Metabolic disorders are a hallmark feature of cancer. However, the evidence for the causality of circulating metabolites to promote or prevent colorectal cancer (CRC) is still lacking. We performed a two‐sample Mendelian randomization (MR) analysis to assess the causality from genetically proxied 486 blood metabolites to CRC.Genome‐wide association study (GWAS) data for exposures were extracted from 7824 Europeans GWAS on metabolite levels. GWAS data for CRC from the GWAS catalog database GCST012879 were used for the preliminary analysis. The random inverse variance weighted (IVW) is the primary analysis for causality analysis while MR‐Egger and weighted median as complementary analyses. Cochran Q test, MR‐Egger intercept test, MR‐PRESSO, Radial MR, and leave‐one‐out analysis were used for sensitivity analyses. For significant associations, additional independent CRC GWAS data GCST012880 were used for replication analysis and meta‐analysis. For the final identification of metabolites, Steiger test, linkage disequilibrium score regression, and colocalization analysis were performed for further evaluation. Multivariable MR was performed to assess the direct effect of metabolites on CRC.p = 0.002), 1,6‐anhydroglucose , nonadecanoate (19:0) , 1‐linoleoylglycerophosphoethanolamine , 2‐hydroxystearate , gamma‐glutamylthreonine  and CRC. MVMR analysis revealed that genetically predicted pyruvate, 1‐linoleoylglycerophosphoethanolamine and gamma‐glutamylthreonine can directly influence CRC independently of other metabolites.The results of this study indicated significant associations between six metabolites pyruvate , 1‐linoleoylglycerophosphoethanolamine, and 2‐hydroxystearate associated with lower colorectal cancer risk while higher levels of 1,6‐anhydroglucose and gamma‐glutamylthreonine increased risk of colorectal cancer. According to updated cancer statistics, over 1.9 million new cases of CRC were diagnosed in 2020, resulting in 93,500 deaths.In recent years, the emergence of metabolomics as a component of systems biology has provided a novel approach to investigating the mechanisms underlying diseases. Specifically, metabolomics can provide insight into the biological mechanisms of diseases by identifying modified metabolites or metabolic pathways.Therefore, exploring the metabolites associated with the development of CRC not only contributes to early screening and prevention of CRC, but also helps to understand the biological mechanisms of CRC for treatment. Unfortunately, the causality between metabolites and CRC is unclear, as there are no prospective studies of metabolites and CRC to date. The unavoidable design weaknesses of conventional observational studies, such as altered metabolites due to deliberate lifestyle changes in patients after the cancer diagnosis, chronic intake of certain drugs, and tumor cell‐induced changes in metabolic substances, resulted in an ambiguous causal relationship between metabolites and CRC. Rigorous randomized controlled trials (RCTs) have the highest credibility in evidence‐based medicine to prove causal effects, but they are difficult to implement due to ethical issues, observation time, costly money, and other constraints. Hence, metabolites that increase the risk of CRC cannot be identified based on the existing evidence.Mendelian randomization (MR) studies have recently gained widespread usage in the investigation of disease etiology. In the absence of RCTs, MR is the most compelling strategy to explore the causality between the exposure of interest and outcome.Since there is a lack of understanding related to the causality between blood metabolites and CRC, further research is required in this area. In this work, we performed MR analysis to comprehensively explore the causal effects of 486 blood metabolites on CRC via genome‐wide association study (GWAS) summary data. Furthermore, we conducted colocalization analysis and metabolic pathway analysis to investigate the underlying biological processes of CRC at the gene and protein levels. This study aims to reveal the metabolism‐related etiology of CRC and to provide insight into its biological processes.22.1A valid MR study should comply with three assumptions: 1) IVs are strongly associated with the exposures of interest; (2) IVs are independent of confounding factors; (3) IVs are not associated with outcome and affect outcome only via exposures. IVs are 2.2https://metabolomics.helmholtz‐muenchen.de/gwas/). Notably, this is the most comprehensive report to date on the genetic loci of blood metabolites, which eventually identified almost 2.1 million SNPs for 486 metabolites associated with human genetic variants by Genome‐wide association scans with high‐throughput metabolic profiling conducted by Shin et al.Genetic data for blood metabolites were accessed from the metabolomics GWAS server (https://www.ebi.ac.uk/gwas/)GWAS summary data for CRC download from GWAS Catalog . This criterion has been widely applied in previous studies.R2 and F statistics for each SNP. R2 and F statistics are calculated as follows: R2 is the instrumental variable that explains the degree of exposure (determinant coefficient of regression equation); N is the sample size of the exposure; k is the number of SNPs . SNPs with F < 10 were defined as poor genetic variants and were removed.p < 1 × 10−5). We further harmonized SNPs for exposure and outcome, and palindromic effects and allelic inconsistent SNPs were removed (e.g. A/G vs. A/C). Then, to satisfy assumption (3), we removed outcome‐related SNPs (p < 1 × 10–5) in the IVs. Finally, we performed MR analysis on metabolites with more than two SNPs.To satisfy assumption (1), IVs associated with blood metabolites were identified by rigorous screening conditions from multiple perspectives. Given the modest number of metabolite‐associated SNPs, we eased the significance threshold 2.4p < 0.05). MR‐Egger and weighted median (WM) methods were defined as complementary analyses. These two methods can provide more robust estimates under lenient conditions. WM allows less than 50% of SNPs to be invalid, while MR‐Egger provides horizontal pleiotropy and heterogeneity detection in the presence of horizontal pleiotropy for all SNPs.The causal effect of blood metabolites and CRC was primarily assessed based on the results of random‐effect inverse variance weighted (IVW). Because IVW estimates are derived from a summary analysis of Wald ratios for all genetic variants.p < 0.05 was considered as heterogeneity of the results.Sensitivity analysis is essential because it examines the horizontal pleiotropy and heterogeneity that can severely violate MR estimates. Horizontal pleiotropy is observed when IVs influence the outcome through other pathways than the exposure of interest. As such, we performed several tests to ensure convincing estimates. In this study, we utilized four methods to detect and correct for heterogeneity and pleiotropy, including the Cochran Q test, MR‐Egger intercept test, MR‐Pleiotropy RESidual Sum and Outlier (MR‐PRESSO), and Radial MR. Cochran Q test‐derived p value for the primary analysis was significant (IVW derived p < 0.05). (2) Consistent direction and magnitude within the three MR methods. (3) No heterogeneity or horizontal pleiotropy in MR results. (4) MR estimates are not severely disturbed by a single SNP.In a word, we rigorously screened blood metabolites with potential causal effects on CRC by multiple criteria: (1) https://shiny.cnsgenomics.com/mRnd/).R2 of IVs, the proportion of cases with an outcome, and the odds ratio (OR) derived from IVW analysis.Moreover, to assess the statistical power of the estimates, we calculated the power with an online website p1 = 1 × 10−4, p2 = 1 × 10−4, and p12 = 1 × 10−5). PP.H4 > 80% colocalization analysis (H4) results provide strong evidence supporting the existence of shared causal variants within specific genomic regions affecting gene expression and CRC risk.To further investigate whether the associations of the identified metabolites between CRC were driven by a locus within a genomic region, we performed colocalization analysis using the coloc R package.2.8http://www.phenoscanner.medschl.cam.ac.uk/) to evaluate whether each SNP was associated with known risk factors for CRC, such as smoking,p < 1 × 10−5), MR analysis would be re‐performed after removing these SNPs to verify the reliability of the results.Although we assessed the horizontal pleiotropy of the MR results through a series of sensitivity analyses to detect any SNPs that violated the MR assumptions,To avoid IVs violating Assumptions 2 and 3 of MR, MR analysis is required to ensure that genetic variants are associated with a single risk factor. In practice, however, some genetic variants are associated with multiple risk factors, which is described as pleiotropy.2.9https://www.metaboanalyst.ca/)To specify the biological mechanisms involved in blood metabolites that have causal effects on CRC, we further performed metabolic pathway analyses using the MetaboAnalyst 5.0 (33.1p = 0.005), 1,6‐anhydroglucose , nonadecanoate (19:0) , taurochenodeoxycholate , 1‐linoleoylglycerophosphoethanolamine , 2‐hydroxystearate , gamma‐glutamylthreonine , X‐11470 , X‐14205 . In summary, the estimates derived from IVW are significant (p < 0.05) while the direction and magnitude of IVW, MR‐Egger, and WM estimates were consistent  and MR‐Egger intercept test (p > 0.05) provided strong evidence for the absence of heterogeneity and pleiotropy . The F statistics of all SNPs associated with metabolites were greater than 10, which indicated a strong power of IVs. The detailed data of IVs are presented in Table t Figure . The Cocpy Table . The sta3.2p = 0.002), nonadecanoate (19:0) , 1‐linoleoylglycerophosphoethanolamine , 2‐hydroxystearate , X‐11470 , X‐14205  decreased risk of CRC, while genetic liability for higher levels of 1,6‐anhydroglucose , gamma‐glutamylthreonine  promoted susceptibility to CRC. Even though it showed a consistent direction in both MR analyses, taurochenodeoxycholate was discarded due to the meta‐analysis revealed non‐significant estimates .To enhance the persuasiveness of the estimates, we replicated the MR analysis with another GWAS data for CRC. As would be expected, similar trends were observed for candidate metabolites in another GWAS data for CRC, although the results were not significant due to the enormous difference in sample size. The results of the meta‐analysis further determined that eight blood metabolites (six known and two unknown) can affect CRC Figure . In deta3.3g = 0.259, Se = 0.218, p = 0.236), 1,6‐anhydroglucose  nonadecanoate (19:0) , 1‐linoleoylglycerophosphoethanolamine , 2‐hydroxystearate , gamma‐glutamylthreonine , X‐11470 , X‐14205  suggesting that MR estimates are not confounded by shared genetic components. Subsequently, we estimated the SNP‐heritability of eight metabolites based on LDSC, and the SNP‐heritability (proportion of variance attributed to genome‐wide SNPs) of metabolites ranged from 0.0318 (1‐linoleoylglycerophosphoethanolamine) to 0.1601 (gamma‐glutamylthreonine) (Table LDSC‐based estimates noted little genetic correlation observed between CRC and pyruvate (Re) Table . In addie) Table .3.4p < 5 × 10−8) from the eQTL consortium and a large‐scale GWAS on blood proteome  regulated pyruvate metabolism and was significantly associated with the development of cancer,me Table S8. Colo3.52 (IVs are independent of confounding factors), we checked whether all SNPs associated with eight metabolites were independent of common risk factors  for CRC in the Phenoscanner one by one. We found gamma‐glutamylthreonine to be independent of any confounding factors. Among the other IVs of seven metabolites, a total of 13 SNPs were observed to be associated with common risk factors for CRC (Table p = 0.027), 1,6‐anhydroglucose , nonadecanoate (19:0) , 1‐linoleoylglycerophosphoethanolamine , 2‐hydroxystearate , X‐11470 , X‐14205 .Although SNPs that violated the estimates have been excluded by sensitivity analysis in this work, to satisfy assumptionRC Table . As expeAfter adjusting for metabolite interactions, MVMR estimates based on IVW Figure  and MR‐P3.6Based on six known metabolites, we identified nine metabolic pathways that may be involved in the mechanism of CRC pathogenesis Table . Citrate4In the current work, we integrated two large‐scale GWAS data to explore the causal effects of genetically proxied 486 blood metabolites on CRC via a rigorous MR design. We finally determined that genetically determined high levels of pyruvate, nonadecanoate (19:0), 1‐linoleoylglycerophosphoethanolamine, 2‐hydroxystearate, X‐11470, and X‐14205 associated with lower CRC risk while genetically predisposition to high levels of 1,6‐anhydroglucose and gamma‐glutamylthreonine increased risk of CRC. MVMR estimates suggested that pyruvate, 1‐linoleoylglycerophosphoethanolamine and gamma‐glutamylthreonine can directly affect CRC independently of other metabolites. Colocalization analysis provided strong evidence that significant loci on GWAS signaling can increase CRC risk by influencing the expression of LDHA. Subsequently, we identified 9 metabolic pathways that may be involved in the biological mechanisms of CRC. To our knowledge, this is the first MR study to date that applied the most comprehensive blood metabolite GWAS data to explore the causality with CRC and incorporates metabolic pathways and colocalization analysis.In recent years, the high incidence and mortality rates of CRC have put a heavy burden on people around the world, making early screening and prevention of CRC an urgent strategy. The emergence of metabolomics technologies has led to an increasing interest in exploring the perceived value of metabolites in CRC. Notably, blood metabolites visually provide a snapshot of biological mechanisms since they simultaneously capture endogenous and exogenous processes.In this work, we found that genetic sensitivity to high levels of pyruvate can keep the body away from CRC, while pyruvate is involved in nine significantly enriched metabolic pathways including the citrate cycle and pyruvate metabolism. Several previous studies are consistent with our results that pyruvate shows great opportunities for the prevention and treatment of cancer. Pyruvate bridges cytoplasmic and mitochondrial metabolism as a hub of cellular metabolism, and it mainly originates from the final process of glycolysis that pyruvate kinase encoding the conversion of phosphoenolpyruvate to pyruvate.This MR study also identified three other blood metabolites (nonadecanoate (19:0), 1‐linoleoylglycerophosphoethanolamine, and 2‐hydroxystearate) that have protective effects on CRC. Unfortunately, there are few previous reports of nonadecanoate‐related effects, and the relationship between nonadecanoate and cancer deserves further study. For 1‐linoleoylglycerophosphoethanolamine, Mika A et al. noted that phosphoethanolamine was highly expressed in CRC tissues but its contribution to CRC was not clear.We also confirmed genetic predisposition to higher levels of gamma‐glutamylthreonine and 1,6‐anhydroglucose were detrimental to CRC. To date, there are no reports of gamma‐glutamylthreonine and CRC, with only one study indicating an increased risk of prostate cancer at high gamma‐glutamylthreonine concentrations.In addition, the high support of colocalization evidence from gene and protein wide was observed between LDHA and CRC. LDH catalyzes pyruvate to lactate, the last step of glycolysis. The elevation of LDH increases the rate of glycolysis, which in turn provides an energy source for cancer cells. Moreover, the acidic microenvironment resulting from the accumulation of lactate also promotes cancer cell invasion and angiogenesis.This MR analysis has several advantages. First and most critically this is the most complete and systematic study to date on exploring the causality between blood metabolites and CRC because in this work we analyzed 486 blood metabolites. Second, rigorous MR analysis was applied to reject the inevitable defects of previous studies such as reverse causality and confounding interference. In detail, to generate convincing estimates a series of methods are implemented to ensure that any factors that violate the MR assumptions are removed. The consistency of the three MR estimates in the direction and the sensitivity analysis demonstrated the robustness of the results. Third, the reliability of the results was further verified by additional GWAS data for replication analysis and meta‐analysis. Although the results of the replication analysis were not significantly attributable to differences in sample size, it showed consistent directionality with the primary analysis, which does not appear to have occurred by accident. Fourth, we assessed the heritability of IVs and the genetic correlation between metabolites and CRC using LDSC, which made the MR estimates more convincing. We also performed colocalization analysis to demonstrate, at both the gene and protein levels, that alterations in significant loci on GWAS signaling can influence CRC by regulating LDHA expression.The current study also has several limitations. One limitation of our study is the limited number of SNPs available for the exposure of interest at the genome‐wide level. To address this, we set slightly relaxed thresholds for our MR analysis, a practice commonly adopted in other studies. However, the F‐statistic value for all selected SNPs exceeded 10, suggesting that our IVs were sufficiently robust. Moreover, the consistent causal direction supported by the results of the Steiger test lends credibility to our relaxed threshold setting. Second, to minimize the impact of ethnic variability, we only used GWAS data from individuals of European ancestry for this MR analysis. Hence, the generalizability of our findings to other populations warrants further exploration and validation. The third limitation of our study is that the precision of MR estimation partly relies on sample size. Therefore, expanding the sample size is necessary to confirm the reliability of our results. Additionally, while MR analysis provides valuable insights into etiology, it is important to note that our findings should be validated through rigorous RCTs and basic research before application in the clinic.5In summary, this MR study revealed that six genetically proxied blood metabolites have causal effects on CRC, and we identified nine metabolic pathways that could be implicated in the development of the CRC. LDHA deserve further investigation as potential therapeutic targets for CRC. The discovery of these serum metabolites provides valuable insights for early screening, prevention, and treatment of CRC, as well as for the design of future clinical studies. Moreover, this MR analysis combining genomics and metabolomics provides a reference direction for the exploration of the etiology and pathogenesis of CRC.Zhangjun Yun: Formal analysis (lead); methodology (lead); software (lead); validation (lead); writing – original draft (lead). Ziwei Guo: Resources (lead); software (lead); validation (lead); writing – original draft (lead). Xiao Li: Formal analysis ; validation ; visualization . Yang Shen: Data curation ; investigation . Mengdie Nan: Investigation . Qing Dong: Conceptualization ; project administration ; supervision ; writing – review and editing . Li Hou: Conceptualization (lead); funding acquisition (lead); methodology (lead); project administration (lead); supervision (lead); writing – review and editing (lead).This study was supported by the National Natural Science Foundation of China Grant Program (Grant/Award Number: 81573959), Capital Health Development Research Fund (Grant/Award Number: 2020–2‐4193 and 2022–1‐4171).The authors declare no conflicts of interest.All data in this study are available in publicly available databases. No additional ethical approval was required.Figure S1. Forest plots for the Mendelian randomization (MR) leave‐one‐out analysis of the significant inverse variance weighted (IVW) estimates.Click here for additional data file.Table S1. List of the identification (ID) for each of the 486 blood metabolites.Table S2.Table S3. Outliers identified by Radial MR.Table S4. The results of MR‐PRESSO.Table S5. The SNP‐based heritability (h2) of the metabolites.Table S6. Estimation of the Steiger direction test from eight blood metabolites to colorectal cancer.Table S7. The expression quantitative trait loci (pQTL) for blood LDHA.Table S8. The protein quantitative trait loci (pQTL) for blood LDHA.Table S9. Confounders identified from Phenoscanner.Table S10. Metabolic pathways with significant enrichment of blood metabolites.Click here for additional data file.


Germ cell tumors complicated by hematological malignancy (HM) are a rare clinical phenomenon. Allogeneic hematopoietic stem cell transplantation  is a potentially effective therapy, but graft‐versus‐host disease (GVHD) is a life‐threatening complication. We report a case of a 13‐year‐old female patient diagnosed with germ cell tumors followed by acute lymphoblastic leukemia. After chemotherapy, she received allo‐HSCT and her chimerism rate decreased rapidly to near zero by 6 months without evidence of HM recurrence. However, she developed severe, multiorgan GVHD‐like manifestations. DNA analysis revealed the pathogenesis of GVHD to be loss of HLA heterozygosity in recipient hematopoietic cells. This paper presents a unique case of a 13‐year‐old female patient diagnosed with concomitant germ cell tumors and acute lymphoblastic leukemia, who underwent allogeneic hematopoietic stem cell transplantation and subsequently developed severe graft‐versus‐host disease (GVHD)‐like manifestations  despite the absence of donor chimerism and recurrent hematological malignancy. The underlying etiology was found to be HLA loss of heterozygosity (HLA‐LOH) in the patient's newly reconstituted hematopoietic lineage. This is the first report of fatal GVHLD caused by HLA‐LOH in the recipient's own immune system without donor chimerism. The paper highlights the importance of understanding the role of HLA‐LOH in GVHLD pathogenesis. ALLacute lymphoblastic leukemiaGCTgerm cell tumorGVHDgraft‐versus‐host diseaseHSCThematopoietic stem cell transplantation1Germ cell tumors (GCTs) are malignancies originating from primordial and pluripotent germ cells. Nonseminoma GCTs of the mediastinum have a unique clinical presentation and pose a high risk for the development of concomitant hematological malignancy (HM) , 2, 3. IGraft‐versus‐host disease (GVHD) is a common complication of allo‐HSCT mediated by donor‐derived lymphocytes attacking recipient tissues because of mismatched HLA expression. It is commonly accepted that the pathogenesis of GVHD depends entirely on successful donor chimerism. Here, we present a special case of a patient diagnosed first with GCTs and then ALL shortly afterward, who received allo‐HSCT and then developed persistent severe GVHD‐like manifestations despite the absence of donor chimerism and recurrent HM cells. The etiology of such contradicting clinical phenomena was eventually found to involve HLA loss of heterozygosity in the newly reconstituted hematopoietic lineage.2A 13‐year‐old Chinese female, with no significant past medical or surgical history, presented with abdominal distension and pain in January 2019. Ultrasound findings included a naive uterus and mixed masses in bilateral adnexa. The patient had an ɑ‐fetoprotein (AFP) level of 4833 ng/mL and a human chorionic gonadotropin level of 56.51 IU/ml. Exploratory laparotomy was performed on January 26, 2019. Pathology showed that the tumor in the ovary was a mixed malignant GCT. The patient underwent one cycle of chemotherapy with the BEP regimen  in February, 2019.9/L had developed after chemotherapy. Bone marrow aspiration was performed on March 21, 2019, and confirmed the diagnosis of B‐cell ALL, chromosome karyotype: 47, XY, +14[20]. The patient was treated with VDLP , high‐dose methotrexate (MTX), and CAM  chemotherapy from April to July 2019. On August 8, 2019, repeat bone marrow aspiration showed complete remission. Twelve days later on August 20, 2019, ultrasound scans showed severe splenomegaly (19.8 cm × 4.4 cm). On August 22, 2019, repeat bone aspiration showed relapse with flow cytometric analysis, indicating that abnormal B lymphoblasts accounted for 14% of cells. Oral trametinib was started in September 2019 and continued until January 2020. On December 5, 2019, a pelvic and abdominal computed tomography (CT) scan revealed an irregular mass of soft tissue in the pelvis and abdomen, measuring 13.2 cm × 8.3 cm × 20.5 cm. The AFP level at this time was 18 293 ng/mL, suggesting GCT recurrence. She underwent resection of the pelvic mass along with splenectomy and partial omentectomy on December 17, 2019. Pathology showed that the right adnexa tissue and mass were an immature teratoma Grade 3) and immunohistochemical staining revealed B lymphocytic leukemia/lymphoma infiltration of the spleen. The patient then received four additional cycles of chemotherapy with the BEP regimen from December 2019 to March 2020.Serum AFP decreased to 10 ng/mL after surgery and the first cycle of chemotherapy. Thrombocytopenia and significant leukocytosis with a leukocyte count as high as 26.49 × 10l‐asparaginase) chemotherapy from April to July 2020. On August 25, 2020, bone marrow aspiration showed morphological remission and flow cytometric analysis produced negative findings for minimal residual disease. The karyotype remained 47, XY, +14 [20].On April 13, 2020, repeat bone marrow aspiration showed 30% lymphoblasts. Flow cytometric analysis indicated that 9.17% of the cells were CD19+, CD45dim+, CD22+, cCD79a+, CD34+, CD10dim+, CD38+, and CD20 partially+, which was consistent with abnormal B lymphoblasts. The karyotype was 47, XY, +14 [19]. She then received three cycles of VICP  chemotherapy and one cycle of HR‐3 , 5 mg/kg/d on Days −5, −4, −3, and −2. On September 8, 2020, the patient received allo‐HSCT. Peripheral blood stem cells were provided by the father as an HLA‐haploidentical donor (blood type B for A) and she was infused with 8.8 × 108/kg mononuclear cells and 3.95 × 106/kg CD34+ cells. She received cyclosporin A, mycophenolatemofetil, and short‐term MTX as prophylaxis for GVHD. Neutrophil and platelet engraftment occurred on Days +18 and +24, respectively. On day +21, bone marrow morphology showed remission with no minimal residual disease detected by flow cytometry. The donor chimeric rate was 99.3%.The patient received an myeloablative conditioning regimen including total body irradiation/idarubicin/cytarabine/cyclophosphamide/antithymocyte globulin‐Fresenius S starting on August 29, 2020. The detailed process was as follows: total body irradiation at 200 cGy bid/d on Days −10, −9, and −8; idarubicin, 8 mg/mThe patient developed cytomegalovirus viremia on Day +40 and Epstein–Barr viremia on Day +43, and after treatment with rituximab and other antiviral therapies, the virus became negative. Bone marrow aspiration was repeated on day +63, and bone marrow morphology continued to show remission with no minimal residual disease detected by flow cytometry. However, the donor chimeric rate was reduced to 64%. On Day +66, the patient developed fever and diarrhea with gastrointestinal bleeding, which was confirmed by colonoscopy and mucosal biopsy as GVHD and improved after treatment with 2 mg/kg methylprednisolone and basiliximab. Bone aspiration repeated on Day +76 showed 99.5% donor chimerism.On Day +141, the patient presented with chest tightness, dyspnea, and hypoxemia. Chest CT showed multiple ground glass opacities in both lungs. Bronchoscopy and bronchioalveolar lavage showed no overt infection. Immune‐mediated lung injury was suspected, and the symptoms and imaging findings improved significantly after methylprednisolone treatment. Follow‐up bone marrow aspiration on Day +155 continued to show remission with negative minimal residual disease by flow cytometry, but the donor chimerism rate was only 9%. Considering the previous transplantation complications, active bone marrow hematopoiesis with good compensation, and a normal leukocyte count, no specialized treatment was administered. The patient had significant jaundice attributed to blood‐type conversion. On Day +183, bone marrow aspiration showed remission with no minimal residual disease by flow cytometry, but the donor chimerism rate was further reduced to 4% and the donor chimerism rate was 0% in peripheral blood CD3+ T cells. On Day +185, the patient developed fever, hypoxemia, hemoptysis, and diarrhea with gastrointestinal bleeding, and chest CT showed bilateral ground glass opacities ; data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); project administration (lead); writing—original draft (lead). Lili Miao: Data curation ; methodology ; writing—original draft (supporting). Zimu Gong: Data curation (supporting); methodology (supporting); writing—review & editing (lead). Wenqiu Huang: Investigation (supporting); writing—original draft (supporting). Yongping Zhang: Investigation (supporting); writing—original draft (supporting). Fuhong Liu: Investigation (supporting); writing—original draft (supporting). Jingbo Wang: Project administration ; supervision .The authors declare no conflict of interest.This retrospective study was approved by the Ethics Committee of Aerospace Center Hospital (No. 20211216‐XYZZ‐01).Written informed consent for publication of this report and accompanying images was provided by the patient's parents.Supporting Information.Click here for additional data file.Supporting Information.Click here for additional data file.


Observational epidemiological studies suggest that lung cancer risk may be raised by gastroesophageal reflux disease (GERD); however, the causal relationship between them remains unknown. Our study performed the two‐sample Mendelian randomization (MR) approach to examine the causal relationship between GERD and lung cancer.n = 129,080). Summary data from genome‐wide association studies (GWAS) data were used to determine outcomes for lung cancer , squamous cell lung cancer (LUSC), and lung adenocarcinoma (LUAD). In this study, three MR statistical techniques (inverse variance weighted (IVW), MR‐Egger, and weighted median) were used to examine the potential causative relationship between GERD and the risk of lung cancer. Cochran's Q test, MR‐Egger intercept test, leave‐one‐out analysis, and the funnel plot were all used in sensitivity analyses.Instrument variables were found to be independent single nucleotide polymorphisms (SNPs) that were highly linked with GERD  and LUAD . Although potential heterogeneity was observed in some studies, random effect IVW was not violated by the heterogeneity, indicating that the causal effect was robust.The main IVW method revealed that GERD substantially increased the risk of lung cancer [odds ratio (OR) = 1.37; 95% CI 1.16–1.63, GERD was positively associated with the risk of lung cancer, for LUSC and LUAD. This study shed light on a new direction for prevent strategy of lung cancer and therapeutic perspectives in patients with GERD. Overview of the current Mendelian randomization(MR) study. GERD, Gastroesophageal reflux disease. SNP, single nucleotide polymorphism; LD, linkage disequilibrium. IVW, Inverse Variance Weighted; WM, weighted median; ILCCO, Lung Cancer Consortium. Meanwhile, the procedure of conducting a RCT is time‐consuming, costly, and occasionally impractical as well as unethical. An approach to address this limitation is Mendelian randomization (MR), a method for evaluating causality that employs genetic instrumental factors to proxy for exposures that might otherwise be confounded or prone to reverse causation.By combining data from published genome‐wide association studies (GWAS), this study first investigated the potential causality between GERD and lung cancer risk through MR analysis.22.1p < 5 × 10−8); (b) genetic instruments are not associated with potential confounders; and (c) genetic instruments affects the outcome only through the risk factors.Using summary statistics from GWAS, we conducted a two‐sample MR analysis to investigate the causal relationship between GERD and lung cancer. There are three assumptions that have to be met in this MR design: (a) genetic instruments predict the exposure of interest (https://ilcco.iarc.fr/) and two large publicly available GWAS abstracts from Jue‐Sheng Ong.https://gwas.mrcieu.ac.uk/) without requesting through the IEU platform. Specifically, the genetic tool for exposure in this study was derived from recent GWAS pooled data on GERD. A total of 129,080 GERD patients and 473,524 controls were analyzed, and 80 single nucleotide polymorphisms (SNPs) were significantly linked to GERD. ILCCO consortium data were accessed for GWAS lung cancer summary , which includes squamous cell lung carcinomas (LUSC) and lung adenocarcinomas (LUAD). The lung cancer patients were classified according to ICD‐O‐3; SQ: 8070/3, 8071/3, 8072/3, 8074/3; AD: 8140/3, 8250/3, 8260/3, 8310/3, 8480/3, 8560/3, 8251/3, 8490/3, 8570/3, 8574/3; with tumors with overlapping histologies classified as mixed. We also performed sub‐type analyses. Lung cancer was subcategorized as LUSC  and LUAD .The primary analysis data were obtained from International Lung Cancer Consortium (ILCCO) , met the Hardy–Weinberg equilibrium (H–W) and genetic distance <10,000 kb. Subsequently, major alleles, allele frequencies, β‐values, p‐values, and standard errors (SEs) for each SNP were collected. Previous MR studies proved that the application of instrumental variables with high strength can effectively improve the accuracy and efficacy of model estimation. To avoid bias caused by weak proxies, the F‐statistic was calculated and there was no instrumental variable with F‐statistic <10.SNPs associated with GERD required ium LD, R2 ≤ 0.001p < 5.0 × 10–8) for other traits. We did not find SNPs strongly associated with the identified carcinogenic factors. A total of 67 SNPs were included in the final MR analysis , 71 SNPs strongly associated with exposure but not with the outcome were obtained. Subsequently, SNPs  were removed from the SNPs because they had moderate allele frequencies of the palindromes. We looked for 67 single nucleotide polymorphisms in Phenoscanner  to assess whether these single nucleotide polymorphisms were associated with genome‐wide significance levels that could affect our results , weighted median (WM), and MR‐Egger regression were used to analyze the causal relationship between GERD and lung cancer in order to enhance the reliability of causal result.−8 to 5 × 10−9 and then repeated the MR estimates, which had been described by the study of Chen et al.In the case that the directions of three MR estimates were inconsistent, we tightened the p thresholds of the SNPs associated with GERD from 5 × 102.4p ≥ 0.05. The funnel plot was also used to detect the heterogeneity, and a symmetry plot indicated the absence of heterogeneity.The P‐value of the Cochran's Q test was used in this study to assess the presence of heterogeneity in the analysis, and it was considered that there was no heterogeneity in the causal analysis when Cochran Q‐derived A fundamental tenet of MR analysis is that instrumental factors may only influence result through exposure; if instrumental variables did not directly affect outcome through altering exposure, the MR assumption would be violated. Thus, the causal relationship between exposure and outcome should be tested for genetic pleiotropy. The bias brought on by genetic pleiotropy may be assessed using MR‐Egger regression analysis, and the pleiotropy's amplitude can be determined using the regression intercept. In addition, to assess the effect of each SNP, the combined effect of each remaining SNP was calculated using the leave‐one‐out method.33.1p = 0. 0003). Meanwhile, the estimates from the weighted median  and MR‐egger regression  analyses showed a consistent direction of the IVW estimates, though the MR‐egger estimate is insignificant.p = 5.35 × 10−5). For lung adenocarcinoma, the MR estimates from the three models were inconsistent . We then tightened the SNP‐GERD p threshold to 5 × 10−9 and repeated MR estimation, and yielded consistent results from the three MR models (Table ).As shown as Table 3.2Q = 122.70, p = 0.0005), the heterogeneity between GERD and LUSC  and LUAD  wasn't existed in our outcomes. Since there were no statistically significant intercepts, the results of MR‐Egger regression indicated that pleiotropy appears to be minimal . Once an individual SNP was utilized as IV, the funnel plot displayed a symmetric distribution of dots indicating causative interactions, demonstrating that there was less chance that possible bias would have an impact on the causal connection . The “Leave‐one‐out” sensitivity analysis suggested that the results of the IVW analysis of the remaining SNPs were similar to those of the analysis with all SNPs included, and no SNPs were introduced to have a significant effect on the causal association estimates after eliminating each SNP in turn for lung cancer and LUSC  is significantly higher than in the common populace, regardless of lung cancer type.However, the available observational studies have been limited by small study size, lack of detailed information on important confounders, such as smoking, related disorders, duration of GERD, and treatment history.The MR method measures causality by linking an “exposure” and an “outcome,” and reduces the risk of confounding from traditional observational studies, which are commonly used to infer causality, but are time consuming and occasionally impracticable. Noteworthy, the two primary subtypes of NSCLC (LUSC and LUAD), the most prevalent and lethal kind of lung cancer with a high death rate .Taking all these findings into account, our results confirmed the notion that GERD increased the risk of lung cancer. Several interpretations could account for this significant causality between GERD and lung cancer. Anatomically, the esophagus is surrounded by the trachea and lungs, and multiples studies have indicated that GERD‐induced reflux content can activate inflammatory cascades in susceptible cells of bronchial and lung tissues.Several limitations should be considered when evaluating our study. First, since the biological mechanisms of GERD and lung cancer are not fully elucidated, applying relevant SNPs with unclear mechanisms as instrumental variables has the possibility of violating the core MR hypothesis. Besides, owing to this ethnic heterogeneity, it should be cautious to generalize the results using GWAS data, which are taken from European people and have diverse cultural traditions, to other ethnic groups. Additionally, there is a lack of a formal mediation analysis to explore the possible pathways underlying GERDs association with lung cancer. Last but not least, given the diversity of lung cancer patients, GERD may be causally related to certain lung cancer subtypes, and a more extensive study of lung cancer subgroups may be considered in the future.In conclusion, this study bolstered the case for a genetic link between GERD susceptibility and lung cancer. Considering the high mortality and morbidity associated with lung cancer patients, it is of significance to recognize and control the risk factor of GERD for lung cancer in order to decrease its prevalence.lin Li: Conceptualization (lead); formal analysis (lead); methodology (lead); resources (lead); visualization (lead); writing – original draft (lead); writing – review and editing (lead). Qiaoya Ren: Formal analysis (lead); investigation ; software (lead); validation ; visualization . Qian Zheng: Formal analysis ; validation . Yuju Bai: Investigation ; project administration ; resources ; visualization . Si Si He: Conceptualization ; resources . Yu Zhang: Investigation ; validation . Hu Ma: Conceptualization (lead); data curation (lead); project administration (lead); supervision (lead); writing – review and editing .This work was supported by grants from the following sources: (a) Research Programs of Science and Technology Commission Foundation of Guizhou Province (Grant Nos. QKHZC[2020]4Y156); (b) Research Programs of Science and Technology Commission Foundation of Guizhou Province (Grant Nos. QKHJC [2020]1Z062); (c)Guizhou Province Science and Technology Project (Qian Ke He[2019]5406).All authors declare no conflict of interests.Ethics approval UK Biobank received ethics approval from the National Health Service National Research Ethics Service. Written informed consent was obtained for all participants electronically.Appendix S1.Click here for additional data file.Appendix S2.Click here for additional data file.


Adequate supplies of donor blood remain a major challenge in sub‐Saharan Africa. This is exacerbated by a lack of confirmatory testing for transfusion‐transmitted infections by blood transfusion services (BTS), leading to significant blood disposal owing to putatively high seroprevalence rates amongst Ugandan blood donors. We aimed to ascertain the false discovery rate of the Architect anti‐hepatitis C virus (HCV) screening assay and categorize screen‐reactive samples into three groups: presumed false positive, active and past infection, and develop an algorithm for confirmatory testing.A total of 470 screen‐reactive HCV blood donations were retested using the Architect anti‐HCV assay, an alternative antibody test (SD Biosensor) and a core antigen (cAg) test. signal‐to cut‐off (S/CO) ratios and pre‐analytical factors  were recorded. Based on the S/CO ratio evaluation, we propose a testing algorithm to guide supplemental tests.The false discovery rate of the Architect anti‐HCV assay was 0.84 as 395/470 (84%) screen‐reactive samples had no evidence of HCV infection (SD Biosensor and cAg negative) , 38/470 (8.1%) were antigenaemic, and 32/470 (6.8%) had evidence of past infection. The median S/CO ratios of the presumed false‐positive and active infection samples were 1.8 and 17.3, respectively. The positive predictive value of HCV positivity in samples with ratios above 12 was 91.8%. On retesting, 104/470 (22.1%) samples became negative.The Architect anti‐HCV assay has a very high false discovery rate in Ugandan BTSs, leading to excessive blood disposal. Pre‐analytical factors likely contribute to this. An introduction of confirmatory testing using an algorithm based on S/CO ratio evaluation could limit unnecessary blood wastage and donor deferral. Eighty‐four per cent of screen‐reactive hepatitis C virus (HCV) blood donations in Ugandan blood transfusion services were identified as presumed false positive.Active HCV infection is only present in 8.1% of screen‐reactive HCV samples.Introduction of confirmatory testing using an algorithm based on signal‐to cut‐off ratio evaluation could limit unnecessary blood wastage and donor deferral.Providing safe blood for transfusion in sub‐Saharan Africa (sSA) remains a challenge in the context of limited resources and infrastructure, suboptimal diagnostics and a high prevalence of transfusion‐transmissible infections (TTIs) . The epiThe mean HCV seroprevalence rate in blood donors attending Ugandan blood transfusion services (BTS) over the last 5 years is 1.8% ; however, the rate ranges between 0% and 8% depending on geographical region and season. National HCV seroprevalence rates for the general population are estimated at 2.7% (0.4–7.0) . There iThe barriers to the implementation of NAT‐based testing are well described and significant in low‐ and middle‐income countries (LMICs) . HCV cAgThis study aimed to determine the false discovery rate of the anti‐HCV screening assay used in two Ugandan BTS centres by retesting screen‐reactive samples with supplemental tests. It aimed to understand the true prevalence of HCV amongst quarantined HCV screen‐reactive blood and categorize this into three groups: active HCV infection, past cleared infection and presumed false‐positive results. Secondary aims were to understand the significance of signal‐to‐cut‐off (S/CO) ratios of the Architect anti‐HCV assay, propose an HCV testing algorithm and investigate pre‐analytical factors affecting results, including haemolysis, time delay and centrifugation speed.g for 10 min before storage to ascertain whether this affected the S/CO ratios on retesting.A total of 470 consecutive anti‐HCV reactive serum samples from voluntary non‐remunerated blood donors were identified between February 2019 and January 2020 in two BTS centres in Uganda: 235 from Nakasero National BTS, Kampala, and 235 from Mbale Regional BTS, Mbale. These samples had been collected, transported, and stored in the respective BTS laboratories, according to routine Ugandan BTS procedures. They had undergone TTI screening as per the National BTS algorithm by anti‐HCV chemiluminescent microparticle immunoassay (CMIA) on the Architect i2000SR analyser  according to BTS Standard Operating Procedures that follow the manufacturer's instructions. A S/CO of ≥1.00 was considered IR and retested in duplicate as per the manufacturer's guidance. The S/CO ratio results for the first and duplicate runs for each sample were recorded directly from the automated system. Both IR and repeatedly reactive (RR) samples were included in the study and are termed ‘screen‐reactive’. Samples with S/CO ratios of 1.00–1.99 were described as borderline reactive in the analysis. A visual haemolysis check was performed using a colour chart on all samples as a proxy for sample quality. The date of blood collection and screening was documented. A delay between sample collection and testing was defined as more than 2 days. Screen‐reactive samples from both sites were prepared into serum aliquots for storage at −80°C. The 235 samples from Mbale underwent an extra centrifugation step (ultracentrifugation) using a microcentrifuge at 10,000 All 470 sera were thawed (after a single freeze–thaw cycle), mixed thoroughly by low‐speed vortexing and retested using the same Architect anti‐HCV assay. S/CO ratios of the repeat testing were recorded to observe any differences in S/CO ratios between the BTS screening and repeat testing, particularly following ultracentrifugation in the Mbale samples.In order to discriminate presumed false‐positive from true‐positive results (both active infection and past infection), samples underwent two supplementary tests: an alternative antibody‐based test and a test to confirm active infection (HCV cAg). The antibody‐based test was the STANDARD Q HCV Ab Test , a rapid chromatographic immunoassay for the qualitative detection of specific antibodies to HCV, prequalified by WHO in 2020 with a quoted sensitivity and specificity of 99.4% (96.6–100) and 99.7% (98.3–100), respectively . The ArcAll samples included in the study were screen‐reactive, that is, were either IR or RR on BTS Architect anti‐HCV screening. Samples also positive for HCV cAg were considered to represent active HCV infection (antigenaemic). Those negative for HCV cAg, with positive SD Biosensor results, were interpreted as past, resolved HCV infection. Samples negative for both SD Biosensor and HCV cAg were regarded as presumed false‐positive anti‐HCV results with no evidence of HCV infection. Samples that fell within the grey zone for the cAg assay or were invalid were categorized as unconfirmed.Based on S/CO ratio evaluation of the Architect anti‐HCV assay, we propose a testing algorithm to guide supplemental tests on IR samples. We applied our dataset to the proposed algorithm.Ethical approval was sought and granted by the Imperial College and Ugandan Research Ethics Committees.χ2 for categorical variables. Significance was defined as a p value of <0.05.Statistical analysis was performed in GraphPad Prism version 9.0 . Median with interquartile range (IQR) was used for S/CO ratios. Median differences in S/CO ratios between BTS screening and repeat testing were analysed using the Wilcoxon signed‐rank test. The presence of haemolysis by time delay between collection and testing was analysed using Following anti‐HCV retesting and additional testing, 395/470 (84.0%) of the screen‐reactive samples tested negative by SD Biosensor and HCV cAg, confirming these as presumed false positives Figure . In thesA S/CO ratio of >15 correlated with a positive cAg in 35/41 (85.4%) samples and a positive SD Biosensor result in 39/41 (95.1%) samples  cAg‐positive samples, the SD Biosensor was negative, suggesting false‐negative SD Biosensor results. The anti‐HCV S/CO ratios of these three samples were 1.4, 3.1 and 17.5 . Two  of the samples with S/CO ratios less than four had confirmed cAg presence. A significant proportion  of samples with S/CO ratios of 3.00–11.99 were classified as presumed false positives. The median S/CO ratio of the presumed false‐positive samples was 1.8 (IQR 1.0–3.2), whereas that of the cAg‐positive samples was 17.3 (IQR 16.3–18.1). The median ratio of the samples displaying past cleared infection was 5.7, with a wider IQR of 3.7–11.3.Z = 7.42, p value > 0.001) and 0.41 in Nakasero . Following retesting of all samples, a statistically significant median increase in S/CO ratio of 0.45 was seen in all samples from Nakasero , whilst a non‐statistically significant median decrease of 0.15 was observed  in Mbale.On the first anti‐HCV screen, a large proportion of samples  were borderline reactive (S/CO ratios 1.00–1.99) .Overall, 262/458 (57.2%) samples showed some level of visual haemolysis, with a higher proportion of Mbale samples showing evidence of haemolysis compared to Nakasero samples (95/235 [40.4%] vs. 167/223 [74.9%]). The median time between donor sample collection and initial screening was 4 days (range 0–33 days) across both sites. There was a significant difference in the proportion of samples with haemolysis in those that were processed after a delay (>2 days)  versus those that were processed without delay (0–2 days)   IR for HCV were discarded unnecessarily. The positive predictive value (PPV) of 0.16 is unacceptably low for a screening test. The BTS report that each year, on average, 224,350 units are donated and screened, with 4038 units discarded on the basis of Architect anti‐HCV positivity . Assuming 84% of these are, in fact, false positive, an estimated 3392 units are discarded unnecessarily each year. Many of these donors were therefore preliminarily classified as HCV seropositive necessitating post‐donation counselling, retesting and donor deferral at a significant economic and personal cost to the BTS and donor, respectively.False positivity in anti‐HCV screening immunoassays is well described and multi‐factorial, with reported false positivity rates of between 10% and 60% , 20. PopStudies have shown a relationship between the S/CO ratio of the anti‐HCV test and the infection status of the individual , 19, 27.We believe that our proposed testing algorithm based on S/CO ratio evaluation presents a pragmatic approach to providing safe blood whilst maintaining judicial use of supplemental tests in LMICs. Importantly, when applied to our dataset, no cAg‐positive samples would have been transfused, and only four false reactive samples (in the S/CO ratio > 12 groups) would have been discarded. The main drawback of omitting confirmatory tests in samples with higher S/CO ratios is that donors would not receive comprehensive results on infection status. By limiting the use of alternative antibody tests to samples with ratios of 3.00–11.99, there is a small but appreciable risk that samples in the lowest ratio category (1.00–2.99) may be anti‐HCV positive (cAg negative) suggesting past cleared infection. These donors would not be accepted for donation in Uganda under current guidelines. We identified 5/183 (2.7%) antibody‐positive cAg‐negative samples in the 1.00–2.99 group, which would have been recommended for transfusion. The risks of transfusing antibody‐positive blood would need to be balanced against resource scarcity, and we acknowledge this as a controversial area. One case of active infection occurred in a sample with a borderline reactive anti‐HCV screening test (S/CO ratio 1.4). This can occur during acute HCV seroconversion or in immunocompromised hosts , and strCurrently, all donors with screen‐reactive (IR or RR) results are deferred, either temporarily or permanently, depending on repeat testing results 3–6 months later and the availability of an alternative anti‐HCV test. The cost of permanent deferral is ~$81  and includes phlebotomy, initial TTI screening, retesting, post‐donation counselling and blood unit disposal. Using the proposed algorithm with supplemental tests (at $10/cAg test and $1/SD Biosensor) to confirm or refute positivity for the 366 donor samples was $1647 compared to the potential cost of permanent deferral of $29,646. In addition, accurate results through confirmatory testing would facilitate linkage to care where appropriate, avoid unnecessary anxiety and return visits for donors and enable those testing falsely positive to return to the donor pool. Furthermore, incorrect seroprevalence data generated as a result of poor diagnostics have repercussions on the accuracy of HCV epidemiology, surveillance, and linkage to care, and therefore, elimination strategies , 21, 30.As described above, both a strength and limitation of this study is that screen‐reactive samples were identified for inclusion after BTS screening; therefore, the process by which they were taken and screened was not according to a research protocol, as such, samples were potentially subject to pre‐analytical errors such as sample haemolysis and testing delays. This may have affected the validity of the assays used in both BTS screening and subsequent testing. It may be a contributing factor to the high false discovery rate. We argue that it is important to present real‐world data in an effort to understand how tests are performing in LMIC laboratory conditions, and therefore, how to improve processes and tailor algorithms appropriately.Our results and the proposed algorithm rely on the alternative antibody test possessing a similar sensitivity to the Architect anti‐HCV assay to reliably categorize screen‐reactive samples as either false positive or past infection. A recent large meta‐analysis showed that RDTs showed comparable sensitivity and specificity profiles compared to EIAs in diverse populations , and speThe gold standard test to detect HCV RNA is NAT. We chose HCV cAg as the confirmatory test as NAT requires a dedicated sample to be taken to maintain RNA integrity, which was not feasible in this study. It is, therefore, conceivable that due to the slightly higher limit of detection of cAg, active infections could theoretically be missed. However, cAg testing is endorsed by WHO as a confirmatory test  and has In conclusion, due to both a high false discovery rate of the Architect anti‐HCV screening assay in this setting and the lack of access to confirmatory testing, a significant amount of blood donated is discarded unnecessarily. Since blood for transfusion is relatively scarce, focusing available resources on obtaining accurate HCV results would result in economic gains from limiting donor deferral and reducing unnecessary blood disposal. Optimization of pre‐analytical factors, including introducing ultra‐centrifugation before testing may reduce false‐positive rates, especially in samples with low positive S/CO ratios. Knowledge of Architect threshold S/CO ratios in predicting anti‐HCV positivity may be helpful in updating testing algorithms and guiding the supplemental tests with which to perform in screen‐reactive samples. We suggest that the algorithm we developed should be validated in other sSA BTS where HCV detection is higher than anticipated in low‐risk blood donor populations. The introduction of confirmatory testing in BTS settings is paramount for accurately identifying HCV‐positive donors who require follow‐up care and confidently distinguishing false‐positive and true‐positive anti‐HCV results to limit blood wastage and unnecessary donor deferrals.The authors declare no conflicts of interest.


Hippocampal theta oscillations have been implicated in associative memory in humans. However, findings from electrophysiological studies using scalp electroencephalography or magnetoencephalography, and those using intracranial electroencephalography are mixed. Here we asked 10 pre-surgical epilepsy patients undergoing intracranial electroencephalography recording, along with 21 participants undergoing magnetoencephalography recordings, to perform an associative memory task, and examined whether hippocampal theta activity during encoding was predictive of subsequent associative memory performance. Across the intracranial electroencephalography and magnetoencephalography studies, we observed that theta power in the hippocampus increased during encoding, and that this increase differed as a function of subsequent memory, with greater theta activity for pairs that were successfully retrieved in their entirety compared with those that were not remembered. This helps to clarify the role of theta oscillations in associative memory formation in humans, and further, demonstrates that findings in epilepsy patients undergoing intracranial electroencephalography recordings can be extended to healthy participants undergoing magnetoencephalography recordings. Oscillatory activity within the hippocampal-entorhinal system has long been hypothesized to play a critical role in cognitive function . In partElectrophysiological studies have demonstrated that theta oscillations are also present during movement in the human hippocampus , albeit The reasons for these discrepancies are perhaps manyfold , but we Here we aimed to assess the role of hippocampal theta activity in subsequent associative memory. To do this, we used a task and memoranda specifically designed to promote associative binding , given tn = 2) and (ii) only 1 or no electrode contacts located in the hippocampus (n = 2). Accordingly, 10 patients  were included in the analyses.Fourteen patients with drug-resistant epilepsy undergoing iEEG monitoring for clinical purposes were asked to perform an associative memory task similar to that used in M ± SD number of contacts = 3.10 ± 1.45), amygdala (M ± SD number of contacts = 2.10 ± 0.99), and temporal neocortex (M ± SD number of contacts = 10.50 ± 2.99). Electrode implantation was unilateral in all patients and dictated by clinical requirements (see Preimplantation MRI and postimplantation CT scans were co-registered to identify electrode locations in the hippocampus (Depth EEG was recorded at 512 Hz (patient 1), 1,024 Hz , or 2,048 Hz (patients 8–9) using a Micromed SD long-term monitoring system (Micromed). The EEG signal was referenced against a common white matter contact that was located remotely from the suspected epileptogenic focus in each patient. Recordings made at a higher sampling rate were downsampled to 512 Hz, to match those recorded with the lowest sampling rate, before any analyses were performed.kitchen), 36 famous people (e.g. Barack Obama), 36 common objects (e.g. hammer), and 36 animals (e.g. dog). From these, 18 randomized location–person–object and 18 randomized location–person–animal events were generated for each patient. For each patient, 9 location-person-object and 9 location-person-animal events were then randomly assigned to be learnt during encoding, and elements from the remaining events were used as foils  during the old/new recognition judgement at test (see below).The stimuli consisted of 36 locations . All pairs were presented on a computer screen as text; 1 element to the left and 1 to the right of fixation. The left/right assignment was randomly chosen on each trial. Each word-pair remained on screen for 6,000 ms. Patients were instructed to imagine, as vividly as possible, the elements interacting in a meaningful way. The word-pair presentation was preceded by a 2,000-ms fixation and followed by a 2,000-ms blank screen.The memory task was similar to that developed by The pairs were presented across 3 blocks with one pair from each event presented during each block, such that the presentation of a pair from one event was interleaved with the presentation of pairs from other events. Within each block, the presentation order of events was randomized. Furthermore, the order of presentations across the 3 blocks was pseudo-randomized such that the presentation order of 1/3 of the events was (i) person–location, location–object, object–person, (ii) location–object, object–person, person–location, and (iii) object–person, person–location, object–person, respectively.Barack Obama) that was drawn from one of the events learnt during encoding or from an equal number of novel events that patients had not seen during encoding. Each cue was presented in the center of the screen and remained on-screen for 3,000 ms. Patients were then asked to indicate whether this was an element that had been seen during encoding using an old/new recognition judgment. Patients had a maximum of 8,000 ms to provide a response. For “old” cues only, the old/new judgment was followed by a forced-choice associative memory task, irrespective of whether the recognition response was correct or incorrect. During the associative memory test, 6 possible associates (1 target and 5 foils) were presented alongside the cue element. The target would be one of the other elements seen together with the cue during encoding. The 5 foils would be elements from the same category as the target. For instance, if the patient had been presented with Barack Obama–kitchen during encoding and cued with Barack Obama during test, then the target would be kitchen and the 5 foils would be other randomly selected locations from other events seen during encoding.During test see , patientBarack Obama and required to retrieve kitchen, they would then be asked to retrieve hammer from among 5 randomly selected objects from other events. The target and foils were presented in 2 rows of 3 below the cue, and the location of the correct target element was randomly selected on each retrieval trial. Patients had a maximum of 10,000 ms to respond with a key press. Responses that fell outside this response window were treated as incorrect (M ± SD% of missing response = 4.91 ± 5.42).For each cue, both elements that were paired with the cue during encoding would be tested in immediate succession before patients were presented with another cue element. For example, if a patient was presented with Barack Obama and asked to retrieve kitchen and then hammer, during block 2 cued with kitchen and required to retrieve hammer and then Barack Obama, and lastly during block 3 cued with hammer and asked to retrieve Barack Obama, then kitchen. Hence, for each event, each element acted as a cue once across the 3 blocks  and as a retrieval target twice across the 3 blocks .Each event was tested with cue–target associations in both directions across 3 blocks. For instance, during block 1, patients could be cued with Barack Obama) and were followed by 2 associative retrieval trials ; and 54 of which related to elements that patients had not seen during encoding and were not followed by any associative retrieval trials.Encoding and test were split into 2 phases, such that all 3 pairs from the first 9 events were encoded  and then tested in both directions , before pairs from the remaining 9 events were encoded and tested. Hence, across the 2 encoding/test phases, patients were presented with a total of 54 encoding trials, 108 old/new recognition trials, and 108 associative memory retrieval trials. Note that retrieval trials followed only after the presentation of cue elements that patients had seen during encoding. As such, during test, patients were required to make a total of 108 old/new recognition judgments; 54 of which related to elements presented during encoding  was preceded by a 2,000-ms fixation and followed by a 2,000-ms blank screen.M ± SD% of excluded trials = 12.87 ± 16.53; M ± SD number of included trials per condition = 15.80 ± 11.34, 16.99 ± 4.08, 14.25 ± 9.37, for 0, 1, and both directions correct, respectively).Estimates of oscillatory power during the encoding period were obtained by convolving the EEG signal with a 7-cycle Morlet wavelet generated using SPM12 . Time-frn = 1) and (ii) insufficient trials to allow for comparisons of subsequent memory performance . Thus, a total of 21 participants  were included in the analyses.Twenty-six participants were recruited to perform the associative memory task. Ethical approval was granted by the local research ethics committee at University College London and all participants gave written informed consent to take part. All participants were compensated for their participation. Five participants were excluded from the analyses for the following reasons: (i) poor data quality (kitchen), 36 famous people (e.g. Barack Obama), 36 common objects (e.g. hammer), and 36 animals (e.g. dog). For each participant, 18 of the 4-element sets were randomly assigned to a closed-loop associative structure. For closed-loops, 9 of the 4-element sets were assigned to be location–person–object events, and the remaining 9 were location–person–animal events. The remaining 18 4-element sets were randomly assigned to an open-loop associative structure, all of which contained all 4 elements.The stimuli consisted of 36 locations  are presented paired with all other elements of the same event  for closed-loops, whereas 4 elements belonging to the same event  are presented as a chain of overlapping pairs  for open-loops. This experimental manipulation has previously been shown to produce greater evidence of pattern completion  for closed- compared with open-loops, both in terms of behavioral responses and BOLD activity . Each of the 36 events were tested with cue–target associations in both directions across 6 retrieval blocks, with one randomly chosen pair from each event tested in each block, making a total of 216 retrieval trials. Each test trial  was preceded by a 3,000-ms fixation and followed by a 1,000-ms blank screen.At test see , particiNote also that in contrast to the iEEG study, encoding and test were not split into 2 encoding/test phases. Instead, participants encoded pairs belonging to all 36 events prior to being tested on all cue–target associations. Again, this difference stems from the fact that the iEEG task was designed to avoid overtaxing the patients.MEG recordings were made using a 275-channel Canadian Thin Films MEG system with SQUID-based axial gradiometers (VSM Med-TECH) while participants sat upright in a magnetically shielded room. Recordings were made at a sampling rate of 480 Hz. Head position coils were attached to nasion and left and right preauricular sites for anatomical coregistration.All data preprocessing and analyses were performed in SPM12  with theM ± SD% of excluded trials = 5.78 ± 8.10; M ± SD number of included trials per condition = 20.00 ± 16.73, 25.39 ± 5.75, 56.48 ± 23.09, for 0, 1, and both directions correct, respectively). Maps were generated on a 10-mm grid, coregistered to MNI coordinates and all SPM images were smoothed using a 12 × 12 × 12-mm full-width half-maximum Gaussian kernel.MEG source localization was conducted using the linearly constrained minimum variance beamformer with a single-shell forward model to generate maps of mean source power differences  for triaPFWE < 0.05 whole-brain corrected.Given our specific hypothesis regarding the hippocampus, hippocampal SPM results are small volume corrected (SVC) within a bilateral hippocampal mask . The masBarack Obama–kitchen). Patients/participants were then required to retrieve the learnt pairs using a forced-choice associative memory test, where a cue element (e.g. Barack Obama) was presented alongside 6 possible targets (e.g. kitchen and 5 foils from the same category as the target). Each cue–target association was retrieved in both directions .Our associative memory task involved patients/participants first encoding a series of pairwise associations (e.g. t(20) = 12.12, P < 0.001, d = 2.65. Mean memory performance in the iEEG study was 48%, which is numerically lower than that seen in prior studies with healthy participants. This is consistent with evidence showing decreases in memory performance in patients with focal epilepsy (t(9) = 5.26, P < 0.001, d = 1.66. Similarly, patients’ old/new recognition performance  was significantly above chance (0.50), t(9) = 18.25, P < 0.001, d = 5.78. Note that for patients who had no false alarms (n = 4), values were set to 1/N, where N is the number of trials corresponding to “new” cues.The mean proportion correct and mean proportion of pairs retrieved correctly in 0, 1, and both directions in the iEEG and MEG study are presented in epilepsy , but we α = 0.05 (1-tailed), #permutations = 1,000) where power increased relative to baseline. Note that because the number of contacts differed across patients, power values were averaged over electrode contacts for each patient prior to analysis. This revealed a significant positive cluster in the theta frequency band (at ~2–7 Hz) from ~0 to 1,500 ms following the onset of the encoding period  = 2.68, P = 0.03, d = 0.85  = 2.70, P = 0.09, pη2 = 0.17. Similarly, paired sample t-tests comparing theta power between pairs that patients did not later retrieve vs those remembered in 1 direction and those remembered in 1 vs both directions revealed no significant effects, ts < 1.93, Ps > 0.08, consistent with the indeterminate status of pairs correctly retrieved in only 1 direction. In sum, we see evidence to suggest that theta oscillations during encoding contribute to later memory, at least in so far as theta activity differs between pairs that were recalled in their entirety and those that patients did not remember.For completeness, we also performed a 1-way ANOVA comparing mean theta power  during the encoding of pairs that were subsequently retrieved correctly in 0 vs 1 vs both directions. It is important to note, however, that activity relating to pairs that were later retrieved correctly in only 1 direction is not a good indicator of subsequent memory, as it reflects a mixture of both successful and unsuccessful encoding. This ANOVA did not reveal a significant effect of subsequent memory, ts < 1.70, Ps > 0.12.Interestingly, some prior studies have shown that pre-encoding theta activity can also be predictive of subsequent memory success . HoweverF = 0.09, P = 0.91, pη2 < 0.01.Having examined the time-frequency spectrograms between encoding and baseline, and across differences in subsequent memory, we now wanted to ascertain whether these differences may be affected by changes in spectral “tilt” . To do st-test, but observed no effect of subsequent memory success, t(9) = 0.44, P = 0.67, d = 0.13. In sum, this suggests that there is no change in spectral tilt during the time window of interest according to subsequent memory performance and is consistent with the proposal that while theta activity supports associative memory, spectral tilt may reflect a more general index of activation , #permutations = 1,000), to identify increases in oscillatory power on electrode contacts in the temporal neocortex or amygdala during encoding. Note that 1 patient had no depth electrodes located in the amygdala and as such this analysis includes only 9 patients (see Ps > 0.15). However, a significant cluster was observed in the amygdala, where power increased relative to baseline (at ~3–6 Hz) at ~500–2,000 ms following the onset of encoding .As theta oscillations have been shown to be widespread across the temporal lobe during encoding , we nextents see . No signt-test revealed no significant difference, t(8) = 0.81, P = 0.94, d = 0.03. Similarly, paired sample t-tests comparing theta power between pairs that were not later retrieved and those remembered in only 1 direction and those remembered in 1 and both directions revealed no significant effects, ts < 0.19, Ps > 0.85.To examine whether power during encoding in the amygdala differed according to subsequent memory performance, we assessed whether increases in theta power  differed between the encoding of pairs that were retrieved correctly in both directions and those that were consistently not retrieved. A paired sample ts < 1.41, Ps > 0.19  and frequency (2–7 Hz) band where hippocampal theta activity increased relative to baseline during encoding, split by subsequent memory performance (i.e. 0 vs 1 vs both directions correct), for electrode contacts located in the amygdala and temporal neocortex. Pairwise comparisons of theta power split by subsequent memory revealed no significant differences in mean theta activity  in contacts located in the temporal neocortex, s > 0.19 , or amygs > 0.31 .F = 6.43, P = 0.04, pη2 = 0.45, but not across electrode contacts located in the hippocampus, temporal neocortex and amygdala, F = 1.23, P = 0.32, pη2 = 0.13, even as an interaction with subsequent memory performance, F = 0.83, P = 0.45, pη2 = 0.09.These results suggest that changes in theta power as a function of subsequent associative memory may show a different pattern within the hippocampus compared with that in the amygdala and temporal neocortex. However, for the 9 patients with electrode contacts in all 3 regions, a 2x3 ANOVA for subsequent memory (0 vs 2 directions correct) and region  showed that theta power differed significantly according to subsequent memory performance, t-tests.To assess this further, we examined the relationship between trial-by-trial variations in mean theta power in the 0 to 1500-ms encoding window within each region. To do so, we first computed power for each electrode contact in each region and then estimated the linear relationship between mean theta power (at 2–7 Hz) on each contact across the regions. We then averaged the beta coefficients for each pair of electrode contacts across the regions and assessed whether the observed fit consistently deviated from 0 using 1-sample t(9) = 4.99, P < 0.001, d = 1.58, and amygdala, t(9) = 4.86, P < 0.01, d = 1.62. Combined, these findings are consistent with the idea that theta oscillations across the temporal lobe may be driven by a single source  and time window  identified in the iEEG study. However, as a control to ascertain whether any effect was specific to the theta frequency band, we also assessed power changes in 3 other canonical frequency bands . Note that the method for computing mean source power used here  requirest-contrast between pairs retrieved correctly in 0 and both directions. This analysis revealed an effect of subsequent memory with greater theta activity within the bilateral hippocampal mask for trials later retrieved correctly in both directions than those not retrieved correctly, with a maxima around the right hippocampus . In addition, there was an effect of subsequent memory at the whole-brain cluster corrected level in the inferior temporal  and cingulate gyrus . No such differences were seen when contrasting source reconstructed theta power for trials associated with pairs retrieved in 0 and 1 directions correctly and those retrieved correctly in 1 and both directions. Similarly, no differences were seen in the alpha, beta, or gamma frequency bands, either at the whole-brain level or within the bilateral hippocampal mask.As a first step, source reconstructed theta power was estimated separately for trials associated with pairs that participants later failed to retrieve, retrieved correctly in 1 direction, and those retrieved correctly in both directions. To assess our main effect of interest, source reconstructed theta power was examined in a second-level general linear model that contained a single F-contrast corresponding to the main effect of subsequent memory. This analysis also revealed an effect of subsequent memory within the bilateral hippocampal mask, with a maxima around the right hippocampus  = 3.57, P < 0.001, d = 0.78  = 3.38, P < 0.01, d = 0.74 (t(20) = 1.74, P = 0.10, d = 0.38.A paired sample d = 0.74 . There wt(20) = 6.01, P < 0.001, d = 1.31, and 1 direction, t(20) = 4.24, P < 0.001, d = 0.92, relative to those pairs that were not remembered, respectively  = 2.33, P = 0.03, d = 0.51. This difference may contribute to the theta power effect seen when contrasting pairs retrieved correctly in 1 and 0 directions in the baseline normalized MEG data during the encoding period. No other significant differences during the baseline period were seen, ts < 1.82, Ps > 0.09.We next assessed whether raw theta activity in the source reconstructed MEG data during the −2,000 to −500-ms baseline period differed between encoding trials that were associated with pairs retrieved correctly in 0, 1, or both directions. To do this, we computed mean source power in the 2–7 Hz theta frequency range during that baseline period, and then extracted power values from the right hippocampal-centered 10-mm sphere specified above. Pairwise comparisons of the extracted power revealed that theta activity was significantly lower for trials associated with pairs that were retrieved correctly in 1 direction relative to those that participants failed to retrieve, In humans, hippocampal theta is thought to be critical for successful memory formation . HoweverInvestigations of the role of theta activity in memory formation have yielded contrasting results, with studies using non-invasive recordings in healthy populations showing that increased theta during e.g.  or priorThe associative nature of the task used here, along with requirements to vividly imagine the items interacting, might be important factors in our finding of positive subsequent memory theta effects in the iEEG and MEG studies, as compared with the less deliberative free recall of single items used elsewhere when negative theta subsequent memory effects have been observed e.g. . It is pWe have also demonstrated that our intracranial results extend to a measure of oscillatory activity recorded using MEG in nonclinical populations. This point is critical because human iEEG studies face the issue of extrapolating findings in patients to the general population, and here we show that averaged effects across patient populations can, at least in this instance, be translated to healthy participants. Although we were able to detect the presence of positive hippocampal theta subsequent memory effects in both the iEEG and MEG study, we are unable to definitively confirm that these effects originate in the hippocampus. Nonetheless, the observation of correlated trial-by-trial variations in theta activity across temporal lobe recording sites in the iEEG study support the proposal that this activity may be driven by a single source . In addiAlthough the findings in the MEG study in large part overlapped with those observed in the iEEG study, they did differ in one aspect. In the iEEG study, we observed that hippocampal theta power during encoding was greater for pairs subsequently retrieved correctly in both directions relative to those not retrieved at all (a finding replicated in the MEG study). However, in the MEG study , we also saw that theta power was greater for those pairs that were retrieved correctly in 1 direction but not the other, compared with those that participants did not subsequently remember. It is possible that epileptic pathology and the relatively small sample size in the iEEG study may have reduced our ability to detect such a difference. Interestingly though, when extracting mean theta power from the same region, we did observe that baseline theta activity in the MEG study was lower for pairs that participants later retrieved correctly in 1 direction relative to those that participants did not remember (an effect not seen in the iEEG study). It is possible that this difference during the baseline period may contribute to the theta power difference seen in the baseline corrected MEG data during encoding.system state prior to encoding, in addition to changes induced by the presentation of the pairs or those evoked by the underlying theta rhythm.Indeed, we cannot rule out some contribution of baseline decreases in theta power to the positive subsequent memory effect we observed here. Except for the above, we saw that baseline activity was not predictive of subsequent memory performance. Nonetheless, even small, nonsignificant, variations in baseline theta activity may lead us to overestimate differences in encoding activity across pairs retrieved correctly in 0, 1, or both directions. In this sense, the results presented here could potentially reflect changes in some We are also unable to specify the functions contributing to subsequent memory, which may include effects of attention or task engagement. Here we assess the contribution of hippocampal theta power to later memory performance by contrasting encoding trials associated with pairs that were correctly retrieved to those in which they were not. As such, our main effect of interest inevitably reflects activity related to the successful formation of associative memories, including activity that is not specific to mnemonic encoding, such as attention or task engagement. Future studies should aim to isolate the neural processes related to successful associative memory formation by controlling for the influence of attention and/or task engagement.Finally, we note that, in contrast to our findings, some previous studies have also described theta subsequent memory effects in medial and lateral temporal lobe regions outside of the hippocampus e.g. . AlthougIn summary, across 2 complementary studies using iEEG and MEG recordings, we have shown that theta activity during encoding in the human hippocampus promotes subsequent associative memory. Importantly, we are able to demonstrate that hippocampal theta effects observed in patients in the iEEG study extrapolate to healthy participants.Joensen-et-al_CerebCortex_SupplMaterial_bhad162Click here for additional data file.


Previous studies have found satisfactory clinical results with the nano‐hydroxyapatite/polyamide 66 (n‐HA/PA66) cage to reconstruct the stability of anterior cervical column. However, studies evaluating the long‐term outcomes of the n‐HA/PA66 cage in multi‐level degenerative cervical myelopathy (MDCM) have not been reported. This study aims to compare the outcomes of corpectomy anterior cervical discectomy and fusion (ACDF) hybrid procedures between the n‐HA/PA66 cage and titanium mesh cage (TMC) to treat MDCM.t test and chi‐square test were applied to compare the differences between groups.After the screening for eligibility, this retrospective study involved 90 patients who underwent corpectomy ACDF hybrid (CACDFH) procedure from June 2013 to June 2018. The CACDFH procedure is the combination of ACDF and anterior cervical corpectomy and fusion (ACCF). According to the cage utilized, we categorized patients into a n‐HA/PA66 cage group and a TMC group. Then, stepwise propensity score matching (PSM) was performed to maintain comparable clinical data between groups. All the patients were followed up ≥4 years and the longest follow‐up time was 65.43 (±11.49) months. Cage subsidence, adjacent segment degeneration (ASD), segmental height (SH), segmental angle (SA), cervical lordosis (CL), and clinical data  was evaluated preoperatively, at 1 week, and at the final surgery follow‐up. The independent student's p = 0.031). Moreover, the cage subsidence rate was significantly higher in the TMC group as compared to the n‐HA/PA 66 group . But there was no significant difference in SH, SA, and CL at any time after surgery as determined through follow‐up. The VAS and JOA scores significantly improved in both groups at 3 months postoperative and at final follow‐up. However, there were no significant differences in the VAS and JOA score at any time between the two groups in preoperative (p > 0.05).Through PSM analysis, 25 patients from the n‐HA/PA66 group were matched to 25 patients in the TMC group. The occurrence of ASD was 16.0% (4/25) in the n‐HA/PA 66 group, which was significantly less than in the TMC group at 44.0% (11/25) (The n‐HA/PA66 cage is associated with lower rate of cage subsidence and ASD than the TMC in the treatment of MDCM. The n‐HA/PA66 cage could be superior to the TMC in corpectomy ACDF hybrid procedures. The findings of this study suggest that the n‐HA/PA66 cage has certain advantages over titanium mesh cage (TMC) in hybrid procedures, including a lower risk of adjacent segment degeneration and cage subsidence. Therefore, the n‐HA/PA66 cage may be more suitable than TMC in multi‐level degenerative cervical myelopathy. The available medical literature has shown that surgical procedures can improve functional status and quality of life by relieving spinal cord compression, especially in multi‐level degenerative cervical myelopathy (MDCM).Autologous bone, allogeneic bone, and titanium mesh with autologous bone are commonly used as struts for cervical stability after CACDFH, but each of these materials has its own advantages and disadvantages.This was a retrospective, single‐center, and consecutive cohort study. We reviewed patients with three‐level MDCM who underwent CACDFH in our hospital from June 2013 to June 2018. This study was approved by the Ethics Committee of the West China hospital (No. 2019–654). Written informed consent was obtained from all participants.Inclusion criteria were as follows: (i) diagnosis of degenerative cervical myelopathy confirmed by imaging and physical examination；(ii) the lesions involved three levels, with ventral spinal cord compression predominating; (iii) patients underwent CACDFH in our hospital from June 2013 to June 2018; and (iv) no previous surgical intervention at cervical. Exclusion criteria were as follows: (i) patients with obvious contraindications to surgery, (ii) patients with tumors, tuberculosis, or infections; (iii) patients with serial ossification of the posterior longitudinal ligament (OPLL) and severe ossification of the ligamentum flavum; (iv) patients lacking complete clinical follow‐up data more than 4 years after surgery; and (v) patients with preoperative adjacent segmental disc degeneration (Kellgren‐Lawrence grade >3 or Pfirrmann grade >3). After exclusion and inclusion criteria were fulfilled, there were 90 patients with complete clinical data enrolled.All the patients underwent routine preoperative examination, including static and lateral flexion/extension X‐ray, computed tomography (CT) scan, and magnetic resonance imaging (MRI). The surgical procedures were performed by one of four senior orthopedic surgeons with more than 20 years of experience. The level of surgery was determined by physical examination and radiological imaging. All preoperative tracheoesophageal push training was performed to prevent postoperative dysphagia. All patients received general anesthesia and a right‐sided anterior cervical approach. ACCF was performed at the severe compression levels, and the TMC or n‐HA/PA66 cage was implanted with autologous bone. Then, a one‐level ACDF was achieved on the adjacent level. Two groups patients were fixed with an Atlantis anterior cervical plate system  to achieve immediate stabilization. All patients were required to wear a cervical collar for 12 weeks after surgery and were followed up regularly.All patient‐related information was obtained from medical records. Clinical information, including age, sex, BMI, concomitant diseases (diabetes and hypertension), surgery level, follow‐up time and other general information was collected preoperatively. Clinical assessments were collected preoperatively, at 3 days postoperatively, and then intermittently until the last follow‐up (at least 4 years of follow‐up). The Japanese Orthopaedic Association (JOA) score was used to assess neurological status, and visual analogue scale (VAS) was used to assess neck pain. All patient follow‐up information was collected during outpatient visits or telephone follow‐up.Static and lateral flexion/extension X‐ray imaging was conducted to assess the cage stability. If necessary, additional CT scan and MRI were performed. Some radiological parameters  were measured preoperatively, 1 week postoperatively, and at the final follow‐up. The following terms are defined, as follows  and p‐values <0.05 were considered statistically significant.PSM analysis was used to select and match patients from n‐HA/PA66 group to patients in the TMC group to eliminate bias in selection. Variables were selected for the PSM multivariable logistic regression model based on their effect on postoperative cage subsidence and ASD. Match tolerance of 0.2 with one‐to‐one nearest neighbor matching technique (greedy matching) without replacement was used using the “MatchIt” package in R , as previously described by Austin.p < 0.001) , including 32 in the n‐HA/PA66 group and 58 in the TMC group. Table ) see Fig and 3.p > 0.05). The detailed demographic data after PSM are displayed in Table After PSM, there were 25 well‐balanced pairs of patients between the n‐HA/PA66 and the TMC group. The mean age for the whole cohort was 57.48 ± 8.87 years old, 32 (64.0%) were female and 18 (36.0%) were male. The average follow‐up time for 50 patients was 68.10 ± 9.11 months. After PSM, all demographic data and clinical characteristics were similar between groups (p = 0.031)  (Table There was a mean SH increase from 7.40 ± 0.72 cm preoperatively to 7.86 ± 0.70 cm postoperatively (1 week) in the n‐HA/PA66 group. There was a mean SH increase from 7.46 ± 0.93 cm preoperatively to 7.76 ± 0.85 cm postoperatively (1 week) in the TMC group. In both groups, the mean SH slightly decreased at the final follow‐up. But, there was no significant difference in SH, SA and CL at any time after surgery. The occurrence of ASD was 16.0% (4/25) in the n‐HA/PA 66 group, which was significantly less than in the TMC group (44.0% (11/25)) (p > 0.05)  Table .Currently, several studies have confirmed that hybrid procedures can reduce surgical complications and improve the bone fusion rate.et al.et al.et al.et al.Preoperative neurological status is an important predictor of functional recovery after anterior cervical spine surgery. Therefore, once spinal cord compression is confirmed by a combination of clinical symptoms and imaging, early decompression should be performed. Wei et al.et al.vs. 40.0%). In addition, a contributing factor to TMC subsidence is that it has a much higher elastic modulus than vertebral trabecular bone. The n‐HA/PA66 cage has a modulus of elasticity similar to that of natural bone, and is close to the PEEK cage in terms of biomechanics, which adequately fits the mechanical strength requirement of intervertebral fusion devices.There are many causes of cage subsidence, including endplate manipulation, cage size, cage position, and the material characteristics of the cage.Continued loss of intervertebral height and cervical curvature may result in secondary compression of the nerve roots and spinal cord, and subsequently increase the risk of reoperation.Adjacent segment degeneration is a common long‐term complication of laminectomy and spinal fusion procedures.Our study evaluated the long‐term outcomes of the two cages used in MDCM, and found that the n‐HA/PA66 cage had a lower risk of adjacent segment degeneration and cage subsidence than the TMC. However, there are several limitations to this study. First, this is a retrospective study that involved a single center that enrolled a relatively small number of patients. Second, selection of the cages for the procedure was not determined by chance or a randomized method, and physician‐related factors could have played a role in the results. This lack of randomization can impact the validity and generalizability of the findings, as it introduces potential biases and confounding factors. Further prospective studies with proper randomization and larger sample sizes are needed to confirm these results and draw more robust conclusions.The findings of this study suggest that the n‐HA/PA66 cage has certain advantages over TMC in hybrid procedures, including a lower risk of adjacent segment degeneration and cage subsidence. Therefore, the n‐HA/PA66 cage may be more suitable than TMC in multi‐level degenerative cervical myelopathy.All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Q.L. and B.H. Acquisition of data: Q.L., B.H., Z.Z., and X.Y. Analysis and interpretation of the data: Q.L. and B.H. Drafting of the manuscript: Q.L., Z.Z., and B.H. Critical revision of the manuscript for important intellectual content: Umar Masood. Statistical analysis: Q.L. and Z.Z. Obtained funding: H.Y., G.F., and Y.S. Study supervision: H.Y. and Y.S.The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of the West China hospital (No. 2019–654). Written informed consent was obtained from the parents.Fig. S1. Characteristic of included patients' baseline before and after propensity score matching.Click here for additional data file.