Crohn’s disease is an inflammatory bowel disease with up to 50 per cent of patients requiring surgery within 10 years of diagnosis. Patient-reported outcome measures (PROMs) are vital to monitor and assess patient health-related quality of life (HRQoL). This systematic review aims to evaluate PROMs within studies for perioperative Crohn's disease patients.Articles from MEDLINE, Embase, Emcare and CINAHL databases were searched to find studies relating to the assessment of HRQoL in perioperative Crohn's disease patients using PROMs and patient-reported experience measures (PREMs) from 1st January 2015 to 22nd October 2023. Bias was assessed using the ROBINS-I tool was used for non-randomized interventional studies and the Cochrane RoB2 tool was used for randomized trials.1714 journal articles were filtered down to eight studies. Six studies focused on ileocaecal resection, one on perianal fistulas and one on the effects of cholecystectomy on patients with Crohn's disease. Within these articles, ten different PROM tools were identified . Overall improvements in patient HRQoL pre- to postoperative for ileocaecal Crohn's disease were found in both paediatric and adult patients. Outcomes were comparable in patients in remission, with or without stoma, but were worse in patients with a stoma and active disease.There are significant variations in how PROMs are used to evaluate perioperative Crohn's disease outcomes and a need for consensus on how tools are used. Routine assessments using an internationally accepted online platform can be used to monitor patients and support areas of treatment pathways that require further support to ensure high standards of care. They also enable future statistical comparisons in quantitative reviews and meta-analyses. Crohn’s disease (CD) is an inflammatory bowel disease that often requires surgical intervention, making patient-reported outcome measures (PROMs) an important tool for monitoring and assessing patient health-related quality of life (HRQoL). This systematic review evaluates the use of PROMs within surgical CD patients and is able to identify eight studies which collectively utilize eight measures of HRQoL and two measures of function. This review highlights the need for a consensus on how these tools are used and to monitor patients, support treatment pathways and enable future quantitative reviews and meta-analyses. CD can manifest with a variety of presentations at multiple sites and surgical management is complex. CD has a high incidence of perianal disease with a 20-year cumulative incidence of up to 42 per cent2. Perianal fistulas are the most common presentation of perianal disease and often require surgical intervention, with a significant proportion of perianal CD patients eventually requiring a permanent stoma4. Despite the medical therapies available for the management of symptoms, surgical intervention remains a mainstay for the treatment pathway of CD, with approximately 50 per cent of patients requiring surgery within 10 years of diagnosis, with historical reports of surgical recurrence being as high as 80 per cent in previously operated patients5. Surgery cannot cure CD, but is indicated for complications of the disease and to improve patients’ quality of life. Despite the high risk of surgery associated with the disease, the regular assessment of patient-reported outcome measures (PROMs) to assess patient health-related quality of life (HRQoL) is lacking7. A multicentre study assessing national perioperative assessments for surgically managed CD patients in Italy found that PROMs and functional outcomes were being assessed in only 1 per cent of patients following surgery8.Crohn’s disease (CD) is a chronic inflammatory bowel disease (IBD) affecting the entirety of the gastrointestinal tract, but most commonly within the distal ileum of the small intestine9. This study aims to investigate and systematically evaluate the use of PROMs within studies across perioperatively managed CD patients.PROMs are crucial to monitor the impact of surgical interventions in CD. These PROMS are becoming increasingly established, not only during perioperative management but also during remission and medical management. The Food and Drug Administration has emphasized the need for the use of patient-reported outcomes as primary outcomes within clinical trialsA comprehensive systematic literature search was performed through the MEDLINE, Embase, Emcare and CINAHL  databases using the Healthcare Databases Advanced Search (HDAS) platform in order to identify relevant articles investigating the use of PROMs, patient-reported experience measures (PREMs) or other HRQoL tools in CD patients following surgical intervention.The search included all English journal articles from 1st January 2015 to the date of the search (23rd October 2023). The main search terms used were: ‘Crohn’s Disease’, ‘Granulomatous Enteritis’, ‘Crohn’s Colitis’, ‘Regional Ileitis’, ‘Terminal Ileitis’, ‘Ileocolitis’, alongside ‘Surg*’, ‘Surg* Manag*’, ‘Operat*’, ‘Colectomy’, ‘Bowel Resection’, and ‘Patient-Reported Outcome’, Patient-Reported Experience’, ‘Patient Experience’, ‘PRO’, ‘PROM’, ‘PREM’.Articles containing PROMs data from surgically managed patients with Crohn’s disease were included, meaning that data encompassing all forms of IBD  that did not distinguish CD patients were excluded. Similarly, results that included both surgical and non-surgical PROMs data that did not separate surgical data only were also excluded.Additional searches were also done into the bibliographies of included studies to identify further articles.Articles were independently screened by two authors (W.J.K. and M.I.), and at full-text screening, disagreements were discussed, and a collective decision was made whether to include or exclude.Articles were subdivided into separate manifestations of Crohn’s disease  and the outcome measures used within these studies were analysed to compare how they are being used, as well as their associated benefits and limitations.Quantitative PROMs outcome data were extracted; however, as PROMs tools within CD surgery are still in their infancy, meaningful statistical analysis was not possible. This is due to differences between what these outcome measures assess; accumulation of data sets and comparisons were difficult to achieve.11, the results of which are summarized in Table S1a & S1b.The ROBINS-I tool was used for non-randomized interventional studies and the Cochrane RoB2 tool was used for randomized trials12 (Table S2) and the review has been registered on PROSPERO (registration identifier: CRD42022349869). A separate review protocol was not prepared.This review was completed following the PRISMA guidelines13–19 were found to contain relevant information and a single further article20 was found through screening bibliographies of articles in the database, leaving eight articles13–20 focusing solely on CD patients following surgery for evaluation for this review . Summaries of the studies, the key findings and information about the PROMs included are represented in the tables below .A total of 1714 articles were identified and were subjected to automated deduplication (using HDAS and Zotero’s deduplication processes) followed by manual deduplication, which removed a total of 510 articles. A final 1204 articles remained for screening, and following title and abstract screening, 1140 articles were excluded, leaving 64 articles for full-text screening; of these, 57 articles were excluded as they lacked PROMs within colorectal surgery, PROMs data were not exclusive to CD , PROMs data were not presented within the paper, or patients were not treated surgically. Seven articleset al.13 and Dipasquale et al.14 were two studies that assessed the outcomes of paediatric patients following ileocaecal resection. Diederen et al.13 assessed the outcomes of 80 patients who had undergone primary ileocaecal resection during childhood (defined by age < 18 years); however, quality of life was only assessed in patients who had reached adulthood at the time of the study ) as there was no single validated, standardized tool for assessing HRQoL in both adult and paediatric patients. The study used the Short Form Health Survey 36 (SF-36) to assess HRQoL, the results of which were compared to mean scores of the general national population. They utilized the patient-reported colorectal function outcome (COREFO) questionnaire to assess colorectal function, which was compared to a separate cohort of patients with normal colorectal function following cholecystectomy; they also assessed the patients’ satisfaction with the surgical procedure13.Diederen et al.14 measured pre- and postoperative HRQoL using a modified IMPACT III questionnaire, an IBD-specific questionnaire for paediatric patients. Scores were obtained 12 and 6 months before surgery and were compared to the 12-month postoperative results of the same patients. Results between 6 months before surgery and 12 months after surgery were compared and statistically analysed14.Dipasquale et al.14 were able to find that median IMPACT III scores from 12 and 6 months preoperatively showed significant improvement across all dimensions of the questionnaire14. When comparing HRQoL outcomes against general populations, Diederen et al.13 found an overall conclusion that paediatric primary ileocaecal resection patients had reported a significantly worse physical-related quality of life. However, mental-related health scores were comparable13.Overall, Dipasquale et al.13 also performed subgroup analysis, dividing the cohort into active disease and remission, and compared scores against the general population. Physical health scores were significantly worse in both active disease and remission against the population, while mental health scores were only significantly worse in patients with active disease; scores were comparable in remissive patients compared to the general population13.Diederen 13.Within patients with active disease, COREFO scores were worse in all domains  compared to a cohort of normal colorectal function. Patients with clinical remission were found to be only significantly worse in social impact domainet al.14 found that patient symptoms such as blood in the stool, abdominal pain and fatigue had significantly improved from 6 months before, 12 months after surgery. Patients also reported having significant improvements in appetite and weight and a better ability to perform activities of daily living14.Dipasquale 14.School absences were reported in almost all patients before surgery, caused by abdominal pain, fatigue and diarrhoea. However, at 1 year following surgery, school absence reduced significantly. Social functioning scores, consisting of a patient’s ability to play, go out, play sport, and travel, significantly improved after surgery14. Dipasquale et al.14 found that emotional functioning results demonstrated a significant reduction of feelings of anger, injustice and embarrassment due to Crohn’s at 12 months and receiving surgery did not affect the patients’ concerns for future health problems (P = 0.202). Diederen et al.13 found that the number of patients who rated their satisfaction following surgery as ‘very satisfied’ was the greatest at 42/80. A further 23 were ‘satisfied’, nine were ‘neutral’, four were ‘dissatisfied’ and two were ‘very dissatisfied’. Lower satisfaction was found to be correlated to a higher level of disease activity13. Dipasquale et al.14 also reported that almost all patients were completely satisfied with the surgery outcomes.Both studies found high levels of satisfaction following surgery15–17 assessed HRQoL in CD patients receiving different treatments. Gerdin et al.15 and Ponsioen et al.16 conducted RCTs comparing medical treatment versus surgical resection. Gerdin et al.15 investigated HRQoL using the SF-36 and the visual analogue scale (VAS), and Ponsioen et al.16 utilized the inflammatory bowel disease questionnaire (IBDQ) as well as the SF-36.Three studieset al.17 compared HRQoL between CD patients with or without a stoma using a shortened version of the IBDQ (SIBDQ) and the Patient-Reported Outcomes Measurement Information System (PROMIS) as well as patient-reported functional outcomes using the Manitoba IBD Index (MIBDI). They recruited patients from a large online cohort of adult patients with IBD and subcategorized them, using MIBDI results, by disease status (remission or active disease) to avoid any confounding caused by this factor17.Abdalla et al.16 found that the surgical treatment group had significantly worse mean IBDQ scores at 2 weeks; however, these scores subsequently improved and became comparable from 6 weeks. Mean IBDQ scores of the surgical treatment group were higher than the medical treatment group at 6, 9 and 12 months; however, these differences were not significant.Ponsioen 16.Similar trends were observed in the mean combined SF-36 scores. The surgical treatment group had significantly lower scores than the medical treatment group at 2 weeks, but these became comparable at 6 weeks. At 6 and 9 months, the surgical treatment group demonstrated significantly better mean combined SF-36 scores, although this significance was lost at 12 months16.When dividing the SF-36 into its physical and mental component scores, physical component scores in the surgical treatment group were significantly worse at 2 weeks but significantly better at 6, 9 and 12 months compared to the medical treatment group. Mental component scores in the surgical treatment group were worse than the medical treatment group at 2 weeks, 6 weeks and 3 months. At 6, 9 and 12 months, the surgical treatment group demonstrated higher mean mental component scores; however, none of these differences were statistically significantet al.15 found that at 1-year follow up, the surgically treated cohort demonstrated significantly better social function SF-36 scores. This difference, however, was no longer observed at 3 and 5 years. While better general health scores were also found in the surgical treatment group, this difference was not significant. Vitality scores were worse in the surgically treated group, but again, this was not statistically significant. Additionally, VAS scores showed no statistical differences15.Gerdin et al.17 found that the mean SIBDQ scores between patients with or without a stoma for both active disease and remission were not significantly different. For those in clinical remission, in all six domains of the PROMIS tool , differences were found to be insignificant. However, in those with active disease, PROMIS scores in the domains fatigue, pain interference, and social satisfaction were significantly worse in those with a stoma than those without.Abdalla 17, as well as assessing and comparing the HRQoL of CD patients with or without a stoma, Abdalla et al.17 also compared outcomes of sexual interest and satisfaction in those with or without a stoma using PROMIS. Results were stratified by gender and, overall, there were no statistical differences found between those with or without a stoma in all domains for all genders17.In this studyet al.20 investigated the effects of bowel resection on the HRQoL of CD patients following their RCT31 comparing the outcomes of patients receiving a standard care arm versus an active care arm . In order to assess patients’ HRQoL, the researchers used the SF-36 as well as the disease-specific IBDQ. Measures of HRQoL were recorded before surgery, as well as at 6, 12 and 18 months after surgery20.Wright 20.The SF-36 results were divided into a physical component score and a mental component score. Results demonstrated a significant improvement of both the mean physical and mental scores from before to after surgery 20.Mean IBDQ scores also demonstrated similar trends, showing a significant pre- to postoperative improvement in HRQoL throughout the follow-up periodet al.18 assessed the PROMs results of CD patients with perianal disease. They investigated the HRQoL outcomes of patients with perianal fistulas following video-assisted anal fistula treatment (VAAFT) using the Measure Your Medical Outcome Profile 2 (MYMOP2) quality-of-life questionnaire as a primary endpoint at 6 weeks following surgery. These results were compared to the patients’ results 6 weeks before surgery, and they also assessed patients’ attitudes and feelings of regret using the Decisional Regret Scale18.The study by Adegbola 18.Median MYMOP2 scores relating to symptoms of pain and discharge demonstrated significant improvements from 6 weeks before to 6 weeks after surgery18.The results also demonstrated overall positive sentiments towards the procedure, with 81 per cent of patients agreeing or strongly agreeing that they had made the correct decision, as well as 71 per cent of patients agreeing or strongly agreeing that they would undergo the procedure again. Furthermore, 95 per cent of patients disagreed or strongly disagreed that the choice they made did them harm32. Koutroumpakis et al.19 sought to investigate the effects of cholecystectomy on the HRQoL of CD patients using the SIBDQ between patients with (151 patients) or without (683 patients) cholecystectomy.CD patients have been found to be associated with a decreased ability to absorb bile acids due to CD-related tissue dysfunction or bowel resection, leading to a higher incidence of gallstones, and this is linked to increased rates of cholecystectomyet al.19 were able to demonstrate that those who underwent cholecystectomy had a significantly worse quality of life compared to those with CD who did not require cholecystectomy19. The cholecystectomy group was associated with significantly lower mean SIBDQ scores upon univariate analysis and after controlling for potential confounders, such as age, gender, smoking status and BMI. Multivariable analysis demonstrated that patients who had a cholecystectomy were associated with significantly worse SIBDQ scores19.Koutroumpakis 19.Subgroup analysis was performed on patients with ileal CD to assess the impact of cholecystectomy on the development of dysplasia because they would have reduced absorption of bile, leading to increased secretion of bile and thus increased spilling into the colon. Within this subgroup, patients who had undergone cholecystectomy were found to have significantly worse SIBDQ scores with uni- and multivariable analysisThis review demonstrated significant heterogeneity in the PROMs tools utilized to assess patient outcomes following CD surgery. Of the ten tools identified, eight are used to assess HRQoL, with the remaining two being used to assess patient-reported functional outcomes. Clearly, there are a wide variety of tools available to assess HRQoL within perioperative CD patients, and this study was able to identify areas within the studies included in this review where improvements could be made.et al.13 was that patient HRQoL scores were compared to the mean values of the general population of adults aged 26–35. A better alternative could have been to compare pre- and postoperative HRQoL if that information had been available, highlighting the need for PROMs-focused prospective studies in CD, which would allow for a clearer representation of the effects of the surgical intervention. The median follow-up time for HRQoL assessment within this cohort was 102.9 months (i.q.r. 46.5–190.0). Due to the extended follow-up period, it may be difficult to draw meaningful conclusions about the direct effects of surgical intervention during childhood on a patient’s HRQoL. While ileocaecal resection during childhood can have lifelong effects, data are lacking regarding subsequent interventions and management steps implemented up until the point of follow-up, which could act as confounding factors. The effects of childhood ileocaecal resection at the time of follow-up on this cohort may be less representative of the effects of the intervention alone.The main limitation found within the study by Diederen 13, patients could have had a significant pre- to postoperative improvement; however, by comparing to a baseline of a general population, these trends would be masked. More frequent/routine recording of PROMs would allow for more representative observations of how the disease progression and various interventions of CD could affect HRQoL over its course.Within this studyet al.14 as, despite being a retrospective analysis, the data were present due to routine records of patient scores within the centre. Through regular close monitoring of PROMs, clinical teams would be able to spot patients that were not coping well and provide further medical, emotional and social support, using the data to potentially signpost patients for further care.This limitation was not present within the study by Dipasquale et al.20 noted the improvement found at 6 months following surgery was maintained at 12 and 18 months. This study was able to demonstrate the sustained improvement in HRQoL following surgical intervention. The conclusion that bowel resection is able to produce a long-term improvement of HRQoL within this trial is significant due to the lack of PROMs data at longer follow-up periods.Wright 20 (which demonstrated an improvement from pre- to postoperative outcomes) to the study by Gerdin et al.15, they were also able to find significantly better outcomes within surgically treated patients when compared to those who were treated with medical therapy at 1- year of follow-up. This benefit, however, was seemingly lost at 3 and 5 years of follow-up.Comparing this study20 highlight the importance of long-term monitoring of patients’ HRQoL through the use of PROMs in CD patients following surgery. CD is a lifelong disease, and the effects of surgery are lifelong too. Routine assessment using PROMs would allow for the analysis of HRQoL outcomes at even longer follow-up periods, allowing researchers to continuously break the ceiling of the definition of the best standard of care, while allowing clinicians and patients to make informed decisions based on long-term empirical data.The two studieset al.16, frequent PROMs assessments were performed, from 2 weeks after baseline up to 12 months of follow-up. Trends of how HRQoL outcomes change over time and differ from medical treatment groups were observed. The study by Gerdin et al.15, which also compares two randomized arms of surgical and medical intervention, was designed so that follow-up would be performed at 1, 3 and 5 years. Although Gerdin et al.15 show significantly better social functioning SF-36 scores for the surgical group, as well as trends to higher general health scores at 1 year, this initial follow-up interval is relatively long. While assessing long-term outcomes is important for such a lifelong disease, it is also important to assess patient outcomes in the short term. The results from Ponsioen et al.16 are able to demonstrate how outcomes may change within that first year after intervention, and that in fact mean IBDQ and SF-36 scores in the surgical treatment group are initially significantly worse, and become comparable over time, becoming significantly better than the medical treatment group at some points. Together, these studies16 are able to demonstrate how regular PROMs data, both short- and long-term, are important in assessing HRQoL in CD patients.Regular PROMs assessments, both short- and long-term, are essential for understanding a patient’s HRQoL. In the study by Ponsioen et al.20 demonstrated consistently lower HRQoL results in women versus men, which is in line with previous literature finding similar results in women with CD34: PCS scores of the SF-36 were found to be significantly lower preoperatively (P = 0.029), at 6 months (P = 0.017) and at 12 months (P < 0.001) following surgery in women compared to men; for the MCS of the SF-36, scores were also significantly lower in women at 6 (P < 0.015) and 12 months (P = 0.006) following surgery; IBDQ scores were also significantly lower in women at 6 (P = 0.007) and 12 months (P = 0.006) after surgery. Women reported a greater impact on body image following surgery for IBD than men, which may negatively impact HRQoL35.Wright et al.17 were unable to find differences stratified by sex in assessments of sexual interest and satisfaction. Despite these results, in other studies stomas have been shown to cause adverse effects on a patient’s sexual function and satisfaction, including self-image, embarrassment, dyspareunia, erectile dysfunction, ejaculation and orgasm. It is important to monitor these adverse effects in order to help provide effective, patient-centred care37. Abdalla et al.17 justify this difference in results as being caused by the indication for surgery, as most patients in these other stoma-related studies receive surgery due to cancer and malignancy as opposed to CD. In patients with malignancy, more radical, extensive resection may be required, leading to worse physiological outcomes as a result of nerve damage17.Abdalla et al.18 is the use of the Decisional Regret Scale. Decisional regret is the measure of the patient’s emotion as a result of feelings that better outcomes could have been achieved from opting for a different intervention or having not received the intervention at all and can be an influential factor within decision-making38. Negative emotions of decisional regret have been previously associated with worse HRQoL, such as having lower levels of satisfaction as well as being linked to depression and thus are an outcome that should be closely monitored in order to help provide further support for those with these risks of poorer HRQoL40.Possibly the most unique outcome of the study by Adegbola 30. In addition to providing further support, it may also allow for the identification of care pathways lacking in patient involvement within the decision-making process, opening that pathway up for improvement, raising the standard of care in order to minimize patient decisional regret, thus improving overall patient HRQoL.Low levels of patient satisfaction and decisional regret are measures that can be targeted with a push for patient-centred care as well as a shared decision-making process between the clinician and patient, allowing for patients to obtain an increased knowledge surrounding the options available, decreasing levels of uncertainty, providing more realistic expectations of the available outcomes, as well as providing feelings of supportet al.19 assessed the outcomes of surgery due to conditions potentially secondary to CD. The authors state how the exploration of this pathological pathway is not well established within research and aimed to explore these patients in further detail19. The study demonstrated the importance of a universal tool within the greater field of colorectal surgery. The study cohort displayed a broad heterogeneity of participants and if a universal tool was to be utilized across colorectal surgery, the HRQoL of patients with co-morbidities requiring multiple surgeries would be able to be assessed throughout their lifetime. A universal tool would enable the comparison of data before and after the development of a secondary condition without having to use a completely separate new tool and having to translate the information in order for a comparison of outcomes.Koutroumpakis 42. Furthermore, the results from patients would be directly uploaded to databases without the need for data input from staff, reducing time and costs, streamlining the entire data collection process42. This would also negate the issue experienced by Dipasquale et al.14 where they were required to modify an existing validated tool in order for its administration via the telephone. The IMPACT III questionnaire, a validated assessment of HRQoL in paediatric IBD patients, is a self-administered tool23. However, to complete the questionnaire via telephone consultation, 20 of the 35 items in the tools were removed. The modified version of the tool has not been validated and may not produce a full, comprehensive picture of the patient’s HRQoL status, hindering the validity of the results of the study. If the PROM had been made available online, the quality of the tool would not have needed to be compromised.With increasing online resources within healthcare due to improvements in technology, catalysed by the coronavirus disease 2019 (COVID-19) pandemic, the advantages of digitally collected PROMs data are becoming more apparent. Patient recruitment and drop-out rates within trials should improve when using digital platforms as less time and effort would be required to recruit patients. They would also be able to access the platform from wherever is convenient, as opposed to being restricted to completing them only when visiting clinics and appointmentsAn online tool with ease of access could lead to a risk of reporting bias, as a result of patients utilizing the tool more frequently when they experience an exacerbation of symptoms. To help reduce this bias, adequate guidance regarding when patients should fill out the tool could prove to be useful. For example, consistent and regular utilization of the tool could be emphasized to patients, and usage patterns could be analysed to identify disproportionate frequency of submissions.41. This digital platform could be expanded from disease-specific to disease-group-specific, not only being used for surgical CD patients but for all IBD patients and even across colorectal surgery, presenting the relevant tools and questionnaires to the specific patient from a single platform .There is no consensus on the best PROM for measuring the HRQoL in patients with CD. The two types of PROMs, generic and disease-specific, targeting different aspects of HRQoL can be complementary if used together. This would produce a holistic view of the patient providing an assessment on how the patient is coping with the specific disease, but also raising red flags for general psychosocial risk. These tools can be collated into a single digital platform and used to gather data from patients with ease, with some suggesting the use of adaptive testing to tailor the relevant questions around the patient using computer algorithmset al.44 is an example of a study utilizing HRQoL as a primary outcome through the use of PROMs to determine quality-adjusted life years within patients undergoing haemorrhoid surgery . Watson et al.44 were able to use the results of the PROMs tools to determine that surgical excision should be considered over stapled haemorrhoidopexy as surgical excision produced significantly better HRQoL outcomes over 24 months post-procedure. Within colorectal surgery, with the impact that CD can have on a patient’s HRQoL, trials should be encouraged to utilize PROMs and patient experience as a primary outcome.The eTHoS trial by Watson 15, making the quality of the data poor. This may also be as a result of this review’s methodology. Searches were limited to only include articles published after 2015. While this might produce data that are within a more consistent timeframe, the absence of older studies could lead to the loss of data as a result.The quality of the systematic review is limited by the types of studies available for analysis. The majority of the articles were of a lower level of scientific evidence, with one of the only three higher-level RCTs that were found being prematurely terminated17. The variation in the PROMs used was also limiting in the fact that comparisons of data between studies could not be easily made. This may be due to a lack of consensus on a single universal tool for providing high-quality, valid data in patients with CD.The tools for measuring HRQoL were also of a limiting nature. Not all of the tools were of the highest quality, with some questionnaires not establishing a minimum clinically important difference for CD, such as PROMISzrad098_Supplementary_DataClick here for additional data file.


Grain boundaries, the two-dimensional defects between differently oriented crystals, tend to preferentially attract solutes for segregation. Solute segregation has a significant effect on the mechanical and transport properties of materials. At the atomic level, however, the interplay of structure and composition of grain boundaries remains elusive, especially with respect to light interstitial solutes like B and C. Here, we use Fe alloyed with B and C to exploit the strong interdependence of interface structure and chemistry via charge-density imaging and atom probe tomography methods. Direct imaging and quantifying of light interstitial solutes at grain boundaries provide insight into decoration tendencies governed by atomic motifs. We find that even a change in the inclination of the grain boundary plane with identical misorientation impacts grain boundary composition and atomic arrangement. Thus, it is the smallest structural hierarchical level, the atomic motifs, that controls the most important chemical properties of the grain boundaries. This insight not only closes a missing link between the structure and chemical composition of such defects but also enables the targeted design and passivation of the chemical state of grain boundaries to free them from their role as entry gates for corrosion, hydrogen embrittlement, or mechanical failure. Interplay between structure and composition of grain boundaries remains elusive, particularly at the atomic level. Here, the authors discover the atomic motifs, which is the smallest structural unit, control the most important chemical properties of grain boundaries. These features qualify GBs as the most important microstructural defects for many high-performance materials.Grain boundaries (GBs) are among the most important features of the microstructure, occupying an area of 500-1000 football fields per cubic meter material with 1 μm grain size. On the one hand, they render positive effects such as the simultaneous increase in strength and ductility7 and atomic motifs10, yet, it is still elusive which of these really matter for structure-property relations. By atomic motifs we mean here certain geometric polyhedra built by atoms that form repeating structural units to constitute the GBs12, e.g., the kite10, domino and pearl8 structures that were described before to decipher the GB’s elementary “genetic” structure. This concept of the atomic motif was inspired by the early works of Gaskell14 and Bernal15: they described liquids and amorphous materials as an arrangement of polyhedral structural units and found that only a surprisingly small number of them is needed to construct such complex materials.Two key factors are missing in our knowledge about GBs. The first one is the systematic and meaningful representation of GBs with pertinent and property-oriented parameters: their characterization requires five geometric degrees of freedom and further parameters to describe them down to their atomistic-scale facetsThe second reason is the difficulty in characterizing both the GB structure and its chemical decoration state down to the atomic scale. For a systematic experimental study, we define five levels of hierarchy with respect to the crystallographic, compositional, and electronic features of GBs. The levels are (a) the macroscale interface alignment and grain misorientation (held constant here); (b) the systematic mesoscopic change in the inclination of the GB plane for the same orientation difference; (c) the facets, atomic motifs, and internal nanoscopic defects within the boundary plane; (d) the GB chemistry; and (e) the electronic structure of the atomic motifs. Identifying key properties of GBs across all these scales at systematic variation of these parameters has not yet been realized.16. On a more refined scale, we find that the GB planes do not generally follow this geometrical criterion, but can further decompose into a sequence of piecewise planar facets that are sets of lower total energy reconstruction motifs17. At the atomic level, local GB structures, including the faceting17, as well as many other features such as secondary defects18, ordering21 and phase transformation22, can play crucial roles in the spatial distribution of solute atoms within GB planes. This complex interplay between GB structure and chemistry leads to a deviation from the Langmuir-McLean type of (sub-)monolayer adsorption behavior23, which takes a thermodynamic view at chemical decoration, yet, is structurally agnostic to these fine atomic details. This complexity shows that—in order to gain a fundamental understanding of the coupling behavior between structure and chemical composition—direct experimental observation of all these structure features conducted across all these scales, together with a mapping of the GBs chemical composition is required. This must be done under conditions, where each of these parameters is varied systematically, step-by-step, while the other parameters are kept constant.The hierarchy of the GB structure across these different features is complex: an adequate representation of a GB requires at least to define the misorientation between adjacent grains and the inclination of the GB plane. At a mesoscopic view energy-favored GB planes are normally the most densely packed ones which comprise a high density of coincidence sites, derived from a virtual lattice that extends over both adjacent crystals16) bicrystal, stabilized by approximately 4 at.% Al and alloyed with C and B atoms24. We have selected this material as Fe-C alloys, also referred to as steels, represent about 1.9 billion tons of material produced each year25, by far the most common metal class, with an uncounted number of safety critical and functional applications, ranging from huge infrastructure to tiny magnets. B has been selected as a second alloy element owing to its peculiar, and often highly beneficial effect on GB cohesion and the resulting material properties. At the smallest scale, we are able to reconstruct the charge-density maps from the differential phase contrast – four dimensional scanning transmission electron microscopy (DPC-4DSTEM) data28 to directly spatially resolve light (low atomic number) interstitial atoms decorating the atomic motifs of GBs. With this multi-scale and multi-physics GB analysis approach we make the surprising observation that it is actually not the macroscopic or mesoscopic geometrical aspects that explain the specific chemical decoration state of an interface as is usually claimed. Instead, features at the smallest length scales, i.e. the atomistic motifs, determine the chemistry of a GB. Analogous to building blocks or Lego bricks, a limited number of atomic motifs with their sequential alignment form complex GB structures with a wide range of microscopic degrees of freedom and varying solubility for accommodating solute atoms.For this study, we have developed a custom-designed workflow along an instrument ensemble which allows to map the structure, chemistry and electronic state of GBs at length scales ranging from macroscopic to atomic. As a model material we have chosen a Bridgeman-produced body-centered cubic (bcc) – Fe Σ5  value24. The macro- and mesoscale study of GB structure was carried out by optical, scanning electron microcopy (SEM), and diffraction imaging. During the growth process, the GB plane changed its orientation with respect to the neighboring grains, see the orange dashed line superimposed on the optical image  image Fig.  show fac28. As shown in Fig. 17), representing the B or C-containing atomic columns. The charge-density map can also provide an opportunity to study the electronic structure of the GB28. Here we focused on applying the charge-density map to the direct imaging of light atoms at the local atomic motifs of Fe GBs in the electron microscope with sub-Ångström-resolution.Using the recently developed DPC-4DSTEM imaging technique, we directly observed the light B and C atomic columns at the local atomic motifs of GBsWe have systematically quantified the GB structures for a series of Σ5-GBs with different mesoscale inclinations using HAADF-STEM images. The analyses include two symmetric GBs, #41 Σ5 (310) // (329. For the Σ5 (11 1 0) // (930 suggests that long-range coherency strains resulting from the misorientation (1.1° deviation from the true Σ5 GB) and inclination can be canceled out by Burgers vectors existing at different junctions of the facetted GB structures. For example, it has been reported by Medlin et al. that an array of (1/5)[310] and (1/5)[120] dislocations can fully accommodate the misorientation and inclination from the true Σ5 GB17. The dislocations that exist at the junction of the facetted structures play a key role in reducing the coherency strain between the two adjacent regions and stabilizing the GB structure. In the last case of the Σ5  electrostatic potential, and the charge-density maps, can be found in Supplementary Fig. 24.We have next studied the above mentioned three types of prevalent atomic motifs with regards to the occurrence of the light elements B and C, using the DPC-4DSTEM method33, the Σ5 (310) //  // (3−2. Our APT results show there is less than a monolayer adsorption of B or C atoms at this Σ5 (310) //  interstitial sites, the segregation energy for interstitial sites approaches zero or even reaches positive values. In these cases, further segregation is energetically unfavorable24.Interestingly, the decoration features with B and C are not strongly correlated but some regions appears to be mutually repulsive, i.e., regions enriched in B can have a low content of C see Fig.  and vice33. Wang et al.32 found that more open GB structures, e.g., with local atomic motifs such as (310) //  segregation behavior has been reported not only for symmetrical GBs, such as {013} and {012}, but also for asymmetrical GBs, e.g., 42. The authors attribute the singular segregation behavior of these GBs to the high values of the interplanar spacing41.Additional in-plane quantitative GB chemistry results are summarized Fig. . The ave34. Wang et al. indicates that the atomic rearrangement from the extended kite structure to the separated kite structure results in a reduction of GB energy by 0.44 32. In this work, we observed both types of local atomic motifs from the previous DPC-4DSTEM analysis  from the original part of the bicrystal see Fig. . The dis37. The wedge extracted by the FIB was mounted on a Si coupon and sharpened at 30 kV into needle-like geometries required for field evaporation, with a subsequent 5 kV cleaning step to remove surface damage implanted with Ga+.We used transmission electron microscopy (TEM) and APT to analyze the GB structure and composition of four selected ROIs, namely #01, #41, #71, and #91. The in-plane lift-out method (electron beam in TEM perpendicular to the common axis [001] of the grains) was used to prepare the TEM lamellae. First, the Cu grid was mounted on the correlative holder and placed horizontally in the FIB chamber for the lift-out process. Then, the chamber was vented for rotating the correlative holder 90° to thin the sample. The TEM lamellae were initially thinned to less than 60 nm at an accelerating voltage of 30 kV and then cleanly polished to a thickness of less than 30 nm at an accelerating voltage of 5 kV. The APT tips were fabricated using the conventional FIB lift-out method44  5000 XS operated with a specimen set point of 40 K and a laser pulse energy of 30 pJ at a pulse repetition rate of 200 kHz for a 0.5% atoms per pulse detection rate. The collected data sets were reconstructed using the AP Suite 6.1 software platform , Fe95.38Al4.45B0.07C0.11, Fe95.37Al4.38B0.11C0.14, Fe95.31Al4.42B0.14C0.14, respectively. Here, the peak decomposition algorithm must be applied to quantify the bulk composition because there is significant peak overlapping between Al+ and Fe2+ at 27 Da . The peak decomposition algorithm is only suitable for statistical compositional analyses. It does not provide spatial resolution, e.g., distinguishing compositional differences between two 1 nm3 cubes 1 nm apart. If we want to quantify the one-dimensional (1D) compositional profile across the GB, we need to assign the 27 Da to either Al+ or Fe2+ ions. In the 1D composition profile of the Supplementary Fig. 2+ ion, resulting in a significantly lower Al content than the value obtained from the wet chemical analysis or the peak decomposition analysis. A more detailed investigation of the Al depletion caused by the co-segregation of B and C can be found in our previous publication24. In the present work, we have mainly focused on the segregation behavior of B and C atoms. It is also worth noting that the local B and C contents obtained from APT composition quantification are significantly higher than the results from the global wet chemical analysis, which is due to GB segregation and a high fraction of GB contained in the reconstructed tip volume.The overall compositions for the reconstructed volume of the tips extracted from ROIs #01, #41, #71, and #91 are Fe36 to quantify the in-plane chemical distribution of B and C atoms at the GB, i.e., the composition map and the interfacial excess map  represent a pair of B and C compositions of the nodes in the GB meshes. We obtain these composition values from the in-plane chemical distribution described in the previous paragraph. The third variable The correlation between the local GB compositions of B and C is evaluated using a weighted Pearson product-moment correlation coefficient. A pair of variables  filter to remove background noise. Additional high-resolution HAADF-STEM images of ROIs #01, #41, #71 and #91 are shown in Supplementary Fig. All high-resolution HAADF-STEM data were acquired using a Cs probe-corrected FEI Titan Themis 60-300 (Thermo Fisher Scientific) equipped with a high-brightness field emission gun and a gun monochromator operating at 300 kV. Images were recorded with a HAADF detector (Fishione Instruments Model 3000) at a probe current of 68 pA using a semi-convergence angle of 23.6 mrad. The semi-collection angle for high-resolution HAADF-STEM images was set to 103-220 mrad. Image series of at least 20 images were acquired with a dwell time of 2 μs at a pixel size of 6 pm. To minimize the effects of instrumental instabilities in the images, we used non-rigid registration and averaging of image series to achieve sub-picometer precision measurement of atomic column positions in high-resolution HAADF-STEM images2. The scanning step size was optimized by comparing the reconstructed 4DSTEM data sets with step sizes of 13-36 pm. The selection took into account the possibility of minimizing distortions due to instrument instability and maximizing spatial resolution to resolve light atoms. We calibrated the CBED pattern in reciprocal space using the standard Au nanoparticle. Here, each pixel in the CBED pattern is 2.0 mrad.The atomic 4DSTEM data were also acquired in the Titan microscope at 300 kV. We collected the entire convergent beam electron diffraction (CBED) pattern as a two-dimensional (2D) image for each probe position during scanning. These images were taken using an electron microscope pixel array detector (EMPAD) with a readout speed of 0.86 ms per frame and a linear electron response of 1,000,000:1. Each CBED image has a size of 128 × 128 47. For Fe, the maximum transferable kinetic energy at 300 kV is 15.25 eV, which is slightly lower than the displacement energy of 16.00 eV47. Theoretically, the radiation damage should not be significant when imaging Fe materials. When acquiring atomic 4DSTEM data sets for imaging light atoms at GBs, a lower dose is preferred.Irradiation of materials with accelerated electrons can initiate ballistic knock-on processes that lead to displacement of atoms from the crystal lattice and produce point defects, e.g., a Frenkel pair consisting of an interstitial and a vacancy55We quantified the dose with the Gatan camera for energy-filtered TEM (EFTEM) by varying the beam via defocusing a monochromator. The measured dose has been shown as a function of the defocus of the monochromator  dark-field image, the electric field map, the (projected) electrostatic potential map, and charge-density map27 49. The script is forked from the py4DSTEM package50. We used py4DSTEM to reconstruct the dark-field image and to calculate center of mass (c.m.) of the transmitted beam for each probe position. Our pyDPC4D script mainly focused on the quantitative reconstruction of the electric field map, the (projected) electrostatic potential map, and the charge-density map. The details of the reconstruction are as follows.The originally collected atomic 4DSTEM data contains the 2D grid of the probe position in real space and the 2D diffraction pattern for each probe position in reciprocal spaceThe dark-field images are reconstructed from 4DSTEM data sets by integrating the intensity of the annular region of 99-122 mrad in the CEBD pattern of each probe position. Supplementary Fig. 54. This method can resolve the structure of weakly interacting phase objects. For example, when an electron probe passes through an electric field, the electron is deflected due to its negative charge. By quantifying the shifts of the transmitted electron probe in the diffraction plane  to the CEBD pattern to calculate the c.m. The purpose of applying a mask is twofold. First, it can eliminate intensity from the high-angle scattering that is less sensitive to the momentum transfer than that from the low-angle scattering. Second, it can also reduce the noise from c.m. calculation. Supplementary Fig.  mrad to 50. Supplementary Fig. We calculated the (projected) electrostatic potential  // (010), (310) // (357. We took the (310) //  package58. Here we have examined some experimental CEBD patterns and compared them with the simulated CEBD patterns. Supplementary Fig. CBED was acquired to determine the thickness of the TEM sample, because the thickness of the TEM sample has been shown to have a strong effect on the CEBD patternWe performed three series of simulations to understand the origin of the contrast appearing in the charge-density map. The benchmark simulation  // (3In the last series, we investigated the influence of atom occupancy on the contrast of the charge-density maps (see Supplementary Fig. Supplementary informationPeer Review File


In multiple logistic regression, a dose–response association from Stage I to Stage III periodontitis for cIMT ≥ 0.8 mm was also found . Leucocyte counts ≥ 7.6 × 103/µL (the highest quintile) was associated with cIMT ≥ 0.8 mm [OR 1.86 (1.11–3.12)], while no association existed for other metabolic risk factors. In conclusion, severe periodontitis and leukocyte counts are independent risk factors of increased cIMT, emphasizing the critical role of inflammation in subclinical atherosclerosis.This study aimed to investigate the association of periodontitis with subclinical atherosclerosis in young adults. In total, 486 non-diabetic military personnel were included in Taiwan. Carotid intima-media thickness (cIMT) was assessed utilizing sonography for subclinical atherosclerosis. Periodontitis severity was defined based on the 2017 US/European consensus. Mean cIMT was compared by analysis of covariance (ANCOVA), and multiple logistic regression model was used to determine the association of periodontitis severity and the highest quintile of cIMT (≥ 0.8 mm) with adjustments for age, sex, metabolic risk factors and leukocyte counts. The mean cIMT increased in those with greater stages : 0.65 mm, Stage I (N = 41): 0.72 mm, Stage II (N = 57): 0.74 mm and Stage III: 0.76 mm, respectively,  Periodontitis is associated with disproportionate host inflammatory immune responses that are induced by an imbalance in the microbial ecological environment in oral cavity; this instigates microbial dysbiosis, along with failed resolution of the chronic destructive inflammation2. The European Federation of Periodontology (EFP) and the American Academy of Periodontology (AAP) joint workshop in 2012 suggests a possible link between atherosclerotic disease and periodontitis3. Two mechanisms are proposed to explain such association. First, the subgingival microbiota and their products may directly access to the circulation and provide a persistent gram-negative bacterial challenge to the host4. Low level bacteremia may initiate host responses that alter coagulability, endothelial and vessel wall integrity, and platelet function, thereby resulting in subsequent atherogenic changes and thromboembolic event in patients with periodontitis7. Second, periodontitis can indirectly alter systemic homeostasis through the release of endotoxins as well as inflammatory cytokines, and lead to endothelial dysfunction8. In addition, in epidemiology, both atherosclerosis and periodontitis may be modified by similar risk factors, e.g., age, elements of tobacco and metabolic disorders10. However, the findings for the association between periodontitis and atherosclerosis markers, i.e., carotid intima-media thickness (cIMT) were inconsistent among middle- and old-aged individuals in previous reports12. The conflicting results might be due in part to many risk factors prevalently presented in the process of atherosclerosis and periodontitis development in older individuals, and the association might be over-adjusted if these risk factors were all included as confounders. It is critical to carry out a study in a population with low vascular risk factors exposure, e.g., military subjects who are healthier and have better physical activity than the general population, and are unlikely to have atherosclerosis, to verify the association. Therefore, this study aimed to investigate the association between periodontitis severity and cIMT, a marker of subclinical atherosclerosis, in a population of military young adults in Taiwan.Atherosclerosis, the main process in atherosclerotic cardiovascular diseases, is a focal thickening of the arterial intima residing between the endothelial lining and smooth muscle layers of arterial blood vessels in response to an immune response16. Participants who underwent both an oral examination and a carotid ultrasonography were included. Participants whose age older than 40 years and total leukocyte counts greater than 11.0 × 103/µL were excluded to avoid a presence of unrecognized infectious diseases in the body. The study protocol was approved by the Ethics Committee of the Mennonite Christian Hospital (No. 16-05-008) in the Hualien City of Taiwan and the study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2013. Participants were informed the nature of the study, and all gave written informed consent to provide the ability to opt out without consequence.This study was a cross-sectional study including participants without taking any antihypertensive medications from the cardiorespiratory fitness and health in eastern armed forces (CHIEF) atherosclerosis study in Hualien City, Taiwan in 2018–2020, which has been described in detail previously17. Blood samples were collected in the morning after an overnight fast for total cholesterol, low density lipoprotein (LDL-C), high density lipoprotein (HDL-C), triglycerides, fasting glucose (FPG), serum uric acid and leucocyte counts. Dyslipidemia was defined as total cholesterol ≥ 200 mg/dL, LDL-C > 100 mg/dL, or triglycerides ≥ 150 mg/dL in both men and women. HDL-C < 40 mg/dL in men and HDL-C < 50 mg/dL in women were also defined as dyslipidemia17. Hyperuricemia was defined as serum uric acid ≥ 7.0 mg/dL in men and ≥ 6.0 mg/dL in women18. Systemic inflammation was defined as elevated leucocyte counts ≥ 7.6 103/µL19. Measurement of systolic blood pressure (SBP)/diastolic blood pressure (DBP) at rest over right upper arm was performed once, using an automatic device . Hypertension was diagnosed as SBP ≥ 130 mmHg and/or DBP ≥ 85 mmHg17. Before the physical examination, participants were asked about their alcohol consumption and tobacco smoking status (active versus former/never)21, and daily tooth brushing frequency.Waist circumference, body height and body weight were assessed in a standing position. The body mass index (BMI) was defined as body weight in kilograms divided by square of body height in meters. Abdominal obesity was defined as waist circumference ≥ 90 cm for men and ≥ 80 cm for women22. Since the study was aimed at physically fit young adults, the extent of periodontitis was merely restricted to the localized (< 30% of teeth involved)22. Stage I periodontitis was defined if either one criterion was fulfilled: (1) interdental clinical attachment loss (CAL) of 1–2 mm at side of greatest loss; (2) radiographic bone loss < 15%; (3) no tooth loss due to periodontitis; (4) maximum probing pocket depth (PD) ≤ 4 mm, or mostly horizontal bone loss. Stage II periodontitis was defined when either one criterion was fulfilled: (1) interdental CAL of 3–4 mm at side of greatest loss; (2) radiographic bone loss of 15–33%; (3) no tooth loss due to periodontitis; (4) maximum PD ≤ 5 mm, or mostly horizontal bone loss. Stage III periodontitis was defined if either one criterion was met: (1) interdental CAL ≥ 5 mm at side of greatest loss; (2) radiographic bone loss extending to mid-third of root and beyond; (3) with tooth loss because of periodontitis; (4) in addition to Stage II periodontitis severity further with PD ≥ 6 mm, or vertical bone loss ≥ 3 mm, or class II/III furcation involvement, or moderate ridge defect. No cases of Stage IV periodontitis were observed in this study. Full mouth oral examination and radiograph of each participant were performed by the same dentist. All subjects returned to the outpatient department for a re-evaluation and a treatment for the oral pathologies within one month. The inter-observer agreement (kappa coefficient) for verifying the severity of periodontitis was estimated 90.6%26.The 2017 AAP/EFP joint workshop on the classification of periodontal diseases were used for the definition of periodontitis stagesp robe of 4–8 MHz on the iE33 machine . Intima–media interface diameter of each participant was manually traced as a continuous line for a calculation13. To verify the cIMT, the measurement procedure was performed repeatedly by the technician, and the coefficient of variation was up to 96.8%14. In whole of the carotid sonographic examinations, no carotid plagues were found. The increased cIMT was defined as those over the highest quintile level, corresponding to the diameter ≥ 0.8 mm, which was modestly greater than the upper normal cIMT level (0.75 mm) in the general population in prior studies27.All ultrasonographic images were acquired at end diastole (defined as the R wave of an electrocardiogram) by the same certificated technician and verified by a cardiologist. The cIMT diameter was quantitatively measured between the leading edges of the media–adventitia interface and the lumen–intima interface over the far wall of the left carotid bulb, using an ultrasound scanner equipped with a linear p < 0.05 was considered to be statistically significance.Periodontitis severity was classified as health, Stage I, Stage II and Stage III. Group differences among the four groups were evaluated using the analysis of variance (ANOVA) for continuous variables and chi-square test for categorical variables. Analysis of covariance (ANCOVA) with adjustments for age, sex, BMI, SBP, DBP, tooth brushing frequency, alcohol intake and tobacco smoking was used to examine if a difference in the mean of cIMT existed among the four groups. We utilized univariate and stepwise multiple linear regression models to assess the association of periodontitis severity with cIMT. In model 1, age, sex, SBP, DBP, waist circumference, lipid profiles, FPG, serum uric acid, tooth brushing frequency, alcohol intake and smoking status and leukocyte counts were adjusted. In model 2, BMI was further adjusted. Univariate and multiple logistic regression models were also utilized to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) of periodontitis severity for increased cIMT ≥ 0.8 mm. In Model 1, age, sex, teeth brush frequency, alcohol intake, tobacco smoking, metabolic syndrome risk factors, e.g., dyslipidemia and hypertension, and elevated leukocyte counts within normal limits were adjusted. In model 2, BMI was additionally adjusted. In addition, participants were divided into four groups, based on the presence( +) or absence(−) of elevated leukocyte counts and severer periodontitis (Stage II or Stage III) to investigate the moderation effect of elevated leukocyte counts in the association between severer periodontitis and increased cIMT ≥ 0.8 mm in the univariate and multiple logistic regression models. Since the sample of the male participants in this study were much more than the female participants, sensitivity analyses were performed for men only with regard to their clinical characteristics and the association of periodontitis severity with increased cIMT ≥ 0.8 mm. The statistical analysis was carried out using SPSS software , and a value of 3/µL (N = 10) were excluded, leaving a sample of 486 subjects for analysis . The clinical characteristics of the men are shown in supplemental Table Of those who underwent a carotid ultrasonography for cIMT measurement N = 1,822), participants who received an oral examination were included (N = 501). Those who were older than 40 years of age (N = 5) and those with total leucocyte counts > 11.0 × 10,822, parp-values < 0.01, respectively). Of the metabolic variables, only total cholesterol and LDL-C were associated with cIMT levels  and − 0.001 , respectively, both p-values < 0.05), while there were no associations for waist circumference, SBP and DBP. The main results were not changed in Model 2.The results of the univariate and multiple linear regression analyses for the association of periodontitis severity with cIMT were revealed in Table p-value for trend = 0.001]. Of the other risk factors, systemic inflammation reflected by total leucocyte counts ≥ 7.6 103/µL was the only risk factor associated with cIMT ≥ 0.8 mm [OR 1.86 ]. Notably, there were no associations for the metabolic syndrome risk factors, e.g., abdominal obesity and hypertension. The main results were not changed in Model 2. With regard to the sensitivity analysis for men only (supplemental Table The results of the univariate and multiple logistic regression analyses models of periodontitis severity, systemic inflammation and metabolic syndrome risk factors for increased cIMT are revealed in Table The moderation effects of systemic inflammation on the association of severer periodontitis for increased cIMT are shown in Table The principal findings of this study were that among young adults who have a low vascular risk burden, periodontitis severity was positively associated with cIMT levels. In addition, there was a dose–response association for clinically increased cIMT with greater periodontitis stages. Systemic inflammation rather than metabolic risk factors and blood pressure was found to be associated with clinically increased cIMT, and systemic inflammation might be a moderator between periodontitis and subclinical atherosclerosis during young ages.28, indicating that age was a moderator for the association between periodontitis and atherosclerotic cardiovascular diseases. In addition, the severity of periodontitis may increase risk of atherosclerosis in some animal studies30. With regard to cIMT, Cairo et al. also revealed that severe periodontitis was associated with cIMT ≥ 0.82 mm in young healthy adults, which was in line with the present study finding31. On the contrary, López-Jornet et al. revealed that periodontitis was associated with carotid atheroma plaques while not cIMT in middle- or old-aged individuals11. Range et al. also found periodontal pathogens and neutrophil activation within atherosclerotic carotid plaques, supporting a link between severe periodontitis and carotid atheroma plaques formation in older individuals32. This study was the first report using physically fit young adults to maximally reduce the effect of age and clarify the association between periodontitis and cIMT.Epidemiological evidence suggested that periodontitis may be associated with a greater risk of incident cardiovascular diseases in adults, and the association was found stronger in younger adults than in older ones3. In previous studies, various mechanisms linking periodontal disease to cardiovascular disease, e.g., increased cytokines due to inflammation, bloodstream infection, dental plaque accumulation and alveolar bone loss have been proposed in middle- and old- aged individuals37. In this study, the mechanisms for the association between periodontitis and subclinical atherosclerosis among young adults were mainly reasoned by systemic inflammation assessed by elevated total leukocyte counts. In patients with periodontitis, cytotoxic neutrophil proteases and histones are responsible to ulcer formations and discontinuity on the sulcular epithelium33. Daily oral hygiene practices, e.g., tooth brushing and flossing might access the pathogens and its lipopolysaccharide from periodontal pocket to the peripheral blood via the sulcular epithelium, which foster bacteremia and endoxemia34. In addition, chronic infection status, e.g., low-grade endoxemia and inflammatory cascade, in which the circulating cytokines bind to the signaling receptors and ligands to generate an inflammatory circle35.Periodontitis was considered as a promoter of many systemic diseases, but the signaling pathways for the interconnection remain elusive38. In addition, a large body of evidence from epidemiologic studies also supported an association between metabolic disorders and atherosclerosis9. Periodontitis contributes to the progress of atherosclerosis, through the subgingival biofilm, either directly modifying lipid structure by bacteria or indirectly stimulation of inflammatory pathways to affect lipid synthesis and atheroma development34. Ylo¨stalo et al. reported an association between periodontal infection and cIMT in middle-aged adults with a low HDL-C level39. Metabolic disorders, however, the present study did not show any association with cIMT, possibly because of merely young fit adults included. Age might play a crucial role in this relevance. Atherosclerosis is a process involving inflammation and lipids interaction that is required decades of time to develop clinically significant diseases. In contrast, this study showed periodontitis and inflammatory markers were independent risk factors of clinically greater cIMT, indicating inflammation status is critical for the development of atherosclerosis in young adults.Metabolic disorders were common non-communicable diseases and previous studies have shown a bidirectional adverse association with periodontitis40 which is highly related to age of military personnel. In this regard, the effects of oral health behaviors on the association between periodontitis and cIMT in military require further investigation.Although oral hygiene was emphasized and obliged in military in Taiwan that all our study participants were found at least once daily tooth brush, some oral unhealth behaviors, e.g., smoking and alcohol intake were prevalent and might increase the risk of periodontitis. In addition, the oral hygiene status in military was found to be associated with rankSome limitations should be considered while interpreting the study findings. First, evidence of the causality of periodontitis for atherosclerosis could not be obtained for the cross-sectional design. Besides, although leucocyte count was a reliable biomarker of inflammation, other inflammatory mediators such as C-reactive protein and interleukin-6 were not available in this study. Moreover, the findings in the military cohort in the present study might not be applicable to the general population of young adults. Finally, more than 90% of the study subjects were men, and thus whether the results were reliable for women may require further study. However, several advantages were present in this study. First, military subjects were characterized by highly homogeneity with unified life style and no regional differences related to access to the dental care, which could reduce unmeasured confounders. Second, there were no participants with diabetes, which has been recognized as an important risk factor for both periodontitis and atherosclerosis.This study suggests that periodontitis and increased leukocyte counts within normal limits are independent risk factors of clinically increased cIMT, whereas no associations were present between other metabolic risk factors and clinically increased cIMT in young adults, highlighting the critical role of inflammation status in subclinical atherosclerosis. In clinical practice, an oral examination for the presence of severer periodontitis incorporating a blood test for the presence of increased leukocyte counts within normal limits could be treated as the signs of increased cIMT in young adults which an early prevention of cardiovascular diseases could be taken by physicians. Future study should be aimed to lower the inflammatory status, i.e., periodontitis therapy, in young adults to prevent the following clinical atherosclerotic cardiovascular diseases development.Supplementary Information.


Photoautotrophs’ environmental responses have been extensively studied at the organism and ecosystem level. However, less is known about their photosynthesis at the single-cell level. This information is needed to understand photosynthetic acclimation processes, as light changes as it penetrates cells, layers of cells, or organs. Furthermore, cells within the same tissue may behave differently, being at different developmental/physiological stages. Here, we describe an approach for single-cell and subcellular photophysiology based on the customization of confocal microscopy to assess chlorophyll fluorescence quenching by the saturation pulse method. We exploit this setup to (1) reassess the specialization of photosynthetic activities in developing tissues of non-vascular plants; (2) identify a specific subpopulation of phytoplankton cells in marine photosymbiosis, which consolidate energetic connections with their hosts; and (3) examine the link between light penetration and photoprotection responses inside the different tissues that constitute a plant leaf anatomy.  •Real-time assessment of chlorophyll fluorescence at confocal microscopy resolution•Study of photosynthesis at single-cell resolution within a living tissue/organism•Relationship between photosynthetic features and cell type or morphology•Mathematical modeling of photosynthesis in a cell volume 3D space Visualizing photosynthetic responses in 3D is essential for understanding most acclimation processes, as light changes within photosynthetic tissues as it penetrates the absorbing/diffusing layers of the cells. To achieve this goal, we developed an imaging workflow merging confocal microscopy and saturating pulse chlorophyll fluorescence detection. This method applies to samples characterized by increasing complexity, and its simplicity will contribute to its widespread use in plant and microalgae photoacclimation studies. Storti et al. develop a method to study photosynthesis at single-cell resolution using confocal microscopy. They use this approach to examine photosynthesis specialization in different tissues and organisms and to understand the relationship between light penetration and photoprotection. The efficiency and regulation of this process is usually assessed in vivo by measuring chlorophyll (Chl) fluorescence,,,,,,,2 assimilation capacity,Photosynthesis is a major bioenergetic process in the biosphere. It feeds most of the food chains on Earth and is responsible for substantial sequestration of CO−2 s−1) to saturate PSII and thus achieve maximum Chl fluorescence emission, Fm.,To develop a 3D saturating pulse confocal /Fm, decreased when the laser intensity increased. The Y decrease reflects the actinic effect of blue laser itself, which increases the F′ parameter. However, the lack of fluorescence quenching during exposure to multiple (four) laser pulses before the saturating pulse  of the moss ng pulse B demonstulse −2, A and 2B,ulse −2, F. On theulse −2, B.Figure 2 assimilation and photoprotective responses: at the beginning of illumination, when CO2 assimilation is largely inactive, part of the absorbed light is dissipated. Conversely, most of the absorbed photons are drained to CO2 assimilation in steady state  and thus NPQ disappears.The 3D-Pulse fluorimeter was fast enough to measure the kinetics of NPQ onset (when the actinic light was switched on) and its relaxation in the dark . We coulP. patens protonema exposed for 6 min to 500 μmol photons m−2 s−1. Time series that acquired with 1 min delay are reproduced at 1 frame per second (fps). The sum of raw fluorescence values for the different stacks is represented in an artificial color scale bar on the top.Four time-course Z-projections (15–20 slices) of P. patens lhcsr1-2 KO protonema exposed for 6 min to 500 μmol photons m−2 s−1. Time series that acquired with 1 min delay are reproduced at 1 frame per second (fps). The sum of raw fluorescence values for the different stacks is represented in an artificial color scale bar on the top.Four time-course Z-projections (15–20 slices) of P. patens: the caulonema and the chloronema. The former has longitudinally elongated cells involved in propagation and nutrient acquisition, the latter has chloroplast-rich cells, usually considered as the photosynthetic part of the moss protonema  were separated based on their NPQ capacity, while the two cell types  could be differentiated because of their plastid cell density. A more refined analysis  revealed subtle heterogeneity in NPQ responses in both caulonema and chloronema, which we could interpret based on 3D imaging. We identified heterogeneous NPQ responses at the cell level  and chlorophyll fluorescence (red) images acquired with an upright Leica DM6 B  optical microscope. Image size = 342.58 × 256.89 μm. Time series acquired with a delay of 30 s between different images and reproduced at 2 frames per second.,,,,Phaeocystis cordata, ,We further investigated the ability of the 3D-Pulse fluorimeter to link NPQ responses to different physiological states focusing on photosymbiosis, a common lifestyle in oceanic plankton between symbiotic microalgae and unicellular eukaryotic hosts.Phaodactylum tricornutumChlamydomonas reinhardtiiRelating plastid volume heterogeneity to single-cell NPQ responses revealed the existence of two symbiont populations: small algae (open symbols) exhibited lower NPQ C and phoPhaeocystis plastids separately because these organelles are around 10 times smaller than the ones found, e.g., in plants and P. patens.,We investigated their NPQ features at the subcellular level. However, it was difficult to track single Plantago lanceolata), we recorded similar NPQ responses on the adaxial and abaxial sides , which harbor an asymmetric mesophyll organization  disentangling heterogeneous cellular responses within a tissue , (2) traHowever, there is a main difficulty associated with this approach. The choice of the laser light intensity is essential to ensure correct measurements of the photosynthetic parameters. In our case, the maximum photosynthetic capacity (the “Y” parameter in dtolleter@gmail.com).Further information and requests for resources and reagents should be direct to and will be fullfilled by the lead contact, Dimitri Tolleter (This study did not generate new unique reagents.Physcomitrium patens Gransden wild-type (WT) strain and lhcsr1-lhcsr2 KO (lhcsr1-2)3)2, 1 mM MgSO4, 50 μM FeSO4, 200 μM KH2PO4 pH 7, traces elements)2 s−1 continuous illumination. 10-days-old moss protonema was employed for confocal microscopy measurement.Non treated seeds of Flaveria robusta, Flaveria bidentis and Plantago lanceolata has been sown on soil  and grown 3–4 weeks in controlled growth chambers in long day conditions (16 h light/8 h dark) at a PPF of 100 μmol photons m−2 s−1. Air temperature was 22°C during the day and 21.0 °C at night. Relative humidity was constant at 70% during the day and night. Young fully developed leaves were chosen for the experiments.Phaeocystis cordata) were gently collected by towing a plankton net of 150 μm in mesh size with a large cod-end (1 L) for 1–2 min in surface waters . After collection, individual cells were isolated under a binocular with a micropipette.2 s−1 controlled illumination. Samples were imaged within 24 h from sampling time. Cultures of the haptophyte P. cordata  of P. patens protonema was endorsed on a 20 × 20 mm coverslip and soaked in 100 μL of water in order to spread the filaments. The coverslip was fixed to the microscopy slide by using a double-side tape of 0.15 mm thickness and finally sealed using VALAP  to prevent water evaporation.P. cordata and Acantharians were settled on a microscopy glass bottom dish  directly on the microscopy plate to avoid media perturbation.F. robusta, F. bidentis and P. lanceolata. 5 × 3 mm sections  were excised using a scalpel. Sections were enclosed between two 20 × 20 mm coverslips allowing to observe both sides of the leaves. A double layer of tape was used to create an enclosure to lodge the leaf section and prevent its crushing. Water was provided to the sample to prevent dehydration during NPQ measurements.All samples were dark adapted for at least 20 min before introduction in the setup.−2 s−1) is determined by a 3-position switch located on the front face of the control box. Electronic diagrams of the control box are shown −2 s−1, duration 1.1 s) to measure of Fm and Fm’. Alternatively, saturating pulses can be switched on manually through a button located on the control box. Refer to The Zeiss LSM 900 microscope was equipped with continuous light and pulses of strong actinic light, provided by a LED module located in front of the biological sample. The module contains 4 red LEDs , equipped with a lens to reduce their divergence. The 4 LEDs were oriented at 42°, to focus their light onto the middle of the sample holding slits. The LEDs deliver actinic light, the intensity of which (100 and 200 and 500 μmols photons m−2. s−1 in white light) to minimize any potential irreversible impact on photosynthesis. Similarly, the determination of z-stacks boundaries was performed at an extremely low intensity level (30 times lower than the one utilized during the experiment) to ensure the integrity of the experiment and prevent undesired effects. The initial z position was set as the first slide where the fluorescence of chloroplasts, derived from chlorophyll fluorescence, could be clearly distinguished from the background. The initial z position was set as the first slide where the fluorescence of chloroplasts, derived from chlorophyll fluorescence, could be clearly distinguished from the background.,Images were acquired with a 20x/0.8 M27 objective (Zeiss) in confocal mode setting with Airyscan 2 detector. This setup has been chosen for fast acquisition without loss of sensitivity, thanks to the use of a collimation optic. Excitation was provided by a blue laser (488 nm) and Chl fluorescence was collected between 650 and 700 nm. Searching for the object under the microscope was conducted under low light intensity conditions (5 μmol photons. mTo estimate maximum photosynthetic capacity (Y), a series of 10 consecutive images was acquired . A saturating pulse was provided by the external LED source after the fourth acquisition A.−2) at which PSII fluorescence reached Fm to avoid excess excitation. The delay for two consecutive acquisitions was set to 1 min to prevent photoinhibion due to laser exposure. 3 points were acquired for dark adapted samples, 6 during exposure to actinic red light (LED module) and 6 points again in the dark to follow NPQ relaxation  were selected to include entire cells and maintain plastids in the acquisition field. The blue laser intensity was set at the minimum value  were scanned for every cells to assess reproducibility.To calculate the fluorescence of each object in a confocal image, images were segmented to obtain the location of a given ROI). Data were used to calculate the sum of the voxel values of a given ROI, and therefore the mean fluorescence value of a given plastid or cell. In the case of We performed PCA considering six observed variables: NPQav, NPQmax, Decay, Induction, Fmax and Area Fraction. The variables used here were calculated in automatized way from NPQ data. NPQav is the mean NPQ when a cell is exposed to light while NPQmax is the maximum of NPQ reached during the light exposition. Decay and Induction are the relaxation and induction rate of NPQ evaluated from the slope of NPQ changes during the first 2 min of the light to dark and the dark to light transitions, respectively. Area Fraction is the area percentage occupied by the chloroplast inside a cell. Fmax is the “Mean gray value” of fluorescence of a given cell chloroplasts for dark adapted samples (Fm).P. patens chloronema and caulonema in the WT or the lhcsr1-2 KO strains. The type of cells (mutant/WT or chloronema/caulonema) did not play a role in the determination of the component and that they are used after to characterise the possible biological role of the components. All data are normalized by subtraction of the mean and division by the standard deviation so that the singular values decomposition is done on the correlation matrix of the data (Variables were measured in 175 cells from the data .http://www.R-project.org). The table of the original observed variables used to construct the six components  giving the largest possible variance of the distribution was selected as the direction for the first principal component. Then, we selected the direction orthogonal to the previous one(s) giving the largest possible variance of the distribution as the direction for the second principal component. By repeating this procedure automatically, we identify vectors representing the scatter of the distribution from major ones to minor ones . Based omponents  provides