Causal relationship between MIF/MCSF and Spontaneous Abortion: Insights from Mendelian Randomization and population Validation

Causal relationship between MIF/MCSF and Spontaneous Abortion: Insights from Mendelian Randomization and population Validation | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 7 August 2025 V1 Latest version Share on Causal relationship between MIF/MCSF and Spontaneous Abortion: Insights from Mendelian Randomization and population Validation Authors : Yilin Fan , Feipeng Cui , Peng Gao , Huan Tang , Liwen Zhou , Jiaxi Chen , Shuhao Yang , … Show All … , Yumin Liu , Zhou Li , Yuqi Li , Xingguang Lin , Jianli Wu , Xun Gong , Xinwei Shi , Jingjing Xu , Yingjia Hu , Wei Tu , and Haiyi Liu [email protected] Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.175454652.28794630/v1 174 views 129 downloads Contents Abstract Abstract Introduction Methods & Materials Results Discussion Conclusion Author contribution Funding statement Conflict of Interest Statement Acknowledgments Ethical statement Data Availability Statement Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Objective : Spontaneous abortion (SA) affects 15-25% of clinically recognized pregnancies. Immune dysregulation is linked to SA pathogenesis, however, causal relationships between specific inflammatory cytokines and SA remain unclear. Methods: Two-sample Mendelian randomization (MR) analysis was performed using SA data from FinnGen (20,775 SA cases, 180,063 controls), and the genetic associations with 40 inflammatory cytokines were obtained from a published GWAS meta-analysis comprising 8,293 European individuals. Further clinical validation was conducted with decidual and villus tissues from 20 SA and 20 controls of Asian patients by quantitative real-time Polymerase Chain Reaction, Western blot, immunohistochemistry, and immunofluorescence. Results: MR analysis identified MIF and MCSF as unidirectional causation of SA, while IL-17 showed bidirectional causality. There were positive associations between SA and both MCSF (OR: 1.04, 95% CI: 1.00-1.08, P =0.040) and MIF (OR: 1.05, 95% CI: 1.01-1.11, P =0.026). Clinical validation revealed significantly elevated MIF and MCSF proteins in SA decidual tissues, with enhanced co-localization with CD68-positive macrophages and CK7-positive trophablasts. Conclusion: Genetic and tissue analyses suggest that MIF and MCSF play causal roles in SA pathogenesis through tissue-specific mechanisms, highlighting their potential as biomarkers and therapeutic targets for clinical intervention. TITLE PAGE Title: Causal relationship between MIF/MCSF and Spontaneous Abortion: Insights from Mendelian Randomization and population Validation Running Head: MIF/MCSF in spontaneous abortion Yilin Fan 1 , Feipeng Cui 1 , Peng Gao 1 , Huan Tang, Liwen Zhou, Jiaxi Chen, Shuhao Yang, Yumin Liu, Zhou Li, Yuqi Li, Xingguang Lin, Jianli Wu, Xun Gong, Xinwei Shi, Jingjing Xu, Yingjia Hu, Wei Tu, Haiyi Liu* \received DD MMMM YYYY \acceptedDD MMMM YYYY Authors: Yilin Fan, MS, Second Clinical School, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, PR China, [email protected] Feipeng Cui, MD, Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, PR China, [email protected] Peng Gao, MD, Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, PR China, [email protected] Co-Authors: Huan Tang, MS, Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, PR China, [email protected] Liwen Zhou, MS, Hubei Provincial Center for Disease Control and Prevention &NHC Specialty Laboratory of Food Safety Risk Assessment and Standard Development, Wuhan, Hubei, PR China, [email protected] Jiaxi Chen, MM, Hubei Provincial Center for Disease Control and Prevention &NHC Specialty Laboratory of Food Safety Risk Assessment and Standard Development, Wuhan, Hubei, PR China, [email protected] Shuhao Yang, MM, Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, PR China, [email protected] Yumin Liu, MD, same as above, Wuhan, Hubei, PR China, [email protected] Zhou Li, MD, same as above, Wuhan, Hubei, PR China, [email protected] Yuqi Li, MD, same as above, Wuhan, Hubei, PR China, [email protected] Xingguang Lin, MD, same as above, Wuhan, Hubei, PR China, [email protected] Jianli Wu, MD, same as above, Wuhan, Hubei, PR China, [email protected] Xun Gong, MD, same as above, Wuhan, Hubei, PR China, [email protected] Xinwei Shi, MD, same as above, Wuhan, Hubei, PR China, [email protected] Jingjing Xu, MD, same as above, Wuhan, Hubei, PR China, [email protected] Yingjia Hu, University of Pittsburgh，School of Public Health, [email protected] Wei Tu, MD, Department of Rheumatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, PR China, [email protected] Corresponding author: Haiyi Liu, MD, Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, PR China Mail: [email protected] , Number: +86 18942916346 Add: No. 1095 Jiefang Avenue, Qiaokou District, Wuhan, Hubei Province, China Word count: 2789 words Abstract Objective : Spontaneous abortion (SA) affects 15-25% of clinically recognized pregnancies. Immune dysregulation is linked to SA pathogenesis, however, causal relationships between specific inflammatory cytokines and SA remain unclear. Methods: Two-sample Mendelian randomization (MR) analysis was performed using SA data from FinnGen (20,775 SA cases, 180,063 controls), and the genetic associations with 40 inflammatory cytokines were obtained from a published GWAS meta-analysis comprising 8,293 European individuals. Further clinical validation was conducted with decidual and villus tissues from 20 SA and 20 controls of Asian patients by quantitative real-time Polymerase Chain Reaction, Western blot, immunohistochemistry, and immunofluorescence. Results: MR analysis identified MIF and MCSF as unidirectional causation of SA, while IL-17 showed bidirectional causality. There were positive associations between SA and both MCSF (OR: 1.04, 95% CI: 1.00-1.08, P =0.040) and MIF (OR: 1.05, 95% CI: 1.01-1.11, P =0.026). Clinical validation revealed significantly elevated MIF and MCSF proteins in SA decidual tissues, with enhanced co-localization with CD68-positive macrophages and CK7-positive trophablasts. Conclusion: Genetic and tissue analyses suggest that MIF and MCSF play causal roles in SA pathogenesis through tissue-specific mechanisms, highlighting their potential as biomarkers and therapeutic targets for clinical intervention. Key words: MIF, MCSF, spontaneous abortion, decidual tissue, villus tissues Introduction Spontaneous abortion (SA) affects 15-25% of clinically recognized pregnancies and remains a significant public health concern 1 . It is associated with profound physical, psychological and economic impacts, ranging from acute complications to long-term sequelae, which significantly affect individual reproductive health and family well-being 2-4 . Despite advances in prenatal care, the management of SA remains challenging, which is largely due to the limited understanding of its underlying mechanisms beyond chromosomal abnormalities. The immune regulation of the maternal-fetal interface has been highlighted in recent evidence in maintaining a successful pregnancy 5 . Maternal immune cells directly interact with fetal trophoblasts and represent a unique immunological environment, and disruption of this precise balance can lead to pregnancy complications including SA 6-8 . Understanding these immune-mediated mechanisms is therefore crucial for developing effective preventive and therapeutic strategies. Previous studies have identified various cytokines as key mediators in pregnancy maintenance. At the maternal-fetal interface, the complex interaction between pro-inflammatory cytokines (such as IL-2, TNF-α, and IFN-γ), anti-inflammatory cytokines (like IL-4 and IL-10), and regulatory factors (such as TGF-β) orchestrate immune responses 9-11 . Decidual macrophages constitute 20-30% of maternal leukocytes 12 . The dynamic polarization states play important roles in maintaining pregnancy 13,14 . Numerous studies showed that the polarization of macrophages is promoted by different pro- and anti-inflammatory cytokines, including TGF-β, IL-10, TNF-α and CXCL16 15-18 . The disruption of normal M1/M2 balance is associated with pregnancy complications such as pre-eclampsia, fetal growth restriction and SA 19,20 . In our previous studies, the NLRP3 inflammasome was found to amplify the inflammatory response cascade through abnormal communication between trophoblasts and macrophages 21 . These researches indicated the important role of cytokines in SA, however, the definitively of causality has not been established between cytokines alterations and pregnancy loss. To address this knowledge gap, Two-Sample Mendelian randomization (MR) analysis was employed to assess the association between inflammatory cytokines and SA. MR is a robust epidemiological approach that utilizes genetic variants as instrumental variables to investigate causal relationships. These genetic variants are randomly assigned during meiosis and independent of potential confounders and reverse causation typically encountered in observational studies 22 . The MR findings were further validated through several molecular experiments using decidual and villous tissues from SA patients and controls, including Quantitative Real-time Polymerase Chain Reaction (RT-qPCR), Western blot (WB) analysis, immunohistochemistry (IHC), and immunofluorescence (IF) co-localization studies. Given potential ethnic differences in immune responses and SA susceptibility, this validation in Asian populations is particularly important. By combining genetic causality assessment with tissue-specific molecular validation and cellular localization studies, our integrated approach aims to not only identify causal cytokines but also elucidate their spatial distribution and potential cellular targets at the maternal-fetal interface. The results are expected to provide robust evidence for the critical role of specific immune mechanisms in SA pathogenesis and lay the foundation for developing mechanism-based interventions. Methods & Materials 1. Study Design A two-sample MR was performed on the bidirectional causality between inflammatory cytokines and spontaneous abortion. Figure 1 presents the overview design of the study. To validate the findings from MR analyses, a prospective cohort of participants was recruited at Tongji Hospital during a three-month period from September to November 2024. The study population consisted of 40 pregnancies in 6-9 gestation ages divided into two groups: the SA case group (n=20) included patients diagnosed with SA with normal fetal chromosome karyotype, while the control group (n=20) comprised healthy pregnant women who underwent elective termination of normal pregnancies. All patients with severe or other major disorders were excluded. The baseline characteristics included maternal age, gravidity, and parity. Based on sample availability and quality, Fresh tissue samples were allocated for different analyses. The study was approved by the institutional ethics committee (approval number: TJ-IRB202401107). The study protocols were approved by the Ethics Committee of Tongji Hospital, Huazhong University of Science and Technology, and all participants provided written informed consent. Data Sources & Instrumental Variables Two-sample MR was conducted using two independent databases. The information about databases is presented in Table S1 . The genetic associations with IC were obtained from a GWAS meta-analysis comprising 8,293 Finnish individuals from three cohorts, consisting of 2,019 unrelated individuals from the 2007 follow-up of The Cardiovascular Risk in Young Finns Study (YFS; mean age 37 years) and participants from FINRISK surveys (1997 and 2002; mean age 60 years) 23 . These genetic associations were adjusted for age, sex, and body mass index. SA data were retrieved from the FinnGen database (R11 version), which defined SA using ICD-10 code O03, including 20,775 cases and 180,063 controls. For genetic instrument selection, significance thresholds of P <1 10 -5 were set considering the limited availability of genetic variants reaching genome-wide significance. The same threshold ( P <1 10 -5 ) was applied when SA was analyzed as the exposure. To ensure independence of genetic instruments, single-nucleotide polymorphisms (SNPs) were clumped using parameters kb=10000 and r2=0.01, and palindromic SNPs were excluded to maintain consistent effect direction. Measurement and protocol Fresh decidual and villous tissues were collected from patients with spontaneous abortion and those undergoing elective abortion (as controls) immediately after surgical evacuation. The samples were divided into three portions: one was fixed in polyformaldehyde and embedded in paraffin, one was preserved in RNA preservation solution for gene expression tests, and the third was stored at -80 ℃, prepared for protein analysis. Rt-qPCR, Wb, IHC, IF, and IF colocalization analyses were performed using standard protocols. For IF co-localization analyses, frozen sections were fixed in neutral fixative for 30 min and underwent antigen retrieval by microwave heating, and after blocking with 3% H 2 O 2 and BSA, they were incubated with primary antibodies (MIF/MCSF combined with CD68 or CK7) overnight at 4 ℃. HRP-conjugated secondary antibodies and tyramide signal amplification (TSA) system were used for fluorescent labeling. Between different antigen detections, microwave treatment was performed to remove previous antibodies while preserving covalently bound fluorophores. Nuclei were counterstained with DAPI, and Images were captured using fluorescence microscopy. All the other detailed conditions and reagents in the different methods are provided in Table S2 . Patient and Public Involvement Considering the content of our research, no patients or the public were involved in the design, conduct, reporting, or dissemination plans of our research. It’s not appropriate or possible. Statistical Analysis MR analyses were performed using the R package ”TwoSampleMR” (version 0.6.8) in RStudio (version 4.4.1). Among the five MR methods available in this package, inverse variance weighted (IVW) was employed as the primary analysis method, which assumes all genetic instruments are valid and independent, using the inverse of the variance as weights. Results are presented as odds ratios (OR) with 95% confidence intervals (CI). MR-Egger regression and weighted median served as complementary methods. MR-Egger accounts for potential directional pleiotropy through an intercept term and assumes instrument strength independent of direct effect, while weighted median provides reliable estimates when at least 50% of the instruments are valid. Simple mode and weighted mode methods were also applied as additional robustness checks 24 . For sensitivity analyses, heterogeneity using Cochran’s Q test ( P >0.05 indicating no significant heterogeneity) was evaluated in both MR-Egger and IVW. Horizontal pleiotropy was assessed through the MR-Egger intercept test ( P >0.05 suggests no significant pleiotropy) and MR-PRESSO analysis (number of simulations=1000) for outlier identification 25 . Leave-one-out analyses were performed to evaluate the influence of individual SNPs on the causal estimates. To address the influence of potential sample overlap between databases, linkage disequilibrium score regression (LDSC) was performed using the R package “MRlap” (version 0.0.3.2). Lamba-gc0.05 suggest no significant weak instrument bias and winner‘s curse 26 . Comparisons between the SA and control groups in baseline characteristics were performed using Student’s t-test based on data distribution. P < 0.05 (2-tailed) was considered statistically significant. For the result of RT-qPCR, gene expression levels were normalized to housekeeping genes and calculated using the 2^ -ΔΔCt method. In WB analysis, protein bands were visualized using enhanced chemiluminescence and quantified by densitometry. For IHC studies, images were captured using confocal microscopy, and co-localization was analyzed using ImageJ software. Results 1. Bidirectional two-sample MR analysis Result of the MR analysis of 40 inflammatory cytokines and SA is depicted in Figure 2 . Three inflammatory cytokines were found causally associated with the risk of SA, including IL-17 (OR: 1.07; 95% CI: 1.01-1.14; P =0.018), MCSF (OR: 1.04; 95% CI: 1.00-1.08; P =0.040), and MIF (OR: 1.05; 95%CI: 1.01-1.11; P =0.026). The results of the anti-direction MR analysis (treating SA as the exposure and inflammatory cytokines as the outcomes) are presented in Figure S1 . SA was associated with three inflammatory cytokines, IL-17 (OR: 1.26; 95%CI: 1.01-1.57; P =0.037), IL-8 (OR:1.41; 95%CI: 1.02-1.96; P =0.039), and GCSF (OR: 1.24; 95%CI: 1.00-1.53; P =0.049). Details of SNPs included in the analysis are presented in Table S3. The trends of each inflammatory cytokine above under the five MR methods were presented by scatter plot in Figure S2 . Among all cytokines, IL-17 demonstrated bidirectional causal relationships with SA, while MIF and MCSF showed only unidirectional associations. These findings highlighted that MIF and MCSF play potential roles as causal factors rather than consequences of SA. Sensitivity Analysis The results of the test of heterogeneity and pleiotropy are presented in Table S4 , respectively, implying that there was no significant heterogeneity or pleiotropy in positive forward-direction results ( P >0.05). The leave-one-out analysis results are depicted in Figure S3 , showing that no SNPs badly influence the main results. LDSC results are presented in Table S5 , indicating no significant sample overlap ( P >0.05). Validation of MIF and MCSF Expression in SA Tissues To validate the MR findings, the expressions of MIF and MCSF were examined in decidual and villous tissues from SA patients and controls using multiple techniques (revealed in Figure 3 ). The Mean maternal age, gravidity, and parity showed no significant differences between the two groups ( Table S6 ). Both MIF and MCSF mRNA levels were quantified by RT-qPCR analysis, and the elevation in SA decidual and villous tissues showed statistically significant ( Figure 3a ). Compared to villous tissues, MIF and MCSF showed higher mRNA expression in decidual tissues regardless of pregnancy outcome. IHC and WB analysis revealed increased expression of both MIF and MCSF in SA decidual and villous tissues ( Figure 3b-d ). In decidual tissues, MIF and MCSF proteins were significantly elevated in SA samples compared to controls ( P <0.05), while both proteins in villous tissues showed increasing trends without reaching statistical significance in WB results. Single-color IF further confirmed these expression patterns ( Figure 3e ). Strong MIF and MCSF fluorescence signals were observed in SA decidual tissues, while the enhancement in villous tissues was more variable. Cellular Localization and Distribution of MIF and MCSF at the Maternal-Fetal Interface To further investigate the expression and distribution patterns of MIF and MCSF at the maternal-fetal interface, dual-color IF co-localization studies were performed using CK7 as a trophoblast-specific marker for villous tissues and CD68 as a macrophage-specific marker for decidual tissues (shown in Figure 4 ). The expression of both CK7 and CD68 remained unchanged across all tissue groups. In villous tissues, MIF and CK7 displayed separate distribution areas in normal samples (with minimal overlap). While in SA tissues, much more co-localization between MIF and CK7 was observed, suggesting increased MIF expression in trophoblast ( Figure 4a merge ). Similarly, normal decidua showed MIF and CD68 distributed in separate regions, while SA decidua demonstrated dramatically increased MIF expression that essentially covered the entire CD68-positive area, indicating MIF extensive co-localization with macrophages ( Figure 4b merge ). MCSF showed co-localization with CK7 in villous tissues of both SA and normal controls, while the expression level significantly increased in SA patients ( Figure 4d merge ). In decidual tissues, MCSF and CD68 displayed scattered, separate distribution in normal controls, whereas clear co-localization trends between MCSF and CD68-positive macrophages were observed in SA decidua ( Figure 4e merge ). Discussion 1. Main findings To date, this is the first study combining Mendelian randomization analysis with comprehensive tissue-based molecular validation to establish causal relationships between inflammatory cytokines and spontaneous abortion. In this study, elevated MIF and MCSF were identified as key players in SA development. Tissue-based validation provided further evidence supporting the MR findings. The strong MIF and MCSF signals in SA decidual tissues highlighted the cytokines’ upregulation at the maternal-fetal interface. These dysregulations of maternal immune imbalance might be the primary driver of pregnancy loss. Interpretation Spontaneous abortion remains a significant reproductive health challenge 1 . Despite numerous observational studies linking cytokines to pregnancy loss, the task of establishing causal relationships with SA remains challenging due to inherent study limitations. Recent research has identified specific cytokine alterations: a study of 116 Asian patients revealed CCL1 enrichment in decidual tissue, while another investigation of 25 participants demonstrated that trophoblast-derived CXCL16 at the maternal-fetal interface induces M2 macrophage polarization and subsequently modulates NK cell function 27,28 . However, observational findings are limited by potential confounding factors and reverse causation, necessitating MR analysis to establish causal relationships. In this study, MR analysis revealed that elevated MIF and MCSF played an important role in SA development. Previous studies indicated that both MIF and MCSF were involved in regulating the M1/M2 macrophage balance, which was crucial for successful pregnancy 29,30 . MIF, a potent pro-inflammatory cytokine, promotes macrophage activation and inflammatory responses, with elevated serum levels documented in various inflammatory diseases 31,32 . During normal pregnancy, serum MIF levels are moderately elevated compared to non-pregnant states but remain lower than in inflammatory conditions, suggesting a delicate balance between its physiological role in pregnancy maintenance and potential pathological effects when over-expressed at the maternal-fetal interface 5,33 . MCSF is fundamental for macrophage homeostasis in vivo and exhibits cyclic elevation in numerous pathological states, including infections, cancer, and chronic inflammatory diseases 34-36 . In an immune atlas study of human decidua, the decreased sub-population of MCSF+ expressing dNK cells was identified in URPL deciduals 7 . It’s reported to be elevated in pre-eclampsia patients, modulating the decidual immune balance by inducing M2 macrophage polarization and phagocytic capacity in response to pro-inflammatory stimuli 30 . A current MR study examining causal associations between cytokines and ten pregnancy-related adverse outcomes identified MCSF as causally linked to spontaneous abortion 37 . While genetic analysis established causal relationships, experimental validation is essential to elucidate the specific mechanisms through which cytokines contribute to pregnancy loss. Furthermore, MIF and MCSF represented obvious elevations in SA decidual and villous tissue in the current study. Notably, enhanced co-localizations of both cytokines with CD68-positive macrophages and CK7-positive trophablasts were observed, without alterations in the number of these cellular populations. Combining those informations, a model of functional rather than quantitative immune dysregulation caused by elevated MIF and MCSF may be tenable in SA pathogenesis. These might suggest that MIF and MCSF could serve as prognostic or early identification biomarkers for SA risk in high-risk pregnancies, and offer a more targeted approach for SA prevention. Given the prevalence of SA, integrating these inflammatory markers into routine prenatal care might substantially reduce the burden of pregnancy loss and associated healthcare costs at the population level. In previous experimental evidence, IL-17 has been reported to cause an increase in abortion rate in mouse models, and observed to be related to recurrent pregnancy loss by interleukin-17-producing T cells 38 . However, in our MR analysis, IL-17 demonstrated bidirectional positive associations, revealing genetically determined two-way causal relationships with SA. While GCSF and IL-8 have been previously associated with SA in observational studies, our bidirectional analysis revealed these cytokines only as consequences of SA 5,8 . The anti-directional nature of these associations suggests that IL-17, GCSF, and IL-8 levels may represent downstream effects of pregnancy loss. Given our study’s focus on the causation of SA, we did not valid all three cytokines from further investigation. Strengths and Limitations Our innovative approach leverages the power of genetic epidemiology through two-sample MR analysis across multiple large-scale European cohorts (totaling over 250,000 individuals), followed by molecular validation in Asian populations using multiple complementary techniques, including RT-qPCR, Western blot, IHC, and dual-color IF co-localization. However, several limitations should be considered when interpreting this finding. While MR analysis provides strong evidence for causal relationships, the genetic instruments explain only a small proportion of cytokine variation, and pleiotropy remains a potential concern despite our sensitivity analyses. Secondly, the correlation between circulating cytokines levels used in MR analysis and local tissue concentrations remains unclear, which may affect the direct translation of genetic findings to tissue pathology. Last, population heterogeneity between European-ancestry MR cohorts and our Asian validation samples may contribute to some inconsistent findings, too. Future studies should include prospective cohort designs with serial cytokine measurements, larger multi-ethnic populations for both genetic and molecular analyses, and functional validation through in vitro and animal models to definitively establish causal mechanisms and therapeutic targets. Conclusion Our study suggests that the abnormal elevation of MIF and MCSF likely represents key causative factors in SA pathogenesis. The specific localization and distribution patterns at the maternal-fetal interface, particularly their enhanced co-localization with decidual macrophages and villous trophoblasts, imply these cytokines may drive SA through aberrant cellular behavior in the maternal-fetal interface. The possibility that MIF and MCSF induce pathological decidual macrophage polarization deserves further investigation as a potential mechanistic pathway in SA development. Declaration Author contribution Yilin Fan: Methodology, Software, Validation, Formal analysis, Investigation, Data curation, Writing - Original Draft, Writing - Review & Editing, Visualization; Feipeng Cui: Conceptualization, Methodology, Software, Formal analysis, Writing - Review & Editing, Supervision, Project administration; Peng Gao: Conceptualization, Methodology, Validation, Writing - Original Draft, Writing - Review & Editing, Supervision, Project administration, Visualization; Huan Tang: Formal analysis, Resources, Investigation, Data curation, Visualization; Liwen Zhou: Formal analysis, Resources, Investigation, Data curation; Jiaxi Chen: Formal analysis, Investigation, Data curation; Shuhao Yang: Formal analysis, Resources, Investigation, Data curation; Yumin Liu: Investigation, Resources; Zhou Li: Investigation, Resources; Yuqi Li: Investigation, Resources; Xingguang Lin: Investigation, Resources; Jianli Wu: Investigation, Resources; Xun Gong: Investigation, Resources; Xinwei Shi: Investigation, Resources; Jingjing Xu: Investigation, Resources; Yingjia Hu: Formal analysis, Data curation; Wei Tu: Investigation, Resources; Haiyi Liu: Conceptualization, Methodology, Resources, Writing - Review & Editing, Supervision, Project administration, Funding acquisition; All authors have read and approved the final manuscript. Funding statement This work was supported by commercial research funds of Tongji Hospital, project number 2023162, and the Postgraduate scientific and technological innovation project funds of Hubei Medical College, project number YC202572. Conflict of Interest Statement No potential conflict of interest was reported by the authors. Acknowledgments We would like to express our sincere gratitude to all those who contributed to this study. Particularly, we are grateful to all the patients who participated in this research. Special thanks go to staff from NHC Specialty Laboratory of Food Safety Risk Assessment and Standard Development and the School of Public Health, Tongji Medical College, for their contributions to the experimental design and data analysis. And we appreciate the technical support from the staff at the Department of Obstetrics and Gynecology, Tongji Hospital, for their assistance with tissue sample processing and immunological analyses. Also, we greatly thank the teams behind the FinnGen and UK Biobank databases for making their valuable data available for research. The computational resources provided by the Tongji Medical College were instrumental in completing our analyses. We would also like to express our appreciation to all the editors and reviewers for their careful consideration of our manuscript and constructive suggestions. Regardless of the outcome, your feedback and comments have helped improve the quality and focus of our paper and made this research better. Ethical statement The study was approved by the institutional ethics committee at 2024.1.23(approval number: TJ-IRB202401107). The study protocols were approved by the Ethics Committee of Tongji Hospital, Huazhong University of Science and Technology, and all participants provided written informed consent. Data Availability Statement The data that support the findings of this study are openly available in the article at http://doi.org/ 10.1016/j.ajhg.2016.11.007 23 , article at http://doi.org/10.1038/s41586-018-0175-2 39 , FinnGen database (R11 version) by reference number ICD-10 code O03 and UK Biobank (UKBB) by reference number data field 2774, and are all able to be accessed from IEU OpenGWAS project at https://gwas.mrcieu.ac.uk/. References 1 Dimitriadis E, Menkhorst E, Saito S, Kutteh WH, Brosens JJ. Recurrent pregnancy loss. Nature reviews. Disease primers. 2020;6:98. 2 Quenby S, Gallos ID, Dhillon-Smith RK, Podesek M, Stephenson MD, Fisher J, et al. Miscarriage matters: the epidemiological, physical, psychological, and economic costs of early pregnancy loss. Lancet (London, England). 2021;397:1658-67. 3 Eschenbach DA. Treating spontaneous and induced septic abortions. Obstet Gynecol. 2015;125:1042-8. 4 Garrido-Gimenez C, Alijotas-Reig J. 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Keywords general obstetrics miscarriage reproductive science: cytokines Authors Affiliations Yilin Fan Huazhong University of Science and Technology Tongji Medical College View all articles by this author Feipeng Cui Huazhong University of Science and Technology Tongji Medical College Tongji Hospital View all articles by this author Peng Gao Huazhong University of Science and Technology Tongji Medical College Tongji Hospital View all articles by this author Huan Tang Huazhong University of Science and Technology Tongji Medical College Tongji Hospital View all articles by this author Liwen Zhou Hubei Provincial Center for Disease Control and Prevention View all articles by this author Jiaxi Chen Hubei Provincial Center for Disease Control and Prevention View all articles by this author Shuhao Yang Huazhong University of Science and Technology Tongji Medical College Tongji Hospital View all articles by this author Yumin Liu Huazhong University of Science and Technology Tongji Medical College Tongji Hospital View all articles by this author Zhou Li Huazhong University of Science and Technology Tongji Medical College Tongji Hospital View all articles by this author Yuqi Li Huazhong University of Science and Technology Tongji Medical College Tongji Hospital View all articles by this author Xingguang Lin Huazhong University of Science and Technology Tongji Medical College Tongji Hospital View all articles by this author Jianli Wu Huazhong University of Science and Technology Tongji Medical College Tongji Hospital View all articles by this author Xun Gong Huazhong University of Science and Technology Tongji Medical College Tongji Hospital View all articles by this author Xinwei Shi Huazhong University of Science and Technology Tongji Medical College Tongji Hospital View all articles by this author Jingjing Xu Huazhong University of Science and Technology Tongji Medical College Tongji Hospital View all articles by this author Yingjia Hu University of Pittsburgh School of Public Health View all articles by this author Wei Tu Huazhong University of Science and Technology Tongji Medical College Tongji Hospital View all articles by this author Haiyi Liu [email protected] Huazhong University of Science and Technology Tongji Medical College Tongji Hospital View all articles by this author Metrics & Citations Metrics Article Usage 174 views 129 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Yilin Fan, Feipeng Cui, Peng Gao, et al. Causal relationship between MIF/MCSF and Spontaneous Abortion: Insights from Mendelian Randomization and population Validation. Authorea . 07 August 2025. DOI: https://doi.org/10.22541/au.175454652.28794630/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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