We anticipate that this research will provide the benefits to clinicians to conduct early screening for thyroid disorders and vitamin D deficiency in female patients of reproductive age and to support the advisability of vitamin D supplementation for all individuals of reproductive age as well as patients with thyroid disorders. Though conducting this study had some limitations which may be taken up in future research like markers of autoimmunity (antithyroid peroxidase) were not tested and the target population was assumed to have sufficient iodine levels.

Notes

Conflict of interest

No potential conflict of interest relevant to this article was reported.

Author contributions

Conceptualization: MM. Methodology: PG. Formal analysis: MM, PG. Data curation: MM. Project administration: PG. Visualization: MM. Software: PG. Validation: MM, PG. Investigation: MM. Writing-original draft: MM. Writing-review & editing: PG. Approval of final manuscript: MM, PG.

Acknowledgments

We extend our gratitude to Dr. Rajiv Ranjan, Dr. Kamlesh, Dr. Manu, and Dr. Bhawna for their contributions to data acquisition and for their moral support.

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