Recurrent pregnancy loss (RPL) is defined as two or more fetal losses and affects approximately 1% - 2% of reproductive women and < 5% of couples (1, 2). Several factors contribute to the pathogenesis of RPL, including chromosomal abnormalities, abnormal uterine anatomy, antiphospholipid antibodies, endocrine factors, infection and autoimmune disorders. However, nearly 50% of RPL cannot be explained by known causes, which are classified as unexplained RPL (3). Although the knowledge of unexplained RPL is limited, emerging studies have shown that unexplained RPL is associated with immunological factors, including peripheral and decidual immune states (4–8). During the menstrual cycle and pregnancy, immune cell subtypes, including natural killer (NK) cells, T cells and gamma-delta (γδ) T cells, in the peripheral and local microenvironments changed dynamically with the fluctuation and secretion of hormones (9, 10). A recent study by Han et al. (11) determined the dynamic alterations of over 370 immune cell features (including cell distribution and functional responses) in maternal blood of normal pregnancy and preeclampsia (PE) by using a high-dimensional mass cytometry immunoassay. They found differentially dynamics of maternal immune system between healthy and preeclamptic pregnancies, suggesting that the determination of the peripheral immune states might lay the groundwork for identifying clinically-relevant immune disorders in women with pathological pregnancy.

The establishment and maintenance of healthy pregnancy depend not only on immune homeostasis between the mother and the growing fetus from the blastocysts to the newborn stage, but also on the appropriate immune response of the maternal immune system (12). Unexplained RPL is still a major clinical challenge for gynecologists worldwide. Growing interest has focused on understanding the immunological mechanisms during pregnancy and how they are relate to pregnancy outcomes. A study has shown that uterine NK and T cells help to create immune tolerance and an embryo-compatible microenvironment to promote blastocyst implantation and invasion into the maternal decidua in early pregnancy, and abnormal uterine NK and T cells are involved in RPL (13). A reference range of uterine NK cell percentages in fertile women has been established by an immunochemistry assay with CD56 staining, and the percentages above and below the reference range may increase the risk of RPL (14). However, in this study, only major NK cells were detected; NK cell subtypes were not measured. Moreover, the detection of uterine NK cells is generally invasive and may be uncomfortable for women. Therefore, it is essential to determine the percentages of immune cell subsets in the peripheral blood and provide guidance for clinicians in the evaluation of RPL patients.

In recent years, emerging studies have focused on the relationship between lymphocytes in the peripheral blood and the pathogenesis of RPL. Several studies have shown that enhanced peripheral blood CD56^dim NK cells and abnormally high circulating NK cells expressing inhibitory cytokines and inhibitory surface receptors contributed to RPL (15–18). A meta-analysis indicated that the numbers of peripheral blood NK (pbNK) cells in infertile women were significantly higher than those in fertile women (19). Moreover, both the numbers and the percentages of pbNK cells in women with RPL were significantly increased compared with those in controls. However, the percentages of uNK cells were not significantly different in women with RPL compared with the controls. In addition, T cell immune balance is also associated with the onset of RPL. Decreased regulatory T (Treg) cells/exhausted T cells and increased Th17 cells/exhausted Treg cells were found in the peripheral blood of RPL patients by flow cytometry (20). A significantly increased Th17/Treg ratio in peripheral blood was observed in women with a history of RPL compared with those without the history of RPL (21, 22). Besides, the cytotoxicity of pbNK cells is negatively correlated with CD8⁺ T cells in normal pregnant women but not in women with the history of RPL (23). Although many prior studies have shown that immune cells in the peripheral blood are involved in RPL, they often focus on single or selected immune cell subsets, and are lack of a systemic and simultaneous assessment of multiple immune cell subsets. Thus, limited information is provided, and sometimes controversial results exist.

γδ T cells also constitute a major lymphocyte population in peripheral blood and constitute 15%-60% of peripheral blood mononuclear cells (PBMCs). γδ T cells are different from NK cells and T cells and known to bridge innate and adaptive immunity (4). Human γδ T cells are divided into two main subtypes according to the rearranged VA chain: Vδ1⁺ and Vδ2⁺ T cells. Vδ2⁺ T cells are distributed mainly in the peripheral blood and possess defense properties against pathogens, whereas Vδ1⁺ T cells are predominant in tissues such as the spleen, intestine, and liver and possess regulatory and effector properties (24). Talukdar et al. (6) reported that a significant decrease in the proportion of CD3⁺CD4^-CD8^-γδ T cells and an increase in the percentage of IFN-γ- and IL-17-producing γδ T cells in peripheral blood were associated with RPL by creating an inflammatory cytokine milieu. However, the relationship between Vδ1⁺ and Vδ2⁺ T cells and RPL remains unclear.

Significant scientific advances in immunological researches have shown that many immune cells participate in the pathogenesis of pregnancy complications; however, due to the diversity of the phenotype and function of immune cells, there is still lacking a simultaneously global readout of multiple immune cell subsets for the evaluation of RPL. Moreover, desperate RPL patients often ask for an “immune cell testing” or “immune therapy”, although there is insufficient evidence to support or ensure the diagnostics and treatment (25). Therefore, it is valuable to develop a set of immune cell panels to examine and evaluate immune cell subsets in RPL patients by noninvasive peripheral blood testing. Herein, we designed a cohort study in nonpregnant women (NPW), women with histories of normal pregnancy (NP) and RPL. The immunological profiles in NPW group were used as baseline controls, which are absent in most published studies. Five milliliters of peripheral blood were donated and analyzed by multiparameter flow cytometry, which can simultaneously detect 63 different immune cell subtypes. The study design and results are shown in Figure 1 . The results showed that 10 kinds of immune cell subsets were specifically different in the RPL group and might be valuable for evaluating the potential risk of RPL. The 10 RPL-related immunological parameters included naïve CD4⁺ T cells (CD3⁺CD4⁺CD45RA⁺CCR7⁺), central memory CD4⁺ T cells (CD3⁺CD4⁺CD45RA^-CCR7⁺), naïve CD8⁺ T cells (CD3⁺CD8⁺CD45RA⁺CCR7⁺), mature NK cells (CD3^- CD56^+lo), Vδ1⁺ T cells (CD3⁺ γδ⁺Vδ2^- T), the ratio of Vδ1⁺ T cells/Vδ2⁺ T cells, terminal differentiated CD4⁺ T cells (CD3⁺ CD4⁺ CD45RA⁺ CCR7^-), effective memory CD4⁺ T cells (CD3⁺CD4⁺CD45RA^-CCR7^-), immature NK cells (CD3^- CD56^+hi) and Vδ2⁺ T cells (CD3⁺ γδ⁺Vδ2⁺ T); the first 6 were found at higher levels and the latter 4 at lower levels in the RPL group than in both the NPW and NP groups. Interestingly, we also found that the percentage of T_PH cells (CD3⁺CD4⁺CXCR5^-PD-1⁺) in the NPW group was higher than that in both the NP and RPL groups. To further analyze these data, we measured the 5^th percentile limit and 95^th percentile limit of these significantly different parameters to set up the reference ranges for diagnosing, predicting and evaluating the risk of RPL and therapeutic efficacy in future; levels above the 95^th percentile limit and below the 5^th percentile limit indicate an increased risk of RPL, which might lay the groundwork for clinicians to identify clinically-relevant immune disorders and create clear and individualized therapeutic plans for RPL patients.

In this study, we simultaneously detected 63 immune cell subsets in the peripheral blood of women without a history of pregnancy and those with histories of NP or RPL, including T cell, NK cell and γδT cell subsets. Also, we defined the reference ranges for each significantly changed parameters. Most prior studies focused on single or selected immune cells, such as T cells and NK cells, in the pathogenesis of RPL (4, 27, 31). In our study, we not only detected the major immune cell subsets as previous studies reported, but also examined the immune cells at different developmental stages, and the special activating and inhibitory receptors and the specific functional molecules expressed on these cells ( Table 2 ). Comparing with the NPW and NP groups, 10 significantly high or low immunological parameters in RPL group were found. Moreover, the percentage of T_PH cells was significantly lower in the NP and RPL groups than that in the NPW group. To our knowledge, this is the first study to simultaneously detect multiple immunological parameters and reveal their hallmarks in RPL, which might provide new insights and individualized evaluation for clinicians to identify clinical-relevant immune dysfunctions in RPL patients.

In our study, peripheral T cell subsets were assessed. The percentages of naïve CD4⁺ T cells, naïve CD8⁺ T cells, and central memory CD4⁺ T cells were significantly increased in RPL. However, the percentages of terminal differentiated CD4⁺ T cells and effective memory CD4⁺ T cells were significantly decreased in RPL. In the development of T cells, primary T cells undergo TCR rearrangement from bone marrow (BM) progenitors lacking CD4⁺ and CD8⁺ coreceptors to generate CD4⁺CD8⁺ double-positive (DP) T cells and then undergo single selection to give rise to CD4⁺ or CD8⁺ single-positive (SP) cells. Ultimately, SP T cells enter the periphery as naïve T cells with CD45RA⁺CCR7⁺ phenotypes (46). When naïve T cells encounter antigen and costimulatory ligands presented by dendritic cells (DCs), they proliferate and differentiate into effector T cells. Naïve T cells can regulate T cell antigen signaling and further differentiate into effector and memory T cells (32, 33). Activated effector T cells are short-lived, and only small proportions survive as memory T cells that persist as heterogeneous subsets (47). Memory T cells are characterized by CD45RA^- (48), whereas effector T cells are by CCR7^-. Effective memory T cells and terminal effector T cells, which are present in the circulation, exhibit CD45RA^-CCR7^- and CD45RA⁺CCR7^- phenotypes, respectively (49, 50).

Compared with pre-pregnancy endometrium, the proportion of naïve T cells in the decidua of full-term pregnancy was significantly reduced, whereas that of memory T cells was significantly increased (51). Memory is the hallmark of the acquired immune system and results from clonal expansion and differentiation of antigen-specific lymphocytes that ultimately persist for a lifetime (35, 52). Effective memory T cells migrate to inflamed peripheral tissues and display immediate effector function, which mediate protective memory, whereas central memory T cells are homing to T cell areas of secondary lymphoid organs and rapidly proliferate and differentiate into effector cells in response to antigenic stimulation, mediating reactive memory (35). Previous study found that effective memory CD4⁺ T cells were increased in normal pregnancy (36). Central memory CD4⁺ T cells were significantly increased in the peripheral blood of preeclamptic women and RPL women compared with the controls (36, 53). It was reported that terminally differentiated CD4⁺ T cells are associated with protection (34), whereas terminally differentiated CD8⁺ T cells play roles in persistent infections, especially cytomegalovirus (CMV) infection (49). In our study, the percentages of both terminal differentiated CD4⁺ T cells and terminal differentiated CD8⁺ T cells were lower in the RPL group than those in the NP group, suggesting that women with RPL might have low immune protection and be susceptible to infections. CD28, as a costimulatory molecule, is mainly expressed in CD8⁺ T cells at birth, and its downregulation is a hallmark of senescence and exhaustion (54). We found that the percentage of exhaustion of CD8⁺ T cells (CD8⁺ CD28^- T cells, immunosuppressive phenotype) was significantly lower, whereas that of potential functional CD8⁺ T cells (CD8⁺ CD28⁺ T cells) were higher in RPL group than in NP group. The results indicated that the immune system in women with a history of RPL might be in a less immunosuppressive state.

Peripheral NK cells consist of approximately 10-15% circulating lymphocytes. Basing on the expression of CD56, NK cells are classically divided into CD3^- CD56^bright (high CD56 intensity) and CD3^- CD56^dim (low CD56 intensity) (55). NK cells belong to the innate immune system and are widely distributed in human tissues. A high frequency of NK cells was found in the circulation, lungs, liver, and uterus (56). CD3^- CD56⁺ NK cells can be differentiated from CD34⁺ common lymphocyte progenitors (CLPs) with CD34 downregulation and CD56 upregulation. CD94 helps the development of CD56^bright NK cells, whereas CD56^bright NK cells subsequently differentiate into CD56^dim NK cells with upregulation of CD16 (57). CD3^-CD56^dim NK cells are the predominant subset of pbNK cells and account for approximately 90% of total pbNK cells, which are functionally well known for their potent cytotoxic activity and are marked as CD3^-CD56^dimCD16⁺ NK cells (58). In line with the previous studies, CD3^-CD56^dimCD16⁺ NK cells possess high cytolytic activity and were increased in RPL, which might be involved in the demise of the conceptus (38, 39). In contrast, CD3^- CD56^bright NK cells account for only 10% of pbNK cells with low cytotoxicity and high production of cytokines and chemokines (59). A previous study showed that decreased peripheral CD3^-CD56^bright NK cells in RPL were associated with the altered cytokine expression profile (37). Our findings further confirmed that CD3^-CD56^+hi NK cells with lower cytotoxicity were significantly lower in the RPL group compared to that in the NPW and NP groups. In addition, many molecules are expressed on NK cells, including natural cytotoxicity receptors, inhibitory receptors, immune checkpoints and inhibitory molecules (60). NK cell functions are also closely associated with the expression of activating and killing inhibitory receptors (KIRs). KIRs can interact with neighboring receptors, such as activating receptors - natural killer group 2, member D (NKG2D), natural cytotoxicity receptors (NCRs) NKp30 (also known as NCR3), NKp44 (also known as NCR2) and NKp46 (also known as NCR1) (61, 62) and KIRs – CD94/NKG2A (KIRD1/CD159a) heterodimers, which belong to HLA-class I (HLA-cl I)-specific inhibitory receptors (60). In our study, the NK cell phenotypes with specific functional molecules were not statistically different among the three groups. These data indicated that NK cells with high cytotoxicity were more enriched than NK cells with low cytotoxicity in RPL, which might be related to RPL. However, there were no differences in the percentages of activated NK cells, early and late inhibitory NK cells, conventional killer NK cells or specific virus-killed NK cells among RPL, NP and NPW groups ( Figures S2A–G ).

Unlike αβ T cells and NK cells, γδ T cells share characteristics of innate and adaptive immune cells (63). γδ T cells represent 3–5% of total T cells in the peripheral blood and can be classified into two main populations, Vδ1 and Vδ2 T cells, according to their TCR variable (V) gene segment usage. Human Vδ2 T cells are the main γδ T cell subtypes in the peripheral blood (64). However, Vδ1 T cells are mainly distributed in epithelial tissues (65). γδ T cells can recognize cells under stressed conditions, particularly infected or transformed cells, and kill them or regulate the immune response against them, paving the way for the development of promising therapeutic strategies for cancer and infectious diseases (66). γδ T cells employ T cell receptors and/or NK cell receptors, including major histocompatibility complex (MHC) molecules and NK cytotoxicity receptors (NCRs) on their surface (such as NKG2D, NKp44 and NKp30), for target cell recognition, which accounts for their remarkable flexibility (67–69). In the maternal-fetal interface, the majority of γδT cells were Vδ1 and produce high levels of TGF-β and IL-10, which might be a key player in maintaining the Th2 bias. Aberrant increase of decidual Vδ2 T cells in early-pregnancy is associated with unexplained spontaneous abortion (40). However, the function of γδT cells is strongly plastic. Previous studies have shown that Vδ1 T cells could efficiently kill myeloma cells and multiple epithelial tumor cell lines, with no significant difference in the specific lysis compared with Vδ2 T cells (70). Vδ1 T cells also have pro-inflammatory properties (41, 42). Interestingly, we found that the percentage of Vδ1⁺ T cells was significantly higher and that of Vδ2⁺ T cells was significantly lower in the RPL group than that in the NPW and NP groups. Additionally, the ratio of Vδ1⁺/Vδ2⁺ T cells was higher in the RPL group. Thus, it is necessary to further elucidate the function of γδT cell subsets in the peripheral blood and maternal-fetal interface of normal pregnancy and pregnancy related diseases including RPL.

In the current study, we also found that the percentage of T_PH cells was significantly higher in the NPW group than that in both the NP and RPL groups. T_PH cells are uniquely antigen-specific T cells with increased expression of genes including IL21 and CXCL13, which play important roles in B cell differentiation and potent B cell chemoattractants, respectively, and thus benefit the B cell function (43–45). In rheumatoid arthritis patients, T_PH cells were expanded in the active phage and decreased with effective treatment. Also, T_PH cells can be expanded in autoantibody-producing diseases (RA, SLE, SSc and coeliac disease), but not in diseases without autoantibodies (seronegative RA and spondyloarthritis), which indicate that T_PH cells augment B cell activation and autoantibody production (71). A previous study showed that IL-10-producing B cells were significantly low, whereas plasma cells were high in women with recurrent implantation failure (RIF). Since IL-10-producing B cells can suppress the production of autoantibodies and contribute to a successful implantation, their downregulation might be related to the occurrence of RIF (72). In this study, we did not detect the B cell populations and T_PH cells in women with RIF, which might limit the understanding of T_PH cells in implantation and needs further investigation.

To the best of our knowledge, this is the first observed finding; the detailed role and underlying mechanism in RPL require further investigations.

Prior studies primarily focused on the assessment of single or selected peripheral immune cell subsets in RPL, which lacks the simultaneous and comprehensive evaluation of immunological profiles in women of childbearing age. Herein, we simultaneously read out the multiple immunological parameters in the peripheral blood of women without any pregnancy (NPW group), or with histories of NP or RPL. A set of 11 statistically changed immunological parameters were found, including naïve CD4⁺ T cells, naïve CD8⁺ T cells, central memory CD4⁺ T cells, terminal differentiated CD4⁺ T cells, effective memory CD8⁺ T cells, immature NK cells, mature NK cells, Vδ1⁺ T cells, Vδ2⁺ T cells, the ratio of Vδ1⁺ T cells/Vδ2⁺ T cells and T_PH cells. Many of these cell types are novel immune cell subtypes involved in pregnancy. Moreover, we also defined the reference ranges based on the 5^th percentile limit and 95^th percentile limit of each immunological parameter to guide clinicians in globally evaluating the immune status of women before pregnancy. Some immune cell subtypes in women with RPL (up to 48%) were above the 95^th percentile limit, whereas some were below the 5^th percentile limit. These novel immunological parameters related to RPL may provide new insight into the immunological pathogenesis of RPL. Nevertheless, there is a limitation. The current study cannot confirm whether the differences in levels of some immune cells are a cause or consequence of RPL. Thus, a prospective cohort study is required to prove the relationship between the significantly changed immune cell subsets and the pregnancy outcomes in RPL.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the Clinical Trial Ethics Committee of Huazhong University of Science and Technology (2018S392). The patients/participants provided their written informed consent to participate in this study.

HL, X-XL, and X-BH were responsible for writing the manuscript, collecting samples, conception, and data analyzing. D-HH, SS, Y-JC, and JT were responsible for clinical sample collecting. DT, Z-NY, and GM were responsible for data analysis and interpretation. A-HL was responsible for data analysis, assembly and interpretation, study design and administration and revision. All authors contributed to the article and approved the submitted version.

This work was supported by National Key Research & Developmental Program of China (2018YFC1003900; 2018YFC1003904).

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.