Prevalence of Thyrotropin Receptor Antibodies and Clinical Profile of Patients with Alopecia Areata: A Cross-Sectional Study

Alopecia areata (AA) is the most common type of autoimmune-related non-scarring hair loss with an annual prevalence of 0.1–0.2% and lifetime incidence of 2% [1]. It affects not only scalp hair but also facial and body hair and is associated with an adverse psychosocial sequelae [2]. Nails can be also affected in more severe cases. Even though the disease is not life threatening, it is associated with several other immune-mediated conditions, such as atopy, autoimmune thyroid disease (AITD), vitiligo, type 1 diabetes, rheumatoid disease, and others [1].

Disturbance in thyroid function is well documented in patients with AA. Thyroid disease is 2 to 3 times more frequent in AA patients compared to the general population [3‒5]. Its incidence is proportional to the severity of AA [4, 6]. The most common dysfunction is subclinical hypothyroidism, followed by subclinical hyperthyroidism, Grave’s disease, and Hashimoto thyroiditis [7]. Even patients with normal thyroid function can present with abnormal levels of thyroid-related autoantibodies. According to a recent case-control study, abnormal concentrations of thyroglobulin antibodies (TgAbs) and thyroid peroxidase antibodies (TPOAbs) were found in 29% of patients, which was 1.4–3 times higher than in healthy controls [4].

Thyrotropin receptor is present on the cell membrane of thyroid follicular cells (thyrocytes) and plays a role in thyroid hormone signaling. Thyrotropin receptor is the target of thyrotropin receptor antibodies (TRAbs) [8]. These antibodies are used in diagnostic tests for hyperthyroidism because they are highly specific and sensitive for Grave’s disease [9]. Abnormal levels can be detected in around 1–2% of healthy individuals [10]. Compared to TgAb and TPOAb, the number of studies measuring TRAb levels in patients with AA is limited [11, 12]. In a recent study, it was discovered that 43% of AA patients exhibited elevated concentrations of TRAb, which was significantly higher compared to the control group, where only 1.2% had elevated TRAb levels [4]. Abnormal TRAb titers were most frequently present in patients with more severe AA but not necessarily with clinically evident thyroid dysfunction. Only 11% of participants were diagnosed with AITD at the point of study. The purpose of this cross-sectional study was to investigate the association between AA and TRAb levels and to assess whether they are correlated with abnormal levels of thyroid hormones and other thyroid antibodies, as well as any relevant variables in the medical history of AA patients.

This cross-sectional study took place at the outpatient department of a university hospital. Patient recruitment occurred between October 2021 and October 2022, with an extension until February 2023 to ensure an adequate sample size. All patients signed an informed consent before their participation to the study. In patients younger than 18 years of age, the informed consent was signed by their accompanying parent, legal guardian, or next of kin to participate in the study. The study was approved by the Institutional Review Board (Ethics Committee) of our hospital (EKVP/56l0l2021), conducted following the STROBE guidelines, and registered with ClinicalTrials.gov as part of a larger study (NCT05098600).

We screened all patients, adults, and children above 12 years of age, with newly presenting, relapsing, or treatment-resistant AA (no hair growth for 6 months) presenting to our outpatient department. Patients with disease in remission at the time of the study, concomitant presence of other types of alopecia (such as androgenetic alopecia stage III and above in Norwood-Hamilton scale or stage II and above according to Ludwig classification, telogen effluvium, trichotilomania, congenital alopecia, tinea capitis, syphilis of the scalp, scarring alopecias), patients opting out of data sharing or refusing to take blood tests and participate in the study were not included.

Because of the lack of epidemiological studies on AA in our region, we assumed a 0.1% annual prevalence, based on population studies from other countries [1]. Using a desired margin of error of 5% and a confidence level of 95%, we calculated a sample size of 139 individuals for this study.

Data were collected from participants during the clinical examination and from the hospital database. Age, gender, place of residence, personal history, medications taken, known allergies, severity and clinical type of alopecia were recorded. The extent of alopecia was measured using the Severity of Alopecia Tool (SALT) [13]. The presence or absence of eyebrows, eyelashes, and body hair was also recorded. According to their SALT score, patients were stratified into the following groups: S1 (0%–24%), S2 (25%–49%), S3 (50%–74%), S4 (75%–99%), and S5 (100%). Blood tests for thyroid function (TSH, FT4, FT3) and antibody activity (TPOAb, TgAb, TRAb) were taken at the end of clinical examination, between 9:00 a.m. and 11:00 a.m. All blood tests were taken on an empty stomach and patients on thyroxine supplementation were instructed to skip their dose on the day of the examination. Those who could not complete the blood tests on the day of the examination were required to visit our hospital by the end of the week between 8:00 a.m. and 11:00 a.m.

All blood analyses were performed at the central laboratory of our hospital. Hormones and autoantibodies were measured using automated immunoassay and clinical chemistry analyzers (Atellica solution Immunoassay, Siemens, Germany, and Cobas e4111 analyzer, Roche diagnostics, Switzerland). In adults older than 20 years, reference ranges for thyrotropin (TSH), free thyroxine (FT4), and free tri-iodothyronine (FT3) were set at 0.55–4.78 mIU/L, 11.5–22.7 pmol/L, and 3.5–6.5 pmol/L, respectively [14]. In patients between 12 and 20 years of age, reference ranges for TSH, FT4, and FT3 were set at 0.48–4.17 mIU/L, 10.7–18.4 pmol/L, and 4.7–7.2 pmol/L, respectively [14]. According to the values set by the manufacturer, levels of TPOAb, TgAb, and TRAb were considered abnormal when they were higher than or equal to 60 kU/L, 4.5 kU/L, and 1.22 U/L, respectively.

Descriptive statistics were used to describe patient characteristics; frequencies and percentages were provided for categorical variables. Quantitative variables were represented as mean and standard deviation for continuous variables or median and interquartile range for non-parametric continuous variables. Proportion test and χ² test were used to compare the prevalence. Comparison of the dependence of very severe AA (S4 and S5 grades) on thyroid function and antibody activity was based on a logistic regression model. The statistical significance level was set to 0.05. All statistical analyses were conducted using Microsoft Excel and IBM SPSS Statistics (version 24.0,IBM Corp.; Armonk, NY, USA).

A total of 139 patients, 97 females and 42 males, were included in the study. An overview of the epidemiological and clinical features of the study cohort is given in Table 1. The average age of onset of AA was 28.86 ± 15.88 years, median 26 years. The average patient age was 35.77 ± 15.18 years, median 36 years. Most patients presented with one (alopecia unilocularis, 38.1%), or multiple (alopecia multilocularis, 20.9%) hairless patches. Total scalp hair loss occurred in 22 patients with 16 of them reporting complete loss of facial and body hair. Around 36.7% of patients appeared with clinically severe or very severe alopecia, defined as a SALT score greater or equal to 50%. One-third of patients (30.9%) were affected by partial or full eyebrow loss.

The prevalence of atopic diathesis and AITD was 33.8% and 15.1%, respectively (online suppl. Table 1; for all online suppl. material, see https://doi.org/10.1159/000540220). AITD and thyroxine supplementation were significantly more common in females than males (20.6% vs. 2.4%, p = 0.006, and 18.6% vs. 2.4%, p = 0.011). Family history of AA was reported by 11.5% of patients and was more frequent in males than females (19.0% vs. 8.2%, p = 0.067).

Values of thyroid hormones and autoantibodies of all our patients together with the descriptive statistics are presented in supplementary Dataset (online suppl. Table 3, 4). Mean TSH value in our sample was 2.39 ± 2.74 mIU/L. The lowest titers were observed in S2 group (1.99 ± 1.34 mIU/L) and the highest in S4 group (3.36 ± 5.30 mIU/L) with difference between males (1.36 ± 0.95 mIU/L) and females (4.24 ± 6.15 mIU/L). Fifteen patients (11.0%) had abnormal TSH values (online suppl. Table 1). Twenty-four patients (17.3%) had elevated TPOAb values, twenty-one (15.1%) had elevated TgAb values, and thirty-five (25.2%) had elevated TRAb values.

Concerning sex differences, elevated levels of TPOAb were present in 20.6% of female patients, whereas in male patients, this figure was lower at 9.5%. Similarly, when examining TgAb levels, we found that 19.6% of female patients had elevated levels, compared to only 4.8% of male patients. Interestingly, the trends were different for TRAb, with titers being higher in almost 43% of males and only higher in 17.5% of females. In total, mean TRAb values were 1.49 ± 2.43 U/L and 1.04 ± 0.33U/L for males and females, respectively.

As for alopecia severity, the highest percentage of patients with abnormal thyroid autoantibodies was observed in S4 group (Table 2). Six patients (37.5%) had elevated TPOAb values, four (25.0%) had abnormal TbAb values, and eight (50.0%) had elevated TRAb titers. Around 40% of patients with elevated values of TPOAb, TgAb, or TRAb autoantibodies had alopecia severity S4 and S5.

We tested a null hypothesis that the prevalence of TRAbs in patients with AA is not different to the general population (1–2% of healthy controls) [9]. The prevalence in our sample was 25.2%, which was significantly higher than the general population (1–2%) (p < 0.001).

When comparing patients based on TRAb titers, we observed notable dichotomies (as shown in online suppl. Table 2). The distribution of gender was significantly different between the two groups. The majority (76.9%) of patients with low TRAb levels were females, whereas high TRAb titers were detected in over 40% of males (18 out of 42) (p = 0.002). Elevated TRAb concentrations were not associated with SALT score (p = 0.202) or TSH (p = 0.625), FT4 (p = 0.986), FT3 (p = 0.393), TPOAb (p = 0.621), or TgAb (p = 0.984) levels. The results of the logistic regression model showed that elevated TRAb levels increased the odds of S4 or S5 grade alopecia with OR = 2.31, 95% CI = 0.958–5.568, p = 0.062. The dependence of the logarithm of TRAb on gender and SALT score is shown in online supplementary Figure 1.

In our study, 1 out of 5 patients presented with abnormally elevated thyroid-specific antibodies. Among our study population, TRAb was the most frequently elevated, especially in males. High TPOAb and TgAb concentrations were more prevalent in female patients. Four out of 10 patients with abnormal concentrations of autoantibodies had very severe or total alopecia (SALT ≥75).

Our findings are in accordance with other studies. In a cross-sectional study by Kasumagic-Halilovic, 18 out of 70 AA patients (25.7%) tested positive for one or both TPOAb and TgAb [15]. Presence of antibodies was not associated with disease severity. On the contrary, in a case-control study by Bin Saif abnormal concentrations of TPOAbs were found in 40% of patients with severe AA but in only 4% of those with mild AA [16]. Ten out of 24 patients with elevated TPOAbs (41.7%) were found to be in the S4 and S5 AA groups; however, we cannot make any direct comparison because Bin Saif classified alopecia severity by the volume and number of hairless patches instead of using the SALT score. In a more recent case-control study by Gao et al. [17], TPOAb concentrations in blood, unlike TgAb, were significantly higher in AA patients compared to their control group (17.26% vs. 3.45%). Their results for TPOAbs are similar to our findings (17.3%).

Unlike TPOAbs and TgAbs, the association of AA with TRAbs has been sparsely examined. A possible etiology is that TRAbs are not routinely tested unless there is persistent hyperthyroidism and suspicion for Graves’ disease [18]. In a case-control study by Noso et al. [4], abnormal TRAb titers were detected in 42.7% of AA patients, which is nearly two times higher than the TRAb prevalence in our study (25.2%). Most patients in their cohort were euthyroid, as were 89.0% of our patients. According to the authors, this finding suggests that TRAbs in euthyroid AA patients were either mostly neutral, or in insufficient amounts to alter thyroid function, or produced as a response to the release of extra-thyroidal TSH receptors from destroyed hair follicles.

Thyrotropin receptors are present on thyrocytes and in various tissues, including periorbital tissue, adipose tissue, bone, skeletal muscle, and skin [19]. The extra-thyroidal receptors contribute to the differentiation of fibroblasts and osteoblasts, lipolysis, and improved insulin sensitivity [19]. They also play a role in the extra-thyroidal manifestations of AITD, particularly Graves’ disease [8, 19]. In the skin, TSH receptors are detected in dermal fibroblasts, dermal papilla cells, epidermal keratinocytes, and melanocytes [19, 20]. Autoantibodies against TSH receptors on fibroblasts, keratinocytes, and melanocytes contribute to Graves’ dermopathy, hyperpigmentation, and vitiligo [19]. Antibodies do not normally target dermal papilla cells because of immune privilege [21]. In AA, the collapse of immune privilege exposes dermal papilla cells and their TSH receptors to the overactive immune system, potentially stimulating the production of TRAbs [20]. These antibodies could cross-react with TSH receptors on thyrocytes, leading to Graves’ disease.

Stimulating TRAbs are pathognomonic of Graves’ disease. It is well documented that patients with AA are prone to develop Graves’ disease and vice versa [22]. A recent systematic review by Lee et al. [11] reported a prevalence of 1.4% of Graves’ disease in AA patients. In our study, we did not collect data on the specific subtypes of AITD to avoid recall and information biases. Many patients were unable to recall their precise AITD diagnosis, and most endocrinology records did not specify the type of AITD. During our study, only 8.6% of AA patients with positive TRAbs had abnormal TSH values, and 11.4% had abnormal FT4 values, indicating that high TRAb titers in our patients did not result in clinical or subclinical hyperthyroidism, at the time of study.

Medications and procedures used to treat AITD can normalize thyroid function and reduce blood concentrations of thyroid autoantibodies potentially distorting the results of our study [23]. We included only patients who were taking treatments for thyroid dysfunction for at least 6 months and recorded their list of medications. No patient received anti-thyroid drugs, such as methimazole, carbimazole, propylthiuracil, and only 1 patient underwent total thyroidectomy in the past. Radioiodine treatment, which may increase TRAb concentration, was not reported by our patients [24]. Nineteen out of 21 patients with confirmed AITD were on long-term treatment with levothyroxine, which does not reduce TRAb levels [25]. The observed increase in TRAb titers was not affected by medications in our cohort.

Thyroid autoantibodies have also been detected in patients with other autoimmune diseases. Prevalence of TPOAbs and TgAbs was significantly higher than controls in patients with type 1 diabetes, vitiligo, myasthenia gravis, autoimmune liver diseases, and connective tissue autoimmune diseases [26, 27]. Abnormal TPOAb and TgAb concentrations have been also associated with atopic diseases [28]. TRAbs are mostly detected in AITD, particularly Graves’ disease (90%) but also Hashimoto thyroiditis [23]. TRAbs were also detected in patients with type 1 diabetes (18–20%) but were not significantly elevated in autoimmune connective tissue diseases, myasthenia gravis, and autoimmune hepatitis [26, 29, 30]. Because levels of thyroid hormones were not measured in most aforementioned studies, no conclusions could be drawn on the effect of high TRAb levels on thyroid function.

We divided our cohort into two groups based on TRAb positivity and compared them using recorded variables. There was a significant disparity in gender prevalence, with relatively more males being diagnosed with elevated TRAb concentrations (p = 0.002). Patients with high TRAb levels were also more frequently diagnosed with atopic diseases and AITD, but the differences were not statistically significant. In this group, abnormal TPOAb levels were also more common (20.3% vs. 16.3%, p = 0.621). Previous studies in patients with Graves’ disease have linked high TRAb levels to male gender and elevated TPOAb levels, which aligns with our findings [31]. However, only 3 of 35 patients in our study had abnormal TSH titers. Although high TRAbs are associated with AITD and Graves’ disease [23], only a small percentage of euthyroid patients with elevated TRAbs eventually develops Graves’ disease [32].

Despite Graves’ disease being more common in females [33], studies have shown that males with the condition often present with higher TRAb titers experience more severe disease and are more frequently diagnosed with additional autoimmune diseases [33]. Genetic and epigenetic factors, such as smoking and infections, contribute to heightened immune reactivity in males, leading to elevated TRAb levels and the manifestation of other autoimmune comorbidities [31, 34, 35]. These factors may explain the higher prevalence of males in the TRAb-positive group observed in our study.

External validity may also be affected by patient demographics, as only patients who visited our metropolitan hospital were recruited, potentially excluding those with mild AA and those living far from the capital. However, half of our participants presented with mild AA (S1), as our hospital includes a walk-in clinic, enabling patients to be examined without a referral from their family doctor. Furthermore, our hospital has a national influence and a well-known hair center that attracts patients from across the country. Despite this, the relative prevalence of alopecia totalis and alopecia universalis was found to be 3 times higher than that reported in a recent systematic review (15.8% vs. 5.2%) [36]. An inevitable recall bias exists when recording personal and family history, but we minimized this by cross-checking the reported information with hospital databases and records provided by general practitioners, when available. Our cross-sectional study design did not enable monitoring of changes in antibodies and AA activity over time, so we were unable to determine whether patients with TRAbs developed Graves’ disease in the future.

Our observational study found that a significant portion (25.2%) of patients with AA had positive TRAbs, with the majority (89.0%) being euthyroid at the time of study. Abnormal TRAbs were more frequent than other thyroid autoantibodies. The presence of high TRAbs was significantly associated with male gender but was not associated with alopecia severity, atopic diathesis, thyroid function, and presence of other thyroid autoantibodies. We believe that patients with AA, especially males, should be also tested for TRAbs and closely monitored for thyroid function, but further research is necessary to verify these findings and explore the potential clinical significance of TRAbs in AA.

We thank Adam Whitley from the department of general surgery, Third Faculty of Medicine, Charles University and University Hospital Královské Vinohrady, for his language review.

The study protocol was reviewed and approved by the Institutional Review Board of our hospital: Ethics Committee of the University Hospital Královské Vinohrady, Approval No. EKVP/56l0l2021. All patients signed an informed consent before their participation to the study. In patients younger than 18 years of age, the informed consent was signed by their accompanying parent, legal guardian, or next of kin to participate in the study.

The authors have no conflicts of interest to declare.

This study was not supported by any sponsor or funder.

A.J.S. was involved in study conception and design, in the acquisition, analysis, and interpretation of data, and drafting of the manuscript. A.F. was involved in the analysis and interpretation of data. A.F. and P.A. were involved in the critical revision of the manuscript.

The data underlying this article are available in the article and in its online supplementary material. Further inquiries can be directed to the corresponding author.