Background. Transcranial direct current stimulation (tDCS) over the left dorsolateral prefrontal cortex (lDLPFC) was reported to promote the recovery of signs of consciousness in some patients in a minimally conscious state (MCS), but its electrophysiological effects on brain activity remain poorly understood. Objective. We aimed to assess behavioral  and neurophysiological effects  of lDLPFC-tDCS in patients with prolonged disorders of consciousness (DOC). Methods. In a double-blind, sham-controlled, crossover design, one active and one sham tDCS  were delivered in a randomized order. Directly before and after tDCS, 10 min of hdEEG were recorded and the CRS-R was administered. Results. Thirteen patients with severe brain injury were enrolled in the study. We found higher relative power at the group level after the active tDCS session in the alpha band in central regions and in the theta band over the frontal and posterior regions (uncorrected results). Higher weighted symbolic mutual information (wSMI) connectivity was found between left and right parietal regions, and higher fronto-parietal weighted phase lag index (wPLI) connectivity was found, both in the alpha band (uncorrected results). At the group level, no significant treatment effect was observed. Three patients showed behavioral improvement after the active session and one patient improved after the sham. Conclusion. We provide preliminary indications that neurophysiological changes can be observed after a single session of tDCS in patients with prolonged DOC, although they are not necessarily paralleled with significant behavioral improvements. Therapeutic options for severely brain-injured patients with disorders of consciousness (DOC) are limited and need to be improved in order to influence long-term outcomes. Among the non-invasive brain stimulation techniques, transcranial direct current stimulation (tDCS) has shown promising results in DOC . This teTo date, only a few studies have investigated tDCS-induced EEG changes in patients with prolonged DOC, and only one used a randomized controlled double-blind design. Cortical excitability changes following lDLPFC tDCS were assessed by Bai et al. in nine UWS patients and seven MCS patients using transcranial magnetic stimulation coupled with EEG . CompareClinicalTrials.gov NCT03823508) and approved by the local ethics committee, and is reported in accordance with CONSORT guidelines.We prospectively enrolled medically stable patients with DOC, who had been hospitalized for one week for a multimodal assessment of their level of consciousness between January 2018 and March 2019. Inclusion criteria were (1) diagnosed as MCS or emergent from the MCS (EMCS) following an acquired traumatic or non-traumatic injury by means of repeated (≥5) CRS-R and (2) more than 3 months post-injury. We excluded patients in UWS and patients with any contraindications to tDCS  or with premorbid neurological or psychiatric conditions  to be completely blind regarding the treatment allocation.Our primary outcome measures were the neurophysiological alterations, as measured by ten minutes of resting-state hdEEG recorded with eyes open directly before and after the stimulation/sham. As a secondary outcome, behavioral improvement was assessed by means of CRS-R assessments performed by an experienced experimenter directly before and after the tDCS/sham . This device offers a built-in placebo mode for the sham session, which is activated by an anonymous code number and includes ramp periods at the beginning and the end of sham stimulation to mimic the feeling of active tDCS. Direct current was applied by a battery-driven constant current stimulator using saline-soaked surface sponge electrodes (7 × 5 cm) with the anode placed over the lDLPFC  [acement)  and the EEG: hdEEG recordings were acquired with a 256-channel saline-solution compatible electrode net  with a sampling rate of either 250 Hz or 500 Hz.CRS-R: The CRS-R consists of 23 hierarchical items  organized into six subscales assessing auditory, visual, motor, oromotor, communication, and arousal functions [unctions . The iteSignals with sampling rates of 500 Hz were down-sampled to 250 Hz. All data were filtered first using an IIR 8th order Butterworth lowpass filter with a cut-off frequency of 45 Hz and then an IIR 4th order Butterworth high pass filter with cut-off frequency of 0.5 Hz. To minimize the effect of electric line noise, a notch filter was applied on the 50 Hz frequency. Bad electrodes  were identified by visual inspection and excluded. Data were segmented into 2 s epochs with a random jitter (50 to 550 ms) between epochs, and then bad epochs  were identified by visual inspection and rejected. Independent components were computed using the infomax algorithm , and thet-test, and to overcome the multiple comparison problem, a non-parametric cluster permutation test was performed [For each session, the power spectral density (PSD) was first estimated for each epoch and each channel using Welch’s method, with 4096 samples per segment and a 200-sample overlap using a 256-sample Hanning window. Then, the power of delta (1–4 Hz), theta (4–8 Hz), alpha (8–12 Hz), and beta (12–30 Hz) was computed using summation of power values over frequency bins. The relative power was calculated at each frequency band by dividing its power by the total power of the broadband  signal. Then, the trimmed mean (20% trim) of the relative power of each frequency band was calculated over the epochs for each electrode. To study the treatment effect of tDCS on spectral power, changes in power values after stimulation in sham and active sessions were analyzed ((post active–pre active) vs. (post sham–pre sham)) at the group level using a related erformed . For thet-tests.Connectivity analyses were performed using the weighted symbolic mutual information (wSMI)  and weighttps://github.com/fraimondo/shock.All preprocessing and processing scripts were written as custom scripts in Python, MNE-Python , and NICStatistical analyses of behavioral data were performed using SPSS version 17.0 . We inveAs exploratory analyses, we looked at the difference in CRS-R total scores between pre- and post-active, as well as pre- and post-sham.Thirteen patients received both active and sham tDCS in a crossover study design. Two patients were excluded due to missing behavioral data. Finally, eleven patients , three traumatic etiologies, three cardiac arrests, four aneurysms, one meningitis) were included in the study. Based on repeated CRS-Rs, six patients were diagnosed as MCS−, four patients as MCS+, and one patient as EMCS. All MCS patients (n = 10) were included in the group-level behavioral analyses. For the EEG analysis, two subjects were excluded due to highly artifacted or missing data, and EEG group-level analyses were finally performed on nine patients  were calculated in theta and alpha frequency bands. After correcting for multiple comparisons, no significant change was observed in any of the frequency bands. However, in uncorrected statistics, an increase in wSMI alpha connectivity was observed in the parietal region , and an increase in wPLI alpha connectivity was observed in the fronto-parietal regions  and we did not find any significant treatment effect either . No significant differences in CRS-R total scores were found between pre- and post-active  or between pre- and post-sham . When subcategorizing by etiology, we did not find any significant treatment effect for TBI  or non-TBI patients .At the group-level (n = 10), the median (IQR) CRS-R total score before active stimulation was 8 (6–10.5) and 8 (6–9.5) after active stimulation. For the sham condition, this was 8.5 (7–12.25) before and 7.5 (4.75–12.5) after. No carry-over effect was identified  is a 40-year-old male who suffered a TBI two years prior. He was diagnosed as MCS+ and he showed object localization only after the active tDCS. The second patient (S04) is a 59 year-old male who had a cardiac arrest 11 months before tDCS. He was diagnosed as MCS- and showed for the first time localization to sound only after the active tDCS. The third patient (S07) is a 63-year-old male diagnosed as MCS+ five months after a TBI, who showed intentional communication only after the active tDCS. None of these new items resulted in a change in diagnosis.We also found six patients who had a lower total score at the post-active CRS-R than at the pre-active CRS-R; nonetheless, none of them lost a sign of consciousness. Five patients obtained a lower total score at the post-sham CRS-R compared to the pre-sham CRS-R. The aim of this pilot study was to explore the direct effects of a single session of tDCS targeting the lDLPFC on the behavioral and neurophysiological outcomes of patients with severe brain injury in a randomized, controlled, double-blind trial. Our preliminary findings show that tDCS tends to modulate the brain activity and connectivity of the stimulated area but also reaches more distant regions in patients with prolonged DOC, even in the absence of behavioral improvement.Considering spectral power analysis, we observed an increase in theta power in the frontal and posterior regions and an increase in alpha power mainly in central regions. However, these results did not survive multiple comparisons correction for the number of electrodes. This is certainly due to the small number of subjects, which leads to insufficient statistical power to reject the null hypothesis but cannot prove that the null hypothesis can be accepted. However, the present findings are in line with previous neurophysiological studies investigating the correlation between EEG metrics and level of consciousness. Normalized theta and alpha power has been shown to efficiently index the states of consciousness . In addiTo explore connectivity, we used wSMI and wPLI, which have been shown to be robust metrics against volume conduction  of the brain are critical for consciousness, even if fronto-parietal connectivity is still determinant . These aPrevious studies have also reported that theta band connectivity is affected by tDCS ,20. AlthBehaviorally, we did not find any significant treatment effect. Nevertheless, three patients displayed a new behavior associated with consciousness according to the CRS-R only after the active tDCS, which falls within the range 21.9–43%) found by previous studies after one single session of tDCS % found b. The absSome limitations in this study should be acknowledged. The first limitation is undoubtedly the small size of our sample. We only included patients who were medically stable and did not present any contraindication to tDCS , corresponding to 54% of the total number of patients that came to our hospital during the recruitment period , more so in a pathological population of severely brain injured patients. These neurophysiological changes occurred in the absence of behavioral improvement at the group level, as quantified with the CRS-R total scores. The results of this pilot, randomized, double-blind, sham-controlled study are encouraging and highlight the potential of tDCS to induce neurophysiological effects in a population of severely brain injured patients.


Thelarche status at the time of interview was self-reported and defined equivalent to Tanner Stage ≥2. We compared geometric means ) of EM and BPA-G using linear regression and assessed whether EM and BPA-G explained any of the association between exposure to the UK and the age at thelarche using hazard ratios and 95% confidence intervals. Average EM decreased with exposure to the UK, whereas BPA-G increased and was significantly higher among white British  and second-generation British-Bangladeshi girls  compared to Bangladeshi girls . Two of four EM ratios  were significantly associated with thelarche. The relationship between exposure to the UK and thelarche did not change appreciably after adding EM and BPA-G to the models. While BPA-G is often considered a ubiquitous exposure, our findings suggest it can vary based on birthplace and growth environment, with increasing levels for girls who were born in or moved to the UK. Our study did not provide statistically significant evidence that BPA-G or EM concentrations explained earlier thelarche among girls who were born or raised in the UK.Timing of breast development (or thelarche) and its endogenous and exogenous determinants may underlie global variation in breast cancer incidence. The study objectives were to characterize endogenous estrogen levels and bisphenol A (BPA) exposure using a migrant study of adolescent girls and test whether concentrations explained differences in thelarche by birthplace and growth environment. Estrogen metabolites (EM) and BPA-glucuronide (BPA-G) were quantified in urine spot samples using liquid chromatography tandem mass spectrometry (LC-MS/MS) from a cross-sectional study of Bangladeshi, first- and second-generation Bangladeshi migrants to the UK, and white British girls aged 5–16 years ( Breast cancer incidence varies widely by country, with some of the highest rates in Western Europe and lowest in Asia . For exaOne hypothesized driver of earlier pubertal timing is exogenous exposure to hormonally active agents, commonly referred to as endocrine disruptors. Bisphenol-A (BPA) is one such agent that is thought to be ubiquitous in industrialized settings through its use in water bottles, food liners, and drinking water pipes, but relatively unknown in low-middle income countries . In BangHistorically, studies of migrant populations have enabled examination of differences in environmental and social factors associated with breast cancer risk . A recenThe first objective of this study then was to assess the relationship between birthplace and growth environment and both endogenous estrogen levels and exogenous BPA exposure within the same cohort of British-Bangladeshi girls. The second objective was to test whether estrogen and BPA concentrations could explain any of the association between exposure to the UK growth environment  and thelarche previously reported . Given tn = 469), details of which are published elsewhere [This analysis was conducted using ABBY data, a cross-sectional study of Bangladeshi, Bangladeshi migrants to the UK, and white British girls aged 5–16 years of age  Bangladeshi (those who were born and reside in Bangladesh), (2) first-generation British-Bangladeshi (those born in Bangladesh but residing in the UK), (3) second-generation British-Bangladeshi (those born in the UK to parents who emigrated from Bangladesh), and (4) white British (those who were born in the UK to parents of white British ethnicity). This scale captures increasing exposure to the UK environment, a proxy for a range of social and environmental exposures. Outcome: Thelarche status was assessed based on self-reporting using a modified version of the pubertal development scale (PDS), and was defined as the PDS equivalent to Tanner Stage 2 or higher ,18. Due Urine Sample Collection: Urine spot samples were provided by UK and Bangladeshi participants between 9:00 and 16:00 h and placed directly on ice. The urine was aliquoted and stored at 20 °C. UK samples could be transported on ice to a local hospital for processing and temporary storage until samples were transferred to Durham University. Samples from Bangladesh were transported on ice to Sylhet M.A.G Osmani Medical College, where they were aliquoted and stored at −20 °C until being shipped on dry ice to the UK. All UK and Bangladesh urine samples were shipped on dry ice to a National Cancer Institute biorepository in the USA and then to the analytical lab. Estrogen Metabolites: The Cancer Research Technology Program, Frederick National Laboratory for Cancer Research conducted liquid chromatography tandem mass spectrometry (LC-MS/MS) on 0.5 mL of urine to measure 15 estrogens and estrogen metabolites (referred to collectively as EM). Details of this method have been published previously ,20. BrieSimilar to previous studies, we examined EM in four groupings: (i) all EM combined, (ii) parent estrogens only (estrone and estradiol), (iii) the 2-, 4-, and 16-pathways, and (iv) ratios of those pathways . The 2-pn = 67) were replaced as 0.01 ng/mL (the limit of detection). All EM and BPA-G values were divided by creatinine to correct for urinary dilution, log2-transformed to account for the skewed distribution, and added to models as continuous variables. We used log-base 2, rather than traditional log-base 10, to improve interpretability since the results can be interpreted as the effect per doubling of the hormone. Creatinine was measured by PPD © using an enzymatic colorimetric assay; all CVs were below 2%.Bisphenol-A: The same urine samples were used to estimate BPA exposure. The urine assay for BPA and BPA-G, the major conjugate excreted in urine, has demonstrated good reproducibility  , and is 2.Other variables: At the time of urine collection, participant age was recorded and anthropometric measurements, including height, weight, and waist circumference were taken while the participant was clothed but without shoes. BMI was calculated in kg/mMissing data: Of the 348 girls with urine samples, 22 (6.3%) were missing data on whether thelarche had occurred, 4 (1.6%) were missing age at urine sample collection, 39 (11.2%) were missing information to calculate BMI, and 24 (6.9%) were missing waist circumference. Additionally, 11% were missing BPA-G as a result of sample volume constraints. In order to explore the potential for selection bias, we examined differences between girls with and without urine samples, and between girls with and without BPA-G. Girls who did not provide urine were more likely to be Bangladeshi in Sylhet or to have a higher BMI Table A1n = 72) from the models and compared results to the model with all girls included.To characterize differences in EM and BPA by migrant scale (objective 1), we estimated differences in the geometric mean and 95% confidence interval (CI) of urinary EM and BPA-G concentrations by the migrant scale using linear regression. Each of the 11 EMs and BPA-Gs served as the dependent variables and were modeled separately with the migrant scale as the primary predictor. Since age and BMI are strongly related to estrogen production and there were known differences in age and BMI between groups, we adjusted all EM models by age and BMI. Regression coefficients were back-transformed for presentation in the tables. In order to assess the impact of menstrual cycle day on urine collection, we performed an additional sensitivity analysis where we removed postmenarche girls  for all analyses, except for the Weibull regression, which was conducted in STATA Version 15.0 .Statistical significance for all analyses was defined as The study population included 161 Bangladeshi, 33 first-generation British-Bangladeshi, 114 second-generation British-Bangladeshi, and 40 white British girls, with an average age of 9.7, 11.5, 10.1, and 9.9 years, respectively . BangladEMs were detected among all study participants. The sum of all EMs combined ranged from 8.6 to 14,719 ng/mL. Second-generation British-Bangladeshi and white British girls had significantly lower levels of 2-pathway  and 0.05 , respectively) and 4-pathway  and 0.01 , respectively) EMs compared to Bangladeshi girls , 4-pathway = 0.01 (95% CI: 0.01–0.02)). Second-generation British-Bangladeshi also had several significantly different EM ratios than Bangladeshi girls, including the ratio of 2-pathway to parent, 4-pathway to parent, 16-pathway to parent, and parent to the 2-, 4-, 16-pathway. In our sensitivity analysis where we removed girls who had reached menarche, the point estimates were similar but no longer significant.p < 0.0001 for trend) with duration of time in the UK, and were nearly 2 times greater in white British compared with Bangladeshi girls  compared to second-generation British-Bangladeshi (4%) or white British girls (15%). BPA-G concentrations increased significantly  were significantly associated with thelarche . The 16-Adding the sum or individual EM or BPA-G concentrations to the model assessing the relationship between migrant scale and thelarche had minimal effects on the association . For exaIn this study of adolescent girls in Bangladesh and the UK, we found there were differences in the urinary concentrations of EM and BPA-G by place of birth and growth environment. In particular, urinary BPA-G concentrations were higher among those living in the UK for longer periods (whether native-born or migrant), while EM concentrations were lower. Our findings represent an important step towards simultaneously characterizing endogenous estrogen hormones and exogenous BPA exposure among adolescents in Bangladesh, an area where little information about BPA exposure is known. While we found some differences in thelarche by EM pathway or route of metabolism, our study did not provide evidence to suggest that variation in EM or BPA-G explained differences in thelarche between groups reported in our prior publication . Recent reviews have highlighted the paucity of information on BPA exposure from low-middle income countries, where packaged foods and beverages containing BPA have become increasingly common but regulation has lagged behind that of developed countries . One of Conversely, we found that EM levels decreased with duration of time in the UK, although not significantly. This runs counter to evidence from adult studies where urinary estrogen levels tend to be higher in Western countries. For example, total estrogen and EMs were three times higher among Asian-American women in the US  than in Shanghai-based Chinese women . We alsoDespite some differences in urinary concentrations of BPA and estrogens between the groups, our study found limited evidence that these concentrations explained the association between exposure to the UK environment and earlier thelarche. Evidence concerning the relationship between BPA and pubertal outcomes has been mixed . SeveralOur study has several limitations to note. First, the data were cross-sectional and thelarche, estrogen, estrogen metabolites, and BPA were only assessed at one point in time. While intraclass correlation coefficients for EMs are high, indicating good reproducibility , BPA varIn response to growing concerns about early-life exposure to endocrine disruptors and pubertal outcomes, we explored the relationship between BPA, estrogen metabolites, and thelarche in a unique, cross-sectional migrant study of adolescent girls from the UK and Bangladesh. By contrasting birthplace and growth environment between countries, we found a clear pattern of increasing BPA-G and decreasing estrogen/estrogen metabolite concentrations with duration of time in the UK. We also found that the onset of thelarche was associated with ratios of parent to pathway estrogen metabolites, particularly the 16-pathway, which has been implicated in breast cancer research. Ultimately, BPA-G and estrogen did not explain earlier onset of thelarche in white British girls, which could indicate that other environmental or social factors were at play, or that the current study was underpowered.


To identify potential microRNA (miRNA) biomarkers of poor, normal and hyperresponse to controlled ovarian stimulation (COS).In the present study, we assessed 40 serum samples from patients undergoing COS. We used ten samples to standardize miRNAs detection in the serum. The remaining 30 samples were split into three groups depending on the patient's response to COS: poor response , normal response , and hyperresponse . Aberrantly expressed miRNAs were identified using a large-scale expression analysis platform. Gene set enrichment analysis was performed to assess the biological processes potentially modulated by the identified miRNAs.Twenty-two miRNAs were detected only in the PR or HR groups when compared with the NR group. From those, 11 presented poor dissociation curves and were excluded from further analysis. A bioinformatics analysis revealed that the selected 11 miRNAs target several genes involved in GnRH, estrogen and prolactin signaling, oocyte maturation, female pregnancy, and meiosis.The large-scale analysis of miRNA expression identified distinct miRNA profiles for poor and hyperresponse to COS, which potentially modulate key processes for human assisted reproduction. All evidence suggests that the serum microRNA profiling may discriminate patients who will respond in an exacerbated manner from those who will respond insufficiently to COS. Further studies may validate these miRNAs, enabling the individualization of treatment and more successful outcomes. The selPrevious registers have reported important data concerning the cancellation rates of IVF cycles (According with the consensus from the European Society of Human Reproduction an Embryology (ESHRE), a poor ovarian response is defined as the collection of fewer than four oocytes in response to a COS protocol of at least 150 IU FSH per day , serum AMH, and other factors have been pointed out as possible predictive factors to ovarian response to COS. However, there is no consensus regarding which factors to take into account or the importance of each factor when determining the dose of gonadotrophins for COS (Caenorhabditis elegans (MicroRNAs (miRNAs) are endogenous, evolutionally conserved, single-strand non-coding RNA molecules of 20-24 nucleotides that post-transcriptionally regulate gene expression in eukaryotes, including mammalian cells . The samples were collected in a private university-affiliated IVF center, between January 2017 and January 2018.We used ten samples to standardize the detection of serum miRNAs. The remaining 30 samples were split into three groups depending on the patient's response to COS: poor response , normal response , and hyperresponse . The aberrantly expressed miRNAs were identified using a large-scale expression analysis platform. In addition, we carried out a gene set enrichment analysis to access the biological processes potentially modulated by the identified miRNAs.The study included only patients undergoing their first or second IVF cycle, using fresh oocytes and/or embryos, and aged <38 years old. We obtained a written informed consent, in which patients agreed to share the outcomes of their cycles for research purposes, and the local institutional review board approved the study.Serum samples were obtained through venipuncture, prior to beginning the COS. After complete filling of a serum separation tube, the blood was carefully mixed with coagulation activating agent and remained standing for 60 minutes. After complete coagulation, the samples were centrifuged at 1500 to 2000xg for 10 minutes until the serum was well separated. Using a Pasteur pipette, the serum was transferred to another tube and stored at -20°C.®, Merck KGaA, Darmstadt, Germany) and pituitary suppression using a GnRH antagonist . Follicular growth was monitored using transvaginal ultrasound starting on day 4 of the gonadotropin administration. When adequate follicular growth and serum E2 levels were observed, recombinant hCG  or leuprolide acetate  was administered to trigger the final follicular maturation. The oocytes were collected 35 hours after hCG administration through transvaginal ultrasound ovum pick-up.Controlled ovarian stimulation was performed using recombinant FSH .A small RNA fraction was extracted from 200µl of serum samples, using a miRNeasy Serum/Plasma extraction kit . ® QIAGEN, Helden, Germany) on a ViiA7 apparatus . We analyzed the data using a PCR Array Data Analysis Web Portal software .The identification and quantification of aberrantly expressed miRNAs, in the pool of samples form PR, NR and HR groups, was performed by using a large-scale quantification PCR-Array platform . For each group, the lists of predicted targets for each miRNA were combined, and those targets predicted by 7 out of the 12 algorithms, or less, were excluded. The remaining target list was then submitted to enrichment analysis using the DAVID Annotation tool (https://david.ncifcrf.gov/).The list of putative targets for the selected miRNAs for each group (HR or PR) was obtained using the miRWalk 2.0 web tool  statistical program. The variables were tested for normality distribution and group homogeneity using the Shapiro Wilk and Levene tests, respectively. When necessary, the samples were normalized using z-scores. The groups were compared by ANOVA, followed by the Bonferroni post-hoc test. Variables are described as mean ± standard deviation and the significance level α was set at 5%.miR-99a-5p, miR-181d-5p, miR-221-3p, miR-92a-1-5p, and miR-1302 were only detected in the HR groups, while miR-150-5p, miR-223-3p, let-7d-3p, miR-891a-5p, miR-99a-3p and, miR-200c-5p were only detected in the PR group  was performed. The GSEA revealed that miRNAs from the HR group potentially modulate key members of GnRH, estrogen, prolactin, and oxytocin signaling pathways, as well as progesterone-mediated oocyte maturation and female pregnancy  and 2. Il cancer  and 2. mThe main objective of treatment individualization in IVF is to offer every single woman the best treatment tailored to her unique characteristics, thus maximizing success, eliminating iatrogenic risks such as OHSS, and minimizing the risk of cycle cancellation. However, the success of individualized COS depends on finding a reliable method for predicting the ovarian response to stimulation.miR-21-5p expression (For the present study, potential miRNA biomarkers of the response to ovarian stimulation were identified in the serum of patients undergoing ART, before COS. Extensive research has been carried out in a search for markers that can be used to facilitate COS individualization. Previous studies have focused on the assessment of miRNAs as molecular markers in assisted reproduction. It has been found that a poor response to COS is associated with altered miRNA expression, specifically with elevated In this manner, the study of altered expression of miRNAs in serum may be more useful. In our study, we aimed at identifying miRNAs in the serum of patients with different responses to COS. Several miRNAs, expressed only in poor and hyperresponders, were identified in a large-scale analysis by the qPCR array platform. miRNAs analyses of target genes indicated that these molecules are key player candidates. Moreover, the levels of this miRNA positively correlate with important clinical aspects, such as the number of retrieved oocytes.miR-181d-5p has been described in various types of cancer, including ovarian carcinoma (miR-181d-5p in ovarian physiology is still to be unveiled.Altered expression of miR-150 expression, detected only in the PR group, is decreased during ovarian development in mice. In addition, it has been shown that miR-150 is expressed in higher levels in atretic rather than healthy follicles in cattle, and may play a role in bovine oocyte maturation (miR-150 expression is decreased in patients with endometriosis treated with simvastatin (A study by Thus, miRNA biomarkers continue to emerge as potential tools for the management of infertility. Future analyses for the detection of these miRNAS in a large number of samples could contribute to their validation as diagnostic tools for the prediction of ovarian response to COS.


The coronavirus disease 2019 (COVID-19) has recently been found to cause cutaneous vasculitis in patients. Granulomatosis with polyangiitis (GPA) is a type of small and medium vessel vasculitis that is often associated with pulmonary issues and has been shown to raise diagnostic complications in COVID-19 infection. In this report, we discuss the first case of new-onset GPA in the setting of active COVID-19 infection. Symptoms often overlap between the two diseases, and while there is no current cure for COVID-19, rapid immunosuppressive initiation can be lifesaving for patients with GPA. Thus, this case is essential in expanding our current knowledge of COVID-19 and its many skin manifestations. The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread across the globe, while clinicians have been compelled to continuously expand their knowledge base of this novel complex disease and its associated symptoms. Several coronavirus patients have been found to have vascular endothelial damage, which is thought to play a role in the development of both skin lesions , 4. HereIn April 2020, a 46-year-old man with a past medical history of diabetes presented to the emergency room with symptoms of cough, shortness of breath, and fevers for four weeks. He was treated for suspected pneumonia with azithromycin two weeks prior to presentation and denied any improvement in respiratory symptoms thereafter. In addition, he developed a painful and pruritic rash that was progressively worsening over the last two weeks  with tachycardia (110 bpm), blood pressure 127/77 mm Hg, respiratory rate 20, and O Figures . He also Figures , as wellμL), decreased hemoglobin (10.3 g/dL), elevated blood urea nitrogen (48 mg/dL), creatine (2.9 mg/dL), and D-dimer . He was cANCA positive, coronavirus PCR negative via nasopharyngeal swab, and coronavirus antibody IgM positive and IgG negative. CT scan of the chest showed patchy ground-glass opacities in the lungs.Laboratory evaluation showed an elevated white blood cell count  . High-poBased upon his clinical presentation, histologic findings, and laboratory studies, the patient was diagnosed with GPA in the setting of suspected coronavirus infection. He was first treated for coronavirus with five-day course of hydroxychloroquine, followed by treatment of GPA with systemic steroids and rituximab infusion. Kidney function improved, and his rash completely resolved. He was discharged on a steroid taper.We discuss a case of c-ANCA-positive granulomatosis with polyangiitis possibly triggered by a COVID-19 infection. Our patient presented with four weeks of symptoms and had negative nasopharyngeal PCR test; however, positivity to RT-PCR begins to decline three weeks after initial symptom onset . FurtherInfections or viruses can trigger GPA , and witGPA can mimic the clinical presentation of COVID-19 infection, and cross-reactivity can complicate interpretation of COVID-19 serology results. Prompt treatment for GPA is indicated to prevent irreversible lung and renal damage. While some immunotherapies may increase the risk of a COVID-19 infection, immunosuppressives have overall shown decreased risk of adverse events associated with infection and better patient outcomes. Our patient with biopsy-proven GPA in the setting of coronavirus infection improved clinically with the use of immunosuppressive therapy. We hope that, by presenting his case, we can further expand clinical knowledge about this complicated viral disease and the use of immunotherapy in a setting where the risk of coronavirus infection is high.


There are few primary care studies of the COVID-19 pandemic. We aimed to identify demographic and clinical risk factors for testing positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within the Oxford Royal College of General Practitioners (RCGP) Research and Surveillance Centre primary care network.We analysed routinely collected, pseudonymised data for patients in the RCGP Research and Surveillance Centre primary care sentinel network who were tested for SARS-CoV-2 between Jan 28 and April 4, 2020. We used multivariable logistic regression models with multiple imputation to identify risk factors for positive SARS-CoV-2 tests within this surveillance network.vs 291 [13·3%] of 2190 women; adjusted odds ratio [OR] 1·55, 95% CI 1·27–1·89). Adults were at increased risk of testing positive for SARS-CoV-2 compared with children, and people aged 40–64 years were at greatest risk in the multivariable model . Compared with white people, the adjusted odds of a positive test were greater in black people . People living in urban areas versus rural areas  and in more deprived areas  were more likely to test positive. People with chronic kidney disease were more likely to test positive in the adjusted analysis , but there was no significant association with other chronic conditions in that analysis. We found increased odds of a positive test among people who are obese . Notably, active smoking was linked with decreased odds of a positive test result .We identified 3802 SARS-CoV-2 test results, of which 587 were positive. In multivariable analysis, male sex was independently associated with testing positive for SARS-CoV-2 (296 [18·4%] of 1612 men A positive SARS-CoV-2 test result in this primary care cohort was associated with similar risk factors as observed for severe outcomes of COVID-19 in hospital settings, except for smoking. We provide evidence of potential sociodemographic factors associated with a positive test, including deprivation, population density, ethnicity, and chronic kidney disease.Wellcome Trust. The world is in the midst of a pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19.Evidence before this studyWe searched PubMed, MEDLINE, and Trip Medical Database from inception to April 14, 2020, for community-based studies that described the epidemiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or the associated illness, COVID-19, using the terms “(COVID-19 or 2019-nCoV or SARS-CoV-2) AND ”, with no language restrictions. We found no relevant studies. Hospital-based studies have reported increasing age, male sex, and certain comorbidities, such as hypertension and diabetes, to be associated with more severe COVID-19 disease. Whether these risk factors apply to the risk of SARS-CoV-2 infection in primary care is unclear.Added value of this studyWe did a cross-sectional study of patients with a SARS-CoV-2 test code result in the Oxford Royal College of General Practitioners Research and Surveillance Centre network between Jan 28 and April 4, 2020. We observed 587 patients with positive results and 3215 with negative results. Since we have sociodemographic and clinical data on patients in our sample, we could assess risk factors for a positive SARS-CoV-2 result, adjusted for potential confounding variables. Increasing age, male sex, population density, more deprived areas, and black ethnicity were associated with an increased risk of a positive SARS-CoV-2 test. Chronic kidney disease and obesity were the only clinical factors associated with a positive test. Current smokers had lower odds of a positive test. To our knowledge, this study is one of the first to investigate risk factors for testing positive for SARS-CoV-2 in the community.Implications of all the available evidenceOur findings suggest some risk factors for SARS-CoV-2 infection in this primary care study are similar to those associated with more severe COVID-19 disease, with men and people older than 40 years at increased risk. Research is needed into the effect of chronic conditions on the risk of infection and disease severity, ethnic variations in COVID-19 incidence, and the risk to smokers.The Oxford Royal College of General Practitioners (RCGP) Research and Surveillance Centre programme is one of the longest established primary care sentinel networks globally. It includes more than 500 urban and non-urban participating general practices, covering a population of over 4 million people .6, 7, 8 We did a cross-sectional study in patients in the Oxford RCGP Research and Surveillance Centre network who were tested for SARS-CoV-2 between Jan 28 and April 4, 2020. Pseudonymised SARS-CoV-2 results and other clinical and sociodemographic data were extracted from computerised primary care medical records of sentinel practices. These data allow estimation of household size,RT-PCR testing for SARS-CoV-2 was done at the Public Health England Colindale Laboratory  using previously described methods.We included patients who were registered at an RCGP Research and Surveillance Centre practice on Sept 30, 2019, who had an entry in their medical record reporting a positive or negative test for SARS-CoV-2. We have developed a COVID-19 surveillance ontology to ensure consistency of case definition and included only people with a coded positive or negative test, and not those with suspected disease .19 PatieThe data used for the analysis were pseudonymised at the point of extraction and encrypted before uploading to the Clinical Informatics Research Group secure server. Personal data were not identifiable during the analysis. The data extraction was done as part of national surveillance work commissioned by Public Health England and approved under Regulation 3 of The Health Service (Control of Patient Information) Regulations 2002.We included the following independent demographic variables: age, sex, and ethnicity, using an ontology to maximise case identification;We used descriptive statistics and reported counts and proportions for categorical data and measures of distribution for continuous data. We described the proportion of participants with missing data for each variable . We testWe used R version 3.5.3 for all analyses; we used the R library mice 3.4.0 for the multiple imputation routine.The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the paper. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.Between Jan 28 and April 4, 2020, we observed 587 patients with positive SARS-CoV-2 results and 3215 with negative results in the surveillance programme. The first positive case presented on Jan 30, 2020, and 100 cases was reached on March 17, 2020. Overall, 2190 (57·6%) of 3802 patients were female and 2497 (65·7%) were white . The medIn univariable analysis, the odds of testing positive for SARS-CoV-2 were higher among older people, men, and people of ethnicity other than white, and people living in more deprived areas . The oddvs least deprived; 2·03, 1·51–2·71) were associated with increased odds of a positive SARS-CoV-2 test.In multivariable analysis, adjusted for all other variables in Active smoking was associated with decreased odds of a positive SARS-CoV-2 test result . People with chronic kidney disease were more likely than those without to test positive for SARS-CoV-2 in the adjusted analysis , but there was no significant association with the other chronic conditions . We founIn sensitivity analyses, we did a complete case analysis  and impuWe report one of the first and largest cross-sectional analyses using primary care data to assess risk factors for testing positive for SARS-CoV-2. In our sample, we found increasing age, male sex, increasing deprivation, urban location, and black ethnicity were associated with increased odds of a positive SARS-CoV-2 test. Current smoking was linked with decreased odds of a positive test. Chronic kidney disease and increased BMI were the only clinical factors independently associated with a positive test.A literature review suggested that COVID-19 has affected more men than women, and principally those aged 30–65 years, with around half of cases being older than 50 years.SARS-CoV-2 transmission is known to be associated with high population density due to increased social mixing,Preliminary evidence has raised concerns regarding the potential increased risk of adverse COVID-19 outcomes among people of Asian and black ethnicities, but few epidemiological studies have assessed risk by ethnic group.Systematic reviews have shown that people with COVID-19 who have chronic comorbidities such as hypertension, diabetes, and cardiovascular disease are at high risk of progressing to severe COVID-19 disease.Previous studies have reported that smoking is associated with increased risk of intensive care unit admission or death among people with COVID-19.To our knowledge, our study is one of the first to report risk factors for testing positive for SARS-CoV-2. Our use of rich primary care surveillance data allowed adjustment for potential confounding factors. The Oxford RCGP Research and Surveillance Centre is an established network of sentinel practices, meaning clinicians are experienced in undertaking surveillance research and use coding ontologies to standardise reporting.Like all routine datasets, some data will be missing from our set. Where data are missing at random, multiple imputation has the potential to reduce bias and improve precision. However, the missing at random assumption is not testable. In certain situations when the missing at random assumption does not hold, we can rely on a complete-case analysis to provide unbiased estimates .Although our study population of primary care patients is likely to be more similar to the general population than that of hospital-based studies, there remains a risk of selection bias because results might reflect the groups of patients who were more likely to present for assessment and be selected for SARS-CoV-2 testing in accordance with guidelines. If certain groups  are only likely to present or be tested when more severely unwell, those who were tested could be more likely to be positive for COVID-19. Conversely, groups with lower thresholds for presentation might be tested with less severe symptoms, and therefore be more likely to test negative. It was not possible to assess the effects of thresholds for presentation and changes in testing guidelines in this analysis. Population-based surveys should ensure consistent levels of testing across subgroups as far as possible to reduce the risk of selection bias.Although RT-PCR testing is the gold standard for SARS-CoV-2 diagnosis, overall test sensitivity in clinical use might be reduced by factors such as swab technique and the timing relative to symptom onset. Therefore, some SARS-CoV-2 cases could have been missed, particularly among patients with lower viral loads, which could bias results if any of the variables that we studied  were associated with differences in viral load, rather than actual infection. Also, the sentinel network changed from in-practice nasopharyngeal swabbing to self-swabbing on March 14, 2020, which nonetheless has been found to be a reliable method when testing for influenza.Further data are needed to establish the epidemiology of SARS-CoV-2, particularly in relation to emerging factors such as ethnicity, deprivation, population density, and smoking. Population-based surveys could help reduce selection bias and ensure adequate inclusion of different population subgroups. Our data from primary care could help monitor incident infections and, therefore, the effect of public health mearues, and we plan analyses of rates of hospitalisation and death as the pandemic unfolds.In conclusion, primary care sentinel network data provide important insights into the epidemiology of SARS-CoV-2, although our study is limited by its small scale and selection of patients presenting for SARS-CoV-2 testing through routing health-care services. Our findings on smoking might be due to presentation confounding and should not encourage people to continue or take up smoking. Increasing age, male sex, socioeconomic deprivation, increased population density, black ethnicity, chronic kidney disease, and obesity were all associated with increased risk of a positive SARS-CoV-2 test.Research and Surveillance Centre dataset can be accessed by researchers. Approval is on a project-by-project basis. Ethical approval by a UK National Health Service Research Ethics Committee is needed before any data release or other appropriate approval. Researchers wishing to directly analyse patient-level pseudonymised data will be required to complete information governance training and work on the data from the secure servers at the University of Surrey . Patient-level data cannot be taken out of the secure network. We encourage interested researchers to attend the short courses on how to analyse primary care or RCGP Research and Surveillance Centre data, which are open to enrolment twice a year.The Royal College of General Practitioners (RCGP)


The primary endpoint was an altered proportion of whole lung lesion volumes from baseline to day 28. Other imaging outcomes, 6-minute walk test (6-MWT), maximum vital capacity, diffusing capacity, and adverse events were recorded and analyzed. In all, 100 COVID-19 patients were finally received either UC-MSCs (n = 65) or placebo (n = 35). UC-MSCs administration exerted numerical improvement in whole lung lesion volume from baseline to day 28 compared with the placebo . UC-MSCs significantly reduced the proportions of solid component lesion volume compared with the placebo . The 6-MWT showed an increased distance in patients treated with UC-MSCs . The incidence of adverse events was similar in the two groups. These results suggest that UC-MSCs treatment is a safe and potentially effective therapeutic approach for COVID-19 patients with lung damage. A phase 3 trial is required to evaluate effects on reducing mortality and preventing long-term pulmonary disability.  is challenging. We performed a phase 2 trial to assess the efficacy and safety of human umbilical cord-mesenchymal stem cells (UC-MSCs) to treat severe COVID-19 patients with lung damage, based on our phase 1 data. In this randomized, double-blind, and placebo-controlled trial, we recruited 101 severe COVID-19 patients with lung damage. They were randomly assigned at a 2:1 ratio to receive either UC-MSCs (4 × 10 At present, ongoing clinical trials of immunotherapeutic approaches include convalescent plasma therapy, monoclonal antibodies against interleukin-6, and cellular therapies.12 Mesenchymal stem cells (MSC) are non-hematopoietic cells with immune modulatory, regenerative, and differentiation properties.13 MSC treatment reduced the pathological changes of the lung and inhibits the cell-mediated immune-inflammatory response induced by the influenza virus in animal models and clinical trials.15 The safety and potential efficacy of MSC have also been evaluated in the patients with acute respiratory distress syndrome (ARDS).19 The immunomodulatory and regenerative properties of MSCs offer potential cellular therapeutic option for lung damage in patients with COVID-19 and require evaluation in randomized controlled trial. In a phase 1 trial we previously demonstrated that intravenous transfusions of human umbilical cord (UC)-MSCs in patients with moderate and severe COVID-19 were safe and well tolerated (NCT04252118).20 We now report results of a randomized, double-blind, placebo-controlled trial performed at two medical centers in Wuhan, China, evaluating the safety and efficacy of intravenous treatment with UC-MSCs in severe COVID-19 patients with lung damage.The Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection,From 5 March 2020 to 28 March 2020, a total of 288 patients were screened at two hospitals in Wuhan city. The majority of severe hospitalized COVID-19 patients were at the convalescent stage and some of them were with progression stage. Among them, 101 eligible patients previously diagnosed as severe COVID-19 type, being referred as the ITT population, were randomized in a 2:1 ratio (66 to the UC-MSC group and 35 to the placebo group). One patient in the treatment group withdrew her previously written informed consent after randomization and did not receive UC-MSC infusion. Therefore, 65 and 35 patients were treated with UC-MSC or placebo, respectively, who were defined as the modified intention-to treat (mITT) population, as shown in Fig. The baseline characteristics were highly consistent between the two groups of patients in the mITT population Table . In brieP = 0.080) . We also observed decrease in the ground-glass lesions in the MSC group than the placebo group, although the difference was not statistically significant. To exclude the effect of missing data on the outcomes of the above results in some mITT cases, we further analyzed the PP population. Almost identical results between the two groups were obtained, as shown in Table To evaluate the difference in the primary endpoint and parts of the secondary endpoints, we analyzed the changes in high-resolution chest CT images and measured the lesions by using centralized imaging interpretation based on the evaluation of both radiologist analyses and lung imaging artificial intelligence software. Through comparison of the Hodges–Lehmann estimator of the total lesion proportion (%) of the whole lung volume, the median change was −19.40%  in the MSC group, and −7.30%  in the placebo group on day 28 from baseline, yielding a difference of −13.31%  Fig. . InteresP = 0.057, Table max) and diffusion lung capacity for carbon monoxide (DLCO), the six-category scale, status of oxygen therapy, and mMRC dyspnea score were similar between the two groups  on the 28th day after the onset of treatment and found that 6-minute walking distance was longer in the MSC group (median 420.00 meters ) than in the placebo group  with a 95% CI of 0.00–57.00  for sensitivity analyses of primary endpoint (Supplementary Table The incidence of adverse events reported during the study was similar in the MSC group 55.38%) and the placebo group (60%)  of the whole lung volume as measured by CT from baseline to day 28. The patients enrolled in this study were all previously diagnosed as severe types, with a longer disease course and older age, compared with the recent studies.25 In particular, all the patients suffered from serious pulmonary damage and needed oxygen inhalation support during the course of disease. Our current trial showed that UC-MSCs therapy improved the resolution of the whole lung damage size, as detected by CT scanning, particularly, the solid component lesions. This finding indicated that UC-MSC administration has a therapeutic benefit for patients with COVID-19, even in the convalescent stage. It is known that the solid component lesions in the lung include the interstitial fibrosis. Thus, the improvements of solid component lesions might also imply the alleviation of lung fibrosis. Long-term follow-up is currently ongoing to monitor the lung lesion of these patients, which will provide more information regarding the patients’ pathological progression including pulmonary fibrosis. Moreover, our previous phase 1 trial has demonstrated that intravenous UC-MSCs transfusion for severe COVID-19 patients with ARDS showed potential improvement of some clinical parameters in part of the treated patients.20 Thus, the effect of MSCs on ARDS in severe COVID-19 patients merits exploration in the future study.COVID-19 is characterized by pathological lung changes in both the parenchyma and interstitium,26 In this trial, the 6-MWT was numerically, but not statistically, improved in the MSC group compared with that in the placebo group. Given that there was no significant difference in cardiovascular diseases between the two groups and no cerebrovascular diseases in both groups at baseline, the findings from the 6-MWT data imply that capacity for aerobic exercise was potentially improved in the UC-MSCs group.The 6-MWT has been used to evaluate patients suffering from a variety of cardiopulmonary diseases. The results reflect the integrated reserve capability of complex physiology, involving the pulmonary and cardiovascular systems, and neuromuscular circulation.29 Cytokines such as serum IL-6 are considered as biologically relevant biomarkers associated with disease progression of COVID-19. In this study, UC-MSC infusion did not result in a significant reduction in the duration of oxygen therapy, mMRC, cytokine, or chemokine levels, which might be in part attributed to the status of the enrolled population, as most of them were not in the acute progressive stage. Other mechanisms of actions have to be explored for MSCs that are measurable in the systemic circulation. We could not, however, measure the local, i.e., intrapulmonary, effects of MSC delivery. It could very well be that the local MSC-mediated effects were not measurable in the systemic circulation, a similar scenario as in MSC treatment of patients with corticosteroid-resistant graft-versus-host-disease. In addition, we and others have found T-cell overactivation existed simultaneously with T-cell exhaustion in severe patients.30 Further study is necessary to investigate how MSC treatment modulate the over-activated T-cell response in vivo.It is hypothesized that the beneficial effect of MSC treatment for patients with severe COVID-19 is mediated via reduction of pro-inflammatory cytokines, that jointly mediate immune pathology and worsen clinical COVID-19 outcomes.7 UC-MSCs were transfused for each patient. No MSC-related predefined haemodynamic or respiratory adverse events were observed. The incidences of adverse events were similar between the MSC group and the placebo group. Only one patient in the MSC group suffered a pneumothorax that was judged to be unrelated to UC-MSC medication. No patient died during the follow-up period. The safety profile of the UC-MSCs confirms the results of our previous phase 1 trial20 and other MSC studies.32 These data suggested that UC-MSC therapy was well tolerated and very safe. In view of the ongoing clinical trials with higher dose of MSCs used in other groups in China, America, and Europe,21 more safety data are expected in the near future.In our trial, a total of three doses of 4 × 1033 patients with COVID-19 require further centralized isolation for 14 days after discharge. In this setting, some of the patients missed the follow-up data at day 28, but they did receive a follow-up check ~7–10 days after the 28-day follow-up window. Importantly, our PP population analysis also revealed similar results compared with mITT population analysis. Whether the cell dosage, interval duration, and cycles of UC-MSC medication were the best regimen for patients with severe COVID-19 were not fully investigated in this study.There were several limitations in this study. A larger sample size could have improved efficacy analyses. According to management guidelines issued by the Chinese National Health Commission (7th edition),To the best of our knowledge, this is the first randomized, double-blind, placebo-controlled trial evaluating the safety and preliminary efficacy of UC-MSCs as a potential treatment for patients with COVID-19 with lung damage, even at the convalescent stage. UC-MSC administration was safe and accelerated resolution of lung solid component lesions and improvement in the integrated reserve capability after UC-MSC administration. UC-MSCs treatment offers a safe and potentially effective therapeutic approach for COVID-19 patients with lung damage. A phase 3 trial is required to further evaluate effects on preventing long-term pulmonary disability, reducing mortality, and determining the underlying mechanisms of UC-MSC treatment for COVID-19 disease.We conducted a randomized, placebo-controlled, double-blind phase 2 trial . The study was done between 5 March 2020 and 28 March 2020. Ethical approval was obtained from the institutional review boards of each participating hospital. The clinical protocol and statistical analysis plan are available in Supplementary Data S33 In brief, patients with any of the following conditions but without invasive ventilation, shock or other organ failure (need organ support therapy) were considered as severe cases: (1) dyspnea (respiratory rate ≥ 30 times/min); (2) oxygen saturation of 93% or lower on room air; (3) arterial oxygen partial pressure (PaO2)/fraction of inspired oxygen ≤ 300 mmHg; (4) pulmonary imaging showing that the foci progressed by > 50% in 24–48 h. The exclusion criteria included patients with shock or COVID-19 combined with any one of other organ failures, those who received invasive ventilation, or patients with any malignant tumor, pregnancy or breastfeeding, or co-infection of other pathogens.Hospitalized patients with severe COVID-19 with laboratory-confirmed SARS-CoV-2 infection by reverse transcription polymerase chain reaction (RT-PCR) were screened. Patients were eligible if they met any of the following criteria: (1) severe COVID-19 diagnosed after onset of disease; (2) chest computed tomography (CT) imaging confirmed pneumonia combined with lung damage. The illness severity of COVID-19 was evaluated in accordance with Guidelines issued by the National Health Commission of China (version 7.0).Eligible patients were randomly assigned in a 2:1 ratio to receive either UC-MSCs or the placebo, in addition to standard care, using an interactive web response management system (IWRS). A permutated-block randomization sequence that was stratified by the trial sites was generated and uploaded to the system. Patients, investigators, and outcome assessors  were all blinded to the treatment allocation. Blinding was also ensured by the product marking, with the UC-MSCs and the placebo having a similar appearance and packaging.A barcode-based product management system  was introduced in this study to manage and track the study products logistics, e.g., preparation, packaging, shipping, storage, and clinical administration to the patients. The application of PIATS could realize the blind label processing by non-informative unique barcodes of the clinical study drug. The concealment of the randomization sequence could be ensured using PIATS and IWRS.20 The UC-MSCs used in this study came from one umbilical cord of full-term deliveries (after consultation with the parents of UC donor). The Wharton’s Jelly (WJ) tissues were cut into ~ 2 mm3 pieces from cord tissue and planted upside down on tissue culture flasks (75 cm2) cultured in DMEM/F12, supplemented with fetal bovine serum  at 37 °C with 5% CO2. The adherent cells were detached with 1× TrypLE  and then re-plated at a density of ~6–8 × 103 cells/cm2 for further expansion. Master cell bank at passage 2 and working cell bank at passage 4 were set separately. A homogenous population of cultured cells at passage 5 were prepared as UC-MSC product. The culture cells were identified by the minimal criteria suggested by International Society for Cellular Therapy (ISCT): (1) plastic adherent under tissue culture flask; (2) >95% of the cell population expressed CD105, CD73, and CD90, and these cells were lack expression (<2% positive) of CD45, CD34, CD11b, CD19, and HLA-DR as measured by flow cytometry ; (3) differentiation potential into osteoblasts, adipocytes, and chondroblasts under standard in vitro differentiating conditions. For each individual MSC batch, cell viability was examined using both trypan blue and 7-AAD/Annexin V staining by flow cytometry after preparation in Tianjin and before intravenous transfusion in Wuhan, respectively . Shipping of cell products by express railway from Tianjin to Wuhan took less than 6 h.The UC-MSC product was an almost colorless suspension containing 4.0 × 107 cells for each procedure, and three procedures were carried out for each patient on day 0, 3, and 6 after randomization. Infusion was started with a standard blood filter tubing set with a pore size of 170 μm. Under electrocardiographic monitoring, the cell product was infused by gravity within 60 min. We also monitored continuous pulse oximetry as well as patient’s physical signs including body temperature, pulse, skin color, respiration, and blood pressure during the infusion period and up to 30 min after infusion.The treatment dose was 4.0 × 10The incidence and nature of all adverse events were reviewed and assessed by the investigators to determine whether they were related to the administration of the study product. Methods for data collection and in-study measurements are described in detail in Supplementary Data SAll patients underwent high-resolution chest CT examination at baseline, day 10, and day 28. The primary outcome was gauged as a change in the total lesion proportion (%) of the whole lung volume from baseline to day 28, as measured by chest CT. It was defined as /total lesion proportion of the whole lung volume at baseline. The secondary imaging outcomes were a change in the total lesion proportion (%) of the whole lung volume from baseline to day 10, a change in solid component and ground-glass lesion proportion from baseline to day 10, 28, and change in lung densitometry at day 10, 28. Lung lesions were evaluated by using the changes in high-resolution chest CT images and measured by centralized imaging interpretation based on both lung radiologist analyses and imaging software. The imaging data were derived from a software-assisted lung volumetry and densitometry procedure (Supplementary Data Smax), DLCO, modified Medical Research Council Dyspnea Scale (mMRC), changes in absolute lymphocyte counts and subsets, as well as plasma cytokine and chemokine levels. Safety evaluation included adverse events and all-cause mortality. Detailed definitions and assessment procedures are described in Supplementary Data SClinical outcomes within 28 days included 6-MWT, status of oxygen therapy maximum forced vital capacity  of the whole lung volume from baseline to day 28 and the difference between the UC-MSC and placebo groups was tested using wilcoxon rank sum test and the median differences were calculated using the Hodges–Lehmann estimation . Six-category scale and MMRC dyspnea score were calculated by using ordinal logistic regression model. The mITT population was considered as the primary analysis population and safety analysis was done in all patients who started their assigned treatment. If the patient missed a chest CT scan, the last scan’s results were carried forward to the missing visit for primary endpoints in the mITT analysis. Other missing values of secondary endpoints and PP analyses were not imputed. Statistical analyses were performed using SAS software, version 9.4 . The figures were generated using GraphPad Prism 7 software .There were no predefined hypotheses made in this study; therefore, we focused on description instead of inference for statistical analyses: all statistical tests, confidence intervals, and This study was approved by the Clinical Trial Ethics Committee of Fifth Medical Center, Chinese PLA General Hospital (2020-013-D), the Medical Ethics Committee of Wuhan Huoshenshan Hospital (HSSLL004), the Medical Ethics Committee of Maternal and Child Hospital of Hubei Province (2020IEC001). Written informed consent was obtained from all the enrolled patients or their legal representatives if they were unable to provide consent.Supplementary Materials


Acute liver failure (ALF) is a potentially life-threatening condition. Liver support therapies can be applied as a bridging-to-transplantation or bridging-to-recovery; however, results of clinical trials are controversial. Our aim was to compare liver support systems in acute and hyperacute liver failure with network meta-analysis. After systematic search, randomized controlled trials (RCT) comparing liver support therapies in adults with acute or hyperacute liver failure were included. In-hospital mortality was the primary outcome, the secondary outcomes were hepatic encephalopathy and mortality-by-aetiology. A Bayesian-method was used to perform network meta-analysis and calculate surface under the cumulative ranking curve (SUCRA) values to rank interventions. Eleven RCTs were included. BioLogic-DT and molecular adsorbent recirculating system (MARS) resulted in the lowest mortality . In non-paracetamol-poisoned patients, BioLogic-DT, charcoal hemoperfusion and MARS may be equally efficient regarding mortality . Considering hepatic encephalopathy, extracorporeal liver assist device (ELAD) may be the most effective option (SUCRA: 78%). However, in pairwise meta-analysis, there were no statistically significant differences between the interventions in the outcomes. In conclusion, MARS therapy seems to be the best available option in reducing mortality. Further research is needed on currently available and new therapeutic modalities. (CRD42020160133). The main causes of acute and hyperacute liver failure are drugs—especially paracetamol overdose (46–65%)—and viruses (29–77%), other etiologies are less frequent (11–23%) like mushroom poisoning, Budd-Chiari syndrome, Wilson-disease or HELLP-syndrome7. Due to the impaired synthetic and detoxification capacities, coagulopathy, jaundice and hepatic encephalopathy may develop8. In hyperacute liver failure considerably elevated transaminase levels and severe coagulopathy can be observed with slightly or not increased bilirubin levels3. Patients with hyperacute liver failure have a greater possibility to spontaneously recover without liver transplantation3.Acute and hyperacute liver failure are potentially life-threatening conditions that can lead to multiorgan failure9. Furthermore, bioartificial liver support therapies that contain hepatocytes can provide synthetic functions as well10. In liver failure when there is a potential for recovery, liver support systems amend the supportive care until the regeneration of the liver. In other cases, the definitive therapy of liver failure is liver transplantation—which is expensive and restricted by the number of organs available—however, liver support therapy can keep these patients alive until a suitable organ is found11. Considering the effectiveness of these therapies the results of clinical trials are controversial, thus, currently they are not recommended by thy European Association for the Study of the Liver (EASL) Clinical Practical Guidelines or the American Association for the Study of Liver Diseases (AASLD) Practice Guidelines outside of clinical trials in acute or hyperacute liver failure13.Extracorporeal liver support systems (ECLS) can be used to aid the liver’s detoxification function by removing albumin-bound toxins and water-soluble substances16.In former meta-analyses in this field, the different interventions were considered equivalent and pooled together in comparison with standard medical therapy (SMT)17. Therefore, we decided to perform a network meta-analysis, in which we are able to assess the different liver support systems’ efficacy and safety in acute and hyperacute liver failure. With the statistical methods of network meta-analysis, we (1) compare the interventions to each other and (2) rank them, to choose the best option regarding the outcome.In conventional meta-analyses two interventions can be compared, however when multiple alternatives exist, network meta-analyses can provide results in a single analysis based on direct and indirect  comparisons as well18.Through the initial searches 2774 citations were identified. After reading the titles and abstracts, 99 articles remained for further assessment. 12 articles could be included for qualitative synthesis and 11 for network meta-analysis , in three of them the Molecular Adsorbent Recirculating System (MARS) was applied25. Through the systematic search we found one study from each modalities analysing high-volume plasma exchange26, exchange transfusion27, Extracorporeal Liver Assist Device (ELAD)28 and charcoal hemoperfusion29. Bioartificial modalities are ELAD therapy  and HepatAssist device . HepatAssist device was included only in the systematic review.All studies included in the quantitative synthesis are parallel randomized controlled trials comparing liver support systems to SMT, published between 1973 and 2016, including 479 patients. Overall, 243 patients were assigned to a liver support therapy and 236 to SMT. In four of the studies BioLogic-DTSeven studies reported detailed demographic characteristics. The mean age was 38.8 years, two studies included adolescents as well. About half of the sample population were female (55.8%—226 of 405). The majority of the studies included patients with different etiologies, however, the distribution of the different etiologic factors was similar to the general population. Seven RCTs recruited patients across Europe (58%), three in the USA (25%) and 2 multicentric trials recruited patients at the study sites across continents 17%)  while SMT seems less effective (43%) in the nonparacetamol-poisoned patient population 25. Considering HVPE, Larsen et al. reported that 3-month overall survival was not improved significantly in the plasma exchange group compared to the control group, however transplant-free survival was significantly better in the HVPE-treated group after 3 months (p = 0.0058)26.We assessed articles in which the follow-up period was at least 30 days. In the trial of Demetriou et al. 30-day survival was 71% in the bioartificial liver-treated group (BAL) and 62% in the control group 26. Ellis et al. examining ELAD therapy reported that 2 patients underwent transplantation and 1 survived in each group28. In the trial published by Wilkinson et al. 2 fulminant hepatic failure patients had liver transplantation, 1 survived and 1 underwent transplantation before the start of the trial period20. In the study from Mazariegos et al. 3 patients from the treatment group had liver transplantation and survived, and no patients were transplanted from control group22.Six trials reported on liver transplantation. Three large RCTs did not find significant differences between the control and treatment groups in the number of patients transplanted and survival rates analysing HepatAssist device, HVPE and MARS21. With ELAD therapy tachypnoea, tachycardia, fever and bleeding occurred in two patients28. In a trial examining HepatAssist device thrombocytopenia was the most frequent adverse event with similar incidences between groups 18. During charcoal hemoperfusion renal failure, cerebral oedema and uncompensated metabolic acidosis were detected29. Examining HVPE, cardiac arrhythmia, acute respiratory distress syndrome (ARDS), pancreatitis, deteriorating in gas exchange, transfusion-related acute lung injury (TRALI), infections confirmed by blood culture and bleeding could be observed. The rate of adverse events were not statistically different in the treatment and control group26.Nine studies reported adverse events. In three trials no adverse events were observed during BioLogic-DT treatment25.In a multi-center RCT MARS was tested, bleeding, death or sepsis did not occur related to MARS therapy, the majority of adverse events were related to liver transplantation and were more frequent in the not paracetamol-poisoned population24.In patients with ALF due to cardiogenic shock after cardiac surgery treated with MARS no bleeding was detected due to thrombocytopenia, other adverse events were not reported23. Three of the trials were adjudicated as overall low risk of bias (33%)26, and nine studies were judged to raise some concerns (67%) , although the significance is not robust given the confidence interval16. Other previous meta-analyses did not find any significant difference between SMT and liver support techniques in the ALF population by subgroup analysis35.Former meta-analyses reported conflicting results considering liver support devices’ effect on mortality in acute liver failure. Zheng et al. found that bioartificial devices reduced mortality in ALF , although from the three studies analysed two represented the same patient population38. Spontaneous recovery is more frequent in this patient population compared to other drug-induced, autoimmune or idiopathic ALF36. Therefore, emergency transplantation as a routine intervention in paracetamol poisoning has been questioned39. We did not have enough data in this patient population for a quantitative synthesis, however in the nonparacetamol-poisoned population no significant difference could be observed between SMT and extracorporeal liver assist devices, and the different liver support therapies applied.Acetaminophen overdose is the leading cause of ALF in the USA, Australia and Europe8. However, because of the disease’s complexity there are several different measurement scales40 and the result is greatly affected by the assessor41. Furthermore, the patients are usually sedated and mechanically ventilated, which makes the evaluation more difficult. In former meta-analyses in populations from both ACLF and ALF patients significant improvement was found in hepatic encephalopathy with ECLS systems34.Hepatic encephalopathy is an important symptom of ALFThe greatest strength of this study is that the different interventions were compared to each other and were not assessed together in comparison with standard medical therapy. However, this study has certain limitations. The most important limitations are the small sample sizes, the heterogeneity of the patient populations, outcomes, and study design and the inconsistency in definitions of liver failure. We were unable to use the node-splitting analysis to examine consistency assumption because there was not enough information from the comparisons in the network. Long-term survival could not be quantitatively analysed, although it is a particularly important factor to assess the efficacy of the interventions. Finally, our network meta-analysis covers a period of more than 40 years, during which SMT has improved remarkably .This network meta-analysis demonstrated that—as BioLogic-DT is not applied in clinical practice anymore—MARS therapy seems to be the best available option in reducing in-hospital mortality, however, no statistically significant differences could be observed among the treatments of acute liver failure considering in-hospital mortality and hepatic encephalopathy. Good-quality randomized trials are needed on currently available and new blood purification modalities to define the role of extracorporeal liver support in patients with acute liver failure.42. We used the classical PICO framework for our clinical question. P: patients with acute or hyperacute liver failure (having regard to the fact that the studies were conducted in a wide range of time (1973–2016) we accepted the articles’ definition of hyperacute and acute liver failure); I and C: artificial, bioartificial liver support therapies, SMT; O: overall in-hospital mortality, mortality-by-aetiology, hepatic encephalopathy, number of patients transplanted, laboratory parameters and adverse events. Our network meta-analysis was registered with the PROSPERO registry (CRD42020160133).The network meta-analysis was reported using the PRISMA Extension Statement for Reporting of Systematic Reviews Incorporating Network Meta-Analyses of Health Care InterventionsFor this network meta-analysis on the 4th of October 2019 we searched Medline (via PubMed), the Cochrane Central Register of Controlled Trials (CENTRAL), Web of Science, Embase and Scopus for RCTs and conference abstracts of RCTs. No restrictions were imposed on the search.We used the following search key in all databases (complemented with the MeSH function in MEDLINE):  AND  AND random*.Randomized controlled trials studying liver support devices in acute-on-chronic liver failure were excluded. In studies in which patients with ALF and ACLF were both involved and provided individual patient data, we only extracted the data of patients with acute liver failure. Transitivity was assessed clinically, based on the eligibility criteria of the included randomized controlled trials. As acute and hyperacute liver failure have mainly similar symptoms despite etiology, we concluded that, regarding the liver support systems’ clinical effect on these symptoms, the conditions of transitivity are satisfied.43.Records from each database were downloaded into EndNote X9 citation manager  and duplicates were removed by the citation manager based on the title of the article, and then manually. The titles then the abstracts and full texts of the identified studies were screened for inclusion against the eligibility criteria by two independent review authors . A third party (ZM) resolved conflicts. Citing and cited articles were revised through Google Scholar, where all the additional sources were identified. The PRISMA flowchart shows the process of the article selection Fig. 43.All data according to study type, author and publication information, demographic data, aetiology, details of the interventions and comparators, mortality, hepatic encephalopathy, number of patients transplanted, laboratory parameters, adverse events and notes were collected in the study database (standardized template). The data from intention-to-treat analyses were extracted independently by the first (AK) and second author (KO), when conflicts arose, a third participant resolved any discrepancies (ZM).The primary outcome of our analysis was in-hospital overall mortality. Secondary outcomes included hepatic encephalopathy (number of patients improved versus worsened plus not improved), mortality-by-aetiology, liver transplantation, long-term survival, and adverse events. We accepted the articles’ definition of adverse events. We planned to analyse changes in laboratory parameters as well but failed to do so because studies reported them in different time instants.44. From the individual studies’ overall RoB assessment, we chose the one which was at the highest risk of bias for each intervention’s (each arm of the network) overall RoB assessment. Then we summarized the interventions’ overall RoB-assessment on the comparison level with the same method. The results of the RoB assessment are depicted in league tables. The colour of the boxes indicates the comparisons’ overall risk of bias assessment . We used the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach to assess the certainty of evidence45. Study limitations were evaluated based on RoB 2 tool, as detailed above. Imprecision was judged based on the sample size calculation of the article of Larsen et al.26. Node splitting could not be performed in any of the networks due to network geometry, consequently inconsistency could not be tested. We compared the individual studies’ populations, interventions and outcomes to rate indirectness. Publication bias was judged by the ‘comparison-adjusted’ funnel plot and Egger’s test. In the league tables we marked the quality of evidence for each comparison. Risk of bias and quality of evidence assessment were performed by two independent review authors , a third party (ZM) resolved conflicts.Risk of bias assessment was first performed on individual study-level according to the Revised Cochrane risk-of-bias tool for randomized trials (RoB 2)A Bayesian-method was used to perform pairwise meta-analyses and network meta-analysis with the random effect model. In case of missing outcome data, we replaced values with the worse outcome, i.e. in case of mortality, death, in case of hepatic encephalopathy, worsening/not improving. We used risk ratios (RR) for dichotomous data with 95% credible intervals (95% CrI). We optimized the model and generated posterior samples using the Monte-Carlo methods running in four chains. We set at least 20,000 adaptation iterations to get convergence and 10,000 simulation iterations. Network estimates (pooled direct and indirect data) of each intervention compared to standard medical therapy and to other interventions are presented in forest plots, summarized in a league table (as shown in the results section). In the network geometry the direct comparisons are presented with edges, and the thickness of the edges is proportional to the number of the head-to-head trials, and the size of the nodes is proportional to the number of studies in which the intervention was applied. We also ranked interventions by their posterior probability via calculating the SUCRA values. ‘Comparison-adjusted’ funnel plot was created with the frequentist approach, and Egger’s tests were performed in the network meta-analysis to assess small-study effect of in-hospital mortality. All calculations were performed with R (V. 3.5.2) package gemtc (V. 0.8-2) along with the Markov Chain Monte Carlo engine JAGS (V. 3.4.0) and STATA 17.0 (StataCorp LLC).Supplementary Information.