A conserved fertilization complex bridges sperm and egg in vertebrates.

Fertilization, the basis for sexual reproduction, culminates in the binding and fusion of sperm and egg. Although several proteins are known to be crucial for this process in vertebrates, the molecular mechanisms remain poorly understood. Using an AlphaFold-Multimer screen, we identified the protein Tmem81 as part of a conserved trimeric sperm complex with the essential fertilization factors Izumo1 and Spaca6. We demonstrate that Tmem81 is essential for male fertility in zebrafish and mice. In line with trimer formation, we show that Izumo1, Spaca6, and Tmem81 interact in zebrafish sperm and that the human orthologs interact in vitro. Notably, complex formation creates the binding site for the egg fertilization factor Bouncer in zebrafish. Together, our work presents a comprehensive model for fertilization across vertebrates, where a conserved sperm complex binds to divergent egg proteins-Bouncer in fish and JUNO in mammals-to mediate sperm-egg interaction.

SnapShot: Enveloped Virus Entry.

In order to initiate successful infection, viruses have to transmit and deliver their genome from one host cell or organism to another. To achieve this, enveloped viruses must first fuse their membrane with those of the target host cell. Here, we describe the sequence of events leading to the entry of representative enveloped viruses, highlighting the strategies they use to gain access to the host cell cytosol.

Structural basis for Marburg virus neutralization by a cross-reactive human antibody.

The filoviruses, including Marburg and Ebola, express a single glycoprotein on their surface, termed GP, which is responsible for attachment and entry of target cells. Filovirus GPs differ by up to 70% in protein sequence, and no antibodies are yet described that cross-react among them. Here, we present the 3.6 Å crystal structure of Marburg virus GP in complex with a cross-reactive antibody from a human survivor, and a lower resolution structure of the antibody bound to Ebola virus GP. The antibody, MR78, recognizes a GP1 epitope conserved across the filovirus family, which likely represents the binding site of their NPC1 receptor. Indeed, MR78 blocks binding of the essential NPC1 domain C. These structures and additional small-angle X-ray scattering of mucin-containing MARV and EBOV GPs suggest why such antibodies were not previously elicited in studies of Ebola virus, and provide critical templates for development of immunotherapeutics and inhibitors of entry.

Global analysis of protein-RNA interactions in SARS-CoV-2-infected cells reveals key regulators of infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control its life cycle remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively the cellular and viral RBPs that are involved in SARS-CoV-2 infection. We reveal that SARS-CoV-2 infection profoundly remodels the cellular RNA-bound proteome, which includes wide-ranging effects on RNA metabolic pathways, non-canonical RBPs, and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Among them are several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19.

Neoadjuvant relatlimab and nivolumab in resectable melanoma.

Relatlimab and nivolumab combination immunotherapy improves progression-free survival over nivolumab monotherapy in patients with unresectable advanced melanoma1. We investigated this regimen in patients with resectable clinical stage III or oligometastatic stage IV melanoma (NCT02519322). Patients received two neoadjuvant doses (nivolumab 480 mg and relatlimab 160 mg intravenously every 4 weeks) followed by surgery, and then ten doses of adjuvant combination therapy. The primary end point was pathologic complete response (pCR) rate2. The combination resulted in 57% pCR rate and 70% overall pathologic response rate among 30 patients treated. The radiographic response rate using Response Evaluation Criteria in Solid Tumors 1.1 was 57%. No grade 3-4 immune-related adverse events were observed in the neoadjuvant setting. The 1- and 2-year recurrence-free survival rate was 100% and 92% for patients with any pathologic response, compared to 88% and 55% for patients who did not have a pathologic response (P = 0.005). Increased immune cell infiltration at baseline, and decrease in M2 macrophages during treatment, were associated with pathologic response. Our results indicate that neoadjuvant relatlimab and nivolumab induces a high pCR rate. Safety during neoadjuvant therapy is favourable compared to other combination immunotherapy regimens. These data, in combination with the results of the RELATIVITY-047 trial1, provide further confirmation of the efficacy and safety of this new immunotherapy regimen.

Androgen receptor blockade promotes response to BRAF/MEK-targeted therapy.

Treatment with therapy targeting BRAF and MEK (BRAF/MEK) has revolutionized care in melanoma and other cancers; however, therapeutic resistance is common and innovative treatment strategies are needed1,2. Here we studied a group of patients with melanoma who were treated with neoadjuvant BRAF/MEK-targeted therapy ( NCT02231775 , n = 51) and observed significantly higher rates of major pathological response (MPR; ≤10% viable tumour at resection) and improved recurrence-free survival (RFS) in female versus male patients (MPR, 66% versus 14%, P = 0.001; RFS, 64% versus 32% at 2 years, P = 0.021). The findings were validated in several additional cohorts2-4 of patients with unresectable metastatic melanoma who were treated with BRAF- and/or MEK-targeted therapy (n = 664 patients in total), demonstrating improved progression-free survival and overall survival in female versus male patients in several of these studies. Studies in preclinical models demonstrated significantly impaired anti-tumour activity in male versus female mice after BRAF/MEK-targeted therapy (P = 0.006), with significantly higher expression of the androgen receptor in tumours of male and female BRAF/MEK-treated mice versus the control (P = 0.0006 and P = 0.0025). Pharmacological inhibition of androgen receptor signalling improved responses to BRAF/MEK-targeted therapy in male and female mice (P = 0.018 and P = 0.003), whereas induction of androgen receptor signalling (through testosterone administration) was associated with a significantly impaired response to BRAF/MEK-targeted therapy in male and female patients (P = 0.021 and P < 0.0001). Together, these results have important implications for therapy.

Time-resolved NMR detection of prolyl-hydroxylation in intrinsically disordered region of HIF-1α.

Prolyl-hydroxylation is an oxygen-dependent posttranslational modification (PTM) that is known to regulate fibril formation of collagenous proteins and modulate cellular expression of hypoxia-inducible factor (HIF) α subunits. However, our understanding of this important but relatively rare PTM has remained incomplete due to the lack of biophysical methodologies that can directly measure multiple prolyl-hydroxylation events within intrinsically disordered proteins. Here, we describe a real-time 13C-direct detection NMR-based assay for studying the hydroxylation of two evolutionarily conserved prolines (P402 and P564) simultaneously in the intrinsically disordered oxygen-dependent degradation domain of hypoxic-inducible factor 1α by exploiting the "proton-less" nature of prolines. We show unambiguously that P564 is rapidly hydroxylated in a time-resolved manner while P402 hydroxylation lags significantly behind that of P564. The differential hydroxylation rate was negligibly influenced by the binding affinity to prolyl-hydroxylase enzyme, but rather by the surrounding amino acid composition, particularly the conserved tyrosine residue at the +1 position to P564. These findings support the unanticipated notion that the evolutionarily conserved P402 seemingly has a minimal impact in normal oxygen-sensing pathway.

The circadian molecular clock in the suprachiasmatic nucleus is necessary but not sufficient for fear entrainment.

We show that nocturnal aversive stimuli presented to mice while they are eating and drinking outside of their safe nest can entrain circadian behaviors, leading to a shift toward daytime activity. We also show that the canonical molecular circadian clock is necessary for fear entrainment and that an intact molecular clockwork in the suprachiasmatic nucleus, the site of the central circadian pacemaker, is necessary but not sufficient to sustain fear entrainment of circadian rhythms. Our results demonstrate that entrainment of a circadian clock by cyclic fearful stimuli can lead to severely mistimed circadian behavior that persists even after the aversive stimulus is removed. Together, our findings support the interpretation that circadian and sleep symptoms associated with fear and anxiety disorders are, in part, the output of a fear-entrained clock, and provide a mechanistic insight into this clock.

B cells and tertiary lymphoid structures promote immunotherapy response.

Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers1-10 and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity11-15, although these have been less well-studied in ICB treatment16. A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling17 that B cell signatures were enriched in the tumours of patients who respond to treatment versus non-responding patients. To build on this, here we performed bulk RNA sequencing and found that B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders. Our findings were corroborated using a computational method (MCP-counter18) to estimate the immune and stromal composition in this and two other ICB-treated cohorts (patients with melanoma and renal cell carcinoma). Histological evaluation highlighted the localization of B cells within tertiary lymphoid structures. We assessed the potential functional contributions of B cells via bulk and single-cell RNA sequencing, which demonstrate clonal expansion and unique functional states of B cells in responders. Mass cytometry showed that switched memory B cells were enriched in the tumours of responders. Together, these data provide insights into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets.

Prophylactic Antibiotic Duration and Infectious Complications in Pancreatoduodenectomy Patients With Biliary Stents: Opportunity for De-escalation.

OBJECTIVE: The aim of this study was to compare infectious complications in pancreatoduodenectomy (PD) patients with biliary stents treated with short, medium, or long durations of prophylactic antibiotics. BACKGROUND: Pre-existing biliary stents have historically been associated with higher infection risk after PD. Patients are administered prophylactic antibiotics, but the optimal duration remains unknown. METHODS: This single-institution retrospective cohort study included consecutive PD patients from October 2016 to April 2022. Antibiotics were continued past the operative dose per surgeon discretion. Infection rates were compared by short (≤24 h), medium (>24 but ≤96 h), and long (>96 h) duration antibiotics. Multivariable regression analysis was performed to evaluate associations with a primary composite outcome of wound infection, organ-space infection, sepsis, or cholangitis. RESULTS: Among 542 PD patients, 310 patients (57%) had biliary stents. The composite outcome occurred in 28% (34/122) short, 25% (27/108) medium, and 29% (23/80) long-duration ( P =0.824) antibiotic patients. There were no differences in other infection rates or mortality. On multivariable analysis, antibiotic duration was not associated with infection rate. Only postoperative pancreatic fistula (odds ratio 33.1, P <0.001) and male sex (odds ratio 1.9, P =0.028) were associated with the composite outcome. CONCLUSIONS: Among 310 PD patients with biliary stents, long-duration prophylactic antibiotics were associated with similar composite infection rates to short and medium durations but were used almost twice as often in high-risk patients. These findings may represent an opportunity to de-escalate antibiotic coverage and promote risk-stratified antibiotic stewardship in stented patients by aligning antibiotic duration with risk-stratified pancreatectomy clinical pathways.

Lead Time and Immortal Time Biases Impact the Calculation of Post-colonoscopy Colorectal Cancer Survival.

The World Endoscopy Organization (WEO) standardized the reporting of post-colonoscopy colorectal cancers (PCCRCs), which account for 7% to 10% of colorectal cancers (CRCs).1 PCCRCs are diagnosed 6 to 36 months after a false negative colonoscopy. Detected CRCs (dCRCs) are diagnosed ≤6 months after an index true positive colonoscopy.2 PCCRC prognosis is unclear, with outcomes reported as comparable,3 superior,4 or inferior5,6 to those of dCRC. Because WEO terminology defines cases relative to the index colonoscopy, conventional survival analyses of PCCRC are susceptible to lead time and immortal time biases. We evaluated the influence of these biases on mortality in a population-based retrospective cohort of 10,938 dCRCs (93.8%) and 717 PCCRCs (6.2%). This study was set within Kaiser Permanente Northern California (KPNC), a large integrated health system, whose members are similar in demographic and socioeconomic characteristics to the Northern California region.7.

Association of Ultra-processed Food and Unprocessed or Minimally Processed Food Consumption With Bowel Habits Among U.S. Adults.

BACKGROUND & AIMS: Ultra-processed foods (UPFs) may have a negative impact on bowel habits. We aimed to assess the association between UPF and unprocessed or minimally processed food (MPF) intake and bowel habits among adults in the United States (U.S.). METHODS: We performed a cross-sectional study using data from the National Health and Nutrition Examination Survey (2005-2010). We used two 24-hour dietary recalls and, based on the Nova classification, calculated intakes of UPFs and MPFs. Constipation and diarrhea were defined using the Bristol Stool Form Scale and stool frequency. We performed survey-weighted logistic regression and substitution analysis to estimate the odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: Among 12,716 U.S. adults, there were 1290 cases of constipation and 1067 cases of diarrhea. Median UPF and MPF intakes were 26.5% and 66.2% of total grams per day, respectively. Greater UPF consumption (in % gram/d) was associated with higher odds of constipation (adjusted OR [aORQ4 vs Q1], 2.20; 95% CI, 1.76-2.74) (Ptrend < .001) but not diarrhea (aORQ4 vs Q1, 0.82; 95% CI, 0.62-1.09) (Ptrend = .12). Increased MPF consumption was associated with lower odds of constipation (aORQ4 vs Q1, 0.46; 95% CI, 0.370-0.57) (Ptrend < .001). Associations with constipation were attenuated after adjusting for diet quality (aORQ4 vs Q1, UPF, 1.53; MPF, 0.69). Substituting 10% of UPF intake with an equivalent proportion of MPFs was associated with lower odds of constipation (aOR, 0.90; 95% CI, 0.87-0.93). CONCLUSIONS: UPF intake was associated with higher odds of constipation, whereas the odds were lower with greater MPF consumption. The effect of food processing on bowel habits was independent of diet quality.

Oral Antibiotic Use in Adulthood and Risk of Early-Onset Colorectal Cancer: A Case-Control Study.

BACKGROUND AND AIMS: Prior antibiotic use may be a factor in the rising incidence of colorectal cancer seen in those under 50 years of age (early-onset colorectal cancer [EOCRC]); however, the few studies to examine this link have reported conflicting results. Therefore, we evaluated the association between oral antibiotic use in adulthood and EOCRC in a large integrated healthcare system in the United States. METHODS: A population-based nested case-control study was conducted among Kaiser Permanente Northern California patients 18-49 years of age diagnosed with EOCRC (adenocarcinoma of the colon or rectum) in 1998-2020 who had ≥2 years of continuous pharmacy benefit prior to diagnosis. Cases were matched 4:1 to healthy controls on birth year, sex, race and ethnicity, medical facility, and duration of pharmacy benefit. Antibiotic exposure >1 year before the diagnosis/index date was assessed using prescribing records. Conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). A sensitivity analysis was performed among those with ≥10 years of continuous prescribing records. RESULTS: 1359 EOCRC cases were matched to 4711 healthy controls. Antibiotic use in adulthood was not significantly associated with EOCRC in unadjusted or adjusted analyses (adjusted OR: 1.04; 95% CI: 0.94-1.26). No associations were seen for cumulative number of oral antibiotic dispensations or for any prior period of antibiotic exposure. CONCLUSIONS: In a large United States healthcare setting, there was no conclusive evidence of an association between oral antibiotic use in adulthood and risk of EOCRC.

Randomized Trial of Patient Outreach Approaches to De-implement Outdated Colonoscopy Surveillance Intervals.

BACKGROUND AND AIMS: Guidelines now recommend patients with low-risk adenomas receive colonoscopy surveillance in 7-10 years and those with the previously recommended 5-year interval be re-evaluated. We tested 3 outreach approaches for transitioning patients to the 10-year interval recommendation. METHODS: This was a 3-arm pragmatic randomized trial comparing telephone, secure messaging, and mailed letter outreach. The setting was Kaiser Permanente Northern California, a large integrated healthcare system. Participants were patients 54-70 years of age with 1-2 small (<10 mm) tubular adenomas at baseline colonoscopy, due for 5-year surveillance in 2022, without high-risk conditions, and with access to all 3 outreach modalities. Patients were randomly assigned to the outreach arm (telephone [n = 200], secure message [n = 203], and mailed letter [n = 201]) stratified by age, sex, and race/ethnicity. Outreach in each arm was performed by trained medical assistants (unblinded) communicating in English with 1 reminder attempt at 2-4 weeks. Participants could change their assigned interval to 10 years or continue their planned 5-year interval. RESULTS: Sixty-day response rates were higher for telephone (64.5%) and secure messaging outreach (51.7%) vs mailed letter (31.3%). Also, more patients adopted the 10-year surveillance interval in the telephone (37.0%) and secure messaging arms (32.0%) compared with mailed letter (18.9%) and rate differences were significant for telephone (18.1%; 97.5% confidence interval: 8.3%-27.9%) and secure message outreach (13.1%; 97.5% confidence interval: 3.5%-22.7%) vs mailed letter outreach. CONCLUSIONS: Telephone and secure messaging were more effective than mailed letter outreach for de-implementing outdated colonoscopy surveillance recommendations among individuals with a history of low-risk adenomas in an integrated healthcare setting. (ClinicalTrials.gov, Number: NCT05389397).

Predicting Risk of Colorectal Cancer After Adenoma Removal in a Large Community-Based Setting.

INTRODUCTION: Colonoscopy surveillance guidelines categorize individuals as high or low risk for future colorectal cancer (CRC) based primarily on their prior polyp characteristics, but this approach is imprecise, and consideration of other risk factors may improve postpolypectomy risk stratification. METHODS: Among patients who underwent a baseline colonoscopy with removal of a conventional adenoma in 2004-2016, we compared the performance for postpolypectomy CRC risk prediction (through 2020) of a comprehensive model featuring patient age, diabetes diagnosis, and baseline colonoscopy indication and prior polyp findings (i.e., adenoma with advanced histology, polyp size ≥10 mm, and sessile serrated adenoma or traditional serrated adenoma) with a polyp model featuring only polyp findings. Models were developed using Cox regression. Performance was assessed using area under the receiver operating characteristic curve (AUC) and calibration by the Hosmer-Lemeshow goodness-of-fit test. RESULTS: Among 95,001 patients randomly divided 70:30 into model development (n = 66,500) and internal validation cohorts (n = 28,501), 495 CRC were subsequently diagnosed; 354 in the development cohort and 141 in the validation cohort. Models demonstrated adequate calibration, and the comprehensive model demonstrated superior predictive performance to the polyp model in the development cohort (AUC 0.71, 95% confidence interval [CI] 0.68-0.74 vs AUC 0.61, 95% CI 0.58-0.64, respectively) and validation cohort (AUC 0.70, 95% CI 0.65-0.75 vs AUC 0.62, 95% CI 0.57-0.67, respectively). DISCUSSION: A comprehensive CRC risk prediction model featuring patient age, diabetes diagnosis, and baseline colonoscopy indication and polyp findings was more accurate at predicting postpolypectomy CRC diagnosis than a model based on polyp findings alone.

Total Venous Control and Vein-to-the-Right Superior Mesenteric Artery Approach in Robotic Pancreatoduodenectomy.

BACKGROUND: Robotic pancreatoduodenectomy is an increasingly accepted alternative for the treatment of pancreatic ductal adenocarcinoma (PDAC).1 However, the ability to perform a meticulous robotic-assisted superior mesenteric artery (SMA) dissection to obtain a margin-negative resection remains unknown.2 PDAC within the head of the pancreas (HOP) that involves the superior mesenteric vein (SMV) and portal vein (PV) requires total venous control (TVC) and a 'vein-to-the-right' (or anterior artery-first) approach to SMA dissection to minimize venous congestion and operative blood loss.3-5 Here, we demonstrate a robotic pancreatoduodenectomy with TVC and a 'vein-to-the-right' approach. METHODS: A 70-year-old woman with cT2N0M0 HOP PDAC with lateral SMV involvement and right gastroepiploic vein occlusion underwent robotic pancreatoduodenectomy after neoadjuvant chemotherapy. After transecting the pancreas, we achieved TVC by dividing the small venous tributaries and encircling the SMV, splenic vein, and PV. We then proceeded with a 'vein-to-the-right' approach. The inferior pancreatoduodenal arteries were divided to minimize HOP inflow and decrease specimen bleeding. Once the specimen was dissected off the periadventitial plane of the distal SMA, the SMV dissection was carefully performed using a partial side-wall vein resection using a vascular stapler. RESULTS: Total operative time was 7.5 h and estimated blood loss was 25 mL. The patient recovered well postoperatively and was discharged on postoperative day 3. Final pathology exhibited a 2.4 cm, moderately to poorly differentiated adenocarcinoma with negative margins (ypT2N1, 2/38 lymph nodes positive). CONCLUSION: For tumors with lateral vein involvement, robotic pancreatoduodenectomy can be safely performed via TVC and a 'vein-to-the-right' approach.

Revisiting the Malignant Masquerade at the Liver Hilum: Have We Made Progress?

BACKGROUND: Distinguishing malignant from benign causes of obstruction at the liver hilum can pose a diagnostic dilemma. This study aimed to determine factors that predict benign causes of hilar obstruction and long-term outcomes after resection. METHODS: Consecutive patients who underwent surgery for hilar obstruction at a single institution between 1997 and 2022 were retrospectively analyzed. Median follow-up was 26 months (range 0-281 months). RESULTS: Among 182 patients who underwent surgery for hilar obstruction, 25 (14%) patients were found to have benign disease. Median CA19-9 level after normalization of serum bilirubin was 80 U/mL (range 1-5779) and 21 U/mL (range 1-681) among patients with malignant and benign strictures, respectively (p = 0.001). Cross-sectional imaging features associated with malignancy were lobar atrophy, soft tissue mass/infiltration, and vascular involvement (all p < 0.05). Factors not correlated with malignancy were jaundice upon presentation, peak serum bilirubin, sex, and race. Preoperative bile duct brushing or biopsy had sensitivity and specificity rates of 82% and 55%, respectively. Among patients who underwent resection with curative intent, grade 3-4 complications occurred in 55% and 29% of patients with malignant and benign strictures, respectively (p = 0.028). Postoperative long-term complications of chronic portal hypertension and recurrent cholangitis occurred in ≥ 10% of patients with both benign and malignant disease (p = non-significant). CONCLUSIONS: Strictures at the liver hilum continue to present diagnostic and management challenges. Postoperative complications and long-term sequelae of portal hypertension and recurrent cholangitis develop in a significant number of patients after resection of both benign and malignant strictures.

Intraoperative renal replacement therapy during liver transplantation in children: Safety, efficacy and impact on survival.

BACKGROUND: Intraoperative Continuous Renal Replacement Therapy (iCRRT) can prevent life-threatening complications, facilitate fluid management, and maintain metabolic homeostasis during liver transplantation (LT) in adults. There is a paucity of data in pediatric LT. We evaluated the safety, efficacy, and impact on survival of iCRRT in pediatric LT. METHODS: We conducted a retrospective cohort study of all children requiring CRRT pre-OLT at a quaternary children's hospital from 2014 to 2022. Demographic characteristics, intraoperative events, and post-LT outcomes were compared between those who received iCRRT and those who did not. RESULTS: Out of 306 patients who received LT, 30 (10%) were supported with CRRT at least 24 h prior to LT, of which 11 (36%) received iCRRT. The two cohorts were similar in demographics, diagnosis of liver disease, and severity of illness. The iCRRT patients experienced massive blood loss and increased transfusion requirements. There was no difference in intraoperative metabolic balance. One-year post-LT mortality rates were similar. CONCLUSION: ICRRT is safe in critically ill children with pre-LT renal dysfunction. It optimizes fluid and blood product resuscitation while maintaining metabolic homeostasis. Candidates need to be carefully chosen for this highly resource-intensive therapy to benefit this fragile population.

Systematic review: risk prediction models for metachronous advanced colorectal neoplasia after polypectomy.

BACKGROUND AND AIM: Colorectal cancer (CRC) is the fourth leading cause of cancer death globally. CRC surveillance is a common indication for colonoscopy, representing a considerable burden for endoscopy services. Accurate identification of high-risk patients who would benefit from more intensive surveillance, as well as low-risk patients suitable for less frequent follow-up, could improve the effectiveness of surveillance protocols and resource use. Our aim was to identify and critically appraise published risk models for the occurrence of metachronous advanced colorectal neoplasia (ACN), defined here as CRC or advanced adenomas detected during surveillance colonoscopy. METHODS: We searched PubMed and EMBASE for primary research studies reporting the development and/or validation of multivariable models that predict metachronous ACN risk. Screening of studies for inclusion, data extraction, and risk of bias assessment were conducted by two researchers independently. RESULTS: We identified nine studies describing nine risk models. Six models were internally validated and two were externally validated. No model underwent both internal and external validation. Good model discrimination (concordance index > 0.7) was reported for two models during internal validation and for one model during external validation. Calibration was acceptable when assessed (n = 4). Methodological limitations and a high risk of bias were observed for all studies. CONCLUSIONS: Several published models predicting metachronous ACN risk showed some promise. However, adherence to methodological standards was limited, and only two models were externally validated. Head-to-head comparisons of existing models using populations independent from model development cohorts should be prioritized to identify models suitable for use in clinical practice.