THE present study utilized the TMT proteomics technique to explore the FF protein expression profile for patients with RSA. To the best of our knowledge, this is the first study using the TMT technique to identify differentially expressed proteins in FF from RSA samples, providing unique insight into the altered reproductive microenvironment of these patients.

The present TMT results revealed upregulation of FcγRII-a and downregulation of FcγRIII-a in FF samples from patients with RSA. Both GO and KEGG analysis indicated significant enrichment of FcγR-mediated pathways, indicating dysregulation of FcγR and its downstream pathways in cases with RSA.

The FcγR was reported to be an important regulator in the maternal–fetal relationship [13, 14]. Like the present study, a recent proteomic study of serum samples from patients with RSA revealed a significant change had occurred in the pathway of FcγR-mediated phagocytosis [7]. These findings indicate that the abnormality of FcγR-related pathways and dysregulation of the immune system in patients with RSA also exist at the level of follicle development.

Complement is a group of proteins that form the core component of the humoral immune system, which protects the host against invading organisms, and initiates inflammation and tissue injury. The complement system is a vital modulator of immune surveillance [15]. A successful pregnancy requires proper immune adaptation of the fetus and placenta, a combination that can be regarded as a semi-allogenic graft. Thus, appropriate complement inhibition is needed to maintain a pregnancy [16, 17], and abnormal activation of complement is reported to be associated with RSA [18, 19]. More than 30 proteins have been recognized as being involved with the complement system [20]. A previous study reported that elevated serum C3 and C4 levels in patients with a history of RSA can be predictors of future miscarriage [21].

In the present study, TMT analysis identified dozens of components of the complement system in FF samples, and several components were found to be significantly upregulated in FF samples from patients with RSA, including C9, C4B, C1QA, and C1QC. Inhibitors of complement activation were downregulated (e.g., CLU), which indicated abnormal complement activation in the microenvironment of follicle growth.

Among the differentially expressed proteins, the peptide-spectrum matches of C4B were the most abundant, indicating it is likely to have higher expression in FF samples. Moreover, C4B is an isotype of native C4 proteins that participates in the classical and mannose-binding lectin complement activation pathways [22]. Therefore, we selected C4B for further study. The results of C4B levels examined in FF samples from a larger sample size of patients were consistent with the TMT analysis, with higher expression levels found in FF samples from patients with RSA compared with control group. ROC analysis of C4B and the reproductive outcomes described above also showed its potential for the prediction of reproductive outcomes; the AUC was 0.710 (p < 0.01). However, a previous proteomics study of serum samples from cases with RSA did not report a significant alteration of components of the complement activation system [7]. We speculate that abnormal component activation is more significant in the follicular microenvironment.

Histidine-rich glycoprotein (HRG) is a 75-kDa single polypeptide, multidomain protein produced by the liver. It can bind to several receptors, including heparin, plasminogen, fibrinogen, and complement components, as well as divalent metal ions [23]. Previous studies revealed the involvement of HRG in coagulation [24], fibrinolysis [25], angiogenesis [26, 27] and the immune system [28, 29].

The present TMT analysis revealed that HRG was significantly downregulated in FF samples from cases with RSA. HRG has been reported to present in the FF, as well as embryos, endometrium, fallopian tube, myometrium, and placenta [30]. Several HRG gene polymorphisms have been reported to be related to the occurrence of RSA [31, 32].

However, the exact role of HRG in the human reproductive system needs further investigation. In the present study, GO analysis of the TMT analysis findings indicated the involvement of HRG in several enriched GO terms, such as serine-type endopeptidase inhibitor activity, endopeptidase inhibitor/activator activity, peptidase inhibitor activity, and positive regulation of immune response.

In addition, HRG has been regarded a regulator of the complement system [23, 29, 33]. It can bind to human C1Q and IgG, and it inhibits the formation of insoluble immune complexes. As mentioned above, C1QA and C1QC were upregulated in the FF samples from patients with RSA. Moreover, HRG has been reported to regulate the expression and function of the FcγR [34, 35]. In summary, HRG has a tight connection with various differentially expressed proteins and pathways, and its downregulation in FF may be an indicator of the dysregulation of various functions, including angiogenesis, coagulation, and the immune system. The correlation between HRG and AMH also revealed its possible relationship with ovarian reserve function.

In the present study, the reduced expression of HRG in FF from patients with RSA was validated by ELISA. Moreover, analysis using ROC curves showed that the expression level of HRG in patients undergoing IVF/ICSI cycles may provide a candidate biomarker to predict reproductive outcomes (e.g., live birth versus not pregnant or miscarriage) in such patients [36]. Consistent with the present study, another study using proteome analysis showed that preconception use of folic acid regulated HRG and downregulated FcγRIII-a in FF samples [37]. Overall, lower HRG expression in ovulating follicles may be an indicator of poor outcomes for future IVF treatment.

However, there are some limitations in our present study: the sample size was relatively small and the unfavorable IVF outcome of RSA patients need to be categorized (no pregnancy; biochemical pregnancy loss, pregnancy loss). Thus, further study is worth pursuing.

In conclusion, the present study identified 98 differentially expressed proteins in the FF of patients with RSA. We also confirmed the abnormal expression of representative proteins, HRG and C4B, in a larger group of patients. Further ROC analysis raised the possibility that HRG protein levels in FF may be used to predict IVF outcomes. This study has provided potential biomarkers to predict the occurrence of RSA, and also demonstrated an abnormal immune system in the follicles of patients with RSA.