In this study, based on a large number of public genetic data by using two-sample Mendelian randomization analysis to explore the causal relationship between immune cells and ovarian dysfunction, we found that 36 immunophenotypes have significant causal effect on ovarian dysfunction, including 18 kinds of immunophenotypes for ovarian function as risk factors and 28 kinds of immunophenotypes as protective factors. Figure 4 shows the effects of immunophenotypes on ovarian function.
Our study found that the risk of ovarian dysfunction with the proportion of Naive CD4 +%CD4 +, but increased with the proportion of Activated Treg%CD4. The main features of CD4 + T cells during aging include loss of proliferation and reduced telomerase activity, TCR restriction, low IL-2 and high IFN- γ production and response. There is evidence that its secreted cytokines play a role in the onset and progression of ovarian dysfunction[22–23]. Naive CD4 + T cells are able to regulate pro-inflammatory and anti-inflammatory signaling through differentiation into various T-helper (Th) cell lineages, with each Th cell lineage having its own unique cytokine profile and functions[24]. Some animal studies show that ovaries of old mice show lower Naive CD4 + T cell infiltration compared to ovaries of young mice. Reduced infiltration of Naive CD4 + T cells indicates a phenotype of immunosenescence, which may partly explain the mechanism of ovarian hypofunction[25]. Alternatively, it has been shown that Activated Treg CD4 cell subsets accumulate in aging and play a key role in the immune system that deteriorate with aging[26], While our results showed that the rising proportion of Activated Treg%CD4 increases the risk of ovarian dysfunction, suggesting a possible further association between this immunephenotypes and the presence and development of ovarian dysfunction.
Conversely, the upregulation of CD39 on CD39 + activated Treg expression in the Treg panel, may be reduced the risk of ovarian dysfunction. CD39 is mainly associated with endothelial cells and immune cell panels, such as B cells, monocytes, macrophages, neutrophils, and regulatory T cells (Treg). CD39 has anti-inflammatory effects, which hydrolyzes ATP to ADP and AMP, thereby reducing pro-inflammatory ATP and increasing the ADP or AMP precursors of anti-inflammatory adenosine[27]. Regulatory T cells (Tregs) play a key role in controlling the immune system and suppressing the inflammatory response[28]. There are many mechanisms for Tregs to inhibit other cell proliferation and cytokine production, including depletion of IL-2, direct toxicity of granzymes or perforin, or direct toxicity of cytokines (eg IL-10, TGFβ, or IL-35)[29]. Pharmacological studies indicated that Tregs degrade ATP to adenosine via CD39, and this adenosine is immunosuppressive[30]. In addition, CD39 + Treg cells are considered to be a unique subpopulation of Treg with highly active and inhibitory,which are responsible for removing extracellular adenosine triphosphate (ATP)[31]. Therefore, we speculate that the mechanism of ovarian dysfunction regulation by CD39 on CD39 + activated Treg is mediated by regulating adenosine to control the effect of inflammation on ovarian function, which is open to being further tested and validated by future animal studies.
In addition, according to the results, BAFF-R on CD20 has positive function on varian dysfunction. CD20 is found only in B cells and is generally not expressed in hematopoietic stem cells, plasma cells and other normal tissues, but in B-cell nausea tumors. BAFF-R is expressed in B cells and embodies different functions in B cells. BAFFR is a binding factor of BAFF on B cells, and the combination of both is an important regulator of B cell survival[32]. The binding of BAFFR-BAFF can activate the signaling pathways such as NF-KB1/2,PI3K,and Akt/ERK[33–34]. Studies have demonstrated that ovarian dysfunction is associated with the inactivation of signaling pathways such as PI3K/AKT[35].At present, some drugs have been proved to improve the oxidative stress status and cell apoptosis by activating the PI3K/AKT pathway, thus delaying the decline of ovarian function[36–37]. This would point to the possibility that upregulation of BAFF-R expression on CD20- could reduce the risk of ovarian dysfunction by activating related pathways such as PI3K or AKT to ameliorate inflammation. BAFF-R on Bcell may become an important target for the treatment of ovarian dysfunctional diseases, which needs more experimental and clinical studies to verify.
Similarly, CD38 on naive-mature B cell also has positive function on ovarian dysfunction.CD38 is a multifunctional cellular protein with signal transduction receptors and enzymatic features that can be expressed in immune populations, including B cells, T cells, macrophages[38]. A previous study showed that CD38 can regulate ovarian function and fecundity through NAD + metabolism, and the expression of CD38 on ovarian macrophages increases with aging, while reducing NAD + levels and increasing the risk of ovarian dysfunction[39–40]. In addition, it has been shown that CD38 expression increases in macrophages, and its expression on B cell is decreased in aged tissues[41]. The above results are not in conflict with this study and suggests that regulating the expression of CD38 on B cell may be an effective way to reduce ovarian dysfunction. Although how many cells driving abnormal CD38 expression during aging are not fully identified, the role of CD38 in aging regions is still pending, and this study provides new ideas for future studies of the underlying mechanisms of the CD38-NAD + axis in the ovary in the context of age-related ovarian dysfunction.
It was recently shown that abnormal expression of HLA DR antigen is associated with ovarian dysfunction[42–43].Among them, the apoptosis of ovarian granulosa cells plays an important role. The follicular granulosa cells are subjected to the continuous infiltration and destruction of the major histocompatibility antigen-Ⅱ (major histocompatibility complex, MHC-Ⅱ), and the granulosa cells are gradually reduced. HLA-DR is the product of MHC-like molecules, which is mainly expressed in monocytes, B lymphocytes and other cells. It can present antigen peptide and play a key role in the initiation of the immune response. The expression level of immune-related cells is known to influence the level of HLA-DR by participating in the immune response[44].However, only a few studies show the association of certain genotyping on HLA DR antigens with ovarian dysfunction[45–46], the phenotype of the HLA DR antigen was not systematically studied. Our results showed that HLA DR on B cell and HLA DR on CD14-CD16 + monocyte increased the risk of ovarian dysfunction, while HLA DR on monocyte reduced the risk of ovarian dysfunction. This result is in line with previous studies, which could facilitate the more accurate application of HLA DR antigen in the assessment of ovarian function[47–48]and helps clinicians identify women with ovarian dysfunction to establish individualized treatment strategies.
Furthermore, the results of this study showed that the risk of ovarian dysfunction was inversely correlated with the expression on CX3CR1 on CD14 + CD16 + monocyte cells. It is evidence that non-classical CD14 + CD16 + monocytes significantly increase with aging, while the expression of the surface of CX3CR1 decreases with aging[49]. CD14 + CD16 + monocytes are low ROS producers and low expression of anti-inflammatory cytokine IL-1, while produce higher levels of reactive nitrogen (RNS), IL-1 β and TNF α, which are closely related to a variety of inflammatory diseases[50]. CX3CR1 Is highly expressed on CD14 + CD16 + monocyte cell surface, which is the only high-affinity receptor for FKN and belongs to the G protein-coupled receptor superfamily[51]. After CX3CR1 binding to FKN, peripheral blood leukocytes can produce chemotaxis and adhesion, achieving rapid mobilization and bringing effector cells to the inflammatory site. When the inflammatory response occurs, the inflammatory factors produced by inflammation can activate the FKN / CX3CR1 signaling to participate in the inflammatory response[52]. Moreover, FKN can adhere to monocytes,which suggested that the FKN/CX3CR1 signaling is able to be important for inflammatory diseases by acting on chemotaxis and adhesion molecules. Several studies have shown that inflammatory monocytes can exert protective effects on the pathomechanism of chronic inflammatory diseases through CX3CR1 dependent adhesion mechanisms[53–55].However, the development of ovarian dysfunction is related with inflammation. Although there are limited data on the involvement of CX3CR1 on CD14 + CD16 + monocyte cells in ovarian dysfunction disease, this suggests that the regulation of CX3CR1 expression may be a target for the control of ovarian dysfunction disease, providing more possibilities for its intervention treatment.
Same as CX3CR1 on CD14 + CD16 + monocyte cells, the rise of CD11c + monocyte in its proportion in the monocyte panel was similarly able to reduce the risk of ovarian dysfunction. Ono et al[56]demonstrated that CD11c + cell depletion caused elimination of PDGF-B signals in ovarian stromal lesions, decreased PDGF-Rβ + pericytes around follicles.CD11c + is expressed in M1 like macrophages, which indicated that PDGF-B may be a mediator for M1 like macrophages to participate in the process of follicular vascularization.There are several studies indicated that tissue-resident macrophages are reduced in the aging ovary, and that the proportion of monocyte-derived macrophages increases which are predominant in the macrophage population.Monocytes are actively recruited to ovarian tissue by trending during inflammation and are more readily polarized into M1-like macrophages[57]. Combined with the results of this study, the fact that more CD11c + monocyte%monocyte is expressed indicates that CD11c + monocyte is recruited to ovarian tissue and polarized as M1 macrophages, which is beneficial to promote follicle development and ovarian vascular formation, and then delay ovarian aging. However, whether M1-like macrophages participate in the regulation of follicle vascularization through PDGF-B and whether monocyte-derived ovarian macrophages can functionally and metabolically fully complement ovarian tissue-resident macrophages are big knowledge gaps for future studies.
It is worth noting that,we found that the occurrence of ovarian dysfunction was associated with CD127 CD8br% CD 8 br, CD16 CD56 on NK, CD19 on CD24 + CD27 +, CD19 on IgD + CD38 naive, CD20 on CD20 CD38, CD24 on IgD + CD24 +, CD25 on CD24 + CD27 +, CD25 on memory B, etc. At present, relevant studies have found an increased concentration of inflammatory factors in the blood of patients with ovarian dysfunction[58–59]. To date, the studies of immune cells in ovarian-related diseases are limited, and the studies of immune cell phenotype on ovarian dysfunction are even more fewer. There is a case-control study found reduced CD4, CD25 +, and hiFoxp3 + Treg cells in patients with POI[60]. The results of our study might provide new ideas for exploring the mechanism of ovarian dysfunction in the future.
Our study has several strengths. We performed a two-sample MR analysis based on published results of a large GWAS cohort with a large sample size and high statistical efficiency. Most previous association studies relied on cross-sectional studies and animal models, thus limiting the ability to establish a causal relationship between systemic immune cells and ovarian dysfunction disease. Our study was based on genetic instrumental variables and performing causal inference using multiple MR analysis methods, reducing the influence of confounding factors and ensuring a valid cause–effect relationship.The MR-STO and MR-EGG tests were used to detect and exclude horizontal pleiotropy.There are also some limitations to our study. First, the study was based on a European database, and whether the conclusions can be extended to other ethnic groups has not been verified. Second, we would evaluate the results with a more relaxed threshold, which may add some false positives. Third, a further stratified analysis cannot be performed in this study due to the lack of personal information.