In China, the occurrence of ≥ 2 consecutive fetal losses is defined as recurrent spontaneous abortion (RSA), a serious reproductive disorder of pregnancy with a prevalence as high as 1 to 5% in women of reproductive age (Practice Committee of the American Society for Reproductive Medicine, 2012). Recent studies have shown that the etiology of RSA is complex and varied, including advanced age, genetic abnormalities, infections, immune disorders, coagulation abnormalities, and endocrine dysfunction (Guo et al., 2020). However, the etiology of most RSA is unknown. Interestingly, more than half of the cases in which RSA occurs present with immune dysfunction (Baek et al., 2007), and these cases are often referred to as immune-related RSA (Alijotas-Reig and Garrido-Gimenez, 2013). Therefore, understanding the early manifestations and mechanisms of RSA will help us to take early steps to prevent pregnancy failure.

The success of pregnancy is based on the completion of an accurate switch of immune defense mechanisms during the early stages of embryo colonization (Wang et al., 2020). The embryo is a semi-allogeneic graft to the mother in early gestation and is therefore antigenic in its initial stages of development. The uterus, as the site of embryo implantation and development, is non-specific in its initial immune response to the embryo. The endometrial stroma contains a large number of immune cells, including NK cells, innate lymphocytes, macrophages, decidual dendritic cells and T cells (Tian et al., 1998). These cells form a pro-inflammatory microenvironment by producing a range of cytokines and chemokines that promote embryonic attachment (Dimitriadis et al., 2005). Subsequently, the Th1-type immune response rapidly changes to a Th2-type immune response, which primarily controls the endocrine and immune response and allows the pregnant woman to continue a safe pregnancy. Available evidence suggests that disturbances in the endometrial immune microenvironment are associated with repeated implantation failure (RIF) and unexplained RSA (Liu et al., 2019). We therefore hypothesize that the cause of RSA may be related to the unsuccessful conversion of Th1 to Th2 immune responses.

Growth stimulation expressed gene 2 (ST2) protein is a member of the interleukin 1 receptor/Toll-like receptor superfamily with two forms, i.e., soluble ST2 (sST2) and trans-model ST2 (ST2L). ST2 was previously considered as a key protein affecting cell proliferation and has an impact on tumor development (Werenskiold et al., 1989). ST2L can be expressed in a variety of cells, such as mast cells, macrophages and some lymphocytes (NK cells and Th2 cells, etc.) (Klemenz et al., 1989; Kumar et al., 1997; Xu et al., 1998; Kuroiwa et al., 2001; Allakhverdi et al., 2007; Smithgall et al., 2008), and it is highly expressed in immune cells that mediate Th2-type immune responses. All these studies suggest that ST2 is inextricably linked to the Th2-type immune response, however, it is unknown whether ST2 plays a role in RSA associated with abnormal Th2-type immune response. Furthermore, as ST2 becomes more widely used, it has been found to be associated with lung disease, sepsis and gastrointestinal disorders (Boga et al., 2016) and is becoming one of the important markers in the management of heart failure to indicate disease progression and prognosis (Daniels and Bayes-Genis, 2014).

Interleukin (IL)-33 is an important ligand for ST2. IL-33 is a dual-function cytokine whose full-length form acts as a transcription factor in the nucleus and inhibits the expression of NF-κB, a gene that regulates pro-inflammatory signaling. When cells are subjected to external stimuli or cellular necrosis, the precursor of IL-33 can be processed by neutrophil-derived proteases into the mature IL-33 form, which is then released as a cytokine into the extracellular compartment (Moussion et al., 2008). Mature IL-33 can bind to ST2L and trigger an inflammatory cascade response. However, sST2 binding to IL-33 prevents its binding to ST2L in immune cells, which in turn inhibits the activation of Th2 cells and the release of anti-inflammatory cytokines (IL-4, IL-5, IL-10, IL-13), and enhances the activation of Th1 cells and the release of pro-inflammatory cytokines (TNF-α, etc.) (Dinarello et al., 2012). Thus, IL-33/ST2 has an immunomodulatory role and this signaling axis may play an important role in a variety of diseases associated with major Th2 immune responses, such as rheumatoid arthritis, sepsis, and ulcerative colitis (Moussion et al., 2008; Dinarello et al., 2012; Daniels and Bayes-Genis, 2014; Pei et al., 2014; Boga et al., 2016).

In summary, we propose the following hypothesis: IL-33 and sST2 in RSA patients may contribute to RSA by attenuating the conversion to Th2-type immune responses. We plan to examine serum levels of IL-33 and sST2 in women with a history of RSA and normal women in the unpregnant and pregnant states to discover they whether are consistent with our hypothesis or have some diagnostic value.

IL-33 is a member of the IL-1 cytokine family and is involved in both the innate and adaptive immune responses through interaction with its receptor ST2. Activation of IL-33/ST2 signaling activates pleiotropic immune functions in a variety of immune cells that can express ST2 protein, including macrophages, neutrophils, eosinophils, basophils, mast cells, Th2 cells, regulatory T (Treg) cells, etc. (Griesenauer and Paczesny, 2017). Recently, it has been shown that IL-33 is involved in the pathogenesis of several inflammatory diseases, including allergic diseases (Liew et al., 2016; Gupta et al., 2017), infectious diseases (Donovan et al., 2016; Xu et al., 2017), and neuropathic pain (Fattori et al., 2017). In addition, sST2 levels are significantly elevated in patients with heart disease and graft-versus-host disease (Mathews et al., 2016; Hartwell et al., 2017). Therefore, IL-33 and its receptor ST2 may be promising new targets for the treatment of RSA.

In this study, patients with RSA were collected as the main study population. Given that RSA is a type of pregnancy loss that may be caused by a failure of transformation of the immune response, we hypothesized that there may be abnormalities in the indicators associated with Th2-type immune transformation during this process. The present study showed a significant increase in serum IL-33 and sST2 in the RSA group compared to the normal pregnancy group (Figure 1), which further validates our hypothesis that RSA is accompanied by abnormal Th2-type immune transformation. Granne et al. (2011) previously reported a significant increase in ST2 expression in patients with preeclampsia, and this increase was detectable prior to the onset of clinical symptoms. During pregnancy, sST2 production may reflect the enhanced Th1-type inflammatory environment of the disease compared to normal pregnancy, as sST2 acts as a decoy receptor for IL-33, competing with membrane-bound ST2L. Thus, the very high circulating sST2 levels seen in preeclampsia would be predicted to reduce the binding of IL-33 to its receptor, thus contributing to the Th1-type bias seen in this disorder. This finding has some similarity to the present study, both suggesting that abnormalities in immune transformation may accompany the abnormal course of pregnancy. In addition, this study also found small amounts of IL-33 and sST2 expression in the normal non-pregnant group and in the normal pregnancy group, and serum sST2 levels were much higher in the RSA group than in the healthy controls (p < 0.0001). It was found that ST2 protein was expressed in alveolar epithelial cells, cardiomyocytes, and also in brain and small intestine cells (Homsak and Gruson, 2020), so the placenta may not be the only source of high levels of sST2 in the serum of patients with RSA.

In this study, serum IL-33 and sST2 were found to have predictive value for RSA for the first time (Figure 2), however, there was no significant correlation between IL-33 and sST2 and gestational age or age in both the normal and RSA groups (Figure 3). Combined with the analysis of Granne et al. (2011), the lower correlation in this study may be related to the small sample size, which does not negate the predictive value of IL-33 and sST2 in RSA.

Further analyzing the relationship between IL-33 and sST2 levels and the number of miscarriage that had occurred in the disease group of patients, we also found that sST2 levels appeared to be higher in patients with a greater number of miscarriages. Given the insufficient sample size, only one patient with a high number of miscarriages seems to be the main factor that enhances the association (Figure 4B), more samples and more rigorous experiments are needed to prove or falsify this hypothesis.

In our clinical case collection work, we found that pregnant women with RSA often have some endocrine disorders, such as diabetes mellitus and thyroid disease. Diabetes mellitus usually causes microangiopathy, which in turn leads to chronic progressive lesions, hypofunction and failure of tissues and organs such as eyes, kidneys, nerves, heart and blood vessels. It has been shown that the IL-33/ST2 signaling pathway is activated in diabetic nephropathy (Elsherbiny et al., 2020), and endothelial dysfunction can be induced during the development of diabetic nephropathy (Xue et al., 2020). Interestingly, ST2, the key protein in this study, can be produced by endothelial cells, which activate immune responses and trigger organismal protective mechanisms by expressing large amounts of ST2 during the sepsis process (Huang et al., 2020). Thus, we speculate whether the high secretion of ST2 by endothelial cells is another influencing factor in the elevation of ST2 in the sera of pregnant women with RSA with diabetes? However, this influencing factor has not been addressed in the present study, and we will continue to investigate this issue in subsequent studies as well.

Since we observed a significant increase in both IL-33 and sST2 in patients with RSA, this phenomenon may be due to the maternal immune system’s response to preeclampsia. In contrast, the elevated serum sST2 may also be associated with the underlying maternal disease itself. Ultimately the maternal incomplete or inadequate conversion of the Th2-type immune response is due to the net effect of increased IL-33 and sST2 levels. It is a pity that the correlation results between IL-33 and sST2 were not obtained in this study, which we will further investigate in our future work.

In conclusion, in this study, IL-33 and sST2 were found to be significantly increased in patients with recurrent miscarriage for the first time. IL-33 and sST2 may become novel biomarkers for early prediction of pregnancy failure and recurrent miscarriage, providing potential new targets for the treatment of RSA.

The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.

Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.

LZ, JF, FD, and JL: conception and design, data analysis and interpretation. LZ and JF: administrative support. FD, JL, LL, QS, and YF: provision of study materials or patients, collection and assembly of the data. All authors wrote the manuscript and approved the final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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