Angiogenesis is a physiological process involving the growth of new blood vessels, which provides oxygen and required nutrients for the development of various pathological conditions. In a tumor microenvironment, this process upregulates the growth and proliferation of tumor cells, thus any stage of angiogenesis can be a potential target for cancer therapies. In the present study, chitosan and his derivatives have been used to design novel polymer-based nanoparticles. The therapeutic potential of these newly designed nanoparticles has been evaluated. The antioxidant and MTT assays were performed to know the antioxidant properties and their biocompatibility. The in vivo antiangiogenic properties of the nanoparticles were evaluated by using a chick Chorioallantoic Membrane (CAM) model. The obtained results demonstrate that chitosan derivatives-based nanostructures strongly enhance the therapeutic effect compared to chitosan alone, which also correlates with antitumor activity, demonstrated by the in vitro MTT assay on human epithelial cervical Hep-2 tumor cells. This study opens up new direction for the use of the chitosan derivatives-based nanoparticles for designing of antiangiogenic nanostructured materials, for future cancer therapy. Angiogenesis is the development of new blood vessels, a process that normally occurs during embryogenesis, wound healing, or uterus function. Besides these normal situations, there are those cases of pathologic angiogenesis found in: diabetic retinopathy, chronic inflammation, ischemic heart, and cancer. With the increase of cancer prevalence among the global population, the discovery of new anti-cancer therapeutics has not been at the same rate. While stimulation of angiogenesis in cancer leads to metastasis, the anti-angiogenic therapy proved to be an important approach against tumor growth. In this respect, it is a well-known fact that the consumption of antioxidants can be recommended to achieve the inhibition of angiogenesis or reversal of unwanted cell–cell adherence . SeveralChitosan is a natural cationic polymer exhibits three chemical reactive sites including a primary amine and two primary or secondary hydroxyl groups for further modification ,6,7. NanDifferent methods for in vivo evaluation of angiogenesis and antiangiogenesis have already been analyzed in the specialized literature . The CAMClostridium, Bifidobacteria and Salmonella) that are capable of colonizing solid tumors in vivo [Angiogenesis inhibitors are part of anticancer drugs that appear to have low haematological toxicity, and in the future to further reduce the frequency and severity of adverse reactions, delivery of antiangiogenic factors to the tumor site is expected by using genetically modified bacteria , acetic acid, sodiumhydroxide, sodium tripolyphosphate (TPP) and organic solvents(p.a.) were purchased from Sigma Aldrich Company. All solvents and chemicals had a high degree of purity.Chitosan-sulfamethoxydiazine, CLB- Chitosan-sulfadiazine, CLC- Chitosan-sulfadimethoxine, and CLD- Chitosan-sulfisoxazole) to obtain innovative nanoparticles’ formulations. The main goal will be to manufacture the nanoparticles by using ionic reticulation as a cross-linking agent sodium tri-polyphosphate (STPP). In this manner, the preparation method will be optimized using pure chitosan, and the established parameters will be further used to obtain nanoparticles based on chitosan low molecular weight derivatives. A similar method was used in one of the previous studies, in obtaining chitosan micro-particles loaded with xanthine derivatives for improvement of type 2 diabetes [In continuation to our previous studies carried out on chitosan ,15, thisdiabetes .Methods of obtaining nanoparticles involves the following technique: each 1 mL 0.1% chitosan derivative solution was dropwise and stirred at 700 rpm in a 10 mL 0.005% TPP (sodium tripolyphosphate) aqueous solution. The wet phase of nanoparticles were analyzed using a Malvern Autosizer 4800 Malvern Instruments, Worcestershire, UK, DLS spectrometer (Dynamic Light Scattering spectroscopy) . For lat−1 [−1 and 32 scans per sample vs. background, using OPUS software . The infrared measurements were acquired with a Bruker ALPHA FT-IR spectrophotometer  in the spectral region of 4000–500 cm−1 ,23. ThisAs stated in the introduction, a high antioxidant potential is beneficial for an antiangiogenic action required in cancer therapy.In our case, the antioxidant effects were determined through in vitro tests by using spectrophotometric methods: total antioxidant capacity, ferric reducing power, and radical scavenging ability. For this, a UV-VIS Spectrophotometer UV-1900, , was used. Similar methods were used in one of the previous studies conducted on polymer matrices in the form of films . In the Thus, the antioxidant capacity was determined based on the phospho-molybdenum method, which includes the preparation of a each nanoparticles stock solution (5 mg/mL in 2% acetic acid), of which 50 μL will be mixed with 2 mL reagent solution. The reagent contains the following substances: sulphuric acid 0.6 M, sodium phosphate 28 mM and ammonium molybdate 4 mM. The absorbance of the sample was measured at 695 nm, using a blank which contains 2% acetic acid (50 μL) and the reagent solution (2 mL); effective concentrations (EC50) were calculated ,25. Ferric reducing power is a method that involves the use of potassium ferricyanide, trichloroacetic acid and ferric chloride as reagents. In this regard, 1 mL of stock solution (5 mg/mL in 2% acetic acid) was mixed with 1 mL of sodium phosphate buffer  and then 1 mL of 1% w/v potassium ferricyanide was added, followed by samples incubation for 20 minutes at 50 °C. To stop the reaction, 1 mL of trichloroacetic acid (10% w/v) was added. The absorbance of each sample was measured at 700 nm, using a blank that contains all reagents and less stock solution. The effective concentrations (EC50) were calculated for this method . c is the absorbance of the control and As for the sample. Radical scavenging ability has been evaluated by using a methanol solution of 1,1-diphenyl-2-picrylhydrazyl . The samples, 50 μL of stock solution (5 mg/mL in 2% acetic acid), were left in the dark for 30 minutes, in contact with 2950 μL DPPH solution and, afterwards, the absorbance at 517 nm was measured using DPPH in methanol as a control solution . The nexAll the experiments were performed in triplicate.2 was made in the shell. Sterile disks of methylcellulose loaded with chitosan derivatives nanoparticles (100 μg/disc) were placed at the junction of two big vessels. The windows were sealed with tape, and the eggs prepared as such, were incubated at 37 °C for 72 h [The CAM model was used to assess the angiogenic activity of chitosan derivatives’ nanoparticles. The main functions of CAM are: respiratory organ for the embryo, storage membrane of excretions and also membranes for mobilization of calcium from the shell to start bone mineralization . The CAMfor 72 h . The embHistopathological study. In order to evaluate the effect of the presence of nanoparticles on the development of angiogenesis, a histopathological study was performed. The first step was to include CAM membrane fragments taken in paraffin. The obtained paraffin blocks were cut into the microtome at 2–3 μm thick, and furthermore the sections were specifically stained with haematoxylin and eosin H & E . The micFor the microscopic exam, each slide was scanned, following the same exposure and calibration procedure. The sections from the identified region which were scanned received a standard name and were outlined. The morphometric study was realized on representative fields of view (FOV) acquired in 5 distinct regions of the digital images, following the same rule of monitoring the structures distributed central and in the four extremities of each FOV. The blood vessels were counted in each distinct FOV/sample and the results were included in our database.In vitro samples cytotoxicity evaluation was performed according to Standard SR-EN ISO 10,993 for cell viability determination, using an MTT assay. The morphology of the cells was examined under the optical microscope after 48 h of samples treatment. 3--2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma Aldrich .The cell lines used for cytotoxicity evaluation were, on the one hand, NCTC clone L929 cell culture of mouse normal fibroblasts and Hep-2 tumor cell culture of human cervix epithelial cells, on the other hand. Both types were purchased from ECACC , together with Minimum Essential Medium (MEM), L-glutamine, and antibiotics penicillin, neomycin and streptomycin. Fetal bovine serum (FBS) was provided by Biochrom . 4 cells/mL for normal fibroblasts NCTC, respectively 6 × 104 cells/mL, for cervix epithelial Hep-2 cells, in 24-well culture plates incubated at 37 °C in a humid atmosphere. Then, the samples (sterile disks of methylcellulose loaded with chitosan derivatives nanoparticles (100 μg/disc)) were added and the plates were incubated for another 24 h, respectively 48 h, at 37 °C and 5% CO2 in a humid atmosphere. The control of each experiment consisted of an untreated cell culture, while the positive control was a solution of 0.003% H2O2 in medium. Each experiment was carried out in triplicate.The cells were cultivated in MEM, supplemented with 10% FBS, 2 mM L-glutamine and a mixture of 100 U/mL penicillin, 100 μg/mL streptomycin, and 500 μg/mL neomycin. In the experiments, the cells were seeding at the density of 4 × 10After 48 h of treatment, the morphology of the cells was examined using a Zeiss optical microscope AXIO-Observer D1 (Germany) and images of the cell morphology were taken.Cell viability was determined using the MTT colorimetric method. Its principle consists of: tetrazolium salt reacted with mitochondrial dehydrogenases, resulting in formation of insoluble blue-violet formazan crystals and soluble in isopropanol [propanol . 2 in a humid atmosphere. The absorbance of the resulting isopropanol solutions was measured using a Mithras LB 940 Multimode Microplate Reader from Berthold Technologies (Germany), at 570 nm wavelength. At the end, cell viability was determined using the next formula:The MTT assay was performed at intervals of 24 and 48 h, after the removing of the samples from the wells, the culture medium was replaced with the tetrazolium salt solution, followed by culture plates incubation for 3 h at 37 °C and 5% CO-one way ANOVA was conducted, for the variables X = blood vessels and Y = study batch, using an alpha of 0.05;-for multiple comparisons of normal distributed series of values, a post-hoc Bonferroni test was applied after one-way ANOVA in case of significance. One-Way ANOVA was significant  and, in these conditions, Bonferroni correction (post-hoc Bonferroni) was performed to reduce the error rate when testing multiple hypotheses.-p < 2) tests are tests of normality in frequentist statistics, available when using the distribution platform to examine a continuous variable.Skewness  and multiple comparisons were made  of the average values—the F test (ANOVA) is used when comparing 2 or more mean values from groups with normal distributions. The obtaining scheme of CLA nanoparticles is presented in Some examples of the DLS measurements of the chitosan derivatives nanoparticles (the size distribution by intensity) are presented in −1 and NH (amide II) in the range 1594–1598 cm−1. At the same time, in the region 3320–3400 cm−1, a broad band was identified attributed to the valence vibration of the alcoholic OH groups. The sulfonamide component is present in the spectrum through the absorption bands characteristic of the sulfonamide group, -SO2-N< in the 1256–1260 cm−1 and 1162–1165 cm−1 ranges. The aromatic nucleus is present through multiple bands, of which the most intense are those from regions 1542–1551 cm−1. The bands were assigned according to the data from the literature [Thus, in the FT-IR spectra of chitosan nanoparticles  as well terature  and suppThe new derivatives of chitosan with sulfonamidic structure were evaluated from the point of view of antioxidant potential.The total antioxidant activity was determined using phosphomolybdenum blue complex with a maximum absorption at 695 nm.EC50 values of chitosan low molecular weight derivatives are comparable (between 3.4 and 9.49 mg/mL) with the one of pure chitosan (10.32 mg/mL). In this context, the most active compounds are: CLA with EC50 = 3.40 mg/mL (three times more active than pure chitosan) and CLD with EC50 = 4.04 mg/mL (2.5 times more active than chitosan)—2+) form.The measurement of reducing power is particularly important for the antioxidant activity of the compounds. The presence of a reducing agent in the obtained nanoparticles results in reduction of the ferric/ferricyanide complex to its ferrous  demonstrate that chitosan derivatives nanoparticles (CLA-CLD) are more active than pure chitosan CL (with EC50 = 1.67 mg/mL) , the amoDPPH is a radical, which demonstrates a strong absorption band at 517 nm, and it passes into colorless or pale yellow, if it is neutralized by antioxidant substances. Thus, this radical is commonly used to evaluate the radical scavenging capacity of antioxidants.Chitosan derivatives (CLA-CLD) have a good scavenging ability with I = 35.86–81.94%, while, for pure chitosan (CL), this value is 13.21%. The most active compounds are CLC (I% = 81.94%) and CLD (I% = 74.13%), these being 6.2 and 5.6 times more active than pure chitosan .The results of antioxidant potential evaluation, through in vitro tests , confirmed the fact that nanoencapsulation of all chitosan derivatives have positively influenced their antioxidant potential, these being more active than pure chitosan.variations from 0 to 8 vessels/field;mean 3.88 ± 2.28 vessels/field;the median value (4 vessels/field) close to the average value and the Skewness test result <2 suggests that the series of values of the number of vessels was homogeneous, so statistical significance tests can be applied.The total number of blood vessels tested/field has the following particularities :variatioAll four types of nanoparticles resulted in reduced angiogenesis , but thep < 0.05) and CLD (p < 0.001) nanoparticles. In these cases, the values achieved were three and one vessels/field. In addition, in the case of CLA and CLB samples, there were slight decreases of these values (5 vessels/field) (p > 0.05) (“p” is corresponding to the correlation between the average level of the vessels and the groups referred to the control (CL)).Thus, in the case of CL control, the median value of the blood vessels registered in the field was 6, which decreased significantly in the case of CLC , at 24 h cell viability values for samples CLA, CLB, CLC, and CLD were between 98.07 and 105.79%, higher compared to the viability obtained in the reference sample CL (95.72%), in a similar way at 48 h of experiment the samples CLA, CLB, CLC and CLD registered viabilities values of 99.28–107.52%, higher than the cell viability 89.29% of the reference sample. The testing in NCTC normal cells at 48 h indicated a good biocompatibility of all tested samples, with values close or higher than the control, with highest viability values for samples CLC and CLD (106.18%. and 107.52%)—Optical microscopy images of normal NCTC fibroblast cells, after 48 h of direct contact with nanoparticles samples, present a morphology similar to control NCTC cell culture, with cells of fusiform or round conformation, with fine intracytoplasmic granules, and having a cell density similar or slightly variable to the control .From MTT assay on human epithelial cervical tumor cells Hep-2 at 24 h, all the viability results of the samples were higher than 85%, indicating lack antitumor activity. At 48 h of testing, the reference sample CL with 87.85% viability had no antitumor effect, while samples CLB and CLC with viability of 79.69% and 69.91% present a weak antitumor activity. The samples CLA and CLD recorded 66.16, respectively, 66.89% viability, showing the most evident antitumor activity on Hep-2 tumor cells , for theThe optical microscopy images of the Hep-2 tumor cells after 48 h of treatment with nanoparticles samples, showed that the cells treated with the CL reference sample have similar aspect with cell morphology of the control culture, with cells of polygonal shape and regular size, indicating the lack of antitumor effect of the sample on Hep-2 culture. In contrast, the images of the cells treated with samples CLA, CLB, CLC and CLD, shows numerous round and/or lysed cells in the culture, or cells detached from the surface of the well as result of an extended cell lysis, indicating the antitumor activity of the respective samples .Optical microscopy images of the morphology of normal NCTC cells and respectively Hep-2 tumor cells treated with tested samples confirm the cell viability results obtained by the MTT assay.Until recently, scientists believed that, due to the rapid development of tumors, they become too numerous to be supplied with blood and, as a result, they die quickly. In fact, cancerous tumors stimulate the development of an entire bundle of new blood vessels that supply the nutrients they need. This process is called angiogenesis, and animal and cell culture studies have shown that, if this process is interrupted, the tumor will "starve." In vitro and in vivo investigations show that, in the absence of blood vessels, tumors can grow until passive diffusion can no longer ensure the supply of nutrients and the elimination of degradation products in the adjacent environment. Tumor angiogenesis results from the interaction of three types of cell populations: tumor cells, stromal cells , and endothelial cells . By the ·O2−), peroxides (H2O2), hydroxyl radicals (·OH), are maintained in basal levels, while it is well known that tumor cells produce higher levels of H2O2. Moreover, studies incriminate peroxides by stimulating pathological angiogenesis. In this respect, different researchers have demonstrated in their studies that antioxidants such as catechins [In general, in normal conditions, reactive oxygen species, such as superoxide  on the one hand, but also on the potential of nanostructures to inhibit angiogenesis through the large contact surface that they provide. According to our findings, free low molecular weight chitosan has a much lower antioxidant activity comparing to its derivatives, this being demonstrated both by the existence of higher effective concentrations and by lower percentages of the ability to inhibit free radicals.<a chick embryo that has not reached the 14th day of its gestation period would not experience pain and can therefore be used for experimentation without any ethical restrictions or prior protocol approval>, which is an advantage. This test also ensures compliance with the second R, which refers to “refinement” to minimize animal suffering, while in terms of the third R, this test would be helpful in “reducing” the number of animals involved [In conventional drug discovery, preclinical animal models which are used to investigate the pharmacological properties of new bioactive substances have the disadvantage of being expensive and time-consuming . Howeverinvolved .Clinacanthus nutans water extract prevents new blood vessel formation in the CAM model. Suramin is known for its antiangiogenic activities, being involved its antioxidant properties, which were found to be higher than in the case of l-ascorbic acid. In this regard, studies have shown that Suramin treatment increases the activity of antioxidant enzymes in the tissues  [The CAM model is a quick and low-cost test that allows analysis of a large number of pharmacological samples in a short period of time. Kue et al.  have app and GR) .In our case, the chorioallantoic membrane model underlined the antiangiogenic effect of antioxidant nanoparticles, the most powerful effect being established for CLC and CLD nanoparticles  , these being the corresponding nanoparticles of chitosan-sulfadimethoxine and chitosan-sulfisoxazole derivatives. Our finding, regarding the fact that the isoxazolic nucleus (for the CLD derivative) and the dimethoxylated aromatic nucleus (for the CLC derivative) generate the most active nanoparticles, is also sustained in the context in which the studies in this field have reported a naturally occurring diarylisoxazole derivative, as a new chemical tool with efficacy against breast cancer cells 22. In addition, in their study, Çalışkan et al.  have synIn the case of the other derivatives, for which the aromatic nucleus is substituted with a single methoxy group (CLA-Chitosan-sulfamethoxydiazine), or even unsubstituted (CLB- Chitosan-sulfadiazine), the antioxidant action is weaker, recorded by higher EC 50 and lower percentages of DPPH radical scavenging ability (I%), compared to other derivatives, which also leads to a lower ability to inhibit angiogenesis, proven by the CAM test. Thus, in the case of these derivatives, if, for the antioxidant action EC values of up to 1179 mg/mL and DPPH radical scavenging ability of up to 35.86% being recorded for the inhibition effect of angiogenesis, five vessels/field were determined.In addition, Hep-2 tumor cells indicated that all chitosan derivatives induced antitumoral effect. At a concentration of 100 μg/disc, all four types of nanoparticles were biocompatible in NCTC cells (with a cell viability of over 99%), while, in Hep-2 tumor cells cultivated in medium, these nanoparticles were anti-proliferative (with a viability of up to 66%) after 48 h of treatment.Regarding the high in vitro biocompatibility of new chitosan derivatives, processed as nanoparticles, due to chitosan, extracted from the exoskeleton of crustaceans, whose non-toxicity, biocompatibility, and bioactivity is well known in the literature, these nanostructures show good biocompatibility, slightly increased compared to those generated by pure chitosan, from 89.29% to over 100%. In one of our previous studies , in whicIn terms of sulfonamides toxicity, Zessel et al. demonstrated in one of his studies , using mHep-2 tumor cells treated with tested samples show a beneficial effect for the modified chitosan with the four sulfonamides and for nano-encapsulation of chitosan derivatives, by recording lower cell viability values compared to the parent chitosan. The MTT assay results for tested nanoparticles in Hep-2 cells exerted a strong cytotoxity at 100 μg/disc (between 66.16% and 79.70% viability). In addition, it was observed that pure chitosan had no significant antitumor activity at this concentration (only 87.85% viability).Currently, the medical relevance of nanomaterials for the prevention and treatment of human cancer remains a continuing challenge. However, the use of appropriate nanostructures, capable of overcoming biological barriers, could be an important strategy for future antitumor therapy. In our study, four types of chitosan derivatives nanostructures were biocompatible for normal NCTC cells (more than 99% viability) and cytotoxic for Hep-2 tumor cells (up to 66% viability), as confirmed by in vitro experiments. In addition, their antioxidant action, demonstrated by three in vitro tests, had a beneficial impact on their ability to inhibit angiogenesis, demonstrated by the CAM test, reducing vascularization from six vessels per field, in the case of pure chitosan (CL), to one vessel per field, in the case of the CLD derivative. In conclusion, our results demonstrate that chitosan derivatives’ nanostructures strongly enhance the therapeutic effect of free chitosan, as far as their antitumor potential is concerned.


We aimed to conduct a meta-analysis to assess the effect of pharmaceutical care on the treatment of coronavirus disease 2019 (COVID-19).All case-controlled studies related to pharmaceutical care on the treatment of COVID-19 will be included in this review. We will use index words related to pharmaceutical care and COVID-19 to perform literature searches in PubMed, Embase, MEDLINE, CNKI, and Wanfang databases, to include articles indexed as of October 20, 2020 in English and Chinese language. Two reviewers will select trials independently for inclusion and assess trial quality. Two pairs of review authors will independently extract information for each included trials. Primary outcomes are clinical outcomes, average hospital stays, costs, patient satisfaction, and incidence of adverse drug reactions. We will evaluate the risk of bias of the included studies based on Cochrane assessment tool. Revman 5.3  will be used for heterogeneity assessment, generating funnel-plots, data synthesis, subgroup analysis, and sensitivity analysis.We will provide targeted and practical results assessing the effect of pharmaceutical care on the treatment of COVID-19.The stronger evidence about the effect of pharmaceutical care on the treatment of COVID-19 will be provided for clinicians.PROSPERO CRD42020214223There is no need for ethical approval, and the review will be reported in a peer-reviewed journal. Many academic institutions and research groups are working collaboratively on beneficial interventions. There are numerous drugs for the treatment of COVID-19, but there is few widely effective drug therapy and many drugs are still only experimental. Timely evaluation and monitoring of drug treatment is particularly important for patients with COVID-19.The novel coronavirus disease 2019 (COVID-19) is caused by infection from the newly emerged, highly contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, an increasing number of studies have evaluated the value of clinical pharmacist participation in clinical work and have confirmed that clinical pharmacists can regulate the use of antibiotics, reduce adverse drug reactions (ADRs), and even reduce treatment costs. To date, the effect of pharmaceutical care on the treatment of COVID-19 has been investigated in several studies,8 and found that pharmaceutical care was benefit for clinical treatment. To provide stronger evidence for the clinical practice, we aim to conduct a meta- analysis of cohort studies to assess the effect of pharmaceutical care on the treatment of COVID-19.Pharmaceutical care is provided by clinical pharmacists to promote health, wellness, and disease prevention.22.1,10 and Cochrane Handbook for Systematic Reviews of Intervention. If we will refine procedures described in this protocol, we will document the amendments in the International Prospective Register of Systematic Reviews database and disclose them in future publications related to this meta-analysis.This protocol has been registered with the International Prospective Register of Systematic Reviews in October 20 as CRD42020214223. In this paper, the protocol will be performed according to the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols guidance2.22.2.1All case-controlled studies, comparing the prognosis of patients COVID-19 received/not received pharmaceutical care will be included in this review.2.2.2The diagnosis of COVID-19 was confirmed as positive result for respiratory pathogen nucleic acid test and nasopharyngeal swab with high-throughput sequencing or real-time reverse transcriptase polymerase chain reaction.2.2.3Patients received/not received pharmaceutical care.2.2.4Any available information about the effect of pharmaceutical care on the treatment of COVID-19 will be assessed. Primary outcomes are clinical outcomes, average hospital stays, costs, patient satisfaction and incidence of ADRs.2.3We will use index words related to pharmaceutical care and COVID-19 to perform literature searches in PubMed, Embase, MEDLINE, CNKI, and Wanfang databases, to include articles indexed as of October 20, 2020 in English and Chinese language. The key search terms will be used are 2.42.4.1Two reviewers will independently select trials for inclusion. We will exclude articals if they meet any of the following criteria:(1)fewer than 10 patients;(2)studies not comparing pharmaceutical care and none pharmaceutical care on the treatment of COVID-19.The specific process of study selection is shown in Figure 2.4.2Two pairs of review authors will independently extract general information for each included trial, including the name of first author, year, country, design, sample size, average age, and sex ratio. The fifth author will check all the data.In the same manner, we will extract data for effect assessments. For each study, we will extract the following information: clinical outcomes, average hospital stay, total hospitalization cost, incidence of ADRs, and patient satisfaction. We will resolve disagreements in the numbers extracted by discussion.2.5The review authors will independently assess the quality of the trials included in the review, in accordance with Chapter 8 of the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011), by(1)random sequence generation,(2)allocation concealment,(3)blinding of participants and personnel,(4)blinding of outcome assessment,(5)incomplete outcome data,(6)selective reporting, and(7)other bias.The fifth author will check all the data. We will use this information to evaluate quality and resolve disagreements by discussion until consensus is reached.2.62.6.12 statistic will be used to assess heterogeneity. It indicates that the heterogeneity exceeds the acceptable range when P < .10 or I2 > 50%. If the heterogeneity is in the acceptable range , the fixed effect model shall be used for data analysis; otherwise, the random effect model will be adopted.The Chi-squared test and I2.6.2P < .05 will be considered statistically significant.Two pairs of review authors will independently extract information for each included trial and whether all the participants are accounted for in the analysis. The fifth author will check all the data. We will use Review Manager 5.3 to assess the risk of bias, heterogeneity, sensitivity and subgroup analysis. We will calculate a weighted estimate of the treatment effect across trials and for the interpretation of the results and we will use 95% CI. 2.6.3We will do the following subgroup analysis to explore the possible causes of high heterogeneity:(1)articles with different impact factors  and(2)trials with low and high risk of bias.2.6.4We will conduct sensitivity analysis by excluding trails one by one and observe whether the synthesis result changes significantly. If there are significant changes, we will make a decision cautiously to decide whether to merge it. If the changes not significantly, it indicates that our synthesized result is firm.2.7If more than 10 articles are available for analysis, funnel plots will be generated to assess publication bias. A symmetrical distribution of funnel plot data indicates that there is no publication bias, otherwise, we will analyze the potential reasons for this outcome and give reasonable interpretation for asymmetric funnel plots.2.8 According to the grading system, the level of evidence will be rated high, moderate, low and very low.We will use the Grades of Recommendations Assessment, Development and Evaluation system to assess the quality of our evidence.3,13 includingPharmaceutical care is the main tasks of clinical pharmacist,(1)participating in the formulation of clinical treatment plans;(2)instructing physicians to use drugs rationally and putting forward suggestions for the irrational use of drugs during the treatment by consulting relevant literature;(3)providing pharmaceutical consulting services, giving the doctor reasonable advice on the medication and treatment;(4),15 andevaluating the drug efficacy and ADRs;(5)educating patients on medication, so that patients have a clear understanding of drug effects and ADRs.–19Studies show that pharmaceutical care can promote the safety, effectiveness, rationality, and economy of drug application in clinical practice.This study will conduct a meta-analysis of related cohort studies, and provide the current evidence on the effect of pharmaceutical care on the treatment of COVID-19, so as to better guide clinical practice.Conceptualization: Jixun Huang, Guangyu Zhao, Hongjun Chen, Jiali Niu, Kaixia Chen, and Yin Liu.Funding acquisition: Jixun Huang and Guangyu Zhao.Investigation: Jiali Niu, Hongjun Chen, Guangyu Zhao, and Jixun Huang.Methodology: Jiali Niu, Yin Liu, Chaoqun Li, Chunxia Yin, Lingyun Jiao, Feng Ju, Ting Xue, and Dengyang Yin.Software: Jiali Niu, Hongjun Chen, Kaixia Chen, and Yin Liu.Supervision: Hongjun Chen, Kaixia Chen, Yin Liu, and Guangyu Zhao.Writing – original draft: Jiali Niu, Hongjun Chen, Kaixia Chen, Yin Liu, Feng Ju, Ting Xue, Dengyang Yin, Chaoqun Li, Chunxia Yin, and Lingyun Jiao.Writing – review & editing: Jixun Huang and Guangyu Zhao.


Idiopathic normal pressure hydrocephalus (iNPH) is a progressive neurological disorder characterized by gait apraxia, cognitive decline, and urinary incontinence. It can be difficult to diagnose iNPH as the symptoms may overlap with other neurodegenerative diseases including cervical spondylotic myelopathy. Cervical spondylotic myelopathy is a progressive degenerative disease in which compression of the cervical spinal cord causes gait disturbances and imbalance, loss of dexterity and strength in the hands, and, at late stages, urinary dysfunction. As with iNPH, increased age is associated with higher incidence and prevalence. Surgical decompression of the cervical spinal cord is the treatment of choice in patients with progressive myelopathy. Accordingly, iNPH and cervical myelopathy may both present with progressive gait impairment and incontinence, especially in the elderly.The case presented here demonstrates that both iNPH and cervical myelopathy may present simultaneously and result in gait disturbances and imbalance in some patients. For patients with suspected iNPH and myelopathic findings on examination, it is prudent to obtain a cervical spine MRI to assess for cervical stenosis. Moreover, cervical stenosis can mask the effect of cerebrospinal fluid diversion in patients with comorbid iNPH and cervical myelopathy. Therefore, the differential for patients who have symptomology suggestive of iNPH should include cervical spine myelopathy, with considerations for possible cervical decompression in addition to placement of a ventriculoperitoneal shunt. The case presented here demonstrates that both idiopathic normal pressure hydrocephalus (iNPH) and cervical myelopathy may present simultaneously and result in gait disturbances and imbalance in some patients. For patients with suspected iNPH and myelopathic findings on examination, a cervical spine MRI should be considered to assess for cervical stenosis. Cervical stenosis can mask the effect of CSF diversion in patients with comorbid iNPH and cervical myelopathy.A 75-year-old man presented with progressive deterioration in gait and balance over a two-year period. One year after symptom onset, he was diagnosed with Parkinson’s disease and was started on carbidopa/levodopa but enjoyed no clinical improvement. Despite the regular use of a walker, he sustained several falls in the month, leading up to his initial evaluation at our institution. He also developed urinary urge incontinence and mild cognitive impairment over a three- to six-month period. The patient denied loss of dexterity or Lhermitte phenomenon. On examination, he scored 29/38 on the Kokmen Short Test of Mental Status, losing points on registration, recall, calculation, and construction. His gait was moderately slow and shuffling, with increased number of steps to turn, reduced arm swing, forward-leaning posture, and Trendelenburg sign. He had full strength in the bilateral upper and lower extremities and no sensory abnormalities. He had symmetric moderate hyperreflexia of the biceps, brachioradialis, and quadriceps, and had moderate spasticity in both lower extremities. There was no Hoffman sign present, and he had mute plantar responses bilaterally.2O. Prior to withdrawal of 30-mL cerebrospinal fluid (CSF), the patient took an average of 20.4 seconds to walk 10 meters  and an average of 5.8 steps for every 180˚ turn. After CSF removal, he walked 10 meters in an average of 17.6 seconds  and, again, took an average of 5.8 steps per 180˚ turn. There was no statistically significant difference in the time it took the patient to walk 10 meters before and after high-volume lumbar tap (p = 0.10) or in the number of steps to turn.MRI of the brain Figures , 1B demoThe differential diagnosis of progressive gait apraxia, mild cognitive impairment, and urge incontinence includes iNPH, cervical spondylotic myelopathy, typical and atypical parkinsonism, hip osteoarthritis, and combinations of these. As described previously, the brain MRI was consistent with iNPH, but the patient had an equivocal response to the high-volume lumbar puncture. The cervical spine MRI revealed diffuse degenerative changes with moderate stenosis at C5-C6, which, in combination with the upper motor neuron findings on examination (even without weakness or loss of dexterity in the hands), suggested cervical myelopathy. The patient had already been treated with carbidopa/levodopa, but without any benefit. Finally, plain radiographs revealed advanced osteoarthritis of the hips and sacroiliac joints, but this diagnosis did not explain the patient’s cognitive impairment or urinary symptoms. Based on the underwhelming response to high-volume CSF removal and mobile spondylolisthesis in the cervical spine, a flexion-extension MRI of the cervical spine was pursued, which demonstrated slight progression of the canal effacement at C5-C6 in extension but, surprisingly, also demonstrated dynamic compression of the spinal cord at C4-C5 Figure .2O. After removing 30-mL CSF, it took the patient an average of 16.0 seconds to walk 10 meters  and 5.0 steps to turn 180˚. The difference in time to complete a 10-meter walk  at C4-C5 and C5-C6 was performed, without complication, to decompress the cervical spinal cord. He continued to suffer progressive deterioration of his gait and balance over the ensuing year, however, and therefore returned for repeat lumbar tap test. Prior to this second high-volume lumbar puncture, the patient took 31.1 seconds on average to walk 10 meters  and an average of 7.6 steps per 180˚ turn. The opening pressure of the lumbar puncture was 194 mmHINPH is a progressive neurological disorder characterized by gait apraxia, cognitive decline, and urinary incontinence . NeuroimCervical spondylotic myelopathy is a progressive degenerative disease in which compression of the cervical spinal cord causes gait disturbances and imbalance, loss of dexterity and strength in the hands, and, at late stages, urinary dysfunction ,8. As wiThe case presented here demonstrates that both iNPH and cervical myelopathy may present simultaneously and result in gait disturbances and imbalance in some patients. For patients with suspected iNPH and myelopathic findings on examination, it is prudent to obtain a cervical spine MRI to assess for cervical stenosis. Moreover, cervical stenosis can mask the effect of CSF diversion in patients with comorbid iNPH and cervical myelopathy. This phenomenon may be, in part, why up to 20% of patients with iNPH fail to achieve full gait resolution after shunting. Therefore, the differential for patients who have symptomology suggestive of iNPH should include cervical spine myelopathy, with considerations for possible cervical decompression in addition to placement of a ventriculoperitoneal shunt.For patients with suspected iNPH and myelopathic findings on examination, it is prudent to obtain a cervical spine MRI to assess for cervical stenosis. The differential for patients who have symptomology suggestive of iNPH should include cervical spine myelopathy, with considerations for possible cervical decompression in addition to placement of a ventriculoperitoneal shunt.


Delayed endolymphatic hydrops (DEH) represents a rare clinical entity characterized by intermittent vertigo attacks mimicking those of Ménière's disease (MD) in a patient with a prior sensorineural hearing loss. Some vestibular tests have been employed in patients with DEH. These tests provide useful diagnostic information and facilitate clinical decision-making. Here, we retrospectively studied the features of video head impulse test (vHIT) and examined its relationship with caloric test used in DEH patients. Included in this study were 17 patients with ipsilateral DEH and 2 with contralateral DEH. Among them, 73.7% (14/19) showed abnormal caloric test response . Meanwhile, only 15.8% (3/19) of patients yielded abnormal horizontal vHIT results . Abnormal caloric response in the presence of a preserved vHIT was common in DEH patients, especially those with ipsilateral DEH. This dissociation might be a distinctive pattern of vestibular deficit in DEH. Vestibular test is designed to assess the status of the vestibular system objectively and quantitatively. Over the last decade, vestibular test has evolved greatly, and the horizontal and vertical semicircular canals (SCC) as well as the otolith organs can be examined separately . TraditiDelayed endolymphatic hydrops (DEH), a rare clinical entity, is characterized by episodic vertigo of delayed onset following profound sensorineural hearing loss (SNHL), and its symptoms mimic those of MD . Kamei eAlthough many vestibular tests have been applied in patients with DEH, including the electronystagmography , caloricA single-center retrospective study was conducted in the Department of Otorhinolaryngology, Union Hospital affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.Nineteen patients with DEH were diagnosed by experienced neurotologists against the diagnostic criteria formulated by the committee of the Japan Society for Equilibrium Research in 1987 : The ipsExclusion criteria included: (1) middle or inner ear infections ; (2) middle or inner ear anomaly ; (3) having received previous ear surgery or intratympanic injections; (4) retro-cochlear lesions ; (5) head trauma; (6) systemic diseases; (7) disorders of central nervous system ; (8) having taken alcohol, caffeine or medications that would affect the results of vestibular tests  within 48 h before testing.This study was conducted in strict accordance with the tenets of the Declaration of Helsinki. Informed consent was obtained from each patient and the project was approved by the ethical committee of Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China.All patients with DEH during the interictal period underwent a battery of tests, including otoscopy, audiometry, videonystagmography, caloric test, horizontal vHIT in the same day. Non-contrast magnetic resonance imaging (MRI) was routinely performed to rule out middle or inner ear infection and malformation. If retro-cochlear lesion was suspected, the patient would receive contrast-enhanced MRI.Pure-tone audiometry was conducted in a soundproof room, at 125, 250, 500, 1,000, 2,000, 4,000, and 8,000 Hz. Pure tone average (PTA) was calculated to be the simple arithmetic mean for frequencies of 500, 1,000, and 2,000 Hz.Vestibular tests were performed by using an infrared videonystagmography system  in accordance with a standard test protocol, including spontaneous nystagmus test, gaze test, saccades, smooth pursuit, and optokinetic test. The Dix-Hallpike and Roll tests were also videonystagmugraphically performed.max) of caloric nystagmus was measured, and the canal paresis (CP) was calculated by using the traditional Jongkees' formula. In this study, if the asymmetry of the caloric nystagmus between the left and right ear was ≥25%, CP was considered significant in the horizontal SCC, indicating an abnormal caloric response. According to the published criteria  would be used to confirm the caloric unresponsiveness.The bithermal caloric test was conducted by using infrared videonystagmography. The subject lay supine with their head and upper trunk elevated at 30°. Each ear was irrigated alternately with a constant flow of air, with the temperature for warm or cool stimulation set at 50 and 24°C, respectively. The duration of each caloric irrigation lasted 60 s. Upon each irrigation, the maximum slow phase velocity  in accordance with the manufacturer's instructions by experienced technicians. Each subject wore a pair of lightweight, tightly-fitting goggles equipped with a small video oculography camera to record and analyze the eye movement. Patient was seated upright facing the wall 1.0 m away and was instructed to fixate a static target on the wall. The patient's head was passively and randomly rotated to the left and right with a low amplitude (5~15°) and at a high peak velocity (150~250°/s) in an abrupt, brief and unpredictable manner. At least 20 head impulses were delivered in each direction. Re-fixation saccades were categorized, in terms of their appearance, as covert and overt. If the velocity of the saccade exceeded 50°/s, they were deemed positive. In the present study, it was taken as abnormal if the horizontal vHIT gain < 0.8 and re-fixation saccades appeared.Nineteen DEH patients were included . According to the diagnostic criteria, 17 patients were diagnosed with ipsilateral DEH and two as having contralateral DEH. All 19 patients reported precedent hearing loss and episodic vertigo attacks. Fifteen patients reported profound hearing loss of unknown etiology in early childhood. In three cases, hearing loss was caused by sudden SNHL in adulthood and in one case deafness was associated with meningitis in childhood . A preciAudio-vestibular test results are presented in In this series, 14 (73.7%) had reduced caloric response, and only three cases (15.8%) had abnormal horizontal vHIT responses. Of the 17 patients with ipsilateral DEH, abnormal CP was observed in 76.5% (13/17) of the patients and 11.8% (2/17) yielded abnormal vHIT result. In 12 cases, abnormal CP was ipsilateral in the ear with preexisting hearing loss and in one patient abnormal CP was in the opposite ear. For the two patients with both abnormal CP and vHIT results, abnormal response occurred in the previously damaged ear in one case, and in the opposite ear in the other case. Of the two patients with contralateral DEH, abnormal CP and vHIT were detected in one case (50%), and abnormalities occurred in the ear with preexisting hearing loss. For all subjects, MRI did not reveal any retro-cochlear lesion.Lifestyle modification was recommended to the patients, including low-sodium diet, low caffeine consumption, and abstinence from stimulants. In all 19 patients, initial medication included the betahistine or diuretics, lasting for at least 3–6 months. Two patients (No. 9 and 14) received one course of intratympanic dexamesone due to unsatisfactory vertigo control. No ablative treatment was administered in this series, especially for the contralateral type of DEH. A follow-up, lasting 3 months−3.0 years, showed that, against the guidelines for MD proposed by American Academy of Otolaryngology-Head and Neck Surgery in 1995 , completIn this study, we investigated the function of horizontal SCC VOR in patients with DEH. In our series, 73.7% (14/19) of the DEH patients showed abnormal caloric response. Meanwhile, only 15.8% (3/19) of the patients yielded abnormal horizontal vHIT results. To the best of our knowledge, this was the first study which examined the VOR function measured by vHIT and identified a discrepancy between the results of vHIT and the caloric test in patients with DEH.Until now, two hypotheses have been put forward to explain this dissociation. First, the hydrostatic temperature dissipation hypothesis attributed the discrepancy to the different test mechanisms between caloric test and vHIT. The distended membranous duct of the horizontal SCC has been suggested to permit convective recirculation within the duct, leading to a diminished thermally- induced pressure gradient across the cupula and thus to less deflection of the cupula and hair cells and less caloric nystagmus. This assumption is supported by the observation that, in MD patients with normal vHIT results, the reduced caloric response was correlated with the severity of vestibular hydrops (as demonstrated by intravenous gadolinium-enhanced MRI of the inner-ear) . Second,Recently, multiple studies have demonstrated this disagreement in patients with MD (idiopathic ELH). McCaslin et al.  and McGaIn this study, we showed that, in patients with ipsilateral DEH, 70.6% (12/17) had abnormal CP in the ear with previous cochlear damage and 5.8% (1/17) had abnormal CP in the opposite ear. Our result was in agreement with that reported by Schuknecht et al. who found that 80% of the ipsilateral DEH patients had unilateral caloric weakness in the hydropic ear and 9% had such hypofunction in the opposite ear . The subIn the present study, 50% (1/2) of the patients with contralateral DEH showed the abnormal CP and vHIT in the previously deaf ear rather than the opposite ear. The pattern of ELH distribution and vestibular deficit is rather complicated in contralateral DEH, indicating that its pathology is more involved compared to its ipsilateral counterpart , 32, 33.We found a vHIT deficit in 15.8% and a caloric weakness in 73.7% of our DEH patients. No other studies investigated the VOR function at these high frequencies in patients with this condition. Alternatively, rotatory chair test can assess the VOR system across the low to middle frequencies (0.01–1.00 Hz). To date, only one published study has investigated the performance of rotary chair and caloric test in DEH patients and revealed that rotatory chair test identified vestibular hypofunction in 3% while caloric test detected the hypofunction in 59% of their patients (In hydropic ear disease with fluctuating nature, the caloric response was speculated to reflect the severity of ELH more than the impairment of VOR function. Clinically, the functional assessment or therapeutic decision may be biased if based on the caloric test alone. This was in agreement of Bodmer et al.  and Lin The study had several limitations. First, the sample size was small, especially for the contralateral cases, because DEH is not a common vestibular disorder. Second, we did not perform gadolinium-enhanced MRI, which might provide further imaging evidence to account for the findings of our VOR tests. Third, this study did not perform the rotatory chair test, in which the VOR time constant has been shown to provide the most reliable assay screening fixed peripheral vestibular loss . Since cAbnormal caloric response in the presence of a preserved vHIT might be a distinctive pattern of vestibular deficit in patients with DEH, a rare variant of Ménière's disease. More evidence is needed to clarify the clinical implication of this dissociation in hydropic ear disease.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Ethical committee of Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.BL: study conception and design, data acquisition, and critical review of the manuscript. YL: data analysis and interpretation, drafting and revision of the manuscript.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.


In this retrospective report, we present five cases of critically ill pregnant or newly delivered women positive for Covid-19 admitted to our obstetrical departments at Karolinska University Hospital. They compose 6% of eighty-three pregnant women that tested positive for SARS-CoV-2 during the period March 25 to May 4, 2020. Three patients were at the time of admission in gestational week between 21 + 4 and 22 + 5 and treated during their antenatal period; meanwhile, the other two were admitted within 1 week postpartum. All of them were in need of intensive care: one was treated with high flow oxygen therapy, the other four with invasive mechanical ventilation . Age above thirty, overweight, and gestational diabetes are notable factors in the cases presented. At the time of admission, they all presented with symptoms such as fever, cough, and dyspnea. Chest imaging with computer tomography scan was performed in each case and demonstrated multifocal pneumonic infiltrates in all of them, but no pulmonary embolism was confirmed in any. Neither did the echocardiogram indicate any cardiomyopathy. Four of the patients have been discharged from the hospital, with an average of 20 hospital days. One antenatal pregnant woman needed prolonged ECMO therapy; in gestational week 27 + 3, she went into cardiac arrest, resulting in an urgent C-section on maternal indication. At the time of writing, she is still hospitalized. In coherence with other published reports, our cases indicate that critically ill pregnant women infected by SARS-Cov-2 may develop severe respiratory distress syndrome requiring prolonged intensive care. The material is limited for conclusions to be made; more detailed information on symptoms, treatment, and outcomes for pregnant and postpartum women managed in intensive care is therefore needed. It is by now known that the pneumonia of unknown cause that was detected in Wuhan, China, reported to the World Health Organization (WHO) County Office on December 31, 2019, was caused by a new coronavirus strain, SARS-CoV-2. The outbreak was declared as a Public Health Emergency of International Concern on January 30, 2020, and the new coronavirus disease was named Covid-19 [Risk factors for developing symptomatic Covid-19 and severe respiratory distress syndrome include age above 70 years and, in addition, a medical condition such as diabetes, chronic respiratory disease, cardiovascular and kidney disease, impaired immune system, obesity, and neuromuscular disease .Initially, it was not clearly known if pregnant women were at increased risk of developing severe symptoms from Covid-19 compared to the nonpregnant female population, as little data and information had been published. Initial studies concluded that pregnant women shared the same clinical characteristics as nonpregnant women and there was not enough evidence to support that pregnant women had an increased risk of developing severe disease compared to the nonpregnant women in the same age groups , 5.As more data has been collected, several reports and case series indicate that pregnant women may be at higher risk of becoming critically ill and requiring higher intensive care. Therefore, careful monitoring of pregnant women with Covid-19 has been warranted . There hIn May 9, 2020, the Swedish Public Health Agency published a report on pregnant and early postpartum (<1 week postpartum) women diagnosed with Covid-19 who required intensive care during the period between March 19 and April 20, 2020. The relative risk for pregnant and postpartum women compared to nonpregnant women was based on the estimated number of pregnant and nonpregnant women in the Swedish population. A total of fifty-three women, nonpregnant and pregnant, between the age of 20 and 45 years, received intensive care according to the Swedish Intensive Care Registry. Thirteen of them were or had recently been pregnant. Although the result was based on a relatively small number of cases, the estimated risk for pregnant women to receive intensive care was significantly higher than the nonpregnant population  for pregnant/postpartum women and 2.5 per 100,000 95% CI 1.8-3.5) for nonpregnant women in the same age group). As a result, pregnant women were recommended to take necessary precautions to avoid infection during pregnancy [-3.5 for The region most affected by Covid-19 in absolute numbers in Sweden is Stockholm . The KarDuring the period between March 25 and May 4, 2020, 885 delivered pregnant women were tested for Covid-19. 7.2%, 64, tested positive; out of these, 76.6%, 49, were asymptomatic and 23.4%, 15, were symptomatic with either mild, moderate, or severe symptoms. Eleven symptomatic patients have been in need of extended hospital care due to moderate or severe symptoms related to Covid-19 disease. Eight had moderate symptoms and were observed on regular medical wards. Three were admitted to the intensive care and in need of invasive mechanical ventilation support. In addition, two antenatal pregnant women who tested positive during the same period needed intensive care due to Covid-19.Since there is a worldwide interest and need of a better understanding on how SARS-CoV-2 affects pregnant women, especially in those that get critically ill, we decided to present a case series on five pregnant women who needed intensive care due to severe or critical Covid-19.This is a retrospective case series of symptomatic pregnant women that tested positive for SARS-CoV-2 with quantitative reverse transcription polymerase chain reaction (qRT-PCR) during the period March 25 and May 4, 2020. Fifteen symptomatic pregnant women required hospital care during the period, at Karolinska University Hospital, Stockholm, Sweden. In this report, we decided to include those pregnant women who at least had entered their second trimester at the time of admission to the hospital or were <1 week postpartum and who developed severe or critical Covid-19 requiring intensive care, resulting in five cases.2)/fraction of inspiration oxygen (FIO2) ≤ 300 mmHg, and/or lung infiltrates > 50% of the lung field within 24-48 hours. Critical disease is described as respiratory failure, septic shock, and/or multiple organ dysfunction/failure [The definition of severe and critical disease is according to the report of the WHO-China Joint Mission on Coronavirus Disease 2019. Severe disease is described as dyspnea, respiratory frequency ≥ 30 breaths/min, blood oxygen saturation ≤ 93% or alveolar oxygen partial pressure  was 25.6 kg/m2) of 94%, respiratory rate (RR) of 24 breaths/minute, and heart rate (HR) 118 beats/minute, and her blood pressure (BP) was 110/78 mmHg. She tested positive for Covid-19 with a PCR test. During the first four hospital days (HD), she did not require any specific medical support or medication besides cough medication. Prophylactic antithrombotic treatment with low molecular- weight-heparin (LMWH) was initiated. Her fever decreased, and the cough got better, but at the fifth HD, the cough got worse, she developed epigastric pain, nausea, and dyspnea and required increased oxygen levels. Her c-reactive protein (CRP) and procalcitonin (PCT) levels increased, and treatment with intravenous antibiotics was induced due to suspected bacterial pneumonia.At the time of admission to the hospital, she was fatigued, had a fever of 39.5°C, oxygen saturation  indicated moderate acute respiratory distress syndrome (ARDS) [2 12 L/min, with endotracheal intubation as a backup. The LMWH dose was doubled as a routine in critically ill pregnant women to prevent thrombotic event. Her potassium level decreased to 2.8 mmol/L, and her aminotransferase levels increased. She was given potassium infusion, in addition to intravenous fluids and parenteral nutrition. Daily fetal heart rate was monitored and normal; no prednisolone or other obstetrical intervention was therefore made at that time. Chest imaging, with CT scan, demonstrated multifocal pneumonic infiltrates, but no pulmonary edema or embolism.At HD 8, the patiente (ARDS) . She wasThe patient became increasingly better; no endotracheal intubation was needed. On HD 11, she was transferred to a medical ward, and on HD 15, gestational week 23 + 6, she was discharged from the hospital. The patient is today in gestational week 28 + 1 with maintained fatigue but no cough. The latest ultrasonographic fetal biometry in gestational week 26 + 3 indicated normal fetal growth, and there were no signs of fetal hypoxia on the Doppler examination.2. Her medication included levothyroxine, insulin, iron, and prenatal vitamins.A 36-year-old, gravida 5, para 2, with African background, and previous normal deliveries, expecting dichorionic/diamniotic duplex (DC/DA) presented at gestational week 22 + 5 at the obstetrical emergency ward with cough, fatigue, arthralgia, myalgia, nausea, and vomiting. The symptoms had first occurred eight days earlier, but now she had worsened; she had not been able to maintain any nutrition and appeared dehydrated. She had developed gestational hypothyroidism and gestational diabetes in this pregnancy. Her BMI was 29.69 kg/m2 98%) and no fever. The ABG indicated acute starvation ketoacidosis and hypokalemia (potassium 2.8 mmol/L); the CRP was elevated, and in addition, she also had proteinuria, ketonuria, leukocyturia, and glucosuria. No other organ dysfunction or signs of preeclampsia was seen on the blood samples. She was transported to the intermediate medical care (IMC: step-down unit from highly intensive care) unit for fluid resuscitation, and prophylactic treatment with LMWH was initiated. She tested positive for Covid-19 with PCR.At the time of admission to the hospital, she had, in addition to the symptoms previously mentioned, tachycardia (HR 130 beats/minute), hypovolemia (BP 94/61 mmHg), and tachypnea (RR 28 breaths/minute) but no hypoxemia , a successful extubation was achieved. On HD 17, step-down from ICU to IMC was performed, and she was transferred to a medical ward for rehabilitation. At HD 22, in gestational week 25 + 5, she was discharged from the hospital.At the moment of writing, the patient has fully recovered from the respiratory insufficiency. She went through several ultrasonographic fetal biometrics which indicated normal growth in both twins, and in gestational week 38 + 1, she was delivered with a planned cesarean section.2. Her medication included insulin, iron, and prenatal vitamins. She had a normal delivery and was discharged from the hospital the following day with no postpartum complications.A 30-year-old, gravida 2, para 1, with Asian background presented at the obstetrical ward in gestational week 37 + 4 with spontaneous start of labor. She had regular contractions and spontaneous rupture of membranes. She tested positive for Covid-19 with PCR, but had no symptoms. She had developed gestational diabetes in this pregnancy. Her BMI was 27.08 kg/m2 88% on 3 L O2→93% on 6 L O2 on oxymask), tachypneic (RR 42→45 breaths/minute), and normotensive (BP 130/71 mmHg) and had a normal HR 95 beats/minute with no fever. The laboratory samples indicated mild leukocytosis and CRP 107 mg/L. The ABG showed respiratory alkalosis. Treatment with antibiotics, LMWH, fluids, and a first dose of chloroquine phosphate was administered before she was transported to an IMC unit.Two days later (postpartum day two), she presented in the emergency ward due to dyspnea. She had developed upper respiratory tract symptoms the same day she was discharged from the hospital and had increased difficulty with breathing. At the time of admission, she was hypoxemic , her clinical condition was stable and extubation from ETI was performed, but due to immediate decompensation, she was immediately reintubated. Although the blood cultures did not show any specific growth of pathogens, the CRP levels continued to be high >100 mg/L and broad-spectrum antibiotics were therefore given.Within three hours, her clinical condition deteriorated with increased RR 50 breaths/minute, SaOOn HD 11 (ICU day 9), she received a tracheostomy, and on HD 14 (ICU day 12), step-down from the ICU to IMC was done. On HD 17, she was decannulated from the tracheostomy and transported to a medical ward for further rehabilitation. At HD 21, she was discharged from the hospital. At the time of writing, the patient has fully recovered from her respiratory insufficiency although fatigue remains as a symptom.2. Her only medication was prenatal vitamins and iron substitution.A 36-year-old, gravida 2, para 1, with Middle Eastern background, presented at the obstetrical ward, in gestational week 40 + 6. She was in a latent phase with regular spontaneous contractions. At the time of admission, she was subfebrile and the cardiotocography indicated fetal tachycardia. She was normotensive (110/70 mmHg), tachycardic (HR 110 beats/minute), and tachypneic (RR 26); the saturation level was not documented. Three days before admission, she had the onset of fever, arthralgia, and upper respiratory tract symptoms. She tested positive with PCR for Covid-19. She had no previous medical history besides migraine, and her BMI was 27.27 kg/mShe was first observed for a couple of hours with regular monitoring and was given intravenous paracetamol, terbutaline, and morphine to release the pain from the contractions during the latent phase. As there was no progress in her labor, the decision for induction with a Foley catheter was taken, and three hours later, there was a spontaneous rupture of membranes with meconium. As there was a clinical suspicion of chorioamnionitis, treatment with intravenous antibiotics was initiated. The progress of labor was slow, and the CTG had an abnormal pattern with continuous tachycardia and episodes with decreased variability, although the fetal scalp blood test indicated normal lactate levels, but as the patient developed fever (38.5°C), the decision for a cesarean section after 20 h from the beginning of induction was made. The operation was uncomplicated, although meconium had contaminated the amniotic fluid; fortunately, the neonate was doing fine with an Apgar of 9 : 10 : 10.2 decreased (89% on air). She received 6 L O2 on oxymask, but due to augmented dyspnea, fever, CRP 234 mg/L, ABG indicating respiratory alkalosis, and an increased demand of oxygen (SaO2 89% on 6 L O2→SaO2 96% on 10 L O2), she was transferred to an IMC for further monitoring. A CT scan was made and demonstrated general multifocal pneumonic infiltrates but no pulmonary embolism.For the first three postpartum days, she was continuously subfebrile, tachycardic, and hypotensive, but with additional fluid, antibiotics, and LMWH, her clinical condition was stable. On HD 6, she suddenly deteriorated in her respiration; her RR increased (23 breath/minute) and SaOOn the following day, HD 7, she developed diarrhea, and the respiratory rate increased to 40-45 breaths/minute. She was transferred to the ICU, and on HD 8, her respiratory insufficiency was defined as moderate ARDS, and ETI was made to secure her ventilation. The echocardiography did not indicate any signs of right or left ventricular failure. The prone position was made for better ventilation.Her clinical condition gradually improved, and on HD 15 (ICU day 9), she was successfully extubated, and on HD 16, she was transferred to a medical ward for further rehabilitation and treatment. On HD 19, she was discharged from the hospital. At the time of writing, she has fully recovered from her respiratory insufficiency.2. Her only medication was iron substitution. For the previous three days, she had developed symptoms including cough, fever, and dyspnea which gradually had worsened. Due to deterioration of her respiration with increased dyspnea, she decided to contact the hospital.A 30-year-old, gravida 4, para 2, with Asian background presented at a general hospital in gestational week 22 + 3. In her obstetrical history, she had one early spontaneous miscarriage, one intrauterine fetal death (IUFD), and one full term delivery. Her medical history related to the current pregnancy included a known protein S deficiency, diet-treated gestational diabetes, and a BMI of 33.75 kg/m2 on air→80% on 15 L O2 oxymask), tachypneic (50-55 breaths/minute), tachycardic (120 beats/minute), and normotensive (120/65 mmHg). She had fever 39.4°C, increased CRP, and hypokalemia, and the ABG indicated acute uncompensated respiratory acidosis. Within an hour, she was intubated and transported to Karolinska University Hospital for further treatment at the ICU. Before the transport, the first dose of prenatal betamethasone and intravenous antibiotics was given. The PCR test for Covid-19 was positive.At the time of admission, she was dyspneic, severely hypoxemic , her clinical condition had improved, and a tracheostomy was performed.On HD 5 (ICU day 5), the patient's CRP levels increased (>200 mg/L) and so did the oxygen demand with episodes of hypercapnia and a low pOOn ICU day 15, she once again deteriorated, and extubation from the ETI could not be performed. In the culture from the tracheostomy, growth of Klebsiella aerogenes was found, and additional antibiotic and antifungal treatments were therefore given. The LMWH doses were increased due to continuously low antifactor Xa levels. On ICU day 18, the mechanical ventilation support was not enough to substitute for her pulmonary insufficiency, indicating the initiation of venovenus extracorporeal membrane oxygenation (v-v ECMO).On ECMO day 5 (HD 22), she was considered to have recovered from Covid-19 due to four negative PCR test in a row, and her pulmonary condition was now assessed to be affected by a “normal” bacterial pneumonia. Over the following ten days, her clinical condition improved, and on ECMO day 14 (HD31), she was decannulated from ECMO. At HD 35, in gestational week 27 + 2, she was transferred from the ECMO ward to the ICU for further treatment.Five hours later she suddenly developed seizures and lost consciousness. A bolus dose of magnesium infusion and diazepam were given with the aim to stop the seizures with little improvement. She did not respond to pain stimulation, and an eye deviation to the right was observed. Urgent preparations for a CT scan and an electroencephalogram (EEG) were made. Just before the transfer, the telemetry signaled arrhythmia with irregular heart rhythm and acute bradycardia, with a loss of pulse. The blood pressure decreased and so did the saturation, resulting in cardiac arrest. Cardiopulmonary resuscitation was initiated and preparation for perimortem caesarean section at the ICU was made. However, she had a return of spontaneous circulation (ROSC) and thereby could be transported to the operation theater. A healthy preterm neonate was delivered in gestational week 27 + 3 and was transferred to the neonatal intensive care unit.After the cesarean section, she was replaced in v-v ECMO (replaced-ECMO day 1) and was in need of several blood and plasma transfusions due to hemodynamic instability. Heparin infusion was therefore temporarily stopped. On HD 38, an exploratory laparotomy was made as a damage control due to continuous need of transfusion and suspicion of intra-abdominal bleeding. The main bleeding came from rectus abdominis muscle tears. Hemostasis was achieved by sutures and diathermy, and the abdominal cavity was packed with several abdominal packs. This was not enough though to maintain hemodynamic stability, and there have been three more reoperations to try to control diffuse bleeding from the abdominal muscles and peritoneum. An angiography during the last relook on HD 43 was made, indicating extravasation from an internal iliac arterial genital branch, and in addition, diffuse bleeding from omental and subcutaneous tissue as from the rectus muscles was still seen. The hemodynamic instability was addressed to severe “clinical” coagulopathy although activated partial thromboplastin time (APTT), rotational thromboelastometry (ROTEM), and fibrinogen levels were normal. Continuous renal replacement therapy (CRRT) was initiated to provide renal support due to acute kidney injury.The bleeding eventually subsided, and her clinical condition gradually improved; on HD 52 (replaced-ECMO day 17), she was successfully decannulated from ECMO and transferred to the ICU for further treatment. On HD 56, decannulation of the tracheostomy was performed, the CRRT was discontinued, and the patient was transferred to a regular medical ward. At the time of writing, her clinical condition is now stable although she is still hospitalized with ongoing rehabilitation.Our aim with presenting these five cases of pregnant or newly delivered women with Covid-19 is to add more knowledge of the clinical outcomes that pregnant women with severe and critical Covid-19 disease may develop. In comparison to other case series and reports that have been published on the subject, Covid-19 and pregnancy, we can also see that there are several similarities in the patient groups when it comes to the terms of clinical presentation, risk factors, and disease progression.In a meta-analysis by Zaigham and Andersson on 108 pregnant women with symptomatic Covid-19, it was found that the mean age for the women was 29-32 years, the main symptom was fever (59%), and elevated CRP above 10 mg/L was seen in more than 70% . This isWhen it comes to imaging diagnostics, Yan et al. found that 96% 104) of their pregnant patients had abnormal findings on the CT scan, described as patchy shadowing or ground glass opacity  of their, and it The role of the echocardiogram in pregnant women with Covid-19 can also be discussed. Arentz et al. published a case series on 21 nonpregnant patients that required intensive care due to Covid-19, and they found that approximately 33% of the patients developed cardiomyopathy with echocardiographic signs of globally reduced left ventricular systolic function . JuuselaDiabetes, hypertension, and high BMI have been announced as risk factors for developing a more severe and critical Covid-19 disease in the nonpregnant population , 16. PreThis report is based on a case series with limited material; in order to make any significant conclusions, more detailed information on symptoms, treatment, and outcomes for pregnant and postpartum women managed in intensive care is needed.


Postpartum depression (PPD), a main cause of maternal suicide, is an important issue in perinatal mental health. Recently, cerebral diffusion tensor imaging (DTI) studies have shown reduced fractional anisotropy (FA) in major depressive disorder (MDD) patients. There are, however, no reports using diffusion kurtosis imaging (DKI) for evaluation of PPD. This was a Japanese single-institutional prospective study from 2016 to 2019 to examine the pathophysiological changes in the brain of PPD patients using DKI. The DKI data from 3.0 T MRI of patients one month after delivery were analyzed; the patients were examined for PPD by a psychiatrist. The mean kurtosis (MK), FA and mean diffusivity (MD) were calculated from the DKI data and compared between PPD and non-PPD groups using tract-based spatial statistics analysis. Of the 75 patients analyzed, eight patients (10.7%) were diagnosed as having PPD. In the PPD group, FA values in the white matter and thalamus were significantly lower and MD values in the white matter and putamen were significantly higher. The area with significant differences in MD value was more extensive (40.8%) than the area with significant differences in FA value (6.5%). These findings may reflect pathophysiological differences of PPD compared with MDD. The incidence of PPD has been reported to be 10–15%2 in developed countries and we reported an incidence of 11.3% in Japan in a previous study3. Depressive symptoms have been reported in more than 25% of perinatal women4. Forty cases of suicide within one year postpartum have been reported for the 10 years from 2005 to 2014 in Tokyo, 36% of which were PPD1. In addition, PPD has been reported to reduce mother-infant relationships including physical contact, laughter, and dialogue, and to affect the development of the child's cognition, emotions, motor functions, and nerve functions5. Thus, early detection of PPD and appropriate interventions are necessary. Currently, the most widely used PPD screening method is the Edinburgh Postnatal Depression Scale (EPDS) developed in 19876. However, EPDS does not assess the pathophysiology of PPD.According to a recent report, 60% of mothers who committed suicide in the first year of childbirth in the 23 wards of Tokyo had mental illness, and postpartum depression (PPD) was the most common (33%)8. Based on these backgrounds, the relevance of neurocircuitry in PPD and differences between PDD and MDD are unknown.Major depressive disorder (MDD) is one of the most prevalent psychiatric disorders. Several neuroimaging studies have suggested that abnormalities of the frontal-subcortical circuits in brain regions such as the lateral orbitofrontal, the dorsolateral prefrontal, and the anterior cingulate are involved in the pathology of MDD10. Herein, we focused on an objective assessment of PPD using diffusion MRI. Diffusion-weighted imaging (DWI) and diffusion tensor imaging (DTI) are expressed using 1st order and 2nd order tensors, respectively11. Conventional DWI and DTI are inadequate to distinguish the actual diffusion because of the narrowing of the intercellular space and the complication of nerve fibers restricting the diffusion of water molecules in the central nervous system13. Therefore, as an advanced form of DTI, diffusion kurtosis imaging (DKI) using 4th-order tensor has been devised. DKI can sharply depict the structure of tissue by correcting the degree of restriction due to the structure of the tissue on the diffusion of water molecules as kurtosis15. This technique represents a promising approach for early diagnosis of various neurodegenerative diseases16. Although many studies have evaluated MDD using DTI 30, few studies have used DKI32. In those studies, the DKI showed a decrease in the mean kurtosis (MK) and radial kurtosis values, which were specific parameters that could not be expressed by DTI, in a wider range than that of fractional anisotropy (FA) value decreased. However, the pathophysiological mechanisms from these findings have not been elucidated. Moreover, the pathophysiological aspects of PPD are also poorly understood. In 2018, Silver et al. used DTI data to show that FA value declines in the brains of patients with PPD as well as MDD33. Although the clinical phenotype of PPD is similar to that of MDD, it is also suggested that PPD and MDD may not be the same34 and it is considered that the pathophysiology of PPD may be different from MDD. With this background in mind, we investigated the pathophysiological feature in the brain of patients with PPD and compared diffusion metrics between patients with and without PPD using DKI.Diffusion MRI is a widely used in-vivo imaging technique that measures the movement of water molecules in tissue, and recently, it has been used for clinical applications to diagnose neurodegenerative or psychiatric disordersThis study was conducted in compliance with the Declaration of Helsinki and according to the Ethical Guidelines for Medical and Health Research Involving Human Subjects established by the Ministry of Health, Labour, and Welfare in Japan. Written informed consent was obtained from all participants. The study protocol was approved by the ethical review committee of the Iwate Medical University Hospital . The protocol for this study was registered with UMIN Clinical Trials Registry (UMIN000037844).Eligible patients in the study were mothers who had delivered at the Iwate Medical University Hospital from August 2016 to February 2019, and were disqualified if they met any of the following exclusion criteria: 1) contraindications for MRI such as metal implants in the body or claustrophobia, 2) history of mental disorder including depression, 3) intracranial organic abnormality, 4) stillbirth delivery, 5) possibly be subjected to mental, physical or social burden by participating in the study, and 6) under 20 years old. Borderline cases and patients for whom it was difficult to determine whether or not they had PPD were excluded from analysis.35, and the diagnostic criteria as listed in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition, was used. Severity was classified as mild, moderate, and severe according to the diagnostic criteria.The purpose of this study was to elucidate the pathophysiology of PPD by comparing the MK, FA, and MD values between patients with and without PPD. Eligible patients underwent an MRI at 1 month postpartum and a psychiatric assessment for PPD within 2 months postpartum (within 1 month of the MRI). According to the American Psychiatric Association, PPD was defined as having a primary symptom of MDD during pregnancy or within 4 weeks after delivery41, are shown in the Table 43. Conventional MR images, including three-dimensional T1-weighted images and axial T2-weighted images, were also obtained.All subjects underwent an MRI examination with a 3-T scanner . DKI source images were obtained using a single-shot spin-echo echo-planar-imaging sequence. Scanning parameters, set by referring to previous reports41. To identify changes in the whole-brain white matter between the PPD and non-PPD groups, we carried out voxel-wise statistical analysis of the MK, FA, and MD maps using tract-based spatial statistics (TBSS) implemented in Functional Magnetic Resonance Imaging of the Brain (FMRIB) Software Library 5.0.945. Regarding the region of interest (ROI) analysis in the basal ganglia of the Johns Hopkins University Eve atlas46, MK, FA, and MD values of the caudate nucleus, putamen, globus pallidus, and thalamus were then automatically measured using coregistered ROIs41.Diffusion metric maps including MK, FA, and MD were calculated from the DKI source data using the in-house software program used in previous studiesThe ratio of patients with PPD to non-PPD was considered to be 1 to 8. Under the conditions of power 0.90, and α 0.05, the sample size required for the analysis was estimated to be 72 in total (8 cases of PPD and 64 cases of non-PPD). Assuming a dropout rate of 5% (4 cases), the number of enrolled patients was determined to be 76.p < 0.05 (two-sided) was used. Statistical analysis was performed using JMP ver. 13.1.0 software .For TBSS, to compare voxel values for MK, FA, and MD between patients with and without PPD, a two-sample t-test using non-parametric statistical inference function included in the FMRIB Software Library was performed with 5,000 permutation sets. All voxels in the brain were corrected using the threshold-free cluster enhancement method with the family-wise error correction. The ratios of the areas in the brain with significant changes in MK, FA, and MD to the mean FA skeleton area were calculated. A linear regression model was performed to investigate the relationship between MK, FA, or MD values and EPDS scores on TBSS. The Mann–Whitney U test was used for comparisons of MK, FA, and MD values obtained by ROI analysis between patients with and without PPD. We tested correlations between MK, FA, or MD values and EPDS scores on ROIs. For the clinical comparisons between the PPD and the non-PPD groups, a Mann–Whitney U test was used for continuous variables and a chi-square test was used for the categorical variables. For all statistical analyses, a significance level of Eighty-nine eligible patients were enrolled from August 2016 to March 2019  and MD values of the putamen in the PPD group were significantly higher (0.73 [0.69–0.73]) than those of the non-PPD group (0.69 [0.64–0.77]) (p = 0.003) . Additionally, there were no significant correlations across groups between MK, FA, or MD values and EPDS scores on TBSS or ROIs.Figure Seven of the eight patients (87.5%) were diagnosed as having mild PPD Table . At one We investigated structural changes in the brain in patients with PPD using DKI. Our major findings were that patients with PPD showed a significant decrease in FA value, a significant increase in MD value, and no significant difference in MK value compared with non-PPD patients. Most interesting, significant differences in MD values were found in a wider area than in areas where FA values were significantly different.47. However, we detected no significant difference in the structure using ROI (data not shown).We found decreased FA in the PPD group within the superior longitudinal fasciculus and the corticospinal tract using TBSS analysis and the thalamus using ROI analysis. A recent study showed no significant differences in FA between the PPD and non-PPD groups using TBSS, while they revealed significantly reduced FA values of the left anterior limb of the internal capsule using ROI29. Recently, the largest multi-site DTI study in MDD showed evidence for global differences in FA and radial diffusivity in multiple WM, including the corpus callosum, internal capsule, corona radiata, cingulum, and fornix, in MDD48. Global lower FA in some WM tracts in MDD was accompanied by global higher radial diffusivity, yet not differences found in MD. Although our results showed significant differences in FA and MD values between the PDD and non-PDD groups, the rate for the area with significant changes in MD voxels relative to the mean FA skeleton voxels (40.8%) was higher than that of FA (6.4%). The increase in MD value may reflect the pathophysiological characteristics of PPD, while decrease in FA value may reflect those of MDD. In this study, most PPD patients had mild PPD; however, if PPD becomes chronic or transitions to MDD, the rate of change in FA may further increase. In order to prove that change in MD value reflects a PPD characteristic, further studies are necessary that compare MD values before, during, and after pregnancy.Many studies of MDD have shown significant differences in FA between MDD and non-MDD patients49. FA is believed to reflect more evident destruction of tissue architecture, such as axonal degeneration and demyelination50, whereas MK is considered to reflect subtle pathological changes including microgliosis, reactive astrogliosis, or decreased myelin, axonal or neuronal density53. Therefore, we hypothesized that an increase in MD value reflects neurogenic vasogenic edema associated with changes in circulating blood volume due to pregnancy and parturition  this is a single-site study and generalizations from the results are not guaranteed, 2) it was difficult to obtain informed consent from patients with unstable mental status, and this might cause selection bias, 3) there is a limit of power depending on the number of patients, and 4) most patients were diagnosed as having mild PPD.20. However, the value was longer that of previous studies on 3 T20, because we used high b-values of 2,500 s/mm2 for the DKI protocol. Hence, we applied four signal averages to maintain sufficient signal-to-noise ratio. Second, the lowest b-values was set to 0 s/mm2. At b-values lower than 200 s/mm2, blood perfusion might affect the measurement signal magnitude, thereby biasing the results57. Third, due to its dense vascular network, the diffusion metrics in the basal ganglia might be somewhat affected by this effect. Although we applied four signal averages to reduce the effects of cardiac pulsations, such as signal loss or signal attenuation, other acquisition techniques, such as cardiac gating, may be needed for further accuracies of the diffusion metrics58. Forth, a set of 20 diffusion gradient directions with four signal averages was chosen. Although accuracies of diffusion metrics are known to depend on the number of directions59, a previous study revealed that DTI data using 6 directions with five or ten averages are comparable to those using 30 or 60 directions with one average60. Therefore, the DTI metrics we obtained are roughly comparable to those that have 80 directions. In addition, MK values we obtained are corresponding to those derived from the optimized scan parameters36. Finally, relatively thick sections, i.e., 4 mm, should be used to maintain an adequate signal-to-noise ratio even on images with high b-values of 2,500 s/mm2. Therefore, the DKI metrics in this study may be include errors due to partial volume effects61, which may deteriorate the accuracy of our results.Another issue is that the choice of acquisition DKI parameters could affect the evaluation of the diffusion weighted data. First, we used a minimum echo time (110 ms), which was comparable to that of previous DTI studies in patients with MDD on 1.5 Tb-values and long acquisition times required by the DKI method, and a future prospective longitudinal study using high-resolution images at 3 T is planned.Our study suggests that PPD strongly affects MD in large sections of the brain, but caused only small changes in FA and MK. To the best of our knowledge, this is a first study that applied recently developed DKI method to PPD. Hence, we need to test the reproducibility of diffusion metrics across different scanners or different field of strengths. In addition, to prove the pathophysiology of PPD further, brain imaging studies that compare MK, FA, and MD values before, during and after pregnancy are needed. Therefore, further studies are needed to clarify whether measuring the MK is valuable on PPD considering the high Although further studies are warranted, our findings exhibit the potency of diffusion MRI for not only early detection but assessment of the PPD recovering process. Furthermore, by elucidating the neurogenic vasogenic edematous status throughout a pregnancy using MRI, a novel therapeutic approach for PPD, such as anti-edema drugs, may result. We hope that the findings herein will contribute to the development of better PPD treatment.Supplementary information