1.Mechanism of Modified Erxian Decoction Regulating Perimenopausal Syndrome via SIRT1/Kisspeptin/GnRH Signaling Pathway
Ruiyu HUANG ; Fang LEI ; Wuchaonan LIU ; Jingjing YANG ; Qianru ZENG ; Shengping LUO ; Yanling CHEN ; Mengge ZHANG ; Fanshun SHEN ; Yihui DENG ; Dingxiang LI
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(22):51-62
		                        		
		                        			
		                        			ObjectiveTo explore the regulation of hypothalamic-pituitary-gonadal (HPG) axis by modified Erxian decoction in rats with perimenopausal syndrome (PMS) and to further analyze the expression of proteins related to the silent information regulator 1 (SIRT1)/hypothalamic kisspeptin (Kisspeptin)/gonadotropin-releasing hormone (GnRH) signaling pathway in the arcuate nucleus region (ARC) of the hypothalamus, so as to reveal the potential target of action and molecular biological mechanism of modified Erxian decoction for the treatment of perimenopausal syndrome. MethodsAn animal model was established via the incomplete castration method, with successful modeling confirmed by the exfoliated cervical cell smear method. The 48 rats were divided into six groups based on the randomization principle after successful modeling, including a sham operation group, a model group, an estradiol valerate group (0.09 mg∙kg-1∙d-1), high-, medium-, and low-dose modified Erxian decoction groups (7.614, 3.807,1.903 5 g∙kg-1∙d-1), with 8 rats in each group. The estradiol valerate group and the high-, medium- and low-dose modified Erxian decoction groups were continuously administered by gavage for 28 days, and the indicators were detected 24 hours after the last administration. Body weights and uterine indices were measured. The pathological changes of the uterus were observed by hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and gonadotropin-releasing hormone (GnRH). Real-time quantitative polymerase chain reaction (Real-time PCR) and Western blot were used to determine the expression levels of SIRT1, Kisspeptin, kisspeptin receptor (GPR54), and GnRH in the ARC region of the hypothalamus and gonadotropin-releasing hormone receptor (GnRH-R) in pituitary. ResultsCompared with the sham operation group, rats in the model group had a significantly increased body weight (P0.01), reduced wet weight and index of uterus (P0.01), endometrial thinning or atrophy, glandular atrophy, and a decreasing number of glands. Additionally, serum levels of E2 and the expression of SIRT1 in the ARC region of the hypothalamus significantly decreased (P0.01). Serum levels of FSH, LH, and GnRH, the expression of Kisspeptin, GPR54, and GnRH in the ARC region of the hypothalamus, and GnRH-R in pituitary significantly increased (P0.01). Compared with the model group, the estradiol valerate group and the high-, medium-dose modified Erxian decoction groups had significantly reduced body weight, serum levels of FSH, LH, and GnRH, and expression of Kisspeptin, GPR54, and GnRH in the ARC region of the hypothalamus and GnRH-R in pituitary (P0.05, P0.01) and significantly increased wet weight and index of uterus, serum level of E2, and expression of SIRT1 in the ARC region of the hypothalamus (P0.05, P0.01). In addition, they showed thickened endometrium, increased number of endometrial glands, and improved glandular atrophy. ConclusionModified Erxian decoction regulates the function of the HPG axis through multi-targets, and its mechanism of action may be related to the up-regulation of the expression of SIRT1 in the ARC region of the hypothalamus, the inhibition of the over-activation of the Kisspeptin/GnRH signaling pathway, the regulation of the expression of GnRH-R in the pituitary, the restoration of secretion balance of gonadotropins, and the elevation of the estrogen level. This study provides an experimental basis for the interpretation of the scientific connotation of modified Erxian decoction in the treatment of perimenopausal syndrome and a theoretical reference for the development of a novel therapeutic strategy based on the SIRT1/Kisspeptin/GnRH pathway. 
		                        		
		                        		
		                        		
		                        	
2.Key biomarkers for the diagnosis of intervertebral disc degeneration associated with oxidative stress:identification based on bioinformatics and machine learning
Yao LAN ; Liuyang CHEN ; Wenhui SONG
Chinese Journal of Tissue Engineering Research 2024;28(35):5591-5597
		                        		
		                        			
		                        			BACKGROUND:Oxidative stress is closely associated with the occurrence and progression of intervertebral disc degeneration,but its underlying mechanisms and effective treatment methods remain unclear. OBJECTIVE:To identify key genes associated with intervertebral disc degeneration accompanied by oxidative stress based on bioinformatics and three machine learning algorithms,as well as to conduct an immune infiltration analysis,followed by experimental validation. METHODS:Gene expression profiles related to intervertebral disc degeneration were obtained from the GEO database and oxidative stress-related genes obtained from the GeneCards database.Differential analysis and weighted gene co-expression networks analysis were performed on the intervertebral disc degeneration dataset.The intersection of the two analyses and the intersection with the oxidative stress-related genes were taken to obtain candidate hub genes.Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses on the candidate hub genes were performed.Machine learning algorithms(LASSO regression,SVM-RFE,and random forest)were used to select the optimal feature genes and perform the receiver operator characteristic curve validation.Simultaneously,immune infiltration analysis was conducted.Nucleus pulposus samples from patients with cervical spondylosis who were treated at the Second Hospital of Shanxi Medical University from July to November 2023 were enrolled as the intervertebral disc degeneration group and nucleus pulposus samples from patients with cervical spinal cord injury as the control group.The relative expression of feature genes in the degenerated intervertebral disc was validated using qPCR method. RESULTS AND CONCLUSION:After differential gene analysis,424 differentially expressed genes were obtained.Weighted gene co-expression networks analysis yielded 5 087 genes,and 1 399 oxidative stress genes were identified,leading to the identification of 23 candidate hub genes.Gene ontology analysis revealed that these candidate hub genes are primarily involved in bacterial defense response,molecular response to bacteria,and other biological processes.In terms of cellular component,they are associated with secretion granule lumen and cytoplasmic vesicle lumen,among others.As for molecular function,they are related to endopeptidase activity and compound binding,including sulfur compounds.Kyoto Encyclopedia of Genes and Genomes analysis demonstrated that these candidate hub genes are associated with neutrophil extracellular trap formation and the renin-angiotensin system pathway,among other signaling pathways.By applying three machine learning algorithms and conducting the receiver operator characteristic curve validation,two key genes,HSPA6 and PKD1,were determined.Immune infiltration analysis revealed a strong correlation between HSPA6 and activated dendritic cells(r=0.88,P<0.001)as well as activated CD4+ T cells(r=-0.72,P<0.01).Similarly,PKD1 showed close associations with effector memory CD8+ T cells(r=0.55,P<0.05)and activated dendritic cells(r=-0.56,P<0.05).qPCR experimental results indicated that the expression level of HSPA6 was lower in the intervertebral disc degeneration group compared with the control group(P<0.000 1),while the expression level of PKD1 was higher in the intervertebral disc degeneration group(P<0.000 1).These findings suggest that HSPA6 and PKD1 can serve as biomarkers for intervertebral disc degeneration accompanied by oxidative stress.Interventions targeting HSPA6 and PKD1 may hold promise for improving intervertebral disc degeneration.
		                        		
		                        		
		                        		
		                        	
3.Analysis of child mortality under 5 years old in Liuyang City from 2013 to 2020.
Huihuang ZHOU ; Manman ZHANG ; Shaoru CHEN ; Ying YANG ; Jianwu WANG
Journal of Central South University(Medical Sciences) 2022;47(3):352-357
		                        		
		                        			OBJECTIVES:
		                        			To provide reference basis for reducing the mortality for children under 5 years old and promote the healthy development, the mortality for children under 5 years old and the main causes for death in Liuyang City from 2013 to 2020 are analyzed.
		                        		
		                        			METHODS:
		                        			The data of 725 cases of death for children under 5 years old in Liuyang City from 2013 to 2020 were collected.The causes and difference of death among the children were analyzed retrospectively by descriptive statistic methods.
		                        		
		                        			RESULTS:
		                        			There were a total of 144 516 live births in Liuyang City from 2013 to 2020. The mortality for children under 5 years old was 5.01‰, for infants was 3.39‰, and for newborns was 1.63‰. The male child mortality was 5.28‰, and the female child mortality rate was 4.72‰, with significant difference (P>0.05). The mortality for children under 5 years old was seasonal fluctuation, without significant difference among seasons (P>0.05). For the past 5 years, the top 3 causes for death among children under 5 years old were preterm birth and low birth weight, congenital heart disease, and pneumonia. Before death, 341 cases (47.04%) were treated in provincial hospitals, 198 cases (27.31%) in county-level hospitals, 56 cases (7.72%) in village-level hospitals, and 130 cases (17.93%) were not treated.
		                        		
		                        			CONCLUSIONS
		                        			The mortality for children under 5 years old in Liuyang City is gradually reduced in the past 5 years. The main causes for death are premature birth and low birth weight, congenital heart disease and pneumonia. We should develop healthy education, improve the rate of prenatal diagnosis, promote the construction of obstetrics and paediatrics, and fundamentally reduce the mortality for children under 5 years old.
		                        		
		                        		
		                        		
		                        			Cause of Death
		                        			;
		                        		
		                        			Child
		                        			;
		                        		
		                        			Child Mortality
		                        			;
		                        		
		                        			Child, Preschool
		                        			;
		                        		
		                        			Female
		                        			;
		                        		
		                        			Heart Defects, Congenital
		                        			;
		                        		
		                        			Humans
		                        			;
		                        		
		                        			Infant
		                        			;
		                        		
		                        			Infant Mortality
		                        			;
		                        		
		                        			Infant, Newborn
		                        			;
		                        		
		                        			Male
		                        			;
		                        		
		                        			Pneumonia/epidemiology*
		                        			;
		                        		
		                        			Pregnancy
		                        			;
		                        		
		                        			Premature Birth
		                        			;
		                        		
		                        			Retrospective Studies
		                        			
		                        		
		                        	
4.Establish and application of scoring scale for trial of labor after cesarean section
Dongmei ZHANG ; Yali WANG ; Wenzhi LIU ; Liuyang XU ; Shumei CHEN
Chinese Journal of Obstetrics and Gynecology 2022;57(5):339-345
		                        		
		                        			
		                        			Objective:To establish a scoring scale for trial of labor after cesarean section (TOLAC), to explore the evaluation ability of this scoring scale for vaginal delivery after cesarean section (VBAC), and to improve the success rate of TOLAC.Methods:The delivery information of 661 TOLAC pregnant women admitted to Zhengzhou Central Hospital Affiliated to Zhengzhou University from 2014 to 2017 was retrospectively analyzed, and the TOLAC scoring scale was established by referring to relevant literatures. A prospective cohort study of pregnant women with TOLAC from January 2018 to December 2019 in Zhengzhou Central Hospital was conducted, including 440 pregnant women who were excluded from contraindications in trial labor. According to TOLAC scoring scale, pregnant women were divided into 3 groups, 0-6 group (94 cases), 7-9 group (234 cases) and 10-15 group (112 cases). The success rate of trial labor, failure reasons and incidence of maternal and neonatal complications were compared among the three groups.Results:(1) The overall success rate of TOLAC in 440 pregnant women was 75.0% (330/440). The success rates of 0-6, 7-9 and 10-15 groups were 53.2% (50/94), 76.9% (180/234) and 89.3% (100/112), respectively. The success rate of 10-15 group were significantly higher than those of 0-6 and 7-9 groups (all P<0.05). (2) Among the causes of trial labor failure, there were statistically significant differences between the three groups in terms of threatened uterine rupture and maternal abandonment (all P<0.05). Pairings showed that the incidences of threatened uterine rupture and maternal abandonment in 0-6 group was lower than those in 7-9 and 10-15 groups, and the differences were statistically significant (all P<0.05). (3) Maternal and neonatal complications mainly included postpartum hemorrhage and neonatal asphyxia, but there were no significant difference in the incidence of TOLAC success or failure among the three groups (all P>0.05). There was no uterine rupture in all groups. (4) The main factors affecting TOLAC score of pregnant women in the three groups included natural labor, estimated weight of the fetus at this time, Bishop score of the cervix at admission and gestational age, and the scores of the above indexes in 10-15 group were significantly higher than those in 0-6 group and 7-9 group (all P<0.05). Conclusions:TOLAC scoring scale has more accurate evaluation ability for VBAC, which could improve the success rate of TOLAC and maternal and child safety. The score of 0-6 is not recommended for vaginal trial labor, the score of 7-9 is recommended for vaginal trial labor, and the score of 10-15 is strongly recommended for vaginal trial labor.
		                        		
		                        		
		                        		
		                        	
5.Protective Effect of Total Ginsenoside Ginseng Root on Learning and Memory Impairment and Anxiety in Rats Induced by Hindlimb Suspension
Yu BAO ; Ying CHEN ; Gui-rong ZENG ; Zi-yu YANG ; Rui-le PAN ; Zhe SHI ; Qin HU ; Jing-wei LYU ; Cong LU ; Yang HE ; Ning JIANG ; Bo PENG ; Xin-min LIU ; Lian-kui WEN
Chinese Journal of Experimental Traditional Medical Formulae 2021;27(7):49-56
		                        		
		                        			
		                        			Objective:To investigate the effect of total ginsenoside ginseng root on the learning and memory impairment and anxiety of hindlimb suspension rats by detecting the performance of rats in the water maze, elevated plus maze, and the expression of hypothalamic-pituitary-adrenal (HPA) axis, inflammatory factors and tryptophan pathway related factors through the intervention of ginsenosides in hindlimb suspension rats. Method:The Wistar male rats were divided into normal group, hindlimb suspension model group, Huperzine A group (0.1 mg·kg-1), and total ginsenoside ginseng root low and high dose groups (100, 200 mg·kg-1), with 8 rats in each group. Except for the normal group, the rats in the other groups maintained a -30° hindlimb suspension state for 24 h. The normal group and the model group received intragastric administration of 10 mL·kg-1 pure water . After 28 days of continuous administration, the water maze and elevated plus maze behavioral tests were performed. After the tests, blood was taken from the abdominal aorta, and the rat brain cortex was peeled off on ice, quenched with liquid nitrogen, and stored at -80 ℃ for later use. LC-MS/MS was used to detect neurotransmitter levels of dopamine, acetylcholine, glutamate, 
		                        		
		                        	
7.Probe into the guiding significance of thyroid biopsy and eluent detection for neck lymph node dissection of thyroid neoplasms
Jixin CHAI ; Liuyang ZHANG ; Bo LI ; Junxing XUE ; Yong CHEN ; Yazhou AO
International Journal of Surgery 2021;48(8):538-542
		                        		
		                        			
		                        			Objective:To explore the advantage of thyroid biopsy and evaluate detection in the application of thyroid cancer cervical lymph node dissection operation guidance, and provide evidence-based basis for guiding PTC patients whether to receive lateral dissection.Methods:The data of 258 patients with thyroid papillary carcinoma admitted to Department of Thyroid Surgery, Affiliated Hospital of Chengde Medical College from April 2018 to December 2019 were retrospectively analyzed. All patients were admitted to the hospital for ultrasonic examination of cervical lymph nodes. According to the examination results, thyroid biopsy and puncture eluent were performed for patients with suspicious signs of metastasis. Patients with positive test results and with metastatic signs in initial ultrasonic examination received lymph node dissection in the cervical region. All patients underwent pathological examination postoperatively to diagnose the lymph node metastasis, and to evaluate the sensitivity, specificity and accuracy of the detection of the patients with lateral clearance.The measurement data was expressed as mean±standard deviation ( Mean± SD), and the count data was expressed as n(%). The software of SPSS21.0 was used to conduct statistical analysis. Results:All 258 patients were successfully completed the diagnosis of various diagnostic methods. Combined with the postoperative pathological diagnosis results, the accuracy rate of ultrasonic diagnosis of lymph node metastasis was 74.42%, the accuracy rate of FNAC diagnosis was 82.95%, the accuracy rate of FNAC-Tg diagnosis was 87.98%, and the accuracy rate of FNAC-Tg diagnosis was 94.96%. The diagnostic sensitivity of ultrasound, FNAC, FNAC-Tg and FNAC-Tg was 73.60%, 81.72%, 91.01% and 95.83%, respectively, and the diagnostic specificity was 76.25%, 86.11%, 79.71% and 88.41%, respectively. In every four months for a time span since April 2018, the average length of hospital stay for patients with each span was (9.17±1.30), (8.39±1.21), (7.94±1.03), (7.46±0.94), (7.33±0.82) d, their neck area incidence of lymph node metastasis were 17.6%, 21.3%, 15.7%, 12.9%, 11.8%, side clear surgical patients accounted for 42.3%, 37.5%, 30.9%, 26.6%, 19.4%. The incidence of lymph node metastasis was 86.8%, 79.4%, 84.5%, 93.2% and 98.1%, respectively.Conclusion:FNAC-Tg method is used in the diagnosis of thyroid papillary carcinoma patients with high sensitivity and specificity of lymph node metastasis, which has certain value in guiding patients whether to perform lymph node dissection.
		                        		
		                        		
		                        		
		                        	
8.Cerebrospinal fluid otorrhea secondary to congenital inner ear dysplasia: diagnosis and management of 18 cases.
Bing WANG ; Wen-Jia DAI ; Xiao-Ting CHENG ; Wen-Yi LIUYANG ; Ya-Sheng YUAN ; Chun-Fu DAI ; Yi-Lai SHU ; Bing CHEN
Journal of Zhejiang University. Science. B 2019;20(2):156-163
		                        		
		                        			OBJECTIVE:
		                        			To describe the characteristics of the clinical presentation, diagnosis, surgical methods, and outcomes of patients with otogenic cerebrospinal fluid (CSF) leakage secondary to congenital inner ear dysplasia.
		                        		
		                        			METHODS:
		                        			A retrospective review was performed of 18 patients with otogenic CSF leakage secondary to inner ear dysplasia who underwent surgery in our group from 2007 to 2017 and had a follow-up of at least 4 months. The average length of follow-up was three years. The characteristics of the clinical presentations of all patients, such as self-reported symptoms, radiographic findings, surgical approaches and methods of repair, position of the leakage during surgery, and postoperative course, including the success rate of surgery, are presented.
		                        		
		                        			RESULTS:
		                        			The patients presented mostly with typical symptoms of meningitis, severe hearing impairment, and CSF otorrhea or rhinorrhea. All 18 patients had at least one previous episode of meningitis accompanied by a severe hearing impairment. The preoperative audiograms of 17 patients showed profound sensorineural hearing loss, and one patient had conductive hearing loss. Twelve patients presented with an initial onset of otorrhea, and two had accompanying rhinorrhea. Six patients complained of rhinorrhea, two of whom were misdiagnosed with CSF rhinorrhea and underwent transnasal endoscopy at another hospital. High-resolution computed tomography (HRCT) images can reveal developments in the inner ear, such as expansion of a vestibular cyst, unclear structure of the semicircular canal or cochlea, or signs of effusion in the middle ear or mastoid, which strongly suggest the possibility of CSF otorrhea. The children in the study suffered more severe dysplasia than adults. All 18 patients had CSF leakage identified during surgery. The most common defect sites were in the stapes footplates (55.6%), and 38.9% of patients had a leak around the oval window. One patient had a return of CSF otorrhea during the postoperative period, which did not re-occur following a second repair.
		                        		
		                        			CONCLUSIONS
		                        			CSF otorrhea due to congenital inner ear dysplasia is more severe in children than in adults. The most common symptoms were meningitis, hearing impairment, and CSF otorrhea or rhinorrhea. HRCT has high diagnostic accuracy for this disease. The most common fistula site was around the oval window, including the stapes footplates and the annular ligament.
		                        		
		                        		
		                        		
		                        			Adolescent
		                        			;
		                        		
		                        			Adult
		                        			;
		                        		
		                        			Cerebrospinal Fluid Otorrhea/therapy*
		                        			;
		                        		
		                        			Child
		                        			;
		                        		
		                        			Child, Preschool
		                        			;
		                        		
		                        			Ear, Inner/abnormalities*
		                        			;
		                        		
		                        			Humans
		                        			;
		                        		
		                        			Infant
		                        			;
		                        		
		                        			Middle Aged
		                        			;
		                        		
		                        			Retrospective Studies
		                        			;
		                        		
		                        			Young Adult
		                        			
		                        		
		                        	
9.Application of PeriCam PSI blood flow imaging perfusion system in rat brain ischemia reperfusion injury models.
Chao WU ; Guoxian CHEN ; Liuyang ZHAO ; Shu LI ; Yun HONG
Journal of Zhejiang University. Medical sciences 2018;47(1):51-56
		                        		
		                        			OBJECTIVE:
		                        			: To study the feasibility and effect of PeriCam PSI system guiding the establishment of ischemia/reperfusion injury model in rats.
		                        		
		                        			METHODS:
		                        			: A total of 70 adult male Sprague-Dawley rats were divided into the control group(=6), PSI monitoring group(=34) and traditional operation group(=30). Ischemia reperfusion model was established with reference to improve Zea-Longa line plug method. After the model established, the blood flow to the brain of control group, PSI monitoring group (ischemic 2 h, 24 h reperfusion) were observed and recorded respectively with PSI. The rats were then executed after 24 h, and the 2,3,5-triphenyltetrazolium chloride (TTC) staining and HE staining were used to observe the brain tissue.
		                        		
		                        			RESULTS:
		                        			: The survival rate and modeling success rate of PSI monitoring group were higher than those of the traditional operation group(all <0.05). The blood perfusion in the brain and the distribution of blood vessels were clearly observed in the control group, and the data were normal. In 2 h ischemic group, the arterial flow was interrupted in the right cerebral artery, and the blood flow in the middle arterial blood supply was significantly decreased than that in the control group(<0.05). After the recovery of 24 h, the artery in the right side of the brain was restored to blood flow, but the blood flow in the partial supply area decreased, unable to recover to normal level. The TTC staining results indicated that there were obvious infarcts in the right brain tissue of PSI monitoring group,and the infarct area was more stable than that of the traditional operation group. The results of HE staining showed that the structure of brain tissue in the control group was normal, and the morphological rules of nerve cells were not change. While in brain tissue from PSI monitoring group, cortex and ischemia half dark stripe, nerve cell degeneration, necrosis and glial fiber disintegration, liquefaction, and light color, screen mesh in ischemic central area were observed.
		                        		
		                        			CONCLUSIONS
		                        			: PSI system can guide ischemia reperfusion model building and improve the success rate of the model.
		                        		
		                        		
		                        		
		                        			Animals
		                        			;
		                        		
		                        			Brain Ischemia
		                        			;
		                        		
		                        			diagnostic imaging
		                        			;
		                        		
		                        			surgery
		                        			;
		                        		
		                        			Disease Models, Animal
		                        			;
		                        		
		                        			Hemodynamics
		                        			;
		                        		
		                        			Male
		                        			;
		                        		
		                        			Rats
		                        			;
		                        		
		                        			Rats, Sprague-Dawley
		                        			;
		                        		
		                        			Reperfusion
		                        			;
		                        		
		                        			instrumentation
		                        			;
		                        		
		                        			Reperfusion Injury
		                        			;
		                        		
		                        			diagnostic imaging
		                        			
		                        		
		                        	
10.Effects of different positions on cerebral blood flow in patients undergoing laparoscopic surgery
Shuo WANG ; Liuyang YU ; Kai CHEN ; Guangzhi SHI ; Ruquan HAN
Chinese Journal of Anesthesiology 2017;37(4):420-422
		                        		
		                        			
		                        			Objective To evaluate the effects of different positions on cerebral blood flow in patients undergoing laparoscopic surgery.Methods Thirty patients of both sexes,aged 23-64 yr,of American Society of Anesthesiologists physical status Ⅰ or Ⅱ,scheduled for elective laparoscopic surgery,were included.Patients' position was changed using a random number table after induction of anesthesia.Patients were placed in the supine position and tilted 15° head-up or 15° head-down,and the parameters of the middle cerebral artery were monitored using the transcranial Doppler ultrasound.After admission to the operating room (baseline),after induction of anesthesia and before pneumoperitoneum,in the supine position,at head-up tilt and at head-down tilt,the mean blood flow velocity,pulsatility index,resistance index,mean arterial blood pressure (MAP) and heart rate were recorded.Results Compared with the baseline in the supine position,the MAP and bilateral mean blood flow velocity were significantly decreased at head-up tilt,and the MAP and bilateral pulsatility and resistance indices were significantly increased at head-down tilt (P<0.05).Conclusion During laparoscopic surgery,head-up tilt can lead to a decrease in cerebral blood flow,and head-down tilt exerts no effect on cerebral blood flow.
		                        		
		                        		
		                        		
		                        	
            
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