Increase in presentations of self-harm to primary care, a risk factor of suicide, has led to a growing interest in identifying at-risk populations.To examine whether osteoporosis or fractures are risk factors for self-harm, suicidal ideation, and suicide.This was a systematic review of observational studies in adults (>18 years) that had examined the role of osteoporosis and/or fractures in subsequent self-harm, suicidal ideation, and/or suicide.Six databases were searched from inception to July 2019. Additional citation tracking of eligible studies was undertaken in November 2022. Screening, data extraction, and quality assessment of full-text articles were performed independently by at least two authors. Where possible, meta-analysis was run on comparable risk estimates.n = 3) reported a significant adjusted negative association for self-harm and suicide.Fifteen studies were included: two examined the outcome of self-harm, three suicidal ideation, and 10 suicide. In approximately half of studies on osteoporosis, the risk of suicidal ideation and suicide remained significant. However, pooling of adjusted odds ratios from three studies indicated no association between osteoporosis and suicide . Nine studies examined the risk of a mixture of fracture types across different outcomes, limiting comparisons. However, all studies examining vertebral fracture (Patients with vertebral fractures, a risk potential factor for suicide, may benefit from clinical case finding for mood disorders with personalised primary care management. However, because of the limited number and quality of studies and mixed findings, further examination of these associations is warranted. Although a history of mental ill health, notably depressive disorders, is an understood risk factor for self-harm,4 pain, and more specifically back pain, are also independent risk factors for self-harm5,6 and suicide, specifically in older adults.7Rates of suicide in the UK have risen since 2017, with a 9% increase by 2019.5,8 Although pain is not a symptom of osteoporosis, pain frequently occurs because of subsequent fractures,9,10 and with one in three women and one in five men aged >50 years experiencing an osteoporotic fracture in their lifetime, osteoporosis has been suggested as a risk factor for self-harm and suicide.5,11 Furthermore, common outcomes of fractures, including osteoporotic fractures, such as isolation and depression are risk factors for suicide.12Recent studies have demonstrated increased risk of self-harm in people with physical health conditions such as fibromyalgia and osteoarthritis.5,13–15 possibly because of a lack of consensus on diagnosis definitions, population variations, and study limitations. The aim of this study was to examine whether osteoporosis and/or fractures are risk factors for self-harm, suicidal ideation, or suicide.Previous research examining the risk of self-harm or suicide in older adults with osteoporosis or fractures has yielded conflicting results,16A systematic review and meta-analysis was conducted. The protocol was registered on PROSPERO (CRD42021272606) and adhered to PRISMA guidance.The population of interest were adults with osteoporosis and/or fractures. The outcomes of interest were risk estimates of self-harm, suicidal ideations , and suicide.studies with a population of <18 years old;studies that could not be translated; andsystematic reviews, case reports, or case series studies.Exclusion criteria were:Searches were tailored and conducted in six electronic databases: MEDLINE, PsycArticles, AMED, CINAHL Plus, PsycINFO, and Web of Science from inception to July 2019. Search strategies utilised database subject headings and text word searching in title, abstract, or keywords, combining terms for: osteoporosis and fractures; and self-harm, suicidal ideation , and suicide (Supplementary Information S1).In addition, the reference lists of included studies and relevant systematic reviews were checked and key studies citation tracked. To account for the delay in publication because of COVID-19, citation tracking was conducted on all included papers in September 2021 and November 2022 to ensure relevant new studies were identified.A two-stage screening of articles against predefined eligibility criteria was implemented: first by titles and abstracts; second by full text.At each stage, screening was conducted by two reviewers independently  and articles were excluded by consensus. Arising disagreements were resolved through discussion.17 It examines the risk of bias across six domains: study population, study attrition, prognostic factor measurement, outcome measurement, study confounding, and statistical analysis and reporting. All included articles were assessed for their quality independently by two pairs of assessors (the first and senior author or the second and sixth author). Any disagreement on scoring was initially discussed and arbitrated by a different reviewer if required.The Quality in Prognosis Studies (QUIPS) tool was used to assess included articles.Data were extracted by two authors (the third and fourth authors) on demographic information , and size of the study sample, numbers of patients with the condition of interest, study setting , exposures , exposure definition, outcome , and method of risk estimates regarding the association between exposure and outcome.Findings were tabulated by their exposure category, that is, articles examining either osteoporosis or fracture were compared separately. Within each of these exposure categories, outcomes were subcategorised by either self-harm, suicidal ideation/behaviour, or suicide. Risk estimates, 95% confidence intervals (CIs), and statistical significance were extracted and reported for each study, and where available separately for men and women.Specifically for fractures, risk estimates were tabulated by location of fracture, where reported. Authors were not contacted if it was not possible to identify certain required information in their publication.I2. Analysis was undertaken in Stata (version 14).Where enough studies (≥3) examining comparable factors were identified, a random-effects meta-analysis was used to pool reported risk of any of the examined exposures along with their 95% CI. Heterogeneity was assessed by Keele Research User Group consists of a diverse group of people with lived experience of osteoporosis, or of caring for people with osteoporosis.In a series of meetings to discuss research, public contributors talked about the significant psychological and social sequelae of osteoporotic fractures. However, there was no direct public and patient involvement in the conduct or interpretation of this study.11,13–15,18–28Searches identified 325 unique articles, of which 306 were excluded and 18 underwent full review (one article could not be retrieved) (Supplementary Table S1). Citation tracking identified a further three eligible studies resulting in a total of 15 included studies .11,13–1511,13,14,18–22 10 studies examined fractures as a risk factor,13–15,20,23–28 and three studies examined both.13,14,20 Of the studies reporting osteoporosis, three reported suicidal ideation as the outcome,18–20 and five reported suicide as the outcome.11,13,14,21,22 Of the studies examining fractures, two reported the outcome of self-harm,23,26 two of suicidal ideation,15,20 six of suicide,13,14,24–28 and one included the outcome of self-harm and suicide.26 In the three studies examining both population groups of osteoporosis and fractures, one reported the outcome suicidal ideation20 and two reported on the risk of suicide .The included studies were categorised into two groups by risk factor, osteoporosis, and fracture. Eight studies reported osteoporosis as a risk factor, suicide .13,14 Mo11,13,20 and East Asia.14,18,19 Study settings included primary care,11 primary and secondary/specialist care,14,21 and general populations.13,18,19,20,22 Four studies did not report average age and/or standard deviation (SD),11,13,19,21 with two only reporting the age ranges used to group participants.13,19 Three studies included participants <45 years of age11,19,21 and four included participants >80 years of age.11,13,19,22 Osteoporosis was predominantly identified utilising disease codes in a range of health record databases (Supplementary Table S2). In terms of outcomes, suicidal ideation was recorded via patient self-report14,19,20 and papers reporting numbers of suicide used healthcare records.11,13,14,21,22Studies were conducted in a range of countries, predominantly Europe18,19Two of the three studies reported significant crude estimates (crude odds ratios [ORs]), ranging from 1.90 (95% CI = 1.38 to 2.62) to 2.13 (95% CI = 1.34 to 3.39) .18,19P<0.05).19 The third study did not report a crude ratio; however, they reported a significant adjusted estimate .20Only one of these reported adjusted ORs and they remained significant .The Kim 11,13,14,21,22 with estimates ranging from a crude OR 1.28 (95% CI = 1.23 to 1.33)14 to 3.12 (95% CI = 1.56–6.21).22 Four studies also reported adjusted RRs, with two retaining significance .13Five of the seven studies relating to osteoporosis as a risk factor for suicide found significant crude results,Adjusted OR data from a total of 4123 primary and secondary care patients across three studies was pooled and found no association between osteoporosis and subsequent suicide  .14,15,24,27,28 and Europe13,20,23 examined fracture as a risk factor. Study settings included primary care,23 a retirement community,25 secondary care,24 across primary and secondary care,14 and the general population . Four studies reported mean ages ranging from 59.8 (SD 14.3) to 75.0 (SD 6.8) years.14,20,24,26 Five studies included participants aged >80 years,13,24,25,27,28 and one included participants below <35 years  reported a grouped fracture risk estimate; however, each utilised a different grouping definition: ‘all fractures’, ‘pathological fractures’, ‘fractures >50 years’, ‘other fractures’, and ‘any fracture in last 5 years’ . Hip and vertebral fractures were the most consistently reported fracture sites, reported in five and four studies, respectively.13,14,20,23,25,28 However, these studies utilised a range of risk estimate types 26 investigated ‘any fractures in the prior 5 years’ as a risk factor for self-harm, whereas Prior et al examined the relationship between vertebral fractures and self-harm categorised by sex.23 Crude and adjusted hazard ratios (HRs) were significant in both men and women. Erlangsen et al (2021) found a significant adjusted incidence ratio (IR) of 1.38 (95% CI = 1.12 to 1.71).26 Prior et al found that men and women with vertebral fractures were almost four and two times more at risk of self-harm than those without such fractures  (Supplementary Table S3).23Erlangsen et al reported significant crude  and adjusted HR  estimates for fractures as a risk factor for suicidal ideation , no adjusted associations were retained. Lutz et al reported a significant adjusted association between experiencing any fractures  and suicidal ideation  (20Tsai ideation .15 Howevto 1.54) .20et al and Turvey et al reported significant crude associations ,14 ‘fractures in those over 50 years of age’ ,25 but only Chang adjusted for confounders, finding significance to be retained  Erlangsen et al (2021)26 examined the adjusted association between self-reported ‘fractures in last 5 years’ and suicide but found no significant association  by Erlangsen et al (in women).14,26,28Three studies examining the role of vertebral fractures on suicide found significant associations in all, with risk estimates ranging from OR 1.40 (95% CI = 1.07 to 1.82) by Jang 14,24–26,28 three of these found a significant crude association. Significant associations were retained in two of these after adjustment.13,14 Although several other fracture sites were examined, conflicting or infrequent examination of these means that no consensus on an association with suicide can be drawn . This was also supported by the one study to have examined the role of vertebral fractures on self-harm.Pooled analysis of three studies found that people with osteoporosis were no more likely to die from suicide than patients without osteoporosis.30 the authors undertook quality assurance to assess such variations . A further limitation of this study is that, because of the variety of definitions, populations, and risk estimates used, it was only possible to pool three studies, which varied widely in quality. However, the fact that the meta-analysis included the study by Chang et al14 that was a very large study, and, that all estimates were adjusted, adds some credibility to these findings and an opportunity for others to build on.This is the first paper, to the authors’ knowledge, to assemble literature on osteoporosis and fractures as risk factors for self-harm, suicidal ideation, and suicide. Despite only 15 studies being included it was possible to conduct a meta-analysis that provides greater reliability of risk estimates. Another strength is the analysis of fracture as a risk factor by bodily location, which highlighted vertebral fractures as a potentially important risk factor. There are, however, several limitations that need to be recognised. Papers were primarily characterised by population group of either osteoporosis or fractures and, although the poor coding of osteoporosis and fractures in primary care data has previously been noted,31,32 The presence of fractures has social implications because of the fear of falling or of recurrent fracture,33,34 with osteoporotic fracture increasing the odds of functional decline by 48%.35 Further poor osteoporotic fracture outcomes include increased social support requirements and diminished quality of life, including depression and deterioration in perceived health.36,37Vertebral fractures have significant long-term physical, psychological, and social sequelae. Pain is the likely mechanism driving the association between the presence of a fracture and subsequent self-harm, suicidal ideation, and suicide; especially as osteoporotic fractures have been reported to induce both acute and chronic nociceptive and neuropathic pain.38 Furthermore, the effects of fractures last long beyond the time the injury was sustained. Hallberg et al reported that, 2 years post-fracture, the social function and mental health components of health-related quality of life were still significantly lower than controls in those with vertebral and hip fractures.39 Although this aligns with spinal, and to a lesser extent hip, fractures being significantly associated with suicide in this review, it does not align with the temporal nature of suicide risk post-fracture as reported by Jang et al.27 They found that risk of suicide was highest in the first 180 days after hip fracture , and then reduced,24 with similar patterns in pelvic and spinal fractures.27,28Suicide risk is multifactorial, with depression, anxiety, and other mood disorders as major risk factors. As such, a fracture’s impact on patient mental health may in part explain the risk identified related to vertebral fractures.9 Despite this common label, a diagnosis of osteoporosis can cause changes in the perception of one’s self that may result in social isolation, low mood, and anxiety.34This lack of association with osteoporosis and suicide may relate to the fact that osteoporosis without fracture is primarily asymptomatic and, although low trauma fracture is an indicator of osteoporosis, not all those who have low bone density have broken a bone. As such, osteoporosis is often referred to as ‘silent’.40 Osteoporosis is commonly diagnosed and patients seek help for fracture care in primary care, which may therefore be an important setting to identify patients at risk of self-harm and suicide and intervene early. New research would enable the assessment of the feasibility, acceptability, and effectiveness of such approaches. Early intervention studies have shown that physical exercise programmes that focus on physical anxiety can decrease fear of falls in those with osteoporosis, and potentially lead to lower rates of isolation,41,42 which might be a causative factor in self-harm and suicide. Such programmes have been shown to be effectively implemented through primary care.43This study indicates that patients diagnosed with vertebral fractures might benefit from case finding for mood disorders, such as depression, a risk factor for suicide, and enquiring about suicide and self-harm.In conclusion, although studies were typically too diverse for pooling of data, one meta-analysis of three studies showed no association between osteoporosis and suicide. Across several individual studies, vertebral fractures were shown to be potential risk factors for suicide. This review demonstrates the potential importance of teasing apart the role of osteoporosis and fracture in research, and provides strong justification for further research around vertebral fracture in this area. Primary care clinicians could implement case finding for mood disorders and suicide risk assessment in patients with vertebral fractures and undertake subsequent management.


Recently, the cases of colorectal cancers has been rising in younger age (<50) individuals. Although current guidelines recommend colorectal cancer screening should be initiated at age 45 instead of 50, the optimal approach of colorectal cancer screening is not clear. This article investigates the efficacy of fecal immunochemical test (FIT), which detects occult blood in stool, in predicting advanced colorectal polyps and tumors among people aged 40–49. The findings suggest FIT is useful to identify such people with high risk to have advanced colorectal lesions. Hence, FIT may be considered as the first-line screening tool for these people, and further comparative study between FIT and colonoscopy will be of great value.Background: The incidence of early-onset colorectal cancer (CRC) is increasing. Many guidelines recommend initiating screening at 45 years. This study investigated the detection rate of advanced colorectal neoplasm (ACRN) by using fecal immunochemical tests (FITs) in individuals aged 40–49 years. Methods: PubMed, Embase, and Cochrane Library databases were searched from inception to May 2022. The primary outcomes were the detection rates and positive predictive values of FITs for ACRN and CRC in people aged 40–49 (younger age group) and ≥50 years (average risk group). Results: Ten studies with 664,159 FITs were included. The FIT positivity rate was 4.9% and 7.3% for the younger age and average risk groups, respectively. Younger individuals with positive FIT results had significantly higher risks of ACRN  or CRC  than did individuals in the average-risk group, regardless of FIT results. Individuals aged 45–49 years with positive FIT results had a similar risk of ACRN  to that of people aged 50–59 years with positive FIT results, although significant heterogeneity was observed. The positive predictive values of the FIT were 10–28.1% for ACRN and 2.7–6.8% for CRC in the younger age group. Conclusion: The detection rate of ACRN and CRC based on FITs in individuals aged 40–49 years is acceptable, and the yield of ACRN might be similar between individuals aged 45–49 and 50–59 years. Further prospective cohort and cost-effective analysis are warranted. Colorectal cancer (CRC) is a leading cause of mortality worldwide ,2,3. BecIn addition to this rapidly rising incidence, early-onset colorectal cancer (EOCRC), CRC occurring before 50 years of age, has become a particular public health problem with several characteristics. Compared with normal CRC, EOCRC tends to be located on the left side of the colon and the rectum , be presSeveral society guidelines recommend an earlier screening age of 45 years in response to increasing EOCRC ,16,17,18The literature searches were conducted on PubMed, Embase, and Cochrane Library from inception to May 2022. The keywords and search strategy are described in Studies were eligible if they investigated colorectal polyps or cancers in individuals aged 40–49 years who received FITs. Studies were excluded if (1) they lacked colonoscopy outcome data, (2) were simulation model studies, (3) had overlapping cohorts with other studies, or (4) were non-English language studies. The following data were then extracted from eligible studies: the name of the first author, year of publication, size, and characteristics of the studies population, FIT brand name, and colonoscopy results. All data were extracted as originally stated or after appropriate calculations. If the necessary data were unavailable, we contacted the corresponding author for additional information.The primary outcomes were the detection rate and positive predictive value (PPV) of FITs for ACRN and CRC. ACRN consisted of CRC with adenomatous polyps with high-risk features such as a size of ≥1 cm, high-grade dysplasia, or a villous component. The secondary outcomes were the detection rate and PPV for overall colorectal neoplasm (CRN), which included low-risk adenomas and ACRN. Comparisons were made between the younger age (40–49 years) and average-risk groups (≥50 years).2 statistic was used to evaluate statistical heterogeneity, which was considered significant if I2 > 50% or if a chi-square test yielded a p-value of <0.1.Comprehensive Meta-Analysis version 3.3.070  with random-effects models were used for all meta-analyses. Odds ratios (ORs) were used for the analysis of the categorical outcome variables. Corresponding 95% confidence intervals (CIs) were used to compare the outcomes of the younger age and average-risk groups. The pooled effect size was significant if the ranges of the 95% CIs of the ORs excluded 1. The IFor all meta-analyses, we evaluated the robustness of the pooled effect estimates by excluding one study at a time. The risk of bias was assessed using the Newcastle–Ottawa scale for retrospective studies. We did not calculate publication bias in this study because the analysis was likely to be underpowered due to relatively few studies in the meta-analyses. The review adhered to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines  Supplem.The search strategy generated 4085 records and 186 potentially eligible studies. After the manual review process, 10 studies were included in the final analysis . The risIn total, 664,159 FITs (35.6% were younger-age group) were included in the studies, and the pooled FIT positivity rate was 4.9% for the younger age group and 7.3% for the average-risk group. Most of the studies used FITs from the OC-SENSOR series ,29,30,31The characteristics of colorectal neoplasms by FIT result are presented in Likewise, CRC was significantly more common among individuals with positive FIT results than among those with negative FIT results in both the younger age  and average-risk groups . Younger individuals with positive FIT results had a higher risk of CRC than did all average-risk individuals  and varied by age and cutoff value . For ACRIn the three studies that reported CRC cases  22,27,3,327,31, CRC screening programs using either colonoscopy or fecal occult blood tests are useful in lowering incidence and mortality ,5,34,35.In this study, positive FIT results indicated a significantly higher risk of ACRN and CRC for people aged 40–49 years. Although the PPV of FITs in this population was lower than that of older individuals, the negative predictive value for CRC was sufficiently high in both age groups to be considered a first-line screening tool. In most studies with healthy participants, sensitivity to ACRN is less than 30% and is even lower for smaller colorectal adenomas. However, this lower sensitivity can be overcome with periodic checkups, similar to screening for average-risk individuals. FITs have lower financial and staff requirements than colonoscopies. Because of the lower absolute incidence of CRC in younger individuals, FITs may be a reasonable modality, with a balance between efficacy and cost.Considering the appropriate timing and possible high-risk candidates for FIT screening for EOCRC is essential. Although guidelines recommend a screening age of 45 years, the subgroup analysis in our study revealed that individuals aged 45–49 years with positive FIT results have a lower risk of CRC than individuals aged 50–59 years with positive FIT results. However, the pooled analysis demonstrated that ACRN incidence might be similar in both age groups with positive FIT results, despite significant statistical heterogeneity. This result might be explained by differences in ethnicity in the younger age group. In this analysis, the outlier study   only incAdditional cost-effectiveness and risk stratification studies are warranted. Our study suggests that a universal FIT-based screening strategy for individuals aged 40–49 years may have a lower yield than the screening of average-risk individuals. To improve diagnostic yielding, the incorporation of other risk factors before enrollment in screening may be helpful. Multiple EOCRC risk factors such as Westernized diets, stress, antibiotic use, a sedentary lifestyle, and first-degree family history  have beeThis study has several strengths. First, it is a systematic review with integrated analysis from up-to-date cohort studies of FIT screening for younger individuals. Moreover, the meta-analyses provides an accurate estimation of the diagnostic efficacy of FIT in these population. Second, the differences related to the age and characteristics of the participants were analyzed. However, this review has several limitations. First, the earliest age for FIT screening in most studies was 40 years; thus, the results cannot be extrapolated to individuals aged younger than 40 years. Among three studies included individuals aged younger than 40 years, the median age of the younger group was 44 years in D’Souza et al. (NICE FIT study) , whereasThis systematic review and meta-analysis indicated that FITs may be a useful tool for the detection of ACRN in individuals aged 40–49 years and should be considered for use as a first-line screening tool from 45 years. Studies addressing cost-effectiveness and special risk factors are warranted to determine the optimal EOCRC screening tool choice.The detection rate of ACRN and CRC based on FITs in individuals aged 40–49 years is acceptable, and the yield of ACRN might be similar between individuals aged 45–49 and 50–59 years. Further comparative and cost-effective analysis of colonoscopies is mandatory to guide its use in EOCRC screening.


P<0.001), CHD , MI , stroke , cardiac death , and all-cause mortality . Sensitivity analyses indicated the pooled conclusions for cardiovascular outcomes and all-cause mortality are robustness. The associations of PD with the risk of ardiovascular outcomes and all-cause mortality could affected by region, study design, PD definition, follow-up duration, and study quality. This study found the risk of cardiovascular outcomes and all-cause mortality were elevated in PD patients, and the intervention for PD should be applied to prevent the risk of cardiovascular outcomes.Studies reported periodontal disease (PD) periodontal disease is associated with many systemic diseases, including cardiovascular outcomes and all-cause mortality. However, the precise mechanistic link for these relationship remained unclear. We therefore performed a meta-analysis of cohort studies to investigate the association of PD with the risk of cardiovascular outcomes and all-cause mortality. We systematically searched the databases of PubMed, EmBase, and the Cochrane library to identify eligible studies until April 2023. The investigated outcomes included major adverse cardiovascular events (MACEs), coronary heart disease (CHD), myocardial infarction (MI), stroke, cardiac death, and all-cause mortality. The summary relative risk (RR) with 95% confidence interval (CI) were calculated using the random-effects model. Thirty-nine cohort studies with 4,389,263 individuals were selected for final meta-analysis. We noted PD were associated with elevated risk of MACEs (RR: 1.24; 95%CI: 1.15–1.34;  Cardiovascular disease (CVD) accounted for 17.8 million deaths worldwide in 2017, and these CVD deaths were caused by ischemic heart disease and stroke were 50% and 35%, respectively [Periodontal disease (PD) is a chronic inflammatory disease, which caused by dental plaque through attacks the immune defense line of periodontal tissue. The clinical manifestations of PD included gingiva bleeding, periodontal pocket, alveolar bone resorption, and tooth loss. PD is highly prevalent accounting for 20–50% of the global population, which could cause a loss of clinical attachment, and the progressive of PD could induce the loosening of teeth or even to the loss of teeth . Study hThe Meta-analysis Of Observational Studies in Epidemiology protocol was applied to perform and report this systematic review and meta-analysis . Cohort The literature search and study selection were independently performed by 2 reviewers following the standardized flow, and conflicts between reviewers was resolved by discussed with a third reviewer until a consensus was reached. The details of inclusion criteria included: (1) Participants: adult population free of CVD at initially; (2) exposure: moderate or severe PD; (3) Control: individuals without PD or mild PD; (4) Outcomes: the study should reported at least 1 of major adverse cardiovascular events (MACE), coronary heart disease (CHD), myocardial infarction (MI), stroke, cardiac death, and all-cause mortality; and (5) Study design: cohort study, irrespective prospective or retrospective cohort design. There was no restriction was placed on the minimum number of participants. The study was excluded if they met: (1) cross-sectional or case-control design; (2) the effect estimate was presented without 95% confidence intervals (CIs); or (3) study did not reported cardiovascular outcome or all-cause mortality.Two reviewers independently performed data collection, and inconsistent results between reviewers was settled by a third reviewer referring to full-text of article. The abstracted information in each included study included first authors’ surname, publication year, region, study design, sample size, range of individuals’ age, male proportion, the definition of PD, reported outcomes, follow-up duration, and adjusted factors. The Newcastle-Ottawa Scale (NOS) was used to assess the methodological quality of observational studies in meta-analysis, which including selection (4 items), comparability (1 item), and outcome (3 items) domains, and the “star system” in individual study ranged from 0–9. Study with 7–9 stars was regarded as high quality, 4–6 stars was defined as moderate quality, and with 0–3 stars was considered as low quality . The quaI2 and Cochren Q statistic was applied to assess the heterogeneity across included studies, and I2 ≥ 50.0% or P < 0.10 was defined as significant heterogeneity [P test, which assuming the data met the normal distribution [P value for pooled conclusions are 2-sided, and P < 0.05 was considered as statistically significant. Statistical analysis in our study was performed by using STATA software .The association of PD with the risk of cardiovascular outcomes and all-cause mortality based on the effect estimate with 95%CI in individual study. The summary relative risk (RR) and 95%CI were calculated using the random-effects model with Mantel-Haenszel Statistics, which considering the underlying varies across included studies ,19. The ogeneity ,21. Sensogeneity . Subgrouribution . Publicaribution ,25. The An initial electronic searches yields 4,532 records, and 3,246 articles were retained after duplicate records were excluded. Then a total of 3,105 studies were removed owing to these studies reported irrelevant topics. Reviewing the reference lists yields 7 potential included studies, and a total of 148 studies were retrieved for full-text evaluations. After this, 106 studies were excluded because of: other design (n = 54), reported other outcomes (n = 37), no sufficient data (n = 15). Finally, 39 cohorts reported in 42 studies were selected for meta-analysis –67. Fig The baseline characteristics of included studies and involved individuals are shown in P<0.001; I2 = 88.9%; P<0.001). Sensitivity analysis was performed to assess the role of single study from the overall analysis, and the results found the pooled conclusion was stability and the heterogeneity was not significantly reduced . The pooled conclusion was robustness and not altered by excluding any particular study. Moreover, the heterogeneity was not significantly changed by removing any individual study (P<0.001), and retrospective cohort studies (P = 0.026) were higher than corresponding subgroups. Although the Begg test indicated no significant publication bias (P = 0.415), while the Egger test suggested potential significant publication bias for CHD (P = 0.029)  . The conP = 0.001; I2 = 80.1%; P<0.001), and this significant heterogeneity was not significantly altered by removing any particular study. Moreover, the pooled conclusion was stability and not changed by removing any single study . Sensitivity analysis indicated the pooled conclusion was stability and the heterogeneity was not significantly changed by sequential removing individual study (P = 0.025), PD definition (P = 0.012), and study quality (P = 0.010) could affect the association between PD and stroke risk. There was no significant publication bias for the association of PD with the risk of stroke . The pooled conclusion and heterogeneity among included studies were not significantly altered by sequential removing individual study (P = 0.012), PD definition (P = 0.014), and follow-up duration (P = 0.010). No significant publication bias for cardiac death was observed . Sensitivity analysis indicated the pooled conclusion was not altered and the heterogeneity was not fully explained by excluding any specific study  The PD pathogens entering the blood can directly invade the cardiovascular endothelial cells and smooth muscle cells, which significantly associated with the risk of vascular diseases. Moreover, the pathogens could colonize at atherosclerotic plaque, pericarditis or myocardial tissue after enter blood, which could affect the occurrence and development of coronary heart disease, occurrence and development of coronary heart disease –71; (2) Subgroup analyses found the associations of PD with the risk of CHD, stroke, and cardiac death could affected by region, study design, PD definition, follow-up duration, and study quality. Several reasons could explained these results: (1) the excess risk of CHD, stroke, and cardiac death related to PD in Western countries might caused by older age and severity of PD. Moreover, large number of included studies were performed in Western countries, and the conclusions was robust with narrow 95%CI; (2) although effect estimate for the relation between PD and CHD in retrospective cohort studies was large than prospective cohort studies, while these results was variable owing to only 3 included studies designed as retrospective cohort. Moreover, prospective cohort studies could eliminate selection and recall bias that might be concerned of retrospective cohort studies; (3) the definition of PD applied clinical diagnosis was more accuracy than self-report, and the severity of PD could affect the effect estimates of cardiovascular outcomes ; (4) conSeveral strengths of this study should be mentioned. First, the analysis on the basis of cohort studies, and the conclusion could proved causality relationship. Second, the analysis based on large sample size, and the conclusions of this study was robust than any single study. Third, the exploratory analyses were performed according to region, study design, gender, PD definition, follow-up duration, adjusted level, and study quality, and the differences between subgroups were compared. Finally, the pooled conclusions in our study was robust and not changed by removing any particular study.The limitations of this study included: (1) the analysis contained both prospective and retrospective cohort studies, and the selection and recall biases were inevitable; (2) the severity of PD were not addressed, which could affect the strength for the associations of PD with the risk of cardiovascular outcomes and all-cause mortality; (3) there were significant heterogeneity across included studies for cardiovascular outcomes and all-cause mortality, which not fully explained by sensitivity and subgroup analyses; (4) the adjusted factors across included studies are differing, which play an important role on the risk of cardiovascular outcomes and all-cause mortality; and (5) inherent limitations for meta-analysis of published data, including inevitable publication bias and restricted detailed analyses.This study found PD were associated with an increased risk of MACEs, CHD, MI, stroke, cardiac death, and all-cause mortality. Moreover, region, study design, PD definition, follow-up duration, and study quality might affect the strengths for the associations of PD with the risk of CHD, stroke, and cardiac death. Thus, individuals with PD should be early identified to prevent the progression of cardiovascular outcomes and all-cause mortality. Further large-scale prospective studies should be performed to assess whether the treatments for PD could reduce the risk of cardiovascular outcomes and all-cause mortality.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


This updated meta-analysis showed that PPI was significantly associated with an increased risk of CKD. Association was observed in the same among moderate-quality studies. Until further randomized control trials (RCTs) and biological studies confirm these results, PPI therapy should not stop patients with gastroesophageal reflux disease (GERD). However, caution should be used when prescribing to patients with high-risk kidney disease.Previous epidemiological studies have raised the concern that the use of proton pump inhibitors (PPIs) is associated with an increased risk of kidney diseases. To date, no comprehensive meta-analysis has been conducted to assess the association between PPIs and the risk of chronic kidney disease (CKD). Therefore, we conducted a systematic review and meta-analysis to address the association between PPIs and CKD. The primary search was conducted in the most popular databases, such as PubMed, Scopus, and Web of Science. All observational studies evaluated the risk of CKD among PPI users, and non-users were considered for inclusion. Two reviewers conducted data extraction and assessed the risk of bias. Random-effect models were used to calculate pooled effect sizes. A total of 6,829,905 participants from 10 observational studies were included. Compared with non-PPI use, PPI use was significantly associated with an increased risk of CKD (RR 1.72, 95% CI: 1.02–2.87,  The global incidence and prevalence of kidney disease are increasing steadily, imposing a significant burden and becoming the eighth-leading cause of morbidity and mortality. Kidney disease is a global public health concern; it is projected to become the 5th most common cause of mortality globally by 2040 ,2. AcuteProton pump inhibitors (PPIs) are one of the most prescribed medications for treating acid-related gastrointestinal disorders ,6. It isThis current study aimed to provide a comprehensive and updated systematic review and meta-analysis to examine the association between PPI and CKD. Moreover, we also aimed to assess whether there is any difference in the association by region, study design, methodological quality, gender, and types of PPI.Study Protocol: Our study was conducted and reported according to the Meta-analyses of Observational Studies in Epidemiology (MOOSE) checklist [hecklist .Search Strategy: We conducted a systematic search for observational studies in PubMed, Scopus, and Web of Science, up to 25 November 2022. The following combination keywords were used: Proton pump inhibitor/s, and chronic kidney disease. We did not restrict language in the initial search. The search strategy was developed with a discussion with experts who have 5 years of experience in conducting systematic reviews and meta-analyses. In addition, a manual search was conducted through the reference lists of previously published reviews and meta-analyses to identify missing studies.Study Eligibility: We considered all types of observational studies that evaluated the association between PPI use and the risk of CKD. Studies were included if they were (i) published in English, (ii) provided clear information about PPI users and inclusion criteria for CKD, and (iii) provided sufficient information to calculate a pooled effect size.Studies were excluded if they were review articles, reports, animal research, conference abstracts, editorials, case reports, or studies without a comparator group. Two authors (CCW and MHL) independently screened all titles, abstracts, and full texts of all included studies. Any discrepancy during the study screening process was resolved through discussion with a third author.Data Extraction: The same two authors developed the data extraction form to collect relevant information from selected full-text articles. The following information was extracted from selected studies: (i) basic information: author name, publication year, and origin; (ii) population: sample size, data source, age, and gender; (iii) methods: study design, inclusion and exclusion criteria, study duration, follow-up time, and adjustments for confounding factors; (iv) outcome: effect sizes with 95% confidence intervals (CIs).Assessment of Risk Bias: We assessed the quality of included studies using the Newcastle–Ottawa Scale recommended by the Cochrane library [ library . It eval library ,12,23.Statistical Analysis: The statistical analysis was performed using Comprehensive Meta-analysis (CMA) software. The pooled risk ratios (RR) with 95% confidence intervals were estimated using a random effects model based on the DerSimonian–Laird method. We drew forest plots to depict the visual interpretation of pooled estimates with 95% CIs. The Cochran Q test and I2 statistic were calculated to assess the degree of heterogeneity among studies. The significance level for the effect size was considered at p < 0.05.Study Identification: Study Characteristics and Quality Assessment: Proton Pump Inhibitor and Chronic Kidney Disease: Ten studies examined the risk of CKD among PPI users. PPI use was significantly associated with an increased risk of CKD compared to non-PPI users. The pooled RR was 1.72 , with a significant heterogeneity among studies Subgroup Analysis: We also conducted comprehensive subgroup analyses of the included 10 studies based on study design, region, methodological quality, gender, comorbidities, comedication, and types of PPI use ( PPI use .p = 0.13). The pooled RR of CKD among PPI users for case-control studies was 1.57 . The heterogeneity among the studies were Q = 7784.31, p < 0.001, and I2 = 99.91% and Q = 83.62, p < 0.001, and I2 = 97.60, respectively.Seven cohort and three case-control studies evaluated the risk of CKD among PPI users. The adjusted pooled analysis of the seven cohort studies showed an increased risk of CKD among PPI users compared to non-PPI users , with significant heterogeneity among the studies . Moreover, the pooled RR for studies from Asia was 2.25 , with significant heterogeneity among studies .Six studies from Western countries examined the impact of PPI therapy on the risk of CKD. The overall pooled RR was 1.28  and 1.97 , respectively. Three studies evaluated the risk of CKD among male PPI users, and the adjusted pooled RR was 1.14 . Moreover, four studies assessed the risk of CKD among female PPI users, and the adjusted pooled RR was 0.95   .p < 0.001), with non-significant heterogeneity . The pooled RR for studies using rabeprazole and esomeprazole were 1.50 , 1.53 .The studies assessed the risk of CKD with esomeprazole; the pooled RR was 1.32 ; therefore, we conducted a sensitivity analysis to observe any change in the findings. In order to evaluate the magnitude of the overall impact of each included study on CKD risk, a sensitivity analysis was performed by excluding studies one by one. However, this study did not observe any difference in overall effect size and heterogeneity among studies (The findings of this study had high heterogeneity (I studies .Publication Bias: p = 0.53).This systematic review with meta-analysis provides a comprehensive estimation of the association between PPI use and the risk of CKD. The findings of this study are based on previously published observational studies. The present study calculated adjusted pooled effect sizes using a random effects model from 10 studies, which included a total of 6,829,905 participants. The results showed that PPI use was significantly associated with a high CKD risk of 72%. These findings are supported by previously published meta-analyses ,34,35,36The biological mechanism underlying the positive association between PPI use and CKD risk is unclear. A previous study suggested that infection and inflammations could partially contribute to the development of CKD among PPI users . PreviouOur subgroup analyses showed that the risk of CKD among PPI users was higher in Asian people than in Western people. Although an insignificant association was observed among Asians, it may be due to a small number of studies. Previous epidemiological studies reported that clinical, metabolic, socioeconomic, and behavioral factors could contribute to a higher risk of CKD in Asian people compared to others ,49,50. HTo our knowledge, this is the largest collection of observational studies on the assessment of PPI use for the risk of CKD, representing more than 10 studies compared with a comprehensive meta-analysis conducted previously. Unlike most recent meta-analyses, which do not have broad subgroup and sensitivity analysis, our study has more comprehensive search methods and analyses. This study also has some limitations. First, this meta-analysis was limited by the quality of the included studies. The pooled evidence from observational studies (cohort and case-control studies) cannot provide an interpretation regarding causation. Second, the findings of this study are prone to selection bias, confounding bias, and exaggeration of associations. However, we considered only the adjusted effect size to calculate a pooled effect size. Third, the study designs, data materials, statistical approaches, duration, and quality of included studies varied. Fourth, no information on the dose and duration of PPI use was available in included studies. Therefore, we were unable to provide the dose and duration effect on CKD risk. Finally, we are unable to classify the risk of CKD based on various stages due to a lack of data.This systematic review and meta-analysis showed that PPI use was associated with an increased risk of CKD. The findings of our study could contribute to a more comprehensive understanding of CKD risk among PPI users. Our study findings highlight the need for early intervention among patients at high risk of CKD and for continuous monitoring of patients with PPI.


CYP2R1, CYP27B1), transport (GC), and metabolism (CYP24A1), as well as in the vitamin D receptor (VDR), are associated with OS and/or PFS in patients with NSCLC. The SNPs in VDR have been the most extensively analyzed. This systematic review summed up the available evidence concerning the association between 13 SNPs in the main genes involved in the vitamin D metabolic pathway and prognosis in NSCLC. It revealed that SNPs in the VDR, CYP27B1, CYP24A1, GC, and CYP2R1 genes could have an impact on survival in this disease. These findings suggest the identification of prognostic biomarkers in NSCLC patients. However, evidence remains sparse for each of the polymorphisms examined, so these findings should be treated with caution.The objective of this systematic review was to provide a compilation of all the literature available on the association between single-nucleotide polymorphisms (SNPs) in the genes involved in the metabolic pathway of vitamin D and overall survival (OS) and progression-free survival (PFS) in patients with non-small cell lung cancer (NSCLC). This systematic review was conducted in accordance with the PRISMA guidelines. It included all the literature published up to 1 November 2022 and was carried out in four databases , using the PICO strategy, with relevant keywords related to the objective. The quality of the studies included was evaluated with an assessment tool derived from the Strengthening the Reporting of Genetic Association Studies (STREGA) statement. Six studies were included in this systematic review. Our findings showed that the BsmI (rs1544410), Cdx-2 (rs11568820), FokI (rs2228570), ApaI (rs7975232), TaqI (rs731236), rs4646536, rs6068816, rs7041, and rs10741657 SNPs in the genes that play a part in vitamin D synthesis ( According to the latest data published by the World Health Organization (WHO), cancer is a leading cause of death worldwide, accounting for approximately six out of every ten deaths. The most common cancers are breast, lung, colon and rectum, and prostate . In the Carcinogenesis can be triggered by a wide range of exogenous and endogenous factors, such as chemical exposure and genetic mutations. Molecular and cellular signaling in carcinogenesis begins with mutations in critical genes , which drives aberrant cell growth, development, proliferation, apoptosis, differentiation, and migration . The lunThe term vitamin D encompasses several liposoluble molecules: ergocalciferol, cholecalciferol, calcidiol, and calcitriol. Cholecalciferol is synthesized in the dermis by the action of UVB radiation 290–320 nm) on 7-dehydrocholesterol, whereas ergocalciferol is supplied to the body through the diet ,14,15. B nm on 7-GC or VDBP, VDR, CYP2R1, CYP27B1, and CYP24A1) [These VDREs enable the expression of a host of genes, which are related to key processes in the inception and development of cancer. This evidence suggests that low serum calcidiol levels or dysfunctionality of the vitamin may favor carcinogenesis, due to the presence of single-nucleotide polymorphisms (SNPs) in the genes that participate in vitamin D metabolism CYP24A1) ,23,24. TA1 ,23,24CYP24A1) ,31,32,33GC (rs7041), CYP2R1 (rs10741657), CYP27B1 , VDR , and CYP24A1 (rs6068816 and rs4809957) genes, according to the information in the dbSNP database [Based on the above, there is a need for continued research toward discovering new biomarkers in NSCLC that facilitate screening and early diagnosis of the disease and make it possible to achieve higher survival rates. This study, therefore, aims to provide a compilation of the existing evidence on the role played in NSCLC survival by SNPs with a minor allele frequency greater than 1% in the database ,35,36.This systematic review was conducted in accordance with the PRISMA guidelines  the PRI. It inclNo language or publication date restrictions were applied. The references in the articles selected were scanned to identify additional relevant articles.VDR, CYP27B1, CYP2R1, GC, and CYP24A1 genes, intermediaries in the vitamin D metabolic process. (III) Studies that reported quantitative results between the SNPs analyzed and the disease outcome. (IV) Studies that assessed general mortality and/or disease progression as outcomes . (V) Cohort-type studies (retrospective and prospective), clinical trials, and randomized clinical trials. (VI) Studies published in any year, with full text availability. (VII) In the case of studies containing a cohort or part of a cohort that had been described previously, the most recent or extensive study was selected. Conversely, studies that presented the same cohort or part of a cohort but evaluated different genes of interest or different outcome measurements were included.The inclusion criteria were as follows: (I) Studies conducted on humans, of any age and any population, who had been diagnosed with NSCLC. (II) Studies that evaluated SNPs in the The exclusion criteria were as follows: (I) Studies based solely on haplotypes. (II) Gene expression studies*. (III) Studies assessing vitamin D levels*. (IV) Studies assessing protein levels*. (V) Vitamin D supplementation studies*. (VI) Mixed cancer cohort studies that do not specifically report the site. (VII) Case report/case series-type articles, conference proceedings, editorials, letters to the editor, clinical guidelines, book chapters, and reviews.* The studies that have been excluded did not analyze single nucleotide polymorphisms.After deleting the duplicates, two researchers (L.E.P.L. and M.I.V.M.) independently screened the titles and abstracts. Disagreements were resolved through discussion with a third researcher (C.P.R). The full texts were retrieved and examined for eligible studies.p value, hazard ratio, and confidence interval (if available). Any discrepancy was resolved through discussion or by consulting a third researcher (A.S.M.). The terminology employed by each author was used to record the outcome measures.Data extraction from the articles included was performed by 2 authors independently (L.E.P.L. and S.R.T.). We developed a data extraction table that included the following information: basic characteristics of the study , description of the sample , and association of gene variants involved in vitamin D metabolism. In addition, the two reviewers extracted the data on measures of association in another table, which included associated SNPs  ; associaTo assess the quality of the studies included, a descriptive analysis was performed by means of a review by two researchers (L.E.P.L. and J.M.G.N.) of 9 items obtained from the Strengthening the Reporting of Genetic Association Studies (STREGA) statement . In the The initial search in the databases consulted produced a total of 396 articles . After tVDR gene [CYP2R1, CYP27B1, GC, and CYP24A1 genes [CYP2R1, CYP27B1, GC, CYP24A1, and VDR, in its analysis [The studies included were published between 2005 and 2021 ,27,28,29VDR gene ,27,28,29A1 genes ,26. Onlyanalysis . All theanalysis ,28,29. TThe polymorphisms associated with OS and PFS in patients diagnosed with NSCLC are summarized in VDR gene: rs1544410 (BsmI), rs11568820 (Cdx-2), rs2228570 (FokI), rs7975232 (ApaI), and rs731236 (TaqI). Five studies included at least three of these in their analyses [p = 0.008; HR = 1.55; 95% CI = 1.09–2.2; CT vs. CC, and p = 0.008; HR = 4.33; 95% CI = 1.34–14.0; TT vs. CC). Similarly, carriers of the rs1544410-T allele showed a higher risk of death compared to the CC genotype  [p = 0.0073; HR = 2.08; 95% CI = 1.22–3.56; TT vs. C) [In this review, information was collected on five SNPs located in the analyses ,27,28,29analyses ,27,28,29p = 0.05; HR = 0.55; 95% CI = 0.32–0.95; AG vs. GG, and p = 0.05; HR = 0.69; 95% CI = 0.16–2.96; AA vs. GG). Likewise, carriers of the A allele had a lower risk of death compared to the GG genotype . Similar results were found when evaluating PFS, where carriers of the A allele showed a lower risk of progression than carriers of the GG genotype  [p = 0.0129; HR = 7.43; 95% CI = 1.53–36.15; AA vs. G) in the multivariate Cox regression. This association maintained a strong trend for PFS in the univariate Cox regression model, where carriers of the AA genotype showed a tendency toward a higher risk of progression compared to carriers of the G allele  [As for the rs11568820 (Cdx-2) polymorphism, two of the four studies that analyzed it found results concerning its association with OS and PFS ,25,27,29A vs. G) .p = 0.04; HR = 1.32; 95% CI = 0.98–1.77; CT vs. CC, and p = 0.04; HR = 1.41; 95% CI = 0.96–2.07; TT vs. CC) [The rs2228570 (FokI) polymorphism is studied in four of the articles included in this review ,25,28,29 vs. CC) . The inf vs. CC) ,28,29.p < 0.001; HR = 2.84; 95% CI = 2.63–3.94; AA vs. CC). This association showed a strong trend in PFS, where the rs7975232-AA genotype displayed a tendency toward a higher risk of progression than the CC genotype  [p = 0.0068; HR = 1.73; 95% CI = 1.16–2.58; AA vs. C). This association was clearer in PFS, wherein the multivariate Cox regression model carriers of the rs7975232-AA genotype presented a higher risk of progression compared to the C allele  [Three of the studies included in this review investigated the influence of the rs7975232 (ApaI) SNP on survival ,27,28. TA vs. C) . p = 0.027; HR = 1.41; 95% CI = 1.00–1.99; AG vs. AA, and p = 0.027; HR = 4.26; 95% CI = 1.32–13.8; GG vs. AA). Likewise, carriers of the rs731236-G allele had a higher risk of death than those with the AA genotype  [p = 0.0005; HR = 2.71; 95% CI = 1.55–4.75; GG vs. A). In addition, this association was maintained in the multivariate Cox regression model for PFS, where carriers of the rs731236-GG genotype showed a higher risk of progression than carriers of the A allele  [The rs731236 (TaqI) SNP was investigated in three of the studies included in this review ,27,28. AG vs. A) . CYP27B1 gene to survival in NSCLC [p = 0.044; HR = 2.05; 95% CI = 1.02–4.14; GG vs. T). The association of this SNP was not observed in the analysis of its influence on OS [p = 0.056; HR = 2.01; 95% CI = 0.98–4.14; A vs. GG). This observation was more evident in PFS, where carriers of the A allele had a higher risk of progression than those with the GG genotype . In addition, in the subgroup analysis, it was found that in 146 patients with non-resected NSCLC, in the multivariate Cox regression model, carriers of the rs4646536-AA genotype presented a higher risk of progression compared to the G allele . The rs3782130 SNP showed a significant association in the univariate Cox regression model with respect to PFS in 146 Caucasian patients (from Spain) with non-resected NSCLC. In particular, carriers of the GG genotype exhibited a higher risk of progression relative to carriers of the C allele  [p = 0.627) [In this review, there were two studies that investigated the relationship of genetic polymorphisms in the in NSCLC ,26. For ce on OS . MoreoveG vs. C) . This asG vs. C) ,26. The CYP24A1 gene and survival in NSCLC [p = 0.0089; HR = 3.47; 95% CI = 1.37–8.79; TT vs. C). This association was maintained in the analysis of the SNP’s influence on PFS since patients carrying the rs6068816-TT genotype had a higher risk of progression than those with the C allele  [p = 0.072; HR = 1.13; 95% CI = 0.86–1.49; CT vs. CC) [Some studies assessed the relationship between the rs6068816 and rs4809957 polymorphisms in the in NSCLC ,26. WithT vs. C) . Likewis vs. CC) ,26 or fo vs. CC)  in patieGC gene was studied in two of the articles included in this review [p = 0.0242; HR = 2.72; 95% CI = 1.14–6.47; GG vs. T). This association was clearer in the analysis of the influence of this SNP on PFS, where in the multivariate Cox regression model, those carrying the rs7041-GG genotype showed a higher risk of progression than those carrying the T allele  [The rs7041 SNP located in the s review ,26. The G vs. T) . On the CYP2R1 gene and OS and PFS [p = 0.033; HR = 0.79; 95% CI = 0.61–1.03; AG vs. GG, and p = 0.033; HR = 0.69; 95% CI = 0.46–0.97; AA vs. GG, respectively). In addition, in the subgroup analysis, they found that in 270 NSCLC patients aged over 60, those carrying the rs10741657-A allele had a lower risk of death than those with the GG genotype . Finally, this association was also observed in the subgroup of 246 patients with NSCLC who did not receive chemotherapy. Patients with the rs10741657-A allele showed a lower risk of death than those carrying the GG genotype  [Two studies included in this review analyzed the relationship between the rs10741657 polymorphism in the  and PFS . Kong et vs. GG) . On the The quality score assigned to each study included in the review is shown in VDR, CYP27B1, CYP24A1, GC, and CYP2R1 genes have proved to be the most significant [This is the first systematic review to have made a specific compilation of all the studies that have so far evaluated the influence of genetic polymorphisms in the main genes involved in the vitamin D metabolic pathway on OS and PFS in patients with NSCLC. The nificant ,46,47,48nificant ,46,47,49VDR gene  is located on chromosome 12 in the 12q13.11 region. It encodes a ligand-dependent transcription factor belonging to the nuclear receptor (NR) superfamily [VDR BsmI (rs1544410) polymorphism were associated with worse OS  [VDR are present in many tissues, including lung tissue [VDR and its ligand regulate 1–3% of all gene expression [VDR in lung cancer is associated with better survival [The erfamily ,51,52,53erfamily . These r = 0.85) . For the = 0.85) , whereasI rs15444 polymorp = 0.85) . This reI rs15444 polymorp = 0.85) . These rI rs15444 polymorp = 0.85) . These rg tissue ,56. It ipression ,58, inclpression ,51,52,59survival ,61. Thissurvival ,61. CYP27B1 gene codes for 1-alpha-hydroxylase, the only enzyme capable of converting vitamin D into its active form. The gene is located at 12q14.1 in the long arm of chromosome 12 [CYP27B1 expression was associated with better OS (p = 0.018). It also found, in turn, that changes in gene expression can be due to SNPs. Specifically, it found that differences in expression were statistically significant in the rs3782130 polymorphism (p = 0.028) [CYP24A1, CYP27B1, and VDR genes in lung cancer is affected by tumor differentiation and characterization. When the tumor is poorly differentiated, expression of CYP24A1 is increased and that of CYP27B1 is reduced [The osome 12 ,63. Thisosome 12 . Likewisosome 12 . However= 0.028) . Moreove reduced ,65.CYP24A1 gene, located on chromosome 20, specifically in the 20q13.2 region [CYP24A1 rs6068816 SNP and increased risk of death and progression in patients with non-resected NSCLC [CYP24A1 were positively associated with a worse prognosis  [CYP24A1 to a worse prognosis, since it has been observed that when the tumor is poorly differentiated, gene expression is greater [CYP24A1, may influence intron splicing. This affects the patterns or efficiency of mRNA splicing, which in turn has an impact on biological activities [CYP24A1 mRNA expression on survival in 3 types of cancer  was assessed in a multiethnic meta-analysis with 1479 cases in 4 studies; it revealed that elevated mRNA expression was associated with worse prognosis  [CYP24A1 expression in lung tumor tissue [Vitamin D metabolism is catalyzed by the 24-hydroxylase enzyme, which is responsible for carrying out a series of hydroxylations to convert calcitriol into calcitroic acid, a molecule with greater polarity to facilitate its elimination via the kidneys ,65,66,672 region ,65. One ed NSCLC . These r greater ,65. Althtivities ,68. Thistivities . Likewis = 38.1) . The resGC gene, located in the 4q13.3 region of chromosome 4, belongs to the albumin superfamily of binding proteins . This gene is characterized by synthesizing the vitamin D binding protein (VDBP), responsible for the blood transport of vitamin D metabolites to the targets of action [GC rs7041 were associated with a higher risk of death and progression [GC rs7041 polymorphism on survival in cancer. The available studies have focused on gene expression and circulating VDBP levels. A study of 148 Caucasian lung cancer patients (from the UK) revealed that low serum VDBP levels predicted lung cancer-specific death (p = 0.04) since VDBP was poorly expressed in tumor tissue cells [GC rs7041-TT genotype was associated with higher 25(OH)D levels (p = 0.007) [p = 0.03; HR = 0.63; 95% CI = 0.44–0.90) and a trend toward greater overall cancer survival . These associations were limited to cases that expressed the Gc2 isoform (rs7041-T/rs4588-T) [The f action ,71,72. Ogression . There iue cells . On the = 0.007) . A studys4588-T) .CYP2R1), located on chromosome 11, in the 11p15.2 region. This enzyme is responsible for synthesizing calcidiol or 25(OH)D from its precursors, ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3) [CYP2R1 rs10741657 polymorphism on survival in NSCLC. First, Pineda et al. in the study performed on 194 Caucasian patients (from Spain), found a strong tendency for higher risk of death in carriers of the rs10741657-AA genotype in the univariate Cox regression model [CYP2R1 rs10741657 was associated with a better prognosis in both stage I-IV NSCLC and subgroup analysis [CYP2R1 and DHCR7. In particular, carriers of the rs10741657 GG genotype were associated with lower 25-hydroxyvitamin D levels [CYP2R1 gene leads to the substitution of proline for leucine at amino acid 99 in the CYP2R1 protein and suppresses the enzyme activity of vitamin D 25-hydroxylase, indicating that genetic variation in this gene may affect 25(OH)D synthesis [The 25-hydroxylase or CYP2R1 enzyme is encoded by the gene of the same name (amin D3) ,46,76. Tanalysis . Differeanalysis . NeverthD levels . Other aynthesis ,78,79. The differences observed between results regarding the impact of certain SNPs on OS or PFS may be mainly due to the high methodological heterogeneity between the studies included, in terms of ethnicity, sample size, and clinical data collection, for example, as well as in the performance of subgroup analyses, where the power of the associations is altered by the small sample size.This review has some limitations, including the following: (I) The inclusion of studies that analyzed the influence of genetic polymorphisms in the vitamin D metabolic pathway on patients with NSCLC, which restricts the possible number of results and prevents them from being extrapolated to other malignancies. (II) Moreover, only the influence of SNPs on OS and PFS was examined, excluding the possible effect of these genetic variants on the risk of developing the disease. (III) Owing to the scarcity of results found and the reporting of results by subgroups, it was not possible to perform a meta-analysis to observe variations in the level of association of the genotypes studied with the disease prognosis. (IV) The studies included were in the low to moderate methodological quality range according to the STREGA statement criteria, and therefore the interpretations of the findings of this review must be treated with caution. Despite these limitations, this study includes an exhaustive compilation of scientific reports that have investigated the association between the main genetic variants related to the vitamin D metabolic pathway and the prognosis of NSCLC. The information endorses the importance of the biological functions of vitamin D and its influence on cancer. The information provided in this systematic review is intended to prepare the ground for conducting randomized clinical trials, with greater statistical power, to evaluate the clinical application of these genetic polymorphisms as prognostic biomarkers of disease, which, together with other biomarkers, such as protein expression, genomics, molecular markers, and clinical biomarkers, may provide a more accurate assessment of prognosis and a better treatment strategy for each patient on an individualized basis.Additionally, future research could focus on designing strategies that adapt to different changes that may arise in the vitamin D metabolic pathway, including evaluating the implementation of vitamin D in its active form as an adjuvant chemotherapeutic agent.CYP2R1, CYP27B1), transport (GC), metabolism (CYP24A1), and binding to the vitamin D receptor (VDR) could be considered predictive biomarkers of OS and PFS in patients with NSCLC. However, the evidence remains scarce for each of the polymorphisms reviewed and the studies display methodological differences and small sample sizes, making it impossible to reach firmer conclusions.The results obtained in this systematic review suggest that the rs10741657, rs4646536, rs7041, rs6068816, BsmI, Cdx-2, FokI, ApaI, and TaqI SNPs in genes related to synthesis (


The performance in evaluating thyroid nodules on ultrasound varies across different risk stratification systems, leading to inconsistency and uncertainty regarding diagnostic sensitivity, specificity, and accuracy.Comparing diagnostic performance of detecting thyroid cancer among distinct ultrasound risk stratification systems proposed in the last five years.Systematic search was conducted on PubMed, EMBASE, and Web of Science databases to find relevant research up to December 8, 2022, whose study contents contained elucidation of diagnostic performance of any one of the above ultrasound risk stratification systems . Based on golden diagnostic standard in histopathology and cytology, single meta-analysis was performed to obtain the optimal cut-off value for each system, and then network meta-analysis was conducted on the best risk stratification category in each system.This network meta-analysis included 88 studies with a total of 59,304 nodules. The most accurate risk category thresholds were TR5 for Eu-TIRADS, TR5 for ACR TIRADS, TR4b and above for C-TIRADS, and possible malignancy for S-Detect. At the best thresholds, sensitivity of these systems ranged from 68% to 82% and specificity ranged from 71% to 81%. It identified the highest sensitivity for C-TIRADS TR4b and the highest specificity for ACR TIRADS TR5. However, sensitivity for ACR TIRADS TR5 was the lowest. The diagnostic odds ratio (DOR) and area under curve (AUC) were ranked first in C-TIRADS.Among four ultrasound risk stratification options, this systemic review preliminarily proved that C-TIRADS possessed favorable diagnostic performance for thyroid nodules.https://www.crd.york.ac.uk/prospero, CRD42022382818. Thyroid nodule is a common occurrence in clinical practice. Ultrasonography plays an irreplaceable role in the early detection of thyroid cancer with merits of being noninvasive, convenient, and affordable . As highPrevious systematic reviews explored the diagnostic performance of various ultrasound risk stratification systems for thyroid nodules , 11, butThis network meta-analysis study was executed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The study protocol was registered with PROSPERO (york.ac.uk) and assigned registration number CRD42022382818. A comprehensive literature search was performed by using the Population, Intervention, Comparison, and Outcome (PICO) format to answer the following questions: How to compare the diagnostic performance among ACR TIRADS, Eu-TIRADS, S-DETECT, and C-TIRADS by taking cytologic or histopathological tests as reference standards in patients who underwent ultrasound detection of thyroid nodules?PubMed, EMBASE, and Web of Science databases were explored from the date of database creation to December 8, 2022, complying with the following search terms: ACR TIRADS, EU-TIRADS, C-TIRADS, S-detect, qualitative diagnosis, and thyroid nodule. The search strategy was mapped out by a researcher with six months of information retrieval experience. Two researchers independently screened the search results, and any discrepancies were resolved through discussion with a third investigator. After removing duplicates and non-research articles, such as reviews, conference reports, and case reports, as well as excluding articles that did not meet research question criteria, a final sample of studies was obtained. These studies provided sufficient data to assess the diagnostic performance of at least one of the four ultrasound risk stratification options.Two investigators were responsible for data extraction, and any conflicting results were decided through discussion with a third investigator. Recorded information covered relevant demographic factors such as country, ethnicity, sex ratio, mean age, the number of patients and nodules, as well as the mean diameter of benign and malignant nodules. Additionally, ultrasound risk stratification methods, optimal cut-off values, and reference criteria in each study were noted. The investigators also extracted the number of true-positive, true-negative, false-positive, and false-negative nodules based on the reference criteria to calculate diagnostic performance of each risk stratification system.In 2017, the TIRADS Committee of the ACR published a white paper introducing a new risk stratification format for thyroid nodules, known as ACR TIRADS. This system categorizes nodules as benign , not suspicious , mildly suspicious , moderately suspicious , or highly suspected malignant  . Eu-TIRAThe risk of bias and applicability concerns of the included studies were assessed based on QUADAS-2 tool, which was routinely employed for analyzing validation studies of diagnostic criteria. Each item was rated as low, high, or unclear risk. This tool estimates the risk of bias across four domains: patient selection, index test, reference standard, as well as flow and timing. Additionally, it assesses applicability concerns, focusing on the first three domains. Two independent reviewers (C.L. and S.H.) commented on included studies, with any disagreement addressed by consensus. The QUADAS-2 scale was then completed based on commentary information.2 was computed to quantify the heterogeneity among inter-studies in terms of sensitivity, specificity, and accuracy for each system, and was considered to be substantial when at least 75%. Then, through included one-arm/two-arm/three-arm studies, direct and indirect comparisons of performance metrics  among four options were performed through random effect network meta-analysis within the frequentist framework, based on risk category with the highest diagnostic accuracy in each option. Next, within-group heterogeneity and between-group heterogeneity were calculated. Finally, the risk stratification system evaluated by included medium-quantity studies was identified as reference option, and odds ratios (ORs) and 95% confidence intervals (CIs) were used to express the sensitivity, specificity, and accuracy with respect to that system for the remaining three systems.The best sensitivity, specificity, and accuracy of each stratification option were determined using Stata13.1 “midas” packages, based on the number of true-positive, false positives, true-negative, and false-negative assessments extracted from the individual studies. Ii1k, Yi2k) referred to true positive and true negative, respectively;  referred to malignant and benign thyroid nodules, respectively; and  referred to “unobserved” sensitivity and specificity, respectively. The binomial distribution describing true positive and true negative between malignant and benign thyroid nodules was as follows:This network meta-analysis was conducted by R package “rstan” . Analysis of Variance (ANOVA) model based on Bayesian algorithm was applied to exhibit network meta-analysis among four systems by utilizing two independent binomial distributions to describe the true positive and true negative rates between benign and malignant thyroid nodules and meantime considering the correlation between sensitivity and specificity . There wi refers to covariate affecting πijk.I = 1,… I; j = 1, 2, k = 1,… K; xMonte Carlo chain number, pre-iterations number, iteration number and step size were set at 3, 10000, 1000, and 5, respectively. Subsequently, absolute effect sizes including sensitivity and specificity, as well as relative effect sizes including risk ratio (RR), OR, and DOR were calculated.Besides, we employed various statistical packages in R4.1.3 to analyze extracted data. Specifically, we utilized “mada” and “reshape” packages to conduct the Summary Receiver Operating Characteristic (SROC) analysis of four optimal categories from risk stratification systems. The forest plots respectively related to ORs of sensitivity and specificity were created using “forestplot” package. To visualize publication bias, we used the “meta” and “metafor” packages.A total of 1998 articles were initially identified through multiple database searches, of which 147 duplicate articles were removed, leaving 1,851 articles for further review. After excluding 1,613 articles based on title and abstract screening, 238 articles were reviewed in full, where 61 articles lacked sufficient data to determine diagnostic performance, and 45 articles were irrelevant to the research topic. Ultimately, 132 studies were included in meta-analysis, comprising 90,451 nodules. A summary of the risk of bias and applicability concerns for each domain was exhibited in 2>75%).As displayed in In the second section, sensitivity for ACR TIRADS TR5  was significantly lower than Eu-TIRADS TR5, and lower  for S-Detect possible malignancy . In contrast, the sensitivity of C-TIRADS  was significantly higher than Eu-TIRADS. The specificity was significantly higher than Eu-TIRADS for ACR TIRADS , and higher  for S-Detect . DOR is commonly applied in systematic meta-analysis to comprehensively assess the performance of diagnostic tests in different studies by combining relationship between true positive rate and false positive rate. DOR close to 1 indicates less accuracy in the test. The DOR of C-TIRADS [12.36 ] ranked first, followed in order by S-Detect [9.87 ], ACR TIRADS [9.20 ], and Eu-TIRADS [8.22 ]. 2>75%, p<0.1). Subgroup analysis found that publication year, age, gender, and race were not contributors to the heterogeneity .In network meta-analysis, within-group heterogeneity and between-group heterogeneity were high ; sensitivity was lowest but specificity was highest for ACR TIRADS TR5 (highly suspicious). This tentative assessment of risk stratification systems for thyroid nodules may assist in future system updates and guide decisions regarding system implementation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.JL and LTY were responsible for the review concept and design. LTY, CL, ZC and SQH drafted the manuscript. JL and LTY provided critical revision of the manuscript for important intellectual content. All authors contributed to the article and approved the submitted version.


Autoimmune thyroid diseases (AITDs) are characterized by unique immune responses against thyroid antigens and persist over time. The most common types of AITDs are Graves' disease (GD) and Hashimoto's thyroiditis (HT). There is mounting evidence that changes in the microbiota may play a role in the onset and development of AITDs.The purpose of this comprehensive literature study was to answer the following query: Is there a difference in microbiota in those who have AITDs?According to the standards set out by the PRISMA statement, 16 studies met the requirements for inclusion after being screened for eligibility.The Simpson index was the only diversity measure shown to be considerably lower in patients with GD compared to healthy participants, whereas all other indices were found to be significantly greater in patients with HT. The latter group, however, showed a greater relative abundance of Bacteroidetes and Actinobacteria at the phylum level, and consequently of Prevotella and Bifidobacterium at the genus level. The strongest positive and negative relationships were seen for thyroid peroxidase antibodies and bacterial load.Overall, both GD and HT patients showed significant changes in the gut microbiota's diversity and composition.https://www.crd.york.ac.uk/PROSPERO/, identifier CRD42023432455. Their aThe importance of microbiota in autoimmune illnesses has come into the spotlight recently. There is no one best pattern of gut bacteria since everyone has a different mix. Nonetheless, Firmicutes and Bacteroidetes are often the most abundant phyla. Factors such as gender, lifestyle age, pharmacological regimens, level of physical activity, and food all influence the microbiome’s makeup , 11.Dysbiosis has been linked to the development of autoimmune illnesses such rheumatoid arthritis, atopic dermatitis, type 1 diabetes, SLE, inflammatory bowel disease, and autoimmune neurological disorders . Patho-mThyroid function is also profoundly influenced by microbiome metabolites. Short-chain fatty acids (SCFAs) have been the primary focus of research because of their ability to improve enterocyte integrity, shield against the invasion of pathogenic microorganisms, affect the immune response, and suppress inflammatory response , 21. FurIn addition, the microbiome has a role in thyroid hormone metabolism, which is part of the gut-thyroid axis. Deiodinases have been found in the human gut in previous research. Animal studies have shown that intestinal bacteria may absorb deconjugated iodothyronine and compete with human albumin for binding thyroid hormones , 23. In 2Tis meta-analysis is reported following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline , with it2.1A systematic literature search for relevant studies published in English was conducted in the databases Scopus, PubMed, and Web of Science from inception up to December 2022. These are some examples of queries looked up for:  OR (Graves OR thyroid) AND (thyroiditis OR disease] AND (Hashimoto OR Graves) AND (microbiota OR microbiome). Studies published after the year 2015 were included in the analysis. Two researchers independently reviewed the findings’ titles, abstracts, and entire texts. PICO criteria was used to determine which studies would be included in this systematic review as shown in 2.2The PICO parameters for this study are as follows. The inclusion criteria are patients with autoimmune thyroid diseases (AITD), including Graves’ Disease (GD) and Hashimoto Thyroiditis (HT), aged 18-65 years, of both genders. The exclusion criteria are individuals with additional autoimmune disorders. Intervention and comparison are not applicable, and the outcomes are changes in the quantity, richness, and diversity of the microbiota and changes in the microbiota’s composition without a clear indication of its diversity. The study design includes cohort, case-control, and cross-sectional studies, while conference reports, expert opinions, literature reviews, letters to the editor, case reports, and studies published before 2015 or not published in English are excluded.2.3The “Study Quality Assessment Tool” developed by the National Heart, Lung, and Blood Institute at the National Institutes of Health was used to evaluate the potential for bias in each of the included studies. Both investigators filled out this form, and any discrepancies were discussed and settled. 2.4Using Comprehensive Meta-analysis Software (CMA version 4) we plotted the meta-analysis findings as forest plots. The GD and HT subsets were used in the meta-analysis. The comparative richness at the phylum and genus levels was computed as a continuous variable, and the pooled standardized mean differences for diversity indices were determined.316 papers were included in this evaluation after the studies were screened using the exclusion and inclusion criteria. As a result, data from approximately 750 human individuals with diagnosed AITDs) and 488 controls were obtained in five different countries. 44.1Specific alpha-diversity indices are used to describe the variety of bacteria in the microbiome and the number of microbial species. Richness can be measured using indices like Chao1 and ACE, whereas diversity can be measured using indices like Simpson and Shannon .Patients with GD had low levels of richness indices than healthy people in all trials included in this meta-analysis. Three studies , 38, 44 Richness indices were observed to be greater in HT participants than in healthy people in two investigations , 33. TheThe majority of the included studies also revealed Good’s coverage indices of more than 99%, indicating that the present sequencing depth correctly captured the state of fecal samples for gut microbiota. It’s also important to remember that factors like food, eating habits, and origin may have a major impact on the variety of microbes living in your digestive tract , 53. How4.2The gut microbiome has been under scrutiny in recent years for its potential involvement in diversity’s emergence and maintenance. Consistent with the findings of the alpha-diversity study, there were substantial differences in the phylum and genus makeup of the intestinal microflora between GD participants and healthy group. The human gut microbiota is dominated by the Firmicutes and Bacteroidetes phyla, which together account for almost 90% of the whole population. The ratio of Firmicutes to Bacteroidetes (F/B) is an important indicator of gut dysbiosis and has been linked to a wide range of clinical states , 60. TheThe variation in the number of the genera was substantially greater between GD patients and healthy controls at lower taxonomic levels. Two investigations , 44 founFurthermore, it was shown that the phylum Actinobacteria was more prevalent in the afflicted than in the healthy, particularly for two genera. Bifidobacterium species may be protective or progressive in autoimmune disorders, making their involvement in immunopathogenesis unclear . AutoimmHowever, compared to patients with GD, the comparative richness of the gut microflora in HT participants was equal to that of healthy participants, demonstrating an opposite pattern of changes. The Firmicutes genus Blautia illustrates the concept of inverse interdependence between GD and HT. Beneficial anti-inflammatory effects may be mediated by these commensal bacteria . BlautiaIn addition to diversity, the type of food also affects the comparative richness of the gut microflora. Previous research has linked the Mediterranean diet to a decrease in Streptococcus and a rise in Bifidobacterium, Prevotella, and Roseburia, all of which degrade dietary fiber , 70. Low4.3Examining the connections between immunological and functional changes in microbiota composition and thyroid functioning parameters might provide light on the involvement of microflora in the prognosis of AITDs. Significant associations were identified for TPOAb, one of the thyroid functioning measures examined. Changes in the composition of the microbiota were also linked to elevations or decreases in TSH and TRAb. A small subset of bacteria were found to have any association with TGAb. However, determining the precise orientations of these relationships is challenging. Bacteroidetes linked inversely with TSH and favorably with TPOAb and TRAb at the phylum level , 41, 43.Collectively, our results provide credence to the hypothesis that microbiota modifications are strongly linked to the onset and progression of AITDs by confirming substantial associations between various gut bacteria and thyroid measures. The oral bacteria Veillonella and Streptococcus cause periodontitis and dental cavities . These tHowever, low levels of Faecalibacterium are associated with an increased risk of developing inflammatory bowel disease and colon cancer in the gut , despitein vitro investigations involving 40 different bacterial strains have documented any effects at all from ATD, and those effects were considered to be rather small (Taking antithyroid medications (ATD) may also alter the bacteria that reside in your intestines. Only er small .Intestinal microbial community composition may also be influenced by gender and sex hormone levels . Women aA similar meta-analysis was conducted in 2021 by Gong et al. and showed a significant correlation between the dynamics of gut microbiota and occurrence of Autoimmune Thyroid Disease. However, research has grown exponentially to determine this relationship since 2021, and a new meta-analysis was warranted to coalesce these findings .5Particularly, this systematic evaluation is constrained by the shortcomings of the papers that were included. These studies sometimes had limited sample numbers, with people of different ages or sexes serving as controls. Most of the studies were done in Asia, but we also included one from Europe, one from Africa, and one from South America. Due to a lack of comprehensive data on diversity indices and relative abundance of the chosen phyla and genera, not all research could be included in the meta-analysis . Despite the increasing availability of data from other, separate groups, only one research has provided an unambiguous description of GO patients. Although modifications in the gut microbiota were determined in all these investigations, the oral microbiome was not studied, which might be of relevance in future research.6This meta-analysis found that people with both GD and HT had significantly different gut microbiota in terms of diversity and composition. Patients with HT had greater diversity indices than healthy participants, whereas those with GD had lower values. Patients with GD also had a greater relative abundance of Bacteroidetes and Actinobacteria. TPOAb levels are often associated with changes in the diversity of microbiota. Additional research is needed to verify these results.DA, NA, US and RS: were responsible for the conception or design of the work, drafting the work, giving final approval, and ensuring accuracy and integrity. SF, MF, FR and RW: were responsible for interpreting the data for the work, drafting the work, giving final approval, and ensuring accuracy and integrity. SG, KA, MA and DT: were responsible for interpreting the data, drafting the work, giving final approval, and ensuring accuracy and integrity. HS AD, MH and AT were responsible for interpreting the data, drafting the work, giving final approval, and ensuring accuracy and integrity. All authors contributed to the article and approved the submitted version.


Zheng et al. Nature Medicine 10.1038/s41591-022-01973-2 (2022).arising from H. Lescinsky et al. Nature Medicine 10.1038/s41591-022-01968-z (2022).arising from J. D. Stanaway et al. Nature Medicine 10.1038/s41591-022-01970-5 (2022).arising from C. Razo et al. Nature Medicine 10.1038/s41591-022-01974-1 (2022).arising from X. Dai et al. Nature Medicine 10.1038/s41591-022-01978-x (2022).5 in which the authors conducted meta-analyses of epidemiological studies to provide an overall conservative quantitative assessment for several important public health questions. For ease of interpretation, they transformed the overall assessment into a star rating (1–5 stars). Examples include five stars for smoking and lung cancer, two stars for low vegetable intake and ischemic heart disease (IHD) and two stars for unprocessed red meat and type 2 diabetes (T2D), colorectal cancer and IHD6. They used this same method to assign just three stars to smoking in relation to IHD and one or two stars to other well-established relationships4. However, we believe there are serious methodological issues with their meta-analyses5; the star rating of evidence strength is overly simplistic and could cast doubt on existing recommendations and policies intended to prevent chronic disease and treat illnesses.We read with interest the Burden of Proof (BoP) studies7. As an example of the BoP analyses1, for unprocessed red meat and T2D 15 studies were included in the meta-analysis, many of which were large and without evidence of small study bias. With what the authors described as a conventional analysis, there is a highly statistically significant and approximately linear positive association and the relative risk for 100 g per day of red meat intake versus no consumption was approximately 1.24. However, due to an approximately 2.5-fold inflation of the width of the conventional CIs due to the BoP methodology, their lower 95% boundary included no association, which was then used to rate evidence and two stars were given. Using the well-established Greenland and Longnecker method8 we estimated the linear association for each study included in Lescinsky et al.1 and then used a standard random effects meta-analysis method (taking into account the extra uncertainty due to estimating the between-study variance) to calculate a summary association relating unprocessed red meat and T2D. A robust positive dose–response is seen (P < 0.001) and the 95% CIs in our analysis  is only modestly wider than that from the conventional fixed effects model. Our analysis, which takes into account between-study heterogeneity in associations, suggests that the UIs provided by the BoP studies are extreme. This issue is further illustrated by their rating of only three stars for smoking and IHD based on meta-analyses of 60 studies, similar to the rating for smoking and lower back pain (only six studies)4. The relationship of smoking to risk of IHD, primarily acute myocardial infarction, is extremely strong and approximately linear with relative risks over 5 for high-intensity smoking; this risk has been recognized as convincing for over four decades by regular reports of the U.S. Surgeon General9. At least six mechanistic pathways have been documented9. On the other hand, the association between smoking and lower back pain lacks clear evidence of causation.In the BoP analyses, the estimated uncertainty intervals (UIs) were several times wider than the 95% confidence intervals (CIs) generated by existing methods that account for heterogeneity among methods1. In addition, there is minimal evidence of publication bias or influence of outlier observations from small studies. These findings suggest that the authors’ assumption about between-study heterogeneity is untenable and their methods that lead to drastically inflated UIs beyond conventional random effects models are not justified.The UIs from the BoP meta-analyses are intended to account for other sources of heterogeneity beyond between-study heterogeneity in mean effects. The authors conducted bias assessment of individual studies, including exposure and outcome measurements, controlled for confounding and selection bias. In the meta-analysis of red meat and six chronic disease outcomes, none of the bias adjustments were statistically significant10. Another assumption made by the BoP authors is that the dose–response relationship is not log-linear. However, previous studies of red meat consumption in relation to risks of diabetes11 and cardiovascular disease12 have concluded that within the range of population intakes, there was no substantive deviation from linearity. We believe that a more appropriate approach is to meta-analyze the estimates from continuous exposures from each study, which can enhance statistical power, reduce between-study heterogeneity and facilitate the interpretation of the data. Still, a critical examination of the studies that contribute most to the conclusions is desirable to evaluate the assessment of exposure and outcome, control of confounding, potential for reverse causation and other aspects of study design.Meta-analyses of studies are commonly used to synthesize published evidence but they cannot overcome variation in the original studies because of different definitions of exposure and outcome, follow-up time, data analyses and covariate adjustment. We believe that a better approach is to obtain individual-level data from all original studies and use standard definitions of the primary exposure and covariates and then model the relationship between exposures and outcomes. This approach is now widely used in nutritional epidemiological studies, providing more robust and informative estimates; these analyses can additionally examine subgroup effects. When using individual-level data to summarize studies, substantive unexplained heterogeneity has been uncommon13. The choice of counterfactuals can make a major difference and substitution analyses are now routinely part of epidemiological analyses13, which could not be addressed in the BoP papers as they require primary data.Furthermore, when evaluating the relationship between one dietary component, such as red meat, and health outcomes, alternative foods such as poultry, fish or plant protein must be considered because a person’s long-term energy intake is tightly regulated within narrow limits without a substantial change in weight or physical activity14, which in turn increases the risk of IHD. Even without prospective cohort evidence linking red meat and IHD, on the basis of these data one could extrapolate that eating large amounts of red meat might be harmful for heart disease. This type of evidence is part of the Bradford Hill criteria15 for evaluating epidemiological evidence and determining in expert reviews whether there is a sufficiently strong basis for translation to recommendations and policies15.Beyond the methodological issues raised here, the totality of the evidence, rather than only epidemiological studies, is critical to consider when drawing conclusions about causality and providing recommendations. The ideal randomized trial in humans is rarely feasible but the totality of evidence could include randomized controlled trials of intermediate risk factors for diseases, salient animal studies and plausible biological mechanisms. For example, in randomized controlled trials, compared with plant protein sources, red meat (high in saturated fat and cholesterol and low in polyunsaturated fat) increases low-density lipoprotein cholesterolConsidering the methodological and conceptual problems we raise with the new meta-analysis methods, the star rating system for the BoP does not seem justifiable. The authors note that the star ratings were designed for policy-makers and individuals to make decisions about their own risk. However, many people may not be aware of the crucial subtleties in studies of diet and other complex environmental exposures and the need to consider other forms of evidence. Although the authors suggest that the precautionary principle may apply when there is some evidence of harm, the BoP estimates imply that these estimates are so uncertain that strong public health recommendations and policies should not be made based on so-called weak evidence. We agree with the authors that further research in many areas is desirable. However, because the CIs in the BoP methodology tend to cover the findings from almost all existing studies, including extreme studies close to the null hypothesis, new studies showing clear associations may have little effect on the BoP conclusions. It is our view that BoP studies are misleading and the star rating system is too simplistic. We believe that reviews by national and international bodies that consider the full range of evidence on these topics are likely to provide a better basis for personal and policy decisions.