Background: The pathogenesis of herniated nucleus pulposus (HNP) is complex and may involve the wide variety of gene polymorphism. However, the reports from the existing studies are inconclusive. The objective of this study was to determine the role of single nucleotide polymorphisms (SNPs) in interleukin 1 alpha (IL-1A), tumor necrosis factor-alpha (TNF-A), and vitamin D receptor (VDR) genes on the susceptibility to herniated nucleus pulposus (HNP).Methods: Four databases  were searched as of April 1st, 2021. Authors, publication year, targeted genes, genotype and allele frequency in each case and control groups were collected. Newcastle-Ottawa scale was used to evaluate the publication quality. The pooled estimates of association ofIL-1A -889C>T (rs1800587),TNF-A -238G>A (rs361525), andVDR TaqI (rs731236) and susceptibility to HNP were assessed using Z test.Results: We screened 3,067 unique studies for eligibility and three, two and nine case-control studies onIL-1A -889C>T,TNF-A -238G>A, andVDR TaqI were included, respectively, in our meta-analysis. The studies consisting 369 HNP cases and 433 controls forIL-1A -889C>T, 252 cases and 259 controls forTNF-A -238G>A and 1130 cases and 2096 controls forVDR TaqI. Our pooled estimates indicated that there was no significant association of those SNPs with the susceptibility to HNP in any genotype, dominant model, recessive model, or allele comparations.Conclusion: Although individual studies suggested the important role of gene expression dysregulation associated with SNPs inIL-1A,TNF-A, andVDR, our data indicated thatIL-1A -889C>T,TNF-A -238G>A, andVDR TaqI had weak association with HNP susceptibility in both genotypes and allele distributions. However, since heterogeneity was identified among studies included in this meta-analysis, further meta-analysis with a larger population and subgroup analysis on specific population are warranted to support this finding. However, it has been suggested that genetic factors also play a vital role in susceptibility to disc degenerative diseases. A study showed that individuals aged younger than 30 years who have a family history of disc herniation have a 14.5 times higher risk of developing disc protrusion than individuals who have no family history.8 Family history is also attributed to a 5.1 times higher risk of disc herniation in people aged between 30-50 years old.8 The Twin Spine Study found that heredity substantially influences disc degeneration by 43-77%.11Herniated nucleus pulposus (HNP) or disc herniation is the most common spinal degenerative disease associated with lower back pain and radicular pain of the lower extremities due to nerve compression.12 where proteoglycans (mostly found in NP) acts as an internal semi-fluid mass and collagen (mostly found in AF) acts as a laminar fibrous container.13 Genes encoding components of IVD such as collagens I,14 collagens IX,15 collagens XI,16 aggrecan,17 cartilage intermediate layer protein (CILP),18 and vitamin D receptor (VDR)19 have previously been studied to determine susceptibility to lumbar disc diseases. Other factors such as increased production of extracellular matrix-degrading enzymes  andMMP-920 and increased expression of inflammatory cytokines such as interleukin-1 alpha (IL-1A), IL-18,21 IL-6, and tumor necrosis factor-alpha (TNF-α)22) are commonly found in disc degeneration. Excessive synthesis, secretion, and biological activity of these inflammatory mediators are associated with tissue destruction and are therefore commonly found in inflammatory disorders including disc degeneration.23The intervertebral disc (IVD) consists of two different components: the nucleus pulposus (NP) and the annulus fibrosus (AF),IL-1A -889C>T (rs1800587).24 People with minor allele ofIL-1A -889C>T  also had a 2.4-fold increased risk of disc bulges24 and a 2.5-fold increased risk of endplate modic change.25 A study in an Iranian population found that among nine SNPs on pro-inflammatory cytokine genes , no association to IVD degeneration was found except for two SNPs in theTNF-A gene (TNF-A−308 G/A andTNF-A −238 G/A).19 TNF-α plays important role in the pathophysiology of HNP such as upregulating the activity and the gene expression of MMP, stimulating other cytokines such as IL-1, IL-6, and IL-8, stimulating cell migration, altering endothelial permeability, and decreasing the synthesis of collagen and proteoglycan.26 A study reported that G allele and GG genotype ofTNF-A 238G>A (rs361525) were 2.51 times and 2.98 times, respectively, more prevalent in patients with HNP compared to healthy controls.19One of the mechanisms that alters the production of protein mediators in the human body are single-nucleotide polymorphisms (SNPs). These genetic variations, single nucleotide changes at specific positions in a gene, may influence gene expression and hence associate to particular disease. A three-fold increase in susceptibility of disc degeneration was observed in individuals with a TT genotype compared to those without the allele (CC genotype) on SNP27 VDR's role in spinal degenerative disorder has been studied in Italian,28 Turkish,29 and Southern European populations.30 A study in a Chinese population suggested that subjects with the t allele ofVDR TaqI (rs731236) had a 2.61 times higher risk to have degenerative disc disease.31 Moreover, individuals aged younger than 40 years who had the t allele were almost six times more likely to develop disc degeneration and 7.17 times more likely to develop disc bulge compared to those without the t allele.31 However, studies in Danish32 and Mexican populations33 contradict previous results suggesting a role ofVDR TaqI in disc degenerative disease. This conflicting role of SNPs inIL-1A, TNF-A andVDR on HNP therefore needs to be further evaluated. This study sought to determine the association ofIL-1A -889C>T (rs1800587),TNF-A 238G>A (rs361525), andVDR TaqI (rs731236) in susceptibility to HNP.Several roles of VDR such as regulating chondrocyte proliferation and differentiation, bone mineralization and remodeling, and matrix production have previously been demonstrated.IL-1A (rs1800587),TNF-A (rs361525), andVDR (rs731236), on susceptibility to HNP. The outcome variable of this study was the risk or susceptivity to have HNP while the response variables were the SNPs in three genes:IL-1A (rs1800587),TNF-A (rs361525), andVDR (rs731236). We searched databases for relevant studies, then extracted and analyzed data from those studies to achieve the pooled odds ratios (ORs) and 95% confidence interval (95%CI) using a random or fixed effect model depending on the data. This study was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guideline.35 The protocol of this study has been registered inPROSPERO (reg. numberCRD42021249187).A systematic review and meta-analysis were conducted to assess the association of three SNPs,st, 2021. The keywords were adapted from Medical Subject Heading (MeSH). Additional studies were also retrieved from the references of relevant papers. If two or more studies with the same study data were identified, the most recent study was used. The processes were conducted by three independent authors .The literature searches were conducted on PubMed, Embase, Cochrane, and Web of Science. The searches were conducted using the keywords:‘degenerative disc disease’ AND (‘IL-1A’ OR ‘rs1800587’ OR ‘-889C>T’) OR (‘TNF-A’ OR ‘rs361525’ OR ‘-238G>A’) OR (‘VDR’ OR ‘rs731236’ OR ‘TaqI’) AND ‘gene polymorphism’, including all results up to April 1IL-1 (rs1800587),TNF-A (rs361525), orVDR (rs731236) on HNP and have case and control groups; and  and consensus established together with senior authors  if discrepancies were found.37 by three independent authors . This evaluation was conducted to ensure the quality of three fundamental methodological parameters of the studies: patient selection (four points), comparability of the groups (two points), and ascertainment of exposure (three points); NOS ranged from 0 to 9. Each study was then categorized based on the NOS: (1) good quality (NOS ≥ 7); (2) moderate quality (NOS ≥ 5); or (3) poor quality (NOS < 5). Consensus was established if discrepancies were found.The quality of the included studies was evaluated using Newcastle-Ottawa Score (NOS)The outcome measure in our study was the incidence of HNP while the predictor covariates were the gene polymorphisms of IL-1A (rs1800587), TNF-A (rs361525), and VDR (rs731236). All genetic models were applied to describe the role of each gene variant in the pathogenesis of HNP. For IL-1A (rs1800587), the allele models were C vs. T and T vs. C; and the genotype models were CC vs. CT + TT, CT vs. CC + TT, and TT vs. CC + CT. For TNF-A (rs361525), the allele models were G vs. A and A vs. G; and the genotype models were GG vs. GA + AA, GA vs. GG + AA, and AA vs. GG + GA. For VDR (rs731236), the allele models were T vs. C and C vs. T; and the genotype models were TT vs. TC + CC, TC vs. TT + CC, and CC vs. TT + TC.IL-1A (rs1800587),TNF-A (rs361525), andVDR (rs731236) on HNP, a Z-test was employed. The Egger test was used to evaluate the publication bias and a p < 0.05 indicated the possibility of publication bias in each calculated result. The Q test was used to evaluate the heterogeneity and decide between random and fixed-effect models for OR calculation. If heterogeneity was indicated , the random effect model was used; otherwise, the fixed-effect model was used. All analyses were performed using ‘meta’,38 ’ metafor’,39 and ‘dmetar’40 packages inR version 4.0.4.41To assess the association ofIL-1A (rs1800587),43 two studies forTNF-A (rs361525),44 and nine studies forVDR (rs731236).51 The summary of studies included in the meta-analysis is presented inThe literature searches yielded 3,199 articles of which 3,067 references were retained after removing duplicates. Screening of the titles and abstracts excluded 2,965 articles as they did not meet the inclusion criteria. After a further screening of full text, an additional 90 studies were excluded due to lack of relevance (n = 90), incomplete data (n = 5), and HWE deviation (n = 6) . 12 studIL-1A -889C>T was 1.37 more frequent in HNP patients than in controls, while the distribution of alleles and other genotypes were similar between patients and healthy controls. CT genotype and T allele ofTNF-A 238G>A were both 1.6 times more frequent in healthy controls than in HNP cases. No difference in the distribution of alleles or genotypes between HNP and controls was observed inVDR TaqI.Our data indicated that the TT genotype ofDR TaqI.IL-1A -889C>T genotypes were associated with the risk of HNPs with CC vs. CT+TT , CT vs. CC+TT , and TT vs. CT+CC   . The pooTNF-A 238G>A also had no significant association with the risk for HNP. No association was observed between genotype models and the risk of HNP: GGvs. GA+AA , GAvs. GG+AA , and AAvs. GG+GA   . DistribVDR TaqI (rs731236) suggested that none of the genotypes were associated with susceptibility to degenerative disc disease HNP with OR: 2.65; 95%CI: 0.60, 11.85 for TTvs. TC+CC, OR: 1.01; 95%CI: 0.55, 1.85 for TC vs. TT+CC and OR: 0.94; 95%CI: 0.69, 1.28 (VDR TaqI (rs731236) were associated with HNP; people with T allele had OD 1.06 with 95%CI: 0.91, 1.22 for HNP.Our estimates for genotypes of9, 1.28 . None of52,53 Another study also found no association between TNF-A gene variants and the risk of bone-joint and spinal tuberculosis.54 Furthermore, a study assessing the role of VDR gene polymorphism in the case of osteoarthritis also failed to show the potential contribution of VDR gene variant in the development of osteoarthritis.55 These evidences suggested that, while theoretically the gene variants of IL-A, TNF-A, and VDR may contribute to the development of bone diseases, the evidence reveals otherwise. For the negative findings of our study, several possible reasons might be proposed. First, HNP is a complex disease and is cause by multiple factors. Thus, no single factor such as a single SNP is responsible for the whole pathogenesis. Second, large variations in the number of samples or allele frequencies among studies in our meta-analysis also contribute to the findings. This probably relates to differences in populations where study data were collected. Finally, the small number of samples significantly influenced the results of our meta-analysis. Therefore, studies with larger sample sizes and sub-analyses for different populations such as Asian, Caucasian, and other populations are warranted whenever more data are available.Our present failed to clarify the role of IL-1A (rs1800587), TNF-A (rs361525), and VDR (rs731236) on the pathogenesis of HNP. To the best of our knowledge, our current study is the first study providing the holistic gene polymorphism in the case of HNP. Therefore, the comprehensive comparison in the context of methodological quality between our study and previous studies was unable to perform. The role of gene polymorphism of IL-1A (rs1800587) was rarely reported. Studies found that IL-1A (rs1800587) has no association with rheumatoid arthritis and systemic sclerosis.56 For example, the degeneration of disc organization could occur during aging as the regulation of the extracellular matrix (ECM), a major component of the disc, is damaged during the aging process.56 The herniated disc or HNP may occur as a result of several pathological mechanisms. Those mechanisms ultimately cause imbalances in disc composition that are directly linked to the quality of the ECM.56 Therefore, the balance between the ECM and its degrading enzymes, such as matrix metalloproteases (MMPs), seems to be the key to maintaining normal disc function. Interestingly, of several mechanical pathways, the regulation of the ECM and the MMPs could also be determined by the occurrence of single nucleotide polymorphisms in genes responsible for ECM regulation as explained below.57HNP occurs when the central part of the intervertebral disc, the nucleus pulposus, herniates through the surrounding part of the disc, the annulus fibrosus. The damage to the annulus fibrosus resulting in the herniated disc may be associated with factors such as gender, age, certain activities such as lifting of weights and carrying, and being overweight.56 The main proteoglycan found in the normal intervertebral disc is aggrecan.58 Although both annulus fibrosus and nucleus pulposus are mainly composed of water, proteoglycan, and collagen, the level of those contents differ between the structures. The annulus fibrosus consists of 70% water, 15% collagen, and 5% proteoglycan, while the composition in the nucleus pulposus is 77% water, 4% collagen, and 14% proteoglycan.56 Any event causing disturbances in those ratios and/or in synthesis and/or degradation of those structural proteins could lead to herniated disc problems. For example, hypoxic and acidic conditions could repress the synthesis of the matrix leading to the dysfunctionalities of the cells of the disc.60Two major structural proteins that are important in the matrix structure of the disc are collagen and proteoglycan.63 A controlled immunohistochemical study observed the accumulation of inflammatory cells, mainly macrophages, in herniated disc cells indicating the role of proinflammatory cytokines in the disease.62 For example, IL-1β could induce the annulus fibrosus to generate inflammatory factors leading to the impairment of proteoglycan aggregation.63 Another cytokine, TNF-α, is also involved in the development of intervertebral disc problems.64 However, it seems that its effect is less significant than IL-1.65 This finding may be related to its relatively lower expression compared to IL-1 in the normal and healthy disc.66The role of inflammation in predisposing the disc to damage has also been revealed. Specifically, proinflammatory cytokines are found to play a specific role in herniated and degenerated discs.67 Therefore, in addition to its pivotal role in maintaining sufficient hydration to the disc, proteoglycan loss could stimulate the movement of cytokines towards the disc activating the inflammation cascade.56Penetration of those inflammatory cells or proteins could be caused by matrix loss. In normal conditions, aggrecan should prevent the penetration of various compounds, especially serum proteins and cytokines.71 The exaggerated activity of MMPs causes excessive degradation of collagen and proteoglycan.58 Another mechanism is associated with the activity of the cytokines in inhibiting tissue inhibitors of MMPs (TIMPs) which are responsible for terminating the action of MMPs.58 Taken together, those actions ultimately impair disc functionality.One of the mechanisms by which the proinflammatory cytokines, such as IL-1α and TNF-α, generate problems in the intervertebral disc is related to their effect on inducing MMP production.56 the nutritional supply to the disc depends on the ability of the nutrients to diffuse from the closest vascularized structure outside the disc which are the vertebral bodies.72 The nutrients then penetrate the cartilaginous endplate and finally reach the annulus fibrosus and nucleus pulposus.73 Accordingly, calcification of the endplate would diminish diffusion of vital nutrients, leading to the death of the disc cells.74 Therefore, VDR plays a critical role as this ligand-dependent transcription factor is involved in regulating calcium homeostasis and bone mineralization in the body, including in the intervertebral disc.76 It has been known that genetic polymorphisms occurring in genes encoding VDR are associated with intervertebral disc problems,76 including herniated disc.As the normal intervertebral disc is relatively avascular and aneural,Our current study had several limitations. First, the pertinent confounding factors that might affect the final findings of our study were not included in analysis, such as mechanical and behavioral factors and the levels of proteoglycans. Second, since the included articles in our current study were non-randomized controlled trials (RCTs), the final findings might have the higher risk of bias. Therefore, the up-coming meta-analysis was expected to involve only RCTs studies. Third, the limited reports on the context of gene polymorphism in HNP had made our study included the limited number of studies. Therefore, our findings should be interpreted with caution.In conclusion, our results suggest that well-regulated IL-1A, TNF-A, and VDR are important for normal intervertebral discs and that dysregulation of these could negatively affect the intervertebral discs. Some individual studies found that SNPs in IL-1A (rs1800587), TNF-A (rs361525), and VDR (rs731236) were associated with the susceptibility to HNP, however, our meta-analysis suggested that the effects are not robust.All data underlying the results are available as part of the article and no additional source data are required.https://doi.org/10.6084/m9.figshare.14479233.77Figshare: PRISMA checklist for ‘The role of single nucleotide polymorphisms of IL-1A -889C>T (rs1800587), TNF-A -238G>A (rs361525), and VDR TaqI (rs731236) on susceptivity of herniated nucleus pulposus’, The title has been added and clarified with the study design “Systematic Review and Meta-Analysis” according to the previous comment. Abstract:No further comment. Introduction:No further comment. Materials and Methods:No further comment. Result and discussion:The discussion has been strengthened with comparison with previous studies that discussed about IL-1A, TNF-A, or VDR generally according to the previous comment. Figures and Tables:No further comment. Conclusion:No further comment.General remark:This current manuscript version may be accepted for indexing. Overall comments:Are the rationale for, and objectives of, the Systematic Review clearly stated?YesIs the statistical analysis and its interpretation appropriate?PartlyAre sufficient details of the methods and analysis provided to allow replication by others?YesAre the conclusions drawn adequately supported by the results presented in the review?YesReviewer Expertise:Stem cell and Tissue EngineeringI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The study design on the title has not been added, I noticed that there is already a design label “Systematic Review” written above the title from the F1000 platform but since this manuscript will be added to some online databases such as Pubmed and Scopus, I recommend to put the design on the title. Also, please clarify if this is a meta-analysis or systematic review. As it is still written “Systematic review” above the title. Abstract:IL-1A), tumor necrosis factor-alpha (TNF-A), and vitamin D receptor (VDR) genes on the susceptibility to herniated nucleus pulposus (HNP)” according to the previous comment.The problem that this review wants to answer is added “this study was to determine the role of single nucleotide polymorphisms (SNPs) in interleukin 1 alpha , TNF-A (rs361525), and VDR (rs731236)”. Please strengthen the discussion with comparison with previous studies that discussed IL-1A, TNF-A, or VDR as general.General remark:No comment. ConclusionThe conclusion has been revised according to the previous comment, answering the research question. The conclusion is current existed studies are not strong enough to answer the question. Hence, further larger population and subgroup analyses are warranted to support these findings. Figures and Tables:Are the rationale for, and objectives of, the Systematic Review clearly stated?YesIs the statistical analysis and its interpretation appropriate?PartlyAre sufficient details of the methods and analysis provided to allow replication by others?YesAre the conclusions drawn adequately supported by the results presented in the review?YesReviewer Expertise:Stem cell and Tissue EngineeringI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The study design on the title has not been added, I noticed that there is already a design label “Systematic Review” written above the title from the F1000 platform but since this manuscript will be added to some online databases such as Pubmed and Scopus, I recommend to put the design on the title. Also, please clarify if this is a meta-analysis or systematic review. As it is still written “Systematic review” above the title. RESPONSE: We have added the a "systematic review and meta-analysis" in our title. General remark:IL-1A), tumor necrosis factor-alpha (TNF-A), and vitamin D receptor (VDR) genes on the susceptibility to herniated nucleus pulposus (HNP)” according to the previous comment.The problem that this review wants to answer is added “this study was to determine the role of single nucleotide polymorphisms (SNPs) in interleukin 1 alpha , TNF-A (rs361525), and VDR (rs731236)”. Please strengthen the discussion with comparison with previous studies that discussed IL-1A, TNF-A, or VDR as general. RESPONSE: Thank you for your suggestion. We have added some discussion based on previous studies.  Result and discussion:No comment. RESPONSE: Thank you. No further edit required.  Figures and Tables:The conclusion has been revised according to the previous comment, answering the research question. The conclusion is current existed studies are not strong enough to answer the question. Hence, further larger population and subgroup analyses are warranted to support these findings. RESPONSE: Thank you. No further edit required. Conclusion This is a systematic review and meta-analysis aiming at evaluating the impact of SNPs on susceptibility to HNP. The research question makes sense. The gap in the literature was well presented and the objective of the review was well formulated.The protocol of the systematic review was prospectively developed and registered in PROSPERO. The search strategy was comprehensive.The screening and study selection process were well described and illustrated in a PRISMA flow diagram. Methodology of quality assessment and statistical analysis was adequate.The results are well presented, summarized in tables, and illustrated in forest plots. The Discussion is comprehensible, well-describes the findings of this systematic review in comparison with the current body of evidence.Conclusions are justified by the findings.Thank you for the opportunity of reviewing this manuscript. It seems like the limitations were presented in the conclusion. I would separate them from the conclusions and move them as the last paragraph of the Discussion. I have one minor comment:Are the rationale for, and objectives of, the Systematic Review clearly stated?YesIs the statistical analysis and its interpretation appropriate?YesAre sufficient details of the methods and analysis provided to allow replication by others?YesAre the conclusions drawn adequately supported by the results presented in the review?YesReviewer Expertise:Clinical outcomes research and evidence synthesisI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. #Reviewer 2I have one minor comment: It seems like the limitations were presented in the conclusion. I would separate them from the conclusions and move them as the last paragraph of the Discussion.Response: The limitation has been provided in the end of discussion section. The conclusion has been revised as suggested. The title is descriptive about the manuscript, however, please state the study design on the title.To draw the interest of the reader, is it possible if the authors simplified the title from “IL-1A-889C>T(rs1800587)" to “IL-1A”, and also the same for TNF-A and VDR?  Abstract:Line 31, please state the problem that this review will answer.Line 35, in the methods section, please state the study design of included samples in this study to show the level of evidence of this meta-analysis.Line 58, for the conclusion, please kindly state the answer and summary of the research question, instead of restating limitation and result. Introduction:The Introduction is already well written. It gives enough information for the reader to understand the context. It also clearly explains why the study was necessary. Materials and Methods:As all the studies included in this review are a case-control, though it is uncommon for a systematic review meta-analysis to use a case control as a sample, please clarify and state that there is no Randomized Controlled Trial and Cohort study about this topic.https://ebm.bmj.com/content/23/2/60 to review the methodological quality of a case series.I noticed the authors used Newcastle Ottawa Score, please kindly checkPlease define the primary outcome in this review. Result and discussion:The results are presented clearly and accessibly, and it also supports the main conclusion.The discussion, has to be more detailed, from line 203- 244, the discussion mainly discusses the pathogenesis of the herniated nucleus pulposus. Please kindly deepen the discussion based on the result of this review, compare it with previous study(s), and explain the finding from the authors' point of view. Figures and Tables:Figures and Tables are adequately explained. Conclusion:The limitations are well stated, however, line 286-288 “Therefore, studies with larger sample sizes and sub-analyses for different populations such as Asian, Caucasian, and other populations are warranted whenever more data are available.” Please put in one paragraph before the conclusion to make the conclusion from your review stronger.General remark:This manuscript may be considered for indexing after Major revisions. Overall comments:Are the rationale for, and objectives of, the Systematic Review clearly stated?YesIs the statistical analysis and its interpretation appropriate?PartlyAre sufficient details of the methods and analysis provided to allow replication by others?YesAre the conclusions drawn adequately supported by the results presented in the review?YesReviewer Expertise:Stem cell and Tissue EngineeringI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. #Reviewer 1Abstract: Line 31, please state the problem that this review will answer.Response: We have added the problem that underly our study in abstract: “The pathogenesis of herniated nucleus pulposus (HNP) is complex, and may involve the wide variety of gene polymorphism. However, the reports from the existing evidence revealed inconclusive findings.”Abstract: Line 35, in the methods section, please state the study design of included samples in this study to show the level of evidence of this meta-analysis.Response: the study design of included articles has been added in the results section of abstract: “We screened 3,067 unique studies for eligibility and three, two and nine case-control studies on IL-1A -889C>T, TNF-A -238G>A, and VDR TaqI were included, respectively, in our meta-analysis.”Abstract: Line 58, for the conclusion, please kindly state the answer and summary of the research question, instead of restating limitation and result.Response: The main findings of our study has been outlined in the conclusion section of abstract: “our data indicated that IL-1A -889C>T, TNF-A -238G>A, and VDR TaqI had weak association with HNP susceptibility in both genotypes and allele distributions.” However, we also described about the clinical implication, limitation and future research direction to prevent the miss-understanding in interpretating the findings of our study.Introduction: The Introduction is already well written. It gives enough information for the reader to understand the context. It also clearly explains why the study was necessary.Response: thank you.Materials and Methods: As all the studies included in this review are a case-control, though it is uncommon for a systematic review meta-analysis to use a case control as a sample, please clarify and state that there is no Randomized Controlled Trial and Cohort study about this topic.Response: We agree that case-control studies are uncommon to include in meta-analysis. However, in the meta-analysis, we cannot manage the findings (the findings of article searching) as we expected if they are not supported by the available data. And unfortunately, in the searching strategy, we only found case-control studies. This point has also been included in the limitations of our study.https://ebm.bmj.com/content/23/2/60 to review the methodological quality of a case series.Materials and Methods: I noticed the authors used Newcastle Ottawa Score, please kindly check Response: We thank you for the suggestion. Case report and or case series studies may be possible to include in systematic review. However, they are not possible  to include in meta-analysis  because they do not have control.Materials and Methods: Please define the primary outcome in this review.Response: The primary outcomes have been provided in the Method section: “Covariates and sub-group analysis. The outcome measure in our study was the incidence of HNP while the predictor covariates were the gene polymorphisms of IL-1A (rs1800587), TNF-A (rs361525), and VDR (rs731236). All genetic models were applied to describe the role of each gene variant in the pathogenesis of HNP. For IL-1A (rs1800587), the allele models were C vs. T and T vs. C; and the genotype models were CC vs. CT + TT, CT vs. CC + TT, and TT vs. CC + CT. For TNF-A (rs361525), the allele models were G vs. A and A vs. G; and the genotype models were GG vs. GA + AA, GA vs. GG + AA, and AA vs. GG + GA. For VDR (rs731236), the allele models were T vs. C and C vs. T; and the genotype models were TT vs. TC + CC, TC vs. TT + CC, and CC vs. TT + TC.”Result and discussion: The results are presented clearly and accessibly, and it also supports the main conclusion.Response: Thank you.The discussion, has to be more detailed, from line 203- 244, the discussion mainly discusses the pathogenesis of the herniated nucleus pulposus. Please kindly deepen the discussion based on the result of this review, compare it with previous study(s), and explain the finding from the authors' point of view.Response: The explanation on our main findings and the holistic comparison including the possible reason of our findings has been provided: “Our present study failed to clarify the role of IL-1A (rs1800587), TNF-A (rs361525), and VDR (rs731236) in the pathogenesis of HNP. To the best of our knowledge, our current study is the first study providing the holistic gene polymorphism in the case of HNP. Therefore, the comprehensive comparison in the context of methodological quality between our study and previous studies was unable to perform. However, several possible reason for the negative findings in our study might be proposed. First, HNP is a complex disease and is cause by multiple factors. Thus, no single factor such as a single SNP is responsible for the whole pathogenesis. Second, large variations in the number of samples or allele frequencies among studies in our meta-analysis also contribute to the findings. This probably relates to differences in populations where study data were collected. Finally, the small number of samples significantly influenced the results of our meta-analysis. Therefore, studies with larger sample sizes and sub-analyses for different populations such as Asian, Caucasian, and other populations are warranted whenever more data are available.”Figures and Tables: Figures and Tables are adequately explained.Response: Thank you.Conclusion: The limitations are well stated, however, line 286-288 “Therefore, studies with larger sample sizes and sub-analyses for different populations such as Asian, Caucasian, and other populations are warranted whenever more data are available.” Please put in one paragraph before the conclusion to make the conclusion from your review stronger.Response: The conclusion has been revised as suggested.Overall comments: This manuscript may be considered for indexing after Major revisions.Response: Thank you.


Epidemiological studies have showed that vitamin D deficiency can increase the risk of liver cancers. Hence, we conducted a meta-analysis to explore the relationship between 25-hydroxyvitamin D [25(OH)D] levels and liver cancer risk. Cochrane Library, Medline, Web of Science, and Embase were searched up to Mar. 2020, and the references of those studies were also searched by hand. A meta-analysis of 11 studies was performed which met the inclusion criteria. Six case–control studies and five cohort studies were included. A total of 11 studies (6 case–control and 5 cohort studies) with 12,895 incident cases were included in the meta-analysis. The meta-analysis showed that liver cancer risk was significantly increased for vitamin D deficiency, and the pooled RR and its 95% CIs was 2.16 . In comparative analyses between 25(OH)D levels in patients with hepatocellular carcinoma(HCC) and those in the control group individuals, the summary RR of liver cancer was -1.11 (95% CI=-1.96 to -0.25). The subgroup analysis of the different geographical region of the population showed that the risk of liver cancer in Asian subgroup, European subgroup and Egyptian subgroup increased for vitamin D deficiency . The results of this meta-analysis indicate that vitamin D deficiency is associated with increased risk of liver cancer. The 25(OH)D3 levels are lower in HCC patients than those in health controls. Maintenance of sufficient serum vitamin D levels would be beneficial for prevention of liver cancer.  Primary liver cancer is the second leading cause of cancer-related death worldwide . Incidence of liver cancer remains highest in Asia, approximately reaching 78% of all, specifically in the East and South-East Asia . Risk factors for liver cancer include chronic infection with hepatitis B or C virus (HBV or HCV), excess alcohol consumption, aflatoxin exposure, diabetes, obesity, and smoking . Many clinical reports have demonstrated that vitamin D deficiency is very common in patients with liver cancer. Experimental studies also confirmed that vitamin D is related to poor tumor response and the prognoses of patients with liver cancer.Vitamin D is an essential nutrient that regulates numerous cellular pathways. Many cell signaling pathways activated by vitamin D are related with cancer development, progression and prognosis . Vitamin D deficiency increases the risk of fracture risk, cardiovascular disease, hypertension, diabetes mellitus, and cancers . Indeed, the biological activity of vitamin D is regulated by a complicated feedback regulation system and depends on receptors of vitamin D(VDR) and other transport proteins. Vitamin D and VDR are not only involved in the immune system , but also playing important roles in the modulation of tumor growth and metastasis . Vitamin D deficiency and insufficiency is a global phenomenon, and it has been estimated that approximately 60% of adults worldwide are vitamin D deficient and insufficient . It is known that Vitamin D deficiency is particularly prevalent in South Asian, including severe deficiency (<12.5 nmol/l) up to 27–60% of individuals . In India, vitamin D deficiency prevails in epidemic proportions, with a prevalence of 70%-100% in the general population . The major causes for the vitamin D deficiency pandemic are the lack of sun exposure and inadequate intake of natural dietary sources . If the status of vitamin D deficiency is not corrected, it may promote liver cancer growth through β-catenin activation and toll-like receptor 7 signaling pathways . In the past decade, many studies have demonstrated that vitamin D deficiency is significantly associated with incident liver cancer , however, the findings remain controversial. Therefore, based on prospective cohort and case-control studies, we conducted a meta-analysis to systematically assess the relationship of vitamin D levels with the risk of liver cancer.Search strategyAll relevant studies were systematically searched in PubMed, Embase, Web of Science and Cochrane Library before March 2020. No restrictions for time of publish were imposed, except for human subjects and English language. Moreover, the reference lists from previous systematic reviews and published meta-analyses were reviewed. Abstracts, meeting proceedings, gray literature, unpublished results, and personal communications were not included. Study selectionStudies were eligible for inclusion if they 1) applied a case–control, cohort study, or clinical trial design and were published as original studies; 2) assessed at least one of the following exposures: vitamin D or 25(OH) D3; 3) use liver cancer or hepatocellular carcinoma as an outcome; and 4) provided at least two groups of relative risks (RRs) or odds ratios (ORs) and 95% confidence intervals (95% CIs) across different categories or could estimate these data by number of cancer cases and population at risk (or controls). To avoid overlap between study populations, we included studies with the largest sample size when studies on the same topic were performed among the same populations. Quality assessmentThe Newcastle–Ottawa Scale (NOS) was used to assess the quality of the included original studies with a case–control or cohort study design . The content of the study was evaluated for four major aspects: selection, comparability, exposure, and results, and thereafter, categorized into high, medium, and low quality. A study with a score > 6 was considered to be well quality. Data extraction The titles and abstracts of the initially retrieved articles were independently screened by two investigators (YZH and WLJ). Disagreements between the two authors will be solved throughdiscussion with the co-author (TXQ) to reach a consensus. The following data from each eligible study were extracted: the first author, publication year, country of the study, study name, study design, characteristics of the study population, follow-up period, sample size, race, age, number of cases, blood vitamin D levels, testing method, maximally adjusted ORs or RRs with 95% CIs, and adjusted covariates.Statistical analysis 2[I2= (Q− df)/Q × 100], with a significance threshold of I2>50 % . A random effects model was used if the I2 statistic was significant. Otherwise, a random effects model was employed. Potential publication bias was assessed by using Begg’s test through using the Stata 16 software. Statistical analysis was performed with Review Manager 5.3  from The Cochrane Collaboration. Continuous variables were analyzed using mean differences (MD) and 95 % confidence intervals (CIs). A P value <0.05 was considered statistically significant. Heterogeneity of the included studies was tested by using Higgins ISelection of studiesA preferred reporting items for meta- analyses flow diagram of literature search is shown in Study characteristics The eleven studies included five cohort studies and six case-control studies. The five cohort studies  consisting of 9,347 incident cases, and 6 case-control studies  consisting 1723 cases and 1825 controls were included. Among these, 4 studies were conducted in the Europe , 2 in the Egypt , 5 in Asia . The included studies, basic characteristics of enrolled patients, and surgical techniques are presented in Overall analyses Vitamin D deficiency and liver cancer risk2=97%)  for case-control studies. This result suggests that vitamin D deficiency could increase the risk of liver cancer.In comparative analyses between vitamin D deficiency and vitamin D sufficiency, significantly increased liver cancer risk was observed for vitamin D deficiency, and the pooled RR and its 95% CIs was 2.16  with moderate heterogeneity (I2=97%) . Six casSerum 25(OH)D levels and liver cancer risk2=98%, P<0.00001 , indicating that serum 25(OH)D level in HCC patients was 1.11nmol/L lower than that in control group , with an evidence of heterogeneity Iol group . AdditioSubgroup analysisWe stratified subgroup analysis based on geographical region of the population . The pooPublication Bias The funnel plot and Begg’s statistical test indicated no evidence of publication bias in the studies included in the meta-analysis (p=0.16) . . Vitamin D deficiency is known to be associated with the increased risk of many human cancers , and extensive epidemiological studies supported vitamin D–cancer hypothesis . Hepatocellular carcinoma is the second most commonly diagnosed cancer and the leading cause of cancer death in men .It’s life expectancy after diagnosis rarely exceeded 3 years in developed countries . Several population-based studies unveiled an inverse correlation between serum 25(OH)D levels and high risk of liver cancer . Moreover, extensive animal and cell culture studies support the anti-tumorigenic effects of vitamin D . As such, it seems that deficiency of vitamin D can contribute to the development and progression of liver cancers.Nevertheless, the evidence of the association of vitamin D with liver cancer risk remains controversial. Fang et al., (2019) shown that 25(OH)D levels were associated with risk of liver cancer in the population-based HCC cohort in China. Therefore, our meta-analysis aimed to explore the correct relationship between vitamin D and liver cancer risk. In this meta-analysis, we obtained summary evidence that vitamin D deficiency will increase the risk of liver cancer, and the pooled relative risk is 2.16. To further analyze the risk of liver cancer in two different research type, we calculated the RRs of cohort subgroup and case-control subgroup respectively. There are five cohort studies that combined in this project for meta-analysis, and the pooled relative risk is 1.46. Six case-control studies were included for subgroup meta-analysis, and the pooled relative risk is 2.97. In addition, our meta-analysis found 1.81 increased risk of liver cancer for the lowest versus highest categories of blood circulating vitamin D levels , that indicated higher blood circulating vitamin D level has a significant inverse association with risk of liver cancer. This result was consistent with Guo’s meta-analysis . Guo et al., (2020) analyzed six prospective studies and reached the conclusion that a higher circulating vitamin D was associated with lower risk of liver cancer. Our meta-analysis results indicated that the negative correlation between vitamin D level and the risk of liver cancer is similar to that of European and Asian population studies .2=97%, Pheterogeneity<0.00001), it may not reflect the true underlying effect. The poor heterogeneity could be caused by the presence of season of blood collection, different races, enrolled individuals with viral hepatitis and the method for measuring blood vitamin D levels. In addition, few studies about this topic were eligible for our current meta-analysis. When we stratified studies by geographical location, the result showed a statistically difference between Asian, European, and Egyptian. Because the presence of poor heterogeneity and the diversity of the studies in the overall analysis . First, vitamin D is metabolized by vitamin D 25-hydroxylase to 25(OH)D, which is the major circulating form of vitamin D in serum . The second step of activation occurs in the proximal tubular of kidney. To date, there are numerous methods to measure the serum 25(OH)D, however the serum 25(OH)D level can be affected by liver function, viral hepatitis and sunlight exposure. That may also affect our heterogeneity of meta-analysis.There are some limitations in the present study. First, although we systematically searched four authoritative databases, there were still some missed studies that could have affected the analysis for unavoidable reasons. Second, We did not analyze the case–control and cohort studies separately. Third, since most of the included studies were performed without American populations, it is not sure whether our findings could be generalized to global populations. In conclusion, our meta-analytical comparison supports the hypothesis that vitamin D deficiency status is related to a higher risk of liver cancer, and thus maintenance of sufficient serum vitamin D levels could be beneficial for prevention of liver cancer. Further multinational, population-based study will be need to resolve the issue of heterogeneity and evaluate the association of blood circulating vitamin D levels and the risk of liver cancer.All authors read, critically reviewed and approved the final manuscript. YZH and WLJ conducted the database searches, screened titles, abstracts and full-texts for eligibility, performed study quality assessments. YZH planed and designed the research; WLJ and TXQ provided methodological support/advice; YZH tested the feasibility of the study; YZH and WLJ extract data; YZH performed the statistical analysis; YZH wrote the manuscript.


NF‐κB1 gene variants to gastrointestinal cancer risk, yet remains conflicting conclusions. Therefore, we conducted this most up‐to‐date meta‐analysis to evaluate the relationship between NF‐κB1 gene insertion (I)/deletion (D) polymorphism, namely −94ins/delATTG or rs28362491, and the susceptibility to gastrointestinal cancers. We searched PubMed, EMBASE and MEDLINE databases updated in April 2021 for relevant studies. Meta‐analysis was carried out by software Stata11.0. The quantification of the relationship was determined by computing the combined odds ratios (ORs) and their corresponding 95% confidence intervals (CIs). Sensitivity analysis, the funnel plot and Begg's rank correlation test were also applied. Our findings indicate that −94ins/delATTG polymorphism could not significantly impact the susceptibility to gastrointestinal cancers. Under any five genetic models, −94ins/delATTG polymorphism was not remarkedly linked to the risk of colorectal, gastric and oesophageal cancer, respectively. The significant role of −94ins/delATTG was only observed in some certain subgroups. Findings here suggest that NF‐κB1 gene −94ins/delATTG polymorphism may not predispose to gastrointestinal cancer susceptibility.Nuclear factor‐kappa B1 (NF‐κB1), a pleiotropic transcription factor, functions as a critical contributor to tumorigenesis. Growing numbers of case‐control studies were carried out to analyse the potential contribution of  Among them, the rs28362491, namely the −94insertion/deletion ATTG polymorphism, ranks the most intensively investigated SNP.NFKB1 gene, thus influencing the stability of mRNA and efficiency of regulating translation. Research regarding NFKB1 gene −94insertion/deletion ATTG polymorphism on its association with gastrointestinal cancer risk was widely performed. However, the conclusions are still contradictory and inconsistent, partly attributed to the underpower and bias of independent studies, especially for small cohorts. Therefore, the exact association between NFKB1 −94insertion/deletion ATTG variant and risk of gastrointestinal cancer awaits to be determined. Here, a renewed meta‐analysis with all potential studies performed before April 2021 was analysed to acquire a clearer impact of NFKB1 −94insertion/deletion ATTG polymorphism on gastrointestinal cancer susceptibility.NF‐κB, short for nuclear factor kappa B, is a pluripotent and critical dimer transcription factor. NF‐κB orchestrates multiple physiological and pathological processes, particularly in cell survival, differentiation, inflammation and carcinogenesis.22.1We reported this meta‐analysis in accordance with the Preferred Reporting Items for Systematic Reviews and Meta‐analyses (PRISMA) and Meta‐analysis of Observational Studies in Epidemiology (MOOSE) reporting guidelines.2.2NFKB1 or NF‐κB1 or nuclear factor kappa B1; (2) −94insertion/deletion ATTG or rs28362491 or SNPs or polymorphisms or polymorphism or variants; and (3) colorectal cancer or gastric cancer or gastrointestinal cancers or oesophageal cancer. To identify all the available studies, we also arranged two authors to screen eligible publications by hand‐searching the references of included publications.We applied an all‐sided literature retrieval using EMBASE, PubMed and MEDLINE up to April 2021. We used the following key words to carry out this procedure: (1) 2.3NFKB1 rs28362491 and gastrointestinal cancer risk; (2) case‐control study; and (3) enough data to calculate odds ratios (ORs) and corresponding 95% confidence intervals (CIs). Editorials, meta‐analyses, reviews, research on animals and repetitively published articles were excluded.Publications are required to meet the following criteria for inclusion: (1) evaluating the relationship between 2.4We arranged two researchers (Hanqiang Wu and Jianrong Liang) to acquire the data independently by adopting the unified data table. The authors acquired the following information from all the studies: surname of the first author, source of control, year of publication, type of cancers, ethnicity of the study subject, numbers of cases and controls, genotyping method, and genotype of SNPs. If the extracted information is disputable, the authors would re‐check the references and make sure which extracted information is right. We assessed the methodologic quality of each study using the quality assessment criteria described by previous studies Table . We judg2.5NFKB1 rs28362491 and gastrointestinal cancer risk was evaluated by identifying the genotype frequencies of all cases and controls. Odds ratio (OR) and 95% confidence interval (CI) were adopted to assess this relationship. The meta‐analysis assessed association by using five different genetic models: homozygote model ; heterozygote model ; recessive model (II vs. ID/DD); dominant model (ID/II vs. DD); and allele contrast model (I vs. D). Stratification analyses were also performed by ethnicity, cancer type, source of control, score and HWE in controls, if applicable. We further analysed the heterogeneity among studies using Cochrane Q test. If p < 0.10 or I2 > 50% for the Q test, the random effects model (DerSimonian‐Laird method) was applied to conduct the analysis; if not, the fixed effects model (Mantel‐Haenszel method) was applied.p value < 0.05 indicates significant finding. Trial sequential analysis (TSA) was performed as described by us previously. Briefly, after adopting a level of significance of 5% for type I error and of 30% for type II error, the required information size was calculated, and TSA monitoring boundaries were built. All statistical analysis was performed using STATA, version 11.0 .We first test if the SNPs in the controls conformed to Hardy‐Weinberg equilibrium (HWE) by goodness‐of‐fit test. The association between 33.1The screening process of the current meta‐analysis was shown in Figure 3.2Among the 16 included studies . In cancer type subgroup analysis, NFKB1 −94ins/delATTG polymorphism still failed to impact the susceptibility in subgroup of colorectal cancer, gastric cancer and oesophageal cancer. Subgroup analysis of ethnicity indicated that significant decreased cancer risk was detected in Caucasians , but not in Asians. Further stratification analysis by source of control revealed that studies conducted as population base protect from cancer risk . Moreover, score subgroup indicated that studies >9 were linked to decreased cancer risk . Further stratification of HWE analysis in controls demonstrated that no significant association was detected in subgroup of both HWE ≤ 0.05 and HWE > 0.05.The detailed results of the meta‐analysis were presented in Table 3.4Here, we also carried out a sensitivity analysis by gradually deleting the included studies in a one‐by‐one manner. The no statistical fluctuation of the pooled OR value suggested that the analytic results were reliable and stable Figure .3.5Begg's funnel plot did not have significant asymmetry Figure . Statist3.6To minimize random errors and strengthen the robustness of our conclusions, we performed TSA Figure . This an4NFKB1 gene −94ins/delATTG polymorphism and gastrointestinal cancer risk. Our findings indicate that NFKB1 gene −94ins/delATTG polymorphism could not modify gastrointestinal cancer susceptibility. Of note, this is the first meta‐analysis performed by far on NFKB1 gene −94ins/delATTG polymorphism and gastrointestinal cancer susceptibility.In the current meta‐analysis, we comprehensively extract information from available epidemiology studies to assess the association between NFKB1 −94ins/delATTG polymorphism analysed here (rs28362491) in cancer susceptibility. Song et al.NFKB1 −94ins/delATTG polymorphism could enhance the susceptibility of colorectal cancer in a Southern Chinese population. However, role of NFKB1 −94ins/delATTG polymorphism in specific cancer is contradictory, namely a decreased cancer susceptibility or a null association. To solve this controversy, several meta‐analyses have been conducted. The first meta‐analysis was carried out in 2011 by Zou et al.NFKB1 −94ins/delATTG polymorphism protects from getting overall cancer in the homozygote model; heterozygote model; dominant model; and allele contrast. Stratified and subgroup analyses indicated decreased susceptibility for prostate cancer, ovarian cancer, lung cancer, nasopharyngeal carcinoma and oral squamous cell carcinoma, and this association also is significant for Asians, especially Chinese subjects, in hospital‐based studies, and in studies with quality score <9. Of note, by far, no available GWAS has identified the significant relationship between NFKB1 −94ins/delATTG polymorphisms and risk of gastrointestinal cancers.Growing evidence has pointed to the involvement of NFKB1 −94ins/delATTG polymorphism impact risk of gastrointestinal cancers. However, NFKB1 −94ins/delATTG polymorphism under any five genetic models has not enough ability to influence susceptibility to overall gastrointestinal cancer. Several merits existed in the current meta‐analysis. First, this is up to now the first and largest meta‐analysis regarding NFKB1 gene −94ins/delATTG polymorphism and gastrointestinal cancers susceptibility. Second, in sensitivity analysis, relative stability and credibility of the results were achieved as no significant changes after deleting each study at a time. Third, there existed no obvious publication bias, indicating the reliability of conclusion. Some limitations in the current meta‐analysis should be acknowledged. First, the number of subjects in the included studies is relatively small, especially in stratified analyses, which might result in a lack of statistical power and prevent a meaningful analysis of the results. Second, the association strength was only evaluated by unadjusted estimates. Adjustment analysis including gene‐gene, gene‐environment factors were not carried out because of the lack of original data. Third, in the included studies, the population sources were generally limited to Caucasians and Asians. Thus, the conclusion here should be interpreted in caution in Africans.Since then, several new relevant case‐control studies on gastrointestinal cancers have emerged. Therefore, we set as a pioneer to determine whether 5NFKB1 gene −94ins/delATTG polymorphism may not predispose to gastrointestinal cancers susceptibility. More attention should be paid to several research directions in future studies. First, more high‐quality studies with expanded sample sizes are necessarily called to further identify the relationship of the NFKB1 gene −94ins/delATTG polymorphism and gastrointestinal cancer risk, especially in Caucasian and African populations. Second, functional studies are needed to clarify the underlying mechanisms of NFKB1 gene −94ins/delATTG in gastrointestinal cancers.In all, our finding has concluded that The authors declare that there are no competing interests associated with the manuscript.Hanqiang Wu: Conceptualization ; Methodology ; Writing‐original draft ; Writing‐review & editing . Jianrong Liang: Conceptualization ; Data curation ; Investigation ; Software ; Supervision ; Validation ; Writing‐original draft ; Writing‐review & editing .Table S1Click here for additional data file.


Background and Objective: Acute kidney injury (AKI) is recognized as an independent risk factor for mortality and long-term poor prognosis in neonates. The objective of the study was to identify the risk factors for AKI in critically ill neonates to provide an important basis for follow-up research studies and early prevention.Methods: The PubMed, Embase, Web of Science, Cochrane Library, China National Knowledge Infrastructure, WanFang Med, SinoMed, and VIP Data were searched for studies of risk factors in critically ill neonates. Studies published from the initiation of the database to November 19, 2020, were included. The quality of studies was assessed by the Newcastle-Ottawa Scale and the Agency for Healthcare Research and Quality (AHRQ) checklist. The meta-analysis was conducted with Stata 15 and drafted according to the guidelines of the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement.Results: Seventeen studies  were included in meta-analysis, with 1,627 cases in the case group and 5,220 cases in the control group. The incidence of AKI fluctuated from 8.4 to 63.3%. Fifteen risk factors were included, nine of which were significantly associated with an increased risk of AKI in critically ill neonates: gestational age , birthweight , 1-min Apgar score , 5-min Apgar score , congenital heart disease (CHD) , hyperbilirubinemia , necrotizing enterocolitis (NEC) , sepsis , and mechanical ventilation . Six of them were not significantly associated with AKI in critically ill neonates: age , male sex , prematurity , cesarean section , prenatal hemorrhage , and vancomycin .Conclusions: This meta-analysis provides a preliminary exploration of risk factors in critically ill neonatal AKI, which may be useful for the prediction of AKI.Systematic Review Registration: PROSPERO (CRD42020188032). Acute kidney injury (AKI) is characterized by an abrupt decrease in kidney function, which is significantly associated with increased mortality in neonates , 2. Due This study was designed to perform a meta-analysis to identify risk factors associated with AKI in critically ill neonates. It may be helpful for the prediction of AKI in critically ill neonates.This meta-analysis was reported in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement . The proWe conducted an electronic search of PubMed, Embase, Web of Science, Cochrane Library, China National Knowledge Infrastructure, WanFang Med, VIP Data, and SinoMed with the keywords including “neonates,” “acute kidney injury,” “risk factors,” and “risk.” Retrieval time was from inception to November 19, 2020. Search terms and Boolean operators included in the search strategies of PubMed and Embase are presented in online Study selection was independently conducted by QH and SJL, with any discrepancies resolved by MW. Inclusion criteria were as follows: 1) Patients are neonates admitted to the neonatal intensive care unit; (2) the risk factors for AKI in neonates are reported; and (3) the definition of AKI is clear, such as Kidney Disease: Improving Global Outcomes (KDIGO) definition, Acute Kidney Injury Network (AKIN) definition, or arbitrary definition  Patients, 12. ExcData were independently extracted by QH and S-JL, with any discrepancies resolved by MW. Data collected included the characteristics of the studies, the demographic characteristics of the patients, accompanying diseases, and therapeutic measures. When full data cannot be obtained from the study, we tried to contact the corresponding author to obtain all the data.Quality assessment was independently conducted by QH and S-JL, with any discrepancies resolved by MW. The quality of cohort and case-control studies was assessed using the Newcastle-Ottawa Scale (NOS), which was widely used in the quality assessment of case-control and cohort studies , 14. TheI2 test, with I2 > 50%, or p-value < 0.1 was considered significant. If there was significant heterogeneity, a random-effects model was used or else a fixed-effects model. Statistical significance was defined as a two-tailed p-value < 0.05. Sensitivity analyses were conducted on each risk factor by removing each individual study from the overall analysis. Subgroup analyses were performed on the risk factors with significant heterogeneity, which were based on the definition of AKI (KDIGO or non-KDIGO) and research method (cohort or non-cohort study)  for dichotomous data and standardized mean difference (SMD) for continuous outcomes. Raw data of continuous variables were converted into mean and standardized difference (SD) wherever possible . Pooled software .Initial screening identified 2,629 publications . FinallyBased on the NOS quality assessment and AHRQ checklist, 14 studies were classified as high quality and three studies as moderate quality –4. The cP = 0.002] , P = 0.000], and 5-min Apgar score . As compared to the non-AKI group, the AKI group had higher incidences of comorbidities such as CHD , hyperbilirubinemia , NEC , and sepsis . Compared to the non-AKI group, the AKI group was more likely to use mechanical ventilation . Age , male sex , prematurity , Cesarean section , prenatal hemorrhage , and vancomycin  were not significantly associated with AKI in critically ill neonates.The analyses of risk factors are shown in = 0.002] , birthweThe sensitivity analyses for each risk factor showed that no individual study significantly altered the results. The results for birthweight and sepsis were shown in Assessment of publication bias using Egger's tests showed that there was no potential publication bias among the included trials in the study except for 1-min Apgar score and sepsis . HoweverThis study revealed that early gestational age and low birthweight were significantly associated with an increased risk of AKI in critically ill neonates. This finding is consistent with the review published by Perico et al. . This maIn this study, we observed that CHD may increase the risk of AKI in neonates by nearly three times, which may be due to the decreased renal perfusion induced by unstable hemodynamics . We wereIn agreement with the findings of van den et al. regarding AKI in critically ill neonates , mechaniConstance et al.  in theirA Manual for Evidence-based Practice  arbitrary definition mainly based on absolute serum creatinine (SCr) ≥1.5 mg/dl;, 24, 27, arbitrarTherefore, subgroup analyses indicated that the results of age, birthweight, and sepsis were not robust. It is necessary to carry out cohort studies to analyze the relationship between risk factors and different stages of AKI in critically ill neonates.First, this analysis was based on cross-sectional, cohort, and case-control studies, whose controls were not community based, so well-designed multicentric cohort studies are needed to explore the above risk factors that are relied on as causal factors associated with AKI in critically ill neonates. Second, some of the risk factors studied, such as antepartum hemorrhage, hyperbilirubinemia, and vancomycin, were assessed in only two publications, which prevented more robust meta-analyses of these factors. Third, birth asphyxia was not included in this analysis for only one study that provided corresponding data . Fourth,In this study, we found the incidence of AKI fluctuates from 8.4 to 63.3%. Gestational age, birthweight, 1-min Apgar score, 5-min Apgar score, CHD, hyperbilirubinemia, NEC, sepsis, and mechanical ventilation were risk factors for AKI in critically ill neonates. Well-designed studies with a considerable number of critically ill neonates are necessary to determine the possible link between these nine risk factors and AKI.The original contributions presented in the study are included in the article/QH, S-JL, and MW contributed to the study concept and design, article selection and quality assessment, data analysis and interpretation, and manuscript writing. Q-LC, HC, and QL contributed to the study concept and design, and manuscript writing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Both the genetic and pathological studies link Alzheimer’s disease (AD) to the triggering receptor expressed on myeloid cells 2 (TREM2). A large number of studies have explored the value of cerebrospinal fluid (CSF) soluble TREM2 (sTREM2) levels as a biomarker for the diagnosis and prediction of AD; however, the findings are inconsistent. We aimed to review the studies that investigated the association of CSF sTREM2 levels and AD risk, and to provide the recommendations for future research.Methods and Results: A systematic literature search was performed using the MEDLINE, EMBASE, and Web of Science  databases. The meta-analysis for the association between the CSF sTREM2 levels and AD risk included 15 studies (17 comparisons) with a total of 1,153 cases and 1,626 controls. The total results showed that the higher CSF sTREM2 levels and AD risk were associated . However, the analysis of the subgroup of “age difference ≤ 2 years” indicated that sTREM2 was not associated with AD  and had a significantly lower heterogeneity. Combining the results of the “age difference of 5–10 years”  and “age difference > 10 years”  subgroups showed that the difference in CSF sTREM2 between the AD and control groups was positively correlated with the age difference. A meta-regression analysis showed that the age difference can explain 33.4% of the between-study variance. By conducting further subgroup analyses of the five age-matched studies (495 cases and 364 controls) according to the measurement method, and whether inclusion criteria containing the requirement for pathological evidence of AD, no changes were observed in the corresponding pooled SMD or in the significance of the results. The meta-analysis result of “age difference ≤ 2 years” group was robust in the sensitivity analysis.Conclusion: The available high-quality evidence does not yet support an association between the CSF sTREM2 levels and AD risk. Age matching between the patients with AD and cognitively unimpaired controls was a major influencing factor in the results. Alzheimer’s disease (AD), as a clinically heterogeneous and complex neurodegenerative disease, is considered the most common type of dementia and induces a heavy burden to the patients and families. The onset of AD is a continuous progressive process that from an asymptomatic state to mild cognitive impairment and to clinical diagnosis of dementia . Early rTypical pathological features in the brain of the patients with AD are beta-amyloid (Aβ) plaques, intracellular neurofibrillary tangles consisting of hyper-phosphorylated tau fibrils, degeneration of neurons, and loss of synapses, neuroinflammation, and glial activation . As the TREM2 increased the risk of AD by almost threefold (APP), presenilin-1 (PSEN1), and apolipoprotein E (APOE). Soluble TREM2 (sTREM2) is generated and released into the extracellular milieu through cleavage of full-length TREM2 by disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) and ADAM10 (A triggering receptor expressed on myeloid cells 2 (TREM2) is a transmembrane protein with V-immunoglobulin extra-cellular domains and cytoplasmic tails . In the hreefold . It is od ADAM10 . A larged ADAM10 . Howeverd ADAM10 . TherefoThis study summarized the studies on the association of CSF sTREM2 and AD risk, with the attempt to find the potential influence factors to interpret the considerable differences of the results reported by these studies and provide recommendations for future research. The previous studies have shown a positive association between the CSF sTREM2 levels and age . MeanwhiThe databases MEDLINE, EMBASE, and Web of Science  were searched up to April 13, 2021 to find the studies evaluating the relationship between CSF sTREM2 level and AD risk. The following keywords were used:  AND (TREM2 OR sTREM2 OR “soluble trigger receptor expressed on myeloid cells 2”) AND [(Alzheimer’s disease) OR (dementia)]. All the reference lists of the resulting primary research reports and relevant reviews were manually searched to identify the additional eligible studies.The eligible studies were included in this meta-analysis: (1) investigated the relationship between the CSF sTREM2 levels and AD risk; (2) included a case group of patients with AD and a group of cognitively unimpaired controls; (3) clinical criteria that used to diagnose AD were qualified; (4) provided data with the mean and SD or median and interquartile range (IQR) or median and the minimum and maximum; (5) the studies performed in humans; (6) full text in English. The articles were excluded if they measured sTREM2 concentrations in the postmortem samples, had a sample size of less than 5, or used the samples that overlapped with other studies. Information regarding the first author, year of publication, study location (country), study design, number of patients and controls, diagnostic criteria, measurement methods, the mean and SD of CSF sTREM2 levels, and information for the potential confounding factors  were extracted.The quality of case-control studies and cohort studies included was evaluated based on the Newcastle-Ottawa Scale (NOS) recommended by the Agency for Healthcare Research and Quality of the United States. Comparability of the cases and controls on the basis of the design or analysis was evaluated based on whether the age and gender matched. The methodological quality of the cross-sectional studies included was assessed using an 11-item checklist which was recommended by Agency for Healthcare Research and Quality (AHRQ). The studies with NOS scores lower than 5, or AHRQ scores lower than 4 were recognized to be of inferior quality and therefore excluded.n = n1 + n2, The disputes were resolved by discussion with a third author during data extraction and quality evaluation. If the case or control groups were further divided into subgroups, the data from the subgroups were merged as IQR/1.35 . If the CI. To analyze the potential influences of age difference at the time of lumbar puncture, measurement method, and inclusion criteria containing the requirement for pathological evidence of AD, we performed the subgroup analysis. Based on the age difference between the AD group and the control group at lumbar puncture, the subjects were divided into three subgroups: (1) age difference ≤ 2 years, (2) age difference of 5–10 years, and (3) age difference > 10 years. Based on the measurement method, the subjects were divided into three subgroups: (1) enzyme-linked immunosorbent assay (ELISA), (2) mesoscale discovery electrochemiluminescence platform-based assay (MSD) or electrochemiluminescence (ECL) immunoassay, and (3) ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS). Additionally, based on the diagnostic criteria, the subjects were divided into two subgroups: (1) with neuropathologic verification, and (2) no relative information. The significance of the pooled SMD was determined using a Z test, and the level was set at p < 0.05.The association between the CSF sTREM2 level and AD risk was estimated by calculating the pooled standardized mean difference (SMD) and the 95% Q test and the I2 statistic. When significant heterogeneity was observed , a random effects model was used for pooling the data from the primary studies. A meta-regression with restricted maximum likelihood estimation (REML) was performed to assess the potentially important covariate exerting substantial impact on the between-study heterogeneity. The age difference  was included in the meta-regression analysis. The sensitivity analysis was performed by sequentially excluding the individual studies to assess the stability of the results. A funnel-plot asymmetry was assessed using Egger’s linear regression test, with p < 0.05 representing significant publication bias. All the analyses were performed using STATA 12.0 software , and all the p values were two-tailed.Heterogeneity across the studies was assessed using the n = 90), conference abstracts (n = 14), the reports of animal studies (n = 7), the reports of studies that investigated either the outcomes irrelevant to this meta-analysis (n = 18), the markers other than sTREM2 (n = 1), review or meta-analysis (n = 12), protocol (n = 1), the studies of which sTREM2 levels was not measured in CSF (n = 2), the reports of studies that only had TREM2 gene information (n = 6), or the studies of which did not have a normal cognitive function group (n = 1).With respect to the association between the CSF sTREM2 levels and AD risk, an initial search identified 184 records of the potentially relevant studies from the databases. Of these, 152 records were excluded based on their title and/or abstract: these were repetitive publications (TREM2 gene study (n = 2), (2) the studies in which the interviewees were not grouped according to the AD clinical diagnosis criteria (n = 4), (3) study in which the sTREM2 levels were not detected in the control group (n = 1), (4) postmortem samples (n = 1), (5) the studies used the samples that overlapped with other studies (n = 5), (6) the studies in which all the participants had Down syndrome (n = 1), and (7) the studies in which data were shown in figures only (n = 3). The 15 studies (17 comparisons) that were ultimately selected for our meta-analysis included 1,153 cases and 1,626 controls (A further 17 articles were excluded after reviewing the full text. These were (1) controls , 2; the CI , 2I = 81.1%]  with a total of 1,153 cases and 1,626 controls. The total results showed that a higher CSF sTREM2 level was associated with increased AD risk  .I2 = 27.4%) and the subgroup of “age differences > 10 years” (I2 = 50.0%). An analysis of the subgroup of “age difference ≤ 2 years” indicated that sTREM2 was not associated with AD . An analysis of the subgroup of “age difference of 5–10 years” , and the subgroup of “age difference > 10 years”  showed that the CSF sTREM2 levels were associated with AD. The results indicated that the greater the difference in age, the greater the difference in CSF sTREM2 levels between the case group and the control group .Of the 17 comparisons, four comparisons had an age difference greater than 10 years, eight comparisons had an age difference from 5–10 years, and five studies had an age difference of 2 years or less. When distinguishing the subgroups by age difference, the heterogeneity decreased substantially in both the subgroup of “age differences ≤ 2 years” , I2 = 81.9%; MSD or ECL immunoassay: SMD = 0.463, 95% CI , I2 = 84.1%; UPLC-MS: SMD = 0.810, 95% CI , 2I = 62.5%; Subgroup analysis of the 17 comparisons was carried out according to the measurement method and the heterogeneity within each subgroup remained high. The results suggested that the measurement methods might impact the results of the difference in the CSF sTREM2 levels between the patients with AD and controls . Neither the subgroup analysis of the five articles distinguished by the measurement method [ELISA: SMD = 0.161, 95% CI , I2 = 0%; others: SMD = 0.001, 95% CI , 2I = 56.2%; CI , 2I = 35.9%; without neuropathologic verification: SMD = 0.083, 95% CI , 2I = 0%; The meta-analysis of the studies with an age difference within 2 years showed that the CSF sTREM2 levels were not associated with AD and there was little heterogeneity between the studies [SMD = 0.090, 95% Z = −0.24, p = 1.000; t = −0.34, p = 0.753).In the sensitivity analysis of the meta-analysis of “age difference ≤ 2 years” group, one eligible study was excluded at a time to assess the influence of each dataset on the pooled SMD. We observed no changes in the corresponding pooled SMD or in the significance of the results , which iZ = 0.45, p = 0.650; Egger’s test: t = 0.62, p = 0.542; In addition, the meta-analysis of 17 comparisons showed robust results with no publication bias  . The pat in mice . In neur in mice .Both the genetic and pathological studies link AD to TREM2. Increased AD risk is thought to be mediated through the reduced TREM2 function . The TRECI of pooled SMD of the subgroup in which the diagnostic criteria were only clinical symptoms was 0.06–0.62, which was very close to “no association.” This suggests that the level of CSF sTREM2 may have a higher value as a biomarker for AD when based on pathological diagnosis. However, it must be noted that the studies used uniform pathological diagnosis index and the data showed a high degree of heterogeneity. The reliability of this conclusion needs further study. On the other hand, the clinical diagnosis focusing on the syndromes is still the routine diagnostic strategy because of good accuracy and the limited standardization and utility of biomarkers (An analysis to investigate whether the diagnostic criteria affected the result revealed that the greater difference of CSF sTREM2 between the AD and control groups was shown in the subgroup of studies contained pathological diagnosis. The 95% omarkers . The valIn this study, when the effect of the measurement methods on the results was analyzed, different results were found for different measurement methods. Negative result was shown in the group of studies using ELISA, the simplest and most practical measurement method. Practicality and standardization of CSF sTREM2 as a complementary marker for the clinical diagnosis are yet to be investigated.This study has certain limitations. First, the heterogeneity within the studies included in the overall analysis was high; therefore, the overall result needs to be interpreted with caution. Second, a total of five studies with approximately equal age between the AD group and the control group were included, involving 495 patients in the AD group and 364 in the control group. It will be necessary to update this meta-analysis in the future when more high-quality literature appears. Last, our study only included the articles written in English, which might have resulted in a language bias.The available high-quality evidence does not support an association between the CSF sTREM2 levels and AD risk. The matching of age between the patients with AD and cognitively unimpaired controls was a major influencing factor in the results.The original contributions presented in the study are included in the article/SH and NP have searched the databases and extracted the information. YW reviewed the processes of literature search and data extraction. SH did the statistical analysis. SH and CL drafted the manuscript. TZ has revised and expanded the manuscript. All the authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Leptomeningeal disease (LMD) is a complication distinguished by progression of metastatic disease into the leptomeninges and subsequent spread via cerebrospinal fluid (CSF). Although treatments for LMD exist, it is considered fatal with a median survival of 2–4 months. A broader overview of the risk factors that increase the brain metastasis (BM) patient's risk of LMD is needed. This meta-analysis aimed to systematically review and quantitatively assess risk factors for LMD after surgical resection for BM.A systematic literature search was performed on 7 May 2021. Pooled effect sizes were calculated using a random-effects model for variables reported by three or more studies.Among 503 studies, thirteen studies met the inclusion criteria with a total surgical sample size of 2105 patients, of which 386 patients developed LMD. The median incidence of LMD across included studies was 16.1%. Eighteen unique risk factors were reported as significantly associated with LMD occurrence, including but not limited to: larger tumor size, infratentorial BM location, proximity of BM to cerebrospinal fluid spaces, ventricle violation during surgery, subtotal or piecemeal resection, and postoperative stereotactic radiosurgery. Pooled results demonstrated that breast cancer as the primary tumor location  and multiple BMs  were significantly associated with a higher risk of LMD occurrence.Breast cancer origin and multiple BMs increase the risk of LMD occurrence after neurosurgery. Several other risk factors which might play a role in LMD development were also identified. Leptomeningeal disease (LMD) is an intractable neuro-oncological complication that is often considered fatal, with a median survival of 2–4 months. The hypothesis is that a relationship exists between LMD and resection due to disruption of anatomical borders, tumor spillage, and thus CSF contamination. Nonetheless, resection has become a cornerstone for treating newly diagnosed brain metastases (BMs). Studies reporting on LMD risk in BM patients were, however, limited in sample size and number of surgically treated patients. This meta-analysis quantitatively assessed what risk factors in surgically treated BM patients were significantly associated with LMD risks and shed light on potential risk factors that are thought to influence LMD occurrence but did not. These results could inform clinicians in deciding what a high-risk profile is for LMD occurrence and initiate future research initiatives to understand LMD occurrence further.1 Adequate treatment of BMs has become increasingly fundamental for these patients' survival and quality of life. Patients with extracranial tumor control and a large, solitary BM benefit most from surgical resection with adjuvant radiation therapy, such as stereotactic radiosurgery (SRS).2The prognosis of newly diagnosed brain metastasis (BM) patients has improved significantly due to systemic therapies and primary tumor control.3–5 LMD is defined as metastatic disease progression into the leptomeninges and cerebrospinal fluid (CSF). Hypothetically, the relation between LMD and surgical resection could exist due to the disruption of anatomical borders in brain tissue and surgical spillage of tumor cells, resulting in CSF contamination. Though treatments for LMD exist, the prognosis is abysmal, with a median survival of 2–4 months.6–8Although resection has become a cornerstone for treating newly diagnosed BMs, studies have suggested that this treatment is associated with a higher risk for developing leptomeningeal disease (LMD).10 However, most of these studies were limited in sample size and number of surgically treated patients, and a comprehensive overview of risk factors for LMD after neurosurgery is currently lacking.Current literature suggests that the LMD risk may vary by BM location, the origin of the primary tumor, treatment modality, and other factors.Therefore, the present study aims to perform a systematic review and meta-analysis of the current literature to summarize risk factors for LMD in BM patients who underwent neurosurgical resection.A systematic literature search was performed in PubMed, Embase, Web of Science, Cochrane, Academic Search Premier, and PsycINFO according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines on 7 May 2021 . ReferenStudies were included if they 1) were randomized controlled trials (RCTs), prospective or retrospective cohorts or case-control studies; 2) reported on BM patients that underwent surgery or a subgroup that underwent surgery; 3) reported on risk factors for developing LMD. This study's outcome was risk factors for the development of LMD. Exclusion criteria were: 1) nonhuman studies, 2) primary brain tumors, 3) studies reporting on <10 LMD patients, 4) non-English publications.11Relevant data were extracted and grouped as follows: study characteristics including study design and sample size, patient characteristics, intracranial tumor characteristics, systemic cancer characteristics, treatment characteristics including previous surgery, chemotherapy, upfront radiotherapy schedule, and outcome measures including risk factors for LMD. The Newcastle-Ottawa scale (NOS) was used for cohort studies and case control studies to assess the risk of bias in all included studies.12 to account for variation between studies, was used to obtain the overall hazard ratio (HR) estimates and 95% confidence intervals (CI). If the standard error was not reported in the included studies, it was calculated using the HR and P-value.13 The meta-analysis was subsequently conducted using the metagen function of the meta package in R.14 The estimated results were visualized using forest plots. The Higgin's & Thompson's I2 was used to assess heterogeneity among studies15; >50% was considered high heterogeneity. A P-value < 0.1 for heterogeneity was considered significant.16Data analysis was performed using R v 3.5.0 . A meta-analysis was performed if more than three studies reported a specific risk factor with poolable effect size metrics. The random-effects model, combined with the DerSimonian-Laird method17–33 No additional studies were identified by the reference check.After completing the search and removing duplicates, 556 publications were identified, of which thirteen studies met the inclusion criteria .10,17–33n = 3),30 and retrospective cohorts (n = 10).31–33 The included studies described a total of 2105 patients, of which 386 (18.3%) developed LMD .Included studies were case-control studies  score21) were analyzed as possible risk factors for LMD, but no significant association was demonstrated ,17 ventricle violation during surgery,19 prior therapy of BM,33 surgical cavity control,18 intracranial failure defined as any residual tumor or other BM after resection,32 time from surgery to SRS,32 gamma knife versus linear accelerator (LINAC) radiosurgery,20 local radiotherapy versus whole brain radiotherapy (WBRT),21 radiation dose,32 sensitivity to hypofractionated radiation,26 single fraction versus multi-fraction SRS,33 preoperative versus postoperative SRS,28 and the year of SRS receipt.33 In total, nine treatment variables were significantly associated with increased LMD risk; Postoperative versus preoperative SRS was significantly increased LMD risk in two studies,28 while hormonal therapy within three months of adjuvant SRS,33 targeted therapy within three months of adjuvant SRS,33 subtotal resection,31 piecemeal resection,17 ventricle violation during surgery,19 intracranial failure,32 and local radiation versus WBRT21 were reported to increase the LMD risk significantly in one study. The year of SRS receipt was significantly associated with increased classical LMD risk specifically.33A total of seventeen treatment variables were reported for LMD: type of systemic therapy,31) and multiple BMs  were associated with a higher risk of LMD; I2 = 0% for both; p-heterogeneity = 0.93 and p-heterogeneity = 0.92 for breast cancer and multiple BMs, respectively). Infratentorial BM location was consistently reported by three studies,32 but was not significantly associated with the occurrence of LMD adjuvant treatment modalities used in the included studies prevents us from ascertaining specific and conclusive recommendations. However, all analyzed study (sub-)groups underwent a resection for their primary BM. Second, relatively few variables could be used for the meta-analysis due to the lack of consistent reporting of effect sizes. Unfortunately, we were unable to retrieve the missing data for the meta-analysis by contacting the corresponding authors. Furthermore, some variables were reported in different studies using noninterchangeable effect sizes, eg relative risk and hazard ratio, which prevented pooling of the results. The uniformity of extracted variables between included studies also differed greatly, which reduced the possible number of meta-analyses.31 To avoid biasing our representation towards positive results, we reported how many studies identified a given variable as a significant risk factor and how many investigated this variable and did not find it significant. Fourth, the included study duration varied from 1998 until 2019; However, the evolution of therapeutic options and the improvement of diagnostic tools may have influenced the course of disease after surgery without or with radiotherapy for brain metastasis. This hypothesis is supported by the significant association of year of performed SRS treatment and increased classical LMD risk in one included study.33Third, a possibly skewed representation of risk factors of LMD is shown as some included studies only reported on statistically significant LMD risk factors.Our study's major strength is that it was the first meta-analysis that focused on the risk of LMD occurrence for surgically treated BM patients. Moreover, the majority of included studies used multivariate analysis, which decreased confounding bias.Future research should investigate the relation of different treatment modalities and the use of surgical instruments and LMD. Studies should investigate potential risk factors that can be mitigated, such as the treatment sequence and choice of surgical technique. Prognostic models for LMD should also be investigated to understand LMD occurrence further. Furthermore, prospective randomized studies should be performed regarding the effect of different treatment modalities on LMD risk and the optimal timing of (adjuvant) treatment(s) to LMD. Finally, more preclinical research should be conducted in BM models to explain the high LMD risk in primary breast cancer patients.Breast cancer as the primary tumor location and multiple BMs increase the risk of LMD occurrence. Important neurosurgical risk factors, including the proximity of BM to CSF structures, ventricle violation during surgery, and the method of resection might influence the occurrence of LMD. Further research should focus on the effect of different treatment modalities on LMD risk, as well as the optimal sequence of treatments.vdab162_suppl_Supplementary_MaterialsClick here for additional data file.