Causal Relationship Between Immune Cells and Female Infertility: A Mendelian Randomisation Study

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However, the precise relationship between distinct subtypes of immune cells and female infertility remains elusive. Method: In this study, we conducted a two-sample Mendelian randomization (MR) analysis to examine the causal relationship between 731 immune phenotypes and female infertility. The inverse variance weighting method was employed as the primary estimator, complemented by MR-Egger and weighted median approaches. To address potential bias from single nucleotide polymorphisms (SNPs), we utilized MR-PRESSO. Additionally, Cochran's Q test and MR-Egger intercept analysis were performed to detect heterogeneity and horizontal pleiotropy. Results: We detected a causal role of fourteen immune phenotypes with female infertility, including CD11c+ CD62L- monocyte AC (OR=1.042,95%CI=1.002-1.083,P=0.037), CD62L-CD86+myeloid DC AC (OR=1.048,95%CI=1.010-1.088,P=0.013),CD62L-CD86+myeloid DC %DC(OR=1.052,95%CI=1.012-1.094,P=0.010), HLA DR++ monocyte AC(OR=1.038,95%CI=1.000-1.078,P=0.049),Activated&restingTregAC(OR=0.900,95%CI=1.838-0.959,P=0.001), Activated&secreting Treg%CD4 Treg(OR=1.009,95%CI=1.001-1.017,P=0.028),CD25hi%CD4+(OR=0.936,95%CI=0.895-0.978,P=0.004),TCRgd AC(OR=1.053,95%CI=1.003-1.106,P=0.039),HLA DR+CD4+%T cell(OR=0.922,95%CI=0.869-0.978,P=0.007),HLA DR+NKAC(OR=0.926,95%CI=0.864-0.993,P=0.031),CD28-DN(CD4-CD8-)%T cell(OR=1.048,95%CI=1.000-1.098,P=0.049),CD19 on IgD+ CD38- unsw mem(OR=1.056; 95%CI=1.026-1.087; p=0.0002),CD24 on IgD+ CD38- unsw mem(OR=0,967,95%CI=0.937-0.997,P=0.033) and CD25 on IgD+CD38- unsw mem(OR=0,977,95%CI=0.964-1.000,P=0.049). Conclusion: The study demonstrates a causal association between 14 genetically determined immune cell phenotypes and female infertility, thereby identifying novel drug targets for the prevention and treatment of this condition. Immune cells Immune phenotypes Female infertility Mendelian randomization Figures Figure 1 Introduction Infertility is defined as the inability to achieve pregnancy following 12 months of regular unprotected sexual intercourse[1]. Approximately 85% of infertile couples have an identifiable cause: the most common causes are ovulatory dysfunction, male factor infertility and tubal disease,the remaining 15% of infertile couples suffer from "unexplained infertility"[2]. According to a report by the World Health Organization, approximately 17% of individuals worldwide experience infertility, affecting one in every six people globally [3]. Observational and retrospective studies have indicated a potential association between immune response imbalances and female infertility[4,5]. Immune cells play crucial roles in folliculogenesis, ovulation, and the regulation of endometrial tolerance [6,7], which are pivotal for women's reproductive health. However, disruption of immune tolerance processes or excessive activation of immunoreactive cells can also contribute to immune-related female infertility. Several retrospective studies have reported an elevated number of peripheral blood NK cells leading to trophoblast antigen activation and subsequent induction of female infertility[8,9]. The relationship between different subtypes of immune cells and infertility remains unclear due to limited sample sizes, flawed study designs, as well as confounding factors beyond the scope of existing research. Therefore, we wished to determine the causal relationship between immune cell and female infertility. While randomized controlled clinical trials (RCTs) are considered the gold standard for determining causality, they are limited by traditional confounding factors such as environmental exposures and behavioral factors [10]. Mendelian randomization (MR) is an approach that utilizes genetic variance indices to measure the causality of disease-related risk factors while minimizing bias caused by confounding or reverse causality inherent in observational studies [10]. Thus, if the underlying assumptions of MR hold true, using genetic variants known to affect immune cells as instrumental variables may provide indirect evidence for the causal effect of immune cells on the risk of female infertility in women. Methods Study design We performed a two-sample MR to assess the causal relationship between 731 immune cell signatures and female infertility. SNP was used as an instrumental variable. To maximize the accuracy of the results, three important assumptions must always be confirmed. First, the selected IVs are associated with risk factors (731 immune cell signatures). Second, IVs is not associated with confounders between exposure (731 immune cell signatures) and outcome(female infertility). Third, IVs has no direct effect on outcome (female infertility), but only affect outcome through exposure (731 immune cell signatures). Data sources The original GWAS for the immune traits was conducted using data from 3,757 individuals of European descent. Summary statistics from the GWAS catalogue [11] were publicly available for 731 immunophenotypes, encompassing 118 absolute cell counts, 389 mean fluorescence intensities (MFIs) of surface antigens, 32 morphological parameters, and 192 relative counts (ratios between cell levels). The MFI, absolute cell and relative counts features contain B cells, CDCs, mature stages of T cells, monocytes, myeloid cells, TBNK (T cells, B cells, natural killer cells), and Treg panels, while the morphological parameters feature contains CDC and TBNK panels. Data on female infertility as an outcome were obtained from the FinnGen Consortium version R10 [14], which included a total of 75,450 Europeans (6,481 cases and 68.969 controls), with information on16.377038 SNPs. SNPs were selected at a threshold significance level of P <5e-8,and all associated data were derived exclusively from studies analyzing populations solely of European origin to eliminate potential bias due to population stratification. Genetic instrumental variable （ IV ） selection criteria The IV significance level for each immunological trait was set at 1×10-5 and these SNPs (linkage disequilibrium [LD] R2 < 0.1, kb = 500) [12], where LD R2 was calculated based on 1000 Genomes Projects as a reference panel. The significance level for infertility was adjusted to 5×10-8.To avoid bias due to weak IV, we use the F-statistic to measure the strength of IV. MR analysis In the MR analysis, we employed three methodologies, namely random effects inverse variance weighting (IVW), MR-Egger, and weighted median. The IVW approach yielded robust causal estimates in the absence of directional pleiotropy and was utilized as the primary method for estimating causal effects between exposure and outcome. MR-Egger and weighted median were employed as supplementary analyses. For instrumental variables (IVs) with P values < 0.05 from the IVW analysis, we conducted MR-PRESSO using the MRPRESSO package to identify and eliminate potentially polymorphic IVs (outliers), thereby providing outlier-adjusted estimates. Sensitivity analysis To ensure the stability of the MR analysis results, we performed tests for heterogeneity and pleiotropy.Cochran's Q test was used to calculate the level of heterogeneity, and MR-Egger regression and IVW methods were used to detect heterogeneity in IVS, with P<0.05 reflecting the presence of significant heterogeneity. We checked for pleiotropy by MR-Egger intercept regression, and a P value < 0.05 was considered significant pleiotropy, and then the study results were unreliable. In addition, we performed a "leave-one-out" sensitivity analysis, in which one SNP was sequentially missing from the MR reanalysis to tease out the potential effects of SNPs. Results To explore the causal effect of immune phenotypes on female infertility, a two-sample MR analysis was used, with the IVW method as the main analysis method. We detected a causal role of fourteen immune phenotypes with female infertility, including CD11c+ CD62L- monocyte AC(cDC panel),CD62L- CD86+ myeloid DC AC(cDC panel),CD62L- CD86+ myeloid DC %DC(cDC panel),HLA DR++ monocyte AC(TBNK panel),Activated & resting Treg AC(Treg panel ),Activated & secreting Treg %CD4 Treg(Treg panel ),CD25hi %CD4+(Treg panel ),TCRgd AC(TBNK panel ),HLA DR+CD4+%T cell(TBNK panel ),HLA DR+NK AC(TBNK panel ),CD28- DN (CD4-CD8-) %T cell(Treg panel ),CD19 on IgD+ CD38- unsw mem(B cell panel),CD24 on IgD+ CD38- unsw mem(B cell panel) and CD25 on IgD+ CD38- unsw mem(B cell panel).Among them, CD11c+ CD62L- monocyte AC increases the risk of female infertility in women(OR=1.042,95%CI=1.002-1.083,P=0.037).The causal relationship between CD62L- CD86+ myeloid DC AC and female infertility(OR=1.048,95%CI=1.010-1.088,P=0.013).CD62L- CD86+ myeloid DC %DC risk for female infertility(OR=1.052,95%CI=1.012-1.094,P=0.010).HLA DR++ monocyte AC risk for female infertility(OR=1.038,95%CI=1.000-1.078,P=0.049).Activated & resting Treg AC risk for female infertility(OR=0.900,95%CI=1.838-0.959,P=0.001).Activated & secreting Treg %CD4 Treg risk for female infertility(OR=1.009,95%CI=1.001-1.017,P=0.028).CD25hi %CD4+ risk for female infertility(OR=0.936,95%CI=0.895-0.978,P=0.004).TCRgd AC risk for female infertility(OR=1.053,95%CI=1.003-1.106,P=0.039).HLA DR+ CD4+ %T cell risk for female infertility(OR=0.922,95%CI=0.869-0.978,P=0.007).HLA DR+ NK AC risk for female infertility(OR=0.926,95%CI=0.864-0.993,P=0.031).The OR estimate for the risk of female infertility S by CD28- DN (CD4-CD8-) %T cell was 1.048(95%CI=1.000-1.098,P=0.049).Two-sample MR analysis showed a strong causal relationship between CD19 on IgD+ CD38- unsw mem and female infertility, random effects model IVW (OR=1.056; 95%CI=1.026-1.087; p=0.0002).Similar results were observed using four other methods，MR Egger(OR=1.052; 95%CI=1.007-1.099; p=0.033),Weighted median(OR=1.051; 95%CI=1.017-1.087; p=0.003),Simple mode(OR=1.057; 95%CI=1.009-1.108; p=0.030),Weighted mode(OR=1.047; 95%CI=1.019-1.076; p=0.003).CD24 on IgD+ CD38- unsw mem AC risk for female infertility(OR=0,967,95%CI=0.937-0.997,P=0.033).The causal relationship between CD25 on IgD+ CD38- unsw mem and female infertility(OR=0,977,95%CI=0.964-1.000,P=0.049). In the sensitivity analysis of the causal relationship between fourteen immune phenotypes and female infertility, the results of both MR Egger and IVW analyses were not statistically significant (p>0.05), and there was no significant heterogeneity. In addition, MR-Egger intercept regression tested for horizontal pleiotropy, and the results showed that horizontal pleiotropy did not exist. In our study, no abnormal SNPs were found using MR-PRESSO. Scatterplots og funnelplots viser også stabiliteten af resultaterne. Discussion This study represents the first utilization of two-sample Mendelian randomization (MR) to investigate the causal relationship between fourteen immune phenotypes and female infertility. The findings indicate that these immune phenotypes may exert an impact on women's reproductive health, ultimately leading to infertility. Previous research has consistently associated dysregulated Tregs with unexplained female infertility in both human and mouse models [13]. In our study, Activated & resting Treg AC, CD25hi %CD4+, Activated & secreting Treg %CD4 Treg, CD28- DN (CD4-CD8-) %T cells in regulatory T cells were significantly and causally associated with female infertility. A study reported a correlation between peripheral blood (PB) proportions of Tregs, which vary throughout the menstrual cycle, and the pathophysiology of infertility [14],suggesting that activated and quiescent Treg AC are associated with infertility.As heterogeneous members of the adaptive immune system crucial for successful pregnancy establishment, CD4+ T cells play a pivotal role. Our results demonstrate that increased expression of CD25hi %CD4+ heightens the risk of female infertility. Notably, this expression is particularly influential during early pregnancy stages such as embryo implantation. Heitmann et al., through culled mice with surface-expressing CD4CD25hi and FoxP3+ transcription factor-defined T regulatory cells (Tregs), discovered reduced litter sizes along with impaired embryo implantation [15]. Furthermore, an elevated frequency of CD4(+) cells was found to elevate the risk of female infertility [16]. CD4+ Treg were found to be focally distributed in the perivascular and periglandular regions of endometrial tissue in women with recurrent miscarriages and repeated implantation failures [17], indirectly supporting a causal relationship between Activated & secreting Treg %CD4 Treg and female infertility. CD28 is expressed on approximately 80% of human CD4 T cells and 50% of CD8 T cells, exerting both proinflammatory and anti-inflammatory effects that are crucial for enabling effector T cells to overcome Treg cell-mediated suppression during immunization [18,19]. A study comparing T-lymphocyte subpopulations in infertile women versus healthy women revealed a significant reduction in serum CD8 T cells among the former group [20]. Our study identified a positive correlation between B cell subsets (CD19+ IgD+ CD38- unsw mem, CD24+ IgD+ CD38- unsw mem, and CD25+ IgD+ CD38- unsw mem) and female infertility. However, limited research exists on B cells in relation to female infertility. One case-control study reported elevated peripheral blood levels of CD19+ B cells，and persistently low levels of memory B cell subsets in a 35-year-old woman with a history of recurrent miscarriage (RPL) and infertility [21]. On the cDC control panel, monocyte AC expressing CD11c+, myeloid DC AC expressing both CD62L- andCD86+, as well as %DC expressing bothCD62L-andCD86+, were found to be associated with an increased risk of infertility in women. Dendritic cells (DCs), which can initiate and orchestrate innate and adaptive immune responses, migrate to draining lymph nodes where they interact with antigen-specific T cells to elicit an inflammatory immune response—a critical factor contributing to infertility[22]. We identified a causal relationship between HLA DR+ NK AC, HLA DR+ CD4+ %T cell, TCRgd AC and female infertility in TBNK cells. TBNK cells refer to the lymphocyte subpopulation comprising T cells, B cells, and NK cells in the human body. NK cells play a crucial role in female reproduction by facilitating embryo acceptance and maintenance, participating in placenta formation, and promoting trophoblast cell growth and differentiation [23]. An elevated number of NK cells can exert cytotoxic effects on trophoblast cells leading to immune-mediated reproductive failure [24]. E Ivanova-Todorova demonstrated that an increase in absolute numbers of NK cells correlates with up-regulation of HLA-DR expression among infertile women. This mechanism may be attributed to increased production of IFN-γ by HLA-DR + NK cells. Elevated IFN-γ disrupts immune homeostasis, impairs granulosa cell growth reducing ovarian reserve, ultimately resulting in infertility [26,27]. A study found higher levels of HLA DR+ CD4+ CD3+%T cell expression both in peripheral blood and endometrium among infertility patients who failed IVF, but, endometrial levels were higher than those observed in peripheral blood [28], suggesting a potential causal relationship between HLA DR+ CD4+%T cell expression and infertility. HLA DR++ monocyte AC expression was causally associated with infection as well as monocyte AC and infertility. Activation of inflammatory pathways along with pro-inflammatory cytokines may subsequently contribute to immune dysfunctions and vascular abnormalities during placenta/peritoneum interactions leading to reproductive disorders and infertility [29]. Currently there is limited research exploring the association between TCRgd AC and infertility which warrants further investigation. In our study, the advantage of two-sample MR is that snp is randomly distributed at conception and the method is not subject to confounding and reverse causality as in traditional epidemiological studies, making the results more reliable. However, the population analysed was limited to Europeans and is not generalisable to other populations, and future research directions should be extended to different ethnicities. The results obtained from this study provide valuable literature support for further investigations into reproductive immunity. However, additional studies are warranted to elucidate the underlying mechanisms involved. Conclusion Our findings expand the understanding of the relationship between immune cell subtypes and female infertility, supporting that 14 immune phenotypes such as Activated & resting Treg AC, CD25hi %CD4+, and Activated & secreting Treg %CD4 Treg are more likely to lead to female infertility and have positively correlated risk ratios. These novel insights contribute to a deeper exploration of immune-related infertility and offer potential drug targets and research directions for the prevention and treatment of female infertility. Declarations Authors’ contributions Yafei Xie, Zelin Zhang and Qiaozhi Yin contributed to the concept and design of the study; Yafei Xie was responsible for statistical analysis and writing of the manuscript; Zelin Zhang and Qiaozhi Yin assisted with the statistical analysis. All authors have read and approved the final manuscript. Funding This study was supported by the Chengdu Science and Technology Bureau(2021-YF05-02042-SN). Data Availability Data can be found in public open access repositories. Data URLs: the GWAS summary statistics for the 731 immune traits are available for download from the GWAS catalogue (study logins: GCST90001001~GCST90002000 https://www.ebi.ac.uk/gwas/home); the summary statistics for female infertility are available from https://r10.finngen .fi/pheno/N14_FEMALEINFERT. All codes used in the study are available from the corresponding author. References Practice Committee of the American Society for Reproductive Medicine. Electronic address: [email protected] . Definitions of infertility and recurrent pregnancy loss: a committee opinion. Fertil Steril. 2020 Mar;113(3):533-535. doi: 10.1016/j.fertnstert.2019.11.025. Epub 2020 Feb 27. PMID: 32115183. Sang Q, Ray PF, Wang L. Understanding the genetics of human infertility. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4007697","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":278015334,"identity":"a96309b2-5d06-4d50-ae31-28eebe62186e","order_by":0,"name":"Yafei XIE","email":"","orcid":"","institution":"Chengdu University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yafei","middleName":"","lastName":"XIE","suffix":""},{"id":278015336,"identity":"9605bbd2-60e0-4b2c-8ce7-51ab866b9691","order_by":1,"name":"Zeling ZHANG","email":"","orcid":"","institution":"Chengdu University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zeling","middleName":"","lastName":"ZHANG","suffix":""},{"id":278015337,"identity":"8f6923c7-d2ab-4695-9097-4081b3186f45","order_by":2,"name":"Qiaozhi YIN","email":"data:image/png;base64,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","orcid":"","institution":"Chengdu University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Qiaozhi","middleName":"","lastName":"YIN","suffix":""}],"badges":[],"createdAt":"2024-03-03 06:32:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4007697/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4007697/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52614976,"identity":"9d4c8a48-1acc-44d7-84b6-7e128195e33e","added_by":"auto","created_at":"2024-03-13 15:48:48","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":228089,"visible":true,"origin":"","legend":"\u003cp\u003eForest plots showed the causal associations between immune cell traits and female infertility by using different methods. IVW: inverse variance weighting; CI: confidence interval\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4007697/v1/e0769649a25818602053a5cf.jpg"},{"id":52814908,"identity":"2278ef71-0fac-4d8a-a29e-e0740bdb8818","added_by":"auto","created_at":"2024-03-16 12:08:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":423723,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4007697/v1/e1bcb3c1-fa5a-4735-8929-cd5bb4c4c17d.pdf"},{"id":52614978,"identity":"02bbe244-81c0-469d-ac1b-807412b64e2f","added_by":"auto","created_at":"2024-03-13 15:48:49","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2522678,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1Causalassociationsbetweenimmunecellsandfemaleinfertility.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4007697/v1/670cd802edbddf26ed648ae4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Causal Relationship Between Immune Cells and Female Infertility: A Mendelian Randomisation Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInfertility is defined as the inability to achieve pregnancy following 12 months of regular unprotected sexual intercourse[1]. Approximately 85% of infertile couples have an identifiable cause: the most common causes are ovulatory dysfunction, male factor infertility and tubal disease,the remaining 15% of infertile couples suffer from \"unexplained infertility\"[2]. \u0026nbsp;According to a report by the World Health Organization, approximately 17% of individuals worldwide experience infertility, affecting one in every six people globally [3].\u003c/p\u003e\n\u003cp\u003eObservational and retrospective studies have indicated a potential association between immune response imbalances and female infertility[4,5]. Immune cells play crucial roles in folliculogenesis, ovulation, and the regulation of endometrial tolerance [6,7], which are pivotal for women's reproductive health. However, disruption of immune tolerance processes or excessive activation of immunoreactive cells can also contribute to immune-related female infertility. Several retrospective studies have reported an elevated number of peripheral blood NK cells leading to trophoblast antigen activation and subsequent induction of female infertility[8,9]. The relationship between different subtypes of immune cells and infertility remains unclear due to limited sample sizes, flawed study designs, as well as confounding factors beyond the scope of existing research.\u003c/p\u003e\n\u003cp\u003eTherefore, we wished to determine the causal relationship between immune cell and female infertility. While randomized controlled clinical trials (RCTs) are considered the gold standard for determining causality, they are limited by traditional confounding factors such as environmental exposures and behavioral factors [10]. Mendelian randomization (MR) is an approach that utilizes genetic variance indices to measure the causality of disease-related risk factors while minimizing bias caused by confounding or reverse causality inherent in observational studies [10]. Thus, if the underlying assumptions of MR hold true, using genetic variants known to affect immune cells as instrumental variables may provide indirect evidence for the causal effect of immune cells on the risk of female infertility in women.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe performed a two-sample MR to assess the causal relationship between 731 immune cell signatures and female infertility. SNP was used as an instrumental variable. To maximize the accuracy of the results, three important assumptions must always be confirmed. First, the selected IVs are associated with risk factors (731 immune cell signatures). Second, IVs is not associated with confounders between exposure (731 immune cell signatures) and outcome(female infertility). Third, IVs has no direct effect on outcome (female infertility), but only affect outcome through exposure (731 immune cell signatures).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData sources\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original GWAS for the immune traits was conducted using data from 3,757 individuals of European descent. Summary statistics from the GWAS catalogue [11] were publicly available for 731 immunophenotypes, encompassing 118 absolute cell counts, 389 mean fluorescence intensities (MFIs) of surface antigens, 32 morphological parameters, and 192 relative counts (ratios between cell levels). The MFI, absolute cell and relative counts features contain B cells, CDCs, mature stages of T cells, monocytes, myeloid cells, TBNK (T cells, B cells, natural killer cells), and Treg panels, while the morphological parameters feature contains CDC and TBNK panels. Data on female infertility as an outcome were obtained from the FinnGen Consortium version R10 [14], which included a total of 75,450 Europeans (6,481 cases and 68.969 controls), with information on16.377038 SNPs. SNPs were selected at a threshold significance level of P \u0026lt;5e-8,and all associated data were derived exclusively from studies analyzing populations solely of European origin to eliminate potential bias due to population stratification.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenetic instrumental variable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eIV\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003cstrong\u003eselection criteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe IV significance level for each immunological trait was set at 1×10-5 and these SNPs (linkage disequilibrium [LD] R2 \u0026lt; 0.1, kb = 500) [12], where LD R2 was calculated based on 1000 Genomes Projects as a reference panel. The significance level for infertility was adjusted to 5×10-8.To avoid bias due to weak IV, we use the F-statistic to measure the strength of IV.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the MR analysis, we employed three methodologies, namely random effects inverse variance weighting (IVW), MR-Egger, and weighted median. The IVW approach yielded robust causal estimates in the absence of directional pleiotropy and was utilized as the primary method for estimating causal effects between exposure and outcome. MR-Egger and weighted median were employed as supplementary analyses. For instrumental variables (IVs) with P values \u0026lt; 0.05 from the IVW analysis, we conducted MR-PRESSO using the MRPRESSO package to identify and eliminate potentially polymorphic IVs (outliers), thereby providing outlier-adjusted estimates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSensitivity analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo ensure the stability of the MR analysis results, we performed tests for heterogeneity and pleiotropy.Cochran's Q test was used to calculate the level of heterogeneity, and MR-Egger regression and IVW methods were used to detect heterogeneity in IVS, with P\u0026lt;0.05 reflecting the presence of significant heterogeneity. We checked for pleiotropy by MR-Egger intercept regression, and a P value \u0026lt; 0.05 was considered significant pleiotropy, and then the study results were unreliable. In addition, we performed a \"leave-one-out\" sensitivity analysis, in which one SNP was sequentially missing from the MR reanalysis to tease out the potential effects of SNPs.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTo explore the causal effect of immune phenotypes on female infertility, a two-sample MR analysis was used, with the IVW method as the main analysis method. We detected a causal role of fourteen immune phenotypes with female infertility, including CD11c+ CD62L- monocyte AC(cDC panel),CD62L- CD86+ myeloid DC AC(cDC panel),CD62L- CD86+ myeloid DC %DC(cDC panel),HLA DR++ monocyte AC(TBNK panel),Activated \u0026amp; resting Treg AC(Treg panel ),Activated \u0026amp; secreting Treg %CD4 Treg(Treg panel ),CD25hi %CD4+(Treg panel ),TCRgd AC(TBNK panel ),HLA DR+CD4+%T cell(TBNK panel ),HLA DR+NK AC(TBNK panel ),CD28- DN (CD4-CD8-) %T cell(Treg panel ),CD19 on IgD+ CD38- unsw mem(B cell panel),CD24 on IgD+ CD38- unsw mem(B cell panel) and CD25 on IgD+ CD38- unsw mem(B cell panel).Among them, CD11c+ CD62L- monocyte AC increases the risk of female infertility in women(OR=1.042,95%CI=1.002-1.083,P=0.037).The causal relationship between CD62L- CD86+ myeloid DC AC and female infertility(OR=1.048,95%CI=1.010-1.088,P=0.013).CD62L- CD86+ myeloid DC %DC risk for female infertility(OR=1.052,95%CI=1.012-1.094,P=0.010).HLA DR++ monocyte AC risk for female infertility(OR=1.038,95%CI=1.000-1.078,P=0.049).Activated \u0026amp; resting Treg AC risk for female infertility(OR=0.900,95%CI=1.838-0.959,P=0.001).Activated \u0026amp; secreting Treg %CD4 Treg risk \u0026nbsp;for female infertility(OR=1.009,95%CI=1.001-1.017,P=0.028).CD25hi %CD4+ risk for female infertility(OR=0.936,95%CI=0.895-0.978,P=0.004).TCRgd AC risk for female infertility(OR=1.053,95%CI=1.003-1.106,P=0.039).HLA DR+ CD4+ %T cell risk for female infertility(OR=0.922,95%CI=0.869-0.978,P=0.007).HLA DR+ NK AC risk for female infertility(OR=0.926,95%CI=0.864-0.993,P=0.031).The OR estimate for the risk of female infertility S by CD28- DN (CD4-CD8-) %T cell was 1.048(95%CI=1.000-1.098,P=0.049).Two-sample MR analysis showed a strong causal relationship between CD19 on IgD+ CD38- unsw mem and female infertility, random effects model IVW (OR=1.056; 95%CI=1.026-1.087; p=0.0002).Similar results were observed using four other methods，MR Egger(OR=1.052; 95%CI=1.007-1.099; p=0.033),Weighted median(OR=1.051; 95%CI=1.017-1.087; p=0.003),Simple mode(OR=1.057; 95%CI=1.009-1.108; p=0.030),Weighted mode(OR=1.047; 95%CI=1.019-1.076; p=0.003).CD24 on IgD+ CD38- unsw mem AC risk for female infertility(OR=0,967,95%CI=0.937-0.997,P=0.033).The causal relationship between CD25 on IgD+ CD38- unsw mem and female infertility(OR=0,977,95%CI=0.964-1.000,P=0.049).\u003c/p\u003e\n\u003cp\u003eIn the sensitivity analysis of the causal relationship between fourteen immune phenotypes and female infertility, the results of both MR Egger and IVW analyses were not statistically significant (p\u0026gt;0.05), and there was no significant heterogeneity. In addition, MR-Egger intercept regression tested for horizontal pleiotropy, and the results showed that horizontal pleiotropy did not exist. In our study, no abnormal SNPs were found using MR-PRESSO. Scatterplots og funnelplots viser ogs\u0026aring; stabiliteten af resultaterne.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study represents the first utilization of two-sample Mendelian randomization (MR) to investigate the causal relationship between fourteen immune phenotypes and female infertility. The findings indicate that these immune phenotypes may exert an impact on women's reproductive health, ultimately leading to infertility.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrevious research has consistently associated dysregulated Tregs with unexplained female infertility in both human and mouse models [13]. In our study, Activated \u0026amp; resting Treg AC, CD25hi %CD4+, Activated \u0026amp; secreting Treg %CD4 Treg, CD28- DN (CD4-CD8-) %T cells in regulatory T cells were significantly and causally associated with female infertility. \u0026nbsp;A study reported a correlation between peripheral blood (PB) proportions of Tregs, which vary throughout the menstrual cycle, and the pathophysiology of infertility [14],suggesting that activated and quiescent Treg AC are associated with infertility.As heterogeneous members of the adaptive immune system crucial for successful pregnancy establishment, CD4+ T cells play a pivotal role. Our results demonstrate that increased expression of CD25hi %CD4+ heightens the risk of female infertility. Notably, this expression is particularly influential during early pregnancy stages such as embryo implantation. Heitmann et al., through culled mice with surface-expressing CD4CD25hi and FoxP3+ transcription factor-defined T regulatory cells (Tregs), discovered reduced litter sizes along with impaired embryo implantation [15]. Furthermore, an elevated frequency of CD4(+) cells was found to elevate the risk of female infertility [16]. CD4+ Treg were found to be focally distributed in the perivascular and periglandular regions of endometrial tissue in women with recurrent miscarriages and repeated implantation failures [17], indirectly supporting a causal relationship between Activated \u0026amp; secreting Treg %CD4 Treg and female infertility.\u003c/p\u003e\n\u003cp\u003eCD28 is expressed on approximately 80% of human CD4 T cells and 50% of CD8 T cells, exerting both proinflammatory and anti-inflammatory effects that are crucial for enabling effector T cells to overcome Treg cell-mediated suppression during immunization [18,19]. A study comparing T-lymphocyte subpopulations in infertile women versus healthy women revealed a significant reduction in serum CD8 T cells among the former group [20].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur study identified a positive correlation between B cell subsets (CD19+ IgD+ CD38- unsw mem, CD24+ IgD+ CD38- unsw mem, and CD25+ IgD+ CD38- unsw mem) and female infertility. However, limited research exists on B cells in relation to female infertility. One case-control study reported elevated peripheral blood levels of CD19+ B cells，and persistently low levels of memory B cell subsets in a 35-year-old woman with a history of recurrent miscarriage (RPL) and infertility [21].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the cDC control panel, monocyte AC expressing CD11c+, myeloid DC AC expressing both CD62L- andCD86+, as well as %DC expressing bothCD62L-andCD86+, were found to be associated with an increased risk of infertility in women. Dendritic cells (DCs), which can initiate and orchestrate innate and adaptive immune responses, migrate to draining lymph nodes where they interact with antigen-specific T cells to elicit an inflammatory immune response—a critical factor contributing to infertility[22].\u003c/p\u003e\n\u003cp\u003eWe identified a causal relationship between HLA DR+ NK AC, HLA DR+ CD4+ %T cell, TCRgd AC and female infertility in TBNK cells. TBNK cells\u0026nbsp;refer to the lymphocyte subpopulation comprising T cells, B cells, and NK cells in the human body. NK cells play a crucial role in female reproduction by facilitating embryo acceptance and maintenance, participating in placenta formation, and promoting trophoblast cell growth and differentiation [23]. An\u0026nbsp;elevated number of NK cells can exert cytotoxic effects on trophoblast cells leading to immune-mediated reproductive failure [24]. E Ivanova-Todorova demonstrated that an increase in absolute numbers of NK cells correlates with up-regulation of HLA-DR expression among infertile women. \u0026nbsp;This mechanism may be attributed to increased production of IFN-γ by HLA-DR + NK cells. Elevated IFN-γ disrupts immune homeostasis, impairs granulosa cell growth reducing ovarian reserve, ultimately resulting in infertility [26,27]. A study found higher levels of HLA DR+ CD4+ CD3+%T cell expression both in peripheral blood and endometrium among infertility patients who failed IVF, but, endometrial levels were higher than those observed in peripheral blood [28], suggesting a potential causal relationship between HLA DR+ CD4+%T cell expression and infertility. HLA DR++ monocyte AC expression was causally associated with infection as well as monocyte AC and infertility. Activation of inflammatory pathways along with pro-inflammatory cytokines may subsequently contribute to immune dysfunctions and vascular abnormalities during placenta/peritoneum interactions leading to reproductive disorders and infertility [29]. Currently there is limited research exploring the association between TCRgd AC and infertility which warrants further investigation.\u003c/p\u003e\n\u003cp\u003eIn our study, the advantage of two-sample MR is that snp is randomly distributed at conception and the method is not subject to confounding and reverse causality as in traditional epidemiological studies, making the results more reliable. However, the population analysed was limited to Europeans and is not generalisable to other populations, and future research directions should be extended to different ethnicities. The results obtained from this study provide valuable literature support for further investigations into reproductive immunity. However, additional studies are warranted to elucidate the underlying mechanisms involved.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur findings expand the understanding of the relationship between immune cell subtypes and female infertility, supporting that 14 immune phenotypes such as Activated \u0026amp; resting Treg AC, CD25hi %CD4+, and Activated \u0026amp; secreting Treg %CD4 Treg are more likely to lead to female infertility and have positively correlated risk ratios. These novel insights contribute to a deeper exploration of immune-related infertility and offer potential drug targets and research directions for the prevention and treatment of female infertility.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYafei Xie, Zelin Zhang and Qiaozhi Yin contributed to the concept and design of the study; Yafei Xie was responsible for statistical analysis and writing of the manuscript; Zelin Zhang and Qiaozhi Yin assisted with the statistical analysis. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Chengdu Science and Technology Bureau(2021-YF05-02042-SN).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData can be found in public open access repositories. Data URLs: the GWAS summary statistics for the 731 immune traits are available for download from the GWAS catalogue (study logins: GCST90001001~GCST90002000 https://www.ebi.ac.uk/gwas/home); the summary statistics for female infertility are available from https://r10.finngen .fi/pheno/N14_FEMALEINFERT. All codes used in the study are available from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePractice Committee of the American Society for Reproductive Medicine. Electronic address: [email protected]. Definitions of infertility and recurrent pregnancy loss: a committee opinion. Fertil Steril. 2020 Mar;113(3):533-535. doi: 10.1016/j.fertnstert.2019.11.025. Epub 2020 Feb 27. PMID: 32115183.\u003c/li\u003e\n\u003cli\u003eSang Q, Ray PF, Wang L. Understanding the genetics of human infertility. Science. 2023 Apr 14;380(6641):158-163. doi: 10.1126/science.adf7760. Epub 2023 Apr 13. PMID: 37053320.\u003c/li\u003e\n\u003cli\u003eHarris E. Infertility Affects 1 in 6 People Globally. JAMA. Published online April 12, 2023. doi:10.1001/jama.2023.6251\u003c/li\u003e\n\u003cli\u003eAmjadi, F., Zandieh, Z., Mehdizadeh, M., Aghajanpour, S., Raoufi, E., Aghamajidi, A., \u0026amp; Aflatoonian, R. (2020). The uterine immunological changes may be responsible for repeated implantation failure. 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What is the role of regulatory T cells in the success of implantation and early pregnancy?. Journal of assisted reproduction and genetics, 24(9), 379\u0026ndash;386. https://doi.org/10.1007/s10815-007-9140-y\u003c/li\u003e\n\u003cli\u003eMal\u0026iacute;čkov\u0026aacute;, K., Luxov\u0026aacute;, \u0026Scaron;., Kr\u0026aacute;tk\u0026aacute;, Z., \u0026amp; Sedl\u0026aacute;čkov\u0026aacute;, L. (2021). Circulating NK and NKT cells in the diagnosis and treatment of immunological causes of female infertility - retrospective data analysis from the tertiary clinical center. Vy\u0026scaron;etřen\u0026iacute; NK a NKT buněk v diagnostice a l\u0026eacute;čbě imunologick\u0026yacute;ch př\u0026iacute;čin žensk\u0026eacute; neplodnosti - retrospektivn\u0026iacute; anal\u0026yacute;za dat terci\u0026aacute;rn\u0026iacute;ho klinick\u0026eacute;ho centra. Casopis lekaru ceskych, 160(1), 27\u0026ndash;32.\u003c/li\u003e\n\u003cli\u003eSauerbrun-Cutler, M. T., Huber, W. J., Krueger, P. 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Human reproduction update, 22(1), 104\u0026ndash;115.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Immune cells, Immune phenotypes, Female infertility, Mendelian randomization","lastPublishedDoi":"10.21203/rs.3.rs-4007697/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4007697/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eObservational and retrospective studies have demonstrated that aberrant immune responses and dysregulation of immune cell populations may be implicated in female infertility. However, the precise relationship between distinct subtypes of immune cells and female infertility remains elusive.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod: \u003c/strong\u003eIn this study, we conducted a two-sample Mendelian randomization (MR) analysis to examine the causal relationship between 731 immune phenotypes and female infertility. The inverse variance weighting method was employed as the primary estimator, complemented by MR-Egger and weighted median approaches. To address potential bias from single nucleotide polymorphisms (SNPs), we utilized MR-PRESSO. Additionally, Cochran's Q test and MR-Egger intercept analysis were performed to detect heterogeneity and horizontal pleiotropy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWe detected a causal role of fourteen immune phenotypes with female infertility, including CD11c+ CD62L- monocyte AC (OR=1.042,95%CI=1.002-1.083,P=0.037), CD62L-CD86+myeloid DC AC (OR=1.048,95%CI=1.010-1.088,P=0.013),CD62L-CD86+myeloid DC %DC(OR=1.052,95%CI=1.012-1.094,P=0.010), HLA DR++ monocyte AC(OR=1.038,95%CI=1.000-1.078,P=0.049),Activated\u0026amp;restingTregAC(OR=0.900,95%CI=1.838-0.959,P=0.001), Activated\u0026amp;secreting Treg%CD4 Treg(OR=1.009,95%CI=1.001-1.017,P=0.028),CD25hi%CD4+(OR=0.936,95%CI=0.895-0.978,P=0.004),TCRgd AC(OR=1.053,95%CI=1.003-1.106,P=0.039),HLA DR+CD4+%T cell(OR=0.922,95%CI=0.869-0.978,P=0.007),HLA DR+NKAC(OR=0.926,95%CI=0.864-0.993,P=0.031),CD28-DN(CD4-CD8-)%T cell(OR=1.048,95%CI=1.000-1.098,P=0.049),CD19 on IgD+ CD38- unsw mem(OR=1.056; 95%CI=1.026-1.087; p=0.0002),CD24 on IgD+ CD38- unsw mem(OR=0,967,95%CI=0.937-0.997,P=0.033) and CD25 on IgD+CD38- unsw mem(OR=0,977,95%CI=0.964-1.000,P=0.049).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003eThe study demonstrates a causal association between 14 genetically determined immune cell phenotypes and female infertility, thereby identifying novel drug targets for the prevention and treatment of this condition.\u003c/p\u003e","manuscriptTitle":"Causal Relationship Between Immune Cells and Female Infertility: A Mendelian Randomisation Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-13 15:48:44","doi":"10.21203/rs.3.rs-4007697/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"500ee94a-ce6f-4854-a1fa-4a38910217fa","owner":[],"postedDate":"March 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-16T11:59:52+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-13 15:48:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4007697","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4007697","identity":"rs-4007697","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}