AIT is the primary autoimmune illness in women of reproductive age [24]. Elevated levels of TPOAb, TgAb, and other non-thyroid tissue-specific autoantibodies are the primary indicators for autoantibody detection. More and more scholars are realizing that this autoimmune response may involve systemic tissues, rather than organ-specific damage. For example, real-world studies have found that women with AIT have a significantly increased risk of developing PCOS, a significant complication that affects reproductive function in women with AIT [4, 6, 10, 25]. PCOS is an endocrine disorder characterized by high levels of androgens, irregular or absent ovulation, and cyst-like formations on the ovaries. It can lead to infertility, miscarriage, and metabolic disorders. At present, it is unclear how AIT patients are prone to developing PCOS, and there are no simple and feasible predictive factors or effective prevention and treatment strategies.

We thoroughly examined the possible risk factors for concurrent PCOS incidence in euthyroid AIT and non-AIT women in this study. Age, BMI, and serum levels of FT4, FT3, TSH, TPOAb, and TgAb are not independent risk factors for concurrent PCOS in the AIT group; instead, age and BMI are the only independent risk factors for the occurrence of PCOS in the non-AIT group. This suggests that other factors may have contributed to the development of PCOS in these AIT women.

Edassery et al. discovered that PDIA3 could be a target antigen in infertile women by analyzing serum anti-ovarian antibodies in women with infertility and premature ovarian failure [13]. PDIA3 is expressed in the cell membrane, cytoplasm, and nucleus, and it has multiple functions [26,27,28]. When expressed on the membrane, it is an important antigen associated with immunogenic cell death (ICD) [26]. Stress can also promote the translocation of PDIA3 to the membrane and increase its autoantibody (PDIA3Ab) production [29]. PDIA3 also serves as a membrane receptor for active Vitamin D, mediating its non-genomic effects [28]. Interestingly, the level of the classic Vitamin D receptor in the ovaries is very low. Vitamin D mainly promotes antral follicle development and estrogen and progesterone synthesis through PDIA3-mediated non-genomic effects [15].

Many scholars have suggested the potential involvement of autoimmune mechanisms in PCOS [8, 9, 30], such as the presence of autoantibodies. Regrettably, only a limited number of studies have verified that PCOS patients exhibit significantly elevated levels of non-organ-specific antibodies. Previous small pilot studies have indicated a higher occurrence of anti-nuclear antibody (ANA) positivity in women with PCOS [31, 32]. However, a study by Petrikova et al. found no significant increases in ANA, anti-Sjögren’s syndrome A (SSA), anti-Sjögren’s syndrome B (SSB), anti-dsDNA, anti-ribonucleoprotein (RNP), antineutrophil cytoplasmic (ANCA)/myeloperoxidase (MPO), or ANCA/proteinase 3 (PR3) in 152 PCOS patients and 76 healthy controls who were tested for these antibodies [30]. Although anti-GnRH antibodies are produced in a few PCOS patients [12], the receptor is not distributed in the thyroid and ovaries. In other words, previous studies had not fully elucidated the relevant target antigens and autoantibodies causing PCOS. We reported high expression of PDIA3Ab in AIT women with PCOS, which is innovative. Other studies have focused on major anti-thyroid antibodies, such as TPOAb and TgAb. TPOAb may destroy follicles through ADCC because of the similarity in structure between antigens in the zona pellucida and the thyroid gland [11]. However, further research has not provided direct evidence that TPOAb and TgAb contribute to the development of PCOS. Consistently, we did not discover a clear and independent association between TPOAb and TgAb titers and an increased risk of concurrent PCOS in AIT.

PDIA3Ab is highly expressed in the serum of patients with rheumatic heart disease, autoimmune hepatitis, and type 2 diabetes [29]. In our earlier investigation, PDIA3Ab levels were higher in the AIT animal model caused solely by mouse Tg immunization of CBA/J mice [16]. PDIA3Ab may result from immune damage to thyroid tissue in AIT individuals, possibly due to the diffusion of the intermolecular epitope, and is not caused by other concurrent autoimmune diseases [17]. In our current study, euthyroid AIT women had a significantly greater level of PDIA3Ab than non-AIT controls [17]. Specifically, the serum PDIA3Ab level was notably elevated in euthyroid AIT women with PCOS compared to those without PCOS. Additionally, the logistic regression analysis results demonstrated that PDIA3Ab total serum IgG expression was an independent risk factor for concurrent PCOS in the euthyroid AIT group but not in the non-AIT group. Trend analysis also showed a positive linear correlation between serum PDIA3Ab total serum IgG level and the concurrent prevalence of PCOS in euthyroid AIT women. All of these findings indicate that PDIA3Ab may contribute to the development of concurrent PCOS in euthyroid AIT women. Furthermore, as an important risk factor, it could also serve as a predictive marker for the disorder.

It is worth noting that, Spearman correlation analysis showed that PDIA3Ab total serum IgG levels were positively associated with LH/FSH ratio and testosterone levels in the AIT-PCOS group. Previous studies revealed that the binding of the active Vitamin D to PDIA3 stimulates the interaction between phospholipase A2 (PLA2) activating protein (PLAA) and PDIA3. Subsequently, PLA2 is activated, triggering the rapid release of arachidonic acid (AA) [33]. In PCOS patients, the activation of PLA2 is reduced, while cyclooxygenase-2 (COX-2) gene expression is significantly up-regulated, which can promote the metabolism of AA to prostaglandin (PG) E2, which can stimulate the production of testosterone [34,35,36]. On the other hand, PDIA3 is present in lipid rafts and cytoplasmic complexes in the membrane of signal transducer and activator of transcription 3 (STAT3)-expressing cells and inhibits the translocation of STAT3 activated by cytokines such as interleukin 6 (IL-6) to the nucleus, making activated STAT3 unable to activate gene transcription [27]. We speculate that PDIA3Ab can prevent the above process, allowing activated STAT3 to enter the nucleus, of course, this needs to be verified by subsequent experiments. Activation of STAT3 can enter the nucleus and also raise COX-2 gene expression eventually leading to an increase in testosterone [34, 36]. Therefore, we speculate that the elevated PDIA3Ab expression in AIT women may contribute to the development of hyperandrogenism through the above mechanisms, which requires further research through in vivo and in vitro experiments.

The association between PDIA3Ab expression and the occurrence of concurrent PCOS in euthyroid AIT and non-AIT women has been systematically assessed in this study. Our findings revealed that the grades of the PDIA3Ab total serum IgG levels (from high to low) were classified as follows: AIT-PCOS group>AIT-non-PCOS group>non-AIT-PCOS group>non-AIT-non-PCOS group. The levels of IgG subclasses in the human body are as follows: IgG1>IgG2>IgG3>IgG4. Among them, IgG1 and IgG3 exhibit the strongest complement activation and ADCC, while IgG2 primarily responds to polysaccharide antigens, and IgG4 demonstrates the lowest biological activity [37, 38]. PDIA3Ab serum IgG3 levels were significantly higher in the AIT-PCOS group compared to the other three groups, while the levels of PDIA3Ab serum IgG2 and IgG4 were not significantly different among the four groups. On one hand, the increased titer of PDIA3Ab may counteract the activation of T cells [16]. On the other hand, PDIA3 is involved in the assembly of major histocompatibility complex (MHC) class I molecules and the antigen presentation process, which helps activate CD8+T cells to exert a cytotoxic effect [39]. PDIA3Ab and its subtypes can damage target cells expressing PDIA3 through complement and humoral immune pathways, but this antibody is not the primary antibody that destroys the thyroid. It is speculated that PDIA3Ab may mainly act on extrathyroidal lesions of AIT such as the ovary. We acknowledge that we have not completed the investigation of the mechanisms of PDIA3Ab in the development of PCOS in euthyroid AIT women. Therefore, their cause-effect relationship awaits further study.

Our study possesses several strengths. It has come to our knowledge that this is the inaugural investigation into the correlation between PDIA3Ab expression and concurrent PCOS in both AIT and non-AIT women. The study has revealed that PDIA3Ab is a significant independent risk factor for concurrent PCOS in euthyroid AIT women. The ROC curve of PDIA3Ab total serum IgG level alone for predicting the occurrence of concurrent PCOS in euthyroid AIT women was 0.700 (95%CI: 0.596–0.804). The corresponding cut-off OD450 value ratio of PDIA3Ab total serum IgG for the maximum Youden index was 0.592, indicating a significant increase in the risk of concurrent PCOS in euthyroid AIT women with a sensitivity of 76.4% and a specificity of 53.5%. Furthermore, and this has never been documented before, the titer-dependent associations between those autoantibody levels and the prevalence of PCOS have been thoroughly evaluated in those euthyroid AIT women alone. PDIA3Ab total serum IgG showed a titer-dependent connection with the prevalence of PCOS, although neither serum TPOAb nor TgAb did. Finally, LH/FSH ratio and serum testosterone were positively related to the serum PDIA3Ab expression in AIT women with PCOS, but not in AIT women without PCOS. All of these results suggest that serum PDIA3Ab expression may be directly connected to a greater risk of concurrent PCOS in euthyroid AIT women, but not serum TPOAb or TgAb expression, and that an elevated level of PDIA3Ab could be a significant factor in AIT-related PCOS. Serum PDIA3Ab level measurement not only provides an independent predictor of concurrent PCOS risk but also helps to explain the mechanism of spontaneous abortion caused by PCOS from a new perspective.

Our study still has limitations: (1) The limited sample size may introduce bias into the findings. To further assess the predictive efficacy of PDIA3Ab on the concurrent PCOS risk among euthyroid AIT women, a bigger sample investigation is required. (2) The predictive value of PDIA3Ab total serum IgG for diagnosing PCOS in AIT patients is limited (AUC = 0.700, 95%CI: 0.596–0.804). Our future work will concentrate on investigating the epitope targeted by PDIA3Ab. Specific antibodies against the antigen epitope may not only enhance the predictive value of PDIA3Ab diagnosis but also potentially intervene to prevent the occurrence and development of PCOS through the induction of immune tolerance. (3) The potential impact of PDIA3Ab on the female reproductive system is complex. Previous studies have demonstrated that PDIA3 affects ovarian and placental function. Our prior research found that PDIA3Ab was linked to spontaneous abortion in euthyroid AIT women during pregnancy. The study focused on the connection between PDIA3Ab and PCOS. It’s important to note that having PCOS does not necessarily conflict with having had a spontaneous abortion. However, it’s worth pointing out that our findings do not rule out patients with a history of spontaneous abortion. This is a limitation of our study. In future research, we aim to explore this topic further.