Introduction

Hashimoto’s thyroiditis (HT), also known as chronic lymphocytic thyroiditis, is one of the most common autoimmune disease worldwide1. It is diagnosed according to the presence of hypoechogenic structure of the thyroid gland in the ultrasonography, and elevated serum concentration of thyroglobulin antibody (TG-Ab) and/or thyroid peroxidase antibody (TPO-Ab)2. Women are more susceptible to the disease than men, with 7–10 times of incidence rate3. As the mistaken attacks on thyroid gland from body’s self-immune system, the thyroid parenchyma is infiltrated with lymphocytes, which can eventually lead to hypothyroidism4,5. Patients with hypothyroidism are usually treated with L-thyroxine (L-T4) replacement therapy, to restore normal levels of thyroid hormone (FT3 and FT4) and thyroid-stimulating hormone (TSH)6. However, no specific treatment exits to improve the immune dysfunction in HT patients and prevent further development of Hashimoto disease.

Patients with HT often report multiple symptoms, such as profound fatigue, memory loss, poor sleep quality, muscle and joint tenderness, dry eyes, and forgetfulness7,8,9. Some symptoms can be relieved by adequate thyroid hormone substitution, while some are persistent despite patients’ euthyroid status9. The persistent symptoms are hypothesized to be associated with autoimmune disease, rather than hypothyroidism10. However, to date, the evidences directly demonstrating the association between abnormally elevated thyroid antibodies in HT patients and their extrathyroidal manifestations are still limited.

Thus, in the present study, the clinical parameters, multiple symptoms and life quality were determined in HT patients who were kept in euthyroid status, as well as in healthy controls. In addition, the association between thyroid antibodies (TPO-Ab and TG-Ab) and inflammatory factors, multiple symptoms and life quality was analyzed to investigate the influence of elevated thyroid antibodies on HT patients.

Methods

Study design and population

A total of 108 patients with Hashimoto’s thyroiditis and 57 healthy subjects were enrolled for the present study. Among them, 51 HT patients and 22 healthy participants were recruited at Affiliated Hospital of Qingdao University in northern China, and the others were recruited at Zhejiang Chinese Medical University in southern China. The study protocol was approved by the Ethics Committee of Qingdao University (Approval Number: QDU-20201107-1). All methods were performed in accordance with relevant guidelines and regulations, as well as the Declaration of Helsinki. Written informed consent has been provided by each participant.

Key inclusion criteria for HT patients were as follows: (a) age between 18–60 years old; (b) diagnosed by at least one endocrinologist according to clinical parameters and color ultrasonography; (c) positive TG-Ab or positive TPO-Ab while in euthyroid status without hormone substitution. The healthy controls were recruited at the physical examination centers of Zhejiang Hospital and Affiliated Hospital of Qingdao University through a health-check program. The main inclusion criteria for the control group were: (a) do not suffer from any type of thyroid disease, such as goiter, hyperthyroidism, hypothyroidism and thyroid tumor; (b) match the age and gender with the patients. Key exclusion criteria, for both groups, included: (a) pregnancy or lactation; (b) alcohol addiction; (c) with other kind of autoimmune disease, such as rheumatoid arthritis or multiple sclerosis; (d) with serious chronic diseases, such as cancer or heart disease; (e) use of anti-inflammatories or immunosuppressant drugs. Demographic data of patients and healthy subjects were obtained with a face-to-face interview. Each participant provided written informed consent and received no financial compensation or gifts.

Blood collection and assessment of biochemical indicators

Fasting blood from participants were centrifuged with 3000 rpm in 4 °C for 10 min to separate serum and erythrocytes. Serum levels of TPO-Ab, TG-Ab, TSH, FT3 and FT4 were determined by chemiluminescent immunoassays (Siemens Healthcare Diagnostics, Munich, Germany). The levels of interleukin (IL)-4, IL-6, IL-10, IL-17A, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were determined by a flow cytometer (BD Biosciences, Franklin Lakes, USA). Serum biomarkers of liver and kidney function and lipid profiles were measured with an automatic analyzer (HITACHI 7020, Sysmex Kobe, Japan). T lymphocyte subpopulations were measured with blood samples by a flow cytometry (BD, USA). Laboratory assays were performed in duplicate to minimize analytical errors, and the presented values are the average of two measurements. All measurements were performed at the end of the study to minimize variability.

Survey of health-related life quality and severity of non-hypothyroid symptom

Health-related life quality was determined with Short Form 36 Health Survey (SF-36), which is made up of 36 questions11. The SF-36 has 8 domains: physical functioning, role physical, role emotional, bodily pain, general health, vitality, mental health and social functioning. The obtained score was transferred as (obtained score—the lowest possible score of the ___domain) / the difference between the highest possible score and the lowest possible score of the ___domain] × 100, so that the score of each ___domain is within (0,100) and comparably reflects health-related life quality. The higher of the score in each ___domain, the better of health status7,12.

Severity of multiple extrathyroidal manifestations was determined with the Hashimoto’s Thyroiditis Symptom Questionnaire. It has 8 domains, related to digestive, respiratory, circulatory, endocrine, neuropsychiatric, movement, reproductive and mucocutaneous system, respectively. Totally, the questionnaire is made up of 49 items, each of which need to be answered with score from 0 to 10 (0 means no symptom, 10 means the symptom seriously affect life quality).

Statistical analysis

Data are described as means ± standard deviations for normally distributed variables, as medians (interquartile ranges) for non-normally distributed variables, and as counts (percentages) for categorical variables. The difference between groups was analyzed by unpaired t test, Mann–Whitney U test and chi-square test, respectively. The correlation between thyroid antibodies and various symptoms as well as health-related quality of life was analyzed using Spearman’s correlation test, with adjustment for age, BMI, marriage, and family history of HT. To control for potential Type I errors due to multiple comparisons, the Benjamini–Hochberg procedure was applied to adjust the p-values, thereby controlling the false discovery rate. P < 0.05 was considered statistically significant, and all analyses were performed with the SPSS version 27.0.

The sample size calculation was based on the formula: \(n=\frac{{Z}_{crit}^{2}p\left(1-p\right)}{{e}^{2}}\)13, where Zcrit is 1.96 for a 5% level of significance in two-sided tests, p represents the estimate of the prevalence rate, and e denotes the margin of error. According to previous studies, the prevalence of Hashimoto’s thyroiditis is approximately 7.5%14. With a tolerable error set at 5%, at least 106 patients with Hashimoto’s thyroiditis were required. Healthy subjects were matched to cases using a 2:1 case-to-control ratio.

Results

Characteristics of participants

Demographic data of participants are shown in Table 1. There was no significant difference between HT patients and healthy subjects in age, gender, body mass index, smoking and drinking. The proportion of married people in HT group was significantly higher than that in healthy controls. Subjects with family history of HT in HT group were more than that in control group.

Table 1 Demographic data of included participants.
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Clinical parameters of participants are shown in Table 2. Without thyroid hormone substitution, parameters related to thyroid function (FT3, FT4 and TSH) of HT patients were similar with healthy controls, only TPO-Ab and TG-Ab in HT group were remarkably higher than that in control group. In addition, lipid metabolism biomarkers (TC and LDL-C), pro-inflammatory factors (TNF-α and IFN-γ), hepatic function index (AST, TP and ALB) and hsCRP levels were significantly increased in HT patients compared with healthy controls, while all of them were still within normal ranges.

Table 2 Clinical parameters of included participants.
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Survey of health-related life quality and severity of non-hypothyroid symptom

Health-related life quality was significantly decreased in HT patients compared with healthy controls, especially in terms of general health, vitality, bodily pain, social functioning and mental health (Table 3). Furthermore, despite patients’ euthyroid status, multiple symptoms were still much more serious in HT patients than in healthy controls, including the symptoms related to digestive system (abdominal distension, constipation, and diarrhea), circulatory system (chest tightness), endocrine system (chilliness, gain weight, and facial edema), neuropsychiatric system (forgetfulness, anxiety, depressed, fatigue, insomnia, irritability, and indifferent), movement system (joint pain and swelling) and mucocutaneous system (dry skin, pruritus, and hair loss) (Table 4).

Table 3 Health-related life quality in HT patients and healthy controls.
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Table 4 Severity of multiple symptoms in HT patients and healthy controls.
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The correlation between TPO-Ab and health-related life quality, inflammatory factors and multiple symptoms

The results of correlation analysis showed serum TPO-Ab was inversely correlated with general health score, and vitality score (Fig. 1A, B, Supplement Table 1), and was positively correlated with TNF-α, and IFN-γ (Fig. 2A, B, Supplement Table 2), and severity of symptoms related to endocrine system, neuropsychiatric system, and movement system (Fig. 3A-C). In detail, with the increase of serum TPO-Ab, symptoms of abdominal distension, diarrhea, chilliness, facial edema, forgetfulness, swelling, gain weight, and dry skin were more serious, while menorrhagia was alleviated (Fig. 4A-I, Supplement Table 3).

Fig. 1
figure 1

The correlation between thyroid antibodies and the 36-Item Short Form Health Survey scores. (A, B): Serum TPO-Ab levels and general health and vitality scores. (C-F): Serum TG-Ab levels and bodily pain, general health, vitality, and social functioning scores. Abbreviations: TPO-Ab, thyroid peroxidase antibody; TG-Ab, thyroglobulin antibody.

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Fig. 2
figure 2

The correlation between thyroid antibodies and inflammatory factors. (A, B): Serum TPO-Ab levels and TNF-α and IFN-γ. (C, D): Serum TG-Ab levels and TNF-α and IFN-γ. Abbreviations: TPO-Ab, thyroid peroxidase antibody; TG-Ab, thyroglobulin antibody; IFN-γ, interferon-γ; TNF-α, tumor necrosis factor-α.

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Fig. 3
figure 3

The correlation between thyroid antibodies and systematic symptom scores. (A-C): Serum TPO-Ab levels and endocrine system symptom scores, neuropsychiatric system symptom scores, and movement system symptom scores. (D-H): Serum TG-Ab levels and endocrine system symptom scores, neuropsychiatric system symptom scores, mucocutaneous system symptom scores, digestive system scores, movement system scores. Abbreviations: TPO-Ab, thyroid peroxidase antibody; TG-Ab, thyroglobulin antibody.

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Fig. 4
figure 4

The correlation between TPO-Ab and multiple symptom scores. Serum TPO-Ab levels and abdominal distension (A), diarrhea (B), chilliness (C), facial edema (D), forgetfulness (E), swelling (F), gain weight (G), dry skin (H) and menorrhagia (I). Abbreviations: TPO-Ab, thyroid peroxidase antibody.

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The correlation between TG-Ab and health-related life quality, inflammatory factors and multiple symptoms

Correlation analysis showed serum TG-Ab level was inversely correlated with scores of bodily pain, general health, vitality, and social functioning (Fig. 1C-F, Supplement Table 1), and was positively correlated with TNF-α, and IFN-γ (Fig. 2C-D, Supplement Table 2), and severity of symptoms related to endocrine system, neuropsychiatric system, mucocutaneous system, digestive system, and movement system (Fig. 3D-H). In detail, with the increase of serum TG-Ab, symptoms of abdominal distension, diarrhea, chilliness, gain weight, forgetfulness, depressed, fatigue, insomnia, and indifferent were more serious (Fig. 5A-I, Supplement Table 3).

Fig. 5
figure 5

The correlation between TG-Ab and multiple symptom scores. Serum TG-Ab levels and abdominal distension (A), diarrhea (B), chilliness (C), gain weight (D), forgetfulness (E), depressed (F), fatigue (G), insomnia (H) and indifferent (I). Abbreviations: TG-Ab, thyroglobulin antibody.

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Discussion

To the best of our knowledge, this is the first study to quantify the relationship between thyroid antibodies (TPO-Ab and TG-Ab) and inflammatory factors, health-related life quality, as well as multiple symptoms in Hashimoto’s thyroiditis patients. We found that both serum TPO-Ab and TG-Ab were inversely associated with life quality and positively associated with various extrathyroidal symptoms and inflammation, even though all the patients were in euthyroid status without hormone substitution.

It has been reported that common symptoms accompanied with HT patients typically included chronic fatigue, dry hair, dysphagia, irritability, and nervousness8,15,16. Clinically, there are still many other symptoms reported by HT patients, even when they were in euthyroid status. However, their relationships with Hashimoto disease have not been confirmed. In the present study, we used the Hashimoto’s Thyroiditis Symptom Questionnaire containing 49 items, which included more detailed symptoms related to digestive, respiratory, circulatory, endocrine, neuropsychiatric, movement, reproductive and mucocutaneous system. The results showed that symptoms of abdominal distension, constipation and diarrhea that related to digestive system, as well as symptoms of forgetfulness, anxiety, depressed, fatigue, insomnia, multiple dreams, irritability and indifferent that related to neuropsychiatric system, were significantly more serious than healthy controls. These findings provided us more comprehensive information about symptoms of HT patients, and also provided guidance for clinician to diagnose the etiology of these extrathyroidal symptoms. Furthermore, due to the euthyroid status of all enrolled participants, these findings also confirmed that these symptoms were caused by autoimmune disease, rather than hypothyroidism.

The precise mechanism of various extrathyroidal symptoms in HT patients was uncertain, but likely associated with elevated TPO-Ab17. It has been proposed that positive TPO-Ab may result in cross-reaction with other tissues, as activated TPO-Ab-producing lymphocytes may leave the thyroid gland and invade other distant tissue, contributing to extrathyroidal symptoms and inflammation18,19. Furthermore, clearance of TPO-Ab always occurs in parallel with a decline in other immunologic mediators20. A previous study has demonstrated that intervention of both levothyroxine and selenomethionine on HT patients for 6 months significantly reduced serum TPO-Ab titers, as well as decreased levels of IFN-γ, TNF-α, IL-1β, IL-2 and high sensitivity C-reactive protein (hsCRP) by about 50%21. Of interest, our study showed that levels of IFN-γ, TNF-α and hsCRP were higher in HT patients than in healthy controls, and increase of IFN-γ and TNF-α were both associated with higher TPO-Ab. Thus, the reduction in serum TPO-Ab is probably a surrogate marker for the modification of the systemic inflammation and extrathyroidal symptoms. In addition, our results also showed that TG-Ab was comparable with TPO-Ab when it come to their relationship with aforementioned proinflammatory factors. However, even though TG-Ab has been known to be associated with HT, its potential function as a predictive marker for inflammation and extrathyroidal symptoms is relatively unexplored.

The symptoms that had been proved to be associated with higher serum TPO-Ab level primarily included fatigue, dry skin, arthralgia, irritability and nervousness22,23. A randomized trial in Norway has showed that normalization of serum TPO-Ab titers of HT patients by total thyroidectomy significantly relieved extrathyroidal symptom burden, especially fatigue, and improved health-related life quality7. However, to date, limited study has given attention to intestinal symptoms in HT patients, let alone its relationship with TPO-Ab. The present study firstly found that abdominal distension, constipation and diarrhea were more serious in HT patients, and both TPO-Ab and TG-Ab were positively associated with severity of abdominal distension and diarrhea, indicating a possibility that reducing serum TPO-Ab and TG-Ab may be an effective way to modify these intestinal symptoms.

Intestinal leakage has been proposed to be an essential condition for the occurrence and development of autoimmune disease24,25,26. Several studies have demonstrated that the leaky gut promotes inflammatory cytokines release, and the latter aggravate an increased intestinal permeability27,28. This vicious circle favors the activation of adaptive and innate immunity in the gut mucosa, and can induce a tolerance breakdown. The activated immune cells may remain in the gut mucosa, or migrate to distant organs, participating in chronic inflammatory and resulting in autoimmune diseases, such as HT29. Furthermore, both abdominal distension and diarrhea are the main common symptoms of intestinal leakage30. Thus, abdominal distension and diarrhea seemed to be unescapable symptoms of Hashimoto disease. However, their close relationship has always been overlooked. The serious intestinal symptoms reported by HT patients in the present study confirmed the assumption, and raised a possibility that both the intestinal symptoms and HT could be cured from the perspective of improving the leaky gut.

In addition, we also found that TPO-Ab and TG-Ab were positively associated with severity of multiple other symptoms related to neuropsychiatric system, such as forgetfulness, depressed, fatigue, insomnia and indifferent. Except fatigue, the relationship between the other symptoms and thyroid antibodies were reported for the first time in this study. However, the mechanisms were uncertain and warranted further investigation.

As the heavy symptom load in HT patients and its positive correlation with thyroid antibodies, it is not a surprise that higher levels of TPO-Ab and TG-Ab were also associated with decreased health-related life quality. For various health parameters in SF-36 questionnaire, bodily pain, general health, vitality, social functioning and mental health were significantly affected by the HT disease. Furthermore, TPO-Ab and TG-Ab were both inversely associated with general health and vitality scores. Obviously, these results reflected an altered health perception of HT patients and may be due to the higher symptom burden.

Strengths of the present study include the study design, where all cases are kept at euthyroid status, thus the observed difference was free of hypothyroidism. Furthermore, not only TPO-Ab, we also paid close attention to TG-Ab, which has been known to be associated with HT, but was seldom involved in other relevant studies. Several limitations also should be noted. First, the present study cannot demonstrate the causal relationship between increased thyroid antibodies and pro-inflammatory factors, as well as multiple symptoms. For example, we observed multiple symptoms related to neuropsychiatric system in HT patients, such as anxiety, depressed, insomnia and irritability, but not sure whether these symptoms were the cause or result of HT disease. Second, the sample size in the study was relatively small, even though this was a multicenter study, which represented participants from both northern and southern China. Future prospective studies with large sample size are still needed. Third, the use of a Likert scale to evaluate specific symptoms, such as diarrhea, asthma and bradycardia, may potentially overstate the significance of certain findings, as the ordinal nature of Likert data may not accurately capture the true intensity of symptoms. Therefore, results from symptom ratings should be interpreted with caution, considering these potential sources of inaccuracy.

In conclusion, the present study demonstrated that HT patients were accompanied with multiple symptoms, especially those related to intestine and mood, even though they were in a euthyroid state. Furthermore, TPO-Ab and TG-Ab levels were inversely associated with general health and vitality scores, and positively associated with pro-inflammatory factors, as well as severity of several intestinal and psychiatric symptoms. These results highlight the symptoms burden that HT patients are suffering from, and are of great significance as they shed light on the clinical picture of HT. Future research that aiming to explore the causes of Hashimoto disease, or develop treatment strategies to decrease antibodies of HT patients is sorely needed.