However, the chemical compositions of the Jinzhong stream water, which is heavily influenced by urban sewage, are dominated by Ca2+ and HCO3–. The Ca2+, Mg2+ and HCO3– in Jinzhong stream are mainly derived from rock weathering, while the Youyu stream is affected by acid mine drainage, and sulfuric acid is involved in the weathering process. Ion sources analysis indicates that the Na+, K+, NO3–, and Cl– in the Jinzhong stream mainly derive from urban sewage discharge; but NO3– and Cl– of the Youyu stream mainly derive from agricultural activities, and Na+, K+ are mainly from natural sources. The element ratios analysis indicates the ratio of SO42–/Mg2+ in Youyu stream (4.61) polluted by coal mine is much higher than that in Jinzhong stream (1.29), and the ratio of (Na++K++Cl–)/Mg2+ in Jinzhong stream (1.81) polluted by urban sewage is higher than Youyu stream (0.64). Moreover, the ratios of NO3−/Na+, NO3−/K+, and NO3−/Cl− in the agriculturally polluted Youyu stream were higher than those in the Jinzhong stream. We can identify the impact of human activities on streams by ion ratios /Mg2+, NO3−/Na+, NO3−/K+, and NO3−/Cl−). The health risk assessment shows the HQT and HQN for children and adults are higher in Jinzhong stream than in Youyu stream and the total HQ value (HQT) of children was higher than one at J1 in the Jinzhong stream, which shows that children in Jinzhong stream basin are threatened by non-carcinogenic pollutants. Each HQ value of F− and NO3− for children was higher than 0.1 in the tributaries into Aha Lake, indicating that the children may also be potentially endangered.To explore the influence of different types of anthropogenic activity on the rivers, we investigate the major ion composition, sources and risk assessment of the karst stream (Youyu stream and Jinzhong stream), which are heavily influenced by mining activities and urban sewage, respectively. The chemical compositions of the Youyu stream water, which is heavily influenced by mining activities, are dominated by Ca They join the Nanming river in Wujiang water system which is a tributary of the Yangtze River in the southwest China . The reg2, which is the tributary with largest discharge of domestic wastewater and the most serious pollution in the Aha Lake basin. The main land use type along the Jinzhong stream are town, village, and industrial land respectively, which accounts for 66.37% of the land area in the watershed; followed by forestland, transportation land, and cultivated land, which, respectively, which account for 11.02%, 9.08%, and 8.45% of land area in the watershed. Gardens, meadows, and other land use types account for 5.08% of the land area in the watershed. The region that the Jinzhong stream flows through is the main urban area of Guiyang City, and the urbanization degree in the watershed is relatively high. It is a typical urban river.The Jinzhong stream flows for a length of 18.5 km, and the catchment area is 5.9 kmin situ using WTW340i multi-parameter water quality meters (made in Germany). The HCO3– was determined by titration with HCl (0.025 mol•L–1) on the day of sampling, and the error was within 5%. Samples were stored and sealed in a 4 °C refrigerator for storage before measurement. Major ions  were analyzed and determined by ion chromatograph (IC) , and SiO2 was measured using the molybdate yellow photometric method. The analytical precision was within ±5%.Sampling campaign implemented occurred during the flood season of rivers in June-August of 2017, with a total of 23 samples . The sampling sites are shown in 34S of sulfate analysis, water was first acidified to pH<2 using distilled 6M HCl. 10% BaCl2 solution were added to water samples to convert the dissolved SO42− into BaSO4 precipitation, and BaSO4 solids were obtained after the samples were filtered through 0.22 μm quantitative filter paper using ultrapure water  to flush the residues until the filtrates did not contain Cl− (as determined using the AgNO3 test). Then, the BaSO4 precipitation on membranes was further calcined  to gain the BaSO4 solid. The δ34SSO4 composition of BaSO4 was determined by analysing SO2 gas using a continuous flow isotope ratio mass spectrometer  with an accuracy better than 0.3‰ was performed in the laboratory of College of Resources and Environmental Engineering, Guizhou University.For δ3– and F– are often used to assess non-carcinogenic health risks in river waters. The hazard quotient (HQ) is usually used to evaluate noncarcinogenic health risks, and its calculation methods refer to Water pollutants mainly through drinking water and skin contact into the human body, damage to human organs, threatening human health . As non-ingestion is the ingestion intake of daily doses, RfD is the average reference dose of daily exposure doses, the reference doses of NO3− and F− are reported as 1.6 and 0.04 ppm/day , and Origin 2022  was used for editing graphics.−1, averaged 746.80 mg·L−1; the TDS of the Jinzhong stream ranges from 428.38–559.77 mg·L−1, with an average of 485.78 mg·L−1. Compared to other rivers that are also located in the karst region, the TDS values of the two streams are higher than the other rivers (−1) (−1)  , and it −1) (−1) , ultimat2+>Mg2+>Na+>K+) . The main cations Ca2+, Mg2+ account for 78.04%, 16.05% (Youyu stream) and 61.42%, 24.03% (Jinzhong stream) of total cations, respectively. However, the main anion in the Youyu stream is SO42–, which accounts for 73.66% of total anions, and HCO3– only accounts for 23.96%; while the main anion in the Jinzhong stream is HCO3–, which accounts for 59.20% of total anions, and SO42- accounts for 30.08%. Moreover, the Youyu stream shows NO3–>Cl–, whereas the Jinzhong stream exhibits Cl−>NO3–. The compositions of Ca2+, Na+, K+, Cl–, HCO3–, and SO42– of the waters in the two stream is obviously different. Ca2+ and SO42– in the Youyu stream are significantly higher than those in the Jinzhong stream, but Na+, K+, Cl–, and HCO3− in the Jinzhong stream are significantly higher than those in the Youyu stream (The trend of main cations in the two streams is the same (both are Ca>Na+>K+) , but difu stream .2+, which is similar to the Wujiang River , which is obviously higher than the Jinzhong stream (SO42−: 0.97 mmol·L−1) and other rivers in the world , oxidation of sulfide, atmospheric input, and industrial activities ; and it is decreased significantly from upstream to downstream, which reveals that the large amount of SO42− in the river water mainly comes from abandoned mine wastewater discharge , is mainly distributed near the sulfide end element, and the SO4–2 ions in the river water are mainly affected by sulfides. In general, the isotope study results are the same as the previous analysis of sulfuric acid source.Previous studies have shown that the chemical composition of areas far from the ocean is basically unaffected by marine input , and theal mines . Which i2+, Mg2+, and HCO3– in karst areas are usually derived from the weathering of carbonate rocks, weathered mainly by carbonic acid and sulfuric acid (2++Mg2+)/HCO3− is close to one while H2CO3 weathers the carbonate rocks; the ratio of 2(Ca2++Mg2+)/HCO3− is close to two and the ratio of 2SO4–2/HCO3– is close to one while H2SO4 weathers the carbonate rocks , but no obvious correlation with SO42−, demonstrated the dissolution of carbonate weathering. It is same as the chemical characteristics of karst rivers. The main anion of the Jinzhong stream is HCO3−, the ratio of 2(Ca2++Mg2+)/HCO3– in the river ranges from 1.40 to 1.44, and the ratio of 2SO4–2/HCO3– ranges from 0.50-0.51, indicated that the Ca2+, Mg2+, and HCO3– were less influenced by sulphuric acid.Caric acid . Generalte rocks . As show2+ and Mg2+ are positively correlated with SO4–2 , whereas Ca2+ and Mg2+ are negatively correlated with HCO3–  (2++Mg2+)/HCO3– in the river water ranges from 2.30 to 18.60, and the 2SO4–2/HCO3– ratio ranges from 1.10 to 19.61, reveals that sulfuric acid apparently participated in the weathering of carbonate rocks in the watershed. The abundant rainfall during the periods of high water causes a large amount of SO42− to flow into the river water, thereby increasing the SO42− concentration in the river water. This makes the equilibrium of chemical reaction (3) proceed to the left, and causes significant CaCO3 deposition  , exhibitposition . Meanwhihe right . especia3–, the existing form of N in waters can be used as the characteristic ion to characterize the influence of anthropogenic activity  which is similar to the Wujiang River (2+-Mg2+ apex is obviously higher than that in Youyu River (0.16 mmol·L−1)  , which smol·L−1)  in which and Na+ . In addiion land . The Jin– content is very low, and Cl– in natural conditions mainly comes from atmospheric precipitation and anthropogenic activity  (– concentration in the Youyu stream is relatively low (0.11 mmol·L−1), which only accounts for 1.69% of total anions in the watershed. The Cl–/Na+ and NO3–/Na+ in the river water have an obvious linear relationship . There is no obvious positive correlation between Na+, K+ and NO3–, Cl–, which indicates that the main source of Na+ and K+ in the Youyu stream is not an agricultural source, and the small amount of Na+ and K+ in the river water likely comes from domestic sewage and industrial wastewater  . The Cl–tionship , which istewater .+, K+, and Cl– , which is obviously higher than the Youyu stream, even though they are located in the same geological background and climatic environment. Its Na+, K+, and Cl− are close to urban rivers  and SO42–/Na+ (0.45) ratios  ratios  of munic2+ is produced by rock weathering and is relatively stable in water /Mg2+ ratios /Mg2+: 5.32–15.93), which are obviously higher than in the Jinzhong stream basin /Mg2+: 3.36–4.44), the Han River  is 3.58 times higher than Jinzhong stream (1.29), and higher than other rivers in the world . Moreover, the ratio of SO42−/Mg2+ was higher in the sampling sites seriously polluted by coal mine wastewater /Mg2+ ratio (1.39–2.31), which similar to the ratio characteristics of the urban Godāvari River, which is subject to an obvious influence by anthropogenic activity (pre-rainy season and rainy season), and the Mekong River, Jinsha River, and lower reach of Amazon River, which are subject to the obvious influence of anthropogenic activity input /Mg2+ can be used to characterize the influence of urban sewage on the rivers. The rivers subject to the obvious influence of acidic mine wastewater have relatively high SO42−/Mg2+ ratio, whereas the rivers subject to the obvious influence of urban sewage has a relatively high (Na++K++Cl−)/Mg2+ ratio.Compared to the Youyu stream (0.20–1.25), the Jinzhong stream has the a high   , which i–/Cl–>1)  Fig. 9)3–, Na+, 3– content of the Jinzhong stream is higher than that of the Youyu stream  are relatively low, and the ratios are obviously lower than those at point Z2 and the Han River and Chishui River, which are subject to the significant influence of agricultural activities (3–/Na+: 0.4) studied by 3–/Na+, NO3–/K+, and NO3–/Cl– ratios can be used to differentiate the characteristics of rivers that are influenced by agricultural activities and urban sewage. If the NO3– concentration is high, the rivers affected by agricultural activities have relatively higher NO3–/Na+, NO3–/K+, and NO3–/Cl– ratios compared to urban rivers that are influenced by urban sewage; whereas the rivers with more urban sewage input have relatively low ratios compared to rivers that are subject to the influence of agricultural activities.The NOu stream , the NO3tivities . But it tivities  and Nanmtivities , and whi3− (HQN) and F− (HQF) in the tributaries into Aha Lake were calculated. The HQ values of children in the Youyu stream exhibited the order of HQF (mean: 0.24) > HQN (mean: 0.23) and those of adults exhibited the order of HQF (mean: 0.15) > HQN (mean: 0.14). The HQ values of children in the Jinzhong stream exhibited the order of HQF (mean: 0.58) > HQN (mean: 0.39) and those of adults exhibited the order of HQF (mean: 0.35) > HQN (mean: 0.24). In addition, both HQT and HQN for children and adults are higher in Jinzhong stream than in Youyu stream, indicating which is affected by the increase of F- and NO3− ion content in river water caused by a large amount of domestic sewage produced by the development of urbanization around Jinzhong stream (T) of children was higher than one at J1 in the Jinzhong stream, which shows that children in Jinzhong stream basin are threatened by non-carcinogenic pollutants and Aha Lake is affected by non-carcinogenic pollutants. Previous studies have shown that children may also be potentially harmful when their HQ value is higher than 0.1 , SO42− (73.66%) and HCO3– (23.96%), belongs to Ca-SO4-HCO3 type; the Jinzhong stream has relatively high Ca2+ (61.42%), Mg2+ (24.03%) and HCO3– (59.20%), belongs to Ca-Mg-HCO3 type. The Youyu stream is subject to an obvious influence by mines has relatively high SO42–; the Jinzhong stream is subject to an obvious influence by urban sewage and has relatively high Na+, K+, and Cl–. The NO3– in the Youyu stream is mainly affected by agricultural activities, whereas NO3– in the Jinzhong stream is mainly affected by urban sewage. The results of ion ratio show that the ratio of SO4–2/Mg2+ in Youyu stream (4.61) polluted by coal mine is much higher than that in Jinzhong stream (1.29), and the ratio of (Na++K++Cl–)/Mg2+ in Jinzhong stream (1.81) polluted by urban sewage is higher than Youyu stream (0.64); moreover, the ratios of NO3−/Na+, NO3−/K+, and NO3−/Cl− in the agriculturally polluted Youyu stream were higher than those in the Jinzhong stream. We can identify the impact of human activities on rivers by ion ratios /Mg2+, NO3−/Na+, NO3−/K+, and NO3−/Cl−). The health risk assessment shows the HQT and HQN for children and adults are higher in Jinzhong stream than in Youyu stream, the total HQ value (HQT) of children was higher than one at J1 in the Jinzhong stream, which shows that children in Jinzhong stream basin are threatened by non-carcinogenic pollutants. In the later period, the water environment management of the tributaries of Aha Lake should be strengthened.The present study evaluated major ion compositions, ratios, sources and risk assessment of karst stream under the influence of anthropogenic activities. The main conclusions of the present study were as follows: The Youyu stream has relatively high Ca10.7717/peerj.15368/supp-1Supplemental Information 1+, K+, Mg2+,Ca2+, F−, Cl-, NO3−, SO42−, HCO3− and δ34S of water in Youyu and Jinzhong streams.DO,EC, T, NaClick here for additional data file.


Thus, this technique opens up the possibility of freezing and storing large volumes of thymus tissue for immediate transplantation into patients with DiGeorge syndrome or atypical (Omenn-like) phenotype.DiGeorge syndrome is a disorder caused by a microdeletion on the long arm of chromosome 22. Approximately 1% of patients diagnosed with DiGeorge syndrome may have an absence of a functional thymus, which characterizes the complete form of the syndrome. These patients require urgent treatment to reconstitute T cell immunity. Thymus transplantation is a promising investigational procedure for reconstitution of thymic function in infants with congenital athymia. Here, we demonstrate a possible optimization of the preparation of thymus slices for transplantation through prior depletion of thymocytes and leukocyte cell lineages followed by cryopreservation with cryoprotective media  while preserving tissue architecture. Thymus fragments were stored in liquid nitrogen at -196°C for 30 days or one year. The tissue architecture of the fragments was preserved, including the distinction between medullary thymic epithelial cells (TECs), cortical TECs, and Hassall bodies. Moreover, depleted thymus fragments cryopreserved for one year were recolonized by intrathymic injections of 3×10 DiGeorge syndrome (DGS) is the most common of the microdeletion syndromes. It occurs in approximately 1:4000 births and affects multiple organs, including the heart, the nervous system, and the kidney . The DGSThe thymus comprises thymic epithelial cells (TEC), mesenchymal cells, fibroblasts, dendritic cells, macrophages, and vascular endothelial cells. TECs are separated into cortical (cTECs) and medullary (mTECs) TECs, which constitute two functionally distinct microenvironments for T cell development .Previously, Ross et al.  showed tHere, we demonstrate a possible optimization of the preparation of thymus slices for transplantation while preserving tissue architecture, including the distinction between mTECs, cTECs, and Hassall's corpuscles.Thirty-five thymus tissue samples from infant donors who underwent thymectomy were prepared as described by Hong and Moore . Donor a©, USA). Of these tissue slices, 15 thymus fragments that were not depleted of leukocytes and thymocytes were used as controls (CTRL), 15 thymus fragments were depleted of leukocytes and thymocytes for 21 days (DT), and 30 thymus fragments were depleted of leukocytes and thymocytes for 21 days followed by cryopreservation in liquid nitrogen at −196°C for 30 days (30-day DCT) or one year (one-year DCT) and thawing. All experimental procedures were performed under sterile conditions in a laminar flow hood. Thymic fragments selected for depletion were placed on a polymeric membrane in a culture plate containing Ham's F-12 culture medium supplemented with 1.34 mM deoxyguanosine (Sigma-Aldrich). Thymic fragments were incubated at 37°C at 5% CO2 for 21 days to complete depletion of thymocytes. The medium was changed every two days over the 21-day culture period.Thymus tissue samples were washed with Ham's F-12 culture medium supplemented with 2 mM L-glutamine , 10% fetal bovine serum (FBS) (Gibco), 25 mM HEPES (Gibco), 100 µg/mL streptomycin sulfate (Gibco), 1 µg/mL gentamicin (Gibco), and 100 µg/mL of antifungal agent amphotericin B  , and decapsulated. Sixty 0.5-mm thick tissue slices measuring 15×15 mm were cut using a Stadie-Riggs tissue slicer  were frozen in cryoprotective media containing Ham's F-12 culture supplemented medium with 2 mM L-glutamine and 5% Dextran FP 40, 5% Mes et al. . To alloAfter storage, thymic fragments were thawed in 5 mL of Ham's F-12 culture medium supplemented with 5% FBS for 10 min at 37°C or until ice had disappeared. After defrosting, thymic fragments were washed twice in supplemented medium for 10 min.2 until use.For recolonization, thymic fragments cryopreserved for one year (one-year DCT) were transferred to a six-well culture plate with 3 mL of supplemented medium and placed in an oven at 37°C with 5% COHuman thymus tissue was fixed in formalin and paraffin-embedded. Tissue was sectioned (5 μm) and evaluated by histological analysis using hematoxylin-eosin (H&E) staining and immunohistochemistry analysis using anti-CKPan  antibody as described by Ross et al. .Thymus tissue samples  were mechanically and enzymatically dissociated using collagenase I  and DNase I  in RPMI 1640 (Gibco) and 2% FBS as described by Shichkin et al. . At the Samples were analyzed by flow cytometry . The gating strategy is shown in it) injections containing 3×106 thymocytes per mL (TR). Thymocytes were obtained from fresh human thymus treated by enzymatic digestion (dissociation method) using collagenase I (2 mg/mL) and DNase I (80 μg/mL) in RPMI 1640 supplemented with 2% FBS as described by Shichkin et al. .Additionally, to assess cell death of CD3+ T cells and CD4+, CD8+, double positive (CD4+CD8+) and double negative (CD4−CD8−) subpopulations, cells were labeled with fluorescence-conjugated antibodies ), stained with 7-aminoactinomycin D  and quantified by flow cytometry before recolonization of thymic fragments.For all experiments, the sample size (n) for each experimental group/condition is provided in the figure legends. Data were analyzed using nonparametric two-way analysis of variance (ANOVA) followed by the Newman-Keuls multiple comparison test. Plots and graphs were generated with GraphPad Prism 8.0 software .Typical histologic features of the normal thymus including thymic cortex, composed of basophilic thymocytes and eosinophilic thymic medulla, and Hassall's corpuscles were observed in fresh human HE-stained thymus slices. DT and DCT samples showed thymic epithelium integrity and eosinophilic discoloration due to thymocytes lost from the tissue after the 21-day culture period or dead thymocytes with nuclear karyolysis showing well defined and eosinophilic Hassall's corpuscles .Medullary epithelial and cortical epithelial cells were analyzed with anti-CKPan (pan-cytokeratin). Cytokeratin-positive cells were detected in CTRL, DT, and DCT samples. Immunohistochemistry analysis of these markers revealed preserved thymic architecture in CTRL, DT, and DCT samples .The degree of viability of thymic epithelial cells is essential in long-term thymus tissue cultures. Thus, the occurrence of cell death was assessed by 7-aminoactinomycin D (7-AAD) staining as described by Shichkin et al. . Cell viDepletion of the thymus tissue was evaluated by loss of expression of CD45+ leukocytes and CD3+ thymocytes in CTRL and DT using the standard procedure of enzymatic dissociation of thymic tissue followed by flow cytometry analysis as described by Shichkin et al. . LeukocyThe CD3+ thymocyte cell population reached 59.8% in CTRL but significantly declined to 0.41% after the 21-day tissue depletion period (P≤0.001) . These rTo compare TEC populations between 30-day and 1-year DCT samples, using DT samples as a control, we used a phenotypic panel for TEC (CD326+ HLA-DR+) as shown in Supplementary Figure S1A. Thymus tissues were prepared as described by Shichkin et al. .The percentage of TECs expressing CD326+ HLA-DR+ was 96.9% in 30-day DCT and 94.3% in one-year DCT samples. There was no significant difference in TEC population between 30-day DCT and 1-year DCT compared to DT 97.4% .it injections of 3×106 CD3+ thymocytes per mL in right and left thymic lobes as described in Materials and Methods. To evaluate the recolonization efficiency and thymocyte cell survival, thymus tissue slices were analyzed by immunohistochemical CD3 staining and thymocyte viability was evaluated in recolonized thymus fragments with 7-AAD staining by flow cytometry in thymocytes cells before recolonization used as control and after 10 days of thymocyte culture in one-year DCT (TR). Anti-CD3 antibody staining confirmed the presence of T cells in one-year DCT samples after 10 days of culture (TRCD3+) , suggesting that recolonization had no effect on thymocyte viability. The frequency of CD4 and CD8 thymocytes in one-year DCT samples was evaluated before and after recolonization. The frequency of thymocyte stages before recolonization was CD4+ 26.4%, CD8+ 10.2%, double-positive (DP) thymocytes 59.6%, and double-negative (DN) thymocytes 3.84% , but on day 10 of recolonization their frequency was: CD4+ 29.8%, CD8+ 14.2%, DP 53.0%, and DN 3.05% . These results showed an increase in frequency of CD4+ and CD8+ mature thymocytes/lymphocytes and a decrease of immature cell profile, suggesting that an extensive positive selection of CD4+ or CD8+ mature cells were formed after thymus tissue recolonization.To assess the functional potential of cryopreserved thymus fragments, 15 depleted thymus fragments cryopreserved for one year were recolonized with FOXN1 deficiency  phenotype, and ficiency ,7,12. Huficiency . The supficiency . These c2SO (penetrating compound), and 5% FBS can support the survival of medullary epithelial cells and cortical epithelial cells with functional thymic epithelial cell architecture for thymic maturation. Structural features of the tissue such as cortex and medullary regions as well as Hassall’s corpuscles were identifiable via hematoxylin-eosin staining, whereas cytokeratin immunohistochemistry revealed that tissue architecture of the cryopreserved slices was similar to that of control fresh human thymus slices. A normal lacy pattern was found in both slices that had been frozen and control slices, again suggesting that the freezing process did not disrupt the architecture of the epithelial cells that constitute the thymic stroma.In this study, we demonstrated that previously decellularized thymus scaffolds preserved in cryoprotective media containing 5% Dextran FP40 (non-penetrating compound), 5% MeThe previously depleted thymus tissue also showed a decrease in CD45+ leukocytes lineage and especially in CD3+ thymocytes. CD45 is expressed differentially in T cells at different stages of development and activation , whereasThe cryoprotective solution was effective in maintaining cell viability after freezing for 30 days and one year at levels similar to controls, as evidenced by negative 7AA-D labeling in flow cytometry analysis . 7AA-D iThe thymus provides multiple microenvironments that are essential for the development and repertoire selection of T lymphocytes. Cortical and medullary TECs constitute the major stromal cells that structurally form and functionally characterize the cortex and the medulla, respectively . In the in vitro in thymus fragments cryopreserved for one year (DCT) and recolonized with CD3+ thymocytes. A significant increase in the frequency of CD4+ and CD8+ mature thymocytes/lymphocytes and a decrease in the profile of immature cells were observed in these fragments, suggesting extensive positive selection for CD4+ or CD8+ mature cells after thymus tissue recolonization.The functional potential of cryopreserved thymus fragments was also observed In summary, thymocyte recolonization in one-year DCT samples demonstrated the capacity to support thymocyte differentiation and contribute with significant numbers of precursors to T cell development.Additional studies using nude mice transplanted  with thyin vitro study showed that cryoprotective media designed specifically for freezing thymus slices for long-term storage in liquid nitrogen preserved TEC architecture and function to support T-lymphocyte development. Thus, this technique opens up the possibility of freezing and storing thymus tissue for immediate transplantation into patients with DiGeorge syndrome, FOXN1 deficiency, and atypical (Omenn-like) phenotype.This preliminary pdf].Click here to view [


Arteriovenous malformation (AVM) leads to a direct connection between arterial and venous networks, in which capillary branches are not involved. Pelvic AVM is a benign and rare condition causing severe pain, hematuria, and rectal or vaginal bleeding.A 36-year-old woman presented with five months history of hematuria. Her medical history was unremarkable, and laboratory findings were all within normal ranges. Abdominopelvic computed tomography (CT) scan revealed a vascular mass in the left lateral pelvis that extended to the bladder neck and was suggestive of an AVM. The patient underwent a laparotomy for the resection of AVM. The first angiography revealed an AVM in the left internal iliac artery. The patient underwent embolization with coil and gel foam. The second angiography revealed complete obstruction of the left internal iliac artery due to multiple coils and AVM of the right internal iliac artery (RIIA), embolized with glue and lipiodol. A week later, venography revealed another left iliac vein malformation embolized with foam sclerotherapy. Forty days later, the third angiography revealed another AVM in the right iliac artery, embolized with three vials of polyvinyl alcohol (PVA). Following two months of follow-up, the symptoms did not return.The present study reported a rare case of recurrent pelvic AVM causing painless hematuria in a female patient. The lesion was treated with several angioembolization sessions.Angioembolization is one of the main therapeutic options for AVM. Appropriate material should be precisely chosen for AVM embolization regarding the AVM's location, size, and condition. •The present study describes the treatment of a rare type of recurrent pelvic arteriovenous malformation (AVM) using several angiography-based embolization sessions.•Angiography can be an effective diagnostic modality for pelvic AVM.•Appropriate material should be precisely chosen for AVM embolization regarding the location, size, and condition of the AVM. TypicalThe occurrence of AVMs in the pelvis is quite rare. Moreover, pelvic AVMs are usually benign lesions. However, they may lead to severe pain, hematuria, and abnormal vaginal or rectal hemorrhage depending on the related organs. Also, the AVMs can affect the adjacent structures ,4. It ha2The present case was a 36-year-old woman presenting with gross hematuria for the last 5 months. The hematuria was intermittent and included large clots. The patient did not report any remarkable medical history, underlying diseases, current illness, ongoing medication use, relevant family history, and any surgical history involving the pelvis and urinary system. Moreover, she did not report any cigarette smoking or alcohol consumption. Regarding obstetric history, she reported 4 previous caesarian sections, with the last one being 2 years ago.No abnormal finding was reported in the physical examination. Moreover, laboratory investigations showed normal Complete Blood Count (CBC), bleeding time, and coagulation tests, except for a low hemoglobin level (7.2 g/dl).The patient underwent cystoscopy twice by the urology department, and several blood clots were discharged from the bladder. However, no abnormality was found in the bladder and urethra, except for a small urethral ulcer. Moreover, an abdominopelvic CT scan with IV contrast was ordered, revealing a vascular mass in the left lateral pelvis that extended to the bladder neck and was suggestive of an AVM . Then, tThe decision to perform a post-operative therapeutic angiography was made regarding the retroperitoneal exploration by the vascular surgeon during the laparotomy . For theThe patient underwent a diagnostic angiography again in another center and was diagnosed with complete obstruction of the left internal iliac artery due to multiple coils. Moreover, an AVM was observed on the right side of the pelvis that was related to the RIIA . The proFollowing 40 days after the last angiography, the patient returned with gross hematuria again. Therefore, another angiography  was perf3As a vascular abnormality, AVM is a snarled tangle of arteries and veins connected without capillaries, thereby disturbing organ perfusion. It has been shown that the global incidence and prevalence of AVMs are 1 and 10 cases per 100,000 individuals, respectively . MoreoveIn most cases of acquired AVM, an iatrogenic history is present, such as a previous cesarean section or Dilation and Curettage (D & C). Many patients with these lesions are women of reproductive age with a history of spontaneous abortion, gestational trophoblastic disease, or infection without any family history. AVMs develop during the process of tissue repair, wherein an improper connection forms between an artery and veins. However, the majority of AVM cases are sporadic and are due to genetic mutations, such as the mutations in ENG and ALK-1 in the hereditary hemorrhagic telangiectasia, RASA-1 in capillary malformation/arteriovenous malformation (CM-AVM) syndrome, or PTEN in Bannayan-Riley-Ruvalcaba or Cowden syndromes .The characteristic presentations of pelvic AVMs are abdominal or pelvic discomfort and pain, rectal pain, tenesmus, low back or sciatic pain (rarely), and genitourinary manifestations, such as hematuria, hydronephrosis, hemospermia, impotence, and orchitis. Moreover, it has been shown that large AVMs may lead to shunts causing congestive heart failure . HoweverNumerous noninvasive diagnostic methods can be used for diagnosing pelvic AVMs, including contrast-enhanced CT scan, Magnetic Resonance Imaging (MRI), and color Doppler ultrasound . NonethePelvic AVMs are generally difficult to treat because of their complex vascular nature . SurgicaThere are multiple treatment options for pelvic AVM, including ligation of the afferent artery, excision, and embolization. Nevertheless, it has been shown that the surgical method is typically ineffective and fraught with complications such as hemorrhage injury to adjacent organs, and a possibility of recurrence . The firA systematic approach might be applied to ascertain the most suitable effective agent. Three critical factors can help the physician to choose the proper agent: a) the size of the vessel, b) the need for temporary or permanent occlusion, and c) the viability of the organ after the embolization. It should be mentioned that every vessel that is visible on angiography is classified as large. In general, there is a negative correlation between particle size and the probability of ischemic events. In this manner, coils plus gel foam can be utilized for permanent occlusion of large vessels. For the small vessels, including AVMs, where tissue death is desirable, proper agents are sclerosant liquid  and glues ,24. As t4The present study reported a case of pelvic AVM causing painless hematuria in a female patient. The lesion was treated with several angioembolization sessions. Angioembolization is one of the main therapeutic options for AVM. However, it is of utmost importance to inform the patient about the nature of the disease and the possibility of several operations because the patient's cooperation is a necessity for achieving a definitive cure. Another important factor in treatment success is the use of appropriate material depending on the location, size, and condition of the AVM. Embolization materials can be permanent, such as coils, or temporary, such as gel foam. Temporary materials are mostly considered in cases with a surgical plan following embolization due to absorption of the material and AVM recurrence.The first angiography showed AVM in the left internal iliac artery.Video and Still 1Second angiography revealed an AVM on the right side of the pelvis related to the right internal iliac artery.Video and Still 2Venography showed malformation related to the left iliac vein.Video and Still 3The third angiography revealed another AVM in the right iliac artery the right iliac artery.Video and Still 4The following are the supplementary data related to this article.No sources of funding were declared for this study.Permission from the ethics committee was not required for case reports at the institution where the research was carried out.Written informed consent for publication of the case report has been signed by the patient and is available upon request from the editors.Iraj Nazari: Involved in the conception and design of the study, revising of the article, and final approval of the version to be submitted and also involved in direct management of the patient.Mohmmad Amin Zargar: Involved in the conception and design of the study, drafting and revising of the article, and final approval of the version to be submitted. Also involved in direct management of the patient.Pegah Panahi: Involved in the design of the study, drafting and revising of the article, and final approval of the version to be submitted.Seyed Mohammad Amin Alavi: Involved in the design of the study, drafting and revising of the article, and final approval of the version to be submitted.Guarantor: Mohammad Amin Zargar.The authors have no conflict of interest to declare.


To better understand the biomechanical profile of direct head impacts and the game scenarios in which they occur in Rugby Union, there is a need for an on-field validation of a new instrumented mouthguard (IMG) against the reference standard. This study considers the potential of a combined biomechanical (IMG) and video analysis approach to direct head impact recognition, both of which in isolation have limitations. The aim of this study is to assess the relationship between an instrumented mouthguard and video analysis in detection of direct head impacts in rugby union.Pilot Study - Observational Cohort designThe instrumented mouthguard was worn by ten  professional Rugby Union players during the 2020–21 Gallagher Premiership (UK) season. Game-day video was synchronized with timestamped head acceleration events captured from the instrumented mouthguard. Direct Head Impacts were recorded in a 2 × 2 contingency table to determine sensitivity. Impact characteristics were also collected for all verified head impacts to further the understanding of head biomechanics during the game.There were 2018 contact events that were reviewed using video analysis. Of those 655 were categorized as direct head impacts which also correlated with a head acceleration event captured by the IMG. Sensitivity analysis showed an overall sensitivity of 93.6% and a positive predictive value (PPV of 92.4%). When false positives were excluded due to ball out of play, mouthguard removal or handling after a scoring situation or stoppage, PPV was improved (98.3%). Most verified head impacts occurred in and around the ruck contest (31.2%) followed by impacts to the primary tackler (28.4%).This pilot validation study demonstrates that this IMG provides a highly accurate measurement device that could be used to complement video verification in the recognition of on-field direct head impacts. The frequency and magnitude of direct head impacts derived from specific game scenarios has been described and allows for greater recognition of high-risk situations. Further studies with larger sample sizes and in different populations of Rugby Union players are required to develop our understanding of head impact and enable strategies for injury mitigation. There is growing interest regarding the acute and chronic health effects of concussive and sub-concussive impacts in contact and collision sports , 2. As aA variety of head impact sensors are now commercially available, with most of the initial validation research arising from studies using instrumented helmets in American football , 6. PrevTo advance the understanding of the potential effects of acute and repetitive head impacts, on-field exposure data is critical to identify the characteristics specific to the game of rugby union. Given the widespread accepted use of mouthguards and the increasing ability to offer minimally intrusive instrumentation, IMGs offer the opportunity to improve the understanding of head impacts and their short, medium and long-term effects. As there has been an appreciation of the effect of load on injury in sport for many years with technology now common place in tracking output predominantly using wearables such as GPS devices, the IMG has the potential to progress the field of head impact load measurement, though requires on-field validation. One approach to validation is the use of video verification of observable impacts.Developments in wearable instrumented technologies offer the potential to provide time-sensitive real-time feedback in a sporting environment to inform exposure monitoring, identify potentially injurious impacts and to provide objective quantification to aid performance and coaching . The majFurther, a clear understanding of the face validity is critical when considering whether head impact telemetry systems, such as the IMG, could be used as an adjunct to a clinical assessment. If the IMG were to be considered as an adjunct to clinical decision making, then having high false positive or high false negative results may undermine the confidence of the practitioner. The consequences of a device that over-estimates impacts could result in a loss of compliance or an overly conservative approach to head impact recognition while under reporting could have more significant consequences whereby a player may remain on the field despite a potentially injurious head impact. Similarly, these compliance and clinical recognition issues may also arise where the IMG is considered to be unreliable in the context of it being used to understand the cumulative kinematic loading sustained throughout the course of a given athletic exposure period .Possessing quantitative biomechanical head impact information may add additional support to the clinical assessment  and furtData were prospectively collected from Gallagher Premiership teams (the premier club Rugby Union competition in England) during the 2020–21 season. A total of 10 players  across two clubs participated in the study. The participants included 3 backs and 7 forwards. There were 24 games requiring video verification of contact events with an average of 3 players per game using the IMG. All participants provided written informed consent to participate in the season-long study. The study was approved by the University of Canberra Human Research Ethics Committee (HREC No 20216912).x, y, z) of acceleration with an “event” recorded if a trigger threshold of 10 g of linear acceleration was reached. The trigger is based on the absolute value across one (ACCO) of the linear accelerometer's three axes. The subsequent “event” was recorded for 20 ms before and for 80 ms after the trigger was reached . The players were afforded the ability to have their unique IMG modified to ensure comfort and to optimise fit. The IMG contained three low-power, high-g triaxial accelerometers each sampling at approximately 3,200 Hz with a 200 g maximum. The IMG also contained a 16-bit triaxial gyroscope with a sampling rate of approximately 800 Hz capable of sampling at 3,200 Hz (down-sampled to 800 Hz). The IMG recorded linear and rotational acceleration, impact location, and duration. The time history incorporated three axes . A contact event was defined as any contact from another player or the ground. Further, the contact event was categorised based on whether it was a direct head impact or not. A direct head impact was defined as any contact above the shoulder with any connection with the head/neck made to the player wearing an IMG by another player or the playing surface.The full game video analysis for all games during the season was conducted at the conclusion of the season by the lead author using Catapult Vision . The video review process consisted of adjusting angles, pausing, replaying, and using slow motion as required. A world-clock was time stamped at the beginning of each video, so that video events could synchronised to time-stamped sensor events. Game footage, sampling at 25 frames per second with a resolution of 1,280 × 720 pixels, was initially analysed using three camera angles  independently of the IMG data, before these two datasets were compared with one another. Video of each game was reviewed and played back at an appropriate speed to verify whether the player was involved in a contact event . The verDuring a secondary analysis, VHIs were also characterised using standardised descriptors and definitions where possible . These iThe lead author, trained in video analysis, completed the verification process. To enable a 2 × 2 contingency table, if the video of a direct head impact matched a head acceleration event captured on the IMG a “true positive” was recorded. Direct head impacts observed on video that did not match a captured head acceleration event on the IMG were identified as false negatives. A contact event with no direct head impact observed on video and no HAE captured was classified as a true negative. Following the initial round of screening for TPs, TNs, and FNs, captured HAEs that did not match with a contact event or direct head impact were identified as false positives.In certain game circumstances the video of the player in question was obstructed by other players or officials. To fully explore the data, we evaluated a “best case” scenario (assuming the “Unclear” are direct head impacts), a “worst case” scenario (assuming the “Unclear” are not direct head impacts), and the “visually verified scenario” . This approach was undertaken to ensure full consideration was given to all events, including those that were “unclear.” All the unclear events were included in separate accuracy analysis, first, entering them all as true positive events, and second, entering them all as false positive events. As a definitive decision against the video verification standard could not be made, unclear events were not included in analysis of game characteristics.To assess inter-rater reliability two reviewers independently completed a verification process on one randomly selected game. Inter-rater reliability was determined using Cohen's K coefficient based on agreement under a two-way mixed effects model comparing the recorded time points separately. The agreement between the two raters when identifying contact events was 84.6% and for direct head impacts was 76.1%. They were blinded to the IMG capture. To assess the intra-rater reliability, the lead author reviewed the same game two weeks apart. The intra-rater reliability was also assessed to ensure consistency across time – for direct head impacts the IRR was 86%.The initial analysis was then carried out on all contact events observed on video regardless of whether a head impact occurred. The sensitivity (TP/(TP + FN) and positive predictive value (PPV = TP/(TP + FP) of the IMG were calculated.p < 0.001 respectively). Based on this outcome and a visual inspection of the resultant histograms IQR, median and non-parametric tests were used to describe the data. For each separate impact, peak linear acceleration (PLA) (g's) and peak rotational acceleration (PRA) (rad/s2) were defined as the maximum values of the resultant time-series data for linear and rotational accelerations respectively . The impact variables related to the characteristics of the impact were assessed for normality to inform the appropriate analyses using a Shapiro-Wilk test. Linear and rotational acceleration were non-normally distributed (W = .48 and W = .46 ectively . PLA andectively .2. The impact magnitudes (PLA and PRA) of unclear results were not statistically different (p = 1 and p = 0.051 respectively)) from verified head-impacts. When using the “visually verified” standard, as shown in the 2 × 2 contingency analysis (n = 43/54), as is recommended .During the video verification process, 2018 contact events were observed in 24 games (61 athlete exposures) across the season, equating to approximately 3,660 playing minutes. There were 257 contact events that were deemed to be unclear and mainly the result of a maul (84.1%) or the player of interest being obscured during a ruck (5.5%). Of those contact events, 95 (37%) had IMG data recorded with a median of 21.4 g and 1,465 rad/sanalysis , an over2  for PRA which were statistically different (p < 0.001) from verified head impacts. There were 1,061 contact events where a direct head impact was not identified nor was a head acceleration event captured from the IMG. These occurred mostly through players being involved in scrums (29.0%), rucks . There were 45 potential false negatives that occurred wherein a direct head impact was identified though no head acceleration event was captured.A total of 54 false positive impacts were observed, 85% of those occurred when the ball was not in play, for example a player removing their mouthguard. Those remaining had full visualisation though with no identified impact. The impact magnitudes for the FP impacts were 62.6 g (24.6–106.7) for PLA and 6,168 rad/s2) were recorded in the 1st half of games and 327  in the second half (p > 0.05). Backs  sustained higher PRAs than forwards , which was statistically significant (p = 0.03) though the PLA between backs (20.3 g) and forwards (21.7 g) was not statistically significant (p > 0.05). Unexpected impacts  had higher PLA than expected impacts  (p = .018). The majority (41.8%) of unexpected contacts were sustained during ruck involvements.Of the 655 VHIs, 328  followed by impacts to the primary tackler (28.4%) and primary ball carrier (13.8%), the remaining (26.6%) were either to the secondary tackler, secondary ball carrier or during scrum and mauls. The highest peak linear accelerations were recorded during maul impacts whereas the highest rotational accelerations were during a secondary tackle .2 1,494–3,100)]. A similar speed related pattern was also seen with moderate arrival speed to a ruck generating higher PLAs and PRAs . There was a difference in PLAs and PRAs if the intent of the primary tackler was that of a driving or “active tackle”  whereas a “passive” tackle was consistently lower .Tackler speed, and speed differential into contact are known risk factors for head impact –27. The This study is one of the first to examine the relationship between video verified direct head impacts and head acceleration events (HAE) captured concurrently by an IMG in Professional Rugby Union Players. A reliable kinematic measurement tool with appropriately set algorithms, should minimise noise (non-impacts) and optimize the quantification of incidence, frequency and severity of “true” head impacts. Combined with a strong internal validation (LCCC – .997) the IMG demonstrated a sensitivity of 93.6% and a PPV of 98.3% during “active minutes” which is indicative of an accurate wearable sensor and comparable to previously analysed instrumented head impact sensors .Previous studies that have relied on head impact sensors in isolation to confirm head impacts have been shown to have high occurrences of false positives resulting in an overestimation of head impact exposure due to the sensitivity and inaccuracies of the sensor used . ConversThis IMG did not have a proximity sensor to filter out recordings where the IMG was not properly placed on the upper dentition. Featuring proximity sensors, such as an infrared sensor to determine contact of the IMG with the teeth, has reportedly been beneficial in reducing the proportion of false-positives . DespiteThe approach was to match IMG captures to video verified events such that head acceleration events have a high likelihood of being matched with direct head impacts. Conversely non-impacts would have a low likelihood of including an IMG capture. In this study, false negatives were considered a direct head impact that did not correspond to a head acceleration event. Although used in the 2 × 2 contingency table to calculate specificity limitations remain due to the challenges that exist in objectively estimating impact magnitude from video review and the set trigger threshold of the IMG. False positives were collected as a secondary analysis. Given the relatively low number, the authors were able to review and consider whether FPs were generated during “ball out of play” time as is usually the case due to players removing their IMGs at a break in play or to talk . ConsistSeveral attempts to define true negatives using machine learning classification have been made , 31 as hPlayers in the current study were exposed to an average of 10 verified impacts per match. A comparison of athlete exposure is relatively problematic due to the relatively new technology, the lack of on-field validation studies using IMGs and the lack of reporting using a standard definition of “athlete exposure”. A comparison to behind ear patches demonstrated an equivalent 10 impacts per player per match  across f2 compared to 1,719 rad/s2 in the current study. This difference may be explained by either the potential of skin patches to over-predict linear and rotational acceleration (The median PLA of 21.5 g 15.6–34.1) was similar to those previously reported kinematic studies completed in Rugby Union . Kieffer–34.1 wasleration  or the lleration .The frequency and magnitude of verified head impacts for various game characteristics have also been reported. The finding of the highest incidence of exposure of verified head impacts occurring during the ruck, an event unique to Rugby Union, has not been previously reported. The unexpected nature of many of these VHIs would seem logical given the players usual body position in the ruck dictates the hips be bent and the head often down contesting or protecting the ball in such a way where anticipation of an impending impact is less likely. Such unexpected impacts at the ruck were shown to be significantly greater than expected events. Previous studies have cited high incidence of concussion during a ruck as well as impact readiness  as assocThe findings related to the tackle contest provide additional quantitative context to several earlier published findings –27. AlthThis pilot validation study contributes important knowledge to head impact recognition strategies expanding into various levels of contact sport. The IMG evaluated in this study represents an improvement over existing instrumentation including helmet based or skin mounted devices. The complimentary approach of independent video verification and comparison to the data captured from an IMG enabled this study to determine how well this new IMG performed during real-world conditions. The accuracy of this IMG means that it could be considered to support the video verification process which lacks the ability to capture the magnitude of impacts. It could also be used as an alarm system where video verification is not readily available or for those discrete impacts obscured from view, which due to the congested nature and game characteristics of rugby union, has been shown to be not insignificant. In the future, advancing technology may enable the IMG to be used in real-time to inform pitch side-clinicians of a potentially injurious head impact by providing reliable data in a timely manner that complements the standard medical assessment and video verification process. This effective recognition, using the best available information and technology is critical to support the removal of a player from training or competition for further assessment.In this study, active minutes or ball in play time was assessed manually to provide two accounts of FP recordings. To limit time waste and the potential for error through data handling, proximity sensing should be added to the IMG to determine whether the IMG is mounted on the teeth. This would enable rejection of data when a player removes the IMG during a break in play or to make a particular tactical call.2  and qualitative  approach for furthering the understanding of game specific head biomechanics in Rugby Union. Further, video verification in isolation lacks the ability to comment on the magnitude of impact and has the potential to overlook discrete, multiple or obscured impacts. Conversely, biomechanical instrumented mouthguard approaches alone without video verification may be prone to false-positive readings and are limited to their predetermined filtering algorithms and trigger thresholds . Given t•Understanding the accuracy of any technology is critical to an assessment of clinical utility. This instrumented mouthguard is a sensitive tool for identifying direct head impacts and could be used at the elite level of Rugby Union in conjunction with a video verification process to improve the clinician's ability to recognise head impacts;•When evaluating validation studies, consideration should be given to how the inclusion or exclusion of head impacts that cannot be discerned through the reference standard (video review) are considered.•The obscured nature of some contact events and the inability of video to consider the magnitude of an impact should ensure that further feasibility studies are completed into the use of IMGs.•At levels of community sport where video verification is not readily available, the use of IMGs should be explored though caution exercises when interpreting the IMG data given the potential issues with false negative results.