Pheochromocytomas (PCCs) are rare neuroendocrine tumors derived from the chromaffin cells of the adrenal medulla. When these tumors have an extra‐adrenal location, they are called paragangliomas (PGLs) and arise from sympathetic and parasympathetic ganglia, particularly of the para‐aortic location. Up to 25% of PCCs/PGLs are associated with inherited genetic disorders. The majority of PCCs/PGLs exhibit indolent behavior. However, according to their affiliation to molecular clusters based on underlying genetic aberrations, their tumorigenesis, location, clinical symptomatology, and potential to metastasize are heterogenous. Thus, PCCs/PGLs are often associated with diagnostic difficulties. In recent years, extensive research revealed a broad genetic background and multiple signaling pathways leading to tumor development. Along with this, the diagnostic and therapeutic options were also expanded. In this review, we focus on the current knowledge and recent advancements in the diagnosis and treatment of PCCs/PGLs with respect to the underlying gene alterations while also discussing future perspectives in this field. Pheochromocytomas are rare neuroendocrine tumors derived from the chromaffin cells of the adrenal medulla. When these tumors have an extra‐adrenal location, they are called paragangliomas and arise from sympathetic and parasympathetic ganglia. According to their affiliation to molecular clusters based on underlying genetic aberrations, their tumorigenesis, location, clinical symptomatology, and potential to metastasize are heterogenous. PGLs arise from the sympathetic and parasympathetic paraganglia outside of the adrenal gland. These ganglia are most commonly localized in the retroperitoneum, pelvis, thorax, head, and neck, but 85% of PGLs occur below the diaphragm.Hypoxia‐inducible factors (HIFs) play an important role in the process of tumorigenesis in PCCs/PGLs. These transcription factors regulate energy, iron metabolism, and erythropoiesis. Thus, their dysregulation may lead to cancer development and progression. HIF‐2α is an isoform, which is commonly expressed in multiple tissues—endothelium, kidney, heart, lung, gastrointestinal epithelium, and neural crest cell derivatives, including chromaffin cells. HIF‐2α also regulates processes in the sympathoadrenal lineage, catecholamine synthesis, and secretion. Overexpression of HIF‐2α was found to be associated with metastatic progression and poor prognosis mainly in cancer types derived from the neural crest, including PCCs/PGLs.2PCCs/PGLs may produce norepinephrine, epinephrine, or dopamine in varying amounts. The clinical manifestation depends on the interaction of norepinephrine or epinephrine with the α‐ and β‐adrenergic receptors in several tissues. Therefore, patients may exhibit variable hormonal manifestations.However, the occurrence of PCCs/PGLs among patients with sustained arterial hypertension is only 0.05%–0.1%. Adrenal PCCs commonly secrete metanephrines and extra‐adrenal PGLs secrete normetanephrines—the respective O‐methylated metabolites of epinephrine, and norepinephrine.SDH) gene and a deficiency in the enzyme dopamine‐β‐hydroxylase, which is responsible for the catalyzation of catecholamine synthesis .Catecholamine production can be independent of tumor size and every patient with a suspicion of PCCs/PGLs should be screened for free metanephrines and normetanephrines in plasma or via 24‐h urine collection. As shown in Table At least 3 days prior to the assessment of normetanephrines and 3‐methoxytyramine in plasma and urine, it is necessary to avoid caffeine, black tea, nicotine, alcohol, bananas, cheese, almonds, nuts, chocolate, eggs, and vanilla.The clinical sensitivity of chromogranin A among patients with PCCs/PGLs is 90%, and this biomarker may serve as a complement to metanephrine assays in a diagnostic setting.3Chest, abdominal, and pelvic multiphasic computed tomography (CT) or magnetic resonance imaging (MRI) scans are considered standard diagnostic procedures. Functional imaging is more specific and can be used for predicting radionuclide therapy. Several radiopharmaceuticals for single‐photon emission computed tomography (SPECT) and positron emission tomography (PET) are available for the diagnosis, staging, and follow‐up of PCCs/PGLs.131I‐MIBG can be indicated.123I or 131I. Both radioisotopes emit gamma radiation detectable by a gamma camera. 123I has a shorter half‐life and is thus the preferred radioisotope for the diagnostic procedure; usually at a dose of 185 MBq. In a therapeutic setting, a high dose level of 131I‐MIBG may emit enough beta and gamma radiation which can lead to lethal damage to tumor cells. In this case, the therapeutic dose may reach 37 GBq.111In‐pentetreotide scintigraphy as being superior to 123I‐MIBG scintigraphy.When tumors appear on a meta‐iodobenzylguanidine (MIBG) scan, treatment with high‐specific activity (HSA) 123I‐MIBG, 111In‐pentetreotide or 99mTc‐hydrazinonicotinyl‐Tyr3‐octreotide) has been shown to be inferior to imaging with PET radiopharmaceuticals.68Ga‐DOTA‐SSA), a dopamine and catecholamines precursor (18F‐FDOPA), a glucose analog (18F‐FDG), or with a norepinephrine analog (11C‐HED).68Ga‐DOTA‐SSA has shown excellent results among patients with PCCs/PGLs. In a systematic review and meta‐analysis, 68Ga‐DOTA‐SSA PET/CT showed a significantly higher detection rate (93%) than 18F‐FDOPA PET/CT (80%), 18F‐FDG PET/CT (74%), or 123/131I‐MIBG scan (38%).The diagnostic performance of imaging with single‐photon agents , 18F‐FDOPA together with 18F‐FDG PET/CT may be used as a second choice alternative to 68Ga‐DOTA‐SSA PET/CT. 18F‐FDG PET/CT has a low specificity but shows a strong diagnostic potential for metastatic PCCs/PGLs, particularly those associated with SDHB mutations.11C‐HED PET/CT demonstrated high sensitivity (96%), specificity (99%), and positive (96%) and negative (99%) predictive values in the diagnostic workup. However, its routine clinical use is limited because of the complex radiopharmaceutical synthesis and short half‐life of 11C.At present, 4GOT2, SLC25A11, DLST, FH, MDH2, IDH1, SDHA, SDHB, SDHC, SDHD4), hypoxia‐sensing pathways , kinase‐signaling pathways , and DNA methylation  have all been associated with susceptibility to PCCs/PGLs.SDHx cluster of genes is associated with high penetrance, early age of onset, intermediate to high risk of metastases, extra‐adrenal location, and poor prognosis.Genetic consultation should be provided, especially in patients ≤45 years with multiple or bilateral lesions. About 25% of PCCs/PGLs are associated with inherited genetic disorders. Germline mutations in genes related to the Krebs cycle . Interestingly, among the cluster 1A/B‐related PCCs/PGLs, there are some differences in primary tumor location.It is worth noting that about 60% of metastatic PCCs/PGLs are associated with cluster 1 mutations .MAX‐related PCCs secrete normetanephrine). These PCCs usually have a better prognosis and low metastatic potential (~4%).In cluster 2, mutations in the MAML3 fusion gene and CSDE1 somatic mutations affect and overactivate the Wnt/β‐catenin pathway, which is responsible for the regulation of metabolism, angiogenesis, proliferation, and invasion.In cluster 3, 5SDHA and SDHB.Despite the fact that most PCCs/PGLs are commonly indolent diseases with nonaggressive behavior, all PCCs/PGLs have a metastatic potential as seen in Table In daily practice, it is difficult to distinguish between benign and aggressive PCCs/PGLs with metastatic potential as the histological features tend to be identical with only minute differences. Similarly, molecular and biochemical markers for metastasis prediction are still limited. Risk stratification systems such as Pheochromocytoma of the Adrenal gland Scaled Score (PASS) and Grading system for Adrenal Pheochromocytoma and Paraganglioma (GAPP) do not reflect the tumor mutational profile, and have an insufficient predictive ability.However, Einsenhofer et al. demonstrated that the plasmatic 3‐methoxytyramine level is 4.7‐fold higher in patients with metastases compared to those without. This biomarker is now considered to be the most accurate discrimination test between localized and metastatic disease.6Despite advancements in systemic treatment, adrenalectomy remains the only curative treatment for locoregional disease. Preoperative administration of α‐adrenergic receptor blockers decreased the postoperative mortality from 40% to 1%–3%.The surgery can be performed as an open procedure  or as a minimally invasive technique (laparoscopic or robotic). The main aims are to avoid catecholamine surges due to mechanical pressure on the tumor during dissection, disruption of the adrenal capsule, and tumor cells dissemination caused by excessive manipulation. With increasing experience, including the application of the principles of “dissecting the patient from the tumor”, the laparoscopic approach was proven to be have similar outcomes in terms of intraoperative hemodynamic instability compared with open resection and even showed less intraoperative blood loss with faster postoperative recovery.Laparoscopic adrenalectomy can be performed via a transabdominal  or retroperitoneal approach. The first one enables earlier ligation of the adrenal vein and a wider working space. However, it requires mobilization of the adjacent organs. The retroperitoneal (posterior retroperitoneoscopic) approach does not include entering the abdominal cavity, which is preferred in patients with suspected adhesions due to previous operations. However, obtaining control during the conversion can be more difficult. The previously recommended “vein first technique”, wherein the adrenal vein is ligated prior to further dissection, does not seem to be necessary anymore.Partial adrenalectomy is reserved only for patients with hereditary syndromes such as in MEN2 or Von Hippel Lindau, in which bilateral adrenalectomy is avoided to prevent steroid dependency.Surgical resection also remains the main treatment for PGLs. While the preoperative medical preparation in functional PGLs is similar to that of PCCs, the risks related to surgery vary according to the localization. Evidence is represented mostly by case series, with no clear guidelines available. Resection of most parasympathetic head and neck PGLs carries the risk of cranial nerve palsy and vascular injury, especially of the carotid artery. Given the rich blood supply, preoperative chemoembolization was suggested to decrease blood loss and operative time. Although this seems to be an efficient method, there is still no consensus on this matter.After resection, adjuvant chemotherapy is not supported by results from clinical trials and is thus not recommended.Open surgery may be also favored if metastatic disease is suspected and in the case of large vessel invasion or regional nodal disease. While surgical debulking in metastatic PCC seemed to be inefficient due to low rates of biochemical response, more recent studies suggest improved overall survival.7Surveillance during the post‐resection period includes regular physical examination, searching for the potential presence of catecholamine‐related symptoms , and biochemical testing (plasma‐free or 24‐h urine fractionated metanephrines). This should be done every 3–12 months during the first year, every 6–12 months in the first 3 years, and then annually up to 10 years after resection. In cluster 1A/B‐ and 3‐related PCCs/PGLs, where the risk of metastatic dissemination is intermediate to high, an MRI from the base of the skull to the pelvis is recommended every 12 months initially, then every 12–24 months. A chest CT is preferred if lung metastases are suspected. In cluster 2‐related PCCs/PGLs, abdominal and pelvic MRI should be performed at least every 5 years.SDHA/B mutations, extra‐adrenal locations, multiplicity, and young age (<20 years).However, the presence of inherited genetic neuroendocrine disorders may modify this scheme.8Approximately, one‐fifth of all patients have a de novo metastatic disease. For unresectable, nonfunctioning tumors, observation is possible, especially if the patient has no tumor‐related symptoms. In this setting, the progression‐free survival (PFS) rate at 1 year is 50%.However, in the metastatic setting, multiple treatment options are available. Cytoreductive surgery, where the goal is to decrease symptoms related to advanced disease and excessive production of catecholamines by chromaffin cells, can be considered. Radiotherapy may follow in order to decrease symptoms. Radiotherapy and radiosurgery are also recommended in a palliative care setting where bone metastases have developed or an oligometastatic disease is present and is associated with pain or neurological symptoms.Currently, radionuclide therapy, chemotherapy, and tyrosine‐kinase inhibitors (TKIs) are the standard care in locally advanced/metastatic diseases. Treatment options for all stages of PCCs/PGLs are illustrated in Figure 8.1131I‐MIBG is dependent upon the expression of NET1, which serves as a target for theranostic agents.131I‐MIBG radionuclide therapy among patients with MIBG‐avid metastatic PCCs/PGLs was demonstrated in multiple trials where complete (CR) or partial responses (PR) were observed in 10% to 34% of patients, whereas the disease control rate (DCR) was observed in up to 92% of treated patients.131I‐MIBG in a single‐arm, multicenter, phase I trial. Twenty‐one patients were enrolled, and the median therapeutic dose was 21.13 GBq. All four patients (19%) who reached a PR received >18.5 GBq. Complete or partial biochemical responses for serum chromogranin A and total metanephrines were observed in 80% and 64% of patients, respectively. Overall survival at 2 years was 61.9%.131I‐MIBG. The median cumulative 131I‐MIBG dose was 35.7 GBq. Seventeen (25%) of them reached a durable reduction of antihypertensive medication. From among 64 evaluable patients, 59 (92%) patients had a PR or a stable disease (SD) as the best response within 12 months. The median overall survival (mOS) was 36.7 months. The most common side effects were nausea, myelosuppression, and fatigue.Uptake of 177Lu‐DOTATATE or treatment with octreotide or lanreotide (in case of symptoms) may be also recommended. There is only limited data available confirming the effect of PRRT in patients with PCCs/PGLs. Preliminary experience suggests low toxicity and favorable efficacy in disease control.131I‐MIBG (14 patients) therapy. PRRT was associated with significantly increased PFS and response to treatment compared with 131I‐MIBG therapy. Toxicity was comparable in both groups. When the subgroup of patients with PGLs was analyzed, OS, PFS, event‐free survival (EFS), and responses to treatment were significantly higher in the PRRT group.If tumors are positive on somatostatin receptor (SSTR) imaging, the peptide receptor radionuclide therapy (PRRT) with 8.22 on day 1, vincristine 1.4 mg/m2 on day 1, and dacarbazine 600 mg/m2 on days 1 and 2, at 21 days intervals). Niemijer et al. analyzed results from four clinical studies concerning a total of 50 patients treated with CVD for metastatic PCCs/PGLs. According to this analysis, the CVD regimen led to a PR or a CR in 46%–55% of patients.Chemotherapy is still a basic option for metastatic disease. It should be considered particularly among tumor subtypes not suitable for radionuclide therapy or in tumors with aggressive growth. Prospective randomized phase II and III trials are not available. Thus, an optimal regimen has not yet been defined. The most studied combination of cytostatics was the Averbuch CVD regimen  was administered. A dose reduction was necessary for all treated patients. The PR was described in eight patients (67%), while CR was confirmed in the next two patients (17%). The median duration of response was 478 days. Median PFS and OS were 930 and 1190 days, respectively. A prolonged therapy resulted in continued incremental tumor reduction. Thus, the CVD regimen may be more effective in the treatment of patients with metastatic PCCs/PGLs with a mutation in the SDHB gene.Tumors with a germline mutation in the 2 on days 1–5, at a 28‐day interval. Metronomic regimen with long‐term and low‐dose temozolomide  is a possible option, which can be considered in case of toxicity during a standard schedule. Hadoux et al. published results from a retrospective analysis of patients with metastatic PCCs/PGLs with SDHB mutation treated by temozolomide monotherapy. Mutation of the SDHB gene was detected in 10 patients (67%). With a median follow‐up of 35 months, mPFS was 13.3 months. PR was observed only in tumors with SDHB mutation and reported in five patients (33%), SD was reported in seven patients (47%), and PD in three patients (20%). The analysis revealed that germline mutation in the SDHB gene was associated with hypermethylation of the O‐6‐methylguanine‐DNA‐methyltrasferase promoter and its low expression. In conclusion, this analysis demonstrates the antitumor activity of temozolomide in patients with PCCs/PGLs and similarly, like the CVD regimen, the efficacy of temozolomide is associated with the presence of SDHB gene mutation.SDHB mutation may serve as a predictive marker for the response to chemotherapy. A summary of chemotherapy regimens used in advanced and metastatic PCCs/PGLs can be found in Table Temozolomide is a DNA‐alkylating agent and oral metabolite of dacarbazine. Its efficacy has been already demonstrated in glioblastomas and neuroendocrine tumors of the gastrointestinal tract. In PCCs/PGLs, temozolomide monotherapy is also effective and it may be considered as a single‐agent treatment, or alternative maintenance treatment after 6–9 cycles of CVD chemotherapy if stabilization or regression was reached. Usual schedule is 150 mg/m8.3TKIs target and inhibit tyrosine kinase enzymes responsible for the activation of proteins by signal transduction cascades inside tumor cells, such as Ras/Raf/MEK/ERK, PI3K/AKT/mTOR, and PLC‐γ/PKC pathways.SDHA, SDHB, and RET genes were detected in 3 patients (13%) and were associated with a PR. No CR was observed in this study. The median time on treatment was 12.4 months and grade 3–4 toxicities were manageable, with the most common being fatigue (16%) and thrombocytopenia (16%). The primary endpoint of the study was DCR, which was met. However, the overall response rate (ORR) in the whole study group was low. The greatest benefit was observed among patients with germline mutations in RET or SDHx genes. The study was closed prematurely due to slow accrual.One of the most investigated TKIs is sunitinib. This oral, small‐molecule multi‐targeted inhibitor of tyrosine kinases blocks the angiogenesis and tumor cell proliferation by targeting multiple receptors, such as the receptors for platelet‐derived growth factors, the receptors for vascular endothelial growth factors (VEGFs) 1 and 2, and RET or KIT receptors. In vitro, sunitinib decreases cell proliferation mostly by targeting the cell cycle, DNA metabolism, and cell organization genes.Cabozantinib is a potent TKI inhibitor that is also under investigation in metastatic settings in phase II trial (NCT02302833). In addition to VEGFRs inhibition, cabozantinib targets the c‐Met receptor. Preliminary results derived from 14 patients have shown ORR of 45% and mPFS of 11.2 months. The side effects of cabozantinib were expectable and similar to those of sunitinib.Pichun et al. conducted a phase II trial with another TKI axitinib. Only nine patients have been enrolled. The primary endpoint was PFS, secondary endpoints were OS, ORR, and safety. The median number of systemic treatments prior to axitinib was 1 (0–3). Three patients achieved a PR, and the next five patients had mild regressions which did not meet PR criteria. Regression has been associated with reductions in plasmatic catecholamines. Only one progression was observed, and grade 3–4 toxicities included hypertension, fatigue, diarrhea, and mucositis. A reduction of the starting dose of axitinib from 5 to 3 mg and 2 mg was necessary for eight patients (89%). Preliminary data of this trial demonstrates that axitinib may result in tumor shrinkage in patients with malignant PCCs/PGLs.Lenvatinib inhibits VEGFRs and fibroblast growth factor receptor pathways and is already approved for the treatment of patients with advanced thyroid cancer, hepatocellular cancer, and in combination with everolimus as a treatment option for clear cell renal cancer. The efficacy of lenvatinib in patients with PCCs/PGLs was evaluated in a prospective phase II trial (NCT03008369), but this study was terminated due to a slow accrual rate. Nelson et al. retrospectively analyzed patients treated with lenvatinib for advanced PCCs/PGLs. Of the 11 evaluated patients, 8 had measurable disease and the DCR was 63% (5 PR and 3 SD).Pazopanib was studied in the phase II trial, where only seven patients with advanced PCCs/PGLs were enrolled. Only one patient had a confirmed PR with a duration of 2.4 years. The treatment of the rest of the patients was discontinued due to disease progression (four patients), withdrawal (one patient), and grade 4 cardiomyopathy (one patient). Median PFS and OS were 6.5 and 14.8 months, respectively. The most common side effects were severe arterial hypertension (50%), diarrhea, hematuria, headache, and fatigue.Everolimus is a mammalian target of rapamycin (mTOR) inhibitor. The efficacy was demonstrated among patients with advanced pancreatic neuroendocrine tumors, renal cancer, and in combination with exemestane in postmenopausal females with hormone‐dependent, HER2‐negative breast cancer. Among patients with PCCs/PGLs, only modest efficacy was demonstrated in the phase II trial.Belzutifan is a second‐generation, small‐molecule HIF‐2α inhibitor with increased potency and an improved pharmacokinetic profile when compared to other HIF‐2α inhibitors, like PT2385 and PT2399.There are other multiple potential targeted therapies, which have already demonstrated efficacy or are currently under study. These include poly(ADP‐ribose) polymerase inhibitors targeting the DNA repair system, SSTR2 analogs (octreotide/lanreotide), inhibitors of Wnt/β‐catenin pathway  for cluster 3‐related tumors, and DNA methyltransferase inhibitors  exhibited no progress at 27 weeks, which was the primary endpoint. The ORR was 9% and the clinical benefit rate was 73%. The other secondary endpoints were PFS and OS, which were 5.7 and 19 months, respectively. Thus, pembrolizumab in monotherapy has presented only modest antitumor activity, regardless of programmed death‐ligand 1 (PD‐L1) expression or hereditary background. No grade 4–5 adverse events were observed.Other checkpoint inhibitors are currently under evaluation in multiple clinical trials for advanced PCCs/PGLs. Programmed cell death inhibiting receptor 1 (PD‐1) inhibitor nivolumab in phase I/II trial (NCT04187404), PD‐L1 inhibitors avelumab in phase I trial (NCT02923466) and atezolizumab in phase II trial (NCT04400474). A combination of nivolumab and cytotoxic T‐lymphocyte antigen 4 (CTLA‐4) inhibitor ipilimumab is currently under study in two phase II trials .968Ga‐DOTA‐SSA PET/CT and 18F‐FDOPA PET/CT, is currently also preferred. Recent research has significantly enriched our knowledge about the molecular background and heterogeneity of these malignancies. Currently, we recognize three different molecular clusters according to the underlying driver mutations. These clusters reflect different genetic backgrounds, tumorigenesis, biochemical, and clinical features of PCCs/PGLs. Apart from the prognostic role of these mutations, they may also become potential predictors for precision and target therapy. Genetic counseling should always be considered due to a relatively high association with inherited genetic disorders.PCCs/PGLs are rare tumors for which clinical symptoms may be attributed most commonly to cardiovascular comorbidities. Similarly, radiological findings may be falsely considered lymphomas or other tumor types. Correct diagnostic workup should be performed without delay. Biochemical tests such as plasma metanephrines, 3‐methoxytyramine, and chromogranin A are useful and recommended when PCCs/PGLs are suspected. Besides CT and MRI scans, functional imaging, such as 131I‐MIBG therapy. Similarly, among patients whose tumors are positive on SSTR imaging, PRRT therapy with 177Lu‐DOTATATE should be considered. Along with recent advances in research, multiple new therapeutic approaches such as targeted therapy are under evaluation and the first results are promising.Whenever it is possible, radical surgery is preferred as it is the only curative option. Cluster 1‐ and 3‐related PCCs/PGLs have intermediate to high metastatic potential and there is a higher risk of recurrence. Moreover, about 20% of patients are diagnosed in an advanced stage, which is usually associated with unpleasant clinical symptoms and a deteriorating quality of life. In the palliative setting, standard chemotherapeutic regimens such as CVD or temozolomide are widely used, but they are not supported by results from randomized phase II/III trials. Patients with MIBG‐avid metastatic PCCs/PGLs benefit from HSA Michal Eid: Conceptualization (lead); data curation ; funding acquisition (lead); resources (lead); writing – original draft (lead). Jakub Foukal: Conceptualization (supporting); data curation ; formal analysis (supporting); validation (supporting); writing – original draft (supporting); writing – review and editing (supporting). Dana Sochorová: Formal analysis (supporting); investigation ; resources (supporting); writing – original draft (supporting); writing – review and editing (supporting). Štěpán Tuček: Methodology (supporting); supervision ; validation ; writing – review and editing . Karel Starý: Supervision (supporting); writing – original draft (supporting); writing – review and editing . Zdeněk Kala: Methodology (supporting); supervision (supporting); writing – review and editing (supporting). Jiří Mayer: Funding acquisition (supporting); methodology (supporting); supervision (supporting). Radim Němeček: Supervision (supporting); writing – original draft (supporting); writing – review and editing (supporting). Jan Trna: Conceptualization (supporting); project administration (supporting); writing – original draft (supporting); writing – review and editing (supporting). Lumír Kunovský: Project administration ; supervision (supporting); writing – review and editing .This work was supported by the Specific University Research (grant no. MUNI/A/1224/2022).The authors declare that they have no conflict of interest.


Real‐world evidence is increasingly used to guide treatment and regulatory decisions for non‐small cell lung cancer (NSCLC). Real‐world treatment patterns and clinical outcomes among patients with advanced/metastatic NSCLC in France, Germany, Italy, Spain, and the UK (EU5) were assessed.This retrospective physician‐completed patient chart review assessed treatment patterns , and clinical outcomes  of patients with stage IIIB/C or IV NSCLC who received pembrolizumab‐based first‐line induction chemotherapy.Overall, 322 patients were included; at first‐line maintenance (1LM), 92% had stage IV NSCLC, 68% had nonsquamous histology, and 89% had no central nervous system (CNS)/brain metastasis. The two most common 1LM regimens were pembrolizumab monotherapy  and pembrolizumab + pemetrexed . Docetaxel monotherapy was the most common second‐line regimen in all countries except Germany . For 1LM therapy, the overall median DOT and DOR were 5 and 10 months, respectively; PFS was 7 months and OS was 8 months. Germany had a longer duration of each outcome except for DOR which was longer in Spain. Clinical outcomes were generally poorer for patients with squamous histology and CNS/brain metastases.This study demonstrated differences in treatment patterns and clinical outcomes in NSCLC across the EU5 and patient subgroups. Improved survival was generally associated with response to first‐line therapy, nonsquamous histology, and CNS/brain metastases absence. These real‐world data provide valuable insights which may aid treatment decision‐making and clinical trial design. This retrospective study provides real‐world treatment patterns and clinical outcomes among patients with advanced/metastatic non‐small cell lung cancer (NSCLC) in France, Germany, Italy, Spain, and the UK (EU5). The most common first line maintenance regimen was pembrolizumab monotherapy, pembrolizumab + pemetrexed was the second most‐common. This study also highlights differences in treatment patterns and clinical outcomes across the EU5, providing insight into real‐world clinical practice which may aid future treatment decision‐making and clinical trial design. Non‐small cell lung cancer (NSCLC) treatment has advanced greatly over the past two decades due to treatments targeting driver alterations in genes including anaplastic lymphoma kinase (ALK), B‐Raf (BRAF), and epidermal growth factor receptor (EGFR).The objective of this study was to analyze real‐world data using retrospective patient chart review to understand demographics, clinical characteristics, treatment patterns, and clinical outcomes for patients with advanced/metastatic NSCLC receiving 1LM therapy in France, Germany, Italy, Spain, and the UK (termed EU5 henceforth).This descriptive retrospective patient chart review – based on a standardized, online physician‐completed case report form (CRF) – captured anonymous patient data and treatment history since diagnosis of NSCLC. Questions on cross‐sectional CRFs were completed by a panel of physicians for each country from July–August, 2021. Index date was the start of 1LM therapy. Treatment history for both living and deceased patients was captured.Physicians were recruited from a proprietary internal IQVIA database of healthcare providers. Screening was based on experience, workload, and management of patients with NSCLC. Physicians included clinical/medical and surgical oncologists, drug‐prescribing radiation oncologists, pulmonologists, and thoracic surgeons, who were required to have personally treated a patient with advanced/metastatic NSCLC on 1LM therapy ≤12‐months prior to or at time of data extraction. A maximum of two physicians from the same center and one physician from the same hospital, per country, were permitted to participate in CRF collection, to avoid cluster effects and patient duplication.Eligible patients were ≥18 years old at index; had histologically/cytologically confirmed diagnosis of squamous, nonsquamous, or mixed‐histology NSCLC  between January 1, 2018 and December 31, 2020; completed 4–6 cycles of platinum‐based 1L induction chemotherapy with pembrolizumab , duration of treatment (DOT), proportion of patients with discontinuation, time to discontinuation (TTD), reasons for discontinuation, and real‐world time to next treatment (rwTTNT). Clinical outcomes included progression‐free survival (PFS), time to CNS/brain progression and overall survival (OS) from start of 1LM as well as objective response rate , and duration of response (DOR) after completion of 1L induction. Exploratory clinical outcomes were time to CNS/brain progression from start of 1L induction therapy and time to death from start of 1LM therapy. Timelines for the assessment of outcomes are depicted in Figure Data collection complied with all applicable laws and regulations, including GDPR , as well as applicable codes and guidelines.All analyses were performed using RStudio version 3.6.2 statistical software. Descriptive statistics were used to report patient demographics and clinical characteristics. For subgroup analyses, patients were stratified into subgroups based on: country, histology (squamous vs. nonsquamous vs. mixed histology), disease stage (advanced vs. metastatic), PD‐L1 status (tumor proportion score [TPS]‐negative [<1%] vs. ≥1% and/or 1–49% vs. ≥50%), best response to induction chemotherapy (CR/PR vs. SD), pemetrexed use in 1LM therapy , and asymptomatic CNS/brain metastases at the start of 1LM therapy (yes vs. no). Where applicable, N (%) of patients with missing data were included in the “unknown/missing” response option. No imputation methods were used to handle missing data. Continuous variables were evaluated to determine normality and to identify outliers. Time to event analyses  were reported using Kaplan–Meier survival methods and analyzed by log‐rank test. Censored events included death and end of follow‐up. Patients were censored at the earliest of these events if they occurred before observation of the outcome of interest.n = 61; Germany, n = 60; Italy, n = 73; Spain, n = 67; and UK, n = 61)  tumors were of nonsquamous histology, with the largest proportion observed in Spain (82%). Patients with squamous histology were almost equally likely to be smokers (48%) or ex‐smokers (46%), while patients with nonsquamous histology were more likely to be ex‐smokers ; carboplatin + pembrolizumab + paclitaxel , and cisplatin + pembrolizumab + pemetrexed  Figure . PatientPatients with a PD‐L1 TPS ≥50% were more commonly treated with carboplatin + pembrolizumab + pemetrexed (57%) than carboplatin + pembrolizumab + paclitaxel (32%); however, these two regimens were relatively balanced across patients with a TPS <1%  and those with a TPS 1–49%  and pembrolizumab + pemetrexed . Among those patients, docetaxel monotherapy was the most used treatment (54%)  Figure .Only two patients progressed to 3L therapy Tables  and S4, p < 0.0001) as well as between patients who did not have asymptomatic CNS/brain metastases at index versus those who did or whose status was unknown  and between patients who did not have asymptomatic CNS/brain metastases at index date versus those who did, or those for whom status was unknown  had significantly shorter (p = 0.002) median time to CNS/brain progression compared with those without CNS/brain metastases at index . Patients with mixed histology had a nonsignificant shorter time to progression than those with squamous or nonsquamous histology . Time to CNS/brain progression was significantly longer (p < 0.0001) in patients who achieved CR/PR to 1L induction therapy versus those who achieved SD .The overall median time to CNS/brain progression was 11 months from start of 1LM Figure . Patientp < 0.0001) in patients without asymptomatic CNS/brain metastases at index (11 months [CI: 9–12]) than those with metastases (1 month [CI: 0.5–1.0]) and those with unknown status (5 months [CI: 4–NA]).The ORR in the EU5 dropped from 73% in 1L induction therapy to 20% in 1LM therapy, with 2%, 18%, and 32% of patients receiving 1LM achieving CR, PR, and SD, respectively Figure . The medp = 0.28). Median PFS was similar between patients with a PD‐L1 TPS ≥50% (6 months) and those with a PD‐L1 TPS of 1%–49% or <1% (7 months). Median PFS was significantly (p < 0.0001) longer for patients who achieved a PR/CR to 1L induction than for those with SD  and was also significantly longer for patients who did not have CNS/brain metastases at index than those who did .The overall median PFS in the EU5 was 7 months, ranging from 5 months (France) to 10 months . No significant difference in OS was observed in patients stratified by PD‐L1 status. OS was significantly longer in patients who achieved a PR or CR to 1L induction therapy than those who achieved SD .The median OS was 8 months in the EU5, ranging from 5 months (France) to 9 months . Patients with squamous or mixed histology had a nonsignificant shorter time to death versus those with nonsquamous histology . The overall time to CNS/brain progression from the start of 1L induction therapy was 16 months (CI: 14–39).This was a retrospective study of treatment patterns and clinical outcomes in patients with advanced/metastatic NSCLC without known driver alterations who had received pembrolizumab as part of 1L induction and 1LM therapy in the EU5. This patient population is of interest given the challenge of treating patients without targetable gene alterations.Overall, carboplatin + pembrolizumab + pemetrexed was the most commonly used 1L regimen; however, this may have been skewed by high usages in Germany and Spain when compared with the other three countries. This is similar to retrospective data (August 2018–December 2019) from the US Flatiron Health oncology database which showed that 75% of patients with metastatic NSCLC and no EGFR or ALK mutations received immunotherapy as 1L treatment, with carboplatin + pembrolizumab + pemetrexed and carboplatin + pembrolizumab + paclitaxel being the two most common 1L regimens.In this study, 1LM therapy was consistent across all 5 countries, with pembrolizumab monotherapy the most used regimen regardless of tumor histology or PD‐L1 status, and docetaxel monotherapy the most common 2L regimen in all but Germany, which had equal usage of docetaxel + nintedanib. Since the introduction of ICIs in 2018, there have been limited real‐world studies of 1LM treatment patterns in advanced/metastatic NSCLC; however, increased adoption of pembrolizumab‐based regimens and decreased use of pemetrexed‐based regimens, regardless of 1L therapy, has been reported,p = 0.008), while median PFS in patients with 1%–49% or >50% PD‐L1 TPS was similar between those who received monotherapy and combination therapy.The overall median PFS on 1LM was 7 months. The median PFS was higher in patients with nonsquamous than squamous histology and was similar to that reported in the KEYNOTE‐189 and KEYNOTE‐407 trials, which had similar populations but only excluded ALK and EGFR driver alterations.Across the EU5, ~57% of patients were recorded to have discontinued 1LM therapy, death, CNS/brain progression/relapse, and local progression/relapse the most common reasons. This is supported by a real‐world study on patients with NSCLC receiving 1L or 1LM therapy, where the main reason for discontinuation was tumor progression.The patient chart review in this study provided a comprehensive treatment history from the time of NSCLC diagnosis. All participating physicians were experienced with the management of NSCLC and had been treating patients with advanced/metastatic NSCLC, receiving 1LM, within 12 months of data extraction. The study design enabled the acquisition of a large amount of data on multiple clinical outcomes, variables, and study‐specific patient populations and subgroups, making it available to physicians and researchers in a shorter time frame than a clinical trial. Trends identified in the data may help guide treatment decisions for various patient populations.Some limitations exist, however, and while the inclusion of both alive and deceased patients allowed analysis of the clinical outcomes of OS and time to death, it may have introduced some bias, including a higher probability of capturing patients with more aggressive disease. However, the quota for the number of deceased patients was conservative and could have led to underestimations of the true proportion of deceased patients. PD‐L1 status would likely have influenced treatment decision‐making; however, sensitivity analyses were not conducted to control for this. Furthermore, certain analyses were limited by the low sample size; for example, as only two patients receiving 3L therapy were included in the study, it was not possible to identify treatment patterns in this subgroup. Additional study limitations include the potential for reporting bias given data was provided by eligible physicians who chose to participate in the study only, and that the study sample may not be representative of the advanced/metastatic NSCLC population of each country, in addition to the inherent limitations that come with retrospective studies, such as the handling of missing data.In conclusion, this study provides detailed real‐world data on treatment patterns and clinical outcomes in patients with advanced/metastatic NSCLC in the EU5 and demonstrates notable differences across countries and patient subgroups. While 1L and 2L regimens differed between the five countries, pembrolizumab monotherapy was the most used 1LM regimen across the EU5. Improved survival outcomes were generally associated with response to 1L therapy, nonsquamous histology, and the absence of CNS/brain metastases; however, survival rates generally remain poor. This valuable insight into real‐world clinical practice highlights the need for efficacious treatments in this patient population and may aid future treatment decision‐making and clinical trial design.Conceptualization, Amine Aziez, Alexander Stojadinovic, Chi‐Chang Chen, and Fil Manuguid; Formal analysis, Amine Aziez, Alexander Slowley, Alexander Stojadinovic, Chi‐Chang Chen, Fil Manuguid, Jessica Chao, Jasjit K. Multani, Kelly F. Bell, Kelesitse Phiri, Marie Yasuda, Sheila Mpima, and Vicky Casey; Investigation, Chi‐Chang Chen, Fil Manuguid, Jasjit K. Multani, Marie Yasuda, Sheila Mpima, and Vicky Casey; Methodology, Amine Aziez, Alexander Stojadinovic, Chi‐Chang Chen, Fil Manuguid, Jessica Chao, Jasjit K. Multani, Marie Yasuda, Sheila Mpima, and Vicky Casey. Writing – original draft, all authors. Writing – Review and editing, all authors. All authors had access to the data in the study and take responsibility for the integrity of the data and the accuracy of the analysis.This study was conducted by IQVIA with funding support from GSK (GSK study 214794).AA and KP are employees of GSK. ASl, ASt, JC, and KFB are employees of, and hold stocks, in GSK. JKM, VC, SM, MY, CCC, and FM are employees of IQVIA. IQVIA was contracted by GSK to conduct this study.Figure S1. Timing of study measures for eligible patients.Click here for additional data file.Figure S2. Treatment regimens used in (a) 1LM by histology, (b) 2L therapy by histology, (c) 1LM by PD‐L1 TPS and (d) 2L therapy by PD‐L1 TPS.Click here for additional data file.Table S1. Patient characteristics by histology and PD‐L1 TPS.Table S2. Physician characteristics.Table S3. Individual treatments and treatment regimens per LOT, in the EU5 and by country.Table S4. Treatment regimens per LOT by histology and PD‐L1 TPS.Table S5. DOT, TTD, and rwTTNT for each LOT, in the EU5 and by country.Table S6. DOT, TTD, and rwTTNT for each LOT by response to 1L induction and by CNS/brain metastases status (at index date).Table S7. DOT, TTD, and rwTTNT for each LOT by histology and PD‐L1 TPS.Table S8. DOT, TTD, and rwTTNT for each LOT in patients with non‐squamous histology, by use of pemetrexed in 1LM regimen.Table S9. Reasons for LOT discontinuation in EU5 and by country.Table S10. Reasons for LOT discontinuation by histology and PD‐L1 TPS.Click here for additional data file.


To determine the risk factors for cesarean section (CS) and adverse fetal outcomes (AFOs) in patients with intrahepatic cholestasis of pregnancy (ICP) based on the severity of maternal hypercholanemia.μmol/L < total bile acids < 40 μmol/L) and severe (≥40 μmol/L). The patients' clinical characteristics and fetal outcomes were assessed.A hospital-based retrospective cohort study was performed between January 1, 2015, and December 31, 2019. A total of 227 nulliparous women with a singleton fetus complicated by ICP were included. The patients were divided into two groups according to the levels of total bile acids, that is, mild (10 p < 0.001). The indications for CS showed that fetal intolerance (65.4% vs. 14.3%) was higher in severe ICP compared with mild ICP (p < 0.001). Severe ICP was associated with an increased risk of preterm delivery (p < 0.001), low birthweight (p = 0.001), and neonatal intensive care unit (NICU) admission (p < 0.001). Women with severe ICP  or preeclampsia  had increased risks of AFOs compared to controls.Among the 227 eligible women, 177 (78.0%) were allocated to the mild group and 50 (22.0%) were in the severe group. Women with severe ICP also had a significantly higher incidence of planned and unplanned CS compared with mild ICP subjects (52.0% vs. 23.7% and 22.0% vs. 6.8%, respectively; Severe ICP and preeclampsia are associated with a higher incidence of AFOs. Intrahepatic cholestasis of pregnancy (ICP) is characterized by unexplained maternal pruritus, raised serum bile acids >10 μmol/L) and/or elevated liver transaminases (ALT > 35 U/L and/or AST > 35 U/L) during the late second and third trimester of pregnancy 0 μmol/L .Although ICP was thought to be a relatively benign condition, recent studies have indicated that ICP has adverse effects for both the mother and fetus, including a notably higher risk of developing preeclampsia and gestational diabetes mellitus (GDM) , 6. Relaμmol/L and <40 μmol/L are considered mild ICP, while severe ICP is defined as maternal total bile acid levels ≥40 μmol/L, which is associated with higher maternal and neonatal complications  between women with severe ICP compared to women with mild ICP.The data for the retrospective cohort study were obtained from January 1, 2015, to December 31, 2019 at the Changzhou Maternal and Child Health Care Hospital in Jiangsu (China), where 10,000–12,000 deliveries per year are performed. Information on maternal demographics, medical comorbidities, and AFOs was obtained from medical records for evaluation. Women with fasting serum bile acid levels of more than 10 μmol/L and pruritus of unexplained cause were included in the study. Since multiparas and women with twin pregnancies might influence the mode of delivery directly, only nulliparous women with a singleton fetus were included in the study. Women were excluded if they had viral or autoimmune hepatitis, liver or biliary disease, and fetal chromosomal or structural abnormalities. The study protocol was approved by the Ethical Committee of the Hospital.μmol/L < total bile acids < 40 μmol/L) or severe (≥40 μmol/L).The patients were screened for ICP in pregnancy utilizing ICD- 10 code (O26.6). The women received ursodeoxycholic acid (UDCA) therapy, and fetal monitoring was performed more than once per day before delivery, except in cases where delivery occurred before ICP diagnosis. For the primary analysis, ICP severity was defined according to maternal total bile acids levels as mild , PROM + anatomic abnormality, and PROM + MSAF.2χ or Fisher's exact test was used to analyze categorical variables. A logistic regression analyse was also performed to assess the risks of AFOs. Our data met the calculated sample size for AFOs which was from 17 to 757 in each group for 80% and 90% power, and a 95% confidence interval. Statistical significance was defined as p-values < 0.05, and the data were analyzed using the statistical software package SPSS 22.0.A two-tailed t-test was used to analyze continuous variables, and the Two hundred twenty-seven pregnancies (177 mild and 50 severe ICP) met the inclusion criteria. The demographic and obstetric characteristics of the patients in the mild group and severe group are presented in p < 0.001, p < 0.001). The indications for planned/unplanned CS in women with mild or severe ICP are shown in p < 0.001). However, the rate of the indications for unplanned CS was not significantly different between the two groups (p > 0.05).There were significant differences in the mode of delivery found between women with severe ICP compared to those with mild ICP (p < 0.001) and low birth weight  compared to those with mild ICP, p < 0.001) and total AFOs  between mild ICP and severe ICP groups was significantly lower  using a logistic regression model (p < 0.001, We discovered a strong association between severe ICP and AFOs (OR 6.397, 95%CI 3.041–13.455, on model . Among GThe present data indicate that women with severe ICP have a greater proportion of CS, including planned and unplanned CS, and a higher incidence of AFOs, compared to women with mild ICP. Percentage of 51.1 more women with severe ICP had fetal intolerance of labor compared to women with mild ICP in planned CS. Preeclampisa might lead to an increase risk of AFOs both in mild and severe ICP.Tetsuya Kawakita et al. reported that the rate of CS in women with mild or severe ICP was less than 40% . In our μmol/L (Although there are no randomized studies investigating the optimal timing of delivery for women with ICP, a majority of researchers have advocated the implementation of elective early delivery for ICP, especially for those patients with total bile acids levels ≥40 μmol/L . Most spRecent studies have reported that an elevated total bile acids level may cause preeclampsia or GDM , 6. PreePrevious studies have reported that high bile acid levels could cause MSAF , 15. MorAs reported in previous studies, severe ICP as well as preeclampsia are risk factors of AFOs , 17, incThe present retrospective study has several limitations. Although the patients in our study were treated with UDCA, except for those who delivered before ICP diagnosis, the lack of data regarding the specific time and dose of UDCA use limited our ability to analyze the effect of UDCA on total bile acids levels. A previous study indicated that UDCA treatment appeared to improve the prognosis of both women with ICP and the fetus . HoweverThe present retrospective study demonstrates a significantly increased incidence of CS, including planned and unplanned, in women with severe ICP. Severe ICP and preeclampsia were associated with AFOs, suggesting that further prospective studies are warranted to identify the proper treatment to improve fetal outcome.


Hypothesis: To evaluate the safety and efficiency of replacing femoral vein pacing with atrial spiral pacing in the right ventricle for temporary cardiac pacing (TCP) during left bundle branch pacemaker (LBBP) implantation.Left bundle branch pacemakers (LBBPs) can better maintain ventricular electrical synchronization than traditional right ventricular pacing (RVP). Temporary cardiac pacing (TCP) is needed to ensure the safety of the operation in patients undergoing LBBP. Currently, there are two methods of installing TCP in conventional permanent pacemaker implantation. n = 76) and the femoral vein electrode group (n = 103). The following were observed: operation time; radiation dose; radiation time; operation expenses; hospitalization time; pacemaker parameters immediately after the operation and at 1 week, 1 month, 3 months, and 6 months after the operation; operation complications and femoral vein puncture point complications were observed in the two groups.A total of 179 patients who underwent TCP during LBBP were selected for retrospective analysis from April 2019 to 2021 and divided into two groups: the atrial spiral electrode group (p < 0.05), radiation doses , and decreased risk of a displaced or dislodged temporary pacing electrode during the operation . The atrial electrode group did not have significant operation costs or material costs associated with femoral vein temporary pacing electrode implantation. In addition, the atrial electrode group did not have an increased risk of pacemaker‐related infections, and the parameters of the pacemaker were unaffected. However, some puncture point complications appeared in the femoral vein electrode group .Compared to the femoral vein electrode group, the atrial electrode group had significantly lower operation times  electrodes were placed into the right ventricular apex through the inferior vena cava, right atrium, and tricuspid valve. Then, the end of the electrode was connected to the program‐controlled instrument to test the parameters satisfiedly for TCP.(a) Ventricular spiral pacing electrode. (b) Femoral vein temporary pacing electrode. (c) Atrial spiral pacing electrode. Patients who underwent TCP during LBBP was divided into an atrial electrode group and femoral vein electrode group. Five surgeons from the two centers who performed, on average, 100 independent pacemaker implantations, performed the TCP and LBBP procedures.The exclusion criteria were as follows: allergy to contrast agents, acute HF, cardiac shock, pulmonary edema, electrolyte disorder, fever and infectious diseases, malignant tumor, acute myocarditis, autoimmune disease, blood coagulation disorders, and severe liver and kidney dysfunction.2.2All methods were carried out in accordance with relevant guidelines and all experimental protocols were approved by by Ningbo Hospital of Zhejiang University (2022RS001). All patients or their family members were informed of the risks associated with TCP and LBBP and signed informed consent forms to undergo the procedures.2.2.1All patients discontinued aspirin, clopidogrel, warfarin, and traditional Chinese medicine 1 week before pacemaker implantation. First‐generation cephalosporin antibiotics were administered to prevent infection 24 h preoperatively and postoperatively. Routine blood counts, electrolyte levels, liver and kidney function tests, blood coagulation function indices, electrocardiograms, echocardiograms, chest computed tomography, and abdominal ultrasounds were examined before the operation.2.2.2atrial active electrode: 5076‐52 (Medtronic), sheath pipe: C315 (Medtronic), ventricular LBBP electrode: 3830 (Medtronic), femoral vein temporary pacemaker electrode: PACEL dominate (St. Jude), the permanent pacemaker models: A3DR01 (Medtronic), SEDRL1 (Medtronic), and RED01 (Medtronic).2.2.3After femoral vein puncture, TCP electrodes were placed into the right ventricular apex through the inferior vena cava, right atrium, and tricuspid valve. Then, the end of the electrode was connected to the program‐controlled instrument to test the parameters satisfiedly for TCP  were inserted through the subclavian vein or axillary vein to a place near the tricuspid valve opening. Then, the ventricular lead  was fed through the C315 sheath pipe. The electrode found the suitable position as a target reference of the tricuspid ring in the right front 30° oblique position under exposure. Then, the electrode distal end stuck out of the sheath pipe near the interval of the tricuspid valve with 2 V pacing. When the incisures appeared in the rising QS wave on the V1 lead, electrode screwing was performed at this site. Once the left bundle branch proved to be capturing the pacing and adjusting parameters satisfactorily, the electrode was fixed clockwise and then placed at the corresponding right atrium location, connected to the pacemaker after adjustment parameters, closing the wound in the end.2.3The primary endpoints of this study were as follows: operation time, radiation dose, radiation time, operation expenses, hospitalization time, complications , and femoral vein puncture point complications .2.4t‐tests were used for comparisons between groups. Classification variables were expressed by frequency or percentage using the χ2 test or Fisher's exact test. p < 0.05 indicates that the difference was statistically significant.SPSS version 22.0 (IBM) was used to statistically analyze the data, and continuous variables are expressed as the mean ± SD. Independent sample 33.1p > 0.05). The patient base data for the two groups are shown in Table One hundred seventy‐nine patients were enrolled; of them, 98 patients were male, 82 patients were female, the mean age was 68.66 ± 12.297 years old, 84 patients had hypertension, 66 patients had hyperlipidemia, 67 patients had a history of diabetes, 16 patients had a history of stroke, and 16 patients had coronary heart disease. There was no significant difference between the two groups in age, sex, body mass index, medical history, pacemaker indications, pacemaker model, heart ejection fraction, creatinine, hemoglobin, or absorbable line used (3.2p < 0.05). TCP was initiated in 33 (43.42%) patients in the atrial spiral electrode group. Meanwhile, TCP was initiated in 42 (40.78%) patients in the femoral vein pacing electrode group. In the atrial electrode group, the X‐ray exposure dose (805.07 ± 132.94) mgy was lower than that in the femoral vein (846.42 ± 87.37) mgy electrode group, and the difference was statistically significant (p < 0.05). Regarding the operation cost, the atrial electrode group did not undergo femoral vein temporary pacing electrode implantation surgery, and surgical consumables cost 2200 Yuan (RMB). The X‐ray exposure time in the two groups was not significantly different (14.76 ± 4.63) versus (15.30 ± 3.29) min (p > 0.05). Regarding surgical complications, there was no temporary pacing electrode dislodgement in the atrial electrode group, while the femoral vein electrode groups had 5 cases of electrode dislodgement, accounting for 4.85%, and the difference was statistically significant (p < 0.05). Regarding other complications, the two groups had no permanent pacing electrode dislodgement. The atrial electrode group had no pneumothorax, but 2 cases of pneumothorax occurred in the femoral vein electrode group, accounting for 1.94%, and there was no significant difference (p > 0.05). Hemothorax did not occur in the atrial electrode group, but 2 cases occurred in the femoral vein electrode group (1.94%), and there was no significant difference (p > 0.05). There were no cardiac perforations or pericardial tamponades in the two groups. The atrial electrode group had 3 cases of pacemaker pouch hematoma, accounting for 3.95%, and 2 cases of pacemaker pouch hematoma occurred in the femoral vein electrode group, accounting for 1.94%; there was no significant difference (p > 0.05). Eight cases of pacemaker‐related infection occurred in the atrial electrode group, accounting for 10.53%, and 12 cases occurred in the femoral vein electrode group, accounting for 11.65%; there was no significant difference (p > 0.05). Regarding complications at the femoral vein puncture point, the femoral vein electrode group had an ecchymoma in 8 cases (7.77%), arteriovenous fistula in 3 cases (2.91%), pseudoaneurysms in 3 cases (2.91%), and no retroperitoneal hematoma; moreover, there were no puncture complications in the atrial electrode group. There were no significant differences in hospitalization time between the two groups (7.27 ± 1.72 vs. 7.39 ± 1.68 days) (p > 0.05). The procedural characteristics in the two groups are shown in Table Both groups of patients were successfully implanted with pacing electrodes and pacemakers. The atrial electrode group had an operation time of 116.86 ± 24.63 min, which was less than that of the femoral vein electrode group (128.94 ± 25.27 min), and the difference was statistically significant  For patients with sick sinus syndrome with normal atrioventricular conduction, atrial sensing, and pacing are completely in line with physiology and do not need LBBP. For patients with permanent AF, only single chamber pacing is needed, and atrial electrode implantation is not needed. Therefore, these two types of patients were not included in this study, so there may be selection bias. (2) This study is a retrospective study; thus, the conclusion may have selection bias. Finally, this study is a two‐center clinical study, the included sample size was large, and the follow‐up time was long. More cases are needed to further verify the conclusion.Zhenwei Li, Jia Su, Qingqing Xu, and Ning Huangfu wrote the main manus cript text. Zewei Sun prepared Figures The authors declare no conflict of interest.


Unstable interpersonal relationships and fear of abandonment are core symptoms of borderline personality disorder (BPD) that often intensify during stress. Psychosocial stress, which includes components of social exclusion and increases cortisol secretion, enhances emotional empathy in healthy individuals. Women with BPD, on the contrary, react with reduced emotional empathy. The aim of the present study was to investigate the effects of perceived social exclusion without accompanying cortisol increase on empathy in women with BPD and healthy women. To induce social exclusion, we randomized 98 women with BPD and 98 healthy women to either an exclusion or an overinclusion (control) condition of Cyberball, a virtual ball game. Subsequently, participants underwent the Multifaceted Empathy Test (MET), which assesses cognitive and emotional empathy. There was no increase in cortisol release after Cyberball. Cognitive empathy did not differ between groups or conditions. Women with BPD reported lower emotional empathy for positive emotions , but not for negative emotions. Exploratory analyses suggested that this effect might be more pronounced after social exclusion. Our results confirm previous findings that cognitive empathy does not differ between women with BPD and healthy women and extend this evidence to social exclusion. Emotional empathy in women with BPD seems to be more sensitive to the effects of stress or ambiguous social situations. Specifically, emotional empathy seems to be reduced for positive emotions, and might further decline after social exclusion. Empathic reactions to emotional stimuli of different valences and to specific emotions should be further investigated.The online version contains supplementary material available at 10.1007/s00406-022-01535-0. Unstable interpersonal relationships and fear of abandonment are among the defining and most debilitating symptoms of borderline personality disorder BPD; , 24). Re. Re24]).Many of the disorder’s core symptoms typically occur in interpersonal contexts  and worsIn line with results of other studies showing enhanced prosocial behavior , 45, 47 In another study, pharmacological stimulation of the mineralocorticoid receptor (MR), one of the cortisol receptors in the brain, resulted in increased emotional empathy in healthy controls (HC) but also in patients with BPD . ResultsAn experimental task frequently used to study social exclusion is the Cyberball paradigm , a virtuWhile one of the TSST’s main purposes is to induce a cortisol increase, most studies have shown no cortisol increase after Cyberball e.g., , 21, 40), 4021, 4The aim of the present study was to investigate why patients with BPD differ in their emotional empathy response to psychosocial stress from healthy controls. In our model, we assume that the inhibition of prosocial behavior seen in patients with BPD is related to a feeling of social exclusion. Therefore, we used Cyberball to induce social exclusion without confounding of a cortisol increase. Subsequently, participants underwent the Multifaceted Empathy Test (MET) as a measure of cognitive and emotional empathy.We expected no changes in cortisol or alpha-amylase release after the Cyberball game in women with BPD and healthy women. We hypothesized that women with BPD and HC would not differ in cognitive and emotional empathy in the control condition (overinclusion). In contrast, we expected that social exclusion would result in enhanced emotional empathy in HC and reduced emotional empathy in patients with BPD.The sample consisted of 98 women with BPD and 98 healthy women, who were matched for age, education, intake of hormonal contraception and menstrual cycle. Women between the ages of 18 and 50 with a BMI between 17.5 and 30 were included. All participants underwent the Structural Clinical Interview for DSM-5 Disorders (SCID)  to asseAt the second session, participants played Cyberball and subsequently performed the Multifaceted Empathy Test  and anoParticipants were randomized to an exclusion or an overinclusion condition of the Cyberball paradigm , a virtuBefore the game, participants read the instructions, stating that they should imagine the game as if it was happening in real life with real people. It also stated that they would be tossing a ball with two real co-players via an internet connection (“cover story”), while in fact these co-players were computer-generated. After completing the whole experiment, all participants were debriefed.We assessed cognitive and emotional empathy with a modified version of the Multifaceted Empathy Test MET; , 52). Th. Th52]).not at all, 9 = very much). We calculated mean scores for overall emotional empathy, emotional empathy for negative emotions and emotional empathy for positive emotions ). Both groups did not differ in empathy for negative emotions (t(194) = 0.80, p = 0.424). Results are shown in Fig. In the analysis of emotional empathy, we found a significant main effect for group,  = 0.13, p = 0.715, η2 = 0.00), but a valence × condition × group interaction effect at trend level  = 3.30, p = 0.071, η2 = 0.02). For exploratory purposes, we calculated post hoc t-tests. Women with BPD reported less empathy for positive emotions in the exclusion condition than in the overinclusion condition, (t(96) = 2.26, p = 0.026, d = 0.46, 95% CI ). In healthy controls, empathy for positive emotions did not differ between conditions, (t(96) = − 0.59, p = 0.560, d = − 0.12, 95% CI ).There was no valence × condition interaction effect  = 2.42, p = 0.122, η2 = 0.01). We found an effect of emotional valence  = 14.40, p < 0.001, η2 = 0.07), an interaction of valence × mood  = 10.52, p = 0.001, η2 = 0.05) and an interaction of group × valence  = 4.37, p = 0.038, η2 = 0.02). There was a valence × group × condition interaction effect at trend level  = 3.37, p = 0.068, η2 = 0.02).To account for the possible influence of mood on emotional empathy, we included baseline mood on the MDMQ good vs. bad scale as a covariate into our main analysis. We did not find a main effect for group anymore, , but not to negative stimuli. This was also significant after including current mood as a covariate into the analysis. In previous studies, our group did not find reduced emotional empathy in women with BPD in the control conditions, but valence was not taken into account , 53. DomThe finding that women with BPD scored lower on emotional empathy in both conditions might be due to a biased perception of ambiguous social situations. In our Cyberball game, co-players were represented by pictograms rather than real pictures, which prevented participants from reading facial expressions or emotions. There is evidence that patients with BPD anticipate social rejection or threat in such ambiguous social situations . AdditioWomen with BPD and HC did not differ in basal cortisol or sAA levels. This matches findings of previous studies that also report no differences between patients with BPD and HC using single rather than continuous measurements of cortisol or sAA , 17. As Based on results after psychosocial stress (TSST) , we had We tested effects of social exclusion in an artificial laboratory setting, which was one of the study’s main limitations. There was a high proportion of patients with comorbid diagnoses and intake of psychotropic medication, however, subgroup analyses yielded no effects on empathy. Patients with depression were excluded from the study, as HPA-axis function and cognition differ between patients with BPD with and without a depressive episode , 54. As We conclude that women with BPD do not differ from healthy women in cognitive empathy and that this is not influenced by stress in general and social exclusion in particular. Emotional empathy, on the other hand, seems to be more sensitive to the effects of stress or ambiguous social situations in women with BPD. Specifically, emotional empathy seems to be reduced for positive emotions, and might further decline after social exclusion. Given these results, empathic reactions to emotional stimuli of different valences and to specific emotions should be further investigated. Future studies investigating the effects of social exclusion should use ecologically more valid Cyberball paradigms for example with virtual reality or a partial exclusion manipulation to increase believability. Next to social exclusion, other components of the TSST, such as social evaluation, one of the TSST’s most stressful components , should Supplementary file1 (DOCX 43 KB)Below is the link to the electronic supplementary material.


To investigate the effect and mechanism of polydatin on bleomycin (BLM)‐induced pulmonary fibrosis in a mouse model. The lung fibrosis model was induced by BLM. The contents of TNF‐α, LPS, IL‐6 and IL‐1β in lung tissue, intestine and serum were detected by ELISA. Gut microbiota diversity was detected by 16S rDNA sequencing; R language was used to analyse species composition, α‐diversity, β‐diversity, species differences and marker species. Mice were fed drinking water mixed with four antibiotics  to build a mouse model of ABx‐induced bacterial depletion; and faecal microbiota from different groups were transplanted into BLM‐treated or untreated ABx mice. The histopathological changes and collagen I and α‐SMA expression were determined. Polydatin effectively reduced the degree of fibrosis in a BLM‐induced pulmonary fibrosis mouse model; BLM and/or polydatin affected the abundance of the dominant gut microbiota in mice. Moreover, faecal microbiota transplantation (FMT) from polydatin‐treated BLM mice effectively alleviated lung fibrosis in BLM‐treated ABx mice compared with FMT from BLM mice. Polydatin can reduce fibrosis and inflammation in a BLM‐induced mouse pulmonary fibrosis model. The alteration of gut microbiota by polydatin may be involved in the therapeutic effect. All mice were housed in specific pathogen‐free conditions in an animal facility with controlled humidity (50%–60%) and temperature (22–24°C) and under a 12 h light–dark cycle (7 AM to 7 PM) with free access to food and drink. All procedures were sanctioned by the ethics committee of Zhejiang Chinese Medical University.2.2The pulmonary fibrosis model was established by intratracheal BLM instillation in mice. Briefly, the mice were anaesthetised by intraperitoneal injection of pentobarbital (50 mg/kg). Anaesthetised mice were placed in a supine position and fixed on a sterile operating table, with the head high and the feet low and the neck skin fully exposed. Ophthalmic scissors were used to cut the neck skin approximately 2 cm along the midline after routine disinfection of the anterior neck area. The trachea of mice was fully exposed after blunt dissection, the trachea was punctured by bending the 1 mL syringe needle at an obtuse angle, the needle was inserted approximately 1.5 cm along the wall of the trachea, and the prepared BLM (5 mg/kg [5000 IU/kg] according to the body weight of micen = 6) with different tracheal instillation solutions and different follow‐up treatments: (1) Control group: intratracheal administration and intragastric administration of normal saline; (2) BLM group: intratracheal administration of BLM as above mentioned and intragastric administration of normal saline; (3) Control + polydatin group: intratracheal administration of normal saline and intragastric administration of polydatin ‐induced bacterial depletion was developed. Mice to be used for faecal transplantation were treated with Abx in advance. Mice were fed with drinking water mixed with four antibiotics  for 3 weeks following the method described previously.2.3The blood sample was collected, and the supernatant (serum) was stored at −80°C for further experiments. The faeces were collected for 16S rDNA sequencing or faecal transplantation. The left lung tissues and/or intestinal tissues were collected for pathological and ELISA analyses. The right lung was collected, and incubated at 60°C for 3–4 days to remove all moisture. Subsequently, the dry weight was measured, and the ratio of wet‐to‐dry weight was calculated.2.4Fresh faeces were collected from the control, BLM, control + polydatin and BLM + polydatin groups. Fresh faeces were collected from mice in the morning using the stress defecation method. The lower abdomen of the mice was gently pressed, and four to five complete faeces were collected from each mouse and stored in a sterilized centrifuge tube. The faeces of all mice in the target group were placed in the same centrifuge tube. The collected fresh faeces were added to sterilized saline at a ratio of 100 mg:1 mL, mixed by vigorous vortexing and centrifuged at 4°C for 3 min at 1200 rpm. Then, 200 μL supernatant was taken for enema administration in Abx‐treated mice (once per day for 2 weeks). The faecal microbiota supernatant from the control group, BLM group, control + polydatin group, and BLM + polydatin group were named control FMT, BLM‐FMT control‐Poly‐FMT, and BLM‐Poly‐FMT, respectively. The Abx‐treated mice were divided into four groups, and each group was named according to the control or BLM model and the origin of faecal microbiota to be transplanted: (1) Control + Control‐FMT; (2) BLM + BLM‐FMT; (3) Control + Control‐Poly‐FMT; and (4) BLM + BLM‐Poly‐FMT.2.5QIAamp Fast DNA Stool Mini Kit  was used for DNA extraction of faecal microbiota. Universal primers were designed using the gene fragment of the conserved region V3V4 of bacterial 16S rDNA, and PCR amplification was performed using the forward primer 341F: CTACGGGNGGCWGCAG and the reverse primer 805R: GACTACHVGGGTWTCTAATCC. The sequence of the V3V4 region was sequenced on the Illumina NovaSeq system.2.6α‐diversity is an indicator of reaction richness, diversity and evenness. α‐diversity was evaluated using QIIME2 software. The observed species, ACE, and Chao1 indices were used in this study. β diversity refers to the replacement rate of species along the environmental gradient among different communities or the dissimilarity of species composition. β‐diversity was evaluated using principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS). The above analysis was completed using the R language (4.02) and QIIME2 (QIME2‐2020.2) as previously described.2.7The mice were euthanized under anaesthesia. For the haematoxylin and eosin staining, the lung tissues were quickly removed after opening the thoracic cavity, washed using precooled PBS solution, fixed in 10% neutral formalin, routinely embedded in paraffin and sectioned 48 h later. The paraffin sections were dewaxed with xylene, rehydrated with gradient concentrations of alcohol, washed with PBS and immersed in haematoxylin staining for 10 min. Stained slices were observed under an inverted microscope. For the Masson staining, paraffin‐embedded sections were deparaffinized with xylene, dehydrated with a gradient of alcohol, washed in PBS, and stained for 10 min with a 1:1 mixed Weigert's haematoxylin solution. After being differentiated for 5–15 s with acidic alcohol differentiation solution, the sections were coloured for 3 min with Masson staining solution and stained for 5–10 min with Ponceau S staining solution. The stained slices were observed under an inverted microscope.2.8The sections were routinely deparaffinized and rehydrated in gradient concentrations of alcohol. Antibody incubation was performed after antigen retrieval, blocking of endogenous peroxidase and blocking of nonspecific binding sites. The primary antibodies used were: collagen I  and α‐SMA . After overnight incubation, the subsequent steps were completed using a Pv 9000 General Purpose Two‐Step Immunohistochemical Detection Kit and DAB chromogenic solution.2.9Cells were lysed using RIPA lysis buffer  to obtain protein samples. After measuring the protein concentration with the BCA Kit (Beyotime), the corresponding volume of protein was added to the loading buffer (Beyotime), mixed well and heated in a boiling water bath for 5 min to denature the protein. Electrophoresis was carried out to separate the proteins. The membrane transfer was carried out on an ice bath with a current of 220 mA for 120 min. The membranes were rinsed for 1–2 min, put into the blocking solution for 60 min at room temperature, and incubated with primary antibodies against collagen I , α‐SMA  or GAPDH  overnight at 4°C. The membranes were washed and incubated with the secondary antibody  for 1 h at room temperature. After dripping the developer solution onto the membrane, signal detection was performed using a chemiluminescence imaging system (Bio‐Rad).2.10TNF‐α, lipopolysaccharides (LPS), IL‐6, and IL‐1β in the lung tissue, intestine and serum of mice were detected in each group according to the instructions of the ELISA kit . All operations were carried out in strict accordance with the instructions.2.11t‐test or one‐way anova, and the Tukey method was used for multiple comparisons. When p < 0.05, the difference was considered statistically significant.The experimental data obtained are expressed as the mean ± SD. GraphPad Prism 8.0 software was used for the 33.1The pathological changes and collagen deposition in mouse lung tissues were evaluated using haematoxylin and eosin and Masson staining. Figure Masson staining showed consistent results, as no collagen deposition was found in the lung tissue of the mice in the control group and the control + polydatin group, while collagen deposition was the most severe in the BLM group. Compared with the BLM group, collagen deposition was significantly reduced in the BLM + PD group. These results indicated that the BLM model was successfully established and that polydatin could effectively ameliorate BLM‐induced lung injury and collagen deposition in the mouse model. The levels of fibrotic markers, collagen I and α‐SMA, were also measured by IHC and Western blot. As shown in Figure 3.2To further evaluate the pulmonary inflammation of the mice in each group, we used ELISA to detect the levels of TNF‐α, LPS, IL‐6 and IL‐1β in the lung tissue, intestine and serum of the mice. Figure 3.3First, rarefaction curves (rank‐abundance) were used to assess whether the sequencing volume was sufficient to cover the species abundance in the sample. Figure 3.4Muribaculaceae, Lactobacillus, Lachnospiraceae, Akkermansia and Allobaculum. The main dominant species in the BLM model group were similar to those in the control group, and the abundance of Pseudomonas, Xylanophilum and Parabacteroides was relatively high in individual samples. The dominant species in the BLM + polydatin group were Muribaculaceae, Lachnospiraceae, Lactobacillus, Dubosiella, Akkermansia, Alistipes, Allobaculum and Parasutterella. Among these microflora, Akkermansia, Lactobacillus and Dubosiella are probiotics, and Lachnospiraceae is a pernicious bacterium. These results indicated that the BLM model and polydatin could alter the dominant microflora in the gut microbiota.Due to the intergroup differences in the gut microbial communities of mice in each group, a Venn diagram was used to analyse shared microflora. As shown in Figure 3.5Muribaculaceae, Lactobacillus, Akkermansia, Alloprevotella and other flora were less abundant in the BLM group. However, compared with the BLM group, the abundance of probiotics Muribaculaceae, Lactobacillus, Akkermansia, Alloprevotella and other flora in the BLM + polydatin group increased significantly  and suppresses the formation of reticular structures in lung tissue.Streptococcus pneumoniae who are receiving antibiotic therapy.Lactobacillus and Bifidobacterium supplementation in children can help improve asthma symptoms and reduce seizure frequency.The gut microbiota is acknowledged as a key innate immune system regulator that is engaged in the immunological development of the intestinal mucosa and impacts immune system development.Dubosiella, a type of probiotic, only existed in the polydatin treatment group. Dubosiella, belonging to the family Erysipelotrichaceae, was found to be significantly reduced in high‐fat diet‐ and STZ‐induced diabetic mice.Dubosiella and decrease the relative expression of activating transcription factor 4 to inhibit blood pressure in mice.Muribaculaceae, Lactobacillus, Akkermansia and Alloprevotella, were detected in all BLM mice but with altered abundance in the presence or absence of polydatin treatment. Polydatin treatment significantly increased the abundance of Muribaculaceae, Lactobacillus, Akkermansia, and Alloprevotella compared with the BLM group, indicating that polydatin could affect the composition of the mouse gut microbiota by increasing probiotic abundance. Lactobacillus was shown to protect mice against influenza virus‐induced pathology and mortality.Muribaculaceae mediated salmon peptide fraction‐induced improvement of metabolic syndrome.Akkermansia muciniphila was associated with the clinical benefit of immune checkpoint inhibitors in patients with non‐small cell lung cancer or kidney cancer.Alloprevotella was reported to be decreased in COPD patients with declining function.BLM stimulation with or without polydatin treatment caused no significant changes in gut microbiota diversity; however, the dominant species and corresponding abundance were altered. Although BLM mice with or without polydatin treatment shared the most dominant species, Complementary probiotics or FMT have been proven to restore the gut microecology and reduce inflammation in acute lung injury and pneumococcal pneumonia.In conclusion, polydatin alleviated BLM‐induced PF and altered gut microbiota species abundance. Moreover, polydatin‐induced alteration of the gut microbiota may also be involved in the therapeutic effect of polydatin in PF.Jia Yang: Conceptualization ; formal analysis ; methodology ; writing – original draft . Xiawei Shi: Data curation ; methodology ; validation ; visualization . Rundi Gao: Data curation ; methodology . Liming Fan: Investigation ; methodology . Ruilin Chen: Investigation ; visualization . Yu Cao: Investigation . Tingzhen Xu: Conceptualization ; writing – review and editing . Junchao Yang: Conceptualization ; funding acquisition ; supervision ; writing – review and editing .The authors declare no conflicts of interest in this work.


Observational studies have reported bidirectional associations between metabolic syndrome (MetS) traits and short leukocyte telomere length (LTL), a TL marker in somatic tissues and a proposed risk factor for age‐related degenerative diseases. However, in Mendelian randomization studies, longer LTL has been paradoxically associated with higher MetS risk. This study investigated the hypothesis that shorter LTL might be a consequence of metabolic dysfunction.This study undertook univariable and multivariable Mendelian randomization. As instrumental variables for MetS traits, all of the genome‐wide significant independent signals identified in genome‐wide association studies for anthropometric, glycemic, lipid, and blood pressure traits conducted in European individuals were used. Summary‐level data for LTL were obtained from a genome‐wide association study conducted in the UK Biobank.β = −0.039, 95% CI: −0.058 to −0.020, p = 5 × 10−5) equivalent to 1.70 years of age‐related LTL change. In contrast, higher low‐density lipoprotein cholesterol was associated with longer LTL  equivalent to 0.96 years of age‐related LTL change. Mechanistically, increased low‐grade systemic inflammation, as measured by circulating C‐reactive protein, and lower circulating linoleic acid levels might link higher BMI to shorter LTL.Higher BMI was associated with shorter LTL (Overweight and obesity might promote the development of aging‐related degenerative diseases by accelerating telomere shortening. Cross‐sectional and longitudinal observational studies have reported bidirectional associations between metabolic syndrome traits and short leukocyte telomere length (LTL), a proposed risk factor for age‐related degenerative diseases.Using two‐sample Mendelian randomization, we previously demonstrated that longer LTL was paradoxically associated with higher metabolic syndrome risk.Higher BMI is associated with shorter LTL, equivalent to approximately 1.7 years of age‐related LTL change.Higher low‐density lipoprotein cholesterol is associated with longer LTL.Increased subclinical inflammation and lower circulating linoleic acid levels might link obesity to shorter LTL.The metabolic syndrome (MetS) is a constellation of interrelated factors that increase the risk for type 2 diabetes and atherosclerosis . These fTelomeres are complexes of repetitive DNA sequences and specialized proteins that cap the ends of eukaryotic chromosomes and function to maintain chromosomal integrity and stability. In proliferating cells, telomeres shorten with each cell division. Shortened telomeres ultimately reach a critical length, which results in DNA damage with ensuing loss of replicative capacity and cell senescence or apoptosis. This, in turn, leads to compromised stem and/or progenitor cell function, tissue atrophy, and functional decline , 4. TeloTL within individuals is generally strongly correlated across tissues  and, conMendelian randomization (MR) is an epidemiological tool using data from genetic studies to estimate the nonconfounded associations between exposures and outcomes. We and others have recently explored the relationships between LTL and MetS traits using MR but failed to detect any causal associations between short telomeres and higher risk of MetS or its constituent traits , 20. On p < 5 × 10−8), independent single‐nucleotide polymorphisms (SNPs) identified in genome‐wide association study (GWAS) meta‐analyses for anthropometric (BMI and WHRadjBMI), glycemic (fasting glucose and insulin), lipid , and BP traits, with MetS as a binary trait, conducted in European individuals pruned for linkage disequilibrium (LD) using ld_clump, with the default set at LD r2 < 0.001 and a genetic distance of 10 megabases (Mb). In the case of fasting insulin, because of the small number of identified sex‐specific GWAS‐significant SNPs , we used the 19 fasting insulin SNPs established by the Meta‐Analyses of Glucose and Insulin‐related traits Consortium (MAGIC) [We investigated the associations of MetS and MetS traits with LTL in men and women of European descent using MR. As exposure instruments, we used all genome‐wide significant ( (MAGIC)  as instrAs outcome data for the sex‐combined analyses, we used summary‐level data for LTL from a GWAS conducted in the UKBB (ieu‐b‐4879)  and, forTo examine reverse causality, we conducted MR analyses using IVs extracted from the LTL GWAS using extract_instruments in the TwoSampleMR package (version 0.5.6) and outcome data using GWAS summary statistics for MetS and MetS traits from the aforementioned studies.https://fuma.ctglab.nl/) [www.ebi.ac.uk/gwas/). Finally, we cross‐referenced the GENE2FUNCTION gene set output with traits and indices of healthy behavior previously shown to be cross‐sectionally associated with LTL [We used Functional Mapping and Annotation of Genome–Wide Association Studies (FUMA) (lab.nl/)  to explowith LTL , 23. Thewith LTL , linoleiwith LTL , years owith LTL , and, inI2) to assess bias for the MR‐Egger method [FADS) gene region in the polyunsaturated FA (PUFA)‐LTL relationships (online Supporting Information Methods). Furthermore, we conducted MR analyses after excluding variants with larger effects on outcome than the exposure trait (Steiger filtering). Results were corrected for multiple testing, with p < 0.0125 (0.05/4) considered significant. This cutoff is based on assessments of the associations of four classes of MetS traits  with LTL. A statistically significant IVW result coupled with directionally consistent associations from all three sensitivity analyses was considered sufficient evidence to claim a causal effect.We used the inverse‐variance weighted (IVW) approach for MR analyses, with MR‐Egger regression, weighted–median MR, and contamination mixture (Conmix) MR as sensitivity analyses. Additional sensitivity analyses for exposures with UKBB participants included calculating the variability in instrument strength  and MendelianRandomization (version 0.6.0) packages in R (version 4.2.1) .To estimate the associations of MetS and its component traits with LTL, we conducted one‐ and two‐sample MR. As IVs for MetS traits, we used all of the genome‐wide significant independent signals identified in GWAS meta‐analyses for anthropometric , glycemic , lipid , and BP  traits conducted in European individuals  and longer  LTL, respectively . However, no significant associations of favorable or unfavorable adiposity with LTL were detected , TAG , systolic BP , and MetS as a binary trait , which were both directionally consistent in sensitivity analyses and robust to Steiger filtering . Finally, we cross‐referenced the GENE2FUNCTION gene set output with traits and indices of healthy behaviors previously shown to be cross‐sectionally associated with LTL ; omega‐6 FAs ; LA , the most prevalent dietary omega‐6 FA in Western diets; and years of schooling  were associated with LTL  but lower omega‐6 FA  and LA  levels and fewer years of schooling . In contrast, higher LDL‐C was positively associated with plasma levels of omega‐6 FAs  and LA  and, in Steiger‐filtered analyses, negatively associated with CRP . Results were robust to sensitivity analyses, although there was evidence of unbalanced pleiotropy, as assessed by the MR‐Egger intercept test, for the associations between LA and LTL and between BMI and years of schooling .Next, we investigated the associations between the aforementioned traits and LTL. As exposure instruments, we used all of the genome‐wide significant independent signals identified from the following: 1) a GWAS of CRP that we newly conducted in the UKBB; 2) the largest GWAS of the plasma metabolome, also conducted in the UKBB  ; and 3) L Figure . MR withL Figure . FinallyI2 = 98.7%, minimum I2 = 98%), suggesting little bias for the MR‐Egger estimates in these analyses . Similarly, the positive association between LDL‐C and LTL was attenuated when adjusted for either omega‐6 FAs or LA. However, neither omega‐6 FAs nor LA influenced LTL independently of LDL‐C. Based on the same analyses, higher CRP and lower LA were estimated to account for ~28.1% and ~9.4%, respectively, of the total detrimental effect of BMI on LTL. A summary of our main findings is shown in Figure We performed mediation analyses using MVMR to decompose the effects of BMI and LDL‐C on LTL, which act directly, and those acting potentially via CRP, omega‐6 FAs, LA, and years of schooling  was associated with shorter mean LTL, which was equivalent to ~1 year of age‐related LTL change (~30 base pairs). Based on the annual LTL change reported by the same group, their estimate compares with ~1.7 years of age‐related LTL change per SD of BMI change in our study, which explored the lifelong consequences of adiposity on LTL. Put in a different way, our data suggest that, compared with an age‐matched lean individual (BMI of 22 kg/m2), a person with obesity (BMI of 32 kg/m2) has a shorter LTL, equivalent to ~3.4 years of age‐related change. Nonetheless, caution is necessary when extrapolating from the data by Bountziouka et al. [https://portals.broadinstitute.org/collaboration/giant/index.php/GIANT_Cohorts_and_Group) is broader (17–90 years) than that of the UKBB (40–69 years). Similar findings were also obtained by a cross‐sectional meta‐analysis capturing data from 146,114 individuals, which estimated that a ~five‐unit difference in BMI was equivalent to a ~25 LTL base pair difference [The finding that BMI was negatively associated with LTL in MR analyses is in accordance with most cross‐sectional epidemiological data , 19, 32.The predominant mechanism through which obesity might shorten LTL is believed to be increased oxidative stress . In animwww.lipidgenetics.org) is also broader than that of the UKBB [Observational studies exploring the relationships between lipid traits and LTL reported uniformly positive baseline and longitudinal associations between HDL‐C and LTL , 18, 40,the UKBB . Consistthe UKBB . Potentithe UKBB  and Hedgthe UKBB  signalinthe UKBB . Again, F score range 21–234). Furthermore, we conducted bidirectional MR and Steiger filtering, which did not provide evidence that our findings were confounded by reverse causality. Finally, based on the MR‐Egger intercept test, none of our significant associations was confounded by unbalanced pleiotropy aside for those between LA and LTL, as well as BMI and years of schooling. However, removal of pleiotropic variants made no changes to the significance or interpretation of the results derived using IVW regression. Regarding limitations, we examined mean LTL length, which does not necessarily translate to TL in tissues and cells relevant to the development of MetS. Second, our findings are based on data from a GWAS conducted in individuals of European ancestry. Therefore, our results and conclusions might not extend to other ancestral populations. Third, MVMR reduces statistical power relative to univariable MR [MR depends on three main assumptions: first, that the IVs associate with the risk factor of interest; second, that they share no common cause with the outcome; and finally, that they do not affect the outcome except through the risk factor. In this regard, all of our exposure instruments were robust .TABLE S3. Over‐representation of genes mapped to GWAS loci for LDL‐C within sets of genes mapped to loci for other GWAS catalogue traits in FUMA (https://fuma.ctglab.nl).TABLE S4. Mendelian randomization estimates of effects of anthropometric, blood pressure, and metabolic traits on leukocyte telomere length .TABLE S5. MR estimates of effects of additional obesity measures on leukocyte telomere length .TABLE S6. MR estimates of effects of anthropometric, blood pressure, and metabolic measures and leukocyte telomere length in women and men.TABLE S7. Two‐sample MR estimates of associations of BMI and body fat percentage with leukocyte telomere length in the ENGAGE cohort .TABLE S8. (A) Bias and type 1 error rate for Mendelian randomization from sample overlap in exposure and outcome GWAS. (B) MRlap results.TABLE S9. Mendelian randomization estimates of effects of leukocyte telomere length  on anthropometric, blood pressure, and metabolic traits.TABLE S10. MR estimates of effects of potential mediators on leukocyte telomere length .TABLE S11. Results of multivariate MR analyses to determine whether the effects of omega‐6 fatty acids and linoleic acid on leukocyte telomere length (sex‐combined) are independent of other polyunsaturated fatty acids.TABLE S12. MR estimates of effects of BMI, and LDL‐C on potential mediators (sex‐combined populations).TABLE S13. MR estimates of effects of additional C‐reactive protein, Fatty acid and educational attainment measures on leukocyte telomere length using non‐overlapping GWAS datasets.TABLE S14. MR estimates of effects of BMI on potential mediators using non‐overlapping GWAS datasets (sex‐combined populations).TABLE S15. Results of multivariate MR analyses to estimate the % mediated effects of BMI, LDL‐C, omega‐6 and linoleic acid, on leukocyte telomere length (sex‐combined).Click here for additional data file.