A forward head and rounded shoulder posture is a poor posture that is widely seen in everyday life. It is known that sitting in such a poor posture with long hours will bring health issues such as muscle pain. However, it is not known whether sitting in this poor posture for a short period of time will affect human activities. This paper investigates the effects of a short-duration poor posture before some typical physical activities such as push-ups. The experiments are set up as follows. Fourteen male subjects are asked to do push-ups until fatigue with two surface electromyography (sEMG) at the upper limb. Two days later, they are asked to sit in this poor posture for 15 min with eight sEMG sensors located at given back muscles. Then they do the push-ups after the short-duration poor posture. The observations from the median frequency of sEMG signals at the upper limb indicate that the short-duration poor posture does affect the fatigue procedure of push-ups. A significant decreasing trend of the performance of push-ups is obtained after sitting in this poor posture. Such effects indicate that some parts of the back muscles indeed get fatigued with only 15 min sitting in this poor posture. By further investigating the time-frequency components of sEMG of back muscles, it is observed that the low and middle frequencies of sEMG signals from the infraspinatus muscle of the dominant side are demonstrated to be more prone to fatigue with the poor posture. Although this study focuses only on push-ups, similar experiments can be arranged for other physical exercises as well. This study provides new insights into the effect of a short-duration poor posture before physical activities. These insights can be used to guide athletes to pay attention to postures before physical activities to improve performance and reduce the risk of injury. Poor posture is regarded as prolonged deviations from the neutral spine, and it usually can be characterized by the forward head posture, rounded shoulders, and increased thoracic kyphosis  was used to collect sEMG signals for analysis. Two sources of sEMG signals were used during the experiment, (1) the sEMG signals were collected from biceps brachii and triceps brachii during push-up exercises in the two sessions. (2) During the poor posture sitting, sEMG signals were collected from eight upper back muscles, including the right upper fibers of trapezius , left upper fibers of trapezius , right middle fibers of trapezius , left middle fibers of trapezius , right lower fibers of trapezius , left lower fibers of trapezius , right infraspinatus , and left infraspinatus . Locations of the eight sensors was illustrated in t-test or Wilcoxon signed rank test was performed to compare the frequency changes of push-up exercise in the two experiment sessions. One-way Analyses of Variance (ANOVA) with repeated measures or Kruskal-Wallis test was employed to measure frequency changes among different levels  during the poor posture sitting. The Tukey post hoc test was used to investigate the significant difference between different levels. The significance value was set at 0.05, and the data was presented as mean value ± standard deviation.Standard statistical analysis was performed on the collected data. Prior to the statistical analysis, data normality was checked with the Shapiro-Wilk test. Depending on whether the data distribution is normal or not, paired The signal processing procedures for the sEMG signal collected from upper limb muscles during the push-up protocol are shown as The MDF and MNF are then calculated for each burst, the Hamming window with length of 100 ms is used to obtain each segment in sEMG signal, the envelope of the windowed signal is illustrated as p = 0.025) smaller than those after poor posture. The statistical analysis of the difference value between the start and the end frequency is shown as p = 0.002), which indicates that the muscle fatigue process has a smaller decreasing percentage after poor posture.The fatigue analysis with the mean MDF for push-up before and after the poor posture protocol is demonstrated as p = 0.007; p = 0.020) smaller than those obtained after poor posture. The similar comparison can be found in Similar results were obtained by calculating the mean MNF values of the triceps brachii. It can be seen from section 3.1 that significant differences are observed for the push-up before and after the short-time poor posture sitting in terms of the frequency change in mean MDF and MNF, which indicates the poor posture has influence on the performance of physical exercise of the group. In this section, a detailed investigation of upper back muscles related to the poor posture is presented to evaluate the changes and sensitive frequencies of upper back muscles during the poor posture sitting.As described in the experiment setup, eight wireless sensors are employed to collect sEMG signal from upper back muscles during the poor posture sitting. An illustration of the raw sEMG signals collected from the LLFT muscle during the 15 min poor posture sitting is demonstrated in As indicated in previous studies  wavelets, the Symlets (sym) wavelets, the Biorthogonal (bior) wavelets, and the Coiflets (coif) wavelets. The decomposition level was set as 7 to obtain a fine resolution in the frequency domain  was calculated for all windows. p = 0.024). As demonstrated in p = 0.023) than the value of level 2 around 37 Hz. The similar frequency differences can be observed in p = 0.0095).It can be seen from p = 0.025), the frequency differences between the start and the end of the push-up (p = 0.025), and the rate of the frequency differences (p = 0.017). Second, it is confirmed from experiments and data analysis from sEMG sensors, some parts of back muscles are affected during the short time of poor posture with a clear indication of fatigue, which has not been observed in literature. As indicated in There are two major findings in this study, first, the short-time poor posture indeed has an effect on the performance of physical exercise. Comparing the subjects' performance of push-ups before and after poor posture, the median frequencies of the two sessions have significant differences after 10 push-ups in terms of the slope of the trend  after the poor posture. People should be mindful of their postures if they will undertake physical activities in the near future.The current study has some limitations. First, only male subjects were recruited for the study, as the experiment requires placing eight sensors on the back muscles, due to the privacy issue and lack of female team members to collect data from female volunteers. We will further investigate whether there has gender difference for the results. Second, due to the limitation of available data, the current study does not include the results from natural posture. The next step research will recruit more subjects to evaluate the effect of poor and natural postures, and investigate the prevention to the immediate and latent effects of muscle fatigue from the poor posture, which will provide know-how and awareness to prevent during sports and most importantly the activities of daily living (ADLs).This study contributed to the understanding of a particular short-duration poor sitting posture on the effect of the performance of physical exercise and the fatigue of back muscles. From statistics of 14 subjects, it was observed that the median frequencies of the sEMG signal of the triceps brachii has significant differences after poor sitting posture, and the median frequencies collected from the RI muscle in the upper back have been shifted in different time levels. The results suggest that the poor posture will lead to a quicker fatigue procedure of the following physical exercise as shown in the median frequency of the corresponding muscles. The results also demonstrated that the proposed frequency analysis techniques were able to identify the frequency range of the individual muscle, which can be used to show a clear shift of median frequencies of noisy sEMG signals in order to represent fatigue.The datasets that support the findings of this study are available from the corresponding author upon reasonable request.The studies involving human participants were reviewed and approved by the Human Research Ethics Committee of the University of Melbourne (#1954575). The patients/participants provided their written informed consent to participate in this study.LL, XG, MR, KG, and YT contributed the experiment design and data collection. LL and YT contributed the data analysis and drafting the manuscript. LL and MR performed the statistical analysis. KG, IM, and DO contributed the manuscript revision and results interpretation. All the authors read and approved the manuscript.IM is employed by the IBM Research - Australia. KG is a sport physiotherapist for the Melbourne University Elite Athlete Unit, and CEO of the Elite Akademy Sports Medicine. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There is limited information about the initiation of triple therapy (TT) in patients with chronic obstructive pulmonary disease (COPD) in primary care. This was an observational, population-based study in patients identified from a primary care electronic medical records database in Catalonia from 2011 to 2015 aimed to identify the use of TT in patients with newly diagnosed COPD. A total of 69,668 newly diagnosed patients were identified of whom 11,524 (16.5%) initiated TT, of whom 8626 initiated TT at or immediately after COPD diagnosis. Among them, 72.3% were GOLD A/B, 14.6% were frequent exacerbators, and 7.1% had asthma–COPD overlap (ACO). Variables associated with TT initiation were: male sex, older age, previous exacerbations, ACO, a previous treatment regimen containing an inhaled corticosteroid, previous pneumonia, and history of lung cancer. A significant number of COPD patients in Primary Care initiated TT shortly after or even before an established COPD diagnosis. Patients with milder forms of the disease are frequently overtreated and, although less frequently, patients with more severe COPD may be undertreated4. In a majority of countries, the initial point of contact for patients with respiratory symptoms is through primary care where targeted detection programs for the early diagnosis of COPD are supported6.Treatments administered for patients with chronic obstructive pulmonary disease (COPD) in primary care do not always follow national or international guidelines7, and initial treatment should be implemented according to guidelines. The Global Strategy for Obstructive Lung Disease (GOLD) recommends classifying patients according to severity of symptoms and history of exacerbations into four categories. Initial treatment is based on one or two long-acting bronchodilators, or the combination of a long-acting beta-2 agonist (LABA) and an inhaled corticosteroid (ICS) for more symptomatic patients with frequent exacerbations8. When treatment with long-acting bronchodilators or with LABA/ICS is not enough to control symptoms or prevent exacerbations, treatment may escalate to triple therapy (TT) with two bronchodilators (a LABA and a long-acting antimuscarinic agent [LAMA]) and ICS. Similarly, the Spanish guidelines also recommend TT as a step up from dual bronchodilation (LABA/LAMA) or from LABA/ICS, but not as initial therapy9.The availability of spirometry in primary care is key for early and accurate diagnosis of COPD2; similarly, data from Switzerland observed a use of TT in 13.8% of GOLD A and 28.2% of GOLD B patients1. It is important to understand whether this represents overtreatment of COPD or whether some of these patients could have been GOLD D patients that improved in the GOLD classification because of the efficacy of TT. In order to understand the reason for TT prescription in primary care, it is important to analyze prescription patterns in large administrative databases. The current study was conducted with the aim of characterizing newly diagnosed patients with COPD initiating TT in primary care and investigating the factors associated with TT prescription in these incident cases with COPD.Studies from different countries have demonstrated a frequent use of TT in patients considered at low risk (patients with GOLD A or B disease). A recent study in the United Kingdom showed that 13.7% of GOLD A and 26.2% of GOLD B received TTA total of 69,668 newly diagnosed (incident) COPD patients were identified; of whom 11,524 (16.5%) initiated TT at some point during the study period. Among these patients, 2898 (25.2%) initiated TT prior to COPD diagnosis, and 8626 (74.8%) at or after COPD diagnosis , with mean age of 68.3 years (standard deviation [SD]: 12.3 years). Less than half (44.3%) had spirometry tests performed within 12 months before and 2 weeks after the index date, mean FEV1 (% predicted) was 65.3% (SD: 19.6%) and 47.6% had at least one exacerbation in the previous year (Table 2 test p < 0.001) and had more previous visits to primary care ; but less frequently had available spirometry records  than patients who did not initiate TT. These differences were also observed between patients who initiated TT before diagnosis, with more exacerbations  and patients with spirometry tests  compared to patients who initiated TT at or after COPD diagnosis. Generally, patients who initiated TT prior to diagnosis also appeared to have a higher prevalence of comorbidities (particularly respiratory related) than patients who initiated TT at or after COPD diagnosis. No significant differences were observed in blood eosinophil concentration (BEC) between groups  were GOLD A/B and 1260 (14.6%) were frequent exacerbators. A total of 615 (7.1%) were classified as asthma–COPD overlap (ACO) (Table 1 (%) values  compared to GOLD A/B . Distribution of comorbidities was similar between subgroups  compared with the 41.2% for all the groups. About a quarter of patients had no previous treatment the year before initiating TT  accounting for all patients who initiated TT at some point during the study period, before or after COPD diagnosis. Restricting the sample to patients who initiated TT at or after COPD diagnosis, median time from COPD diagnosis to TT initiation was 196 days . The median time to TT was shorter for GOLD C/D patients  compared to GOLD A/B  , followed by ACO , and exacerbators  were associated with earlier initiation of TT. Older age, male sex, and pneumonia and lung cancer were also significantly associated with higher probability of early TT initiation  followed by LABA/ICS (15.2%). Mapel et al.19 in the United States also found that the most frequent treatment before TT was LAMA (15.4%) followed by LABA/ICS (7.7%). It might be that incident COPD cases initiating TT may be more symptomatic and, therefore, they are more frequently started already on LABA/ICS (and early stepped up to TT) instead of starting on monotherapies. It is of note that only 34.5% of patients who initiated TT had spirometry recorded in their clinical records. The underuse of spirometry is still a pending issue in primary care that may affect diagnostic accuracy.After excluding patients who initiated TT before COPD diagnosis, the remaining incident patients with COPD initiated TT a median of 196 days after diagnosis, with a shorter period observed in frequent exacerbators and GOLD C/D patients, which is in line with treatment recommendations9.In multivariate analysis, we observed that some variables that define the severity of the disease were significantly associated with the initiation of TT in newly diagnosed patients with COPD. This expected finding included characteristics such as increasing age, more severe GOLD stage, and more frequent exacerbations. Another characteristic significantly associated with TT initiation was the ACO phenotype, which is also in alignment with current recommendations, that recommend the use of ICS in combination with long-acting bronchodilators to prevent exacerbations, in particular in patients with eosinophilic profile or a previous diagnosis of asthma20, since all patients had a diagnosis of COPD. This finding should be analyzed in other studies that investigate whether it is due to a trend toward overprescription in men, or, alternatively an underprescription in women.However, other identified factors associated with the initiation of TT were unexpected. Male sex was significantly associated with TT initiation in patients with incident COPD. This gender bias is difficult to explain and is not linked to the well-described diagnostic bias21 and, therefore, the opposite association would be expected. A hint to explain this paradox can be found in the next characteristic associated with initiation of TT, which is the diagnosis of lung cancer. The possible explanation could be that both patients with episodes of pneumonia and lung cancer may visit the primary care physician more frequently with more respiratory complaints, therefore, stepping up treatment to include ICS may be more likely. However, the association between the initiation of TT and pneumonia suggests that PCP may not be aware of the risk of pneumonia associated with the use of ICS, in particular in patients with more severe disease, underweight, and with low BEC23.Another surprising finding is the significant association of TT initiation and the diagnosis of pneumonia. It is well recognized that the use of ICS is a risk factor for pneumonia in patients with COPD24. Our data and other large database studies25 show that BEC are available for the great majority of patients with COPD in primary care and therefore can be a useful biomarker for the appropriate use of ICS in COPD26.Finally, we did not find an association between TT initiation and BEC. Whilst it must be considered that the relevance of the use of BEC to guide ICS treatment in COPD has only appeared in recent years, the 2012 edition of the Spanish Guideline on treatment of COPD already mentioned the use of BEC to classify patients as ACO and personalize the prescription of ICS predominantly to patients with this phenotypeIn conclusion, we have observed that around 15% of newly diagnosed patients with COPD initiate TT. Among them, one quarter had no previous inhaled therapy. Although these findings may not be consistent with current COPD guidelines, the observation that patients with ACO and frequent exacerbations were more likely to start TT and started TT earlier is in alignment with treatment recommendations.27. This database has been used and validated for epidemiological research in respiratory diseases28.This was a retrospective study with longitudinal follow-up. Data analyzed were obtained from the Information System for the Development of Research in Primary Care (SIDIAP) database; this database includes anonymized electronic medical records from 270 primary care centers with a reference population of 5.8 million people in Catalonia (Spain), which represents more than 80% of the total populationIncluded patients were those over 40 years of age with a first diagnosis of COPD  between January 1, 2011 and December 31, 2015. Patients were required to have at least 12 months of available medical record data prior to diagnosis of COPD. Among incident patients with COPD, those who initiated TT were defined as having prescriptions for LAMA, LABA, and ICS with an overlap of 30 days or more. If a patient had a gap of 60 days for prescription of one or more of the three compounds, they were considered to no longer be persistent on TT.According to the time of initiation of TT, patients were classified into those who initiated TT before the date of COPD diagnosis and those who initiated TT at or after the date of diagnosis.9. Those with a concomitant diagnosis of asthma recorded I clinical records were included in the ACO phenotype30. Patients with two or more exacerbations during the year before initiation of TT were classified as a frequent exacerbator phenotype, patients with only one exacerbation were classified as infrequent exacerbators, and the remaining COPD patients were defined as non-exacerbators. ACO and the exacerbator phenotypes were not mutually exclusive.The phenotypes of the patients were also identified31.Patients were also classified according to the GOLD 2013 categories based on spirometry and exacerbation data as low risk (GOLD A/B) and high risk (GOLD C/D)Detailed information at the time of diagnosis was collected on the following variables: age, sex, comorbidities, smoking history, history of exacerbations, and treatments received during the year before the diagnosis of COPD. Results of spirometry and blood analysis with BEC at diagnosis were also collected.28. Briefly, we used a diagnostic code indicative of a respiratory exacerbation, or receipt of corticosteroids and/or antibiotics used for treating the episode. Exacerbations leading to hospitalizations or treated as part of inpatient care were not available in the data. To avoid misclassification and over-estimation of exacerbations, consecutive episodes with less than 21 days between prescriptions and physician visits were considered as a single event.Exacerbation episodes were identified according to previously published algorithmsThe study was approved by the Research and Ethics Committee of the IDIAP Jordi Gol Institute of Research in Primary Care . Since anonymized data were collected retrospectively, no informed consent was considered necessary.Description of variables was performed with absolute frequencies and corresponding percentages. Continuous variables were described using the mean and SD, while time from diagnosis to TT initiation in days was described using the median and IQR.2 or Fisher exact test where applicable. Quantitative variables were compared using the T-test or Mann–Whitney U test.Categorical variables were compared using the χp values. All statistical analyses were performed using the statistical software package Statistical Package for Social Science (SPSS) version 20.0 . A p value <0.05 was considered significant.The variables significantly and independently associated with time to initiation of TT in newly diagnosed patients were identified by means of a logistic regression analysis. The results were described using hazard ratios with a 95% confidence interval and Further information on research design is available in the Reporting Summary


This study investigates the effects of intrauterine G-CSF on endometrial thickness, clinical pregnancy rate and live birth rate in a recurrent implantation failure (RIF) group with normal endometrium. This study was designed as a prospective randomized controlled trial with the involvement of 157 RIF group pati; ents. The RIF group was formed on the basis of the RIF criteria: “The failure to achieve a clinical pregnancy after the transfer of at least four good-quality embryos in a minimum of three fresh or frozen cycles to a woman under the age of 40 years. The study sample included 82 patients in the G-CSF group who received G-CSF once a day on hCG. The procedure was performed by administering 30 mIU of Leucostim® through slow infusion into the endometrial cavity using a soft embryo transfer catheter. Normal saline of 1 mL was infused into the endometrial cavity in the same way in 75 patients in the control group. The standard ICSI procedure was used for all patients, and fresh cycle embryos were transferred on the third or fifth day. No statistically significant difference was identified in clinical pregnancy rates, miscarriage rates and live birth rates between the G-CSF group and the control group , and no difference was observed between the two groups regarding endometrial thickness (p = 0.965). The intervention of administration G-CSF into the uterine cavity in RIF patients with normal endometrium, did not alter the endometrial thickness, clinical pregnancy rates, or live birth rates. Coughlan et al. proposed that RIF is defined as the failure to achieve a clinical pregnancy after the transfer of at least four good-quality embryos in a minimum of three fresh or frozen cycles to a woman under the age of 40 years2. However, internationally agreed consensus on the definition has yet to be reached. Although the two leading actors in the implantation are the endometrium and the embryo, there are some other influential factors, such as oocyte and sperm parameters, parental chromosome structure, anatomic structure, immunologic factors, thrombophilic conditions and lifestyle, which means that a multidisciplinary approach is required for the management of RIF.Recurrent implantation failure (RIF) refers to a situation in which implantation has repeatedly failed to reach a stage recognizable by pelvic ultrasonography3, although several nonhematopoietic cell types, such as endothelial cells, placental cells, trophoblasts and granulosa‐lutein cells, also express G‐CSF receptors5. G-CSF helps mainly in the mobilization, migration and differentiation of stem cells6, while also facilitates endometrial regeneration by promoting angiogenesis and decreasing cell death by reducing apoptotic activity7. G-CSF plays a role in embryo implantation and the continuation of pregnancy by temporarily suppressing immune response through its effects on lymphocytes, macrophages and T helper-2 cells8. Thus, G-CSF supplementation has been considered as a promising innovative therapy in reproductive medicine. G-CSF has also been reported to play a role in embryonic development, implantation and trophoblastic growth when administered systemically9 and have beneficial effects on endometrial remodeling and receptivity in intra-uterine local administration10.Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic‐specific cytokine produced by bone marrow cells, stromal cells, fibroblasts, and macrophages12. In recent years, studies into recurrent pregnancy loss and recurrent implantation failure have also reported successful outcomes14. In addition to studies that showed that G-CSF administration provides endometrium expansion, particularly in the thin endometrium, and increases implantation and pregnancy rates, there are also studies that found out that G-CSF administration does not change endometrium and pregnancy rates, although it is seen to increase the endometrial thickness17. The relationship between RIF and endometrium, G-CSF administration and endometrium has been studied extensively, although there have not been enough randomized controlled trials on G-CSF administration in the RIF group with normal endometrium. To contribute to the literature, in this study, we investigated the effect of intrauterine G-CSF administration on endometrial thickness, implantation rates and live birth rates in RIF group patients with normal endometrium.Clinical trials that suggest that G-CSF administration may improve the success of the assisted reproductive techniques (ART) have been performed primarily as thin endometrium studieset al. in 2014, being “the failure to achieve a clinical pregnancy after the transfer of at least four good-quality embryos in a minimum of three fresh or frozen cycles to a woman under the age of 40 years2.This study is designed as a prospective randomized controlled trial with 157 patients diagnosed with RIF without endometrial pathology and was carried out in a private infertility center between March 2016 and December 2017. The recurrent implantation failure group was formed on the basis of the RIF criteria defined by Coughlan Women under the age of 40 who met the RIF definition and patients whose follicle-stimulating hormone (FSH) levels were <15 IU/mL were included in this study. Patients with autoimmune diseases, patients with congenital uterine anomalies, patients with Asherman’s syndrome, patients with uterine cavity distorted by myoma or endometrial polyps, patients with confirmed endometriosis or endometrioma, and patients for whom G-CSF was contraindicated  were excluded from this study. The patients with an endometrial thickness less than 7 mm (measured in the pretreatment midcycle) were accepted as thin endometrium and didn’t included this study.17 and the significance level was considered to be 0.05, and samples were measured using the Chi-Square test with a 0.80 power; as a result, findings showed that 74 samples per group and 148 samples in total were sufficient for this study.This study was approved by the Ethics Committee of Turgut Ozal University and was conducted in accordance with the principles of the Declaration of Helsinki. To calculate the sample size, the percentages of clinical pregnancy rates were taken as 37.5% and 14.3% for the groups that received G-CSF and those that did not receive G-CSF respectivelyFor this study, 204 participants were evaluated, and 173 suitable participants were informed in detail about this study. Written informed consent was obtained, and the patients were then randomized using a computer-generated random number sequence (1:1 simple randomization). The patients were blind to which group they were assigned to, the physicians were not. The patients were subjected to randomization only once during this study and were not included in the study for the second time. This study was continued with a total of 157 patients, 82 of whom were in the G-CSF group, and 75 were in the control group. A CONSORT diagram that shows the status of the participants during this study can be found in Fig. All of the patients included in this study were followed by through two cycles. In the pretreatment cycle, age, BMI, infertility duration, FSH and AMH levels, number of antral follicles, sperm count and endometrial thicknesses in the periovulatory period were recorded, and the patients were randomized. In the intracytoplasmic sperm injection (ICSI) cycle, patients in the G-CSF group received G-CSF once a day on hCG day, before hCG injection. The procedure involved the administration of 30 mIU of Leucostim  through slow infusion into the endometrial cavity using a soft embryo transfer catheter. Normal saline of 1 mL was infused into the endometrial cavity of patients in the control group in the same way as the study group. A standard ICSI procedure was used for all patients, and fresh cycle embryos were transferred on the third or fifth day. The ICSI cycle parameters of all the patients and pregnancy outcomes after the ICSI cycle were recorded.Ovarian stimulation protocols and luteal support: In this study, gonadotropin-releasing hormone (GnRH) agonist and GnRH antagonist protocols were administered to patients for ovarian stimulation.In the agonist protocol, GnRH agonist 0.5 mg/day leuprolide acetate  was started in the luteal phase of the previous cycle and recLH treatments were added in addition to recFSH, HMG or recFSH on the second and third days of the cycle. Drugs used in these treatments included recombinant human follicle stimulated hormone (r-hFSH) follitropin-α  or follitropin-β , human menopausal gonadotropin HMG)  or highly purified human menopausal gonadotropin (HP-HMG)  and recLH . In the antagonist protocol, when the gonadotropin was started on second and third days and the dominant follicle was 14 mm, cetrorelix  was added to the treatment as a GnRH antagonist. In both protocols, the development of the follicles was followed by transvaginal USG and E2. When the dominant follicle measurement was 19 mm or more or at least three follicles were 17 mm or more, gonadotropin administration was terminated and ovulation was induced. Ovulation was induced with 250 µg of human recombinant hCG . Oocyte retrieval was performed during transvaginal USG process at between the 36th and 40th hour after the ovulation induction, followed by ICSI with mature oocytes and fresh sperm were done.Luteal phase support started on the day of oocyte retrieval. All patients received vaginal micronized progesterone in gel form  in a single daily administration. Progesterone was used until ten weeks if pregnancy was confirmed.Endometrial thickness determination: A transvaginal ultrasound scan was adopted on the day of hCG injection. The maximum distance between the two outer edges of the endometrial image on a longitudinal section of the uterus observed using a vaginal B-ultrasound  was used to determine endometrial thickness. Measurement of endometrial thickness in the pretreatment cycle was performed in the period following ovulation. In patients without ovulation, the highest endometrial thickness measurement was taken as a baseline for a week from the beginning of the midcycle. In patients in the ICSI cycle, the endometrial thickness measurement on hCG day was performed immediately before G-CSF infusion. No adverse reaction related to intrauterine G-CSF administration was reported in this study.p-value of 0.05 was considered statistically significant.All statistical analyses were performed using SPSS for Windows version 11.5 software . Descriptive statics were expressed as mean±standard deviation (SD) and median (min-max) for numerical variables, and in numerical form (percentage) for categorical variables. Student’s t-test was used to analyze statistically significant differences between the categories of a qualitative variable with two categories regarding a quantitative variable for normally distributed data, while a Mann-Whitney U-test was used for the abnormally distributed data. Chi-square and Fisher exact tests were used to analyze the relationship between two categorical variables. A All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. (Ethics Code: 9 March 2016/99950669/75). The eligible patients signed written informed consent prior to participation in the study.In total, 157 patients with RIF diagnoses were included in this study, with 82 patients (52.2%) in the G-CSF group and 75 patients (47.7%) in the control group. The demographic characteristics and pre-cycle identifiers of these individuals and their spouses are shown in Table In the G-CSF group, 13 couples (15.8%) were secondary infertile, and 69 couples (84.1%) were primary infertile, while in the control group, 14 couples (18.6%) were secondary infertile and 61 couples (81.3%) were primary infertile. No statistically significant difference was identified between the G-CSF and control groups for the primary and secondary infertility rates (p = 0.612).No statistically significant difference was identified between groups for the presence/absence of Polycystic Ovary Syndrome (PCOS) (p = 0.851). The numbers (percentage) of PCOS presence and absence in the G-CSF group was found to be 8 (9.8) and 74 (90.2), respectively, compared to 8 (10.7) and 67 (89.3) respectively in the control group.No statistically significant difference was identified between the two groups regarding the presence/absence of the male factor (p = 0.301). The numbers (percentage) of male factor presence and absence in the G-CSF group was found to be 17 (20.7) and 65 (79.7), respectively, compared to 19 (25.3) and 56 (74.6) respectively in the control group. Testicular sperm extraction procedure (TESE) was performed when no sperm was found in the ejaculate of two patients (2.5%) in the G-CSF group and three patients (4%) in the control group, and no statistically significant difference was identified between the two groups regarding the number of TESE applied (p = 0.672).The characteristics of the ICSI cycle of our study are given in Table In our study, a total of 290 embryos were transferred, including 152 in the G-CSF group and 138 in the control group. In the G-CSF group, 120 (78.9%) cleavage and 32 (21.1%) blastocyst embryo transfers were performed, whereas in the control group, 107 (77.5%) cleavage and 31 (22.5%) blastocyst embryos were transferred. No difference was observed between two groups regarding the cleavage and blastocyst embryo transfer rates (p = 0.809).th weeks of gestation) and early premature birth(birth between 20–28 weeks of gestation). No difference was identified regarding beta hCG positivity, which was considered as per cycle and per embryo transfer, clinical pregnancy and live birth rates between the groups. Chemical pregnancies, miscarriage, early premature birth and term birth rates of pregnancy were also similar between the two groups.Table This prospective randomized controlled trial was designed to determine whether or not intrauterine G-CSF administration before ART in the RIF group had an effect on clinical pregnancy and live birth rates. No statistically significant difference was identified between the G-CSF group and the control group regarding clinical pregnancy rates, miscarriage rates and live birth rates, and no difference was observed between the two groups regarding endometrial thickness.11. In a single pilot cohort study carried out by Gleicher et al.12 in 2013, expansion was reported to be developed in 21 cases with thin endometrium that was resistant to standard therapies, and ongoing pregnancy was reported at a rate of 19.1%. In recent years, pregnancy rates have been reported to increase in the majority of trials in which intrauterine G-CSF was used in thin endometrium cases20. A study by Li et al.21, intrauterine G-CSF administration in the thin endometrium in frozen embryo transfer cycles were analyzed, and it was reported that this did not increase implantation or clinic pregnancy rates. Different from other studies, Eftekhar and Singh reported increased pregnancy rates, although no endometrial thickness increase was shown. In a meta-analysis by Kamath et al., this situation was reported as follows: “The higher pregnancy rates in spite of no apparent benefit in increasing endometrial thickness following G-CSF installation suggests the possibility of an unknown immunological mechanism aiding embryo implantation rather than only endometrial regeneration in both these populations”7. In our study, patients with thin endometrium were excluded, since the embryos were frozen in the event of endometrium at a thickness of less than 7 mm. In the present study, no difference was observed between the groups regarding endometrial thickness in the pretreatment cycle, and no difference was identified between the groups regarding endometrial thicknesses on both the hCG day and the embryo transfer days in the ART cycle. In our study, endometrial thickness did not increase in the group for whom G-CSF was administered compared to the group who did not receive G-CSF. In a study carried out by Barad et al.16 in 2014, the effects of intrauterine G-CSF infusion on a non-selected IVF group with normal endometrial thickness were investigated, and endometrial thickness and IVF outcome were reported to be unaffected by intrauterine G-CSF infusion, which was similar to the findings of the present study.G-CSF in ART was first used by Gleicher on four treatment-resistant cases with thin endometrium, and pregnancy was reported in all four cases23. In our study, G-CSF administration was performed via endometrial installation in the RIF group, although no difference was seen in the implantation and pregnancy rates between the G-CSF and non-G-CSF groups. When administered systematically, G-CSF was reported to have positive effects on oocyte maturation and embryonic development9, while in locally endometrial cavity applications, pregnancy rates may not have changed since the oocyte and embryo were deprived of this positive support. Since thin endometrium was excluded due to the nature of our study, it can be concluded that G-CSF was not beneficial in increasing pregnancy rates in normal endometrium. In a study carried out by Eftekhar et al.24 in 2016, that the findings showed that the intrauterine infusion of G-CSF did not affect the success of ART in normal endometrium cases.In the literature, G-CSF was administered systemically through the subcutaneous route in studies where the G-CSF was administered in the RIF group, and an increase in pregnancy rates was reported in all studies26. The systemic and local use of G-CSF may have different effects, and different mechanisms of action may come into play in different clinical situations. Thus, it would seem plausible that it should be considered separately. Although degradation of endometrial receptors is a common point in all of them, RIF, recurrent pregnancy loss and thin endometrium are very different from each other and are multifactorial entities, which leads us to believe that specific groups should be focused upon in these studies.Literature contains a limited number of studies on the use of G-CSF in ART, and the quality and the level of evidence of the studies that have been made are not very high, including considerable heterogeneity in the selection of the patient population, the means of administration of G-CSF, the dose of G-CSF, application times, and in the statistical interpretations of the study results. Furthermore, in the published studies, the optimal dose, duration of application and route of administration were not identifiedThe present study can be considered to be the first study that investigated the effects of intrauterine G-CSF administration on clinical pregnancy and live birth rates in the RIF group, and designed as a randomized controlled study; it involves well-chosen study and control groups with similar characteristics. These factors stand as the most powerful aspects of our study. Placebo administration was performed for the control group simultaneously with the study group to ensure that the endometrial injury effect (depending on the catheter and instilled fluid) was equal in both groups. The most important limitation of our study our inability to create subgroups due to the limited number of patients. In a larger study, subgroups, such as a thin endometrium group, a systemic G-CSF administered group, and different G-CSF dose administered groups could be applied to make the study much more valuable. Future studies also need to be conducted to ascertain what the most effective dose of G-CSF is when administered by different routes. Additionally; in this study, PGT-A was not performed to the embryos, this is another important limitation of our study. In a study regarding RIF the use of PGT-A would be better in order to avoid the chance that aneuploid embryos are transferred back in uterus.Our study would have been more valuable if we had performed PGT-A to the embryos.In conclusion, intrauterine G-CSF administration in RIF patients with normal endometrium did not alter the endometrial thickness, clinical pregnancy rates, or live birth rates. Further studies are needed to investigate the effect of G-CSF on endometrium and implantation rates in RIF.


The median interval between the procedure leading to CSF hypovolemia and PRES was 4 days. Seizure, altered mental state, and headache were the most frequent presenting symptom. The parietooccipital pattern was most frequent (71.0%). Conservative management remains the mainstay of treatment with excellent outcomes. Three patients had a second episode of PRES. CSF hypovolemia is a plausible cause of PRES via a unique pathophysiologic mechanism including arterial hyperperfusion and venous dysfunction. Patients with CSF hypovolemia is more susceptible to PRES, which is potentially life-threatening. Given that CSF hypovolemia is a common complication of anesthetic, neurological, and neurosurgical procedures, PRES should be early considered for prompt diagnosis and appropriate management.Posterior reversible encephalopathy syndrome (PRES) is a reversible neuroradiological syndrome characterized by reversible vasogenic edema. The pathophysiological mechanism is still unclear, but PRES may be triggered by various etiologies. To date, only a few PRES cases linked to cerebrospinal fluid (CSF) hypovolemia were reported. The association between PRES and CSF hypovolemia needs to be explored. We presented a case of PRES with CSF hypovolemia as a result of an inadvertent dural puncture and reviewed the literature to identify the clinical characterization and pathophysiological mechanism of PRES following CSF hypovolemia. A total of 31 cases of PRES-CSF hypovolemia was included for analysis. The median age was 33 years, with a notable female predominance (87.1%). Fifteen patients (48.4%) didn't have either a history of hypertension nor an episode of hypertension. The most common cause of CSF hypovolemia was epidural or lumbar puncture ( Posterior reversible encephalopathy syndrome (PRES), initially described by Hinchey et al. in 1996 . The typCerebrospinal fluid (CSF) hypovolemia, which is used to be referred to as intracranial hypotension (IH) synonymously, is increasingly recognized as a critical but often a misdiagnosed cause of new-onset cephalalgia , 7. UsuaTo our knowledge, there was no systematic review exploring the pathogenesis, clinical and imaging characteristics, and management of PRES in patients with CSF hypovolemia. Herein, a case of PRES who suffered CSF hypovolemia after an inadvertent dural puncture was presented with potential evidence of hyperperfusion. Then, a systematic analysis of published literature was undertaken to reveal the possible association between PRES and CSF hypovolemia.The information of the patient from the department of Neurology of our hospital was collected for a preliminary analysis. The additional 30 cases (29 articles) in the PubMed and Web of Science database from inception to July 2019 using a combination with “PRES” and various terms related to CSF hypovolemia or high risks of CSF hypovolemia including “cerebrospinal fluid hypovolemia,” “intracranial hypotension,” “CSF leakage,” “epidural puncture,” “epidural anesthesia,” “spinal puncture,” “spinal anesthesia,” “lumbar puncture,” “cerebrospinal fluid shunt,” “spinal surgery,” and “cranial surgery.” A standardized form was applied to collect clinical information from each eligible article including demographic characteristics, related medical history, the probable cause of CSF hypovolemia, clinical manifestations, magnetic resonance (MR) findings (both PRES and CSF hypovolemia), treatment, and clinical outcome. The flow diagram was shown in the Written informed consent for participation, data collection, and publication was obtained from the patient. Because this is a case report and review of literature, no research legal, and ethical approval is required.A 30-year-old woman, gravida 3 para 0, without a previous history of hypertension, presented to the Department of Obstetrics at 40 weeks' gestation. Laboratory investigations at admission remained within the normal range. Epidural analgesia was planned for painless labor. An inadvertent dural puncture occurred in the first procedure. Then, no complication was found in the repeated epidural procedure. Her blood pressure remained consistently normal throughout labor, delivery, and the immediate postpartum period. Two hours after delivery, she complained of mild neck pain that resolved after receiving 2,000 ml Ringer's solution.On postpartum day 2, she developed a moderate postural occipital headache. In the absence of other focal neurological deficits, postdural puncture headache was diagnosed. The patient was managed with non-steroidal anti-inflammatory agents, hydration, and strictly bed rest. The epidural blood patch (EBP) was recommended as the following therapeutic measure, but the patient refused. On postpartum day 3, the patient complained of progressively worsening postural headache, nausea, and photophobia. The patient had to keep a recumbent posture to relief. The blood pressure was noted elevate to an average level of 140/85 mmHg and a highest-level of 178/96 mmHg. Nifedipine was taken to control hypertension. Then, the blood pressure was under 150/90 mmHg. On the early morning of postpartum day 4, the patient became confused when she waked up and turned to a supine position with a blood pressure of 131/90 mmHg. After a few minutes, she had a generalized tonic-clonic seizure which was controlled by diazepam. After she regained consciousness, she complained of diplopia and severe headache in occipital and left frontal region. Neurological examination revealed left abducens nerve palsy, right hemianesthesia, horizontal nystagmus, right tongue paralysis, and right Babinski sign. Diazepam and magnesium sulfate were taken with a concern that the patient was developing postpartum eclampsia. Six hours later, brain magnetic resonance imaging revealed vasogenic edema in the bilateral parieto-occipital regions, basal ganglia, and brainstem . ConvexiIn total, we collected the data on 31 patients (30 patients from literature and our patient) for descriptive analysis. The detailed data of cases were summarized in n = 16), pregnancy (n = 14), pre-eclampsia or eclampsia (n = 5), subarachnoid hemorrhage (n = 2). Five patients had a history of hydrocephalus or intracranial hypertension. Fifteen patients (48.4%) didn't have either a history of hypertension nor an episode of hypertension. The reduction of CSF was resulted from epidural or lumbar puncture (n = 21), CSF shunt (n = 6), spinal surgery (n = 2), head trauma (n = 1). Excluding patient 29 who had no exact date of the onset time of PRES  level of 123 mmHg after delivery. Did hypertension lead to PRES? Our patient complained postural headache before the changes in blood pressure, and the development of hypertension was following the deterioration of headache. On the other hand, the patient only had a slight elevation of averaged MAP. Even the maximum blood pressure didn't exceed the upper MAP limits of autoregulation. Although puerperium might reduce the threshold of PRES, it is likely that hypertension is not pinpointed as the major cause of PRES. In our review, only 16 patients had hypertension , 34, 37,2O). However, nearly half of the IH patients showed normal CSF pressure  secondary to the mechanical stimuli of the sagging of the brain and its affiliations would trigger PRES , 16, 25.In general, PRES is regarded as a benign disease with favorable outcomes , 47. ComClinical managements of PRES are based on the elimination of underlying trigger factors and immediate control of epilepsy. Due to the differences in pathogenesis, the treatment strategy for patients with CSF hypovolemia may differ from those with other etiology. Compared with other etiologies, PRES patients with CSF hypovolemia were likely to have a shorter median time from CSF loss to PRES onset, which support a direct link between the CSF hypovolemia and PRES. The time between the procedure that incited CSF loss and the ictus of the PRES syndrome may depend on the baseline ICP and the speed of the reduction of CSF volume or ICP. We found that seven patients experienced PRES within 1 day. Of them, five patients had intracranial hypertension before PRES onset; all of them had a rapid loss of CSF or a rapid reduction of ICP. One patient with chronic hydrocephalus developed PRES 2 h after a rapid CSF loss of 50 ml. The other patient developed PRES 6 h after a 2 h inadvertent overdrainage of 200 ml CSF. These two patients experienced PRES recurrence rapidly after another rapid reduction of CSF volume. On the other hand, a marked increase in blood pressure may contribute to the development of PRES. Patients who experienced a systolic blood pressure more than 179 mmHg had a shorter interval of PRES onset. Base on the evidence from the reviewed reports, we propose the following recommendations: First, a precipitous reduction of CSF volume or ICP should be avoided. A graded reduction of ICP is strongly recommended in patients with intracranial hypertension, especially in patients with extremely high CSF pressures. Second, in patients with CSF hypovolemia, the treatment of CSF hypovolemia should be initiated at the early stage of the disease . CSF hypThe present case and reviewed literature highlight the pathophysiological link between PRES and CSF hypovolemia. Both arterial hyperperfusion and venous dysfunction may contribute to the development of PRES in patients with CSF hypovolemia. PRES should be early considered in patients with a high risk of CSF hypovolemia when the clinical manifestations can not be explained by CSF hypovolemia or other conditions alone. Precipitous reduction of CSF should be avoided, while appropriate treatments of CSF hypovolemia should be initiated early. The blood pressure should be strictly controlled in patients with CSF hypovolemia to prevent the development of PRES and improve the clinical outcome.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.FF designed the study. YZ and XW collected clinical data and wrote the manuscript. YC and YL searched the literature and edited the pictures. GZ revised the manuscript. All authors contributed to the manuscript revision and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Since Barnard’s first heterotopic heart transplant in 1974, Copeland’s method has been the greatest contribution to heterotopic transplants but has the drawback of donor’s right ventricular atrophy. This new method proposes a modification in the anastomosis of the superior vena cava aiming to pre-serve donor’s right ventricular function by decompressing the pulmonary territory and reducing the pulmonary arterial pressure, as a biological ventricular assist device. Finally, a second intervention is proposed, where a “twist” is performed to place the donor’s heart in an orthotopic position after re-moval of the native heart. A pioneering research on this method received approval from the ethics committee of the Heart Institute of São Paulo. We believe that this method has the potential to im-prove quality of life in a selected group of patients. Barnard’s contributions were followed by the first heterotopic heart transplantation (HHT) with his own technique in 1974. The indications proposed by Barnard for HHT were, at that time: pulmonary hypertension (which is still a routine contraindication for OHT); systemic circulatory support for the native heart in primary graft failure and as circulatory support for the native heart in cases of severe rejection, the main cause of death in heart transplantation in the pre-cyclosporine era,2. It is worth mentioning that most donor hearts that present with those clinical scenarios are excluded for OHT donation.Since these first three indications, two more were added: “small” donor hearts  and compromised donor hearts, also cited in the literature as “marginal” donor hearts, which means hearts that suffered a long ischemia time and are at high risk of complications, for example: hearts who needed high inotropic support, previous cardiac arrest or arrhythmia, abnormal wall motion visualized on echocardiogram and/or an electrocardiogram suggestive of ischemia,2. Then, na end-to-side anastomosis of the ascending aorta, finishing with the pulmonary trunk anastomosis to the respective recipient vessel with an interposed prosthetic tube , otherwise it would not be possible to connect them, at least not without tension  or elevated pulmonary vascular resistance (PVR) and other options for this select group of patients have emerged with time. One of them was the result of researches by Michael Ellis DeBakey and colleagues that culminated with the first successful left ventricular assistance device (LVAD) implantation in 1966.The main obstacle for LVADs remains the high cost, restricting it to a limited number of patients and in developing countries, such as Brazil, this number is even lower. Nonetheless, heartlung transplantation, pioneered by Bruce Reitz and colleagues in 1981, is another option available, but isolated hearts are more readily available than heart-lung blocks, and fewer groups in the world have acceptable success rates with this procedure.Finally, a second surgical technique available for HHT was published by Jake and Hannah Copeland, and for this reason is called Copeland’s technique . This te,2, suggesting as possible causes: poor adhesion to post-transplantation immunosuppressive treatment, mismatch between small donors and recipient’s heart, and marginal donor hearts. The latter is the most probable cause for the inferior results when compared to OHT. Then, a new idea emerged.Analyzing the reported cases, Copeland’s HHT technique had unsatisfactory initial results, such as published in Copeland J and Copeland HThe intention of this new two-stage approach is to assist the left ventricle (LV) by introducing a modification of Copeland's HHT technique, aiming to preserve the donor’s right ventricular function and decompress the pulmonary territory by reducing the pulmonary arterial pressure. After this pressure decrease, we propose the second stage by removing the native heart and “twisting” the donor’s heart to the orthotropic position, releasing the patient from the inherent risks of the dependence on good function of two hearts.The first stage starts with the anastomosis between left atria and ascending aorta, and then the anastomosis from donor's pulmonary trunk and recipient’s right atrium. Considering that the RV is a flow-dependent chamber, its function would be preserved, as it will receive all the flow from the total superior vena cava (SVC) venous return, contrasting the RV atrophy associated with the reduced flow from the side anastomosis of the original Copeland's technique, and this benefit would be attained by the direct end-to-end anastomosis of the donor SVC to the recipient SVC close to the brachiocephalic veins , while c, from the Heart Institute of Sao Paulo, was approved by the institution's ethics committee .These modifications would allow the preservation of the RV function, while decreasing the PVR through the parallel anastomosis of the LV, creating the ideal environment for the previously cited “twist” of the donor heart in the second stage  and 2C. e.g., jugular vein catheter) it will reflect the donor's heart right circulation pressures; alternatively, if measured in the IVC venous return territory , it will reflect the recipient's heart right circulation pressures. Another important remark is that the ideal pathway for the donor's heart biopsy will be the SVC, as it will be directly connected to the donor's right ventricle.It is also interesting to note that the measured central venous pressure will have two distinct values: if measured in the SVC venous return territory (In conclusion, we believe that this new method has the potential to become a viable alternative for surgery in a selected group of patients, reducing the poor long-term outcomes and substantially improving their quality of life in this patient pool, who are mostly in palliative care in developing countries like Brazil.


Thalamocortical posterior nucleus (Po) axons innervating the vibrissal somatosensory (S1) and motor (MC) cortices are key links in the brain neuronal network that allows rodents to explore the environment whisking with their motile snout vibrissae. Thalamocortical posterior nucleus (Po) axons innervating the vibrissal somatosensory (S1) and motor (MC) cortices are key links in the brain neuronal network that allows rodents to explore the environment whisking with their motile snout vibrissae. Here, using fine-scale high-end 3D electron microscopy, we demonstrate in adult male C57BL/6 wild-type mice marked differences between MC versus S1 Po synapses in (1) bouton and active zone size, (2) neurotransmitter vesicle pool size, (3) distribution of mitochondria around synapses, and (4) proportion of synapses established on dendritic spines and dendritic shafts. These differences are as large, or even more pronounced, than those between Po and ventro-posterior thalamic nucleus synapses in S1. Moreover, using single-axon transfection labeling, we demonstrate that the above differences actually occur on the MC versus the S1 branches of individual Po cell axons that innervate both areas. Along with recently-discovered divergences in efficacy and plasticity, the synaptic structure differences reported here thus reveal a new subcellular level of complexity. This is a finding that upends current models of thalamocortical circuitry, and that might as well illuminate the functional logic of other branched projection axon systems.SIGNIFICANCE STATEMENT Many long-distance brain connections depend on neurons whose branched axons target separate regions. Using 3D electron microscopy and single-cell transfection, we investigated the mouse Posterior thalamic nucleus (Po) cell axons that simultaneously innervate motor and sensory areas of the cerebral cortex involved in whisker movement control. We demonstrate significant differences in the size of the boutons made in each area by individual Po axons, as well as in functionally-relevant parameters in the composition of their synapses. In addition, we found similarly large differences between the synapses of Po versus ventral posteromedial thalamic nucleus axons in the whisker sensory cortex. Area-specific synapse structure in individual axons implies a new, unsuspected level of complexity in long-distance brain connections. Rodents explore their environment by rhythmically “whisking” with their motile facial vibrissae. Time-dependent correlations between motor commands and vibrissal follicle mechanoreceptor signals are used for inferring object position and texture. Such computations are performed by multilevel closed-loop neuronal networks encompassing the brainstem, thalamus, and neocortex  axons that innervate focally the L4 the “barrel” domains of the vibrissal primary somatosensory cortex (S1), and the posterior thalamic nucleus (Po) axons that innervate mainly L5a but also L1 of S1 . ImportaActivation of S1–L4 VPM synapses elicits large currents in cortical neurons  and drivIn cortical synapses, bouton and mitochondrial volume, synaptic vesicle pool size, as well as postsynaptic density (PSD) complexity and surface area directly correlate with increased neurotransmitter release probability and synaptic strength . FurtherTo measure identified Po synapses in S1 and MC, we combined single-cell and bulk axonal labeling with light microscopy and fine-scale 3D-electron microscopy . In addition, as Po and VPM synapses have been recently shown to elicit marked different effects on S1 neurons, we reanalyzed our own dataset on VPM S1 synapses  and compad libitum under a 12 h light/dark cycle. Efforts were made to minimize the number of animals required. Six male mice (60–65 d old) were used for the experiments aimed at biotinylated dextranamine (BDA) population-labeling of Po axons for light and electron microscopy. Thirty-two further mice were electroporated with Sindbis Pal-eGFP RNA to individually label thalamic neurons and reveal the full extent of their cell axons.Experiments were performed on adult  male C57BL/6 mice bred in the animal facilities of the School of Medicine of the Autónoma de Madrid University. All procedures involving live animals were conducted under protocols approved by the University ethics committee and the competent Spanish Government agency (PROEX175/16), in accordance with the European Community Council Directive 2010/63/UE. Animals were housed in pairs in cages containing some toys, and provided chow and water For both types of experiment, mice were first anesthetized with an initial intraperitoneal injection of ketamine (0.075 mg/g body weight) and xylazine (0.02 mg/g body weight), and then maintained with oxygenated isoflurane (0.5–2%) throughout the surgical procedure. At the end of the surgery, the isoflurane flow was interrupted and animals recovered promptly. Ibuprofen (120 mg/L) was added to the drinking water to ensure analgesia during the survival period. At the time of kill, animals were overdosed with sodium pentobarbital .Animals were placed in a stereotaxic frame (Kopf Instruments). BDA  was iontophoretically microinjected  with a Midgard Precision Current Source (Stoelting) under stereotaxic guidance  followed by 4% paraformaldehyde (PFA) + 0.1% glutaraldehyde in 0.1 m PB for 30 min. Brains were removed from the skull, and postfixed by immersion for 1 h at 4°C in the same solution. Two series of parallel coronal sections (50 μm thickness) were cut with a Leica VT 1200S vibratome (Leica Microsystems). Sections were cryoprotected by incubation in a sucrose solution (30% in 0.1 m PB) overnight, and were then rapidly freeze-thawed in liquid nitrogen (1 min) and stored in PB until further use.Following a 5 d survival, animals were perfused with PBS  diluted in PB. After washing in PB, labeling was visualized using the glucose oxidase-3–3′diaminobenzidine (DAB) nickel sulfate-enhanced method . They were collected on Pioloform-coated single-slot copper grids (Electron Microscopy Science). Thereafter, they were treated with lead citrate (3 min), and examined with a Libra 120 transmission electron microscope  equipped with a Proscan 2K bottom-mounted digital camera (Albert Tröndle) and the SIS Multi Image Acquisition software package (Olympus). At the EM level, BDA-labeled axons were easily identifiable by the opaque DAB reaction product. Serial digital images were taken at a magnification of 8000× and stored in a database until further use.x–y plane was 5 nm per pixel. Resolution in the z-axis (section thickness) was 20 nm, so the voxel size of the resulting image stack was 5 × 5 × 20 nm.In addition, Durcupan-embedded tissue blocks from the same experiments were used to obtained 3D tissue samples using FIB-SEM. Here, a Crossbeam Neon40 EsB electron microscope equipped with a gallium FIB and a high-resolution scanning emission SEM column (Carl Zeiss) was used. To accurately select the regions-of-interest, a secondary electron microscopic image was acquired from the block surface that was overlaid and collated with previously obtained light microscopic images . Once thWith those resolution parameters, images of 2048 × 1536 pixels (field-of-view of 10.24 × 7.68 μm) were obtained. A total of 20 different stacks of images of the neuropil in the three cortical regions-of-interest were obtained . The number of serial sections per stack ranged from 75 to 478; the total number of serial sections was 4874 (mean: 243.7 sections/stack). Registration  of serial sections was performed with the freely available Fiji software , using aThe distribution in the cortex of thalamocortical axons is spatially selective in highly topographical patterns, in both the radial (laminar) and tangential dimensions of the cortex. Because we specifically wanted to examine the axons arising from Po labeled by means of small anterograde tracer deposits made in this nucleus, we focused on the cortical domains and layers that contained a maximum density of labeled thalamocortical axons . Within The 3D-volume reconstructions and measurements on ssTEM images were performed with the OpenCAR software .The n = 29 in total) in which a lighter staining allowed unambiguous visualization of vesicles . We applied an unbiased stereological sampling method  synaptic sites that were located in smooth or just slightly swollen axon zones and contained some vesicles, yet lacked mitochondria. As placing ends would have been arbitrary, and the FIB-SEM sample of these synapses was small in number, the presynaptic element parameters (volume and surface) of these inter-bouton synapses were not measured.in vivo electroporation  and mounted on a holder (WPI) that has both a pressure port and electrode connection. All procedures were performed over clean, single-use surfaces, and metal instrument tips were briefly exposed to a flame.Micropipettes were pulled from Kwick-Fill borosilicate capillaries . Inner tip diameter was adjusted to 10–15 μm. To eliminate RNase activity, micropipettes were then kept in a stove overnight at 240°C and, after cooling, were backfilled with a RNA stock solution . Two to four 200 Hz trains of 1 ms negative-square pulses at 50 V were then applied through the micropipette tip using a CS20 stimulator (Cibertec). The micropipette tip was left in place for 5 min before removing it from the brain. Finally, the bone defect was closed, the scalp sutured, and animals were returned to their cages.m PB, pH 7.4, for 8 min. Their brains were then removed, immersed overnight in the same fixative at 4°C and cryoprotected by soaking in a sucrose solution (30% in PB) for further 24 h. Serial 50-μm-thick coronal sections were cut on freezing microtome (Leica Microsystems).Following 58–60 h survival, animals were overdosed with pentobarbital and perfused with saline (1 min), followed by 4% PFA in 0.1 To allow intensive high-magnification microscopic analysis, labeling was made stable and opaque by using immunohistochemistry against eGFP and glucose oxidase-nickel enhancement . Free-flSections were examined under bright-field optics at 10–40×. Labeled axons of Po neurons were found to span >30 serial coronal sections. Complete 2D-reconstructions of each axon were drawn using a Nikon camera Lucida, scanned, and redrawn using a vector graphics software .2) we not included.Under bright-field optics, axonal branches appeared as sharply labeled filaments with frequent varicose swellings. To estimate and compare the size of varicosities (putative synaptic boutons), the maximal projection area was measured from live images using a Nikon DMX1200 camera fitted to the microscope and the NIS-Elements imaging software tools . To this end, the major perimeter of each varicosity was focused at 1000× and delineated over the computer screen using the “polyline” and “polygon” software tools. For each cortical area, layer and axon, 50 randomly selected varicosities were measured. Varicosities with cross-sectional areas  near or below the microscope resolution limit  every cell whose axon was found to target the vibrissal domain of the motor cortex (n = 3) were analyzed for this study.The single-axon analysis was performed in animals that had each an individual Po cell electroporated with Sindbis-Pal-eGFP RNA. Of a larger sample of individually labeled and fully reconstructed Po cells . The threshold level of significance was set at *post hoc test, or Fisher's exact test.For each ultrastructural parameter, a mean ± SD, the median, maximum, and minimum values and a coefficient of variation (CV) were calculated. For subsequent data analysis for multiple comparisons we used one-way ANOVA plus T3 Dunnett's as a All synaptic boutons reconstructed were clustered according to six structural parameters investigated, namely: bouton volume, bouton surface area, number of PSDs/bouton, number of mitochondria/bouton, volume of mitochondria, and PSD surface area. The cluster analysis was performed using MATLAB and Statistics Toolbox Release 2016b (MathWorks). The aim was to identify the synaptic parameters that best characterized the synaptic boutons investigated. Thus, a principal component analysis was performed to simplify the dataset and to convert a set of observations of possibly correlated variables into a set of values of linearly uncorrelated variables called principal components PCs; . Then, wU test. Two-sample Kolmogorov–Smirnov test (K–S) was used to compare size distributions of varicosities between areas/layers.For single-cell labeling experiments, comparison of mean sizes of varicosities  between different cortical areas/layers was performed using Mann–Whitney c). In MC, labeled Po axonal arborizations formed a single band from upper L5a to lower L3 . The Golgi-like axon staining revealed frequent varicosities of variable size .To selectively label a sizable population of thalamocortical Po axons, 10 kDa BDA was iontophoretically delivered into Po. Only experiments in which the BDA deposit was restricted to the Po nucleus were analyzed . In S1, Fig. 1EM samples were taken from sections adjacent to those containing the heaviest anterograde labeling in S1 or MC. These were subsequently analyzed using either high-resolution, fine-scale ssTEM or FIB-SEM. In S1, samples were taken from L1 and L5a, which were readily delineated cytoarchitecturally. However, the cytoarchitectonic definition of MC middle layers is ambiguous; thus, our MC samples included L4 and adjacent deep parts of L3; we refer to this neuropil as “MC-L4/3”.The two serial 3D EM techniques yielded in consistent results. The ssTEM analysis allowed a high-resolution 3D-volume reconstruction of the overall geometry of synaptic complexes including the subsequent quantification of PSD surface area and vesicle pool size; however, it sampled only axon varicosities, and a smaller neuropil volume, which not always allowed a full visualization of the spine neck. In turn, FIB-SEM analysis allowed the complete visualization of postsynaptic elements and inter-bouton axonal segments. For efficiency, the FIB-SEM image acquisition was made with a slightly lower resolution than the ssTEM images; for this reason, fine details such as the individual synaptic vesicles could be only unambiguously resolved and counted in the ssTEM samples .bouton and varicosity interchangeably to describe axonal swellings of >0.5 μm of diameter or exceeding by >50% the typical variation of the adjacent axonal segments . This corresponds to asymmetric synapses, regarded as excitatory and glutamatergic .d,f; In the neuropil of the three regions studied, the large majority of Po axon boutons (70–90%) were monosynaptic. Note, in addition, that the FIB-SEM analysis indicates the presence of numerous ∼25%) synapses in the non-varicose inter-bouton segments, which were always monosynaptic. Thus, the actual proportion of total monosynaptic sites in Po axons may be even higher (∼77–96%). Approximately 30% of the boutons in MC-L4/3 and ∼10% in S1 (L5a and L1) simultaneously contacted two different postsynaptic structures  it was even possible to identify and count all the synaptic vesicles.Most Po varicosities 88–92%; , 2 contab,c). Such nonsynaptic boutons were also observed in MC-L4/3 Po axons although less frequently (2%). Such boutons are virtually absent in the VPM S1-L4 axons  of Po axonal varicosities contained a mitochondrion and some synaptic vesicles, yet lacked any evident synaptic contact b,c. Suchequently %. Such bL4 axons .3 of cortical neuropil. At the magnification used, the axonal BDA axonal segments were few and widely scattered. Thus, despite taking our samples from zones that under light microscopy appeared heavily labeled  were established on spines. Synapses onto dendritic shafts . The mean surface area of the S1-L5a PSDs was similar (0.11 μm2) to that previously measured in VPM-L4 synapses (e). In contrast, the mean PSDs surface area of MC-L4/3 and S1-L1 Po synapses were ∼60% larger. In S1-L5a, most PSDs (65%) had disc-like morphologies, whereas most of those in MC-L4/3 (59%) displayed complex horseshoe-shaped, perforated, or fragmented morphologies (a′–c′).Strikingly, the PSDs displayed a wide range in both shape and size a′–c′. Thr 0.11 μm to that synapses e. In conhologies a′–c′.a′,b′), the large majority (70%) of postsynaptic spines in MC-L4/3 displayed this structural sub-element responsible for spine motility but also stabilization of the presynaptic and postsynaptic apposition zone during synaptic transmission. Mitochondria were found inside 2 of the 68 spines postsynaptic to S1-L1 Po boutons, despite the exceedingly rare occurrence of spine mitochondria in rodent somatosensory cortex . Such invaginations were observed in all three cortical domains investigated, but were substantially more frequent in MC-L4 than in the S1 Po synapses . We had observed similar invaginations in mouse S1-L4 VPM synapses; with a prevalence similar to that in Po MC synapses  although were not found near the smallest . Remarkably, the spine PSD was always adjacent to, but outside the protrusion's edge  and we never observe a PSD inside a protrusion. The invaginated intermembrane surface was large (up to 20 times the size of the active zone). The invaginated membranes were smooth, remained closely apposed (∼20–40 nm) and lacked evident membrane specializations . Diffusion of secreted molecules in such an extensive, narrow and isolated intermembrane space is probably nonlinear and free of direct glial scavenging. In addition to, is it even possible that the extensive parallel, tightly apposed membrane surfaces may allow local electric field (ephaptic) conduction . Mitochondrial volume per bouton was ∼33% larger in MC-L4/3 versus S1-L5a Po boutons . Vesicle pools in both MC-L4/3 and S1-L1 boutons were, as a mean, approximately twice as large as those in S1-L5a boutons. Because of the limited number vesicle pools in our MC sample, the statistical significance could be confirmed for S1-L1 versus S1-L5a Po boutons . In addition, both the head volume and the PSD surface area of the spines postsynaptic to Po boutons in MC-L4/3  and S1-L1  were significantly larger (+50%) than those of spines postsynaptic to S1-L5a Po boutons .To substantiate the above observations, structural parameters of Po axon synapses were statistically compared between in the three cortical domains investigated , and ∼50% in mitochondrial volume  and contained more than twice synaptic vesicles  than Po-L5a boutons (a–c). In contrast, the Po MC-L4/3 boutons are statistically indistinguishable from VPM S1-L4 boutons with respect to these three parameters . Overall, these comparisons reveal that axons from two different thalamic nuclei can form structurally different presynaptic specializations in adjacent layers of the same cortical columns as well as those axons from the same thalamic nucleus can form structurally different specializations in separate areas/layers.Next, we compared the structure of synaptic boutons established by Po versus VPM thalamic nuclei axons in S1 by putting side by side the 3D Po bouton measurements and those of VPM S1-L4 boutons , 2. This boutons a–c. In c3 and 0.10 ± 0.05 μm2; p > 0.05 in both cases). In contrast, spines postsynaptic to Po axons were much larger in S1-L1  or MC-L4/3 , consistent with the notion that postsynaptic element differences reflect to a larger extent the specific cell types and/or dendritic domains present in separate cortical layers or areas.The S1 spines postsynaptic to both VPM axons (in L4) and Po axons (in L5a) have almost identical head volumes and PSD sizes (0.06 ± 0.04 μmf). The differences were found to be statistically significant (Fisher's exact test) between VPM-L4 axons and Po axons as a whole , as well as between VPM-L4 axons and Po axons in S1-L1  or S1L5a . We also compared the distribution of synapses situated in inter-bouton segments, > 2 mm away from any mitochondrion . Again the differences were significant between the VPM-L4 axons and Po axons in MC and S1 as a whole , as well as between VPM-L4 axons and Po-L5a axons in S1 . Together, the above results indicate that the axon mitochondria of Po axons in S1 and MC are far less bound to synaptic sites than in VPM S1-L4 axons.In addition to synaptic boutons, we also compared the frequencies of extra-synaptic mitochondria , yet much less so with the size of their vesicle pool  or the total surface of the varicosity PSDs . In each neuropil, presynaptic and postsynaptic parameters clustered independently; for example, the total PSD area per bouton was only weakly correlated with bouton size , but PSD surface area and the volume of the postsynaptic spine head are strongly correlated . Although our samples were relatively small and that we did not include in this analysis the smallest synapses (in inter-bouton segments), the coefficients are in consonance with results by In both Po and VPM boutons the correlations for bouton size with vesicle pool and for PSD surface with bouton volume seem both to be weak b,c. Althin vivo electroporation , three cells were found to have an axonal branch specifically arborizing the vibrissal region of the MC; remarkably, all these three neurons had, in addition, a collateral axon branch arborizing in the vibrissal region of S1.For the 3D EM study, we labeled anterogradely with BDA relatively large populations of Po cell axons. Studies in rat indicate that MC and S1 may be targeted by axonal branches of the same Po neuron , making poration . From a 2, Neuron 2 = 1.13 ± 0.74 μm2, Neuron 3 = 1.42 ± 0.49 μm2) than those formed by the branches of the same axons in S1-L5a  or S1-L1 . Hence, our single-cell analysis demonstrates that the Po cell axons form boutons in MC that are significantly larger than those formed by the same axon in S1.The axonal arborizations in S1 and MC of three such cells are illustrated in We have shown here that individual Po neurons simultaneously innervate MC and S1 through branched axons that in each area have varicosities (putative synaptic sites) of markedly different size. Fine-scale 3D-EM analysis revealed that the differences are even more pronounced at the ultrastructural level: the Po boutons in MC-L4/3 differ significantly from Po boutons in S1 (L1 and L5a) in volume, surface area and mean number of synaptic contacts. Moreover, the MC-L4/3 and S1-L5a Po synapses are markedly different in vesicle pool size, in PSD area and shape, and in the proportion of synapses established onto spines. A comparison with VPM S1-L4 synapses revealed both a sharp contrast between S1 Po and S1 VPM synapses, as well as intriguing similarities between MC Po synapses and S1 VPM synapses ; Fig. 14Individual Po cell axons can innervate both the somatosensory and motor cortices, and often other regions . We rece+2 levels which allow enhanced mobilization and recycling of synaptic vesicles for exocytosis, neurotransmitter release and for the generation of synaptic membrane potentials, specially under repetitive high-frequency firing , and have similar PSD sizes . HoweverThese differences may bear some relation with the recently discovered capacity of Po S1-L5a synapses for delayed yet stable potentiation as a result of conditional adult learning, a capacity that, surprisingly, is lacking in VPM S1-L4 synapses . During 2+ buffering, axonal mitochondria promote the docking and/or fusion of synaptic vesicles (In S1, Po and VPM synapses evoke each responses that have markedly different temporal profiles in cortical cells, in part because of the selective involvement of different glutamate receptors types: the VPM synapses involve only ionotropic receptors and their EPSCs depress markedly by repetitive stimulation , whereasvesicles , as wellvesicles . As a revesicles . A highevesicles  or currevesicles  with lowvesicles , whereasvesicles , which hvesicles .Despite arising from thalamic nuclei that are embryologically and functionally different  and beinIn glutamatergic synapses, synaptic strength relates directly to ultrastructural parameters . AlthougOn a broader perspective, the demonstration of branch-specific synaptic structure and dynamic properties in individual axons innervating separate cortical areas reveals an unsuspected subcellular level of complexity in thalamocortical circuits. This is a finding that upends current models of thalamocortical interaction  and that


Full genome analysis revealed that 16 CSFV strains isolated from wild boars between 2017 and 2019 were identical to the YC16CS strain (sub-genotype 2.1d) isolated from an outbreak in breeding pigs near the border with North Korea in 2016. The rapid increase in CSF in wild boars may be due to a continuously circulating infection within hub area and increased population density. The distribution pattern of CSFV in Korean wild boars moves from west to southeast, affected by external factors, including small-scale hunting, geographical features and highways.There has been a rapid increase in the number of classical swine fever (CSF) sero-positive wild boars captured near the demilitarized zone (DMZ), located the border with North Korea. In 2015–2016, few CSFV-positive antibody boars were detected; however, the number has increased steeply since 2017. Most occurred in the northern region of Gyeonggi before spreading slowly to Gangwon (west to east) in 2018–2019. Multi-distance spatial cluster analysis provided an indirect estimate of the time taken for CSFV to spread among wild boars: 46.7, 2.6, and 2.49 days/km. The average CSF serum neutralization antibody titer was 4–10 (log  Classical swine fever virus (CSFV) is a single-stranded RNA virus belonging to the genus pestivirus . Classical swine fever (CSF) is one of the most important viral diseases affecting domestic pigs and wild boars . Wild bop < 0.01 for 2018 and p < 0.001 for 2019), compared to that of other regions , using two-way analysis of variance (ANOVA) with Bonferroni posttest  of females, 3.6% (123/3362) of males, and 2.3% (19/809) of unknown animals were positive for CSFV antibodies . With reposttest .−4 substitutions/site/year) was 6.19, with a 95% HPD interval of 5.2898–7.2025  in 2017, two (YW18WB and IJ18WB) in 2018, and 11  in 2019. Phylogenetic analysis revealed that all strains belonged to sub-genotype 2.1d . The mea8–7.2025 . Geneticp < 0.05) across South Korea (p < 0.05); from 11th January 2017 to 31th October 2019, 107 cases were identified with high CSFV antibody titers within a 55.15 km (Cluster IV) radius  and Gyeonggi (n = 1249) is 1513  radius A,F. The  is 1513 B. The nu is 1513 A. CSFV-i is 1513 B,C.2 in the serum neutralization antibody test ; this period involved increased contact between susceptible animals and infected hosts. In addition, the radius of spatial–temporal clusters is heavily influenced by habitat fragmentation caused by roads, railways, and pipelines . The aveIn September 2019, in South Korea, African swine fever virus (ASFV) was endemic in breeding pigs; the cause of the disease was believed to be wild boars . For CSFrns vaccine strain [rns vaccine will induce production of anti-CSF E2 antibodies and anti-BVDV Erns antibodies simultaneously, but no anti-CSF Erns antibodies [The effectiveness of using the hunting policy to control the disease spread was still controversial. Several reports suggested that hunting wild boars has reduced CSF occurrence ,15. In 1e strain . Baitingtibodies , which mAfter CSFV spread from west to east, we expected it to descend south along the mountains. We did not observe southward spread in 2018, although it did spread gradually in 2019. We suspect that the main reason for this spread pattern is physical obstacles, e.g., major roads crossing from west to east. The information obtained from wild-boar CSFV-surveillance studies will help to predict the path of spread in wild boars and protect domestic pig farms from CSFV. Moreover, studying the pattern of CSF occurrence in wild boars will be a valuable guide to predicting the route and direction of ASFV spread to the south from the nearby DMZ.In conclusion, a rapid increase of CSF sero-positive in Korean wild boars may result from the circulating infections and increased population density within the hub area. In addition, the distribution of the CSFV in wild boars gradually spread from the west to the southeast and was affected by obstacles, such as small-scale hunting, geographical features, highways, and wire fences.From 2010, wild boars were hunted in co-operation with the Korean Pork Producers Association and the Korean government, to satisfy the OIE requirements for surveillance of wild boars and feral pigs in CSF-free countries. Blood samples were collected from 6970 wild boars hunted in nine provinces , in South Korea, between February 2016 and November 2019 . Blood wSus scrofa); there were 274 sites from 1st January 2016 to 31th December 2019. Space–time cluster analysis was conducted by using SaTScan software version 9.6 , with the minimum time aggregation set at 1 month (accounting for the minimum duration of CSFV antibody persistence), a maximum spatial cluster size set as 50% of the population at risk, and a maximum spatial cluster size set as 50% of the total study period [p = 0.05).Space–time permutation scan statistics  were cal2, with serum samples . PK-15 cells were fixed in prechilled 80% acetone and then reacted with a 3B6 monoclonal antibody specific for CSFV E2 3B6 . PK-15 cells were then stained, using a VECTOR kit , biotinylated anti-mouse IgG (H+L) (Cat. No. BA-9200), ABC solution (Cat. No. PK-4000), and a DAB peroxidase substrate (Cat. No. SK-4100). Staining was observed under a microscope.Serum samples from wild boars were tested in a CSFV E2 Antibody ELISA. The CSFV Ab B-ELISA  is a competition ELISA designed to detect the E2 protein; however, it also provides a PI value (≥40% positive and <40% negative). Serum neutralization (SN) tests based on a neutralizing peroxidase-linked assay (NPLA) were performed to detect CSFV-specific neutralizing antibodies. Briefly, PK-15 cells inoculated with CSFV were incubated for 72 h, at 37 °C/5% COAll statistical analyses were performed by using GraphPad Prism software, version 6.0, for Windows. Data were analyzed by using two-way analysis of variance (ANOVA) with Bonferroni posttest.