Aim: determination of changes in peripheral blood cell counts and EPO and IL-3 levels induced by a series of 10, 20 and 30 standard whole-body cryostimulation (WBC) treatments. The study involved 45 men, experimental group  subjected to 30 WBC treatments  and a control group . Blood samples were collected before the series of treatments and after 10, 20 and 30 treatments. After 10 and 20 treatments we observed lower red blood cell counts and hematocrit and hemoglobin levels compared to baseline (p<0.05) and the control group (p<0.05). Additionally we observed an increase in hemoglobin concentration in plasma (p<0.05), and bilirubin after 10 and 20 treatments, and a decrease in plasma concentration of haptoglobin after 10, 20 and 30 treatments (p<0.05). The number of leukocytes was higher after 10 and 20 WBC treatments compared to baseline and the CON group. EPO concentration in plasma was elevated and the concentration of IL-3 was lower after 10, 20 and 30 WBC treatments. The decrease in indices of the erythrocytic system, plasma hemoglobin and bilirubin, with a simultaneous decrease in haptoglobin concentrations after 10 and 20 WBC treatments, may be due to increased intravascular hemolysis. At the same time there was a small, but statistically significant increase in the concentration of EPO stimulated erythropoiesis which could facilitate a return of erythrocytic system indices to initial levels after 30 WBC treatments. Changes in the white blood cell system showed transient mobilization of this system under the influence of WBC.The effects of extreme cold on the human body are not fully understood, there are also no reports on the effect of cryogenic temperatures on the levels of erythropoietin (EPO) and interleukin 3 (IL-3), two important factors that regulate hematopoiesis.  Recently, cryotherapy and cryostimulation have gained in popularity in the prevention and treatment of musculoskeletal overload in competitive sports and sports medicine, and also the rejuvenation of healthy individuals to accelerate recovery and increase resistance. The initial use of this type of physical therapy in the treatment of acute and chronic soft tissue trauma, pain relief and reduction of inflammatory process activity, expanded the use of cryogenic temperatures for post-operative care after orthopedic surgery, in rheumatoid and nervous system diseases , or as an analgesic and relaxing agent for patients in the terminal stages of cancer Unfortunately, the mechanism and the therapeutic effect of cryogenic temperatures on the human body is still not fully understood. Research is often conducted on small groups, with significant differences in procedures in which cryostimulation (or cryotherapy) is often used in combination with other forms of treatment or as part of physical training. This does not allow unambiguous interpretation of results to assess the impact of cryogenic temperatures on the human body. Therefore literature reports are often contradictory and inadequate. Moreover, very often changes observed after WBC are interpreted with reference to the results obtained after the application of temperatures close to zero, such as immersion or swimming in cold water Whole body cryostimulation (WBC) is an application of extremely low temperatures (from −100°C to −140°C) on the human body. The term “cryostimulation” is increasingly often used to highlight the stimulatory effect of cryogenic temperature, in contrast to cryotherapy where a therapeutic aim is emphasized.It is believed that whole body cryostimulation in healthy individuals is a safe procedure and does not cause adverse changes in the body One very interesting issue is the effect of WBC on peripheral blood and hematological indicators. Changes in peripheral blood indices could influence exercise capacity and general resistance to infectious agents. However, reports on this subject are scarce and sometimes contradictory. There are several papers evaluating the impact of cryogenic temperature on peripheral blood counts in healthy people. Some reports show a decrease in erythrocytic indices Because cytokines are a large group of molecules regulating hematopoiesis and various processes such as cell proliferation and differentiation, inflammatory and immune responses and others, a number of studies have been published over the past years on the effects of WBC on these molecules. The most commonly cytokines determined in these studies include IL-1α, IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12 and TNF Interleukin-3 is a multipotent hematopoietic growth factor, stimulating hematopoiesis by the induction of proliferation and differentiation of early pluripotent stem cells and progenitor myeloid stem cells – myeloid, erythroid and megakaryocytic lineages. An administration of IL-3 to patients increased the number of leukocytes , as well as reticulocytes and platelets As shown in previous studies, the effect of cryotherapy may depend on the number of treatments applied The research involved forty-five healthy male volunteers who had never been subjected to any form of cryotherapy.The subjects were students of the General Tadeusz Kosciuszko Military Academy of Land Forces. Each participant gave a written assent before participating in the study, according to the Declaration of Helsinki. The study was approved by the local Ethics Committee . In addition, the superiors of the respondents agreed to include the WBC cryotherapy series into the daily schedules of the participants.All subjects took part in initial medical qualification in order to eliminate those with potential contraindications to WBC. All the subjects were healthy and normotensive, with a body mass index (BMI) of between 21.12 and 26.3.The men were randomly divided into two groups: an experimental group of 30 (EXP) and a control group of 15 (CON). The groups were homogeneous in terms of age , diet and daily physical activity, due to the scheduled joint activities, accommodation and food provided by the Military Academy. None of them participated in any kind of sports training. The characteristics of the study group are shown in The treatment sessions were performed every day at the same morning hours, five days a week, from Monday to Friday. The subjects entered the chamber in groups of five persons. Each cryostimulation session lasted 3 minutes (−130°C). Entry to the cryo-chamber was preceded by a 30-second adaptation period in the vestibule at a temperature of −60°C, from which the subjects went further to the proper chamber, where they moved slowly in a circle, one after the other, without mutual contact, no additional movement or talking. After a minute, a change in the direction of motion was recommended. Contact with the participants was maintained via a camera in the room and voice contact. Before each treatment systolic and diastolic blood pressures were measured to monitor one of the contraindications to cryostimulation, i.e. high blood pressure.Glasses, contact lenses and all jewelry were removed before entry to the chamber, as well as a thorough drying of the body to eliminate the sensation of cold, noses and mouths were secured with a surgical mask. During the cryostimulation procedure, the subjects were dressed only in shorts, socks, wooden clogs, gloves and a hat covering the auricles against frostbite.During the experimental period, venous blood samples were collected four times to perform hematological and biochemical analyses. Blood was taken in the morning before the first treatment session (T0), in the morning of the day after the 10th treatment session (T1), and after the 20th (T2) and 30th treatment session (T3). To avoid changes in plasma volume and to ensure the proper hydration of the organism, our subject drank 15 ml of water per kg of body mass between 6 p.m. and 10 p.m. a day before blood collection. Blood samples were always obtained after overnight fasting, between 6.00 and 7.30 a.m., after 10 minutes rest in a sitting position, from an antecubital forearm vein using vacutainer tubes with an appropriate K2 EDTA anticoagulant , separately into two tubes; one to determine blood counts (1.2 ml anticoagulated with 1 g/L EDTA) and the other for biochemical analysis of plasma (5 ml EDTA).Directly after blood sampling, the following indicators were determined: the number of erythrocytes (RBC), hemoglobin concentration (Hb), hematocrit value (Ht), red blood cell indices: MCV - mean corpuscular volume, MCH - mean corpuscular hemoglobin, MCHC - mean corpuscular hemoglobin concentration, RDW - red cell distribution width, number of leukocytes, including the share of lymphocytes (LYM), granulocytes (GRA) and monocytes (MON), and the number of thrombocytes (PLT). Hematological parameters were measured by a hematology analyzer (HORIBA ABX Micros 60). Intra-assay precision and accuracy for all hematologic parameters were less than 1.5% and inter-assay precision and accuracy for all hematologic parameters were less than 2.0%. In plasma, the following indicators were measured: the concentration of extra cellular hemoglobin [g/dL] determined spectrophotometrically using Drabkin's reagent; haptoglobin (HAP) concentration [g/L] (CORMAY haptoglobin); bilirubin [mg/dL] (BIOLABO); and levels of IL-3, and erythropoietin (R&D Systems Quantikine ELISA kits). The coefficients of variation of both the intra-assay and inter-assay were less than 10%. The minimum detectable concentration for IL3 was 7.4 pg/mL and less than 0.6 mU/mL for Quantikine IVD ELISA Human Epo.The normality of distribution of dependent variables was tested with the Shapiro-Wilk test. Since in some cases the data distribution was not normal, nonparametric statistical analyses were conducted. Comparison between before and after cryostimulation measurements were done with repeated-measures nonparametric Friedman's ANOVA and Wilcoxon rank sign tests. For comparison between two independent means, the Mann-Whitney U test was used. Correlations between observed variables were calculated by Spearman's coefficient of rank correlation. A p<0.05 was considered statistically significant for all tests. Each studied parameter was characterized by: sample size, arithmetic mean/median, standard deviation. All statistical calculations were conducted using the Statistica 6.0 package.Anthropometrical characteristics of the participants are presented in 0) and after the series of sessions of whole-body cryostimulation treatment (T1–T3) are presented in The hematological profiles of the participants of both groups before (T0E), as well as to the values observed in the control group (CON). There were no changes in mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) in either group during the whole period. However, there was a rise in red cell distribution (RDW) in the EXP group after 20 sessions of WBC indicating an increased variation in the size of individual erythrocytes (anisocitosis).Red blood cells count, hemoglobin concentration and hematocrit dropped significantly after 10 sessions of WBC and remained decreased after 20 sessions in the experimental group (EXP) compared to the resting values , the percentage of monocytes was significantly elevated after 10, 20 and 30 WBC treatments, while the percentage of granulocytes was higher after 10 and 20 WBC sessions.An intensified hemolysis, as confirmed by significantly elevated levels of extraerythrocyte hemoglobin (plasma hemoglobin) and bilirubin as well as by decreased levels of haptoglobin (HAP), was observed after 10 and 20 WBC sessions compared to initial values and values observed in the control group . HaptoglErythropoietin concentration was significantly elevated after 10, 20 and 30 WBC sessions when compared to the initial value and levels observed in the control group . The mosInterleukin 3 (IL-3) decreased after 30 WBC sessions when compared to the initial level, however, concentration of this cytokine in plasma obtained from participants subjected to 10, 20 and 30 WBC was significantly lower compared to concentrations observed in the control group .The mechanisms of the effects of cryogenic temperatures on healthy people are not yet fully understood, although in the case of athletes, changes in peripheral blood may have a huge impact on the immune system (changes in the white blood cell system) or on exercise capacity (changes in the erythrocytes). In recent years there have been only a few reports providing data on the influence of extreme cold on healthy human hematological parameters and these reports are often contradictory and incomplete. Accordingly, we embarked on an investigation of the effects of different numbers of whole-body cryostimulation (WBC) treatments on peripheral blood cell counts and the levels of two cytokines (EPO and IL-3) which have a significant effect on hematopoiesis. To our knowledge this is the first report on the subject.Erythropoiesis stimulating methods, such as high altitude training and hypoxic chambers, are commonly used in sport. After 10 WBC treatments involving active healthy young men, we found a significant decrease in hematological parameters such as the number of red blood cells, hemoglobin and hematocrit compared to baseline and to the values found in the control group. It was observed that these parameters were also similarly reduced after 20 WBC treatments, and after 30 treatments returned to baseline values and values similar to those of the control group. These results are consistent with most previous studies that have observed a significant decrease in the number of erythrocytes, hemoglobin and hematocrit after a series of five to 18 sessions of WBC The increased hemolysis of erythrocytes under the influence of cryostimulation is also suggested by high values of plasma hemoglobin. Increases in plasma hemoglobin were also observed in another work, in blood taken 30 minutes after a single WBC treatment, with a concentration being almost four times higher than before treatment, and still higher the day after Banfi et al. argue, however, that WBC reduces hemolysis of erythrocytes in athletes Firstly, the study by Banfi et al. A similar relationship was observed by Lombardi et al. The erythropoiesis-stimulating effect of WBC treatment may also be indicated by changes in the hematological indices observed by Straburzyńska-Lupa et al. Information on the effects of cryogenic temperatures used during cryostimulation or whole-body cryotherapy on the white blood system in healthy people, are also very sparse, ambiguous and still do not sufficiently fully explain the observed changes.On the basis of this study we can see stimulation of the white blood cells system in young, healthy people, as shown by a statistically significant increase in the number of white blood cells after 10 and 20 WBC treatments compared to baseline and the control group. An increase was also observed in the percentage of neutrophils and lymphocytes, being especially pronounced after 20 WBC treatments. The percentage of monocytes was elevated after 10, 20 and 30 treatments. Our previous reports also confirm that the series of cryostimulations resulted in a statistically significant increase in the total number of leukocytes after 10 No significant increase in leukocytes, however, was reported by Stanek et al. No significant change in the number of leukocytes was also found in professional rugby players by Banfi et al. Lombardi et al. (15) observed a small but statistically significant reduction in the number of leukocytes in rugby players after 14 WBC treatments (2 times daily for 7 days). At the same time they observed no changes in the amount and proportion of different types of white blood cells, suggesting that “the WBC had no effect on the immune system, neither activating nor inhibiting it.”In one very recent study with 10 long distance runners and 10 non-athletes subjected to 12 WBC sessions 3 times a week, significant decreases were noted in white blood cells and neutrophils in the athletes but not in the non-athletes, whereas lymphocytes, monocytes, eosinophils, and basophils remained unchanged both in the athletes and non-athletes. According to the authors it could be supposed that the immune system in athletes is more sensitive to the extreme environmental conditions compared to non-athletes It seems that on the basis of our studies and the observations of other authors, the impact of extremely low temperatures on the white blood system cannot be unambiguously determined. In healthy non-sporting people one can talk about a mobilization of the white blood cells which may have a positive effect on the immune mechanism of the body. Athletes require further well-designed studies on the effects of WBC on the white blood system, taking into account the individual fractions of leukocytes.Platelets play an important role in various stages of blood coagulation, and are also involved in the body's immune processes, wound healing and defense of the organism against some bacteria In this current study we found no changes in the number of blood platelets induced by the WBC treatments, regardless of the number of treatments. The platelet count was not affected by changes in cytokines (EPO and IL-3).On the basis of our present research and other works To our knowledge, there are no reports on the influence of cryogenic temperatures on the level of erythropoietin and interleukin-3, i.e. cytokines which have a significant effect on hematopoiesis.Erythropoietin is the first identified hematopoietic factor that regulates survival, proliferation and differentiation in erythrocytic progenitors and the maturation of erythrocytic precursors. It improves the survival of circulating red blood cells and inhibits their degradation – eryptosis After 10 WBC treatments we observed a significant 4.5% increase in the concentration of EPO. The concentrations of the hormone increased by 10% after 20 and 30 treatments compared to baseline, while no changes in the concentration of this hormone were observed in the control group. The main factor stimulating the synthesis of the hormone is hypoxia. In the period of 60–120 minutes from the stimulus, EPO level in peripheral blood increased, reaching a maximum concentration after about 20 hours Interleukin-3 is a cytokine which interacts with other cytokines, acting on various hematopoietic cell lines In the present study IL-3 concentrations in blood decreased following the series of WBC treatments. The level of this cytokine reduced after 10 treatments, compared to the control group. However, the greatest decrease compared to baseline was observed after 30 WBC treatments. On the basis of our results it cannot be clearly evaluated what the consequences might be of the reduced level of this interleukin for the examined blood counts.Study limitation. For a full explanation of the observed changes in hematological parameters as well as in EPO levels, a measurement or at least a calculation of plasma volume are necessary. Unfortunately, using changes in hemoglobin concentration and hematocrit for calculating changes in plasma volume in this study would be confusing because of hemolysis of erythrocytes observed after a series of WBC. The topic, however, is extremely interesting since there is only one report concerning changes of plasma volume after whole-body cryostimulation calculated on the base of Hb and Ht In summary, the reduced values of the erythrocytic system, an increase in plasma hemoglobin and bilirubin with a simultaneous decrease in haptoglobin concentrations observed after 10 and 20 WBC treatments, may be the result of increased hemolysis of erythrocytes in young healthy men rather than changes in plasma volume. At the same time a small increase in erythropoietin concentration stimulates erythropoiesis, resulting in the red blood cell count and hematocrit and hemoglobin levels returning to normal after 30 WBC treatments. Therefore, in spite of this small rise in EPO concentration, WBC could not be regarded as an unethical method used for stimulation of the erythrocytic system.The increase in the number of leukocytes, the percentage of lymphocytes and granulocytes observed after 10 and 20 WBC treatments may indicate a moderate leukocyte mobilization, although transient after the next ten treatments.


The emergence of a new subtype of the influenza virus in 2009 generated interest in the international medical community, the media, and the general population. Pregnant women are considered to be a group at risk of serious complications related to the H1N1 influenza virus. The aim of this study was to evaluate the outcomes and teratogenic effects of pregnancies exposed to the H1N1 virus during the Influenza A epidemic that occurred in the state of Rio Grande do Sul in 2009.This is an uncontrolled prospective cohort study of pregnant women with suspected symptoms of Influenza A who were reported in the Information System for Notifiable Diseases – Influenza (SINAN-Influenza) during the epidemic of 2009 . There were 589 cases of pregnant women with suspected infection. Among these, 243 were tested by PCR and included in the analysis. The main outcome measures were: maternal deaths, pregnancy outcome, stillbirths, premature births, low birth weight, congenital malformations, and odds ratios for H1N1+ and non-H1N1 pregnant women.There were one hundred and sixty-three (67%) confirmed cases of H1N1, 34 cases (14%) of seasonal Influenza A and 46 (19%) who were negative for Influenza A. There was no difference between the three groups in clinical parameters of the disease. There were 24 maternal deaths — 18 of them had H1N1. There were 8 stillbirths — 5 were children of H1N1 infected mothers. There were no differences in perinatal outcomes.The present data do not indicate an increase in teratogenic risk from exposure to the influenza A (H1N1) virus. These results will help to strengthen the data and clarify the health issues that arose after the pandemic. The emergence of a new subtype of the influenza virus at the end of March 2009 generated interest in the international medical community, the media, and the general population. The influenza A (H1N1) epidemic began in Mexico and rapidly spread to other countries around the world The Influenza A H1N1 virus is the product of multiple genetic rearrangements among strains of influenza that had previously been circulating. Some of these were unique to swine and birds and became capable of infecting humans Pregnant women are considered to be a group at risk of serious complications related to the H1N1 influenza virus, with high morbidity and mortality observed in both previous The only known specific treatment, which is supported by recent evidence, is the use of the neuraminidase inhibitors zanamivir and oseltamivir, of which only the latter is available in Brazil.Rio Grande do Sul is the southernmost state of Brazil. It occupies 3% of the national territory and has a population of 10,914,128 inhabitants (2009), which corresponds to about 6% of the national population. The climate in this state has specific characteristics that are different from the rest of the country. For example, it has the lowest temperatures for the Brazilian winter  During the Influenza A (H1N1) pandemic in 2009 there were 44,544 cases confirmed in Brazil and 2,051 deaths recorded. In Rio Grande do Sul, there were 8,314 reported cases, 3,576 confirmed cases , and 297 deaths  As for effects on the embryo/fetus, there are still few studies on the teratogenic potential of the influenza virus. It has been suggested that there is a potential increase in the risk of defects in neural tube closure associated with maternal hyperthermia Analyses of the effects of Influenza A H1N1 on pregnancies during the pandemic of 2009 have focused primarily on maternal mortality and morbidity, in part due to the immediacy of the pandemic and the speed with which the series of cases were published In South America, only one Brazilian study, which was conducted with 57 hospitalized women, has shown the clinical characteristics and pregnancy outcomes of pregnant women infected with Influenza A (H1N1) during the 2009 pandemic The aim of this study was to evaluate the outcomes,including teratogenic effects, of pregnancies exposed to the H1N1 virus during the Influenza A epidemic that occurred in the state of Rio Grande do Sul in 2009.This is an uncontrolled prospective cohort study that evaluated pregnancies with exposure to the 2009 H1N1 Influenza virus. The sample consisted of pregnant women with suspected symptoms of Influenza A who were reported in the Information System for Notifiable Diseases - Influenza (SINAN-Influenza) during the epidemic of 2009 (database from the state of Rio Grande do Sul).SINAN is maintained by the reporting and investigation of cases of diseases and conditions that appear on the national list of diseases for which notification is compulsory. Besides the Individual Notification Form (FIN), the system also provides an Individual Investigation Form (FII), which is a research guideline that enables the identification of the source of infection and the mechanisms of disease transmission. Most of the information about the patient and his clinical symptoms are collected at the time of notification Confirmation of the infection and detection of the viral subtype was done (when possible) through a biological sample  in order to perform the real time polymerase chain reaction (RT-PCR), in accordance with the RT-PCR protocol for the detection and characterization of Swine Influenza from the Centers for Disease Control and Prevention (CDC) Pregnancy follow-up was conducted by a trained team via telephone, between July 2009 and June 1, 2011. If three phone calls were unsuccessful and a visit to a patient's home could not be arranged, a follow-up was considered to be lost. Data from medical records or the Live Birth Certificate (LBC) would then be used to verify the pregnancy outcome data. The LBC is the standard document of the Ministry of Health and its use is mandatory in Brazil. It includes data to be filled in about the mother , the pregnancy , type of delivery, and the newborn child . The LBC does not include information about pregnancy complications or comorbidities like pregnancy-related hypertension/preeclampsia, gestational diabetes, etc.).A home visit, when possible, was performed by a team member who used a questionnaire identical to that used in the phone interviews. This follow-up by personal contact received direct support from the State Center for Health Surveillance (CEVS).During the Influenza A H1N1 epidemic in 2009, there were 589 cases of pregnant women suspected of infection that were reported to the SINAN-Influenza database for Rio Grande do Sul.Patients were excluded from this study if they did not have a confirmed pregnancy, if it was not possible to contact them after notification, or if they did not have a PCR performed.For data analysis, we included only patients with infection confirmed by PCR laboratory analysis. Follow-ups were done either by phone, home visit, assessment of medical records, or via the LBC information.An influenza-like illness (ILI) was defined as the presence of at least one of the common flu symptoms , in accordance with the widely used definition, and this was reported to the SINAN-Influenza database.A confirmed case was defined as an ILI with a positive Influenza A (H1N1) PCR, and is denoted as H1N1+. Non-H1N1 Influenza cases were those that were seasonal Influenza A or that were negative for Influenza A see . ConsideWe evaluated clinical parameters related to the pregnancy  and the newborn child . A descriptive analysis was done of the samples, with means and standard deviations for both the quantitative variables of the clinical parameters and the frequency of the categorical variables. The analyses were done using Fisher's exact test followed by the Z-test with Bonferroni correction, One-way ANOVA, or the Kruskal-Wallis test, as appropriate. The level of significance was set at 5% for all variables and the program used for the analyses was SPSS for Windows, version 18.0.The research was approved by the Research Ethics Committee of the Research and Post-Graduate Group at the Hospital de Clinicas de Porto Alegre (CEP/GPPG-HCPA).At the beginning of the telephone interview, consent was requested for confirmation of the information available in the records of SINAN-Influenza or for collection of new data, if necessary. Informed consent was conducted verbally, since most of the interviews were conducted via telephone. The patient answered “Yes, I authorize” or “No” to the question “Do you consent to talk by phone with a medical team and provide some data about your flu syndrome episode and your pregnancy?”. The same verbal informed consent was applied to cases of personal interviews. All procedures were approved by GPPG and the clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.During the Influenza A H1N1 epidemic in 2009, there were 589 cases of pregnant women suspected of infection who reported to the SINAN-Influenza database for Rio Grande do Sul.From the follow-ups done, 243 PCR test results were obtained. There were one hundred and sixty three (67%) confirmed cases of Influenza A H1N1, 34 cases (14%) of seasonal Influenza A, and 46 (19%) negative for Influenza A . ContactThe accurate gestational age at the time of infection was obtained in 83 follow-ups. Three women (1.2%) were in the first trimester (0–11 weeks), 27 (11%) were in the second trimester (12–23 weeks) and 53 (21.8%) patients were in the third trimester (≥24 weeks).Use of oseltamivir phosphate was identified in 137 patients (56.3%). Adverse reactions such as vomiting, nausea, abdominal pain, diarrhea, drowsiness, confusion, dizziness, headache, and irritability were reported. There were no serious adverse events in this study population. There was no difference when comparing the three groups in terms of adverse reactions presented. In the analysis of symptoms reported after use of the medication, in comparison with the H1N1+ subgroup only nausea was more frequent in patients without H1N1 confirmed by PCR .Hospitalization was required for 107 of the patients. In the H1N1+ subgroup, 70 (42.9%) patients required hospitalization. The other 37 admissions consisted of 6 (17.6%) in the seasonal Influenza A subgroup and 31 (67.4%) in the Influenza A negative subgroup. The proportion of hospitalizations is significantly different for each group; in other words, there was a predominance of hospitalizations in the Influenza A negative subgroup, followed by the H1N1+ subgroup, and finally the seasonal Influenza A subgroup  were recorded, 18 of these (75%) were H1N1+ patients. Two deaths were in the seasonal Influenza A subgroup and 2 deaths in the Influenza A negative subgroup . The remRegarding pregnancy outcomes, we obtained 236 live births. Eight stillbirths were reported — five of these were for H1N1+ pregnant women, one was from the seasonal Influenza A subgroup, and the other two were for pregnant women for whom research about the virus was not done. There were three miscarriages — one occurred in the Influenza A negative subgroup and two in pregnant women without PCR, so there was no difference between the subgroups . InformaGestational age at birth was obtained in 222 follow-ups. The mean gestational age did not differ between the subgroups. The weight of the newborns was, on average, 3128 grams (±643) for those born to H1N1+ mothers, 2893 grams (±493) for the seasonal Influenza A subgroup, and 2998 grams (±598) for the Influenza A negative subgroup.There were no reports of major complications or malformations for the newborns.By observing the previous pandemics and epidemics, as well as seasonal influenza, pregnant women have been identified as a group at risk of higher morbidity and mortality. During the epidemic of 1957–58, 10% of deaths from influenza in New York (USA) occurred in pregnant women. In Minnesota (USA), 50% of deaths among women of reproductive age occurred during pregnancy In the influenza A (H1N1) pandemic of 2009, pregnant women were among the first cases and first deaths reported, representing 13% of deaths reported to the centers for control and prevention of diseases in the USA In this study, the gestational and maternal outcomes were almost the same when correlated with the Influenza A H1N1 virus. These results differ from other studies which show poor pregnancy outcomes, such as increased perinatal mortality and prematurity The present study may have been unable to detect poor outcomes due to the sample sizes and the severity of maternal disease in the three subgroups used for comparison. It should be noted that in the H1N1 exposed group in our sample the prevalence of deaths , although statistically non-significant, was almost the double of the non-H1N1 influenza groups  . In the Additional information was obtained about the safety of oseltamivir, given that there had been no report of any major complication or abnormality among the outcomes for pregnant women who had used this drug. These data are very important due to the large number of outcomes that could be assessed in this study and they reinforce safety data already published in other papers It is important to emphasize that the follow-ups of 424 pregnant women were done from a sample of 589 pregnant women reported to SINAN-Influenza in 2009. This represents a follow-up rate of 72%, an average that is much higher than for many other studies published on this subject, which have among their limitations a small number of follow-ups  [15], [2One limitation of our study is that we did not collect some clinical information about the respiratory symptoms exhibited by the pregnant women, such as the presence of hyperthermia (and maximum temperature) and viral load, which could account for some of the adverse outcomes reported. However, this limitation is also present in other recent studies on this subject The difficulty in performing follow-ups is due to the number of mobile phones used as the main contact — phone numbers change and phones are lost. Moreover, at the time of the Influenza A (H1N1) pandemic, the consultations were mostly conducted in places adapted for high patient demand, thus it was more difficult and there was a greater margin of error at the time of completing the personal data of the pregnant woman being treated.Previous experience with pregnant women exposed to Influenza A suggests that the maternal outcomes are worse in this subgroup of women. This justified the use of antiviral treatment and vaccinations against the Influenza A H1N1 virus for these patients. Regardless, our results were not sufficient to support this; therefore, it is important to emphasize that more studies are needed, especially ones that correlate maternal exposure to the H1N1 virus with neonatal outcomes, so that we can further clarify the health issues that arose after this pandemic.


Objective. To explore the urinary biochemistry features of syndromes of traditional Chinese medicine (TCM) such as syndrome of stagnation of liver Qi, spleen deficiency, liver Qi stagnation, and spleen deficiency (LSSDS) in sub-optimal health status (SHS). Methods. 12 cases for each syndrome group in SHS were selected, 12 subjects were used as a normal control group, and 1H NMR detection was, respectively, carried out, and the data was corrected by the orthogonal signal correction (OSC) and then adopted a partial least squares (PLS) method for discriminate analysis. Results. The OSC-PLS (ctr) analysis results of the nuclear overhauser enhancement spectroscopy (NOESY) detection indicated that the syndromes in SHS could be differentiated, and there were significant differences in the levels of metabolites of the urine samples of the four groups; the biomarkers of LSSDS in SHS were found out. The contents of citric acid (2.54 and 2.66), trimethylamineoxide (3.26), and hippuric acid  in the urine samples of LSSDS group were lower than that of the normal control group. Conclusion. There are differences in the 1H-NMR metabolic spectrum of the urine samples of the four groups, and the specific metabolic products of the LSSDS in SHS can be identified from metabonomics analysis. Since 1984, when the WHO gave the definition to health as paying attention to “the state of complete physical, mental and social well-being” in its “Charter,” the research on the “third state” has been carried out widely at home and abroad. In 1996, scholar Wang from China named this state as “SHS” [It was found by the clinical epidemiological survey that 60%–70% of people in China are in SHS; the incidences of syndrome of stagnation of liver Qi and spleen deficiency among the population of SHS are high and can be up to 17.25 . In rece1H NMR spectrum of the plasma of model rats with chronic restraint stress-induced syndrome of stagnation of liver Qi and spleen deficiency, the changes in the spectral peak shape of acetic acid, lactic acid, tyrosine acid, low-density lipoprotein, and the unknown compound at 3.44 ppm were very significant. As a result, the metabonomics method was applied in this study in discussing the syndrome of stagnation of liver Qi and spleen deficiency and assisted in revealing its biological changes. The objective of this study is using metabonomics method to differentiate syndromes with clinical urine samples in Suboptimal health status. Currently combination of syndromes classification and biomedical diagnosis becomes a common model in diagnostics in TCM clinical practice. Clinical treatments of a patient rely on the successful differentiation of a specific TCM syndrome. Our previous studies have shown that , throughAmong the personnel from the Chinese Academy of Sciences who had physical examinations in the Medical Center of Beijing Guanghua Hospital from December 2009 to April 2010, 12 cases of syndrome of stagnation of liver Qi and spleen deficiency, syndrome of stagnation of liver Qi, and syndrome of spleen deficiency in SHS who met the inclusion criteria were, respectively, selected, and 12 cases for the healthy group of normal control were selected. 48 subjects were typical permanent residents in Beijing, and it was shown that the four groups were well balanced on age and sex. Both the research protocol and written informed consent were reviewed and approved by an ethics committee of Beijing University of Chinese Medicine prior to the study initiation. In addition, a TCM syndrome questionnaire for measuring SHS was also used in the study , and the(1) Recurrent physical discomfort and decreased efficiency continued for more than three months due to persistent or excessive fatigue. (2) Have no significant organic diseases or psychological disorders.Only those who meet both the above two criteria could be diagnosed as Suboptimal health population.(1) Meet the diagnostic criteria. (2) Age should be between 18 and 49 years old. (3) Informed consent: all subjects should have a consent signed that demonstrates an understanding of this study.Only those who meet all the above three criteria could be included.(1) Subjects who do not meet the inclusion criteria. (2) Women who are pregnant, in lactation period or are attempting to get pregnant. (3) Subjects who do not sign the informed consent form. (4) Subjects diagnosed with metabolic syndrome or who have received any treatment.For the diagnosis of syndrome of stagnation of liver Qi, and spleen deficiency, syndrome of stagnation of liver Qi and syndrome of spleen deficiency, please refer to “GB/T16751.2-1997, Clinic terminology of traditional Chinese medical diagnosis and treatment-Syndromes” of the People's Republic of China .Instruments are INOVA 600 MHz superconducting NMR spectrometer , equipped with pulsed field gradient and a three-resonance gradient probe; Centrifuger: produced by Eppendorf MiniSpin Plus, Germany.Reagents are deutoxide (D2O), produced by Cambridge Isotope Laboratories Inc., USA, 99.9%; 2,2,3,3,-d(4)-3-Trimethylsilyl) propionic acid sodium (TSP), purchased from Merck, Germany.μL of TSP/D2O (1 mg/mL) was added into the 5 mm NMR tube. After the urine samples were thawed at room temperature, 350 μL of phosphate buffer solution and 350 μL of the urine were added into the EP tube, after being well mixed by shaking, centrifuged at 13,000 rpm for 10 min, and 600 μL of the supernatant was added into the NMR tube mentioned above and mixed and was ready for use.About 5 mL of middle-segment fasting morning urine sample of the subjects were collected. Then the samples were centrifuged at 3500 rpm for 10 min after collection. Then 1 mL of the supernatant was taken and placed in the refrigerator with temperature at −20°C for cryopreservation. 30 Detection was carried out in the Magnetic Resonance Laboratory of the Medical Center of Biomedical Analysis of National Academy of Military. Under the conditions of 27°C, Carr-Purcell-Meiboom-Gill (CPMG) and longitudinal eddy delay (LED) were, respectively, adopted for the experiment on the VARIAN UNITYINOVA 600 MHz Superconducting Fourier Transform NMR spectrometer, the presaturated method was used to inhibit the water peak, the saturation time was 2 s, spectral width was 8000 Hz, the sampling point was 32 k, and it accumulated 64 times. The presaturation frequency and central frequency were both at the position of water peak. The free induction decay (FID) signal was converted to NMR spectrum through the Fourier transform at the 128 k point. The peak of the chemical shift of TSP was used as a reference position and set to 0 ppm.The obtained NMR data was converted to a spectrum by the Fourier transform, after phase and baseline correction, the spectrum within a certain range was piece-wisely integrated at a width of 0.04 ppm for each segment, and the segments with solvent peaks and urea peaks (4.6 to 6.2) were excluded at the same time. The integral range of the NOESY data was 0.4 ppm ~ 9.4 ppm. The integral was normalized by the total integral intensity of each spectrum. The obtained data was output and converted to an Excel file and saved.All the integral data was output to Excel files and saved and then entered into the SIMCA-P + software  for analysis. Orthogonal signal corrections (OSC) were performed first, and PLS analysis was then conducted. The analysis results were expressed as scores plot and loadings plot. The degree of separation for each group of data was determined from the scores plot, and the metabolites that contributed to the composition were obtained from the factor loadings plot.1H-NMR spectrums of the four groups—syndrome of stagnation of liver Qi and spleen deficiency (group A), syndrome of stagnation of liver Qi (group B), syndrome of spleen deficiency (group C), and the normal control (group D)—are shown in The OSC-PLS analysis results of t1 axis; it can be said that the group of syndrome of stagnation of liver Qi (Group B) and the group of syndrome of spleen deficiency (Group C) show rough separation trends along the t1 axis.The abscissa of As the diet and lifestyle of the subjects in this study were not strictly restricted, in order to improve the classification results, intergroup pairwise comparisons were using the PLS-DA analysis which was carried out after OSC denoising processing.1H-NMR spectrum of the urine samples of the group of syndrome of stagnation of liver Qi and spleen deficiency (Group A) and the group of normal control (group D) are shown in t1 axis, with no overlap and duplication. This indicates that the two groups have differences in the metabolic phenotypes, and the intergroup difference is significant. We can see from the loadings plot  and the group of syndrome of stagnation of liver Qi (Group B) are shown in The OSC-PLS analysis results of the 1H-NMR spectrum of the urine samples of the group of syndrome of stagnation of liver Qi and spleen deficiency (Group A) and the group of syndrome of spleen deficiency (Group C) are shown in t1 axis, with no overlap and duplication, indicating that the two groups have differences in the metabolic phenotypes, and the intergroup difference is significant. We can see from the loadings plot , trimethylamine oxide (3.26), and hippuric acid .ob-R) at the arcuate nucleus (ARC) in the basal of hypothalamus in the hypothalamus of chronic stressed rats [Syndrome of stagnation of liver Qi and spleen deficiency is one of the common syndromes of SHS and mental disorders in TCM clinic , our recsed rats . Meanwhised rats . ExcessiCreatinine is a metabolite produced during metabolism of muscle tissue and is also a compound related to the renal function. Therefore, the relative increase in creatinine levels suggests the decrease in the metabolic function of syndrome of stagnation of liver Qi and spleen deficiency in SHS. Trimethylamine oxide (TMAO) has a role in promoting the growth of muscle tissue, and the decrease in the content of trimethylamine oxide suggests the decline of the “spleen governing muscles” function of the syndrome of stagnation of liver Qi and spleen deficiency in SHS. Hippuric acid is a small molecule urine toxin, and there is a study indicating that hippuric acid can accurately reflect the energy metabolism of the liver and any reason affecting the oxidative and phosphorylation ability of mitochondria of liver cells; thus, resulting in hepatic energy metabolism disorders can affect the synthesis ability of hippuric acid . TherefoIn this study, we analyzed the metabolic phenotype spectrums of the urine samples of the syndrome of stagnation of liver Qi and spleen deficiency, syndrome of stagnation of liver Qi, syndrome of spleen deficiency in SHS, and the group of normal control from the perspective of metabonomics, and there were significant differences in the metabolites for the syndromes in SHS. These differences are based on the changes in the metabolism of different substances or the metabolic network. The metabonomics technology may be used to identify the specific metabolites for the biological basis of the syndromes of TCM, and the methods of bioinformatics can also be used to analyze the functions of the biomarkers, and to determine the “Metabolic spectrum related to syndrome of TCM” .We also describe in detail the metabolic consequences of syndromes in SHS. In particular, we have shown that the specific metabolic products of the syndrome of stagnation of liver Qi and spleen deficiency in SHS can be identified from metabonomics analysis: creatinine (3.06 and 4.06), trimethylamine oxide (3.26), and hippuric acid . We consider the results of this study to add considerably to our understanding of the biochemistry of TCM syndromes.


To assess the frequency of occurrence of the hypermobility syndrome (HS) in children and teenagers with idiopathic scoliosis (IS). To assess the presence of HS in relation to the angle of curvature, vertebral rotation, length of scoliosis and the treatment used.Joint hypermobility syndrome is diagnosed when the mobility of small and large joints is increased in relation to standard mobility for any given age, gender and race, and after excluding systemic diseases 2]3]. I[3]. I2][. I[3]. I128 children (92 girls and 36 boys) aged 9 to 18 years, comprising 70 IS children (34 single and 36 double IS), Cobb angle from 11 to 53 degrees, and 58 scoliosis-free controls were examined. Beighton scale as well as Hakim and Grahame questionnaire were used to disclose the presence of HS [HS was noted more often in children with scoliosis than in the control group (p<0.0001). The angle of curvature, the apical vertebra rotation, the number of vertebrae of the primary curve and the treatment (brace or physiotherapy) did not influence the frequency of occurrence of HS. In single curve IS, the HS appeared more often than in double curve IS (p=0.03).HS appears more often in children with IS than in healthy controls, especially in single curves. There was no relation of HS with the angle, rotation, length of scoliosis or treatment type. HS should be taken into account when physiotherapy is planned in IS children.


According to the National Heart, Lung, and Blood Institute of the National Institute of Health, hypotension refers to an abnormally low blood pressure (BP). In childhood, hypotension can be determined according to two different definitions: BP below the 5th percentile or below two standard deviations (SDs) of the mean for age and gender . A graphWe used a computerized calculation method to develop high-resolution graphics containing curves with 5,841 points each, to depict the main percentiles associated with low BP for girls from 1 to 17 years old in the 50th percentile of height. Each point represents the calculation of the polynomial equation that includes the statistical processing of the last Report on Blood Pressure in 2004 . We alsoFive monotonic curves of systolic BP for girls representing the 50th, 25th, 10th, 5th, and 2.275th (-2SD) percentiles were built (Figure The correction of systolic BP for height is of little significance, and this updated graphic can be used to diagnose low systolic BP for girls. Clinical studies are necessary to determine the systolic BP percentile that better represents clinically significant hypotension.


Glucose has been reported to have an essential role in the synthesis and secretion of insulin in hepatocytes. As the efflux of glucose is facilitated from the liver cells into the circulation, the mechanism of transportation of glucose into the hepatocytes for the synthesis of insulin was investigated. Grated liver suspension (GLS) was prepared by grating intact liver from adult mice by using a grater. Nitric oxide (NO) was measured by methemoglobin method. Glucose transporter-4 (Glut-4) was measured by immunoblot technique using Glut-4 antibody. Incubation of GLS with different amounts of glucose resulted in the uptake of glucose by the suspension with increased NO synthesis due to the stimulation of a glucose activated nitric oxide synthase that was present in the liver membrane. The inhibition of glucose induced NO synthesis resulted in the inhibition of glucose uptake. Glucose at 0.02M that maximally increased NO synthesis in the hepatocytes led to the translocation and increased synthesis of Glut-4 by 3.3 fold over the control that was inhibited by the inhibition of NO synthesis. The glucose induced NO synthesis was also found to result in the synthesis of insulin, in the presence of glucose due to the expression of both proinsulin genes I and II in the liver cells. It was concluded that glucose itself facilitated its own transportation in the liver cells both via Glut-4 and by the synthesis of NO which had an essential role for insulin synthesis in the presence of glucose in these cells. Glucose has been reported to have a critically important role both in the synthesis and in the secretion of insulin in the islets of Langerhans ,2 which It is also known that although insulin receptors are present on hepatic cells , the uptWe report herein the existence of a novel constitutive form of nitric oxide synthase (cNOS) in the mice liver cell membrane that was found to be stimulated by glucose leading to the synthesis of NO in the mice hepatocytes. The role of glucose induced NO production both in the liver membrane translocation of Glut-4 as well as in the synthesis of Glut-4 in the hepatic cells for the expression of the genetic elements leading to the synthesis of insulin by the sugar in the liver were investigated . The protocol used in this study was approved by the Internal Review Board, Sinha Institute of Medical Science and Technology, Kolkata. The required approval was obtained for the use of animals in the study by the Internal Review Board for Animal Care, Sinha Institute of Medical Science and Technology, Kolkata, consisting of a special committee for animal care and their use that oversaw the welfare, care and nutritional requirements for all the animals used in the study. The committee had a permanent certified veterinarian whose duty is to ensure that the all the animals were free from any diseases as stipulated by the Animal Right Group. All animal related experiments were strictly performed in the presence of a member of the Animal Right Group and under the supervision of the veterinarian, and special care were taken to ensure that no animals were unnecessarily harmed or were subjected to pain during the study. After the termination of the study, the animals were sacrificed by euthanasia in a carbon dioxide chamber.ad libitum. The animals were kept under 12h cycles of light and dark at 23°C.White albino healthy mice (20-25gm each), Swiss strain, irrespective of gender were used for the study . These iThe Goat anti-rabbit immunoglobulin G-alkaline phosphatase, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) were obtained from Sigma Aldrich co, Glut-4 antibody was obtained from Santa Cruz Biotech. Insulin antibody was also obtained from Santa Cruz Biotech (Insulin H-86). ELISA Maxisorb plates were from Nunc, Roskilde, Denmark. All other chemicals were of analytical grade.2 for 5-6 times. The homogenized mass was next centrifuged at 60,000g for 60 min at 0°C. The supernatant fraction was discarded and the pelleted fraction that contained the membranes were suspended in HEPES buffer (pH 7.4) and used for further studies as soon as possible.Typically, adult mice were killed by cervical dislocation. The liver membrane was prepared by gently cutting the whole liver (2.40gm) into small pieces that were collected in HEPES buffer (pH 7.4) without glucose at 0°C and immediately homogenized by freezing and thawing by using liquid N2 in a total volume of 1.0ml. After incubation at 37°C for 30 min, the amount of NO formed in the reaction mixture was determined by the conversion of oxyhemoglobin to methemoglobin by the spectral changes of absorption maxima at 525 and 630 nm under N2 as described  of the membrane preparation Line A,  that ind The treatment of GLS with different amounts of glucose not only resulted in the increased synthesis of NO by the suspension as described , but wasequilibrium reached to 1.11 indicating that there was an appreciable increase of the glucose uptake at equilibrium, due to NO synthesis induced by the sugar in the GLS  units in the ELISA. The use of 0.02M glucose in the same reaction mixture resulted in the increase of the O.D, due to the increased production of the glucose transporter to 0.12±0.006. In other words, the production of Glut-4 at 0.02M glucose was found to be increased by < 12 times when compared to that synthesized in the absence of the added glucose in the reaction mixture . It was in vitro translation of Glut-4 mRNA as described in the Materials and Methods, and the quantitation of Glut-4 was made by immunoblot analysis. It was found that the integrated area of the Glut-4 band in the immunoblot in the absence of added glucose in the incubation mixture was 0.399 mm2 which was increased to 1.285±0.064 mm2 in the presence of 0.02M glucose in the incubation mixture . In contrast, increase of the glucose concentration from 0.02M to 0.03M in the incubation mixture actually resulted in the decreased synthesis of Glut-4, as estimated by the integrated area of the band which was 0.694±0.034mm2  metabolic event. It has been reported that the process is under the regulatory control of a stress induced physiologic inhibitor of NO synthesis, identified to be dermcidin isoform 2, which is abundantly synthesized in hepatocytes, endothelial, muscle cells (unpublished) and in leucocytes . This prIt can be concluded from the results as described above on the glucose induced NO synthesis that the activation of GANOS in the liver cell membrane would play a critically important role both in the transportation of glucose and in the synthesis of bioactive insulin from proinsulin genes products in the liver cells. The presence of glucose alone in the liver cells was not sufficient by itself for the synthesis of insulin, and the activation of the liver membrane nitric oxide synthase by glucose played an essential role in the glucose induced synthesis of insulin, at least in the mice hepatocytes.


One of the etiologic agents of Idiopathic Scoliosis (IS) is the sensorineural and/or neuromuscular factor. J.P. Roll demonstrated that: 1) the muscle spindle is the postural adjustment source, 2) the right balance between agonist and antagonist muscles define the posture, 3) the feet are the gravity reference to the central nervous structures and 4) the sub-occipital muscles connect the head muscles with myofascial body lines. Through the theory of the myofascial lines, T. Myers demonstrated the functional continuity of the muscular system in its entirety, thus justifying the huge sensitivity of the Postural System, and the opportunity to act on the myofascial lines by several receptor inputs. The neuromuscular imbalance is a factor favoring the onset, and conditioning the progress, of the IS. The neuromuscular error manages the irregularity of scoliosis below a 25° Cobb angle, accordingly, neuromuscular rebalancing involves a spine realignment to the gravity axis, making an improvement of the curves by braking the worsening push, and producing a clear improvement in aesthetic damages.To present the results of neuromuscular rebalancing on adolescents with IS.Retrospective longitudinal study. In the group of 308 adolescents  182 completed the therapeutic protocol of 24 months neuromuscular rebalancing. At the starting visit, 50 had an aligned spine, 45 a scoliotic attitude, and 87 an IS . All patients were: 1) checked by x-rays, 2) initially Risser zero and 3) carried out only the Neuromuscular Rebalancing according to B. Bricot.Considering the variations equal to, or greater than, 5° Cobb, among the 24 patients with lumbar scoliosis, only one of them (4.17%) had a curve worsening; among the 33 with a thoracolumbar 4 (12.2%); and among the 30 with a double curve 11 (36.6%). The aesthetic damage improves in all patients.The multi-sensory cocktail managing the automatic balance of the Postural System is certainly one of the idiopathic scoliosis etiological factors, probably the predisposing factor to the onset, and conditioning the disease evolution. The double curve scoliosis remains always the most evolutionary. The aesthetic damage in IS is mainly dependent on the Neuromuscular System failure.


Online hearing tests conducted in home settings on a personal computer (PC) require prior calibration. Biological calibration consists of approximating the reference sound level via the hearing threshold of a person with normal hearing.The objective of this study was to identify the error of the proposed methods of biological calibration, their duration, and the subjective difficulty in conducting these tests via PC.Seven methods have been proposed for measuring the calibration coefficients. All measurements were performed in reference to the hearing threshold of a normal-hearing person. Three methods were proposed for determining the reference sound level on the basis of these calibration coefficients. Methods were compared for the estimated error, duration, and difficulty of the calibration. Web-based self-assessed measurements of the calibration coefficients were carried out in 3 series: (1) at a otolaryngology clinic, (2) at the participant’s home, and (3) again at the clinic. Additionally, in series 1 and 3, pure-tone audiometry was conducted and series 3 was followed by an offline questionnaire concerning the difficulty of the calibration. Participants were recruited offline from coworkers of the Department and Clinic of Otolaryngology, Wroclaw Medical University, Poland.All 25 participants, aged 22-35 years (median 27) completed all tests and filled in the questionnaire. The smallest standard deviation of the calibration coefficient in the test-retest measurement was obtained at the level of 3.87 dB (95% CI 3.52-4.29) for the modulated signal presented in accordance with the rules of Bekesy’s audiometry. The method is characterized by moderate duration time and a relatively simple procedure. The simplest and shortest method was the method of self-adjustment of the sound volume to the barely audible level. In the test-retest measurement, the deviation of this method equaled 4.97 dB (95% CI 4.53-5.51). Among methods determining the reference sound level, the levels determined independently for each frequency revealed the smallest error. The estimated standard deviations of the difference in the hearing threshold between the examination conducted on a biologically calibrated PC and pure-tone audiometry varied from 7.27 dB (95% CI 6.71-7.93) to 10.38 dB (95% CI 9.11-12.03), depending on the calibration method.In this study, an analysis of biological calibration was performed and the presented results included calibration error, calibration time, and calibration difficulty. These values determine potential applications of Web-based hearing tests conducted in home settings and are decisive factors when selecting the calibration method. If there are no substantial time limitations, it is advisable to use Bekesy method and determine the reference sound level independently at each frequency because this approach is characterized by the lowest error. Sound systems of modern home electronic equipment, such as a personal computer (PC), tablet, or smartphone, offer opportunities to conduct hearing examinations at low cost and on a large scale -4. The pHearing tests conducted remotely in home settings on PCs can be divided into 2 groups depending on the necessity of conducting prior calibration. The examinations which do not require prior calibration are usually screening tests represented by speech-in-noise tests ,7-9. TheHowever, most hearing tests, including the basic examination in the form of pure-tone audiometry, require prior calibration of the system, and its omission leads to significant measurement errors . CalibraHoneth et al  used bioThe error analysis of the pure-tone audiometry conducted on a PC calibrated by the biological method showed significant influence of the calibration error . The staThe application of pure-tone audiometry based on biological calibration depends significantly on the measurement error. Because of the much larger error of biological calibration than tolerance required by the standards , the hoThis paper presents 7 methods of measuring the calibration coefficients. All measurements were performed in reference to the hearing threshold of a normal-hearing person. For each method, the measurement error was determined, as well as the timeframe for its calibration and the difficulty level. Next, 3 methods were proposed for determining the reference sound level on the basis of these calibration coefficients and an error analysis was conducted for each.The proposed methods of biological calibration consist in measuring the calibration coefficient that describes the threshold sound level of the reference person. Seven calibration methods were proposed: (1) calibration using an amplitude-modulated signal, (2) calibration using 2 sounds differing by 5 dB, (3) calibration using 2 sounds differing by 2 dB, (4) the ascending method with a 5-dB step, (5) the ascending method with a 2-dB step, (6) calibration based on Bekesy audiometry using the continuous signal, and (7) calibration based on Bekesy audiometry using an amplitude-modulated signal. In methods 1-5, the assessment was conducted for the following frequencies: 125 Hz, 500 Hz, 1 kHz, 2 kHz, 4 kHz, 6 kHz, and 8 kHz. In methods 6 and 7, the frequency was changed in a continuous way from 62.5 Hz to 16 kHz. The sound signal was presented bilaterally.In the calibration with amplitude-modulated signal (method 1), the presented signal was amplitude-modulated using rectangular envelope with frequency of 1 Hz and modulation depth of 100%. The task of the reference person was to set the volume marker in such a way that the generated sound be barely audible. The step of the volume marker was 1 dB.During calibration with 2 sounds differing in intensity (methods 2 and 3), 2 tone signals with a given frequency and a duration of 1 s were presented in turns. The task of the reference person was to set the volume marker in such a way that the louder of the 2 sounds was still audible, and the quieter inaudible. In method 2, the signals differed by 5 dB, whereas in method 3, the difference was 2 dB. The step of the volume marker was 1 dB.The ascending method (methods 4 and 5) was based on the ascending algorithm used for the assessment of the hearing threshold in pure-tone audiometry . A signaDuring calibrations based on Bekesy audiometry, the frequency of a presented signal was increased at the speed of 1 octave/60 s, simultaneously with the change of its intensity. The task of the reference person was to press a button on hearing the signal and keep it pressed for as long as the sound was audible. The intensity of the sound was reduced at a speed of 2 dB/s when the sound was audible, and increased at the same speed when the sound was inaudible. The value of the calibration coefficient was determined as the mean of the values of the sound intensity at which a change in the status of the button occurred, after rejecting the outliers on the basis of the Grubbs’ test . CoefficAll 7 methods were implemented in Java technology in the form of applets embedded in a Web browser. The calibration coefficients expressing the sound intensity in decibels, together with the duration of examinations, were recorded in the database. On completion of all the tests, the participant filled in an offline questionnaire  on the dIn addition to the 7 methods of measuring the calibration coefficients, 3 methods were proposed for determining the reference sound level: (1) the reference sound level determined independently for each frequency depending on the value of calibration coefficient measured at this frequency, (2) the reference sound level estimated by a model fitted to calibration coefficients determined at all frequencies, and (3) as (2) except the model was fitted to a single coefficient determined at the frequency characterized by the smallest measurement error.Participants were recruited offline from coworkers of Department and Clinic of Otolaryngology, Wroclaw Medical University, Poland, using face-to-face prompting from September 2012 to March 2013. The eligibility criteria were age younger than 35 years, lack of previous hearing problems, owning headphones and a PC at home and basic skills to operate it, and the willingness to participate in the research. Each participant performed calibration using all 7 methods 3 times. In series 1, the study was carried out in a sound booth with the use of notebook Dell Vostro 1310 with Microsoft Windows 7 operational system and Technics RP-F290 headphones; in series 2, each person was asked to perform calibration on their own home computer using their own headphones in the quietest conditions possible, preferably late in the evening or at night to minimize background noise level and to create conditions close to those in the sound booth; and series 3 was the repetition of examinations from series 1. Because of the relatively long duration of the series, the participants were informed about the option of taking a break when they felt tired, and most of the participants took advantage of this. In series 1 and 3, pure-tone audiometry was performed with the use of a clinical audiometer Interacoustic AD229e and TDH-39 headphones calibrated in accordance with ISO 389-1:1998. The hearing threshold was determined by using the ascending method in accordance with ISO 8253-1:2010. Additionally, based on the pure-tone audiometry, the bilateral hearing threshold was calculated by choosing for each frequency the threshold of the ear that heard better at this particular frequency.A test-retest analysis of calibration coefficients was conducted, as well as 1-way ANOVA for measurement duration and difficulty. Calibration errors were determined by means of variance estimation. Statistical analyses were performed on the basis of confidence intervals that were estimated in the same way. Estimation of the variance was conducted based on measurement variances and their confidence intervals calculated from the variance and the sample size .P=.05. At P=.01, this relation was insignificant for all the methods. The smallest standard deviation was obtained for the Bekesy (modulated) method.The 25 participants , aged between 22 and 35 years (median 27), who took part in the study completed all the examinations and filled in the questionnaire. All participants were skilled in computer use. On the basis of series 1 and 3, a test-retest analysis was conducted. For each method, a mean difference, standard deviation of the difference and corresponding confidence intervals were calculated . Mean vaP<.001).Durations of calibration in relation to the calibration methods are presented in The shortest times were obtained for the modulated signal method and both dual tone methods (5 and 2 dB), which consisted in self-adjusting the volume marker. The mean duration of calibration based on the ascending method with the step of 5 dB was comparable to the duration of calibration using both Bekesy methods . In the Bekesy methods, the outliers are those examinations that were paused momentarily.P<.001). The easiest method of calibration was the modulated signal method consisting in self-adjusting the volume marker in such a way that the presented tone was barely audible. Subsequently, the easiest methods were based on Bekesy’s audiometry –FR(fi) (1)C) is the mean value of coefficients Ci and FR(fi) is the frequency response of the model.where meani=Ci−ciy (3)i=Mi−ciz (4)X and Y are independent. The standard error of the model fitted to the real calibration coefficients does not depend on the determination error of these coefficients. Therefore, bearing in mind that the variance of the sum of 2 independent random variables is the sum of their variances, the desired variance of the random variable X is:It is worth noting that random variables X)=variance(Z)–variance(Y) (5)variance(c)≈mean(C). Then, on the basis of equation 1 we can calculate that Mi≈mi, where mi is the model estimated on the basis of coefficients ci:The mean value of the determined coefficient is close to the mean value of the real coefficient mean(mi=mean(c)–FR(fi) (6)Mi≈mi, the variance of random variable Z may be calculated on the basis of the difference between coefficients ci and the model mi estimated on their basis, according to equation 7 . Therefore, standard deviation of the random variable Y expressing the standard error of coefficient ci depends on the measurement error of calibration coefficient and the measurement error of the bilateral hearing threshold. Both measurement errors were calculated on the basis of the standard deviation of the differences in test-retest examination – (8)Y) = 2/2+2/2 (9)variance of the residual of the model was calculated (equation 7), and averaged for every calibration method. Next, for each calibration method, variance(Y) was computed on the basis of the standard deviation of the test-retest examination (equation 9 and X) was estimated independently for each calibration method 2+2/2 (11)P=.05.The standard deviation of the bilateral hearing threshold difference in test-retest examination was calculated jointly for all frequencies due to lack of significant differences between frequencies in the 1-way ANOVA at the level of statistical significance Analogical computation were carried out for the mean value of bilateral hearing threshold, assuming the measurement error divided by a square root of 8 as the mean was for 8 frequencies .The error of the independent coefficients method  depends on the distribution of the bilateral hearing threshold in the population and the measurement error of the calibration coefficient. Measurement error of the calibration coefficient can be easily calculated from the standard deviation of the difference in the test-retest examination by dividing its value by the square root of 2. Therefore, the mean error of the independent coefficients method across all frequencies may be expressed in the following equation:2=2+2/2 (12)(independent coefficients SD)where bilateral threshold in the range 125 Hz-8 kHz SD is the standard deviation of the bilateral hearing threshold calculated jointly for all values reduced by the mean at relevant frequencies .The modeled coefficients method consists in estimation of the reference sound level on the basis of the model fitted to the mean value of 8 calibration coefficients determined at various frequencies. Therefore, its error is connected with distribution of the mean bilateral threshold, the error of determining the mean of 8 calibration coefficients, and the standard error of the model. Similarly, as for the independent coefficients method, the error of mean of 8 coefficients can be calculated from the standard deviation of the difference in test-retest examination by dividing its value by the square root of 2, to obtain the error for single coefficient, and by the square root of 8, to obtain the error for the mean. Thus:2=2+2/16+(model SD)2 (13)(modeled coefficients SD)Finally, the error of single frequency method consisting in estimating the reference sound level determined on the basis of the model fitted to 1 calibration coefficient at the frequency with the lowest standard deviation will be:2=2+2/2+(model SD)2 (14)(single frequency SD)The standard errors of each method are presented in This paper presents methods of biological calibration of a PC for hearing examination by determining the reference sound level on the basis of the hearing threshold of the reference person. Seven methods of measuring calibration coefficients and 3 methods of determining reference sound level on the basis of these coefficients were proposed and analyzed. On the basis of 3 series of measurements conducted by 25 participants, the difference between classical pure-tone audiometry and audiometry based on biological calibration was estimated. The smallest standard deviation of the difference was obtained for the Bekesy (modulated) method with the independent coefficients method at the level of 7.27 dB (95% CI 6.71-7.93).The lowest standard deviation in test-retest examination at the level of 3.87 dB (95% CI 3.52-4.29) was obtained using the Bekesy (modulated) method, which entails assessment of the hearing threshold by means of the amplitude-modulated sound according to the rules of Bekesy’s audiometry. This value is in-line with the standard deviation of the test-retest examination of Bekesy’s audiometry . The BekP=.05). The highest error occurred for the single frequency method. However, when compared with the modeled coefficients method, statistical significance was achieved only for the dual tone (5 dB) method (P=.05).The estimated error of determining reference sound level turned out to be the lowest for the independent coefficients method and higher for the modeled coefficients method . This reP=.05).The standard error of the modeled coefficients method was estimated for the model determined in the frequency range 125 Hz-8 kHz. When the range is limited to 250 Hz-8 kHz, the standard error of the model decreases from 6.57 dB (95% CI 5.59-7.54) to 5.98 dB (95% CI 4.45-7.50). This improves the modeled coefficients method, but the independent coefficients method is still more accurate. However, in this case the relation remained statistically significant only for the Bekesy (modulated) method  than the results presented in some studies [Calibration error strongly depends on the hearing threshold of the reference person. This applies especially to the independent coefficients and single frequency methods, in which the sound reference level at a single frequency is determined on the basis of a single measurement, contrary to modeled coefficients method, which uses mean hearing threshold. To verify the obtained results, the distribution of the hearing threshold of the participants was compared with literature data . The sta studies -22, is i studies , and is  studies -27.The examinations in this study were conducted on young employees and interns of the Otolaryngology Clinic . This may have led to better calibration results and shorter duration of the examination than in a population of young people with good hearing without experience with hearing examinations.In the calculations, it was assumed that the examinations conducted on home computers are not burdened with an error resulting from the presence of background noises other than the fan noise. This assumption was made because during home examinations and in those conducted in the sound booth the fan noise was the loudest and the most disturbing sound. Thus, the estimated calibration error takes into account the fan noise. However, in the case of other background noises, the error may turn out to be bigger.Calibration methods presented in the paper were implemented as Java applets embedded in browsers. However, their application is not limited only to Web-based tests, but may also be used for offline determination of the reference sound level or on mobile devices. Moreover, in the case of tablets or smartphones, calibration error may turn out to be smaller because of the lack of fan noises.When conducting examinations on a PC with the use of headphones with very high sensitivity instead of regular ones, interferences of the sound card or other electronic systems may affect the stimulus. During examination at home, such incidents occurred in 2 of 25 cases. As a result, it was impossible to perform the examination. After changing headphones from professional to regular ones, the examination was completed without any problems.Calibration accuracy may be improved if it is conducted by 2 or more reference persons . The greAnother method of improving the accuracy is to introduce additional conditions to reject inaccurate calibrations. For example, calibration using the independent coefficients method may be rejected as the difference between coefficients exceeds the predetermined threshold . In the The final choice of the calibration method will depend on the desired accuracy of calibration and the time for its performance. If considerable accuracy is required, it is advisable to use the independent coefficients method, whereas when quick calibration is the priority, the single frequency method is preferable. The application of the modeled coefficients method is not justified because of higher calibration error than is in the independent coefficients method at the same duration.Two of the 7 methods of measuring calibration coefficients seem worth noting: the modulated signal and Bekesy (modulated) methods. The choice of the better of the 2 is not obvious. The Bekesy (modulated) method is the most accurate at moderate duration, whereas the modulated signal method is the fastest at moderate accuracy. Additionally, the modulated signal method is the easiest, and the Bekesy (modulated) method is the second easiest. However, the methods differ significantly in the complexity of implementation with the Bekesy (modulated) method being more complex. On the other hand, in the case of Bekesy (modulated) method, the measurement can be easily verified on the basis of the differences between the intensities at which the stimulus starts or stops being audible.Therefore, if there are no substantial time limitations, it is advisable to use Bekesy (modulated) method with independent coefficients method, which have the lowest error. When a simple and quick calibration is required, modulated signal method with single frequency method should be chosen.


Our results indicate that the proposed method will offer significant advantages in the investigation of the galvanic coupling intra-body communication.Simulation based on the finite-element (FE) method plays an important role in the investigation of intra-body communication (IBC). In this paper, a finite-element model of the whole body model used for the IBC simulation is proposed and verified, while the FE simulation of the galvanic coupling IBC with different signal transmission paths has been achieved. Firstly, a novel finite-element method for modeling the whole human body is proposed, and a FE model of the whole human body used for IBC simulation was developed. Secondly, the simulations of the galvanic coupling IBC with the different signal transmission paths were implemented. Finally, the feasibility of the proposed method was verified by using  As a result, IBC technology is proposed as a novel and promising technology for Wireless Body Area Networks (WBANs) [etc. On the other hand, galvanic coupling IBC is an important approach to achieve the signal transmission within the human body [Intra-body communication (IBC) is a technology that involves using the human body as a transmission medium for electrical signals . Compare (WBANs) ,2, biome (WBANs) ,4 and suman body ,6. In thman body . Meanwhietc.) or earphones located on the head. Sometimes, signals may transmit from IBC shoes to the other parts of the human body . As a result, the development of the FE model of the whole human body and the investigation of the potential distribution corresponding to different signal transmission paths will help to disclose the signal transmission characteristics of the human body. Unfortunately, due to the complex structure of the human body, it has proved difficult to develop a FE model of the whole human body. For instance, although a precise geometry model of the human body can be achieved by using Magnetic Resonance Imaging (MRI) or Computerized Tomography (CT) technology, the modeling of the whole human body based on these technologies will be a complicated work and result in huge computational cost. Therefore, only the FE model of upper arm has been developed [et al., who first developed an upper arm model and then achieved the simulation of the galvanic coupling IBC [To guarantee the safety of the human body, many IBC experiments were carried out by using simulation methods. Therefore, software simulation serves as an important method in the investigation of intra-body communication. The software simulation of IBC can be implemented by using the transfer function method ,9, in wheveloped –12 by Weling IBC .in vivo measurements, whereby some important conclusions are deduced.In this paper, a finite-element model of the whole body model used for the simulation of the galvanic coupling IBC is firstly proposed and verified, while the FE simulation of the galvanic coupling IBC with the different signal transmission paths has been achieved. First, we discuss the theoretical foundation of the finite-element method for modeling human body. Secondly, the modeling of the whole human body and the corresponding electromagnetic parameters are described, and then the simulation of the galvanic coupling IBC with the different signal transmission paths are implemented and the corresponding potential distributions are discussed in detail. Finally, the feasibility of the proposed model is verified by using in vivo measurements. Section 5 concludes the paper.The rest of the paper is organized as follows: Section 2 focuses on the geometry modeling and the corresponding parameters of the whole human body. The galvanic coupling IBC simulation conditions and the results discussion are presented in Section 3. Section 4 mainly verifies the feasibility of the proposed model by using 2.2.1.The coupling between electromagnetic signals and human body can be explained by the Maxwell equations and the boundary conditions. According to , the indJ̅ = σE̅ and D̅ = εE̅, then the quasistatic electric field of the intra-body communication can be described as follows [In the galvanic coupling IBC application, due to the fact that electronic signal is transmitted and received as electronic form, as a result, only the electric field is of interest ,10. TherBased on the equations mentioned above, the electronic signal transmission within the human body can be simulated by using the finite-element method, which is applied to solve the quasi-static volume conducting boundary problem by the numerical solution of the partial differential equations.2.2.A human body part (such as the upper arm) can be abstracted as a concentric cylinder with multiple layers in the simulation of the interaction between the electromagnetic signal and the human body ,14. SimiThe Modeling of Head and Neck: In our modeling, the head was modeled geometrically as a semi-sphere and a concentric cylinder, which have the same radius of 80 mm, as shown in The Modeling of Torso: According to the geometry of the actual torso of human body, the torso of the developed model was abstracted as an elliptic cylinder, which has a long-axis length of 265 mm, a short-axis length of 195 mm and a height of 590 mm, as shown in The Modeling of Arm and Leg: As shown in The Connection of the Body Part Models: Finally, the models of the human parts  were connected with each other, thereby forming the finite-element model of the whole human body. Generally, the connection of the different FE models can be achieved by using Boolean operations. However, due to the big size and the complexity of the whole human body, this process will generate huge unit numbers and prolong the simulation time. In our modeling, all the nodes within a certain range in the contact surface of the two FE models were combined into one node. As a result, the process time of mesh generation was limited in an acceptable range (30 min).2.3.et al. [σ represents the conductivity and rε represents the relative permittivity. According to the electromagnetic parameters measured in the previous works [The electromagnetic parameters are essential to achieve the IBC simulation based on the finite-element method. The electromagnetic characteristics of human tissues  can be described by their conductivity and relative permittivity. On the other hand, the conductivity and relative permittivity of the different tissues have been investigated by Gabriely et al. ; some ofus works , the con3.3.1.σ) and the relative permittivity (rε) were set as 5.99E7 and 1 [The simulations of the galvanic coupling IBC with different signal transmission paths were carried out using the proposed FE model. The simulations were produced with the electromagnetic analysis package (EMAG) of ANSYS, while the mesh sizes were set between 400,000 and 450,000 elements. To isolate the human body model against air, the normal component of the electric field corresponding to the boundary between the skin and the air was set to zero . AdditioE7 and 1 , respect3.2.Signal transmits from the torso: We can also find from Signal transmits from the arm: The potential distribution when the IBC transmitting electrodes were attached on the arm (A1) of the whole body model is shown in Signal transmits from the leg: 4.4.1.in vivo measurements of the galvanic coupling IBC with the different signal transmission paths were carried out. The measurement setup, as shown in To verify the feasibility of the proposed FE model, 4.2.10(rU/tU) [rU and tU are the input voltage and the receiving voltage of IBC path, respectively. In our calculation of the signal attenuations corresponding to the in vivo measurements, rU is the measured value of the differential voltage between the two receiving electrodes, while tU is the output voltage of the signal generator. Meanwhile, in order to calculate the signal attenuations corresponding to the IBC simulations, rU was represented as the potential difference between the two receiving electrodes, while tU was represented as the potential difference between the two transmitting electrodes.In the investigation of IBC, signal attenuation can be determined by using 20·logSignal transmits from the arm: in vivo measurements and the simulation results corresponding to the paths starting from the right arm (A1), in which in vivo measurement results within the 10 kHz–5 MHz frequency range. Both the simulation results and the measurement results decrease gradually as the signal frequency increases from 10 kHz to 200 kHz. For instance, the absolute attenuation value of the simulation result decrease from 33.86 dB to 23.37 dB within the range of 10 kHz to 200 kHz, while the corresponding value of the measurement also decrease from 32.40 dB to 24.73 dB. Meanwhile, both of the two curves keep a slight variation as the signal frequency increase from 200 kHz to 5 MHz. Additionally, the errors between the simulation and the measurement are limited in an acceptable range, while the maximum absolute value of the errors is only 1.46 dB.in vivo measurement curves corresponding to the A1T2 and A1H1 paths also have similar outlines, while the absolute values of the errors between the simulation and the measurement are limited to 4.03 dB (AlT2) and 6.36 dB (AlH1), respectively, which indicate that the simulation results corresponding to the two paths basically agree with the corresponding measurement results. Moreover, compared with the A1A2 path, the errors between the simulation and the measurement show a slight increase, which is approximately equal to the corresponding errors of the previous results [Similarly, we can find from  results .Signal transmits from the leg: in vivo measurement results decrease as the signal frequency increases from 10 kHz to 500 kHz, while displaying a slight variation as the signal frequency increased from 500 kHz to 5 MHz. For instance, the absolute attenuation value of the simulation result decreases from 30.67 dB to 18.14 dB within the range of 10 kHz–500 kHz, while the corresponding measurement value also decreases from 33.15 dB to 18.49 dB. Meanwhile, the maximum variation range of the simulation result within 500 kHz–5 MHz is 1.75 dB, while that of the measurement results is 3.28 dB. Additionally, the errors between the simulation and the measurement with respect to the frequency range of 10 kHz–5 MHz are limited to 4.10 dB. Similarly, we also can find from On the other hand, due to the fact that both the L1T1 path and the L1A1 path consist of leg and torso, as shown in 5.in vivo measurements have been carried out, and some important conclusions can be drawn as follows: (1) in the galvanic coupling intra-body communication, the relatively higher potentials are mainly focused at the position of the transmitting electrode injecting current signal into the human body, while the potential decreases along the signal transmission path within the body model; (2) according to the simulation results, the relatively higher potential level is mainly focused on the skin layer and the fat layer, while the muscle layer has the relatively lower potential, which is in aggremment with Wegmueller's conclusion [in vivo measurement results within the 10 kHz–5 MHz frequency range; (4) the signal transmission distance has relatively less influence on the signal attenuation within the 10 kHz–100 kHz range, while it begins to influence the signal attenuation within the 100 kHz–500 kHz range and has a relatively high influence on the signal attenuation within the 500 kHz–5 MHz range. Additionally, the distance sensitivity of the proposed FE model is similar to that of the actual human body. The proposed finite-element model of the whole human body has been used for the simulations of the signal transmission among the sensors attached on the different parts of the human body. Our next work will focus on the simulations of the signal transmission within implant sensors.The development of a FE model of the whole human body and the investigation of the potential distribution corresponding to different signal transmission paths will help to clearly disclose the signal transmission characteristics of the human body. In order to meet this requirement, a finite-element method for modeling the whole human body is proposed in this paper, while both the simulations of the galvanic coupling IBC based on the whole human body and the corresponding nclusion ; (3) the


Objectives: In the clinical management of facial pain, a possible cervical origin must be considered. A clinical exploration is therefore essential. The disorder originates in the intimate connections between the cranial portion of the spinal cord and the trigeminal system. Although solid evidence supporting the use of radiofrequency (RF) treatment is lacking, it remains one of the management options to be taken into account. The present study evaluates the efficacy of RF in application to cervicogenic headache.Study design: We present three cases of severe facial pain arising from different cervical structures. Results: In two cases the pain originated in cervical roots C2 and C3, while in the third patient the trigger point was located at the level of the atlantoaxial joint. Pulsed RF was applied for 4 minutes at the dorsal ganglion of C2 and C3 in the first two cases, and for 8 minutes at intraarticular level in the third patient. The pain gradually subsided during the first month in all cases. The first two patients reported 70% improvement after one month, 60% improvement after 6 months, and 30-50% after one year, versus baseline. The third patient reported complete pain resolution lasting approximately 5 months, after which the pain reappeared with the same intensity as before. Conclusions: Radiofrequency is a satisfactory treatment option, affording adequate analgesia, though the effects are sometimes temporary. Key words:Cervicogenic headache, pulsed radiofrequency, analgesia. The idea that headache may originate in different neck structures is not new. Barré and Bärtschi-Rochaix described the condition in the first half of the twentieth century . The keyThe present study describes three cases of severe cervicogenic facial pain subjected to treatment with pulsed RF targeted to the dorsal ganglion of cervical roots C2 and C3 in two cases, and to the atlantoaxial joint in the third patient.Three patients were treated in our Unit due to craniofacial pain of cervical origin, according to the criteria of the International Headache Society .Case 1: A 64-year-old woman presented with a 17-year history of pain that had worsened in the last year. The pain was constant, with attacks that could last days or weeks, and with a mean intensity of 8/10. It was described as being located in the left periorbital zone, and in the last year additional pain had developed at ipsilateral occipital level, with the limitation of neck movement. The condition showed a clear association to stress, and improved with medication (indomethacin and prednisone). Exploration revealed limitation of left lateral flexion and rotation of the neck, with pain in response to palpation of the left greater and lesser occipital nerves. Treatment consisted of pulsed radiofrequency targeted to the left dorsal ganglion of C2 and C3.Case 2: A 37-year-old woman presented with bilateral temporomandibular joint dysfunction and bruxomania. She had suffered constant pain during the past year and a half, with occasional episodes of worsening , and a mean intensity of 8/10. The pain was described as being located in the preauricular zone, irradiating towards the occipital region and mouth, on both sides of the head, and worsened on speaking and chewing. The patient was taking amitriptyline and ketazolam, and did not tolerate antiinflammatory medication. Physical examination revealed pain in response to bilateral palpation of the temporomandibular joint and greater occipital nerve. The pain worsened with movement of the neck. Cervicogenic headache was diagnosed, and treatment was provided in the form of pulsed radiofrequency targeted bilaterally to the dorsal ganglion of C2 and C3  lasting about 7 days, but which had worsened in the last three years, becoming constant with a baseline intensity of 4/10. However, maximum pain scores were reached in the course of 4-5 daily crises. The pain was described as being initially located in the left maxillary and periorbital region, but subsequently displaced towards more posterior cervico-occipital zones, with periorbital irradiation. He had undergone three infiltrations with 4 mg of dexamethasone and 1.5 ml of 3% articaine solution in the sphenopalatine ganglion. The first infiltration afforded total pain relief for 15 days, while the following two infiltrations proved less effective. Treatment was being provided in the form of baclofen (10 mg every 8 hours), indomethacin (25 mg every 8 or 12 hours) and bromazepam (1.5 mg every 24 hours). Physical examination revealed limitation of left rotation and extension of the neck because of the pain, and trigger points were identified in the greater occipital nerve and left atlantoaxial joint region. Treatment consisted of pulsed radiofrequency targeted to the left atlantoaxial joint .The pain gradually subsided during the first month in all cases. Patients 1 and 2, subjected to dorsal ganglion RF treatment, reported 70% improvement after one month, 60% improvement after 6 months, and 30-50% after one year, versus baseline. Patient 3, subjected to intraarticular RF therapy, reported complete pain resolution after two months. This benefit lasted approximately 5 months, after which the pain reappeared with the same intensity as before  innervated by segmental nerves C1, C2 and C3 are possible sources of cervicogenic headache . The preDrug treatment continues to play a fundamental role in these patients. There is no general agreement regarding the treatment of choice for cervicogenic headache, since lack of knowledge of the true mechanism underlying this type of pain obliges us to resort to symptomatic treatments in most cases. There is no solid evidence supporting cervical facet denervation with radiofrequency (RF) or pulsed RF targeted to the dorsal ganglion of the superior cervical roots. At present, the treatment of choice could be regarded as infiltration of the greater occipital nerve (or nerve of Arnold) with local anesthetic and corticosteroids. If this proves ineffective, facet denervation of the highest joints would be the best management choice. A third option in the case of failure of facet denervation would be pulsed RF treatment of the ganglion of the dorsal root of C2 and C3 ,9,10. ThOur patients had received antiinflammatory treatment, though at the time of the visit these treatments were clearly ineffective, and the patients suffered continuous pain associated to variable episodes of worsening. Only one patient had undergone infiltrations of the sphenopalatine ganglion, with uncertain results. This technique is presently not recommended when a cervical origin of facial pain is suspected. If infiltration is performed, the target should be the greater occipital nerve, and even the atlantoaxial joint. As can be deduced from the above recommendations, RF should be targeted to the medial branch of the posterior root of the cervical spinal nerves, with level of evidence 2B+-, rather than to the dorsal ganglion of roots C2 and C3 – for which the level of evidence is 0 , Table 4In the clinical management of facial pain, a possible cervical origin must be considered. A clinical exploration is therefore essen-tial. Regarding the treatment options, radiofrequency affords adequate analgesia (> 50%), though the effects are sometimes temporary.