Monitoring gestational weight gain (GWG) throughout pregnancy among adolescents is important for detecting individuals at risk and timely intervention. However, there are no specific tools or guidelines for GWG monitoring of this group. We aimed to construct GWG charts for pregnant adolescents (10–19 years old) according to pre-pregnancy body mass index (BMI) using a pooled dataset from nine Latin American countries. Datasets from Argentina, Brazil, Chile, Colombia, Mexico, Panama, Paraguay, Peru, and Uruguay collected between 2003 and 2021 were combined after data cleaning and harmonization. Adolescents free of diseases that could affect GWG and who gave birth to newborns weighing between 2,500–4,000 g and free of congenital malformations were included. Multiple imputation techniques were applied to increase the sample size available for underweight and obesity categories. Generalized Additive Models for Location, Scale, and Shape were used to construct the charts of GWG according to gestational age. Internal and external validation procedures were performed to ensure that models were not over-adjusted to the data. The cohort included 6,414 individuals and 29,414 measurements to construct the charts and 1,684 individuals and 8,879 measurements for external validation. The medians (and interquartile ranges) for GWG at 40 weeks according to pre-pregnancy BMI were: underweight, 14.9 (11.9–18.6); normal weight, 14.0 (10.6–17.7); overweight, 11.6 (7.7–15.6); obesity, 10.6 kg (6.7–14.3). Internal and external validation showed that the percentages above/below selected percentiles were close to those expected, except for underweight adolescents. These charts describe the GWG throughout pregnancy among Latin American adolescents and represent a significant contribution to the prenatal care of this group. GWG cut-offs based on values associated with lower risks of unfavorable outcomes for the mother-child binomial should be determined before implementing the charts in clinical practice. Gestational weight gain (GWG) is an important indicator of maternal nutritional status. Weight gain in this period is fundamental to promoting the health of the mother-child binomial, storing the necessary fat for milk production and adequate fetal growth and development, allowing for optimal weight and body composition in the newborn ,2.Deviations in GWG are associated with adverse outcomes for the mother-child binomial. Excessive GWG increases the probability of developing hypertensive disorders of pregnancy, gestational diabetes, cesarean delivery, postpartum weight retention and, in the newborn, it may increase the risk of birth of large-for-gestational-age infants, macrosomia, and, in the long term, childhood obesity –7. In coMonitoring GWG throughout pregnancy is important for detecting at-risk individuals and timely intervention, especially among adolescents who face pregnancy under biological and psychological immaturity conditions, increasing the risk of unfavorable outcomes for mother-child binomial health . AccordiThe World Health Organization (WHO) recommends GWG as an indicator to be monitored during pregnancy . HoweverAn observational, retrospective, longitudinal study was conducted based on secondary data from research projects, information systems, maternal clinical records, and prenatal care institutions in Argentina, Brazil, Chile, Colombia, Mexico, Panama, Paraguay, Peru, and Uruguay. The initial database comprised data from 33,446 women with approximately 150,000 weight measurements. After the data harmonization process by Standardized Site Differences (SSD) technique, 6,414 pregnant adolescents and 34,943 weight records were included in the study .We included women between 10 and 19 years old; with at least one weight measurement (kg) in the second and third trimesters of pregnancy, with their respective gestational age (weeks); without hypertension, preeclampsia, diabetes mellitus or gestational diabetes, tuberculosis, or cardiovascular diseases; and who gave birth to a term newborn (37 to 42 weeks), with birth weight between 2,500 and 4,000 g. In addition, records with a biologically implausible weight gain trajectory (very high weight gains or losses between controls), total GWG ≥ 30 kg, height-for-age z score (HAZ) <-2 according to the WHO charts  (childhohttps://obwheel.quartertone.net/). Gestational age was already calculated in the combined datasets, and it was not possible to know how it was obtained . Pre-pregnancy BMI (kg/m2) was classified according to the WHO criteria for adolescents as underweight: <-2 SD; normal weight: ≥ -2 SD to ≤ +1 SD; overweight: > +1 SD to ≤ +2SD; and obesity: > +2 SD [Maternal age in years was estimated as the difference between the date of birth of the pregnant adolescents and the date of conception. The date of conception was estimated from the gestational ages available in the databases using the ’ob Wheel’ algorithm ( > +2 SD . GWG (kg > +2 SD . Birth wMedians and interquartile ranges (IQR) were calculated for height (cm), pre-pregnancy weight (kg), maternal age (years), gestational age at birth (weeks), birth weight (g), birth length (cm), and GWG (kg) according to the pre-pregnancy BMI category.A proportion of records of GWG that varied from 10.4% (underweight) to 12.8% (overweight) did not present values between the fifth and thirteen weeks of pregnancy (first trimester), which generated an overdispersion (high variance) in the data. Thus, the adjustments of the models to build the GWG charts were complex. The overdispersion of the data was especially present among adolescents with pre-pregnancy under and overweight. Thus, for those women who did not have a weight measurement in the first trimester, it was assumed that the lack of a weight measurement in this gestation period was a real observation that could be imputed. These new observations were assumed as missing not at random (MNAR), and a data imputation approach was considered for GWG in the first trimester. For each pregnant adolescent with missing data, a random week was established between the fifth and thirteenth week to expand the data matrix .Subsequently, the Linear Regression Bootstrap imputation method  was applThe selection of the best imputation method to control for overdispersion in the first trimester  took into account the Generalized Akaike Information Criterion (GAIC) , the AkaTo construct the charts of GWG according to gestational age for each pre-pregnancy BMI, generalized additive models of location, scale, and shape (GAMLSS) were used . The modEq1. Equation for calculation of pre-pregnancy body mass index-specific gestational weight gain z-scores, based on a Box-Cox t or Box-Cox Power Exponential model, where Y is the weight gain at a given gestational age, ν is lambda, μ is mu, and σ is sigma. The random variable Z is assumed to follow a t-distribution with degrees of freedom, τ > 0, treated as a continuous parameter. Parameters of BCT or BCPE model for each pre-pregnancy BMI group are given for rounded gestational ages. To fit the GAMLSS models, the parameters were calculated based on weight gain + 20 kg, so 20 kg must be added to the weight gain when using this equation.th percentile and 10% above the 90th percentile. For the external validation of the model, the machine learning cross-validation methodology was used [The biological plausibility of the predicted GWG values for each model was evaluated, emphasizing the presence of negative GWG for each category of pre-pregnancy BMI and the magnitude of weight loss throughout pregnancy (between the first and third trimesters). The smoothing of the graphs in the first trimester and the percentile amplitude of GWG were also considered in the decision for the final models. Additionally, the internal validation of the model was carried out by calculating the percentage of measurements above/below selected percentiles. For example, if the model has good internal validation, 10% of the observations are expected to be below the 10was used . In thisThis study was approved by the Bioethics Committee of the Faculty of Dentistry of the Universidad de Antioquia, act number 12 of October 18, 2019. Only de-identified data was used in the analyses, which were performed by authorized investigators. The principal investigator of the University of Antioquia signed a confidentiality and custody agreement for the data with the investigator or institutional representative of each country.A total of 6,414 pregnant adolescents aged 10 to 19 and their neonates were included. Most adolescents were classified with pre-pregnancy normal weight (71.8%), and only 1.5% were underweight. The median birth weight increased progressively as the pre-pregnancy BMI category increased . In the th gestational week in the 3rd percentile and sooner (5th week) for the 25th, 50th, 75th, and 97th percentiles. GWG in more advanced weeks presented a gradual and sustained increase  started at the 17increase . A progrks 14–16 .th week for the 3rd percentile. At percentiles 25th, 50th, and 75th, weight losses of a maximum of 1kg were observed between weeks 13 and 19, when GWG was restored. At the 97th percentile, there were always positive values throughout pregnancy  (The median (P50) GWG differed across the pre-pregnancy BMI categories. The highest values for the 50ctively) .th percentile (11.9 for underweight v. 6.7 kg for obesity), 4.3 kg in the 50th percentile (14. 9 kg for underweight v. 10.6 for obesity), and 4.3 kg in the 75th percentile (18.6 kg v. 14.3). At 40 weeks, the largest differences between subsequent categories were observed between normal and overweight, for the 25th, 50th, and 75th percentiles  and IQR (P25 and 75) presented a similar pattern, i.e., the highest values were observed among underweight, followed by normal, overweight, and obesity. The difference between the underweight and obesity categories was 5.2 kg in the 25centiles .th percentile, and the internal validation results were 24.3%. The pre-pregnancy BMI category with the greatest differences between the estimated and expected values was overweight. For the external validation, the differences between observed and expected values were within the 5% accepted difference for normal, overweight, and obesity. For underweight, the differences between observed and expected percentages varied between 0.7% for the 90th percentile and 12.9% for the 50th percentile . However, the lack of charts for the other BMI categories and the fact that the INTERGROWTH-21st standards do not consider the possibility of GWG in the first trimester of pregnancy makes it challenging to use those standards as a tool for GWG monitoring worldwide, particularly for adolescents.International GWG standards were developed by the INTERGROWTH-21st study . These sth and 75th percentiles for individuals with under and normal weight are similar to the GWG ranges recommended in the 2009 IOM guidelines [th percentile was 11.9 kg v. 12.5 kg at the IOM lower limit; and the 75th percentile was 18.6 kg v. 18 kg at the IOM upper limit. For normal weight, the 25th percentile was 10.6 kg v. 11.5 kg for the IOM lower limit, and the 75th percentile was 17.7 kg v. 16 kg for the IOM upper limit. For overweight and obesity, the lower limits of the IOM and the 25th percentile of our charts were similar. However, the upper limits of the IOM value are considerably lower than our 75th percentiles. Although the 2009 IOM guidelines are the most established GWG recommendations, they were designed for adult women from the USA. Therefore, they may not apply to contexts where women are shorter or with limited access to health services [The values observed in our charts for the 25It is necessary to highlight that the charts proposed in this study are only a first step towards establishing a comprehensive system to monitor GWG in adolescents. The next phase shall identify optimal GWG cut-offs associated with the lowest maternal and infant adverse outcomes risk. In addition, there is a need to strengthen the information systems of prenatal control programs and the early start of GWG surveillance to contribute to the timely detection and intervention of maternal-perinatal risks among adolescents from Latin American countries.The development of GWG monitoring charts for Latin American adolescents is a pioneering initiative consolidating data from nine countries. From this harmonized dataset, a significant number of weight measurements were obtained, with their respective gestational age, which, together with an imputation process in the first trimester, made it possible to build GWG charts according to the four pre-pregnancy BMI categories, starting at the fifth week of gestation. Using WHO charts and cut-offs to classify pre-pregnancy BMI is another important strength. In addition, applying several statistical models with various diagnostic measures and internal and external validations to select the best model is aligned with what is recommended when constructing growth charts ,30.As limitations of the study, the insufficient amount of data for underweight adolescents, which resulted in limited satisfactory results in the external validation, needs to be mentioned. In addition, not knowing the origin of pre-pregnancy weight, the lack of a high-quality measurement of gestational age that could be standardized across the datasets, and the lack of information regarding other outcomes, which could have led the charts towards an even more prescriptive approach, are worth mentioning.On the other hand, the study is influenced by the origin of the data, which impacts the quality and inclusion of records because it relies on secondary data. The data acquired from investigative projects provided complete information that met the study’s criteria. In contrast, data obtained from institutional or national information systems exhibited significant data loss due to missing variables and potential errors in weight records. This introduces a selection bias that could impact the generalizability of the data. Consequently, prior validation within each respective country is imperative before utilizing this data. Besides, the charts apply up to the 40th week of gestation.The charts created in this study describe the GWG throughout pregnancy among Latin American adolescents, according to pre-pregnancy BMI, and represent a significant contribution to prenatal control programs. GWG is an indicator that is easy to be understood by women and used by different health professionals. With these new charts, we expect to start changes in the nutritional surveillance of pregnant adolescents, which could positively affect maternal and child health in Latin America during pregnancy and in the medium and long term. The following steps to create final tools to be implemented in prenatal care include defining GWG cut-offs in the graphs based on values associated with lower risks of unfavorable outcomes for the mother-child binomial and disseminating this new tool in the included Latin American countries and others with similar characteristics.S1 Fig*Notes: Diseases considered: Chronic hypertension or hypertensive disorders during pregnancy, diabetes mellitus or gestational diabetes, tuberculosis, or cardiovascular diseases. Abbreviations: BMI: Body mass index; GA: Gestational age; GWG: Gestational weight gain; HAZ: Height-for-age z score.(TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Homotypic entotic figures, which are a form of “cell-in-cell” structures, are considered a potential novel independent prognostic marker in various cancers. Nevertheless, the knowledge concerning the biological role of this phenomenon is still unclear. Since breast cancer cells are remarkably entosis-competent, we aimed to investigate and compare the frequency of entoses in a primary breast tumor and in its lymph node metastasis. Moreover, as there are limited data on defined molecular markers of entosis, we investigated entosis in correlation with classical breast cancer biomarkers used in routine pathomorphological diagnostics . In the study, a cohort of entosis-positive breast cancer samples paired into primary lesions and lymph node metastases was used. The inclusion criteria were a diagnosis of NOS cancer, lymph node metastases, the presence of entotic figures in the primary lesion, and/or lymph node metastases. In a selected, double-negative, HER2-positive NOS breast cancer case, entoses were characterized by a correlation between an epithelial–mesenchymal transition and proliferation markers. We observed that in the investigated cohort entotic figures were positively correlated with Ki67 and HER2, but not with ER or PR markers. Moreover, for the first time, we identified Ki67-positive mitotic inner entotic cells in clinical carcinoma samples. Our study performed on primary and secondary breast cancer specimens indicated that entotic figures, when examined by routine HE histological staining, present potential diagnostic value, since they correlate with two classical prognostic factors of breast cancer. Cancer cells are characterized by aberrant metabolism, which is strengthened by starvation and hypoxic conditions that accompany the rapid growth of tumors . SeveralThere are no automated methods for the identification of entosis. Entotic figures may be identified in tissue sections stained with hematoxylin and eosin (HE) using diagnostic histopathological criteria proposed by Mackay ,9. For cOver the last years, there has been increasing interest in entosis as a potential new prognostic factor associated with worse prognosis for head and neck ,12, tongIn cell cultures, entosis is associated with loss of cell-to-cell and cell-to-matrix connections. In solid tumors, the loss of cell-to-cell connections is also observed during the initiation of metastasis, which is preceded by an epithelial–mesenchymal transition (EMT) . During The mechanism of formation of entotic figures includes the induction of cell detachment by unidentified triggers, which is followed by strong cell-to-cell adhesion via E-cadherin and β-catenin. Subsequently, Rho/ROCK and actomyosin complexes are formed in one of the interacting cells, making it stiffer. This cell is pushed into the engulfing (outer) cell, characterized by the lack of activation of Rho-pathways ,10. It iBreast cancer is the most frequently diagnosed malignancy and the most common cause of cancer-associated death in women . In diagIn the context of breast cancer, it has been reported that prognosis can differ depending on the tumor subtype . RecentlHere, we analyzed and characterized entotic figures in a tissue microarray of 50 selected, entosis-positive breast cancer cases, with specimens being paired into primary lesions and corresponding lymph node metastases (100 tissue samples). We also performed an in-depth analysis of a case of double-negative, HER2-positive breast cancer  with a special focus on the occurrence of selected EMT hallmarks. Routine diagnostic immunochemistry (E-cadherin/vimentin and Ki67 immunostaining) was used for histopathological diagnostics.The three following inclusion criteria were applied: (1) a histopathological diagnosis of NOS cancer, (2) lymph node metastases, and (3) the presence of entotic figures in the primary lesion and/or lymph node metastases. For the study, we selected a tissue microarray containing entotic figure-positive tissues from paired primary lesions and corresponding lymph node metastases from 50 women of a median age of 50 years old . For the statistical analysis, only NOS carcinomas were included. Entotic figures were found in all the analyzed primary lesions and metastatic tissues. The detailed characteristics of the cohort are displayed in Entotic structures were present in all the investigated cases . We invep-value was established to be 0.037 in the metastases. However, we applied a more stringent p-value (p < 0.01) as the limit of statistical significance in this study. We did not observe a statistically significant difference in the entotic density between the paired primary lesions and corresponding metastatic tissues   .As shown in In order to study cell-to-cell interactions within entotic structures, a confocal microscopy analysis of the lymph node metastasis was performed. As shown in Vimentin immunostaining was used as an EMT marker of epithelium-derived cancer cells to visualize connective tissue. As shown in Unlike in the primary lesion, vimentin expression in the lymph node metastasis was strictly limited to the interstitial tissue corresponding to the connective tissue of the lymph node and its capsule E,F. The To visualize proliferating cells, Ki67 immunostaining was conducted. In the primary lesion, Ki67 hotspots corresponded to entotic hotspots A. Ki67-pTo exclude the possibility of misinterpreting entotic figures as cells within lymphatic or blood vessels, immunostaining for classical markers of vessels SMA, CD31 and podoplanin, was performed. As shown in Entosis represents a type of cell-in-cell structure that can be found within a normal epithelium, but is much more prevalent in cancerous tissues ,10. NowaE-cadherin staining helps to distinguish entotic figures from heterotypic cell-in-cell structures . TherefoIn the described cohort, we only analyzed NOS breast cancers positive for entotic figures. The obtained data indicate that entosis is generally independent from patient age, grading, staging, tumor size and hormone receptor status.However, two frequently used markers of breast cancer, Ki67 and HER2, turned out to be significantly correlated with the presence of entotic structures B,C. ThisKi67 is a well-known diagnostic marker of cell proliferation in cancers and is frequently used for the immunohistochemical characterization of breast cancer . The obsHER2 is a classical diagnostic marker in breast cancer . HER2 ovThe correlation of entosis with both Ki67 and HER2 expression connects entosis with cell proliferation. During mitosis, cells detach from neighboring cells, and therefore, mitosis can facilitate cell internalization through entosis . It has During metastasis, cancer cells undergo an EMT, which increases the expression of vimentin, and the expression of E-cadherin is switched to that of N-cadherin . The expOur findings indicate that in the investigated cohort, entotic structures do not occur more frequently in metastases . In contLimitations of the study include the fact that our study only included cases of NOS breast cancer (TNM II and III) positive for entoses. Moreover, our study is a histopathological analysis; thus, we are unable to correlate the number of entotic figures with the survival of patients due to a lack of clinical data. Nevertheless, for the first time, we showed that entosis does not depend on the hormonal status of the cancer, but correlates with two prognostic factors: Ki67 and HER2.For a better understanding of the pattern of entoses in breast cancer, an analysis of human tumor tissue microarray (TMA) slides  was performed.The sample size was estimated with the G*Power software , as described previously . The numhttps://www.tissuearray.com/tissue-arrays/Regional_Lymph_Nodes_Metastasis/BR10010f, assessed on 1 October 2022).For each analyzed case, information regarding age, grading, staging, TNM status, tumor diameter, as well as the expression of Ki67, HER2, ER, and PR receptors was obtained. Detailed data regarding each sample plotted on the slide are provided on the company’s website . The entotic figures within the cohorts were analyzed by four independent researchers, including a certified clinical pathologist, who also verified the specimens. For a coherent evaluation of entoses, the following criteria established by Mackay et al. ,9 were dIn order to ensure the proper quality of the clinical material, standard histopathological procedures were applied. The surgical breast cancer samples (TissueArray.Com/US Biomax) were fixed within 30 min after surgery in 10% neutral formalin, then dehydrated in ethanol, cleared with xylene and embedded in paraffin using a tissue processor . Afterwards, each tissue specimen was individually examined by a certified pathologist and assessed according to the standardizations of diagnosis, classification and pathological grade published by the World Health Organization (WHO). Standard immunohistochemistry (IHC) protocols were performed to ensure the accuracy and specificity of the tissue array products. After deparaffinization and washing in phosphate-buffered saline (PBS), the antigen retrieval step was performed by heating the slides in a 0.01 M sodium citrate buffer (pH 6.0) for 15 min. Next, the specimens were washed in PBS and blocked using normal goat serum for 20 min at RT. Then, an appropriate primary antibody  was applEach collected specimen was consented to be used by both the hospital and the individual. An informed consent form was obtained from every patient, and the rights to hold the research for any purpose or for further commercialized uses were waived.The histopathological specimens were digitalized and analyzed with the use of a self-developed online platform. Each specimen was examined by three independent researchers, who marked entotic structures, according to Mackay’s criteria, on the images. Each selected entotic figure was verified by a separate team consisting of three researchers. For statistical analyses, all necrotic and noncancerous tissue areas were excluded from further studies, and the surface area of cancerous tissue was determined using the self-designed platform. The frequency of entoses was calculated as the number of entotic structures per square millimeter of cancerous tissue.p-value of <0.05. All calculations were performed using the R Project for Statistical Computing software (R Core Team (2020). R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria; .The correlations (patient age and percentage of Ki67-positive cells) were determined using Spearman’s rank correlation test. The differences between groups  were assessed using the Mann–Whitney U test (for two groups) or the Kruskal–Wallis test (for more than two groups). The paired Wilcoxon test was used to test for differences in the frequency of entoses between primary tumors and their corresponding lymph node metastases. Statistical significance was considered at a Archival paraffin tissue specimens from a 75-year-old Caucasian woman with double-negative HER2-positive breast cancer (pT4N3) were obtained.The macroscopic image was as follows:The right mammary gland with the contents of the axilla  was obtained for examination. It was covered with an incised skin ellipse (21.0 × 18.0 cm) and the contents of the armpit (12.5 × 6.0 × 3.0 cm). At the border of the upper quadrants, an ulcerated, bulging tumor (10.5 × 7.5 × 11 cm) was found, with a thoracic margin of 1.5 cm, an axillary margin of 9.0 cm, and a medial margin of 6.0 cm. On the cross-section in the axillary cavity, a pack of fat-infiltrated axillary lymph nodes, measuring 7.5 × 5.5 × 3.5 cm, was identified. On the surface of the skin, an ulcer of a diameter of 1.0 cm was present. The preparations were composed of (1) the skin bump; (2) the tumor with a skin margin; (3) the central part of the tumor; (4) other parts of the tumor; (5) the lower quadrants of the mammary gland outside the tumor; (6) the upper quadrants of the mammary gland outside the tumor; (7) a bundle of lymph nodes; (8) a nipple from the side of the tumor.The histopathological diagnosis was based on the following: invasive cancer NOS, NST NHG 3 (3 + 3 + 3)/28 mitoses/10HPF ER-, PR-, HER2+++, Ki67 positivity in 30% of nuclei, lymphangiosis carcinomatosa, and also present carcinoma ductale in situ foci with high nuclear atypia, constituting about 10% of the cross-sectional area of the tumor.Both the primary breast lesion from the central part and the metastasis into the axillary lymph node, which was a single metastasis at the time of tissue sample collection, were examined.The material underwent standard diagnostic histopathologic procedures, with HE staining and immunohistochemistry using DAKO EnVision™ + System . For the purpose of this study, the slides were specially immunostained for E-cadherin , SMA and CD31 (vascular markers), podoplanin (a marker of lymphatic vessels), and Ki67  for actin visualization, and analyzed by SP5 confocal microscopy . The deparaffinized sections were incubated with Phalloidin-Atto  for 1 h at RT, and subsequently the slides were embedded in VECTASHIELD Antifade Mounting Medium with DAPI .p-value of <0.05.Data analysis was performed using the GraphPad Prism 6.0  software. For statistical purposes, a nonparametric Mann–Whitney U test was used. Statistical significance was considered at a Our results confirmed that homotypic entosis can be found in histopathological examinations of breast cancer, both in the primary lesions and lymph node metastases. In the cohort of NOS breast cancers, we found a positive correlation between the presence of entotic figures, and two well-known and routinely used predictive factors in breast cancer: Ki67 and HER2. For the first time, we identified Ki67-positive inner engulfed entotic cells in a tumor in situ. The presence of Ki67-positive internalized cells, as well as the positive correlation of entoses with the Ki67 proliferation marker, supports the formerly published data that indicate that the inner entotic cell can also divide. However, the fate of entotic cells may differ depending on not yet fully understood conditions. We observed certain immunohistochemical changes characteristic of an EMT (accompanied by entotic figures), which takes place during metastasis. Thus, our findings indicate that entotic figures in breast cancer can have potential diagnostic value, since they correlate with two classical prognostic factors of breast cancer.


Compared with the common grain, Tartary buckwheat enjoys higher nutritional value. Some distinctive nutrition associated with physiological activity of Tartary buckwheat is valuable in medicine. In addition, it’s a good feed crop. In the paper, the main components  and polyphenol bioactive components in Tartary buckwheat and its sprouts were reviewed, and the accumulation of flavonoids in sprouts during germination, especially the methods, synthetic pathways and mechanisms of flavonoid accumulation was summarized. The research on bioactive components and health benefits of Tartary buckwheat also were reviewed. Besides, the applications of innovative physical technology including microwave, magnetic, electromagnetic, ultrasonic, and light were also mentioned and highlighted, which could promote the enrichment of some active substances during seeds germination and growth of Tartary buckwheat sprouts. It would give a good support and benefit for the research and processing of Tartary buckwheat and its sprouts in next day. It focused on the synthesis and enrichment ways of flavonoids, the main active components in Tartary buckwheat, as well as germination processing methods. This paper also reviewed research activities related to the germination of Tartary buckwheat both domestically and abroad. In order to provide reference for full exploitation and utilization of Tartary buckwheat, we collected previous research and summarized this review.Nowadays, more and more people pay attention to their health, for it is one of the most important things we should do to get more natural health benefits from food. Being rich in vitamins and nutrients, whole grain foods such as oat, buckwheat, Tartary buckwheat, barley, wild rice and quinoa have been proven that they can reduce the risk of heart disease, type 2 diabetes, obesity and some cancers. Furthermore, they make great effects in improving gut health and other diseases.in vitro digestibility and functional activities of Tartary buckwheat, which can be improved by heat, humidity, light and cold, etc. As the same time, the flavonoids ingredients can be enriched by increasing the gene expression of key enzymes, thus opening up a new way for the development of new functions of Tartary buckwheat and its applications in food industry  is the highest proportion in all of the carbohydrates, and its structure and properties also are complex. Current research on TBS focus on structural modification and bioactivity studies . Some rein vitro digestibility of TBS using heat-moisture treatment (HMT) and annealing (ANN) was revealed, under different treatments, the amylose content, relative crystallinity, water absorption and gelatinization temperature increased  treatment on Tartary buckwheat starch’s properties were displayed in two similar studies. HHP treatment could cause TBS particles to sag and adhere, and could gradually lose its original shape, but its crystalline type was still typical A-type. Other study reported that HHP treatment could induce the surface roughness of TBS particles, the amylose content and gelatinization temperature increased significantly with the increase of pressure , while treatment .Some studies found that TBS was not easily digested by amylase because of its close association with flavonoid. Therefore, TBS mainly exists in the form of RS, and contains more SDS and less RDS. RS is not digested in the intestine but it is fermented by gut microbes to produce short-chain fatty acids (SCFA) when RS enters the large intestine . Part ofThe changes of properties of Tartary buckwheat starch during germination were studied. The results implied that the starch loss increased (4.4% ~ 5.9%) moderately and scanning electron microscope showed that the porosity around starch granules improved in the process of germination. Germination process had no obvious influence on the X-ray diffraction pattern, while the crystallinity percentage of starch increased with germination rate increasing. Besides, the process of germination upgraded the solubility of buckwheat starch, but had no obvious effect on swelling power. Therefore, a number of functional properties of Tartary buckwheat starch were improved with the treatment of moderate germination . A studyp < 0.05) and random coil contents, and decreased the proportion of α-helix and β-folding in secondary structure. Furthermore, some properties of germinated TBP including solubility (24.37%), water-holding capacity (38.95%), emulsifying activity index (17.21%), emulsifying stability index (11.22%), foaming capacity (71.43%), foaming stability (33.60%), the in vitro protein digestibility (5.56%) and antioxidant capacities were significantly improved (p < 0.05). The functional properties of germinated TBP were optimized by MW treatment of 300 W/50 s  is rich in multiple amino acids and the proportion of amino acids is balanced . The res0 W/50 s .50 was 1.372 mg/mL, which indicated that TBP had good in vitro antioxidant capacity . Among these EAAs, buckwheat varieties were characterized by high contents of leucine, followed by phenylalanine, lysine, and threonine   , and thectively) .The study found, when after 48–72 h of germination of Tartary buckwheat, the content of EAAs of lysine, leucine and phenylalanine and GABA in sprout flour increased 2.3% ~ 24.77, 14.18% ~ 19.19, 10.66% ~ 21.45 and 27.48% ~ 40.89%, respectively . The effTartary buckwheat is rich in flavonoids, phenolic acids, quinones and other polyphenols. Flavonoid is one of the most abundant and important bioactive substances in Tartary buckwheat, mainly including rutin, quercetin and procyanidin . Tartaryin vitro also showed that Tartary buckwheat flavonoid had strong antioxidant capacity. Further studies have shown that increasing the consumption of glucose and the accumulation of glycogen content significantly in HepG2 cells, Tartary buckwheat flavonoid has distinct anti-diabetic influence  and antioxidant capacity of DPPH in sprout was 29.5 mg GAE/g dry weight (DW) and 29.87 TE/g DW, respectively, which was increased by 42.23 and 32.58% compared to hulled seeds. In addition to orientin and vitexin, high levels of other phenolic compounds  were detected in peeled germinated seeds. The data showed that buckwheat seeds, which were peeled and germinated, as a dietary source of phenolic compounds, had great application potential in functional foods and were beneficial to health  UV-C (254 nm) after exposure to blue light and their combination treatments had influenced on flavonoid contents  and total flavonoids in Tartary buckwheat, the rutin content under the combined irradiation of blue light and UV-C (BL + UV-C) was 13.5% higher than that of (UV-C + BL) . SproutsTartary buckwheat is rich in multiple essential fatty acids like linoleic acid and α-linolenic acid. Fatty acids participate in the normal operation and metabolism of cholesterol in human body, inhibit the formation of arterial thrombosis, regulate human body functions and have other many functions such as prevention of cardiovascular disease, antibacterial and anti-inflammatory . The stuTartary buckwheat is rich in a variety of minerals beneficial to human health, especially potassium, sodium, copper, zinc, calcium and iron, which are much higher than other crops. Huang et al. found that the contents of Cu, Zn, Fe, K, and Mg elements were great fluctuation, those ranges were 5.74 ~ 36.01, 8.44 ~ 66.63, 21.8 ~ 3,990, 1737 ~ 5,831, and 729 ~ 3,104 mg/kg in the Tartary buckwheat seeds (water content about 13%) .During germination, the contents of various minerals in Tartary buckwheat will change in different degrees. In the process of plant growth, mineral elements play a significant role in the regulation of cellular permeability and osmotic balance, acting as enzyme cofactors  and formBeing a major source of rutin, buckwheat has a great deal of pharmacological functions, such as antioxidant and hypoglycemic effects . Germina2 induction not only significantly enhanced the antioxidant capacity of buckwheat malt on HepG2 cells, but also protected fibroblasts from oxidative damage. CaCl2 could effectively maintain the accumulation of bioactive compounds in buckwheat sprouts, thus enhancing the sucrose-induced antioxidant capacity  are improved to resist adversity, accelerate metabolism and photosynthesis, and promote the production of secondary metabolites , 97.When Tartary buckwheat was treated with laser light , the results indicated that laser enhanced the antioxidant capacity and anti-inflammatory activities by restraining the activities of cyclooxygenase-2 and lipoxygenase, particularly in sprouts . Another3 increased the DCI content in the sprouts of Tartary buckwheat by up to 9-fold  also could increase α-glycosidase activity to release more free DCI, so as to increase the effect of reducing sugar in buckwheat  of the total phenolic compounds and which were around 6-fold higher than those of common buckwheat sprouts in both at 9 and 12 days after sowing (p < 0.01) and the combination of UV-B, LED blue and red light was more appropriate to the regulation of flavonoids accumulation during the germination of Tartary buckwheat  treatment processes decreased the starch, protein, fat, and ash contents. MW reduced the total flavonoids and essential amino acids and total amino by 19, 3.1 and 4.8%, respectively. Phytic acid and Trypsin inhibitor activity was lowered by 33.4 and 13%. The content of tannin and saponin lost 27.5 and 20.1%, respectively. But the protein digestibility and the ω-6/ω-3 ratio increased by 3.6 and by 4.9, respectively . Above oca.10%), a significant 87% reduction compared to the control group  has deleterious effects to seeds .Due to internal heating, MW treatment usually displays little potential in grain sterilization for sample damage caused by heating . FurtherThe process of germination was accelerated at 300 W MW irradiation for 50 s. After germination, reducing sugar content increased by 7.51 times, starch content decreased by 69.61%, and amylase activity improved obviously. MW irradiation can enhance the activities of PAL, CHI and flavonol synthetase (FLS), and the total flavonoid content achieved the maximum value (5.15 g/100 g DW) after 300 W/50 s irradiation. Therefore, It is valid to improve the functional properties of edible Tartary buckwheat sprouts . Compareca.79.62%) and genistein (ca.70.95%) contents of soybean sprouts , stimulating crop growth, as well as declining crop maturation period. Besides, the non-thermal treatment of MW, like EMF and MF, can improve seed GE and germination rate, enhance the quality and stress resistance of seedlings, and thus raise the yield and metabolic function of crops , 140.Currently, although US, MW, and MF treatments are broadly used as complementary extraction and drying methods, few studies investigate their applications in grain germination . In addiCurrent work indicates that US treatment can improve the edibility and nutritional quality of grain sprouts as an emerging way. These results for regulating the biosynthesis pathway of phenylpropanoid provide a certain reference, which can be used to regulate the nutritional and functional components of sprouted for human health benefits.There are few studies on improving the nutritional value of buckwheat sprouts by laser. Almuhayawi et al.  treated The synthesis and accumulation of flavonoid in plants depends on the activity of key enzymes in the flavonoid pathway, the phenylpropanoid pathway, as shown in Chalcone is formed by the initial substrates 4-coumaryl-CoA and malonyl-CoA under the action of Chalcone Synthetase (CHS), and then CHI catalyzes chalcone to produce 4,5,7-trihydroxyflavanone, as a major product of metabolism, it re-enters other different metabolic pathways and forms different flavonoids . PAL andIt has been shown that PAL and CHI are induced during seed germination by various external factors, such as light, mechanical damage, US, MW, and MF, etc. . In receLight conditions have different effects on the activities of key enzymes for flavonoid enrichment. There are reports showed that UV-A (365 nm) UV-C (254 nm) and their combination treatments significantly increased the activities of key enzymes such as Phenylalanine ammonia-lyase, chalcone isomerase (CHI) and rutin degrading enzyme (RDEs) in Tartary buckwheat, and improved the content of total flavonoids , 94.The appropriate intensity of electromagnetic field treatment can also improve the activities of related enzymes in plants. The changes of PAL and other three key enzyme activities during the germination of Tartary buckwheat seeds under different MF intensities was given , the speIt has been proved that the key enzyme activity and gene expression in the synthetic pathway are closely related to the environment in which plants live. Light quality, salt stress, and EMF all directly affect the expression of genes involved in plant information transduction and biosynthesis . TherefoFtPAL, FtCHS, FtF3H, and FtANS, which promotes the activities of PAL, CHI and other related enzymes in phenylpropanoid metabolic pathway  (Ft4CL in the cotyledons treated with LED blue light was significantly increased by 220.8% (p < 0.01). The expression levels of Ft4CL in cotyledons and hypocotyls were significantly increased by 185.0% (p < 0.05), and the expression levels of FtPAL in hypocotyls were significantly decreased by 9.3% (p < 0.05). Another studied that the effects of light quality on ANR, a key synthetic gene in procyanidin metabolism pathway, the expression of FtANR and FtLAR1 was up-regulated after the treatment of blue light, far red light and ultraviolet light, and the expression of FtLAR3 was up-regulated after the treatment of red light  . Howevered light . This reThe myeloblastosis oncogene (MYB) transcription factor is notable in flavonoid biosynthesis in buckwheat, which is involved in multiple metabolic pathways of flavonoid biosynthesis and can coordinate with multiple genes to regulate flavonoid biosynthesis (FtSAD2 (sensitive to abscisic acid and drought) and FtJAZ1  can interact synergistically with MYB, thus influencing the accumulation of flavones in Tartary buckwheat , thereby affecting flavonoid synthesis  during cultivation. After 300 W/75 s MW irradiation, the maximum rutin content (25064.19 mg/kg DW) was observed in 5-day-old sprouts, while the maximum chlorogenic acid (497.95 mg/kg DW) and vitexin (345.75 mg/kg DW) were observed after 300 W/50 s MW irradiation (FtPAL, FtCHI and FtFlS1 increased by 39.84, 24.78 and 33.72% compared with CK, respectively. Hence, MW irradiation is a valid technique to enhance the functional attributes of edible Tartary buckwheat sprouts, but its power condition and exposure time need to be better optimized and determined whether it can promote seed germination.At present, in Tartary buckwheat germination, the MW technology have been applied, the less research of regulatory mechanism of gene expression of enzymes related to flavonoid synthesis is done. The study confirmed that in Tartary buckwheat sprouts, the transcription of various flavonoid biosynthetic enzyme gene containing adiation . The effadiation . Besidesadiation . And undUS and MF treatments are widely used to cereal germination , but lesAfter buckwheat seeds germinated into sprouts, the nutritional value of buckwheat seeds was greatly increased, the trypsin inhibitor activity was low or even disappeared, the proportion of protein and amino acids was more balanced, the nutritional value of fatty acids was increased, and the activity of rutin-degrading enzyme disappeared, especially the content of flavonoids increased obviously. Under adversity, plants produce a large number of flavonoids, GABA and other secondary metabolites to repair the damage caused by adversity. In recent years, the research on the enrichment of flavonoids in buckwheat is limited to the effect on its content, but the research on its biosynthesis and regulation genes is lacking. Therefore, the key regulatory factors in flavonoid synthesis need to be further studied and explored. At the same time, there is still much room for further research on the physiological functions and mechanisms of flavonoid in plants. In the future, researchers can study the mechanism of buckwheat flavonoids enrichment, such as the activities of related endogenous enzymes, the expression of key enzyme genes and key regulatory factors, so as to accumulate specific secondary metabolites, and thus more effectively play unique health care value of buckwheat flavonoids. Through animal experiments, the health effects of buckwheat sprouts can be further clarified, which provides a theoretical basis for the development of deep-processed sprout vegetables with various health functions. The research on these problems has far-reaching significance for promoting the healthy and rapid development of buckwheat industry in our country.YD and NW: conceptualization. WP, NW, JW, YD, and SW: validation. YD: writing – original draft preparation. SW: writing – review and editing, supervision, project administration, and funding acquisition. WP: visualization. All authors have read and agreed to the published version of the manuscript.This research was funded by National Natural Science Foundation of China, grant number 31772025 and Anhui Natural Science Foundation, grant number 1808085MC93.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Gastric neuroendocrine tumors are a heterogeneous group of neoplasms that produce bioactive substances. Their treatment varies according to staging and classification, using endoscopic techniques, open surgery, chemotherapy, radiotherapy, and drugs analogous to somatostatin.To identify and review cases of gastric neuroendocrine neoplasia submitted to surgical treatment.Review of surgically treated patients from 1983 to 2018.Fifteen patients were included, predominantly female (73.33%), with a mean age of 55.93 years. The most common symptom was epigastric pain (93.3%), and the mean time of symptom onset was 10.07 months. The preoperative upper digestive endoscopy (UDE) indicated a predominance of cases with 0 to 1 lesion (60%), sizing ≥1.5 cm (40%), located in the gastric antrum (53.33%), with ulceration (60%), and Borrmann III (33.33%) classification. The assessment of the surgical specimen indicated a predominance of invasive neuroendocrine tumors (60%), with angiolymphatic invasion in most cases (80%). Immunohistochemistry for chromogranin A was positive in 60% of cases and for synaptophysin in 66.7%, with a predominant Ki-67 index between 0 and 2%. Metastasis was observed in 20% of patients. The surgical procedure most performed was subtotal gastrectomy with Roux-en-Y reconstruction (53.3%). Tumor recurrence occurred in 20% of cases and a new treatment was required in 26.67%.Gastric neuroendocrine tumors have a low incidence in the general population, and surgical treatment is indicated for advanced lesions. The study of its management gains importance in view of the specificities of each case and the need for adequate conduct to prevent recurrences and complications. Its incidence ranges from 3.7 to 30%, and the stomach is the most common site of all NETs. The incidence rates of gastric NETs as well as other NETs are on the rise, possibly due to the great detection in endoscopic and histopathological exams.Neuroendocrine tumors (NET) are uncommon neoplasms and represent about 0.5% of new cancer cases:Regarding histomorphological characteristics, gastric NETs can be classified into four main subgroups.Type I: It is the main type of neuroendocrine tumor, responsible for about 70–80% of cases and usually associated with chronic atrophic gastritis. It presents as multiple tumors, ranging from 1 to 2 cm, and with low metastatic potential.Type II: Tumors are histologically similar to type I but differ in that they are associated with Zollinger-Ellison syndrome or multiple endocrine neoplasia syndromes type 1 (MEN-1). These are rare tumors that, like type I, present as multiple small nodules. The metastatic potential is still low, but it is more significant than type I tumors.Type III: They are sporadic, single, large lesions with a high infiltrative and metastatic potential, especially in cases of poorly differentiated tumors. Usually, preexisting pathological conditions are not observed, as occurs in type I and II.Type IV: They are rare and present as single, large, poorly differentiated lesions with high infiltrative and metastatic potential. In addition to the characteristics already described, type IV tumors differ from the other subtypes, because they are not associated with cells similar to the enterochromaffin cells. They derive from other endocrine cells, which secrete hormones such as serotonin, gastrin and adrenocorticotropic hormone.In 2017, the WHO proposed a new classification of gastrointestinal NETs. They were divided into well-differentiated neuroendocrine neoplasms, subdivided into G1 neuroendocrine tumor (mitotic index <2%) and G2 (mitotic index between 2 and 20%), and poorly-differentiated neuroendocrine neoplasms, which include the G3 neuroendocrine carcinoma (mitotic index >20%), subdivided into small cell, large cell, and mixed neoplasms.The definitive diagnosis of the lesions is by biopsy, usually performed during upper digestive endoscopy (UDE). It is recommended an immunohistochemical analysis, including analysis of chromogranin A and synaptophysin for diagnosis, and the Ki-67 proliferative index, indicative of prognosis.Currently, most cases of gastric NET are treated endoscopically. Surgery is reserved for those in which endoscopic resection cannot be performed or that present poor prognosis factors, such as deep invasion and lymph node metastasesThe objective of the present study was to identify and review the cases of gastric neuroendocrine neoplasia that underwent surgical treatment.The medical records of patients with gastric NETs diagnosed between 1983 and 2018 and treated surgically were reviewed. The study was approved by the Ethics Committee of the State University of Campinas (Unicamp), under CAAE 78447517.0.0000.5404.The study included 15 patients, of which four (26.67%) were male and 11 (73.33%) were female. Age ranged from 20 to 77 years, with an average of 55.93 years.Computed tomography (CT) was performed on 14 patients (93.33%), and liver metastasis was found in three of them (20%). Octreoscan was performed in four cases (26.67%), with liver and bone metastasis detected in one patient (6.67%) and liver and lymph node metastasis in another (6.67%).Comorbidities and other concomitant diseases are described in The time of symptoms onset ranged from 0 to 60 months, with a mean of ten months. The most common reported symptom was epigastric pain (93.33%), followed by bloating (46.67%), and weight loss (46.67%).The macroscopic characteristics visualized in the upper digestive endoscopy (UDE) are summarized in  ; one patient underwent total gastrectomy with Roux-en-Y reconstruction; one was submitted only to Roux-en-Y gastroenterostomy due to advanced tumor (6.67%), another underwent lesion resection plus peritoneal implant resection plus liver metastasis resection (6.67%), and another had subtotal esophagectomy plus partial gastrectomy and reconstruction with gastric tube and cervical esophagogastric anastomosis (6.67%). The characteristics of the 15 cases studied, surgical procedures, recurrence, and follow-up time are summarized in Surgical procedure was indicated in most cases (80%), for curative purposes. The subtotal gastrectomy with Roux-en-Y reconstruction was the technique most applied (53.33%). In two patients, reconstruction was performed through double transit in modified Rosanov techniqueThe histopathological findings of the biopsies performed by UDE, the histopathological results of the surgical specimens, and the immunohistochemical tests are in The immunohistochemical analysis recorded positive chromogranin A in nine patients (60%), and positive synaptophysin in ten (66.67%). The Ki-67 study was carried out in 11 cases showing a rate between 0 and 2%, two cases between 3 and 20%, and four >20%.Postoperative hospitalization time ranged from 8 to 30 days, with an average of 14 days. Nine patients had postoperative complications, and the surgical wound infections were the most common (26.67%). One patient had severe pulmonary complications and died (6.67%).The follow-up time ranged from 1 to 32 years, with a mean of 7.87 years. In the latest follow-up, tomography was performed in 13 cases (86.67%), with liver metastases observed in five patients (33.33%), lymph nodes in two (13.33%), bone metastases in one (6.67%), and peritoneal carcinomatosis in one (6.67%). Octreoscan was performed in five cases (33.33%), showing liver and bone metastases and peritoneal carcinomatosis in one of them (6.67%). During the follow-up period, three patients (20.00%) had tumor recurrence and five (33.33%) were reoperated. Adjuvant treatment with octreotide was performed in two patients (13.33%), chemotherapy in four (26.67%), and combined chemotherapy and radiotherapy in four (26.67%).. The incidence is higher in female patients, over 60 years of age, as observed in this study, due to the hormonal profile, the higher prevalence of atrophic gastritis in women, or associated genetic factors.NETs are rare neoplasms, representing about 2% of gastric tumors in 2019, arterial hypertension was the main comorbidity presented by patients, corroborating this study. This finding is possibly due to the late age of presentation of both diseases.The evaluation of comorbidities is essential in the therapeutic decision. In a study by Darbà and Marsà.The smoking and alcoholism are important risk factors for several neoplasms; their impact on gastric NETs is still uncertain, with weak association or absence. Associated clinical manifestations include abdominal pain, anemia, upper gastrointestinal bleeding, and weight loss. However, a significant number of cases may be asymptomatic. Epigastric pain was the most frequent clinical manifestation, reinforcing the importance of considering this diagnosis in the investigation of symptoms.Gastric NETs present slow growth and are usually non-functional, differing from other gastrointestinal tumors. In this study, there was a predominance of cases with single lesions, larger than 1.5 cm, and located in the gastric antrum – a profile compatible with type III.The UDE is essential in the diagnosis of gastric NETs, and its findings are variable. Type I and II tumors usually present as multiple nodules in the gastric fundus and body, smaller than 1 to 2 cm. Type III tumors are single lesions, generally larger than 2 cm, located mainly in the gastric antrum and fundus. And type IV tumors generally manifest as multiple small lesions. Besides, the immunohistochemical study of chromogranin A and synaptophysin are indispensable markers, considering that the histopathological evaluation can be nonspecific and that false-negative results may occur, with diagnostic doubt about other histological types.Biopsies and histopathological investigation are also essential for treatment. It is recommended that, in addition to the biopsy of the lesion, a biopsy of the surrounding mucosa should be performed to identify atrophic gastritis, intestinal metaplasia, and hyperplasia. Scintigraphy with radioactive octreotide (linked to indium-111), also known as Octreoscan, can help expose primary tumors or metastases not detected in conventional imaging tests.CT and magnetic resonance imaging (MRI) are sufficient for staging in most casesThe Octreoscan was not performed in all cases (only in four in the preoperative period and in five in the follow-up). In the preoperative, bone metastasis was identified in one patient and lymph node metastases in another, which were not diagnosed by CT..Clinical, radiological, as well as histological staging are of great importance in analyzing tumor behavior and determining surgical therapy. The histological grade can be defined by the mitotic rate and/or Ki-67 index, classifying NETs into low-grade tumors (G1), with a Ki-67 index from 0 to 2%; intermediate grade (G2), with a Ki-67 index from 3 to 20%; and high-grade tumors (G3), with a Ki-67 index >20%.In eight cases in this study, the initial diagnosis was gastric adenocarcinoma, without previous evidence of NET. However, in the histopathological evaluation of the surgical specimen, the immunohistochemical examination showed the NETs, predominantly G2 tumors. The difference obtained between the evaluation of the biopsy by UDE and the surgical specimen can be explained by intratumoral heterogeneity, which justifies the need to evaluate more mitotically active areas to minimize evaluation divergences. Initially, the therapeutic definition should consider the classification proposed by Rindi et al., which determines the occurrence of three types of gastric NETs: Type I tumors, with multiple lesions smaller than 1 cm, well-differentiated histology, hypergastrinemia, and association with atrophic gastritis and anemia pernicious; Type II tumors, with clinical and laboratory features similar to type I tumors, differing by their association with Zollinger-Ellison syndrome and multiple endocrine neoplasia type I; Type III tumors, characterized by a single ulcerated lesion and histology ranging from well-differentiated to moderately differentiated; and Type IV tumors, characterized by single and large lesions, poorly differentiated and with a high infiltrative and metastatic potential.Surgery remains the best option for the treatment of gastric NETs. Gastrectomy with lymphadenectomy is indicated for tumors between 1 and 2 cm; for type I and II tumors larger than 2 cm, with six or more lesions, and with muscle layer invasion or lymph node metastasis; and for types III and IV tumors.Types I and II tumors should be evaluated by size, number of lesions, invasion of the muscularis propria layer, and lymph node metastasis. Tumors smaller than 1 cm, with up to five lesions and without muscle or lymph node invasion can be observed or submitted to endoscopic resection. Surgical treatment is the best option when the invasion of the muscularis propria or serosa is diagnosed, and gastrectomy with lymph node resection is recommended.The surgical technique employed may include local excision with endoscopic resection, local resection, subtotal gastrectomy, and total gastrectomy, among others.Type I tumors have low risk of metastases, so conservative treatment is recommended especially in smaller lesions, where endoscopic mucosal resection or endoscopic submucosal dissection should be applied. Antrectomy may be an option, especially in cases of hypergastrinemia or recurrence. Finally, somatostatin analogues, such as octreotide, may be a therapeutic option in inoperable cases or in cases of metastatic disease. In types III and IV tumors, the preferred treatment is subtotal or total gastrectomy with D2 lymphadenectomy, as in gastric adenocarcinomas. Resectable liver metastases must be surgically treated. Arterial embolization, radioablation, and liver transplantation are indicated when tumors are unresectable. Chemotherapy should be considered in cases of extrahepatic metastases or recurrent symptomatic disease. In this study, cases treated with subtotal gastrectomy and Roux-en-Y reconstruction with lymphadenectomy predominated. This profile is mainly due to the definition of a cohort of patients who required surgical treatment and the surgical protocols adopted.In type II tumors, resection of the gastrinoma is the surgical approach of choice, and antrectomy does not have a favorable effect. In this study, only two patients had pulmonary complications and three had anastomotic, predominantly surgical wound infections. Prophylactic measures are important, such as nursing care and antibiotic therapy, as well as new surgical approaches, as minimally invasive surgery, which reduces surgical wound infections and pain.Previous studies showed that patients undergoing a gastrectomy presented pulmonary, anastomotic, and cardiac complications.The follow-up of patients must be individualized, according to tumor type and local guidelines. The recommendation of the National Comprehensive Cancer Network is a clinical history, physical examination, UDE, CT or MRI of the abdomen, and serum chromogranin A every six months, for one to two years, followed by once a year for four years, and biennially up to ten years after surgery. Lin et al. observed tumor recurrence in 47.5% of cases of gastric neuroendocrine carcinoma. In events of recurrence after endoscopic resections, gastrectomy is indicated, in addition to other therapies such as chemotherapy, radiotherapy, and somatostatin analogues.Finally, considering tumor recurrence, we observed, in this study, that 20% of cases had recurrence during follow-up and 33.3% required some reoperation, either due to recurrence or management of complications. Data regarding the recurrence of gastric NETs remain heterogeneous. Studies with type I tumors indicated recurrence in 63.6% and 52% of casesGastric NETs have a low incidence in the general population. However, their study gains importance in view of the specificities of each case and the need for adequate management to prevent recurrences and complications. Although endoscopic treatment and minimally invasive surgery have become relevant in the management of these tumors, conventional surgery should still be considered in a significant number of cases, especially in the presence of type III and IV tumors.


Bracteacoccus, Desmococcus, Geminella, Chlorella, Klebsormidium, and Stichococcus (Chlorophyta) and cyanobacterium Nostoc in the volumetric trap samples and three more in the cultivated samples. The mean annual concentration recorded over 3 years was 19,182 cells*day/m3, with the greatest contribution from the genus Bracteacoccus (57%). Unlike some other bioparticles like pollen grains, autotrophic microorganisms were present in the samples over the course of the whole year, with greatest abundance in February and April. The peak daily concentration reached the highest value (1011 cells/m3) in 2021, while the mean daily concentration during the three analysed years was 56 cells/m3. The analysis of intra-diurnal patterns showed their increased presence in daylight hours, with a peak between 2 and 4 p.m. for most genera, which is especially important due to their potential to trigger allergy symptoms. From the environmental factors, wind speed had a most significant positive association with their concentration, while relative air humidity had a negative influence.Despite their non-negligible representation among the airborne bioparticles and known allergenicity, autotrophic microorganisms—microalgae and cyanobacteria—are not commonly reported or studied by aerobiological monitoring stations due to the challenging identification in their desiccated and fragmented state. Using a gravimetric method with open plates at the same time as Hirst-type volumetric bioparticle sampler, we were able to cultivate the autotrophic microorganisms and use it as a reference for correct retrospective identification of the microalgae and cyanobacteria captured by the volumetric trap. Only in this way, reliable data on their presence in the air of a given area can be obtained and analysed with regard to their temporal variation and environmental factors. We gained these data for an inland temperate region over 3 years , identifying the microalgal genera The online version contains supplementary material available at 10.1007/s11356-023-29341-8. The city is situated between two main geomorphological regions, the Malé Karpaty Mts. (162–559 m a. s. l.) from the north and the Podunajská nížina lowland (200 m a. s. l.) from the south. The Danube River, the second longest river in Europe, flows through the city. Additionally, there are more than 25 natural or artificially created bodies of water in the Bratislava area. These water surfaces, together with various natural  and anthropogenic substrates , represent potential sources of airborne autotrophic microorganisms. Green spaces comprise approximately one-quarter of the 36,759 ha of Bratislava’s area  and January being the coldest one (0.8 °C) . The average annual total precipitation is 806 mm, with May being the rainiest (93 mm) and April the driest (43 mm). The average annual wind speed is 2.5 m/s, with prevailing northwest winds.Airborne microalgae and cyanobacteria samples were taken using gravimetric open-plate and volumetric Hirst-type method Hirst . The comThe volumetric sampling was performed in 2018, 2020, and 2021 and gravimetric sampling in 2020 and 2021. The volumetric sampler (Burkard Manufacturing Co Ltd.) is placed on the roof of Science Park, Comenius University in Bratislava  at the height of 18 m above ground level. Sterile Petri dishes containing culture medium were placed at the same location once a month for 4 h during days with dry and stable weather  with regular watering to prevent the growth of bacteria and fungi. In each of these gravimetric cultivation (GC) samples, the growth of the microalgal and cyanobacterial colonies was examined daily for 3 to 4 weeks, the length of the observation period depending on the growth rate of the colonies. The cells of every colony were observed under the light and epifluorescence microscopes. A microscopic identification of different stages in the life cycle of individual colonies took place several times during the cultivation period of each dish. The collected microalgae and cyanobacteria were identified to the lowest possible taxonomic level, based on morphology according to different determination keys Hindák .3).The volumetric sampler operates at an airflow rate of 10 l/min. The airborne particles suctioned through a horizontally oriented 2 × 14 mm inlet collide with a rotating drum covered with adhesive tape continuously moving under the inlet at the rate of 2 mm/h. After removal from the sampler (once in 7 days), the exposed tape was cut into segments corresponding to 24-h exposure and placed individually on microscope slides coated with a mixture of gelatin, glycerin, phenol, and distilled water. These semi-permanent slides of volumetric trap (VT) samples were stored until their evaluation. Microalgae and cyanobacteria were counted along 12 vertical transects per slide, each representing 2 h of real-time exposure, under a light microscope, at a magnification of 400 × , expressed in μg/m3, were excerpted from the database of the Slovak Hydrometeorological Institute.The meteorological parameters used in this study were mean daily values of air temperature (°C), relative humidity (%) and wind speed (m/s), and daily totals of sunshine (hours) and precipitation (mm). The values were measured in the Meteorological Observatory Bratislava – Mlynská dolina , located close (300 m) to the aerobiological monitoring station. The mean daily concentrations of air pollutants  were studied by establishing non-parametric Spearman’s correlation coefficients. The analysis was performed for the set of all autotrophic microorganisms as well as for the dominant genera in three time periods: (i) all-year-long, (ii) summer months (June–September), and (iii) non-summer months . The distinction between summer and non-summer months was implemented to investigate the impact of individual environmental factors on the concentration of airborne microalgae and cyanobacteria during colder and warmer periods of the year. The data analyses were performed in Statistica 12.The relationships between the mean daily concentration of autotrophic microorganisms and selected environmental variables  and Chroococcus and Phormidium (cyanobacteria). The most frequent genus was Nostoc, which was observed in 67% of all cultivations, followed by Bracteacoccus and Muriella, with a prevalence of 60%. Contrarily, the least frequent genera Desmococcus, Klebsormidium, Chroococcus, and Phormidium occurred only in 7% of cultivations  taxonomic group, namely Bracteacoccus sp., Chlorella sp., Desmococcus sp., and Stichococcus sp.) and two filamentous algae (Klebsormidium sp. and Geminella sp.) (Table Nostoc (cyanobacteria)  Table ; Fig. S1a) Table , as wella) Table  ranged bBracteacoccus, ranging between 64 and 47% of the AMIn in 2020 and 2018, respectively, while the minimum contribution was made by Bacillariophyceae, Nostoc, and Geminella, with the mean annual concentration less than 2% of the total  and April (2598 cells/m3) and lowest in December (450 cells/m3) and November (536 cells/m3)  and at the beginning of February 2021 (667 cells/m3).The mean daily concentrations of microalgae and cyanobacteria ranged from 44 cells/mBracteacoccus, Desmococcus, Geminella, Chlorella, Klebsormidium, and Stichococcus). In most cases, a relatively constant prevalence of microalgae was observed throughout the day , and the selected meteorological factors and air pollutants were assessed during three different periods — (i) all-year-long, (ii) non-summer months, and (iii) summer months — throughout the studied years. The results of the correlation analysis are depicted in Table Desmococcus and Stichococcus during the summer months. The relationships with wind speed were significantly positive, while the associations with relative humidity were significantly negative. Precipitation and air temperature had the least impact on the concentration of microalgae in the air. Among the significant associations with air pollutants identified in this study, ozone exhibited a positive correlation with the levels of all microalgae during non-summer and all-year-long periods, except for the all-year-long concentration of Chlorella. Conversely, the relationships between other air pollutants  and microalgae were negative. PM10 values were significantly associated with all assessed taxonomic groups, although the association was less pronounced in the summer. On the other hand, both CO and NO2 exhibited significant associations with Bracteacoccus and Chlorella. Desmococcus was significantly influenced by CO and NO2 and Stichococcus by CO in non-summer months.Spearman’s correlation coefficients between the total daily concentrations of all autotrophic microorganisms detected in airborne samples, alongside the daily concentrations of four most abundant genera  have multi-layered UV-resistant coats formed by dense cell walls, various antioxidants, and carotenoids  was recorded in February 2020 when the average daily temperature reached 6.2 °C, which is 265% higher than long-term average for this month , and the wind spead reached 2.7 m/s, which is 1.2 and 0.8 m/s higher than in 2018 and 2021, respectively. Similarly to our results, Tormo et al. , Wiśniewska et al.


The nationwide clinical features of Japanese patients with primary nephrotic syndrome (NS), including minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), or membranous nephropathy (MN), have not yet been reported. We collected the clinical personal records of patients with primary NS between 2015 and 2018 from the national registry organized by the Japanese Ministry of Health, Labour, and Welfare. Overall, the demographics, chronic kidney disease classification based on glomerular filtration rate and albuminuria, and treatment of 6036 patients were collected: 3394 (56.2%) with MCD, 677 (11.2%) with FSGS, 1455 (24.1%) with MN, and 510 (8.5%) with others. MN patients were older than MCD and FSGS patients . Steroid-dependent NS or frequently relapsing NS was found in 70.2%, 40.5%, and 24.6%, whereas steroid-resistant NS was found in 6.4%, 36.0%, and 37.9% of patients in the MCD, FSGS, and MN, respectively. The present oral prednisolone use  was 87.2% (21.2), 80.9% (20.0), and 77.5% (18.8) of patients in the MCD, FSGS, and MN, respectively. The national registry of clinical personal records of primary NS could provide an informative insight into the characteristics, clinical features, and treatment approaches for patients with primary NS in Japan. Based on biopsy results, NS is classified into minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), and membranous nephropathy (MN)4 with the global incidence of 0.2–0.8, 0.2–1.1 and 0.3–1.4 per 100,000 person-years, respectively5. The Japan Nephrotic Syndrome Cohort Study (JNSCS), which enrolled 374 Japanese patients with primary NS, reported that the incidence of MCD, FSGS, MN, and others was 41.4%, 10.2%, 39.6% and 8.8%, respectively6, suggesting that their prevalence in Japan is similar to that worldwide. Furthermore, several studies from the Japan Renal Biopsy Registry (J-RBR), a nationwide, web-based registry of renal biopsies, have also described the clinical features of several primary NS in Japan11. These registries are useful for understanding the demographic, clinical characteristics, and treatment outcomes of several kidney diseases14. However, in Japan, most studies on primary NS are either regional or conducted on a small scale. Additionally, no cohort study with more than 1000 patients has reported the distribution of patients with primary NS. Therefore, large-scale demographics, clinical characteristics, and treatment data of Japanese patients with primary NS are needed for better healthcare services, to improve treatment, and to provide a reference for comparisons with future studies and medical administration.Primary nephrotic syndrome (NS), a major course of nephrotic syndrome, is characterized by massive proteinuria, edema, and hypoalbuminuria17. Clinical personal records is a nationwide administrative database of public expenditure on intractable disease maintained by the Japanese Ministry of Health, Labour, and Welfare to register and certify intractable diseases18. This database started to collect data on primary NS data in 2015, which helped investigate the clinical features of routine practice for primary NS. The aim of this study was to describe the demographics, subtypes of glomerulopaties, and treatment of primary NS in Japan using a nationwide registry of clinical personal records.Recent attention in medical and healthcare research has focused on large-scale data from various sources, including administrative or claims data, electronic health records, and data from self-monitoring devicesData were collected from 6036 patients with primary NS from 2015 to 2018  than in FSGS (80.9%) and MN (77.5%) Table . The meaAs for steroid responses, the steroid-resistant NS was significantly higher in MN and FSGS than in MCD . In the steroid-resistant NS, 213 patients had MCD, 236 had FSGS, and 540 had MN  or frequently relapsing nephrotic syndrome (FRNS) was significantly higher in MCD than in FSGS and MN  Table . In the Patients were divided into five age groups: child and adolescent (≤ 19 years), young adult (20–39 years), middle adult (40–64 years), elderly (65–74 years), and very elderly (≥ 75 years). The distribution and risk classification of chronic kidney disease associated with MCD, FSGS, and MN are shown in Supplementary Fig. In patients with MCD, the use of oral prednisolone and intravenous methylprednisolone was significantly lower in the child and adolescent groups as compared with the other age groups Table . In contWith regards to patients with FSGS, the use of mycophenolate mofetil and rituximab was significantly higher in the child and adolescent groups than in the middle adult and elderly groups, but the use of oral prednisolone and intravenous methylprednisolone was lower in the child and adolescent groups than in the other age groups Table . Other iIn patients with MN, the use of prednisolone and other immunosuppressive drugs did not differ much among the five age groups, although the mean dosages of oral prednisolone and cyclosporine were higher in the older age groups  Table . SteroidAs for QIs related to the treatment for primary NS, the use of prednisolone + cyclosporine combination therapy in steroid‑resistant MCD, FSGS, and MN in the present treatment was 42.7%, 47.4%, and 42.2%, respectively, but 51.6%, 67.8%, and 58.6%, respectively, in the previous treatment  was more dominant, whereas MN (24.1%) was less, suggesting that a younger age of onset in MCD than those in previous studies may be due to etiologic differences. Compared to Nephrotic Syndrome Study Network (NEPTUNE) in North America 4, the present study shows that the prevalence of FSGS in Japan was lower (11.4%) and has not changed in the past decade21. These epidemiological differences could be attributed partially to the genetic variants in apolipoprotein L1 (APOL1) among people with sub-Saharan ancestry22.Analysis 1 described the distribution and frequency of glomerulopathies in the national registry of clinical personal records of primary NS by age of onset. MCD, FSGS, and MN median ages were 31, 39, and 60 years, respectively. and younger than those of included in JNSCS 9. Accordingly, future clinical guideline for elderly patients with NS should be developed to reflect new findings.In Analysis 2–1, we compared the demographics, clinical features, and treatment of the three major pathological types of primary NS, which are MCD, FSGS, and MN. To the best of our knowledge, this is the first report to describe both the use and the mean dosage of oral prednisolone was significantly higher in MCD  than in MN , possibly because SDNS or FRNS was significantly higher in MCD than in MN. In addition, since infectious disease is a harmful complication among elderly NS patients, the MN patients were older than the MCD patients and may have received a reduced dose of steroids to prevent infection. These findings are consistent with JNSCS demonstrating better remission rates in elderly patients with NS2. However, in children with steroid-sensitive NS, the clinical presentation is usually sufficiently characteristic to guide initial management without biopsy23. Therefore, the difference between the age of onset and the age at renal biopsy might be greater in patients with MCD than in those with FSGS and MN, especially in younger patients with all three glomerulopathies.The median age of onset and the median age at renal biopsy were lower for MCD than for FSGS and MN . In adult patients with NS, renal biopsy is mandatory for diagnosis if the cause is not apparent on initial evaluation6, the rates of prescription of oral prednisolone (98.7% vs. 87.2%) and intravenous methylprednisolone (31.6% vs. 7.7%) were lower, whereas the rates of prescription of cyclosporine (31.6% vs. 47.2%), mizoribine (5.3% vs. 12.2%), rituximab (2.6% vs. 6.0%), and mycophenolate mofetil (0.7% vs. 2.6%) were higher in patients with MCD in the present study, suggesting that the patients with clinically more acute and severe cases were preferentially included in the JNSCS. Furthermore, the present study might include many patients with remission using multiple immunosuppressive drugs.Compared with the JNSCS11, the prescription rates of immunosuppressive drugs were higher in patients with FSGS in the present study , suggesting that occurrence of steroid-resistant FSGS was higher in the present study than in J-RBR.Compared with MCD, the prescription rates of intravenous methylprednisolone, cyclosporine, tacrolimus, and mycophenolate mofetil were higher in FSGS, as the incidence of steroid-resistant NS was significantly higher in FSGS than in MCD . Compared with J-RBRAnalysis 2–2 in this study included the largest number of elderly (65–74 years) or very elderly (> 75 years) onset patients, i.e. 212 and 118 patients with MCD, 61 and 25 with FSGS, and 363 and 173 with MN, respectively. Elderly and very elderly onset patients with MCD and FSGS have a higher prescription rate and higher dose of steroids and immunosuppressive drugs than younger patients, suggesting that steroid-resistant NS was much common even in the elderly or very elderly patients with MCD and FSGS. In contrast, patients with MN were observed to have similar medications and similar percentages of steroid-resistant NS among the five age groups.24. This study has attemped to revel the real-world use of rituximab for trating primary NS throughout Japan.As for child and adolescent groups of patients with MCD and FSGS, the use of oral prednisolone and intravenous methylprednisolone was lower than that in the other age groups, which might in turn be associated with a higher use of rituximab for SDNS and FRNS in pediatric and adolescent patients with MCD and FSGS as compared with the other age groups. Rituximab has been demonstrated to be effective and safe in patients with difficult-to-treat SDNS and/or FRNS, for reducing the relapse frequency and maintaining remission despite the cessation or tapering of steroids25. The present study has described the QIs related to the primary NS treatment, showing that the use of prednisolone + cyclosporine combination therapy in steroid‑resistant MCD, FSGS, and MN by 42.7%, 47.4%, and 42.2%, respectively, as present treatment, and 51.6%, 67.8%, and 58.6%, respectively, as previous treatment. Moreover, use of the same combination therapy in steroid-dependent or frequently relapsing MCD accountd for 48.8% in the present treatments and 71.0% in the previous treatments. The evaluation of QIs related to the treatment of primary NS is believed to improve the outcome.The Japanese clinical guideline for NS, published in 2014, recommends that patients with relapsing patients of MCD or steroid-resistant NS should be administered the combination treatment of cyclosporine and steroid as effective for reducing urinary protein levels and shortening the duration of achieving remission, compared to steroid-alone treatment (Recommendation grade: C1)This study has several limitations. First, the database does not include clinical characteristics observed at renal biopsy, such as urine protein levels, estimated glomerular filtration rate, serum albumin, and detailed pathological findings including immunostaining. Furthermore, the database does not contain data about complications (hypertension and diabetes), genetic testing, nonpharmacological therapy, cumulative dose of medication, duration from initial renal biopsy, and patient outcomes. We could obtain the renal biopsy date but could not determine whether it was an initial or a repeat biopsy. The data were collected from personal clinical records, and their accuracy depended mainly on those who completed the forms; however, we assessed the validity of the data to the best of our ability. The validity of the analysis itself should be evaluated in further studies using data from other cohorts. Second, we demonstrated the characteristics of primary NS throughout Japan using descriptive analysis. However, a temporal trend analysis would provide more clinically relevant findings. Further studies using temporal trend analysis are needed to reveal longitudinal demographic changes and the rate of guideline-directed medical treatments. Despite these limitations, we believe that the outcomes of this study will help provide an insightful overview of the clinical characteristics and features of patients with primary NS throughout Japan, in addition to developing the optimal management for these patients.In conclusion, by analyzing a nationwide database of more than 6000 patients with primary NS, this study revealed the clinical characteristics of MCD, FSGS, and MN in Japan and provided critical insights for developing optimal management strategies for these glomerulopathies. The gap between current clinical practice and guideline recommendations should be discussed, and further investigations are needed to improve the therapeutic strategies against primary NS in Japan.26. These clinical personal records contain clinically significant information such as symptoms, laboratory test results, and histopathological findings27. Since the Act on Medical Care for Patients with Intractable Diseases was enforced in 2015, data on 338 diseases (as of September 2022), including primary NS (designated intractable disease number 222), IgA nephropathy (designated intractable disease number 66), rapidly progressive glomerulonephritis (designated intractable disease number 220), and polycystic kidney disease (designated intractable disease number 67) collected from clinical personal records have been added to the database.The National Database of Designated Incurable Diseases of Japan was established in Japan and consists of data from the clinical personal records voluntarily submitted when individuals apply for medical support27. However, the database does not include survival data, such as death. The results presented in this study are original and different from the statistics produced or published by the Ministry of Health, Labour and Welfare of Japan27.This cross-sectional study used data from clinical personal records of primary NS, throughout Japan, maintained by the Japanese Ministry of Health, Labour, and Welfare. The records prospectively and annually collected demographic data , histological diagnosis , chronic kidney disease classification based on glomerular filtration rate and albuminuria, date of renal biopsy, medication use , and therapeutic responses . The data were registered only after being reviewed by certified nephrologists1, such as autoimmune diseases , diabetic nephropathy, paraproteinemia , infectious diseases , tumors , drugs , genetic diseases , pregnancy hypertension nephropathy, radiation nephropathy, or rejection of the transplanted kidney.The present study used clinical personal records of patients with primary NS from 2015 to 2018. Primary NS was diagnosed based on massive proteinuria (≥ 3.5 g/day) and hypoalbuminemia  in the absence of any secondary nephrotic syndrome27. If multiple patients had identical data, the data of one patient was included for analysis and the duplicate data was excluded. Erroneous data  were included after likely values were assigned as alternatives. These likely values were determined by referring to the basic statistics of the data27.Data used in stasitical analyses had to be processed and refined in advance to render them suitable for analysis25 as follows. Steroid-resistant NS is defined as never achieved complete remission despite immunosuppressive treatment, including steroid therapy, after diagnosis of NS. SDNS is defined as when steroids cannot be discontinued because of two or more relapses after reducing or discontinuing the dose of steroids. FRNS is defined by frequent relapses (2 or more relapses in 6 months).The clinical characteristics and management of each primary NS in this registry were determined. The use and dosage  of present and previously used steroids and immunosuppressive drugs were also collected. The response of NS to treatment was assessed according to the definition outlined by the Japanese guidelines for NSAnalysis 1: Distribution and frequency of glomerulopathies in the national registry of clinical personal records of primary NS by age (in years) of onset.Analysis 2–1: Comparison of demographics, clinical features, and treatment modalities between MCD, FSGS, and MN after removing patients with multiple diagnoses and missing data , young adult group (20–39 years), middle adult group (40–64 years), elderly group (65–74 years), and very elderly group (≥ 75 years). Clinical features were compared among the age groups.25.CQ5. Is the addition of immunosuppressive agents to steroids recommended for reducing urinary protein levels or preventing renal function decline in frequently relapsing MCD?CQ6. Are additional immunosuppressive agents to steroids recommended for reducing the urinary protein level and preventing renal function decline in steroid-resistant FSGS?CQ9. Is cyclosporine recommended to reduce the urinary protein level and prevent renal function decline in MN?We investigated the QIs related to the treatment for primary NS in some clinical questions (CQs) in Japanese guidelines for nephrotic syndrome “Evidence-based clinical practice guidelines for nephrotic syndrome 2014”Quantitative variables have been expressed as mean (standard deviation) for normally distributed data or median (IQR). Qualitative variables are presented as frequencies (percentages). Differences between groups were compared using chi-squared tests for categorical variables and nonparametric Kruskal–Wallis tests for continuous variables. All data were analyzed using IBM SPSS , and P < 0.05 indicated a significant difference.The study protocol was approved by the Ethics Committee of the Japanese Society of Nephrology , the Institutional Review Board of Niigata University, Asahikawa Medical University, and National Institutes of Biomedical Innovation, Health and Nutrition. Written informed consent to register the data of clinical personal records in the database and to use the data for research and policy-making was obtained from all patients. All procedures performed in the present study were in accordance with the Declaration of Helsinki.Supplementary Information.