Despite the success of antiretrovirals, human immunodeficiency virus (HIV)coinfections continue to cause mortality. We investigated the prevalence ofcoinfections in women with HIV/acquired immunodeficiency syndrome inSergipe, Brazil. We conducted a cross-sectional study. The coinfections investigated weresyphilis, hepatitis B and C, toxoplasmosis, rubella, tuberculosis, andcytomegalovirus. Among the 435 women, 85 (19.5%) had coinfections. The most prevalent wasHIV/syphilis, followed by tuberculosis, toxoplasmosis, hepatitis C,hepatitis B, and rubella. Additionally, 300 (96.2%) were seropositive forcytomegalovirus immunoglobulin G. Despite significant progress in the treatment for people with HIV,coinfections continued to affect this population. Coinfections with human immunodeficiency virus (HIV) continue to be widely discussedworldwideIn Brazil, although the prevalence of HIV is higher in men than that in women, womencomprise an increasing proportion of the population affected by HIV, and approximately4,000 women die from acquired immunodeficiency syndrome (AIDS)-related illnessesannuallyIn this study, we investigated the prevalence of coinfections in women living withHIV/AIDS attended at a reference outpatient clinic for individuals living with HIV/AIDSin Sergipe State, Northeast Brazil.We conducted a cross-sectional study from August 2014 to November 2017 in women livingwith HIV/AIDS who attended the Reference Centre for Sexually Transmitted Diseases, HIV,and AIDS (CRIST/AIDS), the unique reference care center for individuals with HIV inSergipe State, Northeast Brazil.The sample size was calculated based on the number of HIV-positive women registered atthe CRIST/AIDS in 2014 (800 women). The prevalence of the expected coinfections was 50%,confidence interval (CI) was 95%, and maximum error was 5%, which resulted in 260 women.However, at the end of the study, 435 HIV-positive women were included.Data were collected through individual interviews with a structured form. For the viralload and CD4+ T-lymphocyte count, only the data obtained less than 1 year prior to theinterview were included.Patients diagnosed with HIV infection in the CRIST/AIDS underwent initial complementaryexaminations including tests for syphilis, viral hepatitis, toxoplasmosis, tuberculosis(TB), cytomegalovirus, and rubella. In this study, we opted to analyze thesecoinfections that were screened through routine examinations of the service itself. Weretrieved information from all medical records from HIV diagnosis until the interview’sdate. Additionally, the Brazilian Information System for Notifiable Diseases (SINAN)database of TB was used.Toxoplasma gondii and Rubella virus immunoglobulin M(IgM) and immunoglobulin G (IgG) antibodies were recorded; however, only the IgG testresults were considered for cytomegalovirus because IgM test had been requested lessfrequently in the clinic. Syphilis was screened using treponemal and non-treponemaltests. Positive hepatitis B and C diagnosis was established with the followingserological markers: hepatitis B surface antigen (HBsAg) and hepatitis C antibody.Finally, to identify the occurrence of HIV-TB coinfection, record linkage was performedbetween the database of this study and the SINAN database with TB cases among women inSergipe between 2001 and 2017.The characteristics were analyzed using descriptive statistics. The prevalence of HIVcoinfections was described as a simple proportion. Pearson’s chi-squared test andFisher’s exact test were used to compare the association between coinfection and thetime since HIV diagnosis. The significance level was set at 5%. For the associationbetween predictor factors and the occurrence of coinfections, the prevalence ratio (PR)with a 95% CI was used. The data were analyzed using the Statistical Package for theSocial Sciences version 20.0 .This study was approved by the Research Ethics Committee of the Federal University ofSergipe (CAAE No. 92514618.8.0000.5546) and following the Helsinki Declaration. Allparticipants provided written informed consent. Parents or guardians provided writteninformed consent before enrolling their children in the study.The age of the 435 HIV-seropositive women ranged from 13 to 76 years, with a median ageof 38 years ; 38 (88.2%) women had less than 8 yearsof education, 280 were married (67.6%), and 338 (78.3%) had 1-2 minimum wage. Of the 435women, 329 (75.6%) had been infected sexually and 191 (45.4%) had their first sexualintercourse when they were younger than 15 years old. Most of them were diagnosed withHIV infection more than 5 years (228/52.4%), 309 (77.1%) had a CD4+ T-lymphocyte counthigher than 350 cells/μl, 309 (76.7%) had HIV viral load from zero to 999 copies/mL, and414 (95.6%) reported the use of antiretrovirals .Considering only active toxoplasmosis (IgM); rubella (IgM); hepatitis B, hepatitis C, andsyphilis infections; and TB cases from SINAN-Sergipe, 85 (19.5%) of the 435 had cases ofcoinfections. Eighty (94.1%) of the 85 patients had one type of coinfection, and 5(5.9%) had two or more types.The prevalence rates were as follows: syphilis (38/9.1%), TB (17/3.9%), toxoplasmosis(13/3.8%), hepatitis C (10/2.5%), hepatitis B (9/2.3%), and rubella (5/1.8%).Additionally, we identified the seropositivity for the IgG antibody of cytomegalovirus(300/96.2%), rubella (252/90.0%), and toxoplasmosis (242/71.2%). When associating thetype of coinfection with the time of HIV diagnosis, a statistically significant effectwas observed for TB and hepatitis C coinfections. The proportion of HIV-positive womenwho were coinfected and those who were not coinfected with TB and hepatitis C differedaccording to the time of HIV diagnosis .When comparing the data of the HIV-positive women who had at least one coinfection withthose who had no coinfections, it was observed that women who self-reported to be black, those exposed to HIV through sexual intercourse , those who had their first sexual intercourse when they were youngerthan 15 years old , or those who had sex for money  were more likely to have a coinfection .In Sergipe, one-fifth of the participants had some types of coinfection. In individualsliving with HIV/AIDS, coinfection was an expected phenomenon because HIV infectiondeteriorates the immune system. However, this condition can be minimized with the timelyand immediate use of ART. Studies have shown a decrease in opportunistic infections anda reduction in mortality in HIV-infected individuals after the advent of ARTBased on our findings, some interpretations can be offered. A possible explanation forthe high prevalence of syphilis in women living with HIV in this study is due to thenumber of syphilis cases in the Brazilian population that have increased in recentyears. Additionally, both HIV and syphilis diseases share the same risk factors,suggesting a higher likelihood of coinfection occurring, emphasizing the importance oftracking this coinfection in this subpopulation.It is well established that risky behaviors such as needle sharing, abuse of psychoactivesubstances , multiple partners, prostitution, and poor adherence tocondom use increase the chances of acquiring sexually transmitted infections. In thisstudy, more than half of the women reported not using condoms during all sexualencounters. Studies show that not using condoms is mainly due to the belief that it isunnecessary to use condoms among heterosexual HIV-seroconcordant couples and due togender inequality as often the women are in long-term and oppressive relationships,where negotiating condom use with the partner can be difficultDespite not being the most prevalent coinfection in this study, TB remains an importanthealth problem. A national population-based studyIn Sergipe, the relatively high prevalence of TB in women living with HIV may be theresult of the adherence to Brazilian public policies on HIV and TB control, whichinclude timely use of ART, intensive screening for latent TB infection, and earlydiagnosis and immediate treatment of TB with chemoprophylaxis, preventing thedevelopment of active TB infection. In fact, Sergipe is considered as one of theBrazilian states with the highest rates of ART use in coinfected individuals during thetreatment of TB,The effect of ARV on the survival of individuals living with HIV also confirmed thedevelopment of chronic hepatitis caused by viruses B and C in this population,specifically in developing regions and in regions where there is a high endemicity ofHIV infection. In African areas, studies show that the overall prevalence of HBsAg inadult women can range from 7% to 14%,Other findings worth noting were the high frequencies of seropositivity forcytomegalovirus, rubella, and toxoplasmosis IgG antibodies. This is very relevant,specifically for cytomegalovirus and toxoplasmosis because there is a risk of latentreactivation for these infectionsThere have been few published studies regarding active rubella-HIV coinfection; however,several studies have analyzed the seroprevalence of the IgG antibody to evaluate theresponse to the rubella vaccine in individuals living with HIV, which showed similar andconsistent results with our study for rubella IgG positivity. One of them identifiedthat 89.3% were seropositive for rubella IgG and 10.69% were susceptible tocoinfectionIt is clear that it is not only important to understand the epidemiologicalcharacteristics of HIV-positive women but also be aware of the proportion ofcoinfections in this population group. However, this study has some limitations.Although we conducted face-to-face interviews, clinical information was completed frommedical records and surveillance databases. Second, it was difficult to find the recordsof all the test results for coinfections. Conversely, this missing information occurredrandomly; hence, the principal consequence was loss of power. In this sense, it wasassumed that these tests may not have been performed or their results were not includedin the medical records, which may characterize failures in the coinfection screeningprotocol. Third, this study may not have been able to determine the true magnitude ofthe prevalence of these coinfections in the entire female population living with HIV inthe Sergipe State because most of the women evaluated were routinely followed up andreported to be using ART. However, the final sample corresponded to more than half ofthe women enrolled in the service and was almost double the calculated sample size.Despite these limitations, this study determined the prevalence rates and importantcharacteristics that can guide the care provided and the planning of activities forwomen living with HIV.In conclusion, in Sergipe, one-fifth of the women living with HIV had some types ofcoinfection, with prevalence ranging from 1.8 to 3.9, except for syphilis, which had thehighest rate of 9.1. Additionally, high seroprevalence for IgG antibody forcytomegalovirus, rubella, and toxoplasmosis was identified. Our results providepotential strategies to improve control programs for HIV by targeting interventions topopulation, with strengthening of public policies for the prevention, control,diagnosis, and treatment of coinfections in HIV-positive women with differentcharacteristics.


Mycobacterium tuberculosis complex (MTBC), the biosynthetic pathway of the phenolphthiocerol moiety of PGL depends upon the expression of several genes encoding type I polyketide synthases (PKS), namely ppsA-E and pks15/1 which constitute the PDIM + PGL locus, and that are highly conserved in PDIM/PGL-producing strains. Consensus has not been achieved regarding the genetic organization of pks15/1 locus and knowledge is lacking on its transcriptional signature. Here we explore publicly available datasets of transcriptome data (RNA-seq) from more than 100 MTBC experiments in 40 growth conditions to outline the transcriptional structure and signature of pks15/1, using a differential expression approach to infer the regulatory patterns involving these and related genes. We show that pks1 expression is highly correlated with fadD22, Rv2949c, lppX, fadD29 and, also, pks6 and pks12, with the first three putatively integrating into a polycistronic structure. We evidence dynamic transcriptional heterogeneity within the genes involved in phenolphtiocerol and phenolic glycolipid production, most exhibiting up-regulation upon acidic pH and antibiotic exposure and down-regulation under hypoxia, dormancy, and low/high iron concentration. We finally propose a model based on transcriptome data in which σD positively regulates pks1, pks15 and fadD22, while σB and σE factors exert negative regulation at an upper level.One of the most important and exclusive characteristics of mycobacteria is their cell wall. Amongst its constituent components are two related families of glycosylated lipids, diphthioceranates and phthiocerol dimycocerosate (PDIM) and its variant phenolic glycolipids (PGL). PGL have been associated with cell wall impermeability, phagocytosis, defence against nitrosative and oxidative stress and, intriguingly, biofilm formation. In bacteria from the  Mycobacterium tuberculosis (Mtb) that remains a major public health concern. In 2016, approximately 6.3 million new cases of TB were reported , by read counting and through the calculation of RPKM as a proxy for gene expression in each condition. RPKM is a relative value, meaning that it varies according not only to read count, but also to the total number of reads obtained for each experiment. In order to enable comparison across experiments, those values were normalized in relation to sigA housekeeping gene expression.To characterize the transcriptional signature of Mtb strains, that we herein use as our main source of data, it is known that the Mtb H37Rv reference strain, as well as Mtb CDC1551, which are core to our dataset, both present a frameshift mutation in pks15/1, in comparison to Mb BCG Tokyo which retains an intact CDS and is capable of PGL production. To understand the similarity across profiles, a dendrogram was generated, using 80% similarity as a cut-off for cluster formation; this cut-off was established to include the top quarter of similarity values in the analysis. From this, we obtained a total of 53 clusters, 39 being single member clusters. Cluster I included some of our genes of interest (fadD22 and fadD29). Cluster II includes pks1 and pks6, which is a pks1 paralog, and pks12. , fadD22 (0.811), fadD29 (0.757) and also with pks6 (0.807) and pks12 (0.803)  .sigB is correlated with sigE (0.876) and sigH (0.833); sigE with sigH (0.783); sigG with sigJ (0.782); and sigJ with sigK (0.712)  . Both anray data . The exp network . In contmilarity , thus agsigA factor is known to regulate sigG, mostly induced during macrophage infection, which will thus regulate sigJ, known to be overexpressed in late stationary phase of dormant cultures. sigG and sigJ will further regulate sigL, known to be involved in PDIM biosynthesis, that in turn regulates sigK, whose precise functioning remains unclear  when compared to the control. The differential expression of genes reported to play a regulatory role under starvation conditions in Mtb, such as Mb3614c , Mb2076 , relA , prrA , senX3  and regX3 , was also evaluated, whereby after 20 days of starvation up-regulation of these genes was noted . In this integrative analysis, it was also possible to link genes encoding σ factors with the selected genes of interest; e.g. we found a correlation coefficient of 0.8 between sigK and lppX. While these results must be treated with caution, since σ gene expression may not reflect effective factor activity, they offer new insights into the potential function of sigma factors whose functional role remains unclear, such as SigK.By clustering the expression data from more than 100 RNA-seq datasets for lppX, pks1, pks15, fadD22, Rv2949c and fadD29, it was possible to define under which conditions these genes are positively or negatively regulated. We analysed expression levels of strains grown under a diverse set of conditions, namely pH, carbon source, hypoxia and phosphate depletion for Mtb CDC1551, growth phase, exposure to limiting or excessive iron concentration, hypoxia, dormancy, and antibiotic exposure for Mtb H37Rv. This analysis revealed that our selected genes of interest are up-regulated at acidic pH (in Mtb CDC1551) and antibiotic exposure and down-regulated at stationary phase (in Mtb CDC1551), under hypoxia and dormancy, and at both low and high iron concentrations. The combination of two sets of data, i.e. clustering of genes by expression data and differential expression analysis, suggests that fadD29 may be set apart from the other genes in the set. Also, in one of the conditions, fadD29 expression seemed to diverge from that of lppX, pks1, pks15 and Rv2949c, and in another, both Rv2949c and fadD29 expression profiles diverge from pks1 and pks15. Using differential expression analysis, we were also able to confirm that sigK shares the expression profile with the selected genes of interest in 88% of the exploited conditions with significant fold-changes; almost the same percentage is also verified for sigJ. On the contrary, sigE presents approximately 90% of expression profile dissimilarity with the selected panel of genes in the conditions under analysis, with significant fold-changes, as well as sigB.As referred to previously, mycobacteria are subjected to several stress conditions while inside macrophages. By analysing the differential expression of Mtb we were able to gather a robust set of data from previous studies, for M. bovis BCG it was only possible to collect data from seven growth conditions, of which three represent regular in vitro growth, three represent growth under starvation at three time-points, and one represents the addition of vitamin B. This smaller data set led to a lower number of clusters. Interestingly, the members of the putative polycistronic structure, except for lppX, cluster in the same group. This analysis is in full agreement with the one performed for Mtb as most genes of interest are contained in the same cluster. Also, expression values of pks1 appear to be similar in control assays performed in Mb and in Mtb H37Rv for which relative gene expression has a correlation of 0.78, indicating that although PGL production may be abolished in Mtb H37Rv, the pks1 transcript is similarly expressed in both Mtb H37Rv and BCG, suggesting a secondary role for this transcript.While for pks1 and pks15. In this model, we use a conservative approach selecting genes coherently sharing expression patterns and exhibiting functional similarity when considering a polycistronic structure model. For this model, we selected a set of four genes, pks1, pks15, fadD22 and Rv2949c, that fulfil the criteria stated above. Based on differential expression analysis, we also selected a set of three σ factors  that seem to be involved in the regulation of pks1, pks15 and fadD22 expression  that genes encoding for proteins involved in cell wall processes are highly upregulated in complementation mutants, which is in contrast with our data analyses [ΔsigD mutant showed reduced expression of genes involved in the synthesis of phospholipids and fatty acids [K is a regulator of this set of genes, previous analyses of an MtbΔsigK mutant reported no differential expression of our genes of interest [Building on the information from previously published reports and the transcriptome data we retrieved, compiled and analysed here, we propose a regulatory model for pression . Both σKn levels , which i hypoxia . While σanalyses . On the ty acids . Even thinterest , leadingpks1 and pks15 knock-out mutants, and transcriptional fusions with reporter genes of the upstream regions of the constituent genes in this putative polycistronic unit in order to unveil the exact location and activity of the promoter. Analysis of pks1 and pks15 expression in mutant strains of their putative regulators under several growth conditions would further serve to validate the global networks that exert effects on pks1 and pks15 activities, genes that play a crucial role in PGL production and thus act at the interface of host-pathogen interaction.Further experimental validation of our findings and proposed regulatory model could be achieved via ‘classical’ experiments such as Northern blots, construction of S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Background: Implant dentistry has evolved over time, resulting in better treatment outcomes for both patients and clinicians. The aim of this trial was to test whether the immediate loading of implants with a platform-switching design influences the marginal bone level, compared to four-week loading, after one year of follow-up. Moreover, a comparison of clinical data regarding implant survival, implant stability, and patient-reported outcome measures (PROMs) was conducted. Methods: Klockner® VEGA® implants with a ContacTi® surface were placed in partially edentulous patients in the posterior areas. Group A received an immediately loaded prosthesis (one week) and Group B received an early-loaded prosthesis (four weeks). All abutments were placed at the time of surgery. Radiographic and clinical data were recorded. Results: Twenty-one patients were treated (35 implants). No implants were lost during the study. The final marginal bone level did not show differences between groups. The bone loss at 12 months at the implant level was 0.00 mm for both groups (median). The final implant quotient stability (ISQ) values did not differ between groups (median 73 and 70.25), nor did the other clinical parameters or PROMs. Conclusions: The results suggest that neither of the loading protocols with the implants used influenced the marginal bone level—not the osseointegration rate, clinical conditions, or PROMs. Implant dentistry has evolved in recent decades, resulting in better treatment outcomes for both patients and clinicians. This improvement has been possible through progressive changes in each and every aspect of therapy: implant design, implant surface, prosthetic connections, abutments, materials, surgical and loading protocols, and so on . In thisConsidering the factors involved in implant osseointegration, it is generally accepted that the implant surface plays a major role regarding the bone healing process around dental implants. In fact, from the early days of osseointegration, the development of new surfaces has been a main issue in terms of promoting a greater rate of osseointegration, a faster bone apposition, and greater bone–implant contact ,10. In t® . As already described by Aparicio et al. [Furthermore, in vivo studies have observed de novo bone formation and increased bone–implant contact in the early stages of bone healing, in comparison to non-treated surfaces. Based on these investigations, a highly hydrophilic and osteoconductive surface was developed ,27 and ho et al. , once tho et al. .® VEGA® ContacTi® (Vega-CTi) , featuring: (a) a 10° Morse taper internal conical connection, (b) an internal hexagon for index and anti-rotation purposes, (c) a platform-switching design, and (d) enhanced titanium, i.e., grade 4 titanium cold worked at high tension. The implant surface is the already mentioned and previously described ContacTi®.Regarding implant morphology, years ago, the platform-switching design was introduced, meaning that the diameter of the abutment placed onto an implant is narrower than the inner wall of the implant itself . The useConsidering the previous statements, a clinical protocol was developed to investigate the clinical performance of Vega-CTi implants. Therefore, the aim of this randomized clinical trial was, primarily, to test the null hypothesis that the immediate loading of Vega-CTi implants (less than one week) would not influence the marginal bone level, compared to four-week loading, under the concept of OAOT, one year after treatment completion. Additionally, this study obtained and compared clinical data regarding implant survival, implant stability values, and the patient-reported outcome measures (PROMs) under these loading conditions.The present study was originally designed as a prospective, multi-center, single-blinded, controlled clinical trial. This article presents the one-year results of the trial.-Undisturbed, completely healed edentulous sites, at least four months after tooth/teeth extraction;-Adequate bone volume verified by CBCT (cone beam computed tomography) for the placement of a dental implant of at least 8 mm in length and 3.5 mm in diameter; this means at least 9 mm of bone in the vertical dimension and 6.5 mm in the horizontal dimension;-No bone regeneration procedures required;-Adequate prosthetic conditions for proper crown or bridge installation;-Natural antagonist dentition, whether restored or untreated teeth;-Presence of at least 4 mm of keratinized mucosa from the buccal to lingual side of the edentulous ridge;-Absence of oral infections, gingival inflammation, untreated periodontitis, or mucosal diseases.Adult patients, at least 18 years old, missing at least one posterior tooth (molar or premolar) in the mandible or maxilla and willing to receive implant therapy were eligible for screening. In addition, the following inclusion criteria were required:-Uncontrolled systemic diseases;-Pregnancy;-Bone-related disorders such as osteoporosis;-Drug abuse or alcoholism;-Smoking habit of >10 cigarettes per day;-Severe bruxism or severe temporomandibular junction disorders;-Previous history of implant failure.The general exclusion criteria included:In order to enter the study, patients had to be able to understand the nature of the study, to sign an informed written consent form, and to be willing to attend the scheduled follow-up appointments. This study was conducted in accordance with the Declaration of Helsinki (1964) and its amendments for patients participating in clinical studies . The study protocol was reviewed and approved by the ethics committee for clinical studies of the Universitat Internacional de Catalunya with reference number 500077.Three centers in Spain  participated in the study. Three experienced dentists, one at each center, all of them familiarized with the implant system, were responsible for the surgical and prosthetic procedures . Before the study started, the clinicians participated in a calibration meeting to receive all the information  about the study and the assessment of the variables.® Abutment, Klockner Implant System, SOADCO S.L., Andorra). No cemented restorations were used in this study.Vega-CTi implants, as already described, of 3.5, 4.0, and 4.5 mm in diameter and 8, 10, and 12 mm in length were available for the surgeries. The dimensions of the platform-switching for each implant diameter were 0.3, 0.35, and 0.6 mm, respectively. All of the restorations were installed onto abutments of 1 or 2 mm in transmucosal height, designed for screw-retained prostheses , and full-mouth bleeding scores (FMBS). The periodontal chart was completed recording the clinical attachment level (CAL) and probing depth (PD) with a conventional periodontal probe (PCP UNC 15-Hu-Friedy). Periodontal status was assessed according to the classification proposed by Armitage 1999 .®, Dentaid, Spain); later, a 2.0% solution of chlorhexidine  was applied extra-orally to the peri-oral skin. The surgeries were performed under local anesthesia with 40 mg/mL of articaine + 0.01 mg/mL of epinephrine . Then, the full-thickness flaps were raised by means of crestal and intrasulcular incisions, paying attention to preserving at least 2 mm of keratinized mucosa on the buccal and lingual or palatal aspects of the alveolar crest. The crestal bone width was measured buccolingually with a periodontal probe . The bone site preparation was performed according to the manufacturer’s drilling sequence suggested for Vega-CTi implants; however, each clinician was free to adapt the final drilling according to the bone quality in order to ensure optimal primary stability. The implants were inserted using a calibrated torque wrench , taking care to place the implant shoulder at least 1 mm below the crestal level at the most declining part of the bone preparation. Each clinician could decide the optimal diameter and length of the implants, considering the preoperative diagnostic procedures and the surgical evaluation of the bone volume available at the site. Bone quality was registered according to the D1-, D2-, D3-, and D4-type classifications [(A)Manually, by means of a calibrated torsion torque wrench  at the time of placing the implant.(B)Manually, after the use of the torque wrench, according to the following criteria at the time of the abutment connection: Type A: IT > 35 N/cm, and the implant could not be rotated manually; type B: IT < 35 N/cm and the implant could not be rotated manually; type C: IT < 35 N/cm and the implant could be rotated manually; type D: the implant could be rotated manually and moved in a vertical direction. In this case , the patient was excluded from the study, and a delayed loading protocol was carried out.(C)® device and Osstell® transducers (Smart-Pegs®)  in order to obtain of the implant stability quotient (ISQ) values. The transducers were connected to the abutments, screwed at 5 N/cm.By radiofrequency, using the OsstellThe patients received prophylactic antibiotic therapy 1 h prior to surgery . The patients were told to mouthwash for 1 min with a 0.12% solution of chlorhexidine . The selection of the height of the abutment (1 or 2 mm) was subject to the criteria of the clinician at the time of surgery. The ISQ values for the implants in the study were recorded, both buccolingually and mesiodistally. Abutment-level impressions were taken the same day of the surgery, and screw-retained acrylic provisional restorations were prepared for all implants. Finally, healing caps were placed onto the abutments and the surgical flaps were sutured with 5/0 polyamide avoiding any tension. Antibiotic treatment was maintained for seven days (500 mg of amoxicillin or 300 mg of clindamycin for 1/8 h). Non-steroidal anti-inflammatory dexketoprofen (25 mg) was administrated every 8 h if needed, as was a gastric protector (20 mg of pantoprazole) once per day for seven days. At this point, a randomization process was performed in order to allocate the patients to Group A, in which the implants were loaded with provisional restorations within the first week after surgery, or Group B, in which the implants were loaded four weeks after surgery. The randomization was done by the toss of a coin, by an assistant who was not involved in the investigation.Provisional prostheses up to three units and with no cantilever or implant–tooth junctions were inserted and tightened at 20 N/cm within the first week after implant placement in patients of Group A and after four weeks in patients of Group B. Eight weeks after implant placement, the provisional restorations were unscrewed, and definitive impressions were taken. All definitive prostheses  were delivered 10–12 weeks later; the abutments were re-tightened at 30 N/cm and restoration screws at 25 N/cm with a dynamometric wrench. The occlusal screw access hole was filled with a composite material and occlusal adjustment and polishing were carried out if needed. All of the restorations, whether provisional or final, were in occlusion with the opposite dentition in the maximum intercuspation position. If there were any complications with the prostheses, they were recorded .®) in order to obtain a consistent image projection. An independent blinded technician performed the radiographic analysis. A previous calibration procedure was carried out as follows: The same examiner measured a subset of 10 radiographs on three separate occasions, 72 h apart, to determine the intra-examiner reproducibility. An interclass coefficient of 0.99 (p < 0.05) was obtained. The radiographic peri-implant bone level changes in the X-rays were analyzed using software for image analysis . All measurements were performed using 7X magnification.The primary outcome measure was the peri-implant marginal bone level changes. For this purpose, standardized periapical radiographs were taken at five time points: immediately after implant placement (baseline), after the temporary restoration installation (at one or four weeks), after definitive restoration installation (at 10–12 weeks), and at 6 and 12 months. Baseline radiographs were taken with the definitive abutment and the corresponding healing cap screwed. Radiographs for assessing the impression coping fit and the prosthetic components were also performed, but only for clinical purposes. Once the final crowns were in place, a customized standard holder was prepared for each implant, recording the occlusal surfaces of the adjacent and opposing teeth with acrylic resin on a parallel X-ray holder (Dentsply Rinn XCP-DSThe secondary outcome measures were (a) the implant survival at 4, 8, and 12 weeks and 6 and 12 months, by just clinical examination of the presence, stability, and function of the implant/prosthesis; (b) the implant stability, by means of a comparison of the ISQ values at the corresponding follow-up visits; (c) the PROMs. Finally, three clinical variables were recorded: the modified plaque index (mPLI), the modified sulcus bleeding index (mSBI), and PD, at the mesial, distal, vestibular, and palatal/lingual aspects of the implants at 4 weeks and 3, 6, and 12 months.The post-loading status of the provisional and definitive restorations was assessed at each follow-up visit. The following complications were registered in the case of occurrence: mobility of the prostheses, mobility of the abutments, abutment screw loosening, the presence of porcelain chippings, the fracture of the frameworks, and the fracture of the abutments.Adverse events and complications related to the implant surgical treatment (“implant-related adverse events”) such as acute or chronic pain, sensorial alterations, bone fractures, osteomyelitis, loss of osseointegration of the implant, and discomfort and local or systemic infections were recorded. “Non-implant-related adverse events” such as serious illnesses or any condition requiring hospitalization for more than one day were also recorded.The PROMs were assessed by means of a questionnaire regarding comfort, esthetics of the restorations, chewing ability, and overall satisfaction. A four-item rating scale was used to assess the degree of satisfaction: excellent, good, fair, and poor.®, Cork, Ireland) using data from previous studies with a similar design [Sample size was previously calculated with N Query Advisor 4.0 software .Chi-square tests for comparing the categorical variables between two groups were applied. As the variables appeared to be parametric up to the three-month visit, Student’s Since asymmetry or bias of the data was detected, instead of using the mean and standard deviation, the variables with medians and 25th and 75th percentiles were summarized and the non-parametric Mann–Whitney test was used. The follow-up of the repeated measures was carried out with the non-parametric Friedman test in each of the study groups.Twenty-one patients were recruited in this randomized clinical trial from October 2015 to July 2016. A total of 35 implants were included in the study. No implants were lost, and no implant or non-implant related adverse events were observed throughout the study. Two patients (two implants) were not subjected to X-ray exposure at the six-month visit as they were pregnant at that time. Another two patients (two implants) did not attend the one-year visit. p = 0.205 and p = 0.382 for Groups A and B, respectively). Regarding the smoking habit, five patients (20.8%) were smokers and 19 were not (79.2%). From a periodontal point of view, four patients were healthy (16.9%), and 17 were previously treated: five for gingivitis (20.8%), eight for mild chronic periodontitis (33.3%), six for moderate chronic periodontitis (25%), and one for severe chronic periodontitis (4.2%). Again, no statistically significant differences between Groups A and B were observed for any of the parameters at baseline .Twelve of the 21 patients (54.2%) were men and nine (45.8%) were women, with a mean age of 52.4 years (SD: 14.6 years). No statistically significant differences between groups were observed for these variables at baseline (Nine patients (42.8%) and 17 implants (48.6%) were assigned to Group A and 12 patients (57.2%) and 18 implants (51.4%) to Group B (early loading). Fourteen implants (40%) were placed in the maxilla and 21 implants (60%) in the mandible. The locations of the implants by tooth site (FDI nomenclature) are displayed in Twenty-four implants (68.6%) were restored with single crowns, and 11 implants (31.4%) were part of partial fixed prostheses. Additionally, 52.9% and 47.1% of the implants were restored with single crowns and partial fixed prostheses in Group A, respectively, whereas in Group B, the distribution was 83.3% and 16.7%, respectively. Seven of the abutments placed were of 1 mm in height (five in Group A and two in Group B) and 28 were of 2 mm in height (12 in Group A and 16 in Group B).p > 0.05).Regarding the bone quality, 20 implants (57.1%) were inserted into D2 bone  and 15 (49.2%) in D3 bone . The final IT at the implant placement is shown in Regarding adverse events, two patients (two implants) of Group B reported minor surgical postoperative acute pain and inflammation that resolved evenly with prescribed medication.Two patients (two implants) of Group A experienced abutment loosening with provisional restoration at four weeks, and three patients (three implants) of Group B exhibited loosening of the final prosthesis at the three-month visit. The overall survival rate observed at 3, 6, and 12 months was 100% in both groups.p > 0.05) without differences between groups (p > 0.05), nor in the mSBI. An increase in PD (p = 0.142), with no differences between groups (p = 0.716), was observed throughout the study. However, these changes were not relevant from a clinical point of view.p < 0.05). Analyzing the ISQ values according to the moment of measurement, there were statistically significant differences at four weeks, as well as at 6 and 12 months.p = 0.12). Analyzing the MBLc according to the moment when it was measured, there were statistically significant differences between all surgeries and the rest of the moments of registration . There were no statistically significant differences (p = 0.00) between the MBLc at all follow-up visits.Marginal bone level at the crest (MBLc): There were no statistically significant differences between groups (p = 0.195). Analyzing the MBLi according to the moment when it was measured, there were statistically significant differences only between the moment of surgery and 12 weeks. There were no statistically significant differences (p = 0.00) between the MBLi at any other moment.Marginal bone level at the implant (MBLi): There were no statistically significant differences between groups (p = 0.042). Analyzing the MBLa according to the moment when it was measured, there were statistically significant differences (p = 0.024) between the moment of surgery and all of the follow-up visits. Bone contact at the abutment was observed in 51.35% and 61.7% of the implants at 4 and 12 weeks, respectively. These values remained constant after 6 and 12 months with a rate of 63.6% and 67.6%. At the end of the observation period (one year), 66.6% of the implants in Group A and 68.75% in Group B showed bone contact at the abutment. None of these differences were statistically significant.Marginal bone level at the abutment (MBLa): There were statistically significant differences between groups . Whether the subcrestal position of an implant can influence the rate of marginal bone loss is still a matter of discussion ,52,53. R® implants in another clinical trial; however, a higher bone resorption at the crest for subcrestal implants was showed in a radiological CBCT evaluation [Within the limitations of the radiographic evaluation method used in the present study, bone contact at the abutment was observed in 68.75% of the implants at one year. In Group A, an increase in the bone–abutment contact during the study was shown (from 52.9% at one month to 68.75% at one year). The same trend was observed in the test group, from 58.8% at one month to 66.6% at one year) (data not shown). Whether these findings are related to the different positions of the implant shoulders (MBLc) at baseline between the two groups (0.87 ± 0.59 mm in Group A and 1.25 ± 0.39 mm in Group B) or with the different loading protocols (immediate versus early), or both, is something that cannot be answered under this protocol. In this way, Koutozis et al. and Degidi et al. demonstrated a direct correlation between the depth of the implant shoulder and the bone–abutment contact with platform-switched implants ,57. HoweIn our research, the implant level at the time of surgery was at least 1 mm below the bone margin. As seen in the The available literature suggests that primary stability is a relevant factor that may influence the osseointegration of immediately loaded implants ,61,62,63® surface, finding a progressive increase in the ISQ values from surgery up to five years [In order to avoid unscrewing the abutments, and with respect to the “one-abutment–one time” concept, the ISQ value was registered at the abutment level, discarding the ISQ at the implant level. A common finding when assessing implant stability in clinical trials is the initial drop in ISQ values within the first two to four weeks of healing after implant placement ,70,71, wve years . In a sip = 0.002) and D2-type bone (p = 0.003) was observed in the study. These results are in agreement with an investigation where higher ISQ values of implants were found at the time of surgery and at two, four, and eight weeks in areas with bone type D2 compared to bone types D3 and D4 [Across the study period, implant stability in the immediately loaded group was higher compared to the early-loaded group. However, this difference could be associated with a higher proportion of implants placed in the mandible and the D2 bone quality of Group A. Indeed, a positive association between higher ISQ values and mandibular bone . This finding has not been reported in other similar trials [Another interesting finding of the present research was the negative association between ISQ values at the time of surgery and the insertion torque. Lower values of insertion torque were not associated with lower ISQ values. In this way, the correlation between insertion torque and resonance frequency analysis measurement is still controversial in the literature ,79,80,81r trials . The relA limitation of this study was the use of standardized intraoral radiographs to assess marginal bone level, even though this is a well-validated method in the literature , since bAdditionally, the preliminary results of this randomized clinical trial should be interpreted with caution due to the limited sample size and follow-up.Within the limitations of this study, and in relation to the primary objective of this research, it could be concluded that immediate (one week) or early (four weeks) loading of implants a with thermo-chemically treated surface, under the concept of “one abutment–one time,” does not influence the marginal bone level at one year. Regarding the secondary objectives, neither treatment modalities influenced the peri-implant condition, the implant stability over time, or the PROMs. This study suggests that both loading protocols with the mentioned implants and conditions could be considered as treatment options for restoring the posterior areas of the maxilla and mandible. However, these results have to be confirmed by further studies with a larger sample and longer follow-up periods.


The brain tissues were dissected and subjected to biochemical assays to measure the relative expression of stress biomarkers and neurotransmitters to elucidate the underlying mechanisms for behavioral alterations. The results of the behavioral tests showed that chronic exposure to lead could elevate the stress and anxiety levels characterized by elevated freezing and reduced exploratory behaviors. The chronic exposure to PbCl2 at a low concentration also induced a sharp reduction of aggressiveness and short-term memory. However, no significant change was found in predator avoidance, social interaction, shoaling, or color preference. The biochemical assays showed elevated cortisol and reduced serotonin and melatonin levels in the brain, thus, altering the behavior of the PbCl2-exposed zebrafish. In general, this study determined the potential ecotoxicity of long-term lead exposure in adult zebrafish through multiple behavioral assessments. The significant findings were that even at a low concentration, long-term exposure to lead could impair the memory and cause a decrease in the aggressiveness and exploratory activities of zebrafish, which may reduce their survival fitness.Lead and lead-derived compounds have been extensively utilized in industry, and their chronic toxicity towards aquatic animals has not been thoroughly addressed at a behavioral level. In this study, we assessed the risk of exposure to lead at a waterborne environmental concentration in adult zebrafish by behavioral and biochemical analyses. Nine tests, including three-dimension (3D) locomotion, novel tank exploration, mirror biting, predator avoidance, social interaction, shoaling, circadian rhythm locomotor activity, color preference, and a short-term memory test, were performed to assess the behavior of adult zebrafish after the exposure to 50 ppb PbCl Currently, industries are using lead and lead-derived compounds extensively due to their unique chemical properties and economic values. The application of lead has rapidly grown from five million tons per annum in 1970 to approximately 11 million tons in 2016. However, lead can directly enter aquatic environments from urban sources, including households and waste management and sewage treatment plants. Several studies have reported that lead can cause adverse effects even at a low concentration ,4.More evidence has shown the detrimental impacts of lead on human health, especially at a neurobehavioral level. The neurotoxicity of chronic exposure to low concentrations of lead can impair cognitive performance in childhood through to adulthood ,6,7,8,9.Many studies have also been done in rodent and primate models to assess the effects of early lead exposure on tasks of executive performance, such as spatial working memory, response inhibition, cognitive flexibility, and temporal information processing, which are similar to children with ADHD . An incrAlthough mammalian models have been successfully established to explore the neuronal toxicity of heavy metals, several disadvantages of using rodents as animal models have diverted the attention of the scientific community to other animal models in recent years. Due to the practice of the 3R  principle regarding animal welfare, the number of rodents used in each experiment must be reduced. However, due to higher individual variation on behavioral performance, a high number of animals should be used to reach statistical significance in behavioral tests. In addition, due to the large space requirement and relatively expensive instrumental setting, the assessment of ecotoxicity by multiple behavioral endpoints using mice has not yet been widely explored. To overcome these limitations, zebrafish have been proposed to serve as an alternative lower vertebrate model for ecotoxicity research.Danio rerio) has several advantages over rodent models, such as easy maintenance, prolific breeding, rapid development, and external development, making them a practical and robust animal model for human diseases and developmental and toxicological studies [nrxn2a  expression, increased apoptosis, and altered locomotor behavior in zebrafish embryos and larvae [The use of zebrafish  locomotion, novel tank exploration, mirror biting, predator avoidance, conspecific social interaction, shoaling, circadian rhythm locomotor activity, color preference, and short-term memory  locomotion is a sensitive tool for the risk assessment of the alteration of swimming behavior after exposure to chemicals or pollutants . It was ge speed B and avege speed C. The tool group D. The cont ratio G–I. Base2 displayed reduced locomotor activity  as a predator fish to induce the fear response of the zebrafish. In avoiding the predator, any unusual movements of the PbCl2-treated fish compared to the control fish could be conceived as an impact of the treatment. We found that there were no significant differences in behavioral endpoints of the PbCl2-treated fish and the control group  12/12 h setting, the PbClk cycles A. In thek cycles B,G. Irrek cycles D. Meanwhk cycles C,E,F. Fume ratio H,I,L,M. ed group K. On theed group J. Overal2 on short-term memory. A shuttle box is one of the apparatuses that provides stimulus condition. It has been applied to determine memory loss induced by metal poisoning. In this study, we used the passive avoidance test in a shuttle box to assess whether exposure with 50 ppb PbCl2 could impair short-term memory in adult zebrafish. The results showed that the control fish could maintain short-term memory with a latency time around 150–200 s during the testing phase , superoxide dismutase (SOD), and malondialdehyde (MDA) markers. Interestingly, we found that PbClD levels B. In addD levels D in fishperiment A,C.2-exposed fish  was purchased from Shanghai Huayi Company . A PbCl2 stock solution at a concentration of 1000 mg/L (ppm) was prepared in ultrapure water and was stored at 25 °C. Adult fish were exposed to a nominal concentration of 0 (control group), and concentrations of 50 ppb, 100 ppb, and 1000 ppb PbCl2 for 24 h, 96 h (acute treatment), 7 days, 10 days, 14 days (sub-chronic treatment), and 30 days (chronic treatment). The water was changed every two days to maintain the lead concentration during incubation.Lead chloride (PbCl2 exposure), aggression (for acute and sub-chronic effects), predator escape/avoidance, social interaction, shoaling, circadian rhythm locomotor activity, and color preference tests (on day 30 of exposure), which are described below.Eight behavioral assessments were used to evaluate the neurobehavioral disorders associated with the exposure to low concentrations of lead. The behavioral tests included 3D locomotor activity (for acute and sub-chronic effects), passive avoidance  for one minute every 5 min until 30 min passed . Later, Aggressive behavior was determined in adult zebrafish based on the mirror test described in previous studies . The samAmatitlania nigrofasciata) was placed on one side and the zebrafish were put on the other side. After introduction into the tank, the predator avoidance behaviors, indicated by average speed, predator approaching time percentage, average distance to the separator, freezing, swimming, and rapid time movement ratios, were recorded for a period of five minutes.The predator avoidance test is considered as an indicator of predator avoidance behavior in zebrafish. It was carried out based on the previous publication . The samA social interaction test was carried out to assess the zebrafishes’ ability to interact with their conspecific. Similar to the predator avoidance test, a glass separator was used in the same size tank as described in the previous publication . After aAs an innate behavior, shoaling is one of the social-related behaviors to reduce anxiety and the risk to be captured by predators. Based on the previous publication, a shoaling test was conducted to observe the shoaling formation ability of zebrafish . After aTo evaluate lead-induced disorders in zebrafish locomotor activity during the day and night, we performed a circadian rhythm test based on a previously published procedure . To prev2 could impair short-term memory in adult zebrafish, we carried out a passive avoidance test after 4 days (acute treatment), 14 days (sub-chronic treatment) and 30 days (chronic treatment) of exposure (n = 14), referring to previous research with minor modifications [To assess whether PbClications ,62,63. TColor-based experiments are known to be associated with aversion, anxiety, or fear in the zebrafish . The colv/w) of PBS at pH 7.2 using a bullet blender tissue homogenizer . Then, the samples were incubated on ice for 30 min before centrifugation at 12,000× g for 10 min. The crude homogenate was stored in 100 μL aliquots at −80 °C for ELISA. The levels of neurotransmitters—namely, serotonin, dopamine, GABA, and Ach—and AChE were measured by using target-specific ELISA kits purchased from a commercial company . The ROS and SOD levels were also determined . The neuroendocrine marker, melatonin, was determined using commercially available ELISA kits . The target protein content was analyzed according to the manufacturer’s instructions.Five randomly selected zebrafish from the control and treatment group were used for biochemical assays. The whole-brain tissue was homogenized on ice in 50 volumes . Statistical significance was considered to be probability (p) values less than 0.05.All data were presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using the


Grampus griseus) with subsequent rehabilitation attempted. Valuable use of blood samples, and finally necropsy assessments, advances our understanding about the pathology common in live-stranded cetaceans.Free-living cetaceans are threatened, daily, by a wide variety of stressful situations. An example is provided by live-stranding, in which a cetacean is alive on the beach or in shallow water, and unable to free itself and resume its normal activity. This is the first case of capture myopathy and stress cardiomyopathy in a live-stranded juvenile male Risso’s dolphin (Grampus griseus). The animal presented elevated values of creatine kinase, cardiac troponin I and blood urea nitrogen, with some variations during the rehabilitation period. Histologically, we detected vascular changes and acute degenerative lesions analogous to the ones observed in humans with SCMP. We consider this study to be an important contribution to the study of cetaceans since it could help in decision-making and treatment procedures during live-strandings and improve conservation efforts by reducing the mortality of these animals.Capture myopathy (CM) is described in wild animals as a metabolic syndrome resulting from the extreme stress suffered during and after capture, handling, restraint, and transport. Although CM has been characterized in many species of cetaceans, descriptions of cardiac injury—an important component of this syndrome, and, according to previous authors, comparable to the existing human pathology so-called stress cardiomyopathy (SCMP)—are still rare. Therefore, the main aim of this report is to illustrate, for the first time, the biochemical analysis, and gross, histopathological, histochemical and immunohistochemical features of CM, and more specifically of the SCMP involved in this syndrome, caused by the live-stranding and consequent rehabilitation attempt, for a certain period of time, in a juvenile male Risso’s dolphin ( Stress is present in daily life, and all life forms have evolved mechanisms to cope with stressful situations . It is cIn human pathology, despite still under-recognized and often misdiagnosed, an unique reversible cardiac syndrome, triggered by a stressful event, has globally been reported and described as stress cardiomyopathy (SCMP) ,5.The clinical presentation is similar to the acute myocardial infarction. Diagnostic criteria for SCMP has been established by the Heart Failure Association . Regarding the biomarkers, cardiac troponin I (cTnI) and/or creatine kinase (CK) have small, rapid increases to above normal levels ,5,6,7.Histological findings are consistent with ischemia-reperfusion injury, such as vascular changes  and acute necrotic degenerative lesions  ,5,8. Cetaceans are exposed daily to a wide variety of stressful situations  that influence their well-being. Efforts have been made to reduce the impact of these situations, but whales and dolphins continue to be threatened ,13,14,15Odontoceti in which specific cardiac markers have been analyzed. Histopathological changes consisting of ischemia-reperfusion injuries may be observed. These changes result in local-to-generalized vasospasms and vasodilation (as seen in human SCMP), as well as in direct traumatic injury to muscles (rhabdomyolysis), with acute renal failure associated with myoglobinuric nephrosis secondary to muscle damage [The acute and stressful deaths of live-stranded cetaceans might be attributed to the “stress response syndrome” or “alarm reaction” which are thought to be comparable to those described in capture myopathy (CM), in which the cardiac damage, due to the extreme stress, seems to have an important role. According to some authors, cetaceans are especially predisposed to develop SCMP, similarly to the SCMP in humans. It is the most devastating form of acute stress described in several animals that may occur during and after the capture, handling, restraint, and transport ,10,14. Ae damage ,15,20,21Grampus griseus).The aim of the present report is to illustrate, for the first time, the biochemical analysis, gross, histopathological, histochemical and immunohistochemical features of the CM, and more specifically of the SCMP involved in CM, caused by the live-stranding and consequent rehabilitation attempt in a juvenile male Risso’s dolphin (Grampus griseus) which was stranded alive on the coast of Gran Canaria , on April 26, 2019 (notification at 8:30 a.m.). After receiving specialized first aid  at the location, the animal was transferred to the Wildlife Recovery Center (Gran Canaria), where it was monitored 24 h a day. From day 0 (26 April 2019) to day 5 (1 May 12019), several diagnostic tests  were performed to establish the most appropriate treatment. Around 10 p.m. on 29 April 2019 (day 3), the animal began to have muscle spasms. The next day, a lateral body curvature began to develop at the caudal peduncle, with increasing muscle tremors. Due to the worsening prognosis the animal was euthanized on 1 May 2019 (day 5). The therapeutic treatment and the euthanasia protocol applied in this animal are described in the This study is of a juvenile male Risso’s dolphin , were allowed to coagulate, and then centrifuged twice, each time at 3500 rpm for 5 min to obtain the serum for analysis. The first blood sample was collected 4 h after the warning (approximately midday), the remaining samples every 24 h during rehabilitation, and also post-mortem (1:30 h after euthanasia). The biomarkers for acute skeletal and heart muscle damage (CK and cTnI) and the kidney function (BUN and creatinine) were analyzed. longissimus dorsi and rectus abdominis) and heart muscles (both atria and ventricles), atrioventricular valves , semilunar valves (sigmoid aortic and sigmoid pulmonary with the corresponding arteries), and kidneys were fully studied. Tissue sections (4- and 5-µm-thick) were used for hematoxylin and eosin (HE) and Masson’s trichrome technique, respectively, while 3-µm-thick layers were immunolabeled with anti-myoglobin, anti-fibrinogen, anti-cardiac troponin I, and anti-cardiac troponin C primary antibodies. The followed protocol was presented in a previous publication, and it can be accessed in Prior to the necropsy, and after the euthanasia, an entire body scan was carried out, using helical computer tomography (CT) (Toshiba Astellion 16) setting the protocol on 120kV, 300 mA and a reconstruction slice thickness/slice interval of 1/0.80 mm. All the DICOM files obtained were processed with the Horos software. Thereafter, a standard protocol full necropsy was performed, where representative tissue samples were collected and fixed in 10% formalin for approximately 48 h and processed using standard protocol . Due to The different measurements obtained during the rehabilitation time and after euthanasia, are presented in It was possible to observe that every single result obtained for CK was high. It is important to remark the new peak of the value on day 3. This animal presented one value within the normal range on day 0 for cTnI, being the rest of the measurements elevated. As noticed in the CK, the cTnI also presented a peak on day 4 and 5–1:30 h after euthanasia. The animal demonstrated higher BUN values and lower creatinine level than normal values.The 105 kg juvenile male, measured 205.5 cm, had a very poor body condition, and displayed several lacerations, distributed in a multifocal manner, on the rostrum, dorsal and pectoral fins, tail flukes and ventral part of the body, attributed to live-stranding. External and internal signs of a spinal lateral curvature (S-shape) were identified at the level of the caudal peduncle .longissimus dorsi and the rectus abdominis demonstrated a multifocal, severe contraction band necrosis and myonecrosis consisting of segmentary fibrilar degeneration with hyalinized eosinophilic sarcoplasm and hypercontraction , terrestrial , and marine wild mammals , are the CK and BUN elevation ,19,21,27After an injury to the muscle , the serum level of CK begins to rise in 4–9 h, peaks at 24 h, and returns to baseline 48–72 h, unless a new injury or permanent damage occurs . Every sAlthough CK is considered a sensitive marker of myocardial damage, it is present in the skeletal muscles in high concentrations. Due to its poor specificity to detect specific heart damage, troponins have been adopted as the new gold standard for necrosis, more specifically cTnI, since it is detectable in very low amounts (0.01 µg/L) in the blood of healthy individuals ,29. ThusOdontoceti that analyze specific cardiac markers to detect the resulting damage to the heart in these animals. Compared to the recommended values in humans (≤0.1 µg/L) and in dogs (≤0.03–0.07 µg/L), this animal presented one value within the normal range on day 0 (0.035 µg/L), being the rest of the measurements elevated [At present, there are no published studies on elevated ,24,25. Aelevated ,26,30. AThe chronology of events described in this case report is concurring with previous cases of live-stranded cetaceans, being the scoliosis a common problem in these animals, occurring during the rehabilitation process secondary to lack of swimming in a compromised animal ,31. The histopathological findings present in animals who suffered from CM consist of acute to subacute degeneration (rhabdomyolysis) and acute renal failure associated with myoglobinuric nephrosis secondary to muscle damage. The alterations related with the SCMP present in CM are associated with morphological alterations namely acute degenerative lesions , vascular changes  and infiltration by inflammatory cells, based on the histological analysis of myocardial tissue ,19,21,27We consider this article to be an important contribution to clinical biochemistry in cetaceans. Valuable use of blood samples and finally necropsy assessment advances our understanding about the pathology in live-stranded cetaceans.


To the Editor—In a Viewpoint article in the Journal of the American Medical Association (JAMA) regarding the third novel coronavirus (SARS-CoV2) outbreak in Wuhan, China, in December 2019, Drs Wu and McGoogan presented the key findings in a large case series of 72,314 infected individuals, refining the estimates in China to severe disease in 14% of cases and a case-fatality rate of 2.3%.1 Among the total case records, only 889 (1%) were classified as asymptomatic , >5 times lower than those observed in Italy.2 After February 21, 2020, Italy became the hardest-hit country, with the highest death toll of 17,127 (>5 times as much as China) and 135,586 confirmed cases on April 7, 2020.3 It has now become evident that the percentage of mild or asymptomatic cases in developed societies, such as China and Italy, prior to major restrictions or control, was much higher than previously recognized. Although challenging without strict containment measures, identification of these subjects may slow the spread of SARS-CoV2. The cases of Veneto and Lombardia, contemporaneously affected neighboring regions in Italy, provide 2 telling examples with strikingly different endings. As of April 15, 2020, a total of 44,107 tests per million people have been performed in Veneto, which is double the number of tests conducted in Lombardia (Fig. 4 This strategy may have avoided overwhelming the health system, with a consequent positive impact on the case fatality rate in Veneto, which is currently almost 3 times lower than that in Lombardia.Recent evidence shows that 86% of all infections were undocumented prior to the Wuhan shutdown on January 23, 2020, representing the infection source for 79% of documented cases.5 Lombardia did run out of intensive care beds at the end of March, resulting in the need to transfer critical patients to other Italian regions or European countries. Furthermore, between one-half and one-third of the daily national deaths are still being recorded in this region at this time.In fact, in line with previous modeling predictions,At a time when Italy is facing its biggest challenge since World War II, the ‘Veneto model’ indicates that early mass screening for SARS-CoV-2 can make a positive difference, and it should be recommended to other countries responding to the COVID-19 pandemic.


Piaractus mesopotamicus) following fasting, using in vitro and in vivo strategies. Isolated muscle cells of the pacu were subjected to 72 h of nutrient restriction, followed by 24 h of incubation with nutrients or nutrients and AA (200 µM). Fish were fasted for 15 days, followed by 6 h and 15 and 30 days of refeeding with 100, 200, and 400 mg/kg of AA supplementation. AA addition increased cell diameter and the expression of anabolic and cell proliferation genes in vitro. In vivo, 400 mg/kg of AA increased anabolic and proliferative genes expression at 6 h of refeeding, the fiber diameter and the expression of genes related to cell proliferation at 15 days, and the expression of catabolic and oxidative metabolism genes at 30 days. Catalase activity remained low in the higher supplementation group. In conclusion, AA directly affected the isolated muscle cells, and the higher AA supplementation positively influenced muscle growth after fasting.In fish, fasting leads to loss of muscle mass. This condition triggers oxidative stress, and therefore, antioxidants can be an alternative to muscle recovery. We investigated the effects of antioxidant ascorbic acid (AA) on the morphology, antioxidant enzyme activity, and gene expression in the skeletal muscle of pacu ( Skeletal muscle is a tissue that responds to different stimuli, and in fish, it represents approximately 60% of their body weight, being a vital source of amino acid ,2. MoreoThroughout their lives, fish are subjected, both in natural and artificial environments, to periods of food restriction that lead to muscle degradation ,6. In faSalmo salar  [Oncorhynchus mykiss (rainbow trout) [Ctenopharyngodon idellus (grass carp) [Piaractus mesopotamicus (pacu) [Paralichthys adspersus (fine flounder) [Sparus aurata (sea bream) [In aquaculture, given that muscle loss can affect the final profits, a search for strategies to encourage muscle recovery has been undertaken in different species. The central approach in this situation is the refeeding process, which can lead to compensatory growth. In this condition, refed fish can reach the size of non-fasted animals and can even surpass their weight and length . Several salmon) , Oncorhyw trout) ,18,19, Css carp) , and Pias (pacu) ,20,21. As (pacu)  and Paralounder) , as welllounder) , rainbowlounder)  and Spara bream) . Althouga bream) ,21,23.2O2 production in aged rats submitted to repetitive loading exercises [Feed restriction can also increase oxidative stress and the production of reactive oxygen species (ROS) . Animalsxercises . In isolxercises  and myotxercises .The global value of aquaculture has ascended from 68.5 to 148.6 billion US dollars from 2000 until 2014 . Only inThe impact of investigations using fish’s muscle cell culture has increased in the last years (reviewed in ). This aGiven this scenario, the present study evaluated the effects of AA supplementation on the morphology, antioxidant enzyme activity, and gene expression in the skeletal muscle of pacu following fasting, using both in vitro and in vivo strategies. We demonstrated that AA supplementation directly affects isolated muscle cells, thereby increasing the cell diameter and the expression of genes related to anabolism and cell proliferation. Moreover, high levels of AA increased the muscle fiber diameter and the expression of genes related to anabolism, cell proliferation, and oxidative metabolism, as well as decreased Cat activity, which positively influenced muscle growth resumption after food restriction.Since in vitro studies allow the control of the environment by the modification of the culture media to evaluate the effect of a specific nutrient (reviewed in ), we isoWe also evaluated the diameter of muscle fibers, in vivo, to confirm our fasting protocol’s ability to promote a reduction in muscle fiber area and to elucidate the impact of AA supplementation during refeeding. After fasting, there was a decrease in the frequency of fibers with a higher diameter . We alsoConsidering the stressful effects of nutrient deprivation and fasting  and the igf1, mtor, and rps6kb1a, in vitro . Nutriein vitro A, and fa in vivo B–E. In vDp group A, indicapression  but thatrragc and rps6kb1a expression in the H-AA group compared to the other refeeding groups  [Fasting periods can happen due to factors such as migration, reproduction, and food shortages, and it is commonly found at some stage throughout the lifetime of different fish species. Metabolic reserve mobilization allows the survival of animals in response to this challenge . In thistilapia) , grass ctilapia) , as welltilapia) ,24, gilttilapia) , and raitilapia) . These atilapia) ,20,21, rtilapia) , reviewetilapia) ), it seetilapia) ). This cROS production typically occurs in aerobic organisms mainly due to mitochondrial metabolism, and in high concentrations, these molecules can cause cellular damage . Conditimafbx and murf1a), myogenesis (myod and myog), and oxidative metabolism (pgc1α and sdha). These results may be related mainly to the refeeding process than to the AA supplementation, since the expression of those genes were similar between the refeeding groups, and the feeding can modulate the expression of genes of different signaling pathways even in the initial hours of refeeding as seen in Atlantic salmon [Siniperca chuatsi) [Vitamin C, or AA, is considered one of the most effective antioxidant agents that can inhibit a series of harmful molecules . In the c salmon  and Chinchuatsi) .igf1 and mtor was similar between the groups, which is probably related to the moment of evaluation considering the dynamic gene expression in signaling pathways and that the initial transcriptional response can happen in minutes .While the expression of some genes was not affected by the AA level, we found interesting changes in the expression of anabolic genes at 6 h of refeeding. Although the expression of the iewed in ), we fourragc gene codifies a protein that compounds the heterodimer RagA/RagB–RagC/RagD, which in the presence of amino acids can stimulate protein synthesis through the allocation of the mtorc1 complex near the lysosomal membrane . The si targets . The rpsiewed in ). An inciewed in , and theiewed in . Consideiewed in ). It is iewed in ,69.Umbrina cirrosa subjected to fasting has been shown to experience a decrease in general metabolic activity in skeletal muscle and a decrease in muscle mass [The faster response in the H-AA group at 6 h of refeeding may have influenced the muscle phenotype at 15 days. At this time, we observed that the H-AA group had a decrease in the frequency of muscle fibers with a small diameter and an increase in the frequency of muscle fibers with a higher diameter. Moreover, we observed a general decrease in the expression of anabolic, catabolic, cell proliferation, and mitochondrial metabolism genes in the L-AA group. In addition to the elevated antioxidant enzyme activity in this group throughout the experimental period, this fact may indicate a prolonged effect of fasting, affecting the metabolic process and the recovery of the muscle mass with the lower AA level. In accordance, cle mass . Besidescle mass ), in linpcna and myod, genes highly expressed in proliferative fish muscle cells  and anabolic genes (rragc and rps6kb1a) may explain our morphological results observed in the H-AA group at 15 days of refeeding. In fact, in our in vitro results, although in the absence of AA (Dp + N group), the cells showed improvements in the anabolic and hypertrophic processes, the supplemented cells showed higher diameters; higher expression of igf1, rps6kb1a, and pcna; and increased Gpx activity, indicating an even better and faster re-establishment of growth both by protein synthesis and cell proliferation. Similar results were found in pacu muscle cells cultivated with AA since isolation, showing that under both stressful or normal growth conditions, the antioxidant increased anabolic and decreased catabolic gene expression [Interestingly, we observed increased expression of e cells . Miciewed in . After mmodeling . Thus, tigf1, mtor, murf1a, myog, and pgc1α may be related to higher food consumption in the L-AA group during refeeding. Although the food supply was the same between the groups, the consumption was not measured, which could be a limitation of the present work. Despite this fact, our molecular, enzymatic, and morphological results indicate that a higher amount of food could be needed in the absence of AA to stimulate anabolic processes.Surprisingly, considering the final experimental period, the L-AA group showed some molecular similarities to the H-AA group. In nutrient restriction situations, fish can increase food ingestion as a strategy to supply energetic needs. This behavior can recover the weight, length, and muscle growth parameters ,76,77. IIn summary, we demonstrated that higher AA supplementation improves anabolic, myogenic, and oxidative metabolism gene expression, as well as antioxidant enzymatic activity, thereby enhancing pacu muscle growth in the initial phases of refeeding in vivo. Moreover, we confirmed that AA supplementation also positively affects the growth of isolated muscle cells. Our findings may represent a strategy in aquaculture systems to enable faster muscle recovery after stressful conditions, thus reducing the costs of this situation and improving the final profits.3 tanks with 35 fish in each tank (density of 140 fish/m3) at 28 °C under a 12 h light/12 h dark photoperiod. The experimental groups were kept in independent systems to avoid AA cross contaminations in the water. Each group was kept in three tanks, resulting in three replicates per groups.The experiments were conducted according to the ARRIVE guidelines  and the ®, Stay-C®35, DSM Nutritional Products, Basel, Switzerland, 35% AA activity). The AA amounts in each diet were established following previous work using the same fish species [Fish were fed ad libitum, three times daily with diets described in  species ,82,83. T6 cells/mL in twelve well plates, previously treated with poly-L-lysine and laminin . Cells were cultured at 28 °C in complete growth medium , and 1% antibiotics; Sigma-Aldrich, Saint Louis, MO, USA) for 10 days until they reached 80–90% confluency. Next, cells were divided into the groups: Dp—cultured for 96 h in deprivation medium ; Dp + Nted from ,33,45.n = 6 animals/per group) in a cryostat and stained with hematoxylin and eosin. Images were obtained from six animals per group. The area of eight hundred fibers per animal was measured in ImageJ® [® [For in vivo determination of muscle fibers diameter, histological muscle cryosections (10 µm) were obtained from fast muscle tissue of pacu  and muscle cells in vitro (n = 5 independent cell cultures/per group) using Trizol and following the manufacturer recommendations ). The RNA was quantified in the spectrophotometer NanoVue™ Plus , and the integrity was ensured through electrophorese in 1% agarose gel. To avoid DNA contamination, the samples were treated with the RQ1 RNase-Free DNase kit  following the manufacturer recommendations. The total of 1 microgram/microliter of RNA was used to the synthesis of the cDNA, which was performed using the GoScript™ Reverse Transcription System  following the protocol recommendations. Gene expression levels were detected by RT-qPCR through QuantStudio™ 12 K Flex Real-Time PCR System  using the GoTaq® qPCR Master Mix kit ) and primers , anabolism , catabolism , and oxidative metabolism  was evaluated. The comparative Ct methods, 2Ct∆∆ [rpl13 gene was selected as reference due to its stability and similarity between the groups.The total RNA was extracted from fast muscle tissue in vivo  and in vitro muscle cells (n = 4 independent cell cultures/per group) in an extraction medium  with protease inhibitor . The samples were centrifuged for 20 min at 4 °C and 1968× g. The supernatant was collected and quantified following the Bradford method [The total protein was extracted in vivo from fast muscle samples  activity was measured following the Beauchamp and Fridovich method . Briefly2O2 presence, as previously described in [2O2 and potassium dichromate diluted in acetic acid. The activity was measured in a spectrophotometer at 620 nm.The Cat (EC 1.11.1.6) activity was determined through the reduction of the dichromate diluted in acetic acid to chromic acetate in Hribed in . Samples2O2 were added to samples at 3 microgram/microliter concentration. The mixture was heated at 37 °C for 15 min, and 5% trichloroacetic acid was added. Next, the samples were centrifuged for 5 min at 327× g, and the supernatant was collected. For each 100 µL of the collected supernatant, 700 µL of DTNB (0.4 mg/mL) were added. The activity was measured in a spectrophotometer at 320 nm. The results of the activity of the antioxidant enzymes were expressed as U.mg pt−1.The Gpx (EC 1.11.1.9) activity was quantified, as described in . Phosphawww.graphpad.com, accessed on 10 November 2020). The data were represented as mean ± SD. Statistical significance was considered when the p-value was <0.05.For in vitro analyzes, the statistical significance related to gene expression and antioxidant enzyme activity was assessed using one-way ANOVA followed by Tukey’s post hoc test. For the differences related to muscle cells’ diameter, the Kruskal–Wallis followed by Dunn’s post hoc test was performed. For in vivo analyzes, an unpaired T-test was performed to assess the statistical significance between C and F groups in gene expression, antioxidant enzyme activity, and length. The statistical differences between these groups in weight and muscle fibers diameter were assessed using the Mann-Whitney test. After refeeding, the statistical significance related to body length and weight was tested, performing the Kruskal-Wallis followed by Dunn’s post hoc test. One-way ANOVA was followed by Tukey’s post hoc test to assess the statistical differences in gene expression, antioxidant enzyme activity, and muscle fibers diameters between the refeeding groups. All in vivo comparisons were performed between the groups at each experimental timepoint. The differences between timepoints were not assessed. Data were analyzed using GraphPad Prism v.6 (GraphPad Software, La Jolla, CA, USA,


Previous studies have given an inaccurate assessment of the role of acupuncture in in vitro fertilization (IVF). We will use acupuncture doses as an entry point, discussing the dose-related effects of acupuncture therapy in women undergoing IVF.This study will search the following database: EMBASE, PubMed, Web of Science, the Cochrane Central Register of Controlled Trials (CENTRAL), and 4 Chinese databases. All databases will be searched from the date of database establishment to January 31, 2019. In addition, we will search possible studies which were included in previous meta-analyses. The primary outcomes are the clinical pregnancy rate (CPR) and the live birth rate (LBR). The secondary outcomes involved the biochemical pregnancy rate (BPR), the ongoing pregnancy rate (OPR), serum hormone level, the incidence of ovarian hyper-stimulation syndrome (OHSS), the cycle cancellation rates, and adverse events (AEs). After checking and integrating the raw data, we will use a 2-step to conduct the meta-analysis. Firstly, we will assess the effect of acupuncture on in vitro fertilization and embryo transfer (IVF-ET). Secondly, the meta-analysis will be performed for studies with similar total number of treatment sessions to investigate the dose-related effects of acupuncture. RevMan V.5.3 statistical software will be used for meta-analysis. If it is not appropriate for a meta-analysis, then a descriptive analysis will be conducted.This study will investigate the relationship between pregnancy outcomes and the doses of acupuncture therapy in women undergoing IVF, and answer whether a higher-doses of acupuncture treatment will contribute to a better outcome of IVF-ET.The funding of this meta-analysis may provide convincing evidence for clinicians, benefitting more patients who crave children.INPLASY202070072 A World Health Organization's (WHO) report has shown that, in 2010, there were estimated 48.5 million couples were infertile around the world. In recent years, significant advances have been made in the treatment of infertility due to the advent of assisted reproductive technologies (ART). In vitro fertilization and embryo transfer (IVF-ET) is an important component of assisted reproductive technology. Over 300,000 ART cycles were performed in Europe during 2013. For all in vitro fertilization (IVF) cycles, the clinical pregnancy rate (CPR) per aspiration and per transfer were stable with 29.6% and 34.5%, respectively. However, due to lower endometrial receptivity, poorer embryo development, and a combination of other factors, CPR following IVF-ET remain low. Even some couples repeatedly failed in implantation.As a global public issue, infertility is explain as a disease of the reproductive system defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse. and Paulus et al reported that acupuncture could improves CPR in women undergoing IVF-ET. Studies–11 have shown that acupuncture can significantly improve endometrial tolerance, regulate local blood circulation, and relieve anxiety, creating favorable conditions for embryo implantation. The application of acupuncture in the field of assisted reproduction has been receiving increasing international attention.Acupuncture, as a characteristic treatment of traditional medicine in China, is becoming more and more accepted worldwide because of its safety, effectiveness, and low side effects in clinical practice. Since 1990s, Stener-Victorin et al–14 showed that there was no significant difference in the pregnancy outcomes between acupuncture and sham acupuncture during IVF. Some systematic review–17 have also been controversial about whether acupuncture can improve CPR and LBR in women who undergoing IVF. In summary, the impact of acupuncture on pregnancy outcomes remains uncertain.However, a confirmative and consistent result cannot be achieved by previous studies. Some studies–14,18 the number of acupuncture treatments was generally 1 to 3 times. However, women undergoing IVF have complex conditions and most of their primary illnesses require long-term acupuncture treatment. Some reviews,20 suggested that during an ART cycle, the frequency, number of treatments, and timing of acupuncture were identified as important, at the same time, patients should start acupuncture treatment 3 to 6 months before IVF and receive >6 times treatments. Studies have shown that acupuncture treatment for 3 menstrual cycles can effectively improve the tolerance of the endometrium and create conditions for successful conception in women who have repeatedly failed to grow.,21 We believe that the effectiveness of acupuncture is related to the courses as well as the frequency for the time of onset and accumulative effects. Therefore, we will investigate the relationship between pregnancy outcomes and the doses of acupuncture therapy in women undergoing IVF, and answer whether a higher-doses of acupuncture treatment will contribute to a better outcome of IVF-ET.We found that in studies with negative results,22.1,23This study protocol was registered in International Platform of Registered Systematic Review and Meta-Analysis Protocols (INPLASY) as INPLASY202070072. The protocol is structured in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analysis Protocols and Cochrane handbook.2.2This study will systematically search EMBASE, PubMed, Web of Science, and the Cochrane Central Register of Controlled Trials (CENTRAL). In addition, we will also search 4 Chinese databases: China National Knowledge Infrastructure (CNKI), China Biomedical Literature Database (CBM), Wanfang Database, and China Science Journal Database (VIP database). All databases will be searched from the date of database establishment to January 31, 2019. Because of language limitation, we will only search and screen studies published in English or Chinese. Full articles or abstracts will be included. The following search terms will be combined for systematic search:(1)“acupuncture”, “acupuncture therapy”, “electroacupuncture”, “auricular acupuncture”, “acupressure”, “transcutaneous electrostimulation”;(2)“in vitro fertilization/IVF”, “intracytoplasmic sperm injection/ICSI”, “infertility”, “embryo transfer”, “assisted reproduction technology”;(3)“randomized/randomised controlled trial”, “controlled clinical trial”.The search strategy will be adjusted for different database requirements. Terms in Chinese will be used in Chinese databases. The search strategy of PubMed is shown in Table 2.32.3.1To investigate the effectiveness of acupuncture in improving the IVF outcomes, we will include para-, randomized controlled trials (RCTs) that compared acupuncture therapy with sham acupuncture or placebo or no treatments (waiting list control). Studies assessing other effects of acupuncture in the period of IVF cycles, such as pain-relieving during the egg retrieval will not be included. To guarantee the quality, only the sample size was over 30 in each group will be included. We will exclude the following studies: retrospective studies, studies with crossover design, animal or in vivo experimental studies, case reports, or comments.2.3.2We will include women undergoing IVF and age between 18 and 45. Patients underwent IVF with or without intracytoplasmic sperm injection (ICSI) will be included, no matter who were experiencing a fresh cycle of IVF, or having history of failure cycles before. Patients having other gynaecological diseases  will also be included.2.3.3The source of stimulation could be manual acupuncture, electroacupuncture, auricular acupuncture, warm needling, scalp acupuncture, intradermal needle, and transcutaneous electrostimulation (TENS). To control the heterogeneity, we will exclude studies reporting acupuncture therapy combined with Chinese medicine. Because the effectiveness of acupuncture therapy could not be evaluated.2.3.4(1)Placebo controls: including needling in the control groups could either be with a sham needle device, such as Streitberger, Park placebo acupuncture devices, etc. Where skin penetration does not occur because the tip of the needle is blunted, normal acupuncture needles were applying at an area not recommended by TCM practitioners for fertility treatment, such as non-acupoints or non-specific points for fertility treatment, sham laser, placebo drugs, or other sham interventions.(2)No acupuncture treatment, such as waiting list, which means that patients in the control group did not receive acupuncture treatment. Or patients received general care or usual care, which means that patients in the control group only receive advice and/or health education, such as exercise recommendations.We will exclude the studies which applying Chinese medicine, or other methods that we can’t define the therapeutic effects as a control.2.42.4.1(1)Clinical pregnancy rate (CPR): defined by the presence of a fetal heartbeat at 6 to 7 weeks of pregnancy;(2)Live birth rate (LBR): a baby born alive after 24 weeks gestation.2.4.2(1)Biochemical pregnancy rate (BPR): a positive human chorionic gonadotropin (hCG) serum or urine test 11 days after embryo transfer (ET);(2)Ongoing pregnancy rate (OPR): pregnancy beyond 12 weeks of gestation which is confirmed by fetal heart activity on ultrasound;(3)Endometria condition, such as receptibility, resistance index (RI) or pulsatility index (PI);(4)Serum hormone level, such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), FSH/LH ratio);(5)The incidence of hyperstimulation ovarian syndrome (OHSS);(6)Cycle cancellation rates;(7)Adverse events (AEs).2.52.5.1We will import the title, author, and abstract of the retrieved article from the database into Endnote V.X9  and delete repetitive articles after checking by this software. Two reviewers (ZZH and WX) will independently go through article titles, abstracts, and keywords one by one to filter out articles that meet the inclusion criteria. Eligible full text will be further assessed while excluded studies will be recorded and interpreted. The results will be cross-checked by 2 reviewers. Any disagreements will be resolved by consensus. Further arguments will be arbitrated by a third reviewer (ZQH). The study flow chart is shown in Fig. 2.5.2 which includes the following information: the general information of the studies, study characteristics, participants, interventions and controls, outcomes, results, adverse events, main conclusions, ethical approval, and other information. In order to investigate the doses related effects of acupuncture, we specifically document the number and frequency of acupuncture sessions, duration of treatment and the time window of acupuncture treatment. All the information from extraction form will be recorded in an Excel file. Reviewers will be trained before extraction and reach a common consensus to guarantee the consistency. Any discrepancy will be resolved through discussion between the 2 reviewers, or judged by a third reviewer (ZQH). If the reported data are insufficient, we will contact the author of the studies for consultation and solution.Two reviewers (YZQ and HTH) will independently check the eligibility of the included studies and extract data according to a predesigned data extraction form according to the Reporting Interventions in Clinical Trials of Acupuncture (STRICTA),2.5.3Two reviewers (ZZH and WX) will independently evaluate the methodological quality of included studies by using the Cochrane Collaboration's tool for assessing risk of bias . The following domains will be accessed: random sequence generation, allocation sequence concealment, blinding, data integrity, selective reporting, and other sources of bias . The assessment results will be divided into 3 levels: low risk, high risk, and uncertain risk. In the process, the discrepancy will be discussed by the 2 reviewers to reach an agreement, or judged by a third reviewer (ZQH).2.5.4For dichotomous data, such as CPR, LBR, BPR, OPR, and the rate of AEs, we expressed the results for each study as the risk ratios (RRs) with 95% confidence intervals (CIs). And for the continuous data, such as the number of retrieval eggs, blood hormone level, and so on, we expressed the results as the difference or standard mean difference (SWD) with 95% CI.2.5.5We will attempt to obtain missing or inadequate trial data by contacting the first author or correspondent author of the included study by email. If no additional data is available, we will analyze the existing data and discuss the potential impact of missing data.2.5.6I2 test and P value of the chi-square test of heterogeneity. When the I2 test is <50%, the study is not considered to have a large heterogeneity. When the I2 values are above 50%, there is significant heterogeneity between trials. To investigate the contributors to the heterogeneity, we will use sensitivity and subgroups analysis. If the heterogeneity is too high, the meta-analysis will not be performed, we will conduct a descriptive systematic review.The heterogeneity will be evaluated by using both the 2.5.7We will use the contour-enhanced funnel plot to assess the risk of publication bias within each pairwise comparison. If a sufficient number of studies are included , we will use funnel plots to assess reporting bias. If the funnel plot is asymmetric, Egger regression test will be used.2.5.8I2 value is <50%; otherwise, we will use a random-effects model for data synthesis.In our review, the effect of acupuncture on IVF-ET will be assessed in first step. In order to investigate the dose-related effects of acupuncture, the meta-analysis will be performed for studies with similar total number of treatment sessions. Among the included studies, the total number of acupuncture treatment sessions will be divided as follow: low dose stimulation (1–3 treatments); medium dose stimulation (4–10 treatments); medium-high dose stimulation (11–30 treatments); high dose stimulation (>30 treatments). RevMan V.5.3 statistical software will be applied for data synthesis. Statistical analyses will be performed with RevMan V.5.3 statistical software to present direct and indirect comparisons between acupuncture treatment and controls. For dichotomous data, such as CPR, LBR, BPR, OPR, and the rate of AEs, we expressed the results for each study as the risk ratios (RRs) with 95% confidence interval (CIs). And for the continuous data, such as the number of retrieval eggs, blood hormone level, we expressed the results as the difference or standard mean difference (SWD) with 95% CI. According to the suggestions from the Cochrane Handbook, we will use a fixed-effects model for data synthesis if the 2.5.9We plan to conduct the following subgroup analyses of the main results to explore the stability of the results across different study subgroups.(1)Different time points of acupuncture treatment: acupuncture began 3 menstrual cycles before transfer; acupuncture began 3 menstrual cycles before transfer; acupuncture began from day 21 of the previous cycle until the day of human chorionic gonadotrophin (HCG) administration; acupuncture on the day of embryo transfer, etc;(2)Different types of acupuncture treatment: such as manual acupuncture, electroacupuncture, TENS, etc;(3)Different types of control: such as sham acupuncture or placebo;(4)Different underlying diseases: such as PCOS, POF, etc2.5.10To ensure the stability and reliability of the results, a sensitivity analysis will be performed. These will be based on different statistical approach, different heterogeneity quality, and different sample size. Excluding the studies which were poor quality or potential contributors to heterogeneity, the meta-analysis will be reused. We will compare the results and discuss.2.5.11The grading of recommendations assessment, development, and evaluation will be used to evaluate the quality of SRs. We will assess the risk of bias, inconsistency, indirectness, inaccuracy, and publication bias. The results of the evaluation were divided into 4 levels: high, moderate, low, or very low.3The results of a single clinical study are often insufficient to draw definitive conclusions and remain inaccurate estimates of the true effect of the hypothesis. Meta-analysis is a great way to resolve uncertainty when report disagree and assist the applicability of the findings to a wider range of patients and treatment options.–29 and that all of these chronic conditions need multiple courses of acupuncture treatment.,30 At the same time, menstruation, as a physiological phenomenon of the female cycle, is closely related to ovulation and has a natural cycle rhythm. Therefore, the acupuncture treatment of diseases related to the menstrual cycle should also be combined with the menstrual cycle. Previous studies have ignored the cumulative effects of acupuncture. Our study will address this question and attempt to explain whether acupuncture can achieve the goal of improving pregnancy outcomes through the treatment of underlying conditions. There are also some limitations in this systematic review. Firstly, due to language limitations, we can only include articles in Chinese and English; secondly, because some articles do not give the exact number of acupuncture treatments, we can only estimate the approximate range based on the descriptions in the text. Thirdly, we may have overlooked the discussion of the relationship between acupoints compatibility and the therapeutic role of meridian acupoints based on traditional Chinese medicine theory. Lastly, there is a high possibility that a large heterogeneity comes from the different acupuncture methods, treatment durations. Therefore, descriptive SR but not quantitative SR will be necessary.Most infertility patients tend to have underlying conditions. Several studies have shown that acupuncture is effective for conditions that cause infertility,With the development of complementary alternative medicine, acupuncture has been widely used in the field of assisted reproduction. We sincerely hope that the funding of this review could provide convincing evidence for clinicians, benefitting more patients who crave children.Conceptualization: Qianhua Zheng, Zihao Zou, Ying Li.Data curation: Xin Wen, Zihao Zou, Zuoqin Yang, Tinghui Hou, Qianhua ZhengFormal analysis: Qianhua Zheng, Siyuan Zhou.Investigation: Qianhua Zheng, Ying Li, Xinyu Hao.Methodology: Qianhua Zheng, Xinyu Hao.Project administration: Qianhua Zheng, Ying Li.Supervision: Ying Li.Writing – original draft: Zihao Zou, Qianhua Zheng.Writing – review & editing: Xinyu Hao, Ying Li