Since the triglyceride glucose (TyG) index can reflect insulin resistance, it has been proven to be an efficient predictor of glycolipid-metabolism-related diseases. Therefore, this study aimed to investigate the predictive value of the TyG index for visceral obesity (VO) and body fat distribution in patients with type 2 diabetes mellitus (T2DM).2 for males and > 115 cm2 for females). Logistic regression was performed to identify independent factors of VO, and receiver operating characteristic (ROC) curves were used to compare the diagnostic performance according to the area under the ROC curve (AUC).Abdominal adipose tissue characteristics in patients with T2DM, including visceral adipose area (VAA), subcutaneous adipose area (SAA), VAA-to-SAA ratio (VSR), visceral adipose density (VAD), and subcutaneous adipose density (SAD), were obtained through analyses of computed tomography images at the lumbar 2/3 level. VO was diagnosed according to the VAA  and females (9.59 vs. 9.01). The TyG index showed significant positive correlations with VAA, SAA, and VSR and negative correlations with VAD and SAD. The TyG index was an independent factor for VO in both males (odds ratio [OR] = 2.997) and females (OR = 2.233). The TyG index ranked second to body mass index (BMI) for predicting VO in male (AUC = 0.770) and female patients (AUC = 0.720). Patients with higher BMI and TyG index values showed a significantly higher risk of VO than the other patients. TyG-BMI, the combination index of TyG and BMI, showed significantly higher predictive power than BMI for VO in male patients  but showed no significance when compared with BMI in female patients .. TyG is a comprehensive indicator of adipose volume, density, and distribution in patients with T2DM and is a valuable predictor for VO in combination with anthropometric indices, such as BMI.The online version contains supplementary material available at 10.1186/s12933-023-01834-3. Type 2 diabetes mellitus (T2DM), a complex endocrine-metabolic disease driven by a chronic positive energy balance, has become a major global public health crisis globally . From 20Although body mass index (BMI) might be a convenient and simple index to monitor the growth in obesity prevalence at the population level, studies have shown that obesity defined by BMI cannot account for the extreme variation in intra-visceral fat distribution between individuals . FurtherComputed tomography (CT) is considered the gold standard for independently assessing visceral and subcutaneous abdominal fat mass . In addiThe triglyceride glucose (TyG) index, which is calculated using triglyceride (TG) and fasting blood glucose (FBG), has become an attractive option for predicting insulin resistance . It was Therefore, in this study, we aimed to investigate whether the TyG index can adequately assess the fat distribution and predict VO in patients with T2DM.T2DM patients hospitalized in the Department of Endocrinology, Affiliated Hospital of Jining Medical University, From January 2017–December 2021, were included in this study. All patients underwent CT scans, including the center plane of the third lumbar vertebra on axial images and blood analyses to determine FBG and TG levels. The exclusion criteria were as follows: (1) missing the major anthropometric measures, such as height and weight; (2) patients with malignant tumors, hepatic damage, immune disease, abnormal thyroid function, or stage V diabetic nephropathy; and (3) patients with acute complications or infections. On admission, all patients were informed that their medical records could be used for research unless they indicated their opposition. In the present study, none of the patients indicated opposition. This study was approved by the Ethics Committee of the Affiliated Hospital of Jining Medical University (No. 2021C041).All biochemical and immune indices in the present study were measured at our hospital laboratory. FBG and TG levels were measured using an automatic analyzer . The TyG index was calculated as the natural logarithm (Ln) of [TG (mg/dl) × FBG (mg/dl)/2]. The TyG-BMI, TyG-WC and TyG-WHR were calculated as TyG multiplied by BMI, WC and WHR, respectively. Both systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured using a sphygmomanometer after at least 5 min of seated rest. Body mass index (BMI) was calculated by dividing the body weight in kilograms by height in meters squared.2 for men and > 115 cm2 for women at the L2/3 level [Abdominal CT was performed using a Dual-Source Flash CT scanner . Two authors identified axial CT images at the L3 level, where the spinous and two transverse processes could be visualized. Abdominal adipose fat was assessed using abdominal axial CT images at the L3 level and Slice-O-Matic software , as described in our previous study . The CT /3 level , 23. TheHypertension was defined as systolic blood pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg, and the use of antihypertensive medications. Dyslipidemia was defined as a disorder of lipoprotein metabolism and the use of lipid medications. Alcohol intake was defined as the consumption of at least 30 g of alcohol per week for at least a year. Cigarette smoking was defined as smoking at least 100 cigarettes in a lifetime .Continuous variables are summarized using the median and interquartile range if not normally distributed and the mean ± SD if normally distributed. Categorical variables were summarized as frequency counts and percentages. These variables were compared between the two groups using an independent samples t-test or Mann–Whitney U test. Categorical variables were analyzed using the χ2 test. Univariable correlations between variables were assessed using Pearson’s correlation coefficients. Variables with significant differences between the VO and non-VO groups were enrolled in the logistic regression analysis to identify independent factors of VO. Receiver operating characteristic (ROC) curve analyses were used to compare the diagnostic performance of the independent factors for VO according to the area under the ROC curve (AUC). The overall predictive power for VO was further analyzed by comparing their AUCs using the Z test . Since W2 vs. 116.10 cm2) but a lower level of SAA (131.05 cm2 vs. 153.51 cm2) than female patients.A total of 976 patients (614 men and 362 women) were enrolled in this study. Their adipose tissue and clinical characteristics are shown in Tables Except for higher areas of visceral and subcutaneous adipose tissues, VO patients showed higher levels of VSR and lower levels of VAD and SAD. Regarding other clinical features, VO patients showed higher BMI, SBP, DBP, hemoglobin, creatinine, alanine transaminase, TG, FBG and TyG index levels and higher percentages of dyslipidemia and hypertension. On an individual level, patients with the same BMI may have a different level of VAA. As shown in Fig. The correlations between adipose tissue and other clinical characteristics are shown in Fig. 2 and 24.05 kg/m2, and of TyG index were 8.91 and 9.07, respectively  and females  Fig. . In maleThe scatter plot demonstrates the distribution of VO and non-VO patients according to their BMI and TyG index Fig. . The patAs shown in Fig. Only 218 patients (125 men and 93 women) have the recorder of WC and WHR. Among all variables, WC showed the highest correlation with VAA in males (r = 0.746) and females (r = 0.716). As shown in Supplemental Fig. 1, WC showed strong predictive power for VO in males (AUC = 0.831) and females (AUC = 0.896). TyG-WC, the combination index of TyG and WC, showed higher diagnostic performance for VO in males (AUC = 0.860) but not in females (AUC = 0.890).2 in males, ＞24.05 kg/m2 in females, respectively) and TyG index  are more likely to have VO, which means that further examination is warranted.In this study, we demonstrated an association between VO and the TyG index in patients with T2DM. The TyG index is a more efficient predictor of VO in individuals with T2DM than other biochemical indicators. The statistical cut-off value of the TyG index was 8.91 in males and 9.07 in females for VO prediction. These findings are clinically relevant because VO is an important health issue that needs to be detected early. However, current equipment is unsuitable for screening at the population level in primary care. According to our study, patients with higher values of both BMI , and cardiovascular disease –47. The Several pieces of evidence have suggested strong sex specificity for regional adipose tissue distributions, with females having a higher proportion of gluteal-femoral body (subcutaneous) fat; however, males have more in the abdominal  region , 49. SimThis study has several limitations. First, more than 80% of the patients were under antidiabetic treatment, nearly 20% of the patients received statins, and a few subjects took fibrates, which inevitably affected the stability of our results. Second, we continuously collected data from all participants at a particular location over time; thus, our participants represent hospitalized patients with T2DM but not the general population with T2DM. Third, only 25% of included patients had a record of WC and WHR. The predictive ability of TyG, in combination with these anthropometric indices for VO, needs to be confirmed by further large sample studies. Fourth, the predictive ability of HOMA-IR for VO was not compared because serum C-peptide and insulin were susceptible to antidiabetic drugs and not routine inspection items in our hospital. Finally, because our study was conducted in Chinese adults with T2DM, the findings may not be readily generalizable to other populations or ethnicities.In conclusion, we showed a significant association between an increased TyG index and VO in patients with T2DM. The TyG index is a comprehensive indicator of adipose volume, density, and distribution in patients with T2DM. It is a valuable predictor of VO in combination with anthropometric indices, such as BMI.Below is the link to the electronic supplementary material.Supplemental Table 1. The adipose tissue and clinical characteristics of male patients with different levels of BMI and TyG index Supplemental Table 2. The adipose tissue and clinical characteristics of female patients with different levels of BMI and TyG index Supplemental Fig. 1. ROC analyses of TyG-WC and TyG-WHR for VO (A) male patients; (B) female patients


To study the degree of morphological and functional changes in thyroid-associated ophthalmology (TAO) patients using optical coherence tomography and electrophysiological studies and investigate their clinical correlation.A cross-sectional study including 40 patients with TAO and a control group with age- and sex-matched healthy subjects. All subjects underwent a complete ophthalmological examination, proptosis evaluation, spectral domain optical coherence tomography, and electrophysiological tests .β =  −  0.56 and 0.39 and p = 0.03 and 0.01, respectively); while duration of the disease, and clinical activity score (CAS) were the most important predictors of average RNFL thickness .On multiple regression analysis, the degree of proptosis and P50 amplitude were the most important predictors of central foveal thickness (CFT) (In the absence of fundus changes, macular thinning along with functional alterations noticed by pattern and multifocal electroretinogram could indicate presence of subclinical retinopathy in TAO cases. Thyroid-associated ophthalmopathy (TAO) is an autoimmune disorder characterized by infiltration of the orbital fat and interstitial tissues by inflammatory cells including lymphocytes, plasma cells, mast cells, and macrophages, and by retention of fluid and build-up of glycosaminoglycan. The ensuing expansion in the volume of orbital constituents results in increased intraorbital pressure , 2. WhenIt has been demonstrated that DON can occur in TAO patients who do not experience extraocular muscle swelling –6. EvaluElectrophysiological studies (EPS) that have been shown to provide objective evaluation of DON include VEP and pattern electroretinogram (PERG). Their results have also been shown to correlate with the clinical presentation and staging of the patients , 12. VEPThis cross-sectional study was conducted in the ophthalmology department of a tertiary care center during the 1-year interval of March 2018 to March 2019. We included the right eyes of 40 patients diagnosed with TAO with clinically apparent proptosis, in addition to the right eyes of 40 healthy individuals as controls.We excluded patients with pathological myopia  or with a known retinal disorder such as diabetic retinopathy. Patients suffering from uveitis, drusen, glaucoma, previous eye trauma, retinal vascular abnormalities, age-related macular degeneration, opaque media, previous optic nerve disease or other chronic eye diseases were also excluded, as well as those with history of intraocular surgery, refractive surgery, intravitreal injection, or systemic diseases other than thyroid disease.All participants underwent full medical and ophthalmological history taking. Validation of the diagnosis of hyperthyroidism was by measuring serum levels of thyroid stimulating hormone (TSH), free triiodothyronine (T3), and thyroxine levels (T4). All subjects underwent ophthalmological assessment by best-corrected visual acuity (BCVA) measurement using Snellen chart (and transformed into logMAR), slit-lamp examination, Goldman applanation tonometry, indirect ophthalmoscopy, ocular motility examination, and evaluation of the proptosis using Hertel's exophthalmometer. The following seven criteria were used to calculate clinical activity score (CAS): spontaneous orbital pain, gaze-evoked orbital pain, active (inflammatory phase) TAO-related eyelid swelling, eyelid erythema, active (inflammatory phase) TAO-related conjunctival redness, chemosis, and inflammation of caruncle or plica, with a score ≥ 3/7 indicating active TAO as reported previously . A-scan The spectral domain optical coherence tomography (SD-OCT) was used to perform the OCT . For macular thickness and RNFL measurement, mydriasis was used to prepare the eyes using tropicamide 0.5% eye drops. Our findings were represented by a macular map, which consisted of nine sectorial thickness measurements arranged in three concentric circles with sizes of 1, 3, and 6 mm, making three rings. The superior, nasal, inferior, and temporal quadrants were separated into the two outer rings. RNFL thickness was measured using the Disc Map Protocol (6 × 6 mm). The previously described procedure was performed by the same experienced operator for all patients.Electrophysiological tests were assessed with the RETI port/scan 21; . The parameters of the pattern visual evoked potential (PVEP) test and the recordings of the multifocal electroretinogram (mfERG) were in accordance with the International Society for Clinical Electrophysiology of Vision (ISCEV) standard , 16. Alsχ2) was used to assess gender difference between the two studied groups. The data were statistically analyzed using an independent sample t test. The different variables that can affect the macular thickness were assessed using regression analysis and Pearson's correlation analysis. The significance of the results was determined at p < 0.05 levels.The Statistica software, version 12, was used to analyze the data. The mean and standard deviation (SD) of quantitative variables were calculated. The chi-square (χ2 = 0.25 and p = 0.62). The age range of patients group was between 25 and 72 years old (mean ± SD = 46.35 ± 13.32), which was age and gender matched with the control group (mean ± SD = 46.63 ± 13.63). Table p < 0.001).In this study, a total of 80 eyes of 80 subjects were included, 40 in each group. They belonged to 29 males and 11 females without a statistically significant difference in the two studied groups. The male/female ratio was 2.3:1 in the control group and 3:1 in TAO group . The mean disease duration was 1.63 ± 0.70 years  and the clinical severity score ranged from 2 to 5.The results of OCT analysis are shown in Table p = 0.02). Regarding the results of mfERG analysis  showed a considerably lower value of ring1 than controls. Furthermore, in the cases, PERG P50 had a statistically significant lower amplitude. This may indicate early macular insult in these patients.We noted that the four-quadrant N1 amplitude is smaller, despite the fact that there was no statistical difference between the two groups when it came to the mean quadrant N1 amplitude. It is worth noting that the electrophysiologic tests as mfERG and P50 wave of PERG, which assess macular function, revealed substantial differences between patients and controls. While PVEP and the N95 wave of the PERG, which test visual pathway and optic nerve function, found no statistically significant differences between the groups. These findings suggest that TAO may have an impact on retinal and macular function.Retinal atrophy and lower macular thickness on OCT with corresponding diminished central response on mfERG were tested by Abazari et al.  similar According to Meirovitch et al. , OCT abnOur study has various limitations, the most significant of which is the small sample size multicenter studies may be more valuable, and a cross-sectional design would not provide more thorough data. Antithyroid antibody testing was not undertaken in our cohort, with sole reliance on serum thyroid hormonal levels and clinical examination for confirmation of the diagnosis. Finally, evaluation of the retinal microvasculature and their association with clinical and electrophysiologic variables was not conducted. A future study utilizing optical coherence tomography-angiography in TAO patients to investigate such subject is currently underway.Macular thinning along with functional alterations noticed by PERG and mfERG could be associated with subclinical retinopathy in TAO cases in the absence of fundus abnormalities.


The relationships between periodontitis and non-communicable diseases (NCDs) have been investigated through several different case-definitions. The differences in methodology may have hindered the basis of comparison between these studies. The classification from the 2017 World Workshop on the Classification of Periodontal and Peri-implant Diseases and Conditions offers a unison platform that may facilitate future comparison of such research. The present study aimed to reproduce associations between periodontitis and other NCDs using the 2017 Classification, in the Trøndelag Health Study (HUNT).The fourth HUNT-survey was carried out between 2017 and 2019. Clinical variables, blood samples and answers to questionnaires were collected from 4933 participants. Periodontal status was assessed based on the latest staging system, and its associations with NCDs were estimated by logistic regression models adjusted for potential confounders.Compared to no or Stage I periodontitis, participants with Stage III/IV periodontitis (radiographic bone loss exceeding 33%) were associated with cardiovascular disease, hyperglycemia in participants with diabetes and chronic obstructive pulmonary disease (COPD)/emphysema. Associations with hyperglycemia in participants with diabetes and COPD/emphysema were also observed in participants with Stage II periodontitis. The only observed association when considering never-smokers alone, was with COPD/emphysema.Periodontitis Stage II and III/IV were associated with major NCDs. Effect sizes increased with increasing periodontitis stages, which implies greater occurrence of coincident comorbidities in patients with severe periodontitis.The online version contains supplementary material available at 10.1186/s12903-023-03743-z. Periodontitis is a destructive inflammatory disease, causing loss of supporting bone and soft tissue surrounding the teeth, and may lead to tooth loss if left untreated. Periodontitis has been reported in 40% of the US adult population , and sevPeriodontitis is associated with several non-communicable diseases (NCDs) –6. ShareP. gingivalis has been shown to accelerate atherosclerosis in animal models, and to induce aorta fatty streaks after bacteremia [The supporting evidence of an association between periodontitis and systemic diseases includes several mechanisms. It is suggested that elevated systemic inflammation, observed through acute-phase proteins and oxidative stress biomarkers is a result of organisms entering the circulation. AGE-RAGE interactions are central in the plausible mechanistic links between periodontitis and diabetes, and leads to the exaggerated inflammatory response and periodontal tissue destruction seen in diabetics. Moreover, diabetes is associated with elevated levels of several cytokines and other mediators in saliva and gingival crevicular fluid (GCF) . Translocteremia , 9. Furtcteremia , 10.The 2017 Classification redefines the outline of case criteria and disease severities. The aim of this cross-sectional study was to reproduce associations between periodontitis with the use of the 2017 Classification and several prevalent NCDs.n = 103,800), were invited of whom 56,042 (54.0%) participated [The investigated population was a part of The Trøndelag Health Study (HUNT). HUNT is a longitudinal population-based study which constitutes a large database of questionnaire data, clinical measurements and biological samples –13. The n = 19) performed all clinical examinations, as described in a previous publication [Periodontal and systemic health status was assessed by clinical and radiological examination, answers to questionnaires and by collection of blood samples. Trained, calibrated dentists/dental hygienists . For participants with only BW radiographic examination available (n = 22), the percentage of bone loss was determined according to a study of average root lengths [n = 71).A description of case definition and periodontitis classification has been published previously , in shor lengths . All par lengths –20. Dete lengths , 22, bit lengths . The comSelf-reported history of NCDs  and hyperglycemia in self-reported diabetics, were the study endpoints. CVD was defined as a composite of myocardial infarction and/or angina pectoris and/or apoplexia . RheumatThe exposure periodontitis stage was grouped into three categories .2, continuous), hypertension (> 140/90 mmHg) and HbA1c-level (continuous) [Selected confounders included age (continuous), sex, smoking , years of education , total gross household income , BMI (kg/mtinuous) –27. InfoDescriptive statistics are presented as mean and standard deviation for continuous variables, and percentages with 95% confidence intervals for categorical variables. The one-way ANOVA test was used to assess significant differences across group means for continuous variables and Pearson chi-square test was used for categorical variables. Logistic regression analyses were used to assess associations between periodontitis stages and CVD , hyperglycemia in diabetics  in self-reported diabetics), rheumatoid disorders  and COPD/emphysema . All models were adjusted for potential confounders, listed in footnotes of Table Inter-rater reliability for clinical measurement (PPD) was calculated using intraclass correlation coefficient (ICC) two-way mixed effect model, assessing absolute agreement.ICC (95% CI) for the clinical examiners were:Generalized stage: ICC (95% CI) = 0.92 (0.88–0.95)Localized stage: ICC (95% CI) = 0.94 (0.91–0.96)0.57 (0.52–0.60), 0.68 (0.65–0.71), 0.71 (0.68–0.74), 0.77 (0.75–0.79), 0.77 (0.74–0.79), and 0.79 (0.75–0.82). Inter-rater reliability for evaluation of generalized stage and localized stage were calculated using ICC two-way mixed effect model, assessing absolute agreement.Inter-rater reliability for radiographic bone loss assessment (percentage of root length) was calculated using ICC two-way mixed effects, assessing consistency.Periodontal bone loss: ICC (95% CI) = 0.95 (0.918–0.964). All statistical analyses were based on complete cases and were performed using Stata/MP 16.0 .The HUNT4 Survey was approved by the Norwegian Data Protection Authority. Informed consent was obtained from all participants and/or their legal guardians. The current study was performed in accordance with relevant guidelines and regulations and was evaluated and approved by the Norwegian Regional Committees for Medical and Health Research Ethics during the conception of the study and when the data collection commenced . The paper was prepared following the STROBE guidelines.Characteristics of the total population by periodontitis stages are presented in Table The associations between periodontitis stages and NCDs are presented in Table The analyses of participants below 75 years and of non-diabetics, corresponded to the results of the main analyses  and Stage III/IV  in participants younger than 75 years, are in line with a report from the PAROKRANK case–control study , despite, while other studies report comparable periodontal status in patients with controlled diabetes to that of the general population [There is abundant literature on the relationship between periodontitis and diabetes. It has been stated that diabetic patients in general have higher severity of periodontal disease compared to non-diabeticspulation . A systepulation  concludePeriodontitis was not associated with rheumatoid disorders in the present study, which is in contrast to a recent systematic review and meta-analysis . Hussainn = 104) of the total population reported COPD/emphysema, and 51 of these 104 individuals (49%) were classified with periodontitis Stage III/IV.The present analysis of COPD/emphysema is supportive of a Japanese 5-year cohort study . The autThe stages of the 2017 classification reflect severity of periodontitis. Accordingly, it may be used to explore a dose-dependent relationship between periodontitis stages and associated conditions, and perhaps more easily identify if there are certain stages or individuals with increased likelihood of coincident diseases. This is of relevance for patients and dental professionals and may be important in risk factor modification as part of periodontal therapy. Conversely, individuals with systemic diseases may present more severe stages of periodontitis, and awareness of this association for patients and medical doctors is encouraged. The latest staging and grading system may facilitate research on how established and potential risk indicators or determinants of periodontitis are distributed between the different stages of disease. Nevertheless, the suitability of the new classification system in epidemiological research, and its potential benefits for patients’ periodontal health has yet to be explored. The cross-sectional design of the present study will not fully reflect disadvantages of the classification in this respect.The investigated population is representative of smaller cities and rural areas in Norway , 13 and Moreover, the magnitude of the crude ORs compared to the adjusted ORs of the statistical estimates suggests that some of the independent variables have a significant effect as determinants of the outcomes, together with periodontitis stages. This should be taken into consideration when interpreting the results.Self-reported systemic illness is a limitation in the present investigation. HUNT cohort profiles  indicateThe present investigation demonstrates that associations between periodontitis and history of CVD, diabetes and COPD/emphysema, increases with periodontitis stage severity.Additional file 1: Supplementary table 1. Association between periodontitis stages and cardiovascular disease, diabetes, rheumatoid disorders and COPD/emphysema, in participants below 75 years.Additional file 2: Supplementary table 2. Association between periodontitis stages and cardiovascular disease, rheumatoid disorders and COPD/emphysema, in non-diabetics.Additional file 3: Supplementary table 3. Association between periodontitis stages and diabetes, without consideration of HbA1c-levels.Additional file 4: Supplementary table 4. Association between periodontitis stages and cardiovascular disease, diabetes, rheumatoid disorders and COPD/emphysema, in never-smokers.Additional file 5: Supplementary table 5. Association between periodontitis stages and NCDs, by logistic regression analysis. Adjusted by five levels of income


Small intestine neuroendocrine neoplasms (siNENs) will attain more importance due to their increasing incidence. Moreover, siNENs might lead to a desmoplastic reaction (DR) of the mesentery causing severe complications and deteriorating prognosis. The expression of fibrosis-related proteins appears to be the key mechanisms for the development of this desmoplastic reaction. Therefore, this study aimed to investigate the association of the desmoplastic mesentery with specific fibrosis-related protein expression levels.By immunohistochemistry, the protein expression levels of four fibrosis-related markers  were investigated in primary tumors of 128 siNENs. The expression levels were correlated with the presence of a desmoplastic reaction and clinico-pathological parameters.n = 38), 64.9% (n = 83), 92.2% (n = 118) and 80.5% (n = 103), respectively. There was no significant correlation of a single marker or the complete marker panel to the manifestation of a desmoplastic mesentery. The desmoplastic mesentery was significantly associated with clinical symptoms, such as flushing and diarrhea. However, neither the fibrosis-related marker panel nor single marker expressions were associated with clinical symptoms.In the primary tumor, APLP2, BNIP3L, CD59 and DKK3 were highly expressed in 29.7% (The expression rates of four fibrosis-related markers in the primary tumor display a distinct pattern. However, the expression patterns are not associated with desmoplastic altered mesenteric lymph node metastases and the expression patterns did not correlate with prognosis. These findings suggest alternative mechanisms being responsible for the desmoplastic reaction. Small intestine neuroendocrine neoplasms (siNENs) have an increasing incidence , BNIP3L , CD59  and DKK3  . Every punch was categorized semi-quantitatively into 0: negative; 1: weakly positive; 2: moderately positive; 3: strongly positive  software. Categorical variables were tested for independence using the Fisher’s exact test. Survival was calculated from the date of surgery. End points for observed and disease-free survival were death of any cause and diagnosis of first recurrence, respectively. Significant and independent predictors of overall survival and disease-free survival were identified by Cox proportional hazard analysis. The procedure was set to a threshold of 0.05. Statistical significance was defined as a n = 86) of patients had a G1 tumor. DR was significantly associated with distant metastases and with a perineural tissue infiltration (p < 0.05). However, there was neither a significant correlation of DR with the tumor size nor its grading or Ki67 index (p > 0.05).In the present analysis, the primary tumors of 128 siNEN patients were analyzed. The clinicopathological characteristics are displayed in (Table n = 118), 80.5% (n = 103) and 64.9% (n = 83), while only 38 tumors (29.7%) highly expressed APLP2. However, there was no significant correlation of the expression rates of either marker with the presence of a DR (p > 0.05). In a further analysis, the predictive value of the combination of the four markers was tested. This analysis revealed that only 20 cases (15.6%) had high expression rates of all four markers. Thus, there was no significant correlation of the complete marker panel to DR accordingly (p > 0.05). High APLP2 was seen significantly more often in tumors with infiltration of the perineural tissue (p < 0.05) . Nonetheless, DR was not an independent risk factor for overall survival in the multivariate analysis (p > 0.05). Patients with a DR developed recurrent disease significantly earlier (median 18 months) than patients without a DR (median 42 months) (p < 0.001). DR was an independent risk factor for recurrence-free survival. Additionally, the presence of distant metastases at diagnosis was an independent risk factor for a significantly shorter recurrence-free survival . However, DR was significantly associated with flushing and diarrhea. Flushing was present in 60% (n = 21) of patients with DR and 59% of patients with DR (n = 23) suffered of diarrhea. CgA blood levels did not correlate significantly with a fibrosis-related marker or the presence of DR. On the contrary, 5-HIAA blood levels were significantly increased in patients with either a DR or a siNEN highly expressing APLP2 (p < 0.05).Data of preoperatively measured biomarkers, such as Chromogranin A (CgA), serotonin and 5-hydroxyindoleacetic acid (5-HIAA), were analyzed. The values of CgA were available for 100 patients (78.1%), of serotonin for 30 patients (23.4%) and of 5-HIAA for 84 patients (65.6%). The information about clinical symptoms, such as flushing and diarrhea, was conclusive for the entire cohort. Therefore, only the clinical symptoms and CgA as well as 5-HIAA were further investigated. None of the four fibrosis-related markers showed a significant correlation to clinical symptoms  retrospective studies and the most important prognostic factors remain tumor size and grading  in the receiver operating characteristic analysis of 0.962–0.969 compared to the other two fibrosome genes with an AUC of only 0.377 and 0.785  of siNENs expressing APLP2 (Arvidsson et al. SiNEN patients with a desmoplastic mesentery are significantly more prone to flushing and diarrhea. Thus, a possible correlation of the marker panel with clinical symptoms and biomarkers, such as CgA, serotonin and 5-HIAA, was investigated. Due to incomplete data sets, only CgA and 5-HIAA could be analyzed sufficiently. Nonetheless, the protein expression levels of the fibrosis-related markers were not associated to CgA and 5-HIAA blood levels. This lack of significance might be due to the fact that the present study analyzed the fibrosis-related markers in the primary tumor. Furthermore, only less than a third of patients expressed APLP2, which is contradictory to Arvidsson et al. who found 81% (Although this is the largest study of a well-described siNEN collective so far, there are limitations as well. First, this is a retrospective cohort study of a surgical collective introducing a bias. Since siNENs are still a rare disease and diagnostic as well as predictive tools need to be discovered, retrospective studies are inevitable. The present study presents comprehensive data of more than 120 siNEN patients representing the largest collective in this field. Second, the present analysis investigated histological sections on a TMA which represents only a small part of the tumor. Thus, to minimize a bias, two punch biopsies of each primary tumor were analyzed and using a TMA is well established (Kidd et al. In conclusion, this is the first study elucidating the association of four well-known fibrosis-related markers to the presence of a desmoplastic mesentery. However, there was no significant correlation of the marker panel to the prognostic relevant DR. Thus, the induction of DR remains ill-defined and further research is urgently necessary.


Z=3.66, P<0.001)), higher PI ) and higher resistive index ), compared to neonates who did not develop NEC. However, our findings do not support a strong association between the Doppler ultrasound indices and development of NEC at the time of disease onset. This meta-analysis suggests that first postnatal day SMA Doppler parameters, namely peak systolic velocity, PI and resistive index, are higher in neonates who develop NEC. On the other hand, the aforementioned indices are of uncertain significance once the diagnosis of NEC has been established.The role of postnatal Doppler measurements of the superior mesenteric artery (SMA) in detecting neonates at risk of necrotizing enterocolitis (NEC) remains uncertain; therefore, we systematically reviewed and meta-analyzed the existing evidence regarding the usefulness of SMA Doppler measurements in detecting neonates at risk for NEC. We used the Preferred Reporting Items for Systematic reviews and Meta-Analyses guidelines, and we included studies which reported the following Doppler ultrasonography indices: peak systolic velocity, end-diastolic velocity, time average mean velocity, differential velocity, pulsatility index (PI) and resistive index. Eight studies were eligible for inclusion in the meta-analysis. Evidence suggested that, during the first postnatal day, neonates who developed NEC had a significantly higher peak systolic velocity (mean difference of 2.65 cm/s (95% confidence interval [CI] 1.23, 4.06, overall effect Supplementary material is available at 10.1007/s00247-023-05695-6. Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease in neonates and is characterized by intestinal injury, systemic inflammation and multisystem organ failure . ExtremePrenatal placental failure is a risk factor for antenatal NEC due to increased placental vascular resistance which results in a reduction of end-diastolic blood flow in the umbilical arteries, ultimately progressing to absent or reversed end-diastolic flow . Fetal aTo date, studies have mainly focused on the role of prenatal examination of umbilical artery Doppler indices in the evaluation of the risk of NEC; however, the evidence regarding the role of postnatal measurement of SMA Doppler indices remains inconsistent. Therefore, we aimed to perform a systematic review and meta-analysis of the literature regarding the value of SMA Doppler indices for the early detection of neonates who are at increased risk of developing NEC.A pre-specified search protocol was formulated by two authors  to examine whether Doppler ultrasonography of the SMA can assist in the early prediction of NEC. The study protocol was registered in PROSPERO  (CRD42022316568). We formulated eligibility criteria using the P (Populations), I (Intervention), C (Comparison), O (Outcome) worksheet and search strategy. In detail, the population consisted of neonates, the intervention was defined by the performance of SMA Doppler ultrasonography, comparisons were performed between neonates who developed NEC and counterparts who did not develop NEC and the outcome was the manifestation of NEC.1. What is the Doppler ultrasonography difference on the first postnatal day between neonates who developed NEC in comparison to those who did not?2. What is the Doppler ultrasonography difference at disease onset between neonates with and without NEC?A series of clinical questions were formulated, including the following:The questions were elucidated based on the results of the systematic literature search.The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines were adopted for this systematic review. Two authors (D.R. and P.M.) independently performed a literature search in PubMed, Google Scholar and ScienceDirect. Disagreements were resolved by consensus or after consultation with a third reviewer . The literature was searched from 1 January 1990 to 31 January 2022, with filters [‘humans’] and search terms ‘necrotizing enterocolitis’ OR ‘NEC’ plus Boolean operator ‘AND,’ ‘superior mesenteric artery’ OR ‘SMA’ plus Boolean operator ‘AND,’ ‘Doppler’ or ‘ultrasound’ or ‘sonography.’ Additionally, the references of the identified studies were searched to ensure that no study was missed.Eligible studies were those that included humans, with a full text available and published in English and with available neonatal data regarding Doppler ultrasonography indices  independently reviewed the title, abstract and full text of the included studies. Data were extracted independently by the two reviewers on a Microsoft Excel spreadsheet using a predefined checklist. Extracted data included but were not limited to the following: author name, year, journal name, the country where the study was conducted, study design, study population, sample size and details of main findings, such as the differences between the subgroups of subjects regarding the Doppler ultrasonography, the risk of NEC and the risk of feeding intolerance.Two authors (D.R. and P.M.) independently assessed the risk of bias using the Newcastle–Ottawa Scale for evaluating the methodological quality of studies, which is composed of three factors: selection, comparability and outcome/exposure . For cohSelected studies were subdivided into two groups: (a) studies that evaluated Doppler ultrasonography indices on the first postnatal day when neonates had no obvious signs or symptoms of NEC; (b) studies that evaluated Doppler ultrasonography indices at the onset of NEC.Cochrane Handbook for Systematic Review of Interventions , with heterogeneity I2=83%  in end-diastolic velocity between the two groups of 0.05 cm/s , with heterogeneity I2=66%  compared to those who did not, with heterogeneity I2=87%  between the two groups, with heterogeneity I2 = 37%  in peak systolic velocity of  − 0.10 cm/s  between the two groups, with heterogeneity I2=88%  in end-diastolic velocity of  − 0.64 cm/s  between the two groups, with heterogeneity I2=66%  in time average mean velocity of 8.26 cm/s  between the two groups, with heterogeneity I2=91%  of  − 0.63  between neonates who developed NEC compared to those who did not, with heterogeneity I2=90%  in resistive index of  − 1.19  between the two groups, with heterogeneity I2=97% Supplementary file2 (PDF 812 KB)Supplementary file3 (PDF 734 KB)Supplementary file4 (PDF 816 KB)Supplementary file5 (PDF 809 KB)Supplementary file6 (PDF 834 KB)Below is the link to the electronic supplementary material.


Although most patellar dislocations are associated with medial patellofemoral ligament (MPFL) injury, many patients also sustain concomitant patellar osteochondral fractures following a patellar dislocation. Few prior studies have described or evaluated risk factors for patellar osteochondral fractures in pediatric patients. The purpose of the present study was to describe the incidenceand location of patellar osteochondral fractures following acute patellar dislocation in pediatric patients. In addition, we described risk factors for patellar osteochondral fractures in this population. We hypothesized that most fractures would occur at the inferomedial quadrant of the patella following a traumatic injury mechanism.Following Institutional Review Board approval, the electronic medical record was queried to identify pediatric patients ≤ 18 years old who underwent MPFL reconstruction (MPFLR) or non-operative treatment for patellar instability between July 2016 and February 2020. Osteochondral fractures were defined as full-thickness chondral injuries with attached subchondral bone or purely osseous injuries measuring ≥ 3 mm (mm) in at least two magnetic resonance imaging (MRI) planes. Patients were included in the study if they had complete preoperative x-ray and MRI studies with minimum 6-month follow-up. Patients were excluded if they had incomplete imaging, isolated chondral fractures, or less than 6 months follow-up. Univariate analysis was used to identify patient factors associated with osteochondral fractures. Multivariate regression analysis was used to identify risk factors for osteochondral fractures.p = 0.041), skeletal immaturity (p = 0.028), and decreased patellar tilt (p = 0.021) and patellar osteochondral fractures. Multivariate regression analysis identified male sex as an independent risk factor for osteochondral fractures . No patients had recurrent dislocation at minimum 6-month follow-up. All patients with osteochondral fractures returned to sports at most recent follow up.Sixty patients (63 knees) were included in the study, 15 (23.8%) of whom had a patellar osteochondral fracture. The majority of osteochondral fractures (87%) involved the inferomedial quadrant of the patella. Univariate analysis showed an association between male sex (In this study, 23% of pediatric patients with acute patellar dislocations have a concomitant patellar osteochondral fracture. The majority of patellar osteochondral fractures involve the inferomedial quadrant of the patella. Male sex is an independent risk factor for patellar osteochondral fractures, and skeletal immaturity is associated with patellar osteochondral fractures in this population.Level III. Acute patellar dislocations (APDs) are a common cause of knee pain in pediatric patients . Twenty-The majority (96–97%) of APDs are associated with injury to the medial patellofemoral ligament MPFL , 24), wh, wh24]),The incidence and characteristic patterns of concomitant patellar osteochondral fractures in adults with APDs are well-described. Eleven to 76% of adults sustain osteochondral fractures following patellar dislocation, the majority of which 42 – 63%) involve the patella , 29, 30.2 – 63% iPatellar osteochondral fractures are a relatively common sequela of APD in children, with a reported incidence of 29% in first-time dislocation events . A previThe primary aim of the present study was to describe the incidenceand location of patellar osteochondral fractures in pediatric patients with acute patellar dislocations. The secondary aim of the study was to describe risk factors for concomitant patellar osteochondral fracture, surgical management, and recurrent dislocation rate in this patient population at mid-term follow-up. The authors hypothesized that the majority of fractures would involve the inferomedial quadrant of the patella and occur more frequently following traumatic injury mechanisms.Institutional review board (IRB) approval was obtained at our institution prior to initiation of the study (IRB number: 2020–0203). We conducted a retrospective review of a consecutive series of pediatric patients treated for acute patellar dislocation at a single tertiary care center between July 2016 and February 2020. All surgical procedures were performed by a single pediatric orthopaedic sports medicine-trained orthopaedic surgeon (D.W.G.). Eligible patients were identified either by query of the electronic medical record by procedure type (MPFL reconstruction) and surgeon (D.W.G.) or ICD-10 code for patellar instability  from July 2016 to February 2020.Patients were included in the study if they were ≤ 18 years old and underwent pre-operative anteroposterior, lateral, and Merchant view plain radiographs and standard magnetic resonance imaging (MRI) of the affected knee. Inclusion criteria also consisted of minimum 6-month postoperative follow-up for patients who underwent surgery or minimum 6-month clinical follow-up post-diagnosis if they elected to pursue non-operative treatment. Patients with incomplete imaging, isolated chondral fractures, or less than 6 months of postoperative/post-diagnosis clinical follow-up were excluded from analysis.Hamstring autograft was used for all patients. A 2 cm posteromedial incision is made over the proximal tibia, and a tendon harvester is used for semitendinosus tendon harvest. The graft is prepared for double-bundle reconstruction, with a target final graft length of 60 mm, including 20 mm in the femoral socket. A 3 cm anteromedial incision is made over the patella, and the medial border of the patella is exposed. Two patellar sockets are drilled in the proximal half of the patella to a depth of 12-15 mm. A 2 cm incision is made over the femoral MPFL insertion site. A guidewire is placed at Schöttle’s point  in the eA femoral socket is drilled to a depth of 20 mm. The doubled end of the graft is placed into the femoral socket and secured with an interference screw. The graft is then passed between layers 2 and 3 of the knee, and the graft length is determined based on the appropriate tension at 30 degrees of knee flexion. The two free ends of the graft are secured to the patellar sockets with suture anchors. The knee is brought through full range of motion for a final check of graft isometry .Like the skeletally immature patients, hamstring autograft was used for skeletally mature patients who had closed distal femoral physes on magnetic resonance imaging (MRI) using the same technique described above. The graft is prepared for double-bundle reconstruction, and the patellar sockets are prepared in the same fashion. A 2 cm incision is used at the MPFL insertion site on the femur, and a guidewire is placed at Schottle’s point . HoweverPatellar fractures were defined as either full-thickness chondral injuries with attached subchondral bone  or purely osseous injuries (without associated articular cartilage) measuring ≥ 3 mm in at least two MRI planes . Patellar fractures without at least 3 mm of bone in two or more MRI planes were excluded from analysis. Patellar osteochondral fractures were categorized by location Fig. . PatientPatellar tilt was calculated as the angle between a line parallel to the articular surface of the patella and a line connecting the summits of the medial and lateral femoral condyles on Merchant view radiographs Fig.  13]. Th. Th13]. Descriptive statistics were used to evaluate patient and fracture characteristics. Means and standard deviations were used for continuous data, whereas categorical data was reported as frequencies and percentages. Data for age, TT-TG, CDI, and patellar tilt were normally distributed per the results of the Shapiro–Wilk test; therefore, unpaired t-tests were used for analysis. Data for BMI was not normally distributed based on Shapiro–Wilk test results, and Wilcoxon Rank-Sum test was used for analysis. Categorical data were analyzed with Fisher’s exact tests. Poisson regressions with robust error variance  were used to determine association between each patient characteristic and occurrence of a fracture. Two-way mixed inter- and intraclass correlation coefficients were calculated for each measurement. Data analysis was performed with STATA . Statistical significance was determined at the α < 0.05 level.A total of 80 patients (84 knees) were identified, 60 of whom (63 knees) remained following application of exclusion criteria. Of the patients included in the study, the mean age was 14.2 ± 1.8 years, and mean BMI was 21.9 ± 7.8. Thirty-two patients (53.3%) were female, and 30 (47.6%) were right knees  were visible on AP knee XR (Table Fifteen knees 23.8%) sustained osteochondral fractures, and the majority of fractures involved the inferomedial quadrant  and skeletally immature . There was no difference in age between patients with osteochondral fractures (average age 13.6 ± 1.7 years) compared to those without osteochondral fractures , laterality , or average follow-up  between the two groups. There were no differences between the two groups with regard to injury mechanism  or history of prior dislocation (Table p = 0.23). There was also no association between sex and skeletal maturity (p = 0.25) or sex and age (p = 0.34) in the entire cohort.Patients with osteochondral fractures were more likely to be male (p = 0.079) or TT-TG values between the two groups . Patients without osteochondral fractures had increased patellar tilt (17.8 ± 6.8°) compared to those with osteochondral fractures .There were no differences in average CDI values . Among those with osteochondral fractures who underwent surgery (n = 13), 5 patients (38.5%) underwent MPFL reconstruction (MPFLR) with patellar open reduction and internal fixation (ORIF) and 8 (61.5%) underwent MPFLR only , whereas 62.5% (5 patients) of those who underwent MPFLR only had articular cartilage involvement (Table p = 0.061). There were no recurrent patellar dislocations at most recent follow-up in either group. All patients with osteochondral fractures (n = 15) returned to sports at most recent follow up.The majority of patients with and without osteochondral fractures underwent surgical intervention , whereas patellar tilt was no longer significant following multivariate regression analysis  4.8, 95% confidence interval [CI]: 1.08–20.9, Two-way mixed intraclass correlation coefficient values were as follows: CDI: 0.75, TTTG: 0.90, patellar tilt: 0.81, osteochondral fracture location: 0.77. Two-way mixed interclass correlation coefficient values were as follows: CDI: 0.77, TTTG: 0.76, patellar tilt: 0.90, osteochondral fracture location: 0.77.We found that patellar osteochondral fractures occurred in 23% of pediatric patients with APD, and the majority of osteochondral fractures involved the inferomedial quadrant of the patella. Male sex was an independent risk factor for patellar osteochondral fractures, as males were 4.8 times more likely to sustain an osteochondral fracture than their female counterparts. In addition, skeletal immaturity was associated with osteochondral fractures in our cohort. No patients in either group had a recurrent dislocation at minimum 6-month follow-up.Although there is a wide range of reported rates of associated osteochondral fractures in adults with APD reported in the literature (11–76%) , 29, 30,Like Jungesblut et al.  and MochThe present study builds on the existing literature by including a skeletally immature patients (average age 14.2 ± 1.8 years) who sustained patellar osteochondral fractures with articular cartilage involvement. Mochizuki et al. , by contResults of a previous histological study by Flachsmann et al.  may explMales were 4.8 times more likely to sustain concomitant patellar osteochondral fractures than females in our cohort. This result is in line with Zheng et al. , who fouWith regard to radiographic parameters, patients without osteochondral fractures had increased patellar tilt compared to patients with osteochondral fractures. In addition, TT-TG and CDI values tended to be higher on average in the no fracture group compared to the fracture group, although this finding was not significant. Given that increased patellar tilt, CDI, and TT-TG are risk factors for patellar dislocations , it coulThere were two purely osseous fractures in our cohort, both of which occurred in zone IV. It is likely that these purely osseous zone IV fractures occurred as a result of a medial patellotibial ligament (MPTL) avulsion, as opposed to a medial patellofemoral ligament avulsion. The MPTL originates on the distal third of the medial border of the patella and inserts just medial to the patellar tendon insertion approximately 15 mm distal to the joint line , 20, andThere are several limitations to the present study. Given the retrospective nature of this study, our results are limited by selection bias, as the type and timing of surgical management were not randomized. In addition, our small sample size renders our study susceptible to type II error, particularly for variables which approached significance, such as CDI. There was also heterogeneity in our study population, as more than one third of the patients included in our study had a history of at least two patellar dislocations. However, we argue that rate of recurrent patellar dislocations in our study population is representative of pediatric patients with APD. Moreover, given the underlying assumptions of Poisson regression model, we were unable to evaluate skeletal immaturity as a risk factor for osteochondral fractures, as there were no skeletally mature patients with osteochondral fractures in our cohort. However, skeletal maturity was associated with osteochondral fractures in univariate analysis, and there was no association between skeletal maturity and gender in univariate analysis. Finally, the present study only included short-term follow-up (minimum 6 months), and future studies can include longer-term follow-up.In conclusion, male sex is an independent risk factor for patellar osteochondral fractures, and skeletal immaturity is associated with patellar osteochondral fractures in pediatric patients. The majority of patients have good outcomes, including a low recurrence rate, at short-term follow-up.


African swine fever virus (ASFV) is a substantial threat to pig populations worldwide, contributing to economic disruption and food security challenges. Its spread is attributed to the oronasal transmission route, particularly in animals with acute ASF. Our study addresses the understudied role of nasal mucosa in ASFV infection, using a nasal explant model. The explants remained viable and revealed a discernible ASFV infection in nasal septum and turbinates post-inoculation. Interestingly, more infected cells were found in the turbinates despite its thinner structure. Further analyses showed (i) a higher replication of genotype II strain BEL18 than genotype I strain E70 in the epithelial cell layer, (ii) a preference of ASFV infection for the lamina propria and a tropism of ASFV for various susceptible cell types in different areas in the nasal mucosa, including epithelial cells, macrophages, and endothelial cells. Using porcine respiratory epithelial cells (PoRECs), isolated from nasal tissue, we found a difference in infection mechanism between the two genotypes, with genotype I favoring the basolateral surface and genotype II preferring the apical surface. Moreover, disruption of intercellular junctions enhanced infection for genotype I. This study demonstrated that ASFV may use the respiratory mucosa for entry using different cell types for replication with a genotype difference in their infection of respiratory epithelial cells.The online version contains supplementary material available at 10.1186/s13567-023-01249-8. Asfarviridae  was calculated. Mock-inoculated samples from 0, 24, 48, and 72 hpi were analyzed Figure A. Cells 2; BEL18: 22.80 ± 1.45 cells/mm2) than in the septum explant . To study the pattern of infection in different areas of the nasal mucosa, infected cells were quantified and the proportion of infected cells in each area was calculated as percentage , by immunofluorescence (IF) staining against CD163 and ASFV major capsid protein p72 , SWC3 (marker for myeloid cell), Sn (marker for macrophage), CD1c (marker for antigen presenting cell), cytokeratin , vimentin , MHCII (marker for antigen presenting cell), and von Willebrand factor     and CD163  , employing both apical and basolateral side-inoculation strategies. To determine whether intercellular junction (ICJ) influences ASFV infection, cells were treated with 25 mM EGTA before inoculation at the apical surface.2), than after apical inoculation (72 hpi: 2.22 ± 2.22 cells/cm2) . However, subsequent incubation resulted in a progressive decrease in infection (48 hpi: 113.70 ± 29.66 cells/cm2 and 72 hpi: 84.81 ± 24.48 cells/cm2) , compared to basolateral inoculation (1.85 ± 3.21 cells/cm2)  and significantly decreased at 48 hpi (58.15 ± 1.28 cells/cm2) and slightly increased at 72 hpi  , whereas BEL18-infected cells, upon apical inoculation, appeared to be dispersed individually Figure B. In thed Figure B. Furthed Figure .ASFV continues to pose a severe threat to global pig populations, causing substantial economic damage, disrupting food security, and giving issues with animal welfare. Among the several ASFV transmission routes, the oronasal route is a significant factor in understanding the viral spread, especially in animals with acute ASF . The nasWe first analyzed the viability of nasal mucosa explants during in vitro cultivation. Our findings demonstrated that cells in the epithelium (EP) and the lamina propria (LP) maintained high levels of viability after 72 h of incubation. We observed an increase in the number of apoptotic cells within the submucosa (SM). However, these cells were mainly restricted at the edge of the tissue, suggesting the potential damage during the cutting of the explant. Grossly, the nasal explants appeared to be viable without substantial morphological change.Our results, obtained via immunofluorescence staining for the major ASFV capsid protein p72 and monocyte/macrophage marker CD163, uncovered key aspects of ASFV infection within the nasal explant. Intriguingly, infections by both genotypes (genotype I strain E70 and genotype II strain BEL18) were discerned within the nasal septum and turbinates. Over the incubation period after inoculation, we noted a rise in the total number of infected cells in both nasal tissues for both strains. Although the difference between E70 and BEL18 strains was not significant, we observed a higher number of infected cells in the turbinate explant compared to the septum explant. We additionally measured the thickness of the septum and turbinate explants, and surprisingly, the septum was significantly thicker than the turbinate, which appeared to be due to the thicker submucosal layer , revealed that the lamina propria is a preferential site for ASFV replication, irrespective of the viral strain. The infected cells were found in clusters, predominantly in the EP and LP implying a spread in a cell-associated way. Although it is generally accepted that the main target cells of ASFV are monocytes and macrophages , a signiBuilding further upon our findings, which revealed a significant proportion of cytokeratin-positive infected cells in the epithelium, we expanded our investigation by inoculating two ASFV strains in the respiratory epithelial cells isolated from the nasal tissue (PoRECs). Intercellular junctions (ICJ) play a pivotal role in maintaining the integrity and polarity of the ciliated pseudostratified columnar epithelium by providing a distinct barrier between the apical and basolateral domains of the cell. Certain viruses, such as respiratory syncytial virus, hepatitis A virus, and simian virus 40, display a preference for infecting via the apical surface of the cells –38, whilWe also identified cytokeratin-negative infected cells in our PoRECs. Additional triple IF staining against ASFV, vimentin, and CD163 revealed that infected cells are neither mesenchymal/fibroblast cells nor monocytes/macrophages. This suggests that these cytokeratin-negative infected cells are probably non-differentiated epithelial cells. In the future study, an approach with various epithelial cell markers is necessary to better characterize these cells. Based on these findings, one may conclude that genotype I and genotype II use different receptors to enter the nasal mucosa. Future work will focus on the identification of these receptors.In summary, the present study is the first that investigated ASFV infection in the nasal mucosa. The findings from our ex vivo model have revealed that ASFV demonstrates a broad cell tropism in the nasal mucosa. Furthermore, our in vitro model using primary respiratory epithelial cells highlights that epithelial cells are susceptible to ASFV and that the two genotypes use different surfaces for infection: genotype I basolaterally and genotype II apically. While our study provides valuable insights into the molecular and cellular aspects of ASFV infection within the nasal mucosa, they have inherent limitations as they cannot fully replicate the complex environment within a living organism. Therefore, future in vivo studies will be crucial for gaining a more comprehensive understanding of ASFV pathogenesis in the nasal mucosa at early timepoints in infection. The knowledge gained from our current research will significantly facilitates the interpretation of subsequent in vivo results. Our study shed new light on the pathogenesis of ASFV in the respiratory system and emphasizes the possible role of the nasal route for ASFV transmission.Additional file 1. Reactivity of selected cell markers in the lung and nasal tissue sections. Immunofluorescence staining for the selected cell markers was performed on the lung and nasal tissue sections. Reactivity signal is presented in plus and minus symbols. Abbreviations—Mo: monocyte, Mf: macrophage, Mc: myeloid cell, APC: antigen presenting cell, Ep: epithelial cell, MSc: mesenchymal cell, Fi: fibroblast, En: endothelial cell.Additional file 2. Immunofluorescence staining of the lung tissue section. All the cell markers were positive in the lung tissue except vWF. Cell markers (red), and nuclei (blue). Scale bar: 50 μm.Additional file 3. Percentage of cell marker positive cells within ASFV-infected cells. Quantification and calculation were made in three regions in the nasal septum (A) and turbinate (B) explants: epithelium (EP), lamina propria (LP), and submucosa (SM). Septum-E70: E70 inoculated septum explant, septum-BEL18: BEL18 inoculated septum explant, turbinate-E70: E70 inoculated turbinate explant, turbinate-BEL18: BEL18 inoculated turbinate explant. Values are presented as mean value of three animals ± SD.Additional file 4. Characterization of ASFV infected PoRECs. Triple immunofluorescence staining for ASFV p72 (green), vimentin (red), and CD163  was performed on respiratory epithelial cells after inoculation with E70 and BEL18 strains. Cell nuclei were presented in blue. Scale bar: 50 μm.Additional file 5. Measurement of the nasal explant thickness. (A) Areas measured in the nasal septum and turbinate explants. (B) The width and thickness of the nasal explants were measured. The total thickness of the septum tissue was significantly higher than that of the turbinate tissue. (C) The thickness of different areas in the septum and turbinate explants was measured. The submucosa was significantly thicker in the septum compared to that of the turbinates. Statistical analysis for (B) and (C) was performed using two-way ANOVA followed by Šidák’s multiple comparison test (****p < 0.0001).


RMST) as one of the most downregulated lncRNA in ATC. RMST has been reported to be deregulated in a series of human cancers, to play an anti-oncogenic role in triple-negative breast cancer, and to modulate neurogenesis by interacting with SOX2. Therefore, these findings prompted us to investigate the role of RMST in ATC development. In this study we show that RMST levels are strongly decreased in ATC, but only slightly in DTC, indicating that the loss of this lncRNA could be related to the loss of the differentiation and high aggressiveness. We also report a concomitant increase of SOX2 levels in the same subset of ATC, that inversely correlated with RMST levels, further supporting the RMST/SOX2 relationship. Finally, functional studies demonstrate that the restoration of RMST in ATC cells reduces cell growth, migration and the stemness properties of ATC stem cells. In conclusion, these findings support a critical role of RMST downregulation in ATC development.Thyroid cancer is the most prevalent endocrine malignancy and comprises a wide range of lesions subdivided into differentiated (DTC) and undifferentiated thyroid cancer (UTC), mainly represented by the anaplastic thyroid carcinoma (ATC). This is one of the most lethal malignancies in humankind leading invariably to patient death in few months. Then, a better comprehension of the mechanisms underlying the development of ATC is required to set up new therapeutic approaches. Long non-coding RNAs (lncRNAs) are transcripts over 200 nucleotides in length that do not code for proteins. They show a strong regulatory function at both transcriptional and post-transcriptional level and are emerging as key players in regulating developmental processes. Their aberrant expression has been linked to several biological processes, including cancer, making them potential diagnostic and prognostic markers. We have recently analyzed the lncRNA expression profile in ATC through a microarray technique and have identified rhabdomyosarcoma 2-associated transcript ( New hotspots in cancer research are represented by long non-coding RNAs (lncRNAs). LncRNAs, by interacting with DNA, RNA or proteins, serve as regulators at transcriptional and post-transcriptional levels. Their ability to impact on angiogenesis, proliferation, migration, apoptosis, and differentiation has been associated to both oncogenic and tumor suppressor functions. Furthermore, their presence in the peripheral blood and urine identified lncRNAs as candidate biomarkers in cancer diagnosis and prognosis .Thyroid cancer (TC) represents the most common malignant endocrine tumor. Differentiated forms of thyroid cancer (DTC) comprise the common thyroid cancer types, papillary (PTC) and follicular (FTC), and present an excellent outcome in most cases. Conversely, anaplastic thyroid cancers (ATC) are the undifferentiated and most aggressive forms of TC. ATC is still a deadly cancer with an overall survival rate of 3–5 months after initial diagnosis .NAMA (noncoding RNA associated with MAP kinase pathway and growth arrest) that targets the MAPK signaling pathway . As. AsRmst  4) Fig. .RMST has been described as indispensable for physiologic neurogenesis. More in details, it interacts with SOX2, a transcription factor, thus regulating in a coordinated manner many downstream genes implicated in neurogenesis [ogenesis . In the ogenesis . SOX2 haogenesis , 26 and ogenesis .SOX2 expression levels in undifferentiated human and mouse thyroid lesions and analyzed them in relation to the RMST levels in the experimental systems above described  samples compared with the control tissues (n = 4)  Fig. . These f 4) Fig. .RMST is strongly downregulated in undifferentiated thyroid lesions, and further support the hypothesis of a critical role of RMST downregulation in stemness and in progression of ATC.Our expression data on both human and mouse samples indicate that RMST role in ATC from a functional point of view, we first of all evaluated by qRT-PCR its expression levels in a panel of PTC-derived (B-CPAP and TPC-1), FTC-derived (WRO) and ATC-derived  cell lines, including in our analysis as a control the Nthy-ori, an immortalized cell line derived from NT epithelial cells [RMST expression levels compared to Nthy-ori  vector   Fig. .Fig. 3RMRMST on cell cycle and apoptosis in order to understand how it inhibits the proliferation of TC cells. We analyzed the cell cycle distribution in serum-deprived RMST-transfected cells compared to the corresponding empty vector control (pLenti) cells. Supplementary Fig. RMST cells did not display differences in cell cycle phases compartmentalization compared to the relative pLenti-TC cells  and AnxV-PI+ or AnxV+PI+ (necrotic/dead) cells  represents the main program activated in cancer cells to ensure the acquisition of an aggressive phenotype . Since tRMST on EMT in ATC cells we analyzed the expression of master regulators of EMT and tested the invasion properties of the ATC cells overexpressing RMST. As reported in Fig. RMST was able to sustain a trend in mRNA downregulation of several EMT markers  in both FRO and SW1736 cell lines, being only NES1 levels significantly downregulated in SW1736-RMST cells, compared to that transfected with the empty vector. Consistently, the FACS analysis performed on permeabilized TC cells transfected with RMST revealed a significant reduction in the protein levels of both SLUG and SNAIL, two critical EMT master regulators [To evaluate biochemical and functional impact of gulators , compareRMST effects on functional EMT, we first of all evaluated the morphology of FRO and SW1736 cells transfected with the empty vector or with RMST. Supplementary Fig. RMST-transfected cells, looking well adherent one to the other and with a classic epithelial morphology   Fig. .RMST physiological and functional roles involve its ability to interact with SOX2 transcription factor [RMST in comparison to SOX2 levels in human and mice TC samples was observed, we investigated the levels of SOX2 in cells with or without enforced expression of RMST, together with the expression of several other stemness markers  since SOX2 in TC is a master regulator of cancer stem cells [RMST overexpression in both FRO and SW1736 cells significantly reduced the mRNA of SOX2 and of several other stemness markers, such as NANOG, OCT4, ABCG2, ALDH, CD38 and CD133, as assessed by qRT-PCR. The reduction in SOX2 and OCT4 expression was then confirmed also at protein level by intracellular cytofluorimetric analysis  was carried out. Interestingly, as shown in Fig. RMST overexpression, when compared to relative controls, as assessed by the number of the thyrospheres and average sphere diameter. Furthermore, we employed a limiting dilution assay on FRO and SW1736 cells cultured as thyrospheres to evaluate the impact of RMST on the clonogenic potential of TC cells. RMST transfection significantly reduced the clonogenic potential of TC cells compared to the relative controls . Therefore, it requires a better comprehension of the molecular mechanisms underlying its development to make an early diagnosis, prevent TC progression, put on effective therapeutic treatments, thus improving the survival of ATC patients.RMST lncRNA downregulation in ATC  is deregulated in a series of human cancers [LncRNAs are new emerging players in human cancers and, because of their aberrant expression in this pathology, they could represent important tools for the diagnosis and novel targets for the approach of innovative therapies. In this study we aimed at characterizing the role of the nt study ) since s cancers , 15, (b) cancers , (c) reg cancers , and (d) cancers .RMST expression in a panel of TC samples confirming its drastic downregulation in ATC, whereas just a slight reduction was observed in PTC and FTC. Interestingly, SOX2 expression was inversely correlated with that of RMST in the same neoplastic tissues. Subsequently, these results were further confirmed through the analysis of two publicly available databases, thereby reinforcing the hypothesis of a critical role of RMST downregulation in TC progression. Interestingly, Rmst downregulation, associated with an increase of Sox2 expression, was also observed in UTC induced in transgenic animals carrying a mutated form of the BRAF gene.Firstly, we evaluated RMST was considerably lower in adjacent NT tissues than it was in PTC tissues, and this downregulation was correlated with the TNM stage and lymph node metastases [RMST was specifically downregulated in ATC [RMST role in thyroid carcinogenesis remains unknown.According to a recent study, the expression of tastases . A more d in ATC , but, deRMST expression in FRO and SW1736 cells. RMST repressed the main aggressive features of ATC cells, such as cell proliferation, migration and invasion. Moreover, a reduction of several mesenchymal markers was also observed in RMST overexpressing ATC cells when compared to the control empty vector transfected cells, indicating the involvement of RMST in EMT. Noteworthy, RMST expression is able to repress the expression of mesenchymal markers such as SNAIL, SLUG and TWIST1 [RMST can also regulate them by acting on their upstream regulatory pathways [Then, its role in TC progression was investigated by restoring d TWIST1 , 34. Thed TWIST1 , 35. Intpathways .RMST restoration led also to a significant reduction in the expression of the stemness markers OCT4, SOX2 and NANOG. It is known from the literature that these three transcription factors play a key role in regulating the stem phenotype in several human cancers including TC [RMST confirms the key role of the latter in repressing the stem characteristics of TC. Furthermore, once again, as observed in the case of mesenchymal markers, the Wnt/β-catenin, Notch and Hedgehog signaling pathways are also involved in the regulation of these stemness markers [RMST can actually interfere with one of these three pathways in order to repress the expression of either aforementioned mesenchymal or stemness markers. Interestingly, the restored RMST expression is also capable of suppressing genes such as ALDH1 and ABCG2, whose role has been seen to be associated with the acquisition of a stem phenotype in TC [CD133 stem cell marker, which has been reported to characterize CSCs and to be associated with a poor prognosis in different tumor types, and CD38 [RMST, thus confirming once again its involvement in the modulation of the stem phenotype [Noteworthy, uding TC , 37, andpe in TC , 39. Simand CD38  are alsohenotype .RMST expression in stem-like features, we carried out a sphere forming assay in FRO and SW1736 cells. Interestingly, thyrospheres deriving from RMST-TC cells were fewer and smaller than those deriving from pLenti-TC cells. Overall, these findings highlighting the involvement of RMST in the sphere formation, self-renewal and tumor-initiating ability of thyroid CSCs, may provide novel therapeutic strategies for the cure of ATC patients.To better elucidate the consequence of RMST expression in adult tissues . The GSE65144 contains 45 NT and 11 ATC samples, whereas the GSE33630 contains 13 NT and 12 ATC samples. Deregulated lncRNAs were filtered using GEO2R (http://www.ncbi.nlm.nih.gov/geo/geo2r/), a tool used for identifying differentially expressed genes. LncRNAs with FDR adjusted p ≤ 0.05 and fold change (FC) ≥ 1.2 were considered being differentially expressed. Venn diagrams of deregulated lncRNAs were generated using FunRich 2.1.2 software.The gene expression datasets GSE65144 and GSE33630 have been derived from GPL570 Platform  , provided samples of both healthy and malignant human thyroid tissue. All patients provided their informed consent. Under the reference number DC-2011-1437, the Ministry of Research, the South-East IV People’s Protection Committee, and the Health Regional Agency were notified of the activity of conserving biological material. The Ministry of Health approved the biological material cession operation and assigned it the reference number AC-2013-1867.Using Trizol reagent , total RNA was isolated from cell lines and normal or malignant thyroid tissues. The QuantiTect Reverse Transcription Kit  was used to generate double stranded cDNA from 1 µg of total RNA from each sample. Quantitative Real-Time PCR (qRT-PCR) experiments were carried out using CFX96 thermocycler . Each PCR reaction contained 10 µl of 2 × Sybr Green (Bio-Rad), 200 nM of each primer, and 20 ng of the previously produced cDNA. The Primer-BLAST program was used to design the oligonucleotides for qRT-PCR .The Dulbecco’s modified Eagle’s medium  was supplemented with 10% fetal bovine serum , 1% penicillin/streptomycin and 1% L-glutamine (Sigma-Aldrich) to grow the FRO and SW1736 cancer cell lines. Cells were kept at 37 °C in a 5% COIn accordance with the manufacturer’s instructions, cells were transfected using Life Technologies’ Lipofectamine 2000 reagent. Applied Biological Materials Inc.  sold the human RMST expression plasmid. qRT-PCR testing verified RMST expression. 1.5 µg/ml of puromycin (Life Technologies) was used to generate the FRO and SW1736 stable-transfected cells.2 environment. After 14 days spheres were counted. The formula (number of developed spheres/number of wells carrying cells) × 100 was used to calculate the number of spheres [http://bioinf.wehi.edu.au/software/elda/index.html) [Cells were plated in serum-free DMEM/Nutrient Mixture F-12 Ham  enriched with 2% B27, 10 ng/ml of epidermal growth factor and 20 ng/ml of basic fibroblast growth factor , at a density of 7 cells per well on ultra-low-attachment 96-well , for the spheroid-forming test. Cells were kept at 37 °C in a humidified 5% CO spheres . Cells wex.html) .4 or 6 × 104 cells for migration and invasion tests, respectively) on the upper side of the Transwell chamber. A volume corresponding to 300 μl of complete medium was then delivered to the inferior chamber as a chemoattractant. After 24 h, a crystal violet solution  was used to stain the cells that had moved to the lower side of the chambers. The invasion assay used the same approach after 48 h. FRO and SW1736 cells were properly seeded in a 96-well plate to standardize the cell count. After 4 h, the absorbance at 490 nm was measured using a microplate reader  using a CellTiter assay . The chamber’s crystal violet was then de-stained with a PBS-0.1% SDS solution before being read at 570 nm. By normalizing the values of crystal violet to those of CellTiter, the results were obtained.Transwell migration and invasion tests were carried out in Transwell chambers . Matrigel (Sigma-Aldrich) was applied to the upper side of the Transwell chamber for the invasion assay. In summary, serum-deprived media was used to plate FRO and SW1736 cells (3 × 10Confluent TC cell monolayers were treated with mitomycin (2 μg/ml for 2 h) (Sigma-Aldrich) for wound-healing tests. With the use of a p10 pipet tip, a "scratch" was made on the cell monolayer. For 24 h, cells were permitted to migrate at 37 °C in an incubator. A phase-contrast microscope was used to acquire multiple photograms, which were then quantitatively analyzed using specialized software  . The wouwww.bdbiosciences.com). Each sample covered at least 104 events that were recorded. Cells were permeabilized when required using the Cytofix/Cytoperm kit from BD Biosciences [Cells were treated with isotype control or specific antibodies for 30 min at 4 °C. Cell Signaling  provided the human SOX2 antibody, while the anti-OCT4-PE antibody came from Miltenyi Biotec . CellQuesciences .Cells were permeabilized with cold 70% ethanol in PBS for cell cycle analysis and stored at −20 °C until next day. Cells were then resuspended in PBS supplemented with 50 μg/ml propidium iodide (PI) and 100 mg/ml RNase, and were kept for 30 min at 30 °C. CellQuest software was used to analyze the data obtained through the FACS Calibur .6 cells, following a serum starvation of 18 h. Prior to analysis employing CellQuest software and the FACS Calibur, 5 μl of PI solution was added [Dead cells were identified and distinguished using the Annexin V-FITC Kit from Miltenyi Biotec. Cell staining was performed in a dark environment for 15 min at room temperature by adding 10 μl of Annexin V-FITC to 10as added .BRAFV600E double transgenic mice were kept in the University Federico II’s  animal facility, which has received approval from the Italian Ministry (D.M. 78/213-A and 12/2018-UT), under pathogen-free conditions with controlled humidity, light and temperature. They were also given access to standard food and water as well as 2500 mg/kg of doxycycline for 1 week. According to the 3Rs principle and in order to minimize the mouse sample number, we employed four (n = 4) mice either for the control and for the treated group, achieving statistically significant differences. No randomization and blinding criteria were applied. The Institutional Animal Care and Use Committee (IACUC) of University Federico II and the Italian Ministry of Health have both given their approval for the study, which has 2013/0078506 as protocol number.Tg-rtTA/TetO 2, 0.3% tween 20, and 5% normal goat serum . Incubation with primary antibodies was performed at 4 °C overnight in blocking solution. Sections were then washed for 5 min with PBS-0.2% triton and with PBS twice for 5 min and subsequently incubated at room temperature for 1 h with a fresh blocking solution containing 1:100 secondary antibody (biotinylated anti-rabbit and anti-mouse IgG (H + L); Vector Laboratories). After secondary antibodies incubation and washing (5 min with PBS-0.2% triton and twice for 5 min with PBS), section were incubated for 30 min at room temperature with ABC . Finally, DAB substrate  was added after washing with PBS-0.2% triton for 5 min and thrice for 5 min with PBS. After staining with eosin , sections were de-hydrated, treated with DPX Mountant  and covered with glasses. Axioskop microscope equipped with an Axiocam 105 color digital camera  was employed to acquire images, that were subsequently analyzed with the Axio Vision software [Thyroids were embedded in paraffin after being fixed in 4% PFA and dehydrated. From paraffin blocks, sections 7 μm-thick were cut, then being processed for the subsequent analysis. The sections were permeabilized for 5 min with PBS-0.2% triton and then washed with PBS twice for 5 min. Subsequently, they were put in the microwave for 15 min in citrate buffer  for antigen unmasking. Endogenous peroxidases were subsequently blocked with 1.5% oxygen peroxide and methanol and tissues were made permeable with PBS-0.2% triton for 5 min, washed with PBS twice for 5 min, and then blocked at room temperature for 1 h with a PBS solution (blocking solution) consisting of 3% BSA, 20 mM MgClsoftware .T-test and Anova test were employed to assess the collected data’s statistical significance. We used the Bonferroni post-test to evaluate group differences when the Anova test was significant (p < 0.05). In each experiment, the significance was evaluated at p < 0.05. The mean values and standard deviation (SD) of the data are presented. The cBioPortal database was used to depict clinical-pathological characteristics with respect to RMST expression levels [GraphPad Prism was utilized to conduct the statistical evaluations. n levels .Supplementary figure legendsSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3


Information available to date regarding the pregnancy outcomes of intrauterine insemination (IUI) without ovarian stimulation (OS) in infertile patients with unilateral tubal occlusion remains scarce. The objectives of this study were to investigate for couples affected by unilateral tubal occlusion /transvaginal real-time three-dimensional hysterosalpingo-contrast sonography (TVS RT-3D-HyCoSy)) and male infertility: (1) whether significant differences exist in pregnancy outcomes between IUI with or without OS cycles, and (2) whether the pregnancy outcomes of IUI without OS in women with unilateral tubal occlusion were similar to those of women with bilateral patent tubes.258 couples affected by male infertility completed 399 IUI cycles. The cycles were divided into three groups: group A, IUI without OS in women with unilateral tubal occlusion; group B, IUI with OS in women with unilateral tubal occlusion; and group C, IUI without OS in women with bilateral patent tubes. The main outcome measures, including clinical pregnancy rate (CPR), live birth rate (LBR), and first trimester miscarriage rate, were compared between either groups A and B or groups A and C.P < 0.001), the CPR, LBR, and first trimester miscarriage rate were comparable between these two groups. When comparing group C to group A, the duration of infertility was significantly longer in group C than that in group A (group A vs. group C: 2.3 ± 1.2 (year) vs. 2.9 ± 2.1 (year), P = 0.017). Except for the first trimester miscarriage rate, which was significantly higher in group A  than that in group C  (P = 0.044), no significant differences were observed in the CPR and LBR in these two groups. After adjusting for female age, body mass index, and the duration of infertility, similar results were obtained between groups A and C.Although the number of dominant follicles > 16 mm were significantly higher in group B than that in group A  and male infertility, IUI without OS might be an alternative treatment strategy. However, when compared to patients with bilateral patent tubes, the patients with unilateral tubal occlusion showed a higher first trimester miscarriage rate following IUI without OS cycles. Further studies are warranted to clarify this relationship.The online version contains supplementary material available at 10.1186/s12884-023-05705-3. Intrauterine insemination (IUI) is an effective and frequently used fertility treatment worldwide in the management of couples with unexplained and mild male factor infertility. It is less invasive, less stressful, more cost-effective, and more easily accepted by patients than other available treatments –3. IUI cFor IUI, at least one patent fallopian tube is required. There are multiple approaches to diagnose tubal patency, including hysterosalpingography (HSG), hysterosalpingo-contrast sonography (HyCoSy), and laparoscopy with dye chromopertubation. Although laparoscopy with dye chromopertubation remains the gold-standard test in the diagnosis of tubal pathologies, HSG and HyCoSy are widely used as the first-line approach to assess the anatomy of the uterus and the patency of the fallopian tubes in women with infertility –7.Compared to bilateral patent tubes or bilateral tubal occlusion, the diagnosis of unilateral tubal pathology has more limited prognostic significance and management strategies. One management strategy is IUI with OS through the available patent tube. Several studies –12 have 6/ejaculation or sperm concentration < 15 × 106/mL; progressive motility < 32%; and normal morphology < 4%, according to World Health Organization criteria (5th version); (2) female patients aged 20–40 years with a normal fertility status; (3) female patients with unilateral tubal occlusion; and (4) females with bilateral patent tubes (only those undergoing IUI without OS cycles were included). Patients were excluded if tubal patency was assessed by laparoscopy with dye chromopertubation. In addition, patients with a history of previous surgery (except for caesarean section) were excluded.This retrospective study reviewed the clinical data of 838 couples who underwent IUI treatment for infertility at the Center for Reproductive Medicine of Zhongshan City People’s Hospital between January 2014 and October 2021. Patients with fallopian tubal patency assessed by HSG or transvaginal real-time three-dimensional hysterosalpingo-contrast sonography (TVS RT-3D-HyCoSy) were investigated. The inclusion criteria were as follows: (1) couples affected by male infertility, which was defined as one or more subnormal semen variables: total sperm < 39 × 10The study protocol was approved by the institutional ethics committee of Zhongshan City People’s Hospital, and informed consent was obtained from all participants for their clinical data.The TVS RT-3D-HyCoSy procedure was performed by an experienced sonographer. All ultrasound examinations were performed using the Voluson E8 Expert  ultrasound machine with dedicated 3D imaging software and coded contrast imaging (CCI) mode. The detailed process of TVS RT-3D-HyCoSy, as described in the report by Zhou et al. , was carFor TVS RT-3D-HyCoSy, a direct sign of tubal patency , 14 is vIn our center, mild OS (1–2 follicles) were recommended. The OS protocols, as documented in our previous study , were asBiochemical pregnancy was confirmed by measuring serum β-human chorionic gonadotropin levels on days 14–16 after IUI. In women with positive β-human chorionic gonadotropin levels, transvaginal ultrasound examination was performed 2 weeks later to confirm clinical pregnancy. The outcomes of clinical pregnancy were subsequently recorded, including spontaneous abortion, ectopic pregnancy, multiple pregnancies, and live birth. The main outcome measures were CPR, live birth rate (LBR), and first trimester miscarriage rate. Clinical pregnancy was defined as the observation of a gestational sac inside or outside the uterine cavity using transvaginal ultrasonography. Live birth was defined as the birth of an infant after at least 28 weeks of gestation, with postnatal evidence of life. First trimester miscarriage was defined as spontaneous pregnancy loss within the first 12 weeks of gestation.P < 0.05.Statistical analysis was performed using the Statistical Package for Social Science . Continuous variables were expressed as mean ± standard deviation (SD). The normality of data was evaluated. Independent-Samples T-test was used for the comparison of normally distributed data, while Mann-Whitney U-test was used for non-normally distributed data. Categorical data, described as frequencies and percentages, were compared by the chi-square test or Fisher’s exact test, as appropriate. A binary logistic regression model was used to adjust for potential confounders to explore whether unilateral tubal occlusion was related to the pregnancy outcomes  of IUI without OS. Statistical significance was set at A total of 258 couples were recruited for this study. Among them, 80 women had unilateral tubal occlusion, and the remainder were diagnosed with bilateral patent tubes. Together they completed 399 IUI cycles. The CPR/cycle was 10.3% (41/399), the LBR/cycle was 8.3% (33/399), and the first trimester miscarriage rate was 12.2% (5/41). No ectopic pregnancies or multiple pregnancies occurred. The cycles were divided into three groups: group A (n = 75), IUI without OS in patients with unilateral tubal occlusion; group B (n = 66), IUI with OS in patients with unilateral tubal occlusion; and group C (n = 258), IUI without OS in patients with bilateral patent tubes.P < 0.001), no significant differences were found. The CPR, LBR, and first trimester miscarriage rate were also comparable between the two groups.The basic clinical parameters of groups A and B were compared Table . BesidesP = 0.017) (Table P = 0.044). In the binary logistic regression model, only body mass index (BMI) was found to be significantly related to clinical pregnancy  and live birth , while female age  was shown to be significantly related to miscarriage in the first trimester. After adjusting for female age, BMI, and the duration of infertility, no significant differences were observed in CPR or LBR between groups A and C, except for the first trimester miscarriage rate   vs. 2.9 ± 2.1 (year), To eliminate the potential influence of repeated cycles on the results, the same analyses were performed including only the first cycle. Similar results were observed between groups A and B and between groups A and C  and male infertility. The results showed that in patients with unilateral tubal occlusion diagnosed by HSG or TVS RT-3D-HyCoSy, although the number of dominant follicles > 16 mm was significantly higher in IUI with OS cycles, the pregnancy outcomes of IUI without OS cycles, including CPR, LBR, and first trimester miscarriage rate, were comparable to those of IUI with OS cycles.Assessing tubal patency is fundamental to evaluate female fertility, as it determines the treatment course. The management of patients with normal bilateral patent tubes or bilateral tubal occlusion diagnosed by HSG/HyCoSy is clear and straightforward. However, this is different when it comes to women with only one confirmed patent tube. Three possible approaches have been suggested : (1) furRecently, the results from a network meta-analysis  showed tBesides, the cost-effectiveness and patient preferences should also be taken into account. A study on the cost-effectiveness of OS agents  for IUI in couples with unexplained subfertility showed that over four IUI-OS cycles conducted within one year, both the cumulative LBR and the average costs per four cycles were highest in cycles with Gn . And in cycles with CC and LE, the cumulative LBRs were 29.4% and 32%, respectively, and the average costs per four cycles were €362 and €434, respectively . HoweverP = 0.018), followed by women with bilateral occlusion (22.5%) and women with bilateral tubal patency (5.2%). Another study [P = 0.028). A retrospective cross-sectional study [P = 0.080). In the present study, all included patients had two fallopian tubes, and tubal patency was just diagnosed using HSG/TVS RT-3D-HyCoSy. Those patients with a history of previous surgery (except for caesarean section) were excluded. Therefore, it is difficult to rule out the existence of CE and endometriosis in these patients, as both often presents asymptomatically.When comparing the pregnancy outcomes of IUI without OS in patients with unilateral tubal occlusion and patients with bilateral patent tubes, the results showed that the CPR and LBR were comparable, but, interestingly, the first trimester miscarriage rate was significantly higher in patients with unilateral tubal occlusion. After adjusting for female age, BMI, and the duration of infertility, a similar result was obtained. We speculate that in patients with unilateral tubal occlusion, there might be some potential risk factors that could affect pregnancy outcomes, such as chronic endometritis (CE) and endometriosis, which have been suggested to be associated with pregnancy loss. A recent study  showed tcclusion .5% and wal study  demonstral study  reportedal study , patientIn our study, in the binary logistic regression model, BMI was found to be significantly related to clinical pregnancy and live birth. It is widely accepted that elevated BMI can lead to multiple health issues, including problems with reproduction for women, such as infertility, and pregnancy complications. An abundance of research suggests that obesity has a profound detrimental impact on in vitro fertilization (IVF) pregnancy outcomes. A 2019 meta-analysis  that incThere are some limitations in our study. First, the retrospective nature and small sample size of our study may have led to potential inherent biases. Besides, as we mentioned above, IUI without OS might be more cost-effective, and more easily accepted by patients with a spontaneous ovulatory menstrual cycle. But of note, for infertile patients with only one patent tube, if ovulation occurs on the contralateral ovary of the patent fallopian tube, the cycle would be cancelled, considering the potential impact on pregnancy rate and patient preference. Within a certain period time, repeated cycle cancellation might lead to an increase in the cost. Therefore, the cycle cancellation rate should be taken into account. Due to the retrospective nature of our study, the cancellation cycle was not recorded, thus the comparisons of cancellation rates and cost-effectiveness between IUI with or without OS in patients with unilateral tubal occlusion were not possible. And, to our knowledge, no such data have been reported thus far. However, we deduce that in this study, the cycle cancellation rate might be similar between IUI with or without OS in patients with unilateral tubal occlusion. We explain it as follows: Firstly, mild OS is recommended in our center, which means 1–2 dominant follicles developed in most OS cycles. In this study, among 66 OS cycles, 29 OS cycles generated 1 follicle (account for 43.9%), and 32 cycles generated 2 follicles (account for 48.5%). In this case, we infer that in patients with unilateral tubal occlusion, the cancellation rate would not be too much lower in IUI with OS cycles than that in IUI without OS cycles. Additionally, strict cancellation criteria also increased the cancellation rate of OS cycle to some extent. Further studies are warranted to confirm our speculation.In conclusion, our study showed that IUI without OS might be an alternative treatment strategy for couples affected by unilateral tubal occlusion (diagnosed by HSG or TVS RT-3D-HyCoSy) and male infertility. The pregnancy outcomes were comparable between IUI with or without OS cycles in these patients. However, in IUI without OS cycles, although CPR and LBR were comparable, the first trimester miscarriage rate might be higher in patients with unilateral tubal occlusion than in those with bilateral patent tubes. Further studies are warranted to clarify this relationship.Below is the link to the electronic supplementary material.Additional file 1