This study is aimed at raising the question of the use of several TIRADS systems that stratify the risk of thyroid nodule malignancy. Approximately 5–20% of thyroid nodules are malignant, but most nodules are benign, and they are scored by FNA biopsy. One of the goals is to reduce the number of unnecessary FNA and the associated with-it possible complications for the patient and financial cost. Most TIRADS systems are based on the fact that one suspicious feature of a thyroid nodule classifies it as malignant, but there is a modified Kwak et al. system that is based on the count of malignant features. Therefore, this study is intended to estimate the specificity, sensitivity, and accuracy of the systems and, in the future, think about reducing the number of FNA biopsies. The result of this study can be important for all doctors who face thyroid changes, such as radiologists, ultrasonography specialists, and endocrinologists, those who must decide about the need for an FNA.Background: Various Thyroid Imaging and Reporting data systems (TIRADS) are used worldwide for risk stratification of thyroid nodules. Their sensitivity is high, while the specificity is suboptimal. The aim of the study was to compare several TIRADS systems and evaluate the effect of hypoechogenicity as a sign of risk of malignancy on the overall assessment of diagnostic accuracy. Methods: The prospective study includes 274 patients with 289 thyroid nodules to whom US and risk of malignancy were assessed according to four TIRADS systems—European (EU-TIRADS), Korean (K-TIRADS), TIRADS by American College of Radiology (ACR TIRADS), and modified Kwak et al. TIRADS (L-TIRADS) systems, in which mild hypoechogenicity is not included in malignancy risk suggestive signs. For all thyroid nodules, a fine needle aspiration (FNA) biopsy was performed and evaluated according to the Bethesda system. For all systems, diagnostic accuracy was calculated. Results: Assessing the echogenicity of the thyroid nodules: from 81 of isoechogenic nodules, 2 were malignant (2.1%), from 151 mild hypoechogenic, 18 (12%) were malignant, and from 48 marked hypoechogenic nodules, 16 (33%) were malignant. In 80 thyroid nodules, mild hypoechogenicity was the only sign of malignancy and none appeared malignant. Assessing various TIRADS systems on the same cohort, sensitivity, specificity, PPV, NPV, and accuracy, firstly for EU-TIRADS, they were 97.2%; 39.9%; 18.7%; 99.0%, and 73.3%, respectively; for K-TIRADS they were 97.2%; 46.6%; 20.6%; 99.2%, and 53.9%; for ACR-TIRADS they were 97.2%; 41.1%, 19.0%; 99.0%, and 48.0%, respectively; finally, for L-TIRADS they were 80.6%; 72.7%; 29.6%; 96.3%, and 73.3%. Conclusions: This comparative research has highlighted that applying different TIRADS systems can alter the number of necessary biopsies by re-categorization of the thyroid nodules. The main pattern that affected differences was inconsistent hypoechogenicity interpretation, giving the accuracy superiority to the systems that raise the malignancy risk with marked hypoechogenicity, at the same time with minor compensation for sensitivity. The thyroid nodules are very common in the general population, and with the increase of imaging techniques, more thyroid nodules are accidentally detected . CompariNodular thyroid disease is relatively common. Most of the thyroid nodules are benign and asymptomatic, and their prevalence varies and accounts for about 85–93% in the population; in addition, 20% of them decrease in size over a lifetime . About 8Adenomas account for only 5% to 10% of all nodular thyroid diseases. In most cases, thyroid dysfunction is not observed, and less than 10% have hyperfunction, which can lead to thyrotoxicosis. Usually adenomas are solitary, but they can also develop as part of a multinodular formation .Benign follicular adenoma is a true neoplasm characterized by compression of adjacent tissues and fibrous encapsulation. Cytologically, the features of follicular adenoma are usually indistinguishable from those of follicular carcinoma. Signs of follicular carcinoma are vascular and capsule infestations that can be detected during histology, so fine needle aspiration (FNA) is not considered as a reliable method to distinguish adenoma from carcinoma; therefore, tumors are usually removed surgically .However, thyroid cancer is rare and accounts for less than 1% of all malignancies . It accoFollicular carcinoma is the second most common thyroid cancer type and accounts for 10 to 15% of all thyroid cancer .Medullary thyroid carcinoma accounts for 3.5 to 10% of all thyroid cancers and is characterized by early lymphatic metastatic spread, aggressive invasion in adjacent tissues and organs, and has a poor prognosis. On the ultrasound examination, medullary carcinoma usually looks like papillary carcinoma and is perceived as a solid, hypoechogenic nodule, often with calcifications, which may look more visually coarser than in papillary carcinoma. Calcifications are observed in the primary tumor as well as in the metastatic lymph nodes .Anaplastic thyroid carcinoma usually affects elderly people and is one of the deadliest solid thyroids tumors. It accounts for less than 2% of all thyroid cancers but has the worst prognosis, with 5-year mortality above 95%. The tumor manifests with a rapidly growing mass that extends beyond the gland and grows into adjacent structures. It does not tend to lymphogenic dissemination, but it aggressively grows in adjacent muscles and fat tissue. Sonographically, anaplastic cancer is usually hypoechogenic and often surrounds or grows into blood vessels and neck muscles. Often, these tumors cannot be properly evaluated by the ultrasound due to their large size. In this case, the extent of damage can be assessed more accurately by computed tomography or magnetic resonance imaging .In this regard, the clinical challenge is to distinguish clinically significant malignant thyroid nodules and thus to identify patients who will require surgery for benign thyroid nodules and who will require long-term observation. That is why the main purpose of US thyroid nodule examination is to differentiate malignant nodules from benign ,13,14.In the past decade, several professional societies and research groups have implemented guidelines to provide a standardized assessment of ultrasonographic features of thyroid nodules, such as Thyroid Imaging Reporting and Data System (TIRADS), to assess the need for fine needle aspiration biopsy (FNA) .Initially, in 2009, Horvath et al. introduced a thyroid malignancy risk stratification system—TIRADS , but it Most variants of the TIRADS systems include mild hypoechogenic nodules in the potential risk group or TIRADS 4, however, several studies report that most thyroid nodules are mild hypoechogenic, both benign and malignant ,18,19,20Several studies have been performed comparing thyroid malignancy risk assessment systems. The sensitivity of these systems is high enough, but specificity is quite low. Therefore, in this study we compare four TIRADS systems—Kwak et al., hereafter, referred to as L-TIRADS, European TIRADS (EU-TIRADS), Korean TIRADS (K-TIRADS) and American College of Radiology TIRADS (ACR-TIRADS) systems—including the sensitivity and specificity and evaluate the effect of hypoechogenicity as a sign of risk of malignancy on the overall assessment of diagnostic accuracy.This was a prospective multicentre study that was conducted in the period from 2019 to 2021. In total, 274 patients with 289 thyroid nodules underwent the conventional ultrasound (US) examination and all patients with clinical indication for fine needle aspiration biopsy (FNA) were included in the study. The study was approved by the institutional review boards and the responsible ethics committee.Without a regular clinical description of ultrasonographic examination, a standard protocol was additionally developed, with included patient data, suspicious ultrasound features of thyroid nodules, as well as all the necessary features for such systems as the TIRADS system used in Latvia (L-TIRADS), European (EU-TIRADS), Korean (K-TIRADS), and ACR TIRADS system. In addition, the protocol included such indicators as multinodular goiter, thyroiditis, suspicious lymph nodes in four groups of the neck—lateral and medial lymph node groups of right and left side.In the study, thyroid ultrasonography was performed with an ultrasonography device “Canon Aplio i800” with a linear probe i18LX5 (5–18 MHz), and with “Philips EPIQ 5G” with a linear probe eL 18-4. The ultrasound examination was performed by one of five certified radiologists with five and more years of experience. All ultrasound and biopsies, as well as documentation, were carried out by one radiologist. After ultrasound, the description documented the localization of the thyroid nodules, its size, contour, borders, shape, internal components, echogenicity, calcifications, vascularization, as well as the appearance of the thyroid parenchyma and lymph nodes of the neck.Based on ultrasonographic features, each thyroid nodule was evaluated according to four TIRADS systems.The modified Kwak et al. TIRADS system  that is According to the European TIRADS system  (EU-TIRAIn accordance with the Korean TIRADS system  (K-TIRADAmerican College of Radiology TIRADS (ACR TIRADS)  is basedThe US-guided FNA biopsy was performed with a 23-gauge needle, three aspirations were performed according to the local standards, and the material was applied onto glass slides and sent for cytology evaluation ,26. Sampp-value < 0.05.IBM SPSS software, 22.0 version , MS Excel 2010 software were used to analyse statistical data and create graphs. Pearson correlation was used for correlation between Bethesda categories and all four TIRADS systems that were included in the study. Sensitivity (SEN), specificity (SPE), positive predictive value (PPV), negative predictive value (NPV), accuracy (ACC), 95% confidence interval (CI), area under curve (AUC) were calculated for each TIRADS system—L-TIRADS, EU-TIRADS, K-TIRADS, and ACR-TIRADS. Cronbach’s Alpha was calculated for reliability between L-TIRADS and EU-TIRADS, L-TIRADS and K-TIRADS, L-TIRADS and ACR-TIRADS. The statistical significance level of this study was assumed to be p < 0.001). The age of the patients included ranged from 23 to 85 years, with a mean age of 55 years. The mean age was 55 years for women and 52 years for men. Most patients were in the age group from 60 to 69 years—69 patients (23 %).For a duration of 18 months, 289 thyroid nodule ultrasounds and fine-needle aspirations in 274 patients were analysed. Most patients were women , and only 41 (14.9%) men were included in this study. Using the binomial test, it was concluded that in our study the proportion of women and men (85.1%/14.9%) was statistically significantly different .In the study, 151 (52.6%) thyroid nodules were mild hypoechogenic compared to thyroid parenchyma, of which 133 (88.0%) were benign and 18 (12.0%) were malignant. A total of 48 thyroid nodules were marked hypoechogenic, compared to the neck muscles of which 32 (66.(6)%) were benign and 16 (33.(3)%) were malignant. From 81 (28.2%) isoechoic nodules 79 (97.5%) were benign, and only 2 (2.5%) were malignant, and all hyperechoic nodules  were benign . Using Sp <0.001).In the study of 231 thyroid nodules, the maximum diameter was noted, from which the majority  of thyroid nodules were more than 1 cm diameter (>1 cm), and 39 (16.9%) thyroid nodules were less than 1 cm diameter (<1 cm). From 192 thyroid nodules that were >1 cm diameter, 176 (91.6%) were benign, and 16 (8.4%) were malignant. Of the 39 thyroid nodules that were <1 cm diameter, 26 (66.(6)%) were benign, and 13 (33.(3)%) were malignant . Using SA summary of thyroid nodules by cytological Bethesda category and L-TIRADS, EU-TIRADS, K-TIRADS, and ACR-TIRADS is shown in Category I or non-diagnostic results were observed in 20 (6.9%) fine needle aspirations, category III of atypia of undetermined significance were in 17 (5.9%) cases.IV category or suspicious of follicular neoplasm were in 4 (1.4%) cases, only the L-TIRADS system did not categorized these nodules as TIRADS 5, and the most accurate evaluation was performed by EU-TIRADS and ACR-TIRADS systems.V category or suspicious of malignancy were in 10 (3.5%) cases, and malignant nodules or category VI were identified in 26 (9.0%) cases. Comparing EU-TIRADS, K-TIRADS, and ACR-TIRADS that showed similar rates of TIRADS 5 nodules in the Bethesda VI category (more than 8%), L-TIRADS categorized the Bethesda VI nodules in TIRADS category 4 A to C, resulting in the reduced sensitivity for TIRADS 5 (2.1%).p < 0.001), as well as between Bethesda classification and EU-TIRADS , between Bethesda classification and K-TIRADS , and between Bethesda classification and ACR-TIRADS  systems there was also a positive, statistically significant, moderately strong correlation.Using Pearson correlation, it was found that there was a positive, statistically significant, moderate correlation between Bethesda classification and L-TIRADS  than other systems—EU-TIRADS , K-TIRADS  and ACR-TIRADS . All TIRADS systems showed similar area under the ROC curve, but the L-TIRADS AUC value was better 0.766, followed by K-TIRADS with an AUC value 0.719 , however L-TIRADS showed higher specificity (SPE = 72.7%) and a better accuracy  and including predominantly women (85.1%), in accordance with the several studies that also noted a higher prevalence of thyroid nodules in women and noted that the incidence of thyroid nodules increases after the age of 50 ,30. A toVarious TIRADS systems  are used to define thyroid nodule risk of malignancy and to select patients for FNA. All systems are based on thyroid nodule malignancy features evaluation such as hypoechogenicity, microcalcifications, shape proportions (AP > LL), ill-defined and irregular margin. For these systems, diagnostic accuracy is similar, with high sensitivity and low specificity ,32. In oHypoechogenicity is one of the main but unspecific features of thyroid nodules on US . The posIn the L-TIRADS system, which is the Kwak et al. modified version, mild hypoechogenicity is not included in the malignancy features. Mild hypoechogenic thyroid nodules, which do not have any other malignant features, are stratified as thyroid nodules with low risk of malignancy or TIRADS 3 category nodules, which results in higher specificity and diagnostic accuracy, 72.7% and 73.3%, respectively, and therefore showed different performance in comparison to the other systems with remaining high NPV 96.3% and a comprehensive decrease in sensitivity 80.6%. Based on the cytological response, among the nodules categorized as L-TIRADS 5, 58% were cytologically malignant (Bethesda 5 and 6), while from the nodules categorized as K-TIRADS, 5–31% were cytologically malignant, from nodules categorized as EU-TIRADS, 5–29% were cytologically malignant, and from thyroid nodules that were categorized as ACR-TIRADS 5, only 13% were cytologically malignant. Comparing EU-TIRADS, K-TIRADS, and ACR-TIRADS that showed similar rates of TIRADS 5 nodules in the Bethesda VI category (more than 8%), L-TIRADS categorized the Bethesda VI nodules in TIRADS category 4 A to C, resulting in the reduced sensitivity for TIRADS 5 to only 2.1%. This trade-off of the raised suspicion and lack of clear categorization of malignancy is compensated by the L-TIRADS system’s high performance in the TIRADS 3 category with two-times higher true benign nodules (Bethesda 2).The aim of TIRADS systems is to evaluate thyroid nodule risk of malignancy and to select patients for FNA. In these TIRADS systems, indications for FNA are based on thyroid nodule malignancy risk assessment or TIRADS category and size of the nodule ,24,35. LAnalyzing echogenicity as malignancy risk evaluation criteria, it seems that only 12.0% of the mild hypoechogenic nodules were malignant, which means that this feature is not specific by itself, while among the marked hypoechogenic nodules, 66.6% were malignant, which means that every two out of three marked hypoechogenic nodules are malignant and this proves that this feature is specific for malignancy. The result of our study shows that mild hypoechogenicity cannot serve solely as a malignancy risk feature and may increase FNA rates unnecessarily. From 80 thyroid nodules, in which mild hypoechogenicity was the only possible malignancy sign, no cytological malignancy (Bethesda 5 and 6) by FNA was found. This approved the concept of mild hypoechogenicity with lower values to be considered as a sign of malignancy, as described in several TIRADS systems: ACR TI-RADS non-marked hypoechogenicity without other signs is classified as TIRADS 3 (mild suspicious), EU-TIRADS category 4 (intermediate risk) and K-TIRADS also as category 4 (intermediate suspicion) ,23,24.Regarding the size of the nodule—one of three nodules with a diameter less than 1 cm was malignant, but it should be noted that most of the small nodules firstly appear as relatively hypoechogenic and for these sub-centimeter nodules, FNA biopsy may be advised only if they have several signs of malignancy.There were some limitations in our study, such as the examination of the thyroid gland was performed using two ultrasound machines, and in our study, there were several ultrasound specialists who performed examination and FNA, which could lead to different assessments and decisions about FNA of thyroid nodules. Furthermore, some of the patients had to be excluded from the study due to non-informative results of cytology, which may be due to imperfect ultrasound control during FNA, as well as changes in image quality. Lack of histology results in our cohort should be noted as a major limitation.In conclusion, EU-TIRADS, K-TIRADS, and ACR-TIRADS showed high sensitivity, compared to L-TIRADS; however, the specificity and accuracy were higher for the L-TIRADS system. The main pattern that affected differences was inconsistent hypoechogenicity interpretation, giving accuracy superiority to the systems that raise the malignancy risk with marked hypoechogenicity and at the same time with minor compensation for sensitivity. This comparative research has highlighted that applying different TIRADS systems can alter the number of necessary biopsies by categorization of the mild hypoechogenic thyroid nodules as a low malignancy risk pattern.


Computer-assisted jaw reconstruction (CAJR) has benefits in reducing operation time and improving reconstruction accuracy, compared to conventional freehand jaw reconstruction. However, no information is available regarding learning curves in CAJR with the use of 3D-printed patient-specific surgical plates (PSSP). The purpose of this study was to assess surgical outcomes and learning curve for the first 58 consecutive CAJR using 3D-printed PSSP performed by a single surgical team in a single institution.In a prospective study, consecutive patients who underwent free flap CAJR using 3D-printed PSSP were included. The determination of proficiency, based on the cumulative sum of surgical success  passing the acceptable boundary line of cumulative sum analysis, was the primary outcome. To find out any potential factors influencing the learning curve, baseline characteristics of patients were compared before and after proficiency achievement. Secondary outcomes included inflexion points of the total operation time, blood loss, length of hospital stay, and bone graft deviation, measured by the cumulative sum analysis.p = 0.046 and p < 0.001, respectively). Mean values of operation time, intraoperative blood loss, length of hospital stay, and bone graft deviation were 532.5 ± 119.2 min, 1,006.8 ± 547.2 ml, 16.1 ± 6.3 days, and 0.9 ± 1.2 mm, respectively. Two trends of learning curve were observed in the CUSUM analyses of total operation time, length of hospital stay, and bone graft deviation, in which the first and second inflexion points occurred between 8 and 17 cases and between 43 and 46 cases, respectively.From December 2016 to November 2020, 58 consecutive cases underwent surgery performed by a single surgical team. The overall surgical success rate was 94.8% (55/58). A three-stage learning curve of primary outcome was observed. The proficiency was achieved after 23 cases. The proportions of advanced tumor staging and concomitant surgery after obtaining proficiency were significantly higher than those before achieving proficiency (Our results revealed a three-stage learning curve of CAJR with the use of PSSP, including initial learning, plateau, and overlearning. Based on CUSUM analysis, the surgical proficiency was achieved after 23 cases, and total operation time, length of hospital stay, and bone graft deviation stabilized after 8–17 cases. The emerging technique of computer-assisted jaw reconstruction (CAJR), which facilitates preoperative surgery simulation and transfers the virtual plan to a real operation, significantly impacted conventional surgical approaches , 2. StudWith the increasing popularization of CAJR surgery, the needs of standardizing surgical training and optimizing patient outcomes are urgent. The cumulative sum (CUSUM) control chart is a sequential analysis technique in statistical quality control, typically used for monitoring change detection . Now, thOur team started performing CAJR using 3D-printed patient-specific surgical plates (PSSP) in December 2016. We reported our first experience in 2018 and indicated that CAJR with PSSP is feasible, safe, and precise . This stwww.HKUCTR.com).This study was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (No. UW 16-315), registered in ClinicalTrials.gov with a No. of NCT03057223 and in The University of Hong Kong Clinical Trials Centre with a study identifier of HKUCTR-2113  and flap survival. Cases were divided into two groups according to the achievement of proficiency, and the baseline characteristics were compared to analyze the potential factors influencing the learning curves.The secondary outcomes were the stabilization of total operation time, intraoperative blood loss, length of hospital stay, and reconstruction accuracy to a steady state. The determination of stabilization was based on the inflection point of the cumulative graph. The accuracy of jaw reconstruction 1 month postoperatively was measured by calculating the deviation distance between preoperative planned and postoperative achieved bone graft positions using the same method described previously .CUSUM analysis was performed to detect subtle deviations of the surgeon’s performance in primary and secondary outcomes .n = 161/2,305) by the latest systematic review and meta-analysis (n = 6/136) (1) or unacceptable (H0). Once the line reached H1 or H0, proficiency was obtained or lost, respectively.The overall fibula flap failure rate was revealed as 7.0%  , 23–27. = 6/136) . All cal= 6/136) . BoundarThe sequential differences of total operation time, intraoperative blood loss, length of hospital stay, and bone graft deviation between each case and the mean value were also detected by CUSUM . The meat test was used to detect differences in the means in the patients’ age and donor bone length. Chi-square test was used to detect differences in the proportions of gender, diagnosis, TNM classification, surgical site, bone segments, and concomitant surgery. p < 0.05 was considered statistically significant.All statistics were calculated using SPSS Statistics . Data were presented as number (percentage) for categorical data and mean ± standard deviation for continuous data. Independent samples A total of 58 consecutive patients who underwent CAJR using PSSP were included. Baseline characteristics of total cases are shown in Altogether, surgical failure occurred in 5.2% of patients (3/58). One was intraoperative flap failure due to sclerotic vessels and recurrent arterial thrombosis. The other two cases were postoperative flap failures caused by venous compromise of flap at postoperative day 4 and late-stage artery thrombosis at postoperative day 10. 3D-printed PSSP were successfully used in all the patients. p = 0.046) and concomitant surgery (p < 0.001). From the 24th to 58th cases, the proportions of stage III & IV malignancy and concomitant surgery were significantly higher than the first 23 cases, indicating an increased proportion of cases with advanced stage malignancy and more complex surgery after first achieving the proficiency.According to the achievement of proficiency, we divided the patients into two groups. From the baseline characteristics of the two groups in The mean operation time was 532.5 ± 119.2 min. The inflexion point is the 8th case, from which the operation time started to diminish, although there was a slight trend of increasing operation time from the 28th case to the 46th case. The linear and CUSUM analysis graphs of total operation time are shown in Mean intraoperative blood loss was 1,006.8 ± 547.2 ml. There was a trend of decreasing blood loss in the first 22 cases. After that, the intraoperative blood loss was increasing. The linear and CUSUM analysis graphs of intraoperative blood loss are shown in Mean length of hospital stay was 16.1 ± 6.3 days. Four inflexion points were presented at the 5th, 17th, 28th, and 45th cases, respectively. The linear and CUSUM analysis graphs of hospital stay length are shown in Mean bone graft deviation was 0.9 ± 1.2 mm. After 15 cases, the bone graft deviation started to diminish as a general trend. However, from the 31st to 43rd cases, a slightly increasing trend of bone graft deviation was observed. The linear and CUSUM analysis graphs of bone grafts deviation are shown in The concept of learning curve in medicine was commonly defined as the time taken and/or the number of procedures an average surgeon needs to be able to perform a procedure independently with a reasonable outcome . As modeNowadays, computer-assisted surgery is increasingly utilized in reconstructive surgery, while no literature has reported the learning curve for this surgical procedure. In the present study, we performed CUSUM analysis on the learning curve of CAJR using 3D-printed PSSP by a single surgical team. Although operation time is the most common determinant for the learning curve on surgery, the assessment on the basis of a single parameter might be simplistic , 34. We For the primary outcome, our study revealed a three-stage learning pattern of CAJR with the use of PSSP, including initial learning, plateau, and overlearning. The first stage is initial learning. The learning curve regarding surgical success stabilized after 11 cases, and first achieved proficiency after 23 cases. The second stage is plateau. When surgeons felt competent in this surgical procedure, the significantly increased proportions of tumor staging and concomitant surgery suggested that the complexity of surgery after 23 cases was higher than the earlier cases. The concomitant surgery, simultaneous dental implant placement with the aid of a “three-in-one” patient-specific surgical guide , was alsFor the secondary outcomes, we found that although the four graphs of CUSUM analysis had different curve shapes, a similar ascending trend occurred from 23–31 cases to 43–46 cases. The increased complexity after 23 cases could also reasonably explain the increased total operation time and intraoperative blood loss. As a result, we actually observed two phenomena of “learning curve” in the CUSUM analysis graphs of the total operation time, length of hospital stay, and bone graft deviation. In the first learning curve (from 1st to 28th–31st cases), the inflexion points occurred between 8 and 17 cases, from which operation time, length of hospital stay, and bone graft deviation started to diminish. In the second learning curve (from 28th–31st to 58th cases), stabilizations of the operation time, length of hospital stay, and bone graft deviation occurred between 43 and 46 cases.The main novelty of this study was using the CUSUM technique to analyze the learning curve of CAJR with PSSP. We utilized a chosen level of surgical failure rate as strict as two times the currently acceptable level (11.1%) to determine proficiency, which was one strength of our study . The cohThere are certain limitations of the present study that need to be addressed. First, the CUSUM analysis included different diseases , surgeries (maxillary and mandibular reconstructions), and free flaps (fibular and iliac flaps), which influenced the homogeneity of enrolled cases. A multi-center clinical trial, with a big enough sample size of the same disease and surgery, might be needed to overcome this limitation. However, it will lead to other limitations of different expertise of multiple surgical teams and hospital setting. Second, post-operative oral function and quality of life are important outcomes for jaw reconstruction. However, there are a lot of confounding factors influencing these outcomes, which will need a well-designed prospective randomized control trial for further investigation. Thus, we did not include them in the present analysis. Last, the learning curve of time spent on preoperative preparation, such as virtual surgical planning and PSSP design, was not reported in this study. Previously, we reported that the time spent on virtual surgery and plate design was 18.8 ± 13.2 h, and the time taken for 3D printing, post-processing, and product delivery was 162.9 ± 55.2 h . The conA three-stage learning pattern of CAJR with the use of PSSP was revealed, including initial learning, plateau, and overlearning, which may guide the clinical teaching and training of CAJR using PSSP. Based on CUSUM analysis, surgical proficiency was obtained after 23 cases. Stabilization of total operation time, length of hospital stay, and bone graft deviation occurred after 8–17 cases. Our study provided evidence to guide the training of this new surgical procedure to ensure patient safety and clinical outcomes.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster. The patients/participants provided their written informed consent to participate in this study.W-yZ and Y-xS: study design. Y-xS: study supervision. W-yZ, WC, JP and W-fY: data collection. W-yZ and MW: manuscript preparation, statistical analysis, and data interpretation. All authors contributed to the article and approved the submitted version.The study was funded by the Health and Medical Research Fund (No. 08192096), Hong Kong SAR, Guangdong Science and Technology Department (No. 2019A050516001), University Research Committee Platform Technology Funding 2021, and HKU Seed Fund for Translational and Applied Research (202010160032).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


LILR region and evolutionary equivalents in some rodent and bird species have been thoroughly characterized. In non-human primates, the LILR region is annotated, but a thorough comparison between humans and non-human primates has not yet been documented. Therefore, it was decided to undertake a comprehensive comparison of the human and non-human primate LILR region at the genomic level. During primate evolution the organization of the LILR region remained largely conserved. One major exception, however, is provided by the common marmoset, a New World monkey species, which seems to feature a substantial contraction of the number of LILR genes in both the centromeric and the telomeric region. Furthermore, genomic analysis revealed that the killer-cell immunoglobulin-like receptor gene KIR3DX1, which maps in the LILR region, features one copy in humans and great ape species. A second copy, which might have been introduced by a duplication event, was observed in the lesser apes, and in Old and New World monkey species. The highly conserved gene organization allowed us to standardize the LILR gene nomenclature for non-human primate species, and implies that most of the receptors encoded by these genes likely fulfill highly preserved functions.The genes of the leukocyte immunoglobulin-like receptor (LILR) family map to the leukocyte receptor complex (LRC) on chromosome 19, and consist of both activating and inhibiting entities. These receptors are often involved in regulating immune responses, and are considered to play a role in health and disease. The human  LILR, KIR, and LAIR gene families, has been reviewed on a regular basis (The human immunoglobulin superfamily (IgSF) represents more than 700 cell-surface and secreted receptors, which are characterized by the presence of one or more immunoglobulin-like (Ig) domains . Severalar basis , 6, 7.KIR region, located on the telomeric side of the LILR gene cluster, is known to be highly dynamic, and, at population level, haplotypes may show considerable diversity in gene architecture and allelic content  and a soluble (LAIR2) receptor, and are located in the center of the LILR region , the first ancestral mammalian species started to roam the earth. As mentioned previously, the presence of a  lineage Figure 2 lineage .PIR region is comparable to the LILR region in humans  are encountered in mice, and the genomic architecture of the n humans , 45. Thec region . In goatnterpart –44. ThesKIR and LILR Ig-like domains were identified  (Data obtained from the class of ray-finned fish (Actinopterygii) evidenced that the origin of the  450 mya Figure 2nctatus) . In thisnctatus) .LILR-like system, in a way similar to that of the MHC complex, emerged before the major expansion of the vertebrate lineage, approximately 450 mya. Some of the LILR receptors have MHC class I molecules as their ligands  is divided into the centromeric region (~ 154 kb) and the telomeric region (~ 211 kb). Notably, KIR3DX1, previously known as KIR3DL0 or LENG12, is embedded between LAIR2 and LILRA2 in the telomeric region  (LILR region has an average gene distance of 18 kb (8 kb – 27 kb) between the stop codon and the start codon of adjacent genes, while the telomeric region has an average gene distance of 23 kb (9 kb – 30 kb)  –63. The ic genes , 64.LILR genes in humans  (LILR in non-human primates (NHP), the human LILR gene nomenclature is loosely followed by annotation algorithms such as those from NCBI and ENSEMBL. Due to the high levels of similarity between the different LILR genes, difficulties may arise in phasing the genomic regions, and therefore it may accidently happen that orthologous genes are not given the corresponding name. To root out such errors, we have selected the latest and freely accessible genomes, which were sequenced by the latest next generation platforms  against human genomic sequences. However, different intron length resulted in alignment issues. Therefore, all available primate mRNA sequences were aligned, and a phylogenetic tree analysis was performed using the Geneious tree builder . Each cluster with mRNA sequences was aligned and compared to identify transcripts with a potentially incorrect gene name. These sequences were then aligned against the relevant human reference transcripts to sort out whether the genes were annotated correctly or incorrectly in the public database. Next the genomic gene location was defined in relation to the human LILR region, before adjusting the gene nomenclature in the relevant non-human primate species. Using this approach, we have explored all available LILR data on different nonhuman primate species: namely, chimpanzee, bonobo, gorilla, orangutan, gibbon, rhesus and cynomolgus macaque, and common marmoset, and have introduced a naming of LILR genes based on the orthologous positions in the human genome, which was taken as a reference . This gene locates in the centromeric region between LILRB2 and LILRA3, and shares 99.3% similarity between the two species. Comparative sequence analysis revealed that the domain and intermediate intron sequences of this additional LILRA2-like gene are approximately 94% similar to the adjacent LILRB2 gene. The sequence encoding the domains is comparable with LILRB2, while the stem and transmembrane region are more similar to activating LILR receptors; therefore, we have designated this gene as LILRAB2. Although, the stem and transmembrane region is comparable to activating LILR receptors, we were not able to pinpoint which recombination event(s) generated the LILRAB2 gene. There is no evidence for the presence of an additional, maybe disrupted, human LILRA2 gene in the centromeric region. The reannotation of LOC109024105 for gorilla, LOC100432416 for orangutan, and LOC718403 for rhesus macaque revealed evidence for the presence of a LILRAB2 gene in these species , leukocyte Ig-like receptors (LIR), and CD85 were different names used to denote n humans , 66. In e (HGNC) , 67. LILlatforms Table S2KIR nomenclature has already been standardized in humans and NHP  and chimpanzee LILR region (~ 605 kb) are highly similar  is similar to that of the chimpanzee, but the transcription status of several genes, including LILRA6, LILRB5 and LILRA7, could not be confirmed. The LILRA8 gene appears to be non-functional, as sequence analysis revealed two frameshift mutations that result in the introduction of a premature stop codon, and, therefore, this gene is considered to be a pseudogene  most likely represents an assembly error introduced by the shotgun approach that is used to sequence the reference genome and is therefore not illustrated in In great apes, the  similar Figure 5d LILRB5 , 79. In LILR region is located on chromosome 10, and comprises approximately 494 kb. As compared to other primate species, gibbons frequently feature chromosome re-arrangements , shared a common ancestor with humans approximately 25-33 mya Figure 4 make-up , 77. BotLILR region , LILRB2, and LILRA4. In the telomeric region LILRA1, LILRB1, and LILRB4 were encountered. LILRB2 and LILRB4 are, however, defined as low quality sequence. Furthermore, a tandem of KIR3DX1 and KIR3DX2 genes was observed. Adjacent to LILRB4, an unannotated stretch of ~ 280 kb is identified, which does not contain a LILR or KIR equivalent. This might reflect an error in the assembly of the reference genome.Platyrrhini, or New World monkeys (NWM), shared with humans a common ancestor that lived approximately 27-52 mya Figure 4R region , 77. Theosome 19 . In marmLILR region in different primate species. We should note, however, that for most non-human primate species only one or a few complete genomes have been sequenced. The LILR organization in this study is based on a single reference genome per species. Although the LILR cluster seems to be an ancient and conserved set of genes, subtle variation in the gene organization might exist per individual or per population, as is demonstrated for the null haplotype in humans that lacks the presence of LILRA3. Moreover, the transcription status of several genes was also not confirmed on the current reference genomes and requires additional characterization studies before definitive conclusions can be drawn.To the best of our knowledge, we present here the first comprehensive comparison of the KIR gene clades, KIR3DL and KIR3DX, have been defined , suggesting that transcription of the gene may occur , both of which inhabit Africa.In the genomes of several NHP species analyzed, we observed a second  KIR3DX2 6. The N product . HoweverKIR3DX1 exon sequences are compared with chimpanzee KIR3DX1 using Geneious Prime 2020.2.4, since the full-length human KIR3DX1 transcripts would contain the 7 bp deletion, and the chimpanzee is most closely related to humans (KIR3DX1 for gorilla (97.9%), orangutan (95.1%), gibbon (94.3%), rhesus macaque (90.8%), and cynomolgus macaque (91.0%). In contrast, KIR3DX1 in common marmoset is 68.4% similar to chimpanzee KIR3DX1. Furthermore, the coding region of KIR3DX1 in rhesus and cynomolgus macaque is 99.4% similar, indicating that this gene is highly conservated in macaque species. When introns are included in the sequence similarity comparison, higher levels of diversity are determined. In orangutans, for instance, the genomic KIR3DX1 sequence displays 83.5% similarity to chimpanzees, and the difference are mainly explained by insertions and deletions in the introns. The genomic sequence of chimpanzee KIR3DX1 is 76.6% and 78.7% similar to the sequences of rhesus and cynomolgus macaques, respectively. These observations suggest a selective pressure to largely conserve the KIR3DX1 exons, whereas the introns might be more prone to diversification, which may impact the regulation of expression levels and transcript splicing. Overall, the KIR3DX1 coding sequence is highly conserved between human, great apes, lesser apes, and OWM, whereas the KIR3DX1 gene in NWM diverged during evolution. The function of KIR3DX1 is still unknown. However, it has an inhibitory potential due to the presence of two ITIM motifs in the cytoplasmic tail  and genomic (99.5%) sequence regions. In marmosets, the KIR3DX2 gene largely deviated at the genomic DNA level, with 57.5% similarity to rhesus macaques, whereas the exon sequences were more conserved (80.4%).Comparing the sequences of KIR3DX gene in lesser apes, OWM and NWM is a duplication of KIR3DX1 in a common ancestor that is lost during radiation towards human and great apes. The KIR3DX2 sequence in gibbons is, however, far from similar to KIR3DX1 in chimpanzees (59.4% on exons and 40.6% on gDNA). In contrast, the coding regions of KIR3DX2 in both macaque species and in the common marmoset seem to be more conserved, with 83.7/83.8% and 85.7% sequence similarity to the coding sequence of KIR3DX1 in chimpanzees, respectively. This substantiates an ancient KIR3DX1 duplication event, which remained more conserved in OWM, while the duplicated gene diverged during evolution in gibbons.The most likely explanation for an additional In humans, LILR receptors are expressed both on myeloid and lymphoid immune cells, including monocytes, B and T lymphocytes, natural killer (NK) cells, neutrophils, and dendritic cells (DC), and it is generally accepted that they control a variety of immune responses and maintain homeostasis , 87, 88.Activating and inhibitory LILR receptors consist of two or four extracellular Ig-like domains . In addivia a positively charged arginine residue in the transmembrane region of the LILRA receptor  toward IL-10 . LILRB1 rculosis . It is pignaling .Plasmodium falciparum, the causative agent of malaria, produces repetitive interspersed family (RIFIN) proteins, which are displayed on infected erythrocytes  infection, which expresses UL18, an MHC class I homolog, on infected cells. The engagement of UL18 and LILRB1 may inhibit the clearance of CMV-infected cells, and, therefore, CMV might escape the innate immune response . During hrocytes . Some RIhrocytes .LILRA3 SNP rs103294 and LILRB1 SNP rs16985478 may be a risk factor for prostate cancer and non-small cell lung cancer, respectively , but it is not evident whether it influenced LILR gene evolution as well (KIR genes, appeared later in evolution, and are able to scan for the presence of polymorphic epitopes on MHC class I molecules. In humans, KIR3DX1 is classified as a pseudogene, while KIR3DX1 in the non-human primates is seen to code for a functional gene product. A duplication of KIR3DX1 was observed in the lesser apes, OWMs, and NWMs, suggesting that this duplication might be present in the ancestor and was lost in great apes and humans. As far as we know, the function and ligand of KIR3DX1 is not yet resolved. At last, LILR receptors are placed in context for the role they may play in health and disease. It is tempting to speculate that old genes are frequently associated with diseases. However, one would expect that serious disease associations linked to old genes have been weeded out during evolution. The other issue is that disease association in a highly conserved region with limited levels are hard to pick up due to linkage phenomena. Since the LILR region in primates is remarkably conserved, non-human primates are an excellent tool to thoroughly study the functional aspects of LILR genes. This type of undertaking might enhance the available non-human primate disease models in order to improve the health both of humans and animals.In this communication, we provided an overview on the genomic organization of the volution Figure 5 as well , 55, 168LS drafted the manuscript. JB, NG, and RB edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


After 24 months of MedDiet and daily PA, a significant decrease in body weight  and BMI  was observed in the total population with respect to baseline. Serum inflammatory biomarkers  after 24 months of intervention were significantly reduced. Adipokine profile (adiponectin and resistin) did not improve with the intervention, as adiponectin levels significantly decreased and resistin levels increased in all the population. Inflammatory biomarkers and adipokine profile had a significant correlation with anthropometric parameters, body composition, and physical activity. (4) Conclusions: After 24 months of lifestyle modification, our MHOPp reduced their Z-score of BMI, leading to an improvement of inflammatory biomarkers but inducing deterioration in the adipokine profile, which does not improve with MedDiet and physical activity intervention. An adequate education within the family about healthier habits is necessary to prevent and reduce an excessive increase in obesity in childhood.(1) Background and aims: Obesity and high body max index (BMI) have been linked to elevated levels of inflammation serum markers such as C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha , adiponectin, and resistin. It has been described that adipose tissue presents a high production and secretion of these diverse pro-inflammatory molecules, which may have local effects on the physiology of the fat cell and also systemic effects on other organs. Our aim was to evaluate the impact that lifestyle modifications, following a Mediterranean Diet (MedDiet) program and physical activity (PA) training, would have on inflammatory biomarkers in a metabolically healthy prepubertal population with obesity (MHOPp) from Malaga . (2) Methods: 144 MHOPp subjects (aged 5–9 years) were included in this study as they met ≤1 of the following criteria: waist circumference and blood pressure ≥ 90 percentile, triglycerides > 90 mg/dL, high-density lipoprotein cholesterol (HDL-c) < 40 mg/dL, or impaired fasting glucose (≥100 md/dL). Selected subjects followed a personalized intensive lifestyle modification. Anthropometric measurements, inflammation biomarkers, and adipokine profile were analyzed after 12 and 24 months of intervention. (3) Results: 144 MHOPp participants (75 boys—52% and 69 girls—48%;  Obesity and overweight are the abnormal or the excessive fat accumulation, as defined by the World Health Organization (WHO). According to the WHO estimations, 41 million children under 5 years of age and more than 340 million children and adolescents (ages 5 to 19) were overweight or obese in 2016. The prevalence of overweight and obesity in children and adolescents has increased from 4% in 1975 to more than 18% in 2016 [Adipose tissue is a specialized organ that secretes different biomarkers that behave as paracrine and endocrine regulators . These b2) do not have metabolic complications. These subjects are known as metabolically healthy with obesity (MHO). Obesity and its associated diseases have been deeply studied. However, data from the MHO population are limited, particularly from the MHO prepubertal population (MHOPp) [Obesity is associated with chronic and cardiovascular diseases, but there are some people who despite presenting obesity (body mass index (BMI) ≥ 30 kg/m (MHOPp) . Althoug (MHOPp) . MedDiet (MHOPp) . Youth h (MHOPp) . Childho (MHOPp) .The current study purpose was to explore the impact that 12 and 24 months of lifestyle modifications, by following a MedDiet program and daily PA training, would have on inflammatory biomarkers in MHOPp Spanish from Malaga .This study is a cross-sectional study of a MHOPp (4–9 years aged) that included participants of both sexes during a total period of 24 months. Inclusion criteria were boys (from 4 years to testicular volume < 3 mL) and girls (from 4 years to Tanner S2 breast bud elevation) with obesity (percentile ≥ 95)  and 1 orThe recruitment period was from November 2016 to May 2017 in different schools from the city of Malaga . These schools were located in the urban environment of the city and, in order not to bias the results, subjects with different socioeconomic statuses  and educational levels  were included in the study. Recruitment was carried out by performing preschool and elementary school classroom visits at different schools. Families of the possible selected participants were contacted to provide them with information about the study and were invited to participate in the program. The steps for the inclusion of children in the study were as follows: (a) acceptation of inclusion in the study by parents/legal representatives of the children; (b) verification that children met inclusion criteria; (c) interview with a nutritionist.The parents/legal representatives and the selected subjects were summoned to the Department of Internal Medicine in the Regional University Hospital of Malaga so that they could be informed about the design and objectives of the study, as well as in order to request their written informed consent for the voluntary participation of their children.All parents/legal representatives gave their informed consent to participate in this study, and protocols were approved by the institutional ethics committee .The subjects who participated in the study had different nurse and nutritionist visits; an initial visit, 12 months after, and at 24 months of intervention. Anthropometric measurements  and blood pressure were obtained by trained sanitary personnel. Food intake and PA questionnaires were completed in order to analyze the lifestyle intervention impact. A nonconsecutive, 3-day dietary record (two workdays and one weekend day), containing detailed information about food composition and cooking recipes over 72 h . Adheren2). Obesity was defined as a BMI ≥ 25 kg/m2 for children. Waist circumference was measured halfway between the last rib and the iliac crest by using an anthropometric tape. Blood pressure was measured with a validated automated electronic sphygmomanometer  after 5 min of rest while the participant was in a seated position. Blood samples were collected in order to know the inclusion criteria after an overnight fast, and biochemical analyses were performed in local laboratories using standard enzymatic methods.Anthropometric variables were measured by trained personnel according to the protocol. Weight and height were obtained through high-quality electronic calibrated scales and a wall-mounted stadiometer, respectively. BMI was calculated by dividing weight (kg) by height squared . Serum adipokine and inflammatory biomarkers levels  were measured using an enzyme-linked immunosorbent assay . For adiponectin levels, the minimum detectable concentration was 0.246 ng/mL. The intra- and inter-assay coefficients of variation were 3.5% and 6.5%, respectively. For resistin levels, the minimum detectable concentration was 0.026 ng/mL. The intra- and inter-assay coefficients of variation were 4.7% and 8.4%, respectively. For IL-6 levels, the minimum detectable concentration was 0.70 pg/mL. The intra- and inter-assay coefficients of variation were 2.6% and 4.5%, respectively. Lastly, for TNF-alpha levels, the minimum detectable concentration was 1.6 pg/mL. The intra- and inter-assay coefficients of variation were 4.7% and 5.8%, respectively. High-sensitivity CRP levels were measured using ELISA . The minimum detectable concentration was 0.1 mg/mL. The intra- and inter-assay coefficients of variation were 4.4% and 3.3%, respectively.After having evaluated eating behaviors, families and children were guided to carry out lifestyle modifications to promote and incorporate a healthy diet. In this case, a caloric restriction was not implemented, due to the fact that the participants were growing children. The MedDiet recommended to the studied subjects included extra virgin olive oil and nuts consumption. The recommended caloric intake was 1500 kcal/day , distribA daily PA was also recommended following the internationally accepted PA guidelines . In addiTotal body and regional body composition were estimated using dual-energy X-ray absorptiometry . Each subject was scanned distinguishing between bone and soft tissue. Edge detection and regional demarcations were done by computer algorithms . The abdominal region was delineated by an upper horizontal border located at half of the distance between acromions and external end of iliac crests, a lower border determined by the external end of iliac crests, and the lateral borders extending to the edge of the abdominal soft tissue. The percentage of body fat, fat mass, lean mass, total mass, and total fat were indicated.t-test was used to compare quantitative variables, whereas the chi-square and Mann–Whitney tests were used for qualitative variables in order to contrast variables measured within each group at different time periods. Relationships between adipokine profile and inflammatory biomarkers (serum levels) and anthropometric and body composition parameters, adherence to the MedDiet, different physical activity intensities, and adipokine profile and inflammatory biomarkers (between them) were examined by means of Pearson correlation analysis.Quantitative variables were expressed as mean ± standard deviation (SD), and qualitative variables were expressed as percentages. SD Z-scores for anthropometric parameters  were calculated according to Spanish Child Growth Standards . Studentα error of 0.5%), 80% statistical power, and a 5% loss rate, a sample of 110 MHO subjects is required. A recruitment of at least 130 participants was planned to take in account the possible nonparticipation or loss to follow-up. Statistical analysis was performed using SPSS for Windows, version 22.0 .To calculate the sample size , we relied on other clinical studies that have shown the metabolic benefits of weight loss in the MHOPp , and assThe initial recruitment of population from 36 schools in Malaga included a total of 949 MHOPp, but only 172 participants came to the first visit at the Regional University Hospital of Malaga. Finally, after the participant rejection and the exclusion of participants due to not meeting the inclusion criteria, 144 MHOPp participants finally joined the study .p = 0.62), with an average age of 7.8 ± 1.4 years. All families presented a low–middle socioeconomic status (97.3%), with 42.3% of them showing a medium–high educational level and 57.7% of them a low one.From these 144 participants, 75 (52%) were male and 69 (48%) female  and SDS BMI  was observed, and in the total population  with respect to baseline. Moreover, after 24 months of the intervention, children, both in the total population and according to gender, significantly decreased their SDS weight  and their SDS BMI .SD (Z)-scores for weight, height, BMI, and blood pressure (systolic and diastolic) for all participants are shown in p < 0.0001) and 24 months of intervention . TNF-alpha serum levels also decreased after the intervention . Concerning the adipokine profile, adiponectin serum levels decreased after intervention  and resistin serum levels increased in both sexes after 12 and 24 months of follow-up .Regarding analytical parameters , hsCRP sp < 0.0001) and after 24 months of intervention . They also reduced their total fat intake  and increased their fiber intake. Adherence to MedDiet improved (+3 points) after 24 months of intervention, especially due to the reduction in the consumption of red meat, sausages, hamburgers, and commercial pastries, and thanks to the increase in the daily consumption of vegetable and fruits. A decrease in butter, margarine, and cream consume was observed, with olive oil being the main source of fat used in cooking.Changes in food intake and MedDiet adherence were also analyzed. Our participants decreased their energy intake after 12 months of follow-up  showed association with adiponectin levels but for resistin levels no associations were found. After 24 months of intervention, for adiponectin serum levels, no associations were found and resistin levels only showed a significant association with SD (Z)-BMI .Multiple regression models adjusted for sex, baseline age, baseline BMI Z-score, and abdominal fat mass in baseline conditions for both adipokines were tested. For adiponectin and resistin serum levels, no associations were found. After 12 months of intervention, only delta SD BMI Z-score (p < 0.0001 in both periods of time) and their vigorous daily PA .The PA of our subjects also improved due to the intervention . All of On the other hand, participants increased significantly the fat mass, lean mass, and total mass. However, regarding the percentage of total fat, a significant decrement was observed in all the population, especially in girls, after 12 and 24 months of intervention .Correlation analyses between body composition and metabolic parameters are shown in After 12 months of intervention, glycemic profile  was positively associated with abdominal body composition. It was replicated with triglycerides levels. After 24 months of intervention, only HDL-c presented a negative correlation with total fat.Correlation analyses are showed in After 12 months of intervention, hsCRP was positively associated with WHI and abdominal fat mass. However, there were negative associations between IL-6 and the adherence to the MedDiet and PA, and between TNF-alpha and WHI, and adiponectin with anthropometric parameters, body composition, and PA.After 24 months of intervention, there were positive associations between hsCRP and IL6 with WHI, and between TNF-alpha with SDS weight and resistin with all anthropometric parameters and body composition. On the other hand, adiponectin negatively correlated with weight, waist circumference, and WHI.Positive correlations were observed between inflammatory biomarkers and adipokine profile, and IL-6 and TNF-alpha, and for resistin with hsCRP, IL-6, and TNF-alpha, in baseline conditions.After 12 months of intervention, only IL-6 correlated positively with TNF-alpha, and after 24 months of intervention, only hsCRP presented a positive correlation with resistin levels and a negative correlation with adiponectin levels.The purpose of this study was to determine whether adherence to MedDiet and regular PA promote modifications in serum inflammatory biomarkers and adipokines levels after 12 and 24 months of lifestyle modification. The results obtained in our study show that, according to Z-score, the participants, as a whole and regardless of gender, lost weight and reduced their BMI, although obesity degree, measured by BMI, remained. Inflammatory biomarkers hsCRP, IL-6, and TNF-alpha significantly decreased after 12 and 24 months of intervention; however, adipokines profile did not improve despite the intervention. Nevertheless, these changes in BMI Z-score could be likely to translate into clinically important differences for subjects with obesity. BMI reduction can improve health status as Lycett K et al. showed . Their fThe presence of obesity is conditioned by the socioeconomic level of the population. The families of our prepubertal population are characterized by presenting a medium–low socioeconomic level (97.3%) and an educational level with primary studies (57.7%). These conditions, together with the place of residence , have favored the presence of obesity in our population; data in agreement with Papadimitriou A et al. ,32 The gIt should be taken into account that over 35% of children with obesity under 10 years old, even under 5 years old, present one or more cardiovascular risk factor or MetS, such as arterial hypertension, dyslipidemia, disturbed glucose tolerance, or insulin resistance (IR) . The MetOn the other hand, inflammatory biomarkers hsCRP, IL-6, and TNF-alpha are associated with obesity and other diseases, including inflammation, infection, heart attack risk, and cancer . In our In addition, TNF-alpha is produced by macrophages/monocytes during acute inflammation and is responsible for several signaling events within cells, leading to necrosis or apoptosis. This protein is also important for resistance to infection and cancers . In our IL-6 is a marker of the MetS, but TNF-alpha is involved in insulin resistance and dyslipidemia. During physical activity, IL-6 is produced by muscle fibers and inhibits the production of TNF-alpha . It is oOn the other side, human obesity is also associated with a pro-inflammatory state, reflected in the elevation of others biomarkers such as CRP. Some studies provide evidence supporting a positive association between body composition and CRP . Normal Adipose tissue is not only involved in fat storage, it also has a crucial role as an endocrine organ, since it produces several bioactive substances, collectively called adipokines. Excess adipose tissue results in a deregulated production of these adipokines, which is related to the pathogenesis of various cardiovascular diseases. One of the most important adipokines is adiponectin, which is mostly expressed in subcutaneous adipose tissue. Its expression and blood concentration decrease as adiposity increases. This cytokine takes part in the regulation of glucose level and in the lipidic metabolism . It is aκB pathway.Resistin, a cysteine-rich adipokine induced during adipogenesis, is proposed as a link between obesity and T2DM and may modulate numerous steps in the insulin-signaling pathway leading to insulin resistance and promote inflammatory processes . ResistiBoth adiponectin and resistin act on TNF-alpha, which may define a synergistic response between both adipokines. This pro-inflammatory biomarker decreased significantly after 12 months of intervention, but increased after 24 months of lifestyle modification, in line with what was observed in the expression levels of the adipokine profile. Although MHOPp subjects in our study remained with obesity after 24 months of intervention, according to Z-score, they decreased their weight and BMI, which translates into a significant reduction of inflammatory biomarkers. These data were corroborated by Gomez-Huelgas R et al. in MHO adult population  and implThis study does not present random measurements or control subjects due to the lack of an international consensus in the definition of childhood MetS. Despite this, we believe that it is important that each participant compares his or her results at the end of follow-up to his or her conditions at baseline, rather than to others. The comparison of the data of the different parameters studied has been used to verify the efficacy of the 24-month lifestyle intervention in MHOPp.Lifestyle intervention in prepubertal population based on consumption of MedDiet and daily PA training decreases pro-inflammatory biomarkers . In contrast, an increment of adiponectin levels was observed, although without reaching the basal concentration levels, after 24 months of lifestyle modification in MHOPp. An adequate education within the family about healthier habits to prevent and reduce an excessive increase in obesity in childhood is necessary.


Objective: To analyze conditional survival (CS) in patients with advanced epithelial ovarian cancer (EOC) and investigate prognostic factors that affect the CS rate to provide more accurate survival information.Methods: Patients with advanced EOC between 2004 and 2015 were identified from the Surveillance, Epidemiology, and End Results (SEER) database. CS analysis was performed to depict exact survival for patients who had already survived a specific number of years. Cox proportional hazards regression was performed to ascertain the individual contribution of factors associated with actuarial overall survival (OS) at diagnosis and CS at 1, 3, and 5 years after diagnosis.Results: Of 11,773 patients, OS decreased from 32.2% at 6 years to 25.1% at 8 years, while the corresponding 5 year CS (CS5) increased from 37.5% at 1 year to 43.9% at 3 years. Subgroup analysis stratified by clinicopathological characteristics showed that CS5 was always higher than the corresponding actuarial survival . Based on multivariate analysis at diagnosis, age, race, marital status, histological type, tumor grade, size, T stage, M stage, surgery, radiation therapy, and chemotherapy were independent prognostic factors for OS. Five years after diagnosis, however, only age, histological type, tumor grade, and laterality were persistently significant independent prognostic factors . Furthermore, patients with poor pathological prognostic factors achieved greater improvements in CS5 rates, and the survival gaps between OS and CS were more obvious.Conclusion: CS of advanced EOC was dynamic and increased over time. Age, histology, tumor grade, and laterality were significant prognostic factors even 5 years after diagnosis. Thus, the availability of updated prognoses at various time points will allow clinicians to better guide their patients. Ovarian cancer is one of the most common tumors in women, with the highest mortality rate among gynecology malignancies , 2. EpitThe prognosis of patients with cancer mainly depends on stage and the individual's response to treatment, but prognosis can also change for each individual over time. Furthermore, with increased survival, there is also increasing interest in quality of life for advanced epithelial ovarian cancer survivors and their survivorship care. However, traditional Kaplan–Meier assessment can only be used to determine survival and prognosis at the time of diagnosis and does not change with passage of time , 6, whicRecently, conditional survival (CS) assessment has become an accurate and informative assessment method to better predict survival time in patients with cancer . CS reprTo the best of our knowledge, no study has specifically examined the conditional survival of patients with advanced EOC. Thus, in this study, we used the Surveillance, Epidemiology and End Results (SEER) database and performed conditional survival analysis to predict more accurate survival for patients with advanced EOC. In addition, changes over time in the prognostic significance of clinicopathological and treatment-related factors were also analyzed.https://seer.cancer.gov/seerstat/). The SEER database is a national population-based cancer registry that is globally recognized for its accuracy and completeness. Information about cancer incidence and survival for approximately 26% of American people is collected and published by this institute. Since the data from the SEER registry are deidentified and publicly available, no IRB approval was necessary.The data were obtained from the SEER registry of the National Cancer Institute (*Stat (version 8.3.8) software (Reference number: 10579-Nov2019). The inclusion criteria were as follows: (1) patients were pathologically diagnosed with advanced EOC between 2004 and 2015 (FIGO stage III and IV); (2) patients were at least 18 years of age; (3) ovarian cancer was the only primary carcinoma; and (4) histological code was in accordance with the International Classification of Tumor Diseases, Third Edition (ICD-O-3) (Patient data were extracted from the latest version of the SEER database using SEERICD-O-3) . PatientICD-O-3) .In this study, clinicopathological features included age, race, marital status, tumor size, laterality, grade, histological type, T stage, N stage, M stage, surgical information, radiotherapy information, and chemotherapy information. In addition, TNM status in this study was redefined according to the American Joint Committee on Cancer seventh edition (AJCC). The optimal cutoff point of tumor size was determined by the median value.Continuous variables are expressed as the means and standard deviation (SD), and categorical variables are expressed as numbers. OS was calculated from the day of diagnosis until the day of death from any cause (event) or the last day of follow-up (censored). Survival rates and median overall survival were calculated using Kaplan-Meier analysis. The log-rank test was performed for comparisons between groups.P <0.050. Statistical analyses were performed using SPSS® version 22.0  and R version 3.3.3 .CS is defined as the probability of surviving an additional number of y years given that a patient has already survived for x years and can be calculated from Kaplan-Meier survival data. For instance, an additional 5-year survival (CS5) was calculated as CS5 = S(x +5)/S(x), which means that CS5 among patients who survived 3 years from the date of diagnosis was calculated by dividing the survival at 8 years by the survival at 3 years. Multivariate Cox proportional hazards regression was performed to evaluate the hazard of the CSS rate at the time of diagnosis and CS rates for multiple survival periods  . All tesIn total, 11,773 patients were included in our cohort. The median age of these patients was 61 (IQR: 52–69). Most patients were white (84.8%) and married (57.5%). Most subjects were T3 stage (91.0%), grade III & IV (85%), serous histologic type (76.4%), and bilateral (57.8%). There were 4,675 (39.7%) patients with lymph node metastasis and 3,252 (27.6%) patients with distant metastasis. Regarding treatment schemes, 11,608 (98.6%) patients underwent surgery, 10,121 (86.0%) patients received chemotherapy, and 157 (1.3%) patients underwent radiation during their treatment courses .In this cohort, the 3 and 5 year survival rates were 57.2 and 37.8%, respectively. The median survival was 44  months . ConditiP < 0.05, P < 0.05).Upon multivariate analysis at the time of diagnosis, patients who were older, black, unmarried, had mucinous or clear cell histology, had low-grade tumors, had low T and M stages, and underwent radiation therapy had significantly lower OS rates than the controls , mucinous histology , advanced tumor grade , and bilateral tumors . The actuarial survival for patients with M0 at the tenth year was 23.7 vs. 56.2% for patients who had already survived for 5 years (Δ = 32.5%).The actuarial survival rates and corresponding CS5 for all patients and according to clinical and tumoral characteristics are shown in The relationship between the changes in CS5 and pathological prognostic factors  is shown in To the best of our knowledge, this is the first study to focus on CS in patients with advanced EOC. In this study, the CS of advanced EOC increased over time, while the actuarial OS declined. The improvements in CS5 rates and the survival gaps between OS and CS were more clear for patients with poor pathological prognostic factors. Furthermore, multivariate analysis confirmed that age, tumor grade and histological type were significant prognostic factors at the time of diagnosis, and their prognostic effects persisted until 5 years after diagnosis. Our results demonstrated that CS analysis could provide more comprehensive prognostic information and help the clinical follow-up of patients with advanced EOC.EOC is the most common type of ovarian cancer, and approximately 70% of cases are advanced disease (FIGO stage III and IV) at the time of diagnosis . HoweverIn the present study, we found that older age, black race, mucinous histology, larger tumor size, advanced grade, higher T stage, and M1 stage were independent risk factors for actual OS. However, marriage, surgery and chemotherapy were independent protective factors for advanced EOC, which was consistent with previous studies –18. PrimSubgroup analysis stratified by the clinicopathological characteristics found that CS was higher than actuarial survival in each subgroup. For patients with poor prognostic factors, such as advanced tumor grade, advanced T stage and M1 stage, the growth trend of CS was significantly better than that of the control group, which was similar to the other tumors , 21, 22.This study has several limitations. First, this was a retrospective study; thus, there was a certain degree of selection bias. Second, due to the lack of information on tumor dissemination, residual tumors after surgery, the type of chemotherapy and maintenance therapy in the SEER database, the impact of these factors on CS was not further analyzed in this study. Last, because the SEER database lacked information on Asian and European patients, and no other database was included, the universality of our conclusions was reduced. Nonetheless, the SEER database is a well-known national database that provides both a large cohort size and long-term follow-up, two necessary components for studying conditional survival. This study is the first to focus on CS of advanced EOC, which could support dynamic prognosis assessment and enable accurate and individualized follow-up strategies.In conclusion, CS estimates of advanced EOC generally increase over time, especially for patients with poor pathological factors at baseline. Age, histological type, tumor grade and laterality remained significant prognostic factors even 5 years after diagnosis. Our study provides an effective way to dynamically evaluate the long-term survival of advanced EOC and recommends the need for continuing surveillance and care in long-term survivors.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.All authors have signed the SEER Research Data Agreement to protect the privacy of patients, which is consistent with ethical principles.PeZ and GC designed the experiments and wrote the manuscript. PiZ collected the data. PeZ and PiZ contributed to the statistical analysis of the data. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The Covid-19 pandemic caused by the novel coronavirus SARS-CoV-2 has completely reshaped the lives of people around the world, including higher education students. Beyond serious health consequences for a proportion of those directly affected by the virus, the pandemic holds important implications for the life and work of higher education students, considerably affecting their physical and mental well-being. To capture how students perceived the first wave of the pandemic's impact, one of the most comprehensive and large-scale online surveys across the world was conducted. Carried out between 5 May 2020 and 15 June 2020, the survey came at a time when most countries were experiencing the arduous lockdown restrictions. The online questionnaire was prepared in seven different languages  and covered various aspects of higher education students’ life, including socio-demographic and academic characteristics, academic life, infrastructure and skills for studying from home, social life, emotional life and life circumstances. Using the convenience sampling method, the online questionnaire was distributed to higher education students aged 18 and over and enrolled in a higher education institution. The final dataset consisted of 31,212 responses from 133 countries and 6 continents. The relationships between selected socio-demographic characteristics and elements of student life were tested by using a chi-squared test. The data may prove useful for researchers studying the pandemic's impacts on various aspects of student life. Policymakers can utilize the data to determine the best solutions as they formulate policy recommendations and strategies to support students during this and any future pandemic. It comprised 39 mainly closed-ended questions divided into seven sections, namely socio-demographic and academic characteristics, academic life, infrastructure and skills for studying from home, social life, emotional life, life circumstances as well as personal reflections on Covid-19 Certain answer choices (“other” and “not applicable”), open-ended questions  and questions raising anonymity issues (the question on a student's e-mail address) are excluded from the dataset. Hence, it covers a total of 160 items from across various aspects of student life. Detailed information on the measured aspects of student life during the first wave of the Covid-19 pandemic is presented in The final sample consists of 31,212 respondents who shared their perceptions of the first wave of the Covid-19 pandemic's impact. The response rate was 33.1% since 94,246 respondents actually opened the link to the online questionnaire. The participation was unequally distributed throughout the 133 countries from the 6 continents The respondents ranged in age from 18 to 70 years with a mean age of 23.6 years and standard deviation of 5.6 years, with most being female (65.4%). Most respondents were full-time (87.8%) and first-level (80.1%) students. They were mostly studying in the field of the social sciences (37.0%) The content of the dataset can be divided into 10 different aspects of student life . Carried out between 5 May 2020 and 15 June 2020, the data collection came at a time when most countries were experiencing the arduous lockdown restrictions. The data preparation, aggregation, cleaning process, and calculations were performed in the Python programming language utilizing the Pandas and Numpy libraries The survey targeted all higher education students. With the aim of reaching a wide range of students, a non-probabilistic sampling technique, specifically convenience sampling, was utilized All participants were informed about the survey, including its details, and provided their informed consent before participating. Participants agreed to participate in the survey by clicking on a “next page” button, as was explicitly stated on the initial page of the online questionnaire. Participation in the survey was anonymous and voluntary, implying participants could withdraw from the survey without any consequences. Given the data-protection rules, the survey was available to participants being at least 18 years old and enrolled in a higher education institution. The procedures of this survey comply with the provisions of the Declaration of Helsinki regarding research on human participants. Ethical Committees of several of the higher education institutions involved approved this study, such as the University of Verona (protocol number: 152951), ISPA – Instituto Universitário , University of Arkansas (IRB protocol number: 2005267431), Walter Sisulu University  and Fiji National University (CHREC ID: 252.20).Aleksander Aristovnik: Conceptualization, Investigation, Writing – review & editing, Supervision, Project administration, Funding acquisition. Damijana Keržič: Investigation, Writing – review & editing. Dejan Ravšelj: Investigation, Data curation, Writing – original draft, Writing – review & editing, Project administration. Nina Tomaževič: Investigation, Writing – review & editing. Lan Umek: Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data curation, Writing – review & editing, Visualization.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.