Effective management of patients with chronic kidney disease (CKD) relies on timely detection of clinical deterioration towards end stage kidney failure. We aimed to design an electronic Patient-Reported Outcome Measure (ePROM) system, which would allow patients with advanced CKD  to: (i) remotely self-report their symptoms using a simple and secure online platform; (ii) share the data with the clinical team in real-time via the electronic patient record to help optimise care. We adopted a staged development process which included: a systematic review of PROMs used in CKD; formation of a co-design team; prototype system design/development, user acceptance testing and refinement; finalisation of the system for testing in a pilot/feasibility trial.A co-design team was convened, including patients with lived experience of CKD; clinical team members; IT/Informatics experts; academics; and Birmingham Clinical Trials Unit representatives. A prototype system was developed and iterative changes made before finalisation during a series of operational meetings. The system allows patients to remotely self-report their symptoms; provides tailored self-management advice; allows monitoring of real-time patient ePROM data; sends automated notifications to the patient/clinical team in the advent of a severe symptom report; and incorporates longitudinal ePROM symptom data into the electronic patient record. Feasibility of the system will be evaluated as part of the National Institute for Health Research funded RePROM  pilot trial (ISRCTN12669006).Routine ePROM collection with real-time feedback has the potential to improve outcomes and reduce health service costs. We have successfully developed a trial-ready ePROM system for advanced CKD, the feasibility of which is currently being explored in a pilot trial. Assuming feasibility is demonstrated, formal evaluation of efficacy will take place in a future multi-centre randomised controlled trial. The global burden of chronic kidney disease (CKD) continues to increase, with age-standardised death and disability-adjusted life years rising over recent decades . PatientWith increasing use of digital healthcare, there has been much interest in the potential of harnessing electronic Patient-Reported Outcome Measures (ePROMs) to aid the management of symptom burden and optimise use of limited healthcare resources . These mEvidence in an oncology setting suggests patients are willing to complete ePROM symptom questionnaires on a regular basis, and that the data can be integrated into the electronic patient record (EPR), with beneficial results. Studies in cancer populations suggest that ePROM symptom monitoring may be associated with enhanced patient-clinician communication and patient activation; earlier detection of adverse events; improved patient quality of life; reduced use of accident and emergency services; fewer inpatient hospital episodes; and improved survival, even for ‘computer-inexperienced’ patients –14.Whilst disease-specific PROMs are commonly used in pre-dialysis renal research, for example the Kidney Disease Quality of Life—36 (KDQOL- 36) and KDQOL-SF measures , 16, theDevelopment of the RePROM symptom reporting system is reported according to the Criteria for Reporting the Development and Evaluation of Complex Interventions in healthcare: revised guideline (CReDECI 2) .The RePROM study was undertaken within the Centre for Patient-Reported Outcomes Research at the University of Birmingham and the Queen Elizabeth Hospital Birmingham (QEHB) within the UK National Health Service (NHS) University Hospitals Birmingham Foundation Trust (UHBFT).https://www.invo.org.uk).Development of the study design was informed by a series of meetings held with the study Patient Advisory Group (PAG), which included people with lived experience of CKD. PAG members were recruited via a targeted invitation distributed by renal staff at QEHB and academic staff within the Centre for Patient-Reported Outcome Research (CPROR) at the University of Birmingham. Members contributed to the design of the project prior to the application for funding, and the subsequent refinement of the protocol and patient-facing documentation, and were also involved in the ePROM co-design group, as outlined below. The PAG received compensation for their involvement in line with guidelines provided by the National Institute for Health Research INVOLVE UK national advisory group  and cancer (n = 1), who were members of the study patient advisory group; members of the broader renal clinical team at QEHB (n = 7), UHBFT IT/Informatics experts (n = 3); academics (n = 3); and members of the Birmingham Clinical Trials Unit (BCTU) (n = 5). In addition, design input was sought from international ePROM experts Profs Ethan Basch , Niels Hjöllund  and Galina Velikova .In parallel, a co-design team was convened, comprising: patients with lived experience of CKD  or via the EPR (clinicians). This identical view was suggested by both patients and clinicians in the co-design team as an essential feature aimed at fostering a shared understanding of the patient’s symptom burden.A system algorithm highlights severe symptoms that may require clinical follow-up, delivering pertinent information to the patient via an on screen ‘pop-up’, and sending an automated email to the kidney care team containing details of the symptom(s) triggering the notification and providing a direct link with which to access all of the patient’s ePROM reports Fig. .Feasibility of the system will be evaluated as part of the National Institute for Health Research (NIHR) funded RePROM  pilot trial (reference: PDF-2016-09-009). A protocol for the study is available . The ful2 inclusive; or a projected risk of progression to end-stage renal failure within 2-years ≥20% using the 4-variable Tangri renal risk calculator [In summary, the RePROM pilot trial is an investigator led, single-centre, open-label, two-arm randomised controlled pilot trial of 66 participants aged 18 years or over with advanced CKD  ≥6 and ≤ 15 mL/min/1.73mlculator ). The trTest and pilot the trial protocol ;Assess the willingness of clinicians to randomise participants into the trial;Assess the willingness of people with advanced CKD to be randomised into the trial;Assess the acceptability of the ePROM system;Explore the need for a non-web-based intervention platform for participants who are unable to use the online ePROM;Inform selection of the most appropriate primary outcome measure for the full-scale RCT;Provide data to help estimate the sample size for the full-scale RCT;Provide a platform to develop and pilot the processes to capture costs and outcomes to inform the health economic evaluation for the full-scale RCT;Determine key participation criteria for centre involvement in the full-scale RCT.The primary aims of the study are to pilot the trial protocol and assess the feasibility of undertaking a full-scale RCT exploring the use of an ePROM in the management of advanced CKD. This will include assessment of both quantitative and qualitative data. The pilot study will:A range of trial outcomes will be captured 3, 6, 9 and 12-months post randomisation (assessment window +/− 3 weeks). These include clinical data e.g.: Serum Creatinine, Calcium, Phosphate, Bicarbonate, Albumin, eGFR, Albumin Creatinine Ratio, blood pressure, and for participants with diabetes: glucose and HbA1c. Patient quality of life will be assessed using a paper version of the EQ-5D-5L questionnaire, a reliable/validated generic measure of health status commonly used in CKD trials research . HealthcWe have successfully developed an ePROM system which allows patients with advanced CKD to remotely self-report their symptoms using a simple and secure online platform. The system integrates symptom data into the hospital EPR and: (i) allows real-time monitoring aimed at facilitating optimal care; (ii) includes automated self-management advice for patients; and (iii) can trigger automated notification of both the patient and the clinical team in the advent of a severe symptom report.To date, research exploring ePROM efficacy has primarily focused on cancer. Basch and colleagues conducted an RCT in the US, involving patients receiving outpatient chemotherapy for advanced solid tumours; with participants in the intervention arm asked to report 12 common symptoms on a weekly basis using the web-based STAR (Symptom Tracking and Reporting) ePROM system . Fourth,n = 60 simultaneous users). Moreover, this study was the first to evaluate cost-effectiveness of an ePROM-based symptom monitoring approach  [A survival benefit was replicated in a similar ePROM RCT, conducted by Denis et al., in a cohort of patients undergoing treatment for lung cancer in France using the ‘e-FAP’ (e-Follow-Up Application)  , 14. Agafe Year) .Both the STAR and e-FAP systems appeared to facilitate early detection of symptom deterioration and supportive care needs, improving patient outcomes and offering health provider/societal cost savings. In theory, such a system could lead to similar benefits in a CKD population, where effective management relies on the timely detection of clinical deterioration towards end stage kidney failure .n = 99 patients on attendance at outpatient home dialysis clinics in two cities on the west coast of Canada over a 6-month timeframe [Feasibility of ePROM capture in patients with CKD has been demonstrated in Canada and the UK. Schick-Makaroff and Molzahn successfully used tablet computers to capture ePROM data from imeframe . Pittmanimeframe . Eighty imeframe , 13.Our system has adopted these additional real-time features and is currently being evaluated in the RePROM pilot trial in a pre-dialysis population. We intend to explore the experiences of patients using RePROM, compared to those documented using the aforementioned ePROM systems in renal disease , 18 and As mentioned, our co-design group were unable to endorse an existing, validated, PROM for use within our system as planned. Systematic review demonstrated that renal PROMs frequently lacked validation in English-speaking populations (particularly in the UK) and were missing evidence to support important measurement properties including measurement error, structural validity, responsiveness and patient acceptability . There iRoutine ePROM collection and real-time feedback in patients with advanced CKD has the potential to improve outcomes and reduce health service costs. We have successfully developed a trial-ready ePROM system, the feasibility of which is currently being explored in a pilot study. An updated system, incorporating a validated renal symptom item bank currently in development, will be subjected to evaluation in a future UK-based multi-centre RCT.


While the luting gap/marginal opening between the TiN-cap and the ceramic crown was within the clinically acceptable range, the SEM analysis failed to identify a measurable microgap at the cone-in-cone junction. Within the limits of the in-vitro study it can be concluded that the Acuris conometric interface does not allow for bacterial translocation under non-dynamic loading conditions.Encouraging clinical results were reported on a novel cone-in-cone coupling for the fixation of dental implant-supported crowns . However, the presence or absence of a microgap and a potential bacterial leakage at the conometric joint has not yet been investigated. A misfit and a resulting gap between the conometric components could potentially serve as a bacterial reservoir that promotes plaque formation, which in turn may lead to inflammation of the peri-implant tissues. Thus, a two-fold study set-up was designed in order to evaluate the bidirectional translocation of bacteria along conometrically seated single crowns. On conometric abutments filled with a culture suspension of anaerobic bacteria, the corresponding titanium nitride-coated (TiN) caps were fixed by friction. Each system was sterilized and immersed in culture medium to provide an optimal environment for microbial growth. Positive and negative controls were prepared. Specimens were stored in an anaerobic workstation, and total and viable bacterial counts were determined. Every 48 h, samples were taken from the reaction tubes to inoculate blood agar plates and to isolate bacterial DNA for quantification using qrt-PCR. In addition, one Acuris test system was subjected to scanning electron microscopy (SEM) to evaluate the precision of fit of the conometric coupling and marginal crown opening. Throughout the observational period of one week, blood agar plates of the specimens showed no viable bacterial growth. qrt-PCR, likewise, yielded a result approaching zero with an amount of about 0.53 × 10 Due to their favorable long-term clinical results, fixed implant-supported restorations are considered a valuable treatment alternative for the replacement of single or multiple teeth ,2. In prIn order to overcome the aforementioned problems associated with the attachment of fixed implant-supported restorations, the use of a conical coupling retention to support single crowns has recently been introduced . In thisThe employment of conically coupled abutments with different designs indicated promising prosthetic results. Hybrid-acrylic prostheses and lithium disilicate or monolithic zirconia fixed partial dentures (FPDs) and single crowns (SCs) retained by conical friction showed minimal technical problems and healthy peri-implant soft-tissues ,14,15,16While encouraging in-vitro and in-vivo results have been reported for cone-in-cone morse-tapered connections used to retain implant-supported FPDs and SCs ,18,19, l−4 µg/mL DNA for the specimens and 2.65 µg/mL DNA for the positive control (p < 0.001) (p = 0.99). Possible deviations for the test days caused no significant difference on the mean bacterial count (p = 0.69)  , whereas = 0.69) .All specific primers tested in qrt-PCR revealed no bacterial DNA for the specimens, in contrast to the positive control. Due to an inadequate seating of the TiN cap on the abutment of specimen No. 6, only poor conometric coupling could be achieved resulting in an additional positive control with an increased amount of DNA.While the luting gap/marginal opening between the TiN-cap and the ceramic crown (P2) was within the clinically acceptable range of approximately 115 μm, the SEM analysis failed to identify a measurable microgap at the cone-in-cone Acuris junction (P1) a–f.A dental implant system comprises the endosseous portion and connects to a superstructure that ultimately restores function and esthetics. Although both are intimately joined in one-piece implants, for two-part implant systems, a microgap at the interface between implant and abutment and at the interface between abutment and prosthesis seems unavoidable. The latter include a removable transmucosal abutment that is attached on one side to the implant and on the other side to the restoration. As a result, an implant–abutment interface and an abutment–restoration interface are generated. The size of the latter depends on the respective manufacturer and seems to be limited to less than 50 μm for current implant systems . Larger While encouraging clinical results have been reported for cone-in-cone morse-tapered connections at the implant abutment junction , little An improved seal in terms of microbial leakage may be achieved by using prefabricated components. The recently introduced Acuris conometric coupling examined in the current in-vitro study uses the friction between the abutment and a prefabricated titanium nitride (TiN) coping for retaining a crown. A full-ceramic crown is luted extraorally onto this prefabricated final TiN coping and then attached intraorally to the abutment to provide a conometric friction retention . The fin2 copies/μL in real-time PCR for all positive assemblies  [The specific qrt-PCR analysis and agar plate spreading of the present in vitro investigation revealed no bacterial transfer from and into the prosthetic connection of conometrically seated Acuris single crown-units. The null hypothesis that the conometric interface does not allow for bacterial translocation can thus be considered as accepted. Comparison of the present findings can only be made with caution, as the available literature data are limited, and bacterial translocation appears to be dependent on the particular geometry and accuracy of the prefabricated components of each system. The results of a recent in vitro study with two other conometric systems using different materials showed a minimal microbial concentration of less than 1 × 10It has been suggested that the sealing performance of conometric morse taper systems and their retention capacity is achieved by the wedge effect. Conical coupling retention is, however, only achieved when the coping is fully seated on the abutment. During the in vitro study-setup, very little room for movement was available in the anaerobic chamber, which resulted in suboptimal conditions for the assembly of the TiN copings. As a result, one coping of implant sample No. 6 was incorrectly retained and consequently showed increased DNA values. It is important to note that this increase was clearly due to inappropriate handling of the TiN cap rather than a system relevant bacterial leakage. This sample was therefore only used as an additional positive control. In a clinical scenario, incomplete seating of the TiN coping-crown unit would result in retention deficiency and instant crown detachment. The seating of the coupling is thus checked immediately after insertion by means of a periapical radiograph. Tight proximal contacts or excessive submucosal emergence profiles may be other reasons for inadequate crown fit and poor retention in a clinical scenario. A drawback of the present in-vitro study is the relatively small number of samples, which should be expanded in future investigations. A further limitation relates to the fact that the contribution of loading on bacterial leakage at the conometric interface was not evaluated. It has been shown that testing of the implant–abutment connection under dynamic-loading conditions is an important part of the experimental design to evaluate the bacterial colonization of dental implants ,31. The p < 0.001). Hence, a holistic examination of bacterial colonization for the cone-in-cone coupling showed no microbial leakage. SEM analysis revealed a marginal luting gap between the Acuris TiN-cap and ceramic crown within a clinically acceptable range of 115 μm, but no microgap at the cone-in-cone Acuris junction. A further study is planned to expand comparability and to reveal clinical implications for practitioners. We included the four most prevalent oral microbes in our bacterial mixed culture solution used for bacterial entry and exit out of the implant system . These bacteria are usually benign—however, under certain conditions, they may cause caries, parodontitis, mucositis or perimplantitis [Referring back to our hypothesis, the microbial sealing ability of the conical prosthetic connection was evaluated in a double-verification study set-up. No bacterial translocation was detected, neither from nor into the Acuris abutment system. Amounts of both living and dead bacteria were measured using agar plate spreading and qrt-PCR. Agar palates exhibited no bacterial growth for the implant specimens according to the negative control. Results of qrt-PCR measurement for the bacterial exit out of the system confirmed no significant difference between the implant specimens and the negative control , moderate colonizing Actinomyces naeslundii , Fusobacterium nucleatum  and late colonizing Porphyromonas gingivalis  strains was prepared.A two-fold study set-up was designed in order to evaluate the bidirectional translocation of bacteria through the sealed conometric system. A total of 10 conometric abutments  were joined to screw implants  with a new titanium abutment screw and torqued to 24 Ncm, as recommended by the manufacturer . The expn = 8) were used as test specimens, one implant was used as the negative control (n = 1), and for the positive control, 4 µL pure bacterial mixed culture solution was utilized (n = 1). One of the total ten implant systems was previously used for a pretest and could not be included in this set-up. The occlusal opening of eight Acuris abutments that were previously mounted on the respective implants was filled with 4 µL of bacterial mixed culture of anaerobes. Corresponding TiN-caps were seated on the abutments and fixed according to the manufacturer’s recommendation , one implant was used as a negative control (n = 1), and for the positive control, 4 µL pure bacterial mixed culture solution was utilized (n = 1). For this purpose, after completion of the first part of the study design, all implant–abutment conometric samples were decontaminated using a DNA AWAY Kit for surface and device decontaminations . The process was followed by autoclave sterilization. Cleaning success was controlled with qrt-PCR.In order to confirm the bacterial exit results, specimens were tested for bacterial leakage into the conometric system. In this test set-up, nine implant specimens were tested  was subject to scanning electron microscopy . Both, the accuracy of fit of the morse taper coupling and the marginal opening of a monolithic zirconia crown  that had been cemented extraorally on the Acuris TiN-Cap before its conometrical seating, were analyzed microscopically. The measurements were determined at two predefined reference points  according to the characteristic design of the components . The Acun = 4—four amplification values per positive control), negative controls  and implant specimens  were used to calculate mean bacterial DNA amount in mg/mL . The mean value of qrt-PCR amplification results for positive controls (in mg/mL  counts. Within the limits of the in-vitro study it could be concluded that the Acuris conometric interface did not allow for bacterial translocation under non-dynamic loading conditions. While the luting gap between the prefabricated TiN-cap and the ceramic crown was within the clinically acceptable range, no microgap could be detected at the cone-in-cone Acuris junction by SEM analysis.


On the other hand, interactions across different chromosomes  have received less attention, being often regarded as background noise or technical artifacts. This viewpoint has been challenged by emerging evidence of functional relationships between specific trans chromatin interactions and epigenetic control, transcription, and splicing. Therefore, it is an intriguing possibility that the key processes involved in the biogenesis of RNAs may both shape and be in turn influenced by inter-chromosomal genome architecture. Here I present the rationale behind this hypothesis, and discuss a potential experimental framework aimed at its formal testing. I present a specific example in the cardiac myocyte, a well-studied post-mitotic cell whose development and response to stress are associated with marked rearrangements of chromatin topology both in cis and in trans. I argue that RNA polymerase II clusters  and foci of the cardiac-specific splicing regulator RBM20  exemplify the existence of trans-interacting chromatin domains (TIDs) with important roles in cellular homeostasis. Overall, I propose that inter-molecular 3D proximity between co-regulated nucleic acids may be a pervasive functional mechanism in biology.Three-dimensional (3D) genome organization has emerged as an important layer of gene regulation in development and disease. The functional properties of chromatin folding within individual chromosomes (i.e., intra-chromosomal or in  N6-methyladenosine (m6A), but also N1-methyladenosine (m1A), 5-methylcytosine (m5C), and pseudouridine  are 5'-capped, spliced, and finally 3'-polyadenylated and cleaved, altogether generating mature mRNAs. It is well established that these central steps of mRNA biogenesis occur in close proximity and at the same time and place as transcription  involving loci on different chromosomes whose nucleic acids are co-regulated by the same set of protein factors. I further propose that the resulting architecture is not just practical  but also functional, by which I mean that it promotes the efficacy and fidelity of RNA biogenesis.With regards to mRNA biogenesis, over 20years ago Peter Cook and Ana Pombo proposed that transcription may be largely spatially restricted to “transcription factories” . These ctrans, and with some specific examples borrowed from cardiomyocyte biology. Having outlined the supporting evidence, I then flesh out my hypothesis and propose an experimental framework for its formal testing. I conclude by discussing open questions in the area. Throughout I focus on mammalian genome regulation and architecture since it is well established that it involves unique mechanisms not seen in other model organisms such as invertebrates, plants, and yeasts (which in turn have their own peculiarities). Nevertheless, I present selected examples from the broader spectrum of eukaryotic life, as I speculate that the general principle behind the notion of TIDs may be broadly applicable.I begin by extensively reviewing the main features of inter-chromosomal genome architecture. I follow this with a shorter discussion of the possible role of genetic variants in cis) interactions resulting from a variety of hierarchical features at different genomic scales. These include various kinds of DNA loops (such as promoter-enhancer pairing), topologically-associating domains , and A/B compartmentalization (chromosome-wide separation of active/inactive chromatin due to inter-TAD contacts). A large body of work has recently emerged to show that while a large fraction of intra-chromosomal genome structure is largely invariant across cell types and states, dynamic changes do occur at all levels and can contribute to gene regulation both during development and in disease  interactions are much less understood and studied. The motivations are at least three-fold. First, there is a widespread belief in the community that most inter-chromosomal interactions are just noise resulting from Brownian movements of chromatin, and/or technical artifacts arising from proximity ligation-based methods . Indeed, one commonly used quality control measure for 3C-type studies is the ratio of cis/trans interactions, which is expected to be high. While trans interactions are certainly more noisy than cis ones, as discussed below, abundant evidence indicates that there is nevertheless signal worth measuring. Which brings us to the second problem, namely the relative immaturity of statistical and computational approaches to confidently identify trans interactions. Indeed, due to their different biophysical nature trans interactions cannot be simply treated analytically with the now well-established methods developed for cis ones . Finally, compounding on this limitation is the fact that trans interactions are less frequent than cis ones by a factor of ~2–5 (depending on the cell type and assay), while being also spread across a much wider contact matrix 2D space (and even a broader one if we consider three-way interactions). This results in data that is very sparse unless a prohibitively expensive sequencing depth is utilized, particularly in the case of genome-wide studies. Statistical approaches to detect trans interactions have therefore either low power (few data points) or low resolution (genomic bins of large size are used to aggregate the sparse interaction counts). As a result of these three main factors, trans interactions are often dismissed early during the analysis or only used for very basic broad assessments of chromosome territories.In contrast to this, inter-chromosomal , this would still account for 1/20th of the data. Is it not worth to analyze it beyond just confirming the well-established concept of chromosome territories?The well-established concept of chromosome territories is perhaps the motivation leading many to dismiss inter-chromosomal architecture, relegating its possible relevance to “exotic” examples such as sperm cells , this same feat can now be achieved from “conventionally-sized” datasets with moderate coverage using the SNIPER algorithm  each human chromosome possesses a few regions collectively explaining the majority of trans interactions; (2) such regions are characterized by high transcriptional activity; and (3) they interact with each other in a largely indiscriminate fashion. This led the authors to speculate that such domains intermingle chiefly because they happen to all be accessible within the nuclear interior, “escaping” the constraints of their respective chromosome compartments. However, refinements in the quantitative analyses of 3D structure population models showed that some inter-chromosomal interactions are actually relatively specific and liked to gene co-regulation , can extrude to the surface and engage with each other in the proximity of transcription factories, tRNA factories, polycomb domains, the nucleolus, nuclear speckles, and/or other yet-to-be clarified nuclear subcompartments .In the following subsections, I provide a more in-depth review of the nuclear subcompartments that are most relevant to the hypothesis of this article. For this, I focus on structures involved in the biogenesis of mRNAs. The relationship between chromatin organization and other nuclear subcompartments involved in non-coding RNA biogenesis, such as the nucleolus and tRNA factories, is reviewed extensively elsewhere .Trans interactions between active genes may be indiscriminate and dictated by the random motion of open chromatin in the euchromatic nuclear interior, or could be highly specific. A few examples of the latter category have accumulated over the years, indicating that both mechanisms likely contribute to inter-chromosomal architecture.As just discussed, highly transcribed genomic domains are those most often involved in inter-chromosomal interactions. Accordingly, inhibition of transcription reduces chromosome intermingling . Moreovecis- and in trans- often colocalize at the site of transcription factories (Hbb (beta-globin) gene, and 7% for the inter-chromosomal interaction between Hbb and Hba , which is substantially higher than the expected frequency for a random interaction (~1%). In this context cis interactions involving Hbb require initiation of transcription, arguing for an active recruitment model . Remarkably, both the recruitment of Hbb to transcription factories and its colocalization with its trans partners Hba and Epb4.9 requires the erythroid transcription factor Klf1  and a third gene on a different chromosome (SLC6A5) can associate as part of a NF-κB-dependent multigene cluster  had no effect on the formation of RBM20 foci and on the splicing of CAMK2D and CACNA1C. This excludes the possibility that titin is somehow involved in the control of RBM20 localization and/or activity, and indicates that the TTN pre-mRNA has specific trans-acting effects.While much less established, the role of mRNA biogenesis steps besides transcription in inter-chromosomal regulation is beginning to emerge. Indeed, my colleagues and I recently showed that in cardiomyocytes derived from hESCs (hESC-CMs) foci of the muscle-specific splicing factor RBM20 mediate inter-chromosomal interactions between some of its key target loci . We alsong sites . In celltrans-acting “zip coded splicing factory.” Whether this is an exceptional case or the first of many examples of this type of regulation is yet to be determined.Overall, we concluded that RBM20 foci represent the first example, to the best of our knowledge, of a nuclear subcompartment specialized in the regulation of splicing for a defined subset of genes located across multiple chromosomes, in other words, a trans interactions involving a single gene and one or more regulatory sequences that either repress or promote its expression. Repressive regulations include the inter-chromosomal interaction between the T helper cell 2 locus control region (TH2 LCR) and the IFN-γ gene  to an active intra-chromosomal engagement with Sox17 , an important regulator of gene silencing through the deposition of the repressive histone mark histone 3 lysine 27 trimethylation (H3K27me3). PcG factors such as the catalytic subunit EZH2 are involved in both intra- and inter-chromosomal interactions  identified by genome-wide association studies (GWAS) representing non-synonymous substitutions in protein-coding genes . It is gis eQTLs . Neverth30 genes . An even58 genes .trans eQTLs remains unclear, but three major possibilities have been proposed so far (trans eGene), perhaps as a result of an inter-chromosomal regulatory interaction such as those described in the above section “Trans-Acting Regulatory Regions”. Secondly, the gene variant may act indirectly through a third actor: a proximal gene (cis eGene) whose expression is directly regulated by the SNP and that in turn can directly modulate expression of the trans eGene. Proposed mechanisms behind the mediating action of such a cis eGene include its function as a transcription factor, micro RNA, or another regulatory non-coding RNA  or through modulation of a cis eGene. Indirect regulation or reverse causality can be tested through mediation analysis, which assesses whether a third factor (either a cis eGene or a phenotype) significantly accounts for the observed trans eQTL-trans eGene relationship. Using this approach it was found that 20–35% of trans eQTLs can be explained by the alteration of a mediating cis eGene  consortium reported similar observations, as 31.6% of lead trans eQTL identified across 27 tissues proved to be also cis eQTL, and 77% of these were predicted to contribute to the trans effect by mediation analysis . On the other hand, the authors found that approximately one third (13/37) of these cis eGenes shared the same regulatory motif with the trans eGenes. A similar enrichment of transcription factor motifs in the promoter of trans eGenes linked to trans eQTL has also been reported elsewhere  some TRHs mediate trans eQTL effects. Along these lines, trans eQTLs identified by the GTEx consortium are specifically enriched for CTCF binding sites , suggesting that trans contacts were not inflated by suboptimal capture of the native chromatin environment. A high proportion of trans interactions in cardiomyocytes was observed also in other in situ Hi-C experiments in cardiomyocytes from other hESC lines . Such correlation was even more remarkable for left ventricle trans eQTL gene clusters, reaching 77.2%. Therefore, cardiomyocyte gene expression seems to be particularly affected by genetic variation in trans, providing further evidence for the possible role of inter-chromosomal genome organization in this cell type.As discussed in the above section “ samples . On the 50 genes . A simil samples . Notablyper se. In fact, the contribution of transcription factories to 3D genome topology has been hypothesized at least a decade ago  since some of the underlying mechanisms (described in the next subsection) likely translate to the specific regulations involved in the biogenesis of various classes of RNAs. Moreover, while this hypothesis is articulated specifically for inter-chromosomal regulations, it likely also extends for intra-chromosomal ones. Indeed, there is abundant evidence that ed genes .Trans-Acting Regulatory Regions” and “Polycomb Domains”) and of “zip coded” transcription factories (section “Transcription Factories”), but are not limited to this. Further regulation can be achieved by pre-mRNA processing steps in both specialized nuclear speckles (section “Nuclear Speckles”) and tissue-specific splicing factories (section “Splicing Factories”). Moreover, I anticipate that future studies will reveal how other aspects of RNA biogenesis, such as RNA post-transcriptional modifications, are similarly involved in regulating inter-chromosomal architecture.With regards to the first statement, relevant RNA biogenesis steps include the regulation of transcription, as exemplified by the existence of specific regulatory inter-chromosomal interactions , chromatin modifiers , genome organizers , the transcriptional machinery , splicing regulators , and other protein factors involved in various steps of RNA biogenesis. RNA species with a structural and/or enzymatic role are also likely involved, though this will need to be determined by further studies.trans-interacting (chromatin) domain” (TID). This acronym purposely reminisces TAD in order to stress the conceptual similarity between these structural features of the genome. Similarly to TADs, TIDs are statistical constructs that can be observed only at a population level , the probability of intra- and inter-chromosomal interactions becomes similar. Therefore, very long-range intra-chromosomal interactions may be effectively considered analogous to inter-chromosomal ones when computing and studying TIDs.While the term “RNA factory” describes the overall subnuclear structure, I propose that the genomic loci involved could be referred to as a “on level . In indiI propose a three-step process :Trans-acting regulatory factors bind onto core co-regulated nucleic acids.Regulatory factors from multiple nucleic acids aggregate to form new clusters and/or enrich pre-existing ones.Accessory nucleic acids are brought into proximity the cluster until a dynamic equilibrium is reached.trans-acting regulators include transcription factors, epigenetic remodelers, genome organizing proteins, and Pol II and its cofactors, which engage with DNA either directly or through its associated histones. For splicing factories, the key trans-acting factors, namely splicing regulators, require prior initiation of transcription to generate the target RNA molecules. In both cases, multiple binding sites are found on either the same or multiple molecules (in the case of RNAs). The model stipulates that one or few nucleic acids with the highest density of binding sites function as “core” element(s), dictating the local accumulation of the regulators.Step one involves different factors for distinct classes of RNA factories. For transcription factories, Step two is the result of either active mechanisms  and/or Brownian movements of nucleic acids extruding from the core of chromosomal territories. Changes in the local chromatin environment may facilitate this process and increase intermixing in the inter-chromosomal space. Stabilization of core inter-chromosomal interactions involve either high-affinity interactions between factors that can form dimers and/or higher-order multimers , as well as weaker interactions such as those involving low-complexity regions characteristic of proteins that can undergo liquid-liquid phase separation . In someIn step three, large and growing clusters of regulators stabilize additional inter-chromosomal interactions with co-regulated nucleic acids characterized by lower binding density for the regulators. These “accessory” DNA or RNA molecules are those that most strongly benefit from interfacing with the RNA factory, which augments the efficiency of their regulation. As the RNA factory grows in size, it eventually hits steric and thermodynamic limitations that stabilize its further growth to a state of dynamic equilibrium.Throughout the process, regulators can be either general and housekeeping, binding to nucleic acids with low degree of specificity or no specificity at all , or highly specific and responsive to cell states . The resulting RNA factory is more or less cell state-specific depending on the relative balance of these two classes. “Zip coded” transcription and/or splicing factories result from highly specific regulators enriching for their target nucleic acids.Besides descriptive investigations, at least three types of perturbations can be used to test the model -D:Depletion of core nucleic acids.trans-acting regulatory factors.Addition of clustered binding sites for trans-acting regulatory factors.Depletion of trans-acting regulatory factor(s) to one or more of the core co-regulated nucleic acids. For DNA loci, this can be achieved by mutations or larger deletions. For RNAs, this can be achieved by mutations of the originating genes to prevent their transcription, or through epigenetic silencing. For both DNA and RNA perturbations, an important control experiment is to reduce the protein levels of the gene(s) of interest by a similar magnitude as the test perturbation, but without affecting the abundance of binding sites for the trans-acting regulatory factors. This is key to exclude that any observed effect is not simply due to impairment of intermediate protein-mediated regulations. For instance, one can generate knockout and/or hypomorph mutations that do not alter the DNA sequence in the regions of interest and/or do not alter RNA levels. The predicted outcomes of this perturbation are: (1) impaired clustering of the trans-acting regulators; (2) impaired inter-chromosomal interactions between loci in the TID; and (3) decreased efficiency in the regulation for accessory nucleic acids (The first experiment involves reducing or completely preventing the binding of ic acids .trans-acting regulatory factor(s). For instance, one can express transgenic copies of a core nucleic acid or an artificial sequence containing arrayed binding sites. In the former case, the transgene can be mutated to prevent protein translation and bypass the need to control for this aspect. An RNA can also be upregulated through epigenetic mechanisms. The predicted outcomes of this perturbation are opposite to those outlined above . Unfortunately, most of the analytical tools for 3C-type data developed so far have focused only on intra-chromosomal interactions (reviewed in cis and trans interactions (trans interactions in mammalian systems, such as their enrichment for loci in the A compartment and their different distribution across small and large chromosomes. A similar limitation applies to existing network-based strategies to study inter-chromosomal interactions both from bulk Hi-C (trans interactions resulting from the random intermixing of neighboring chromatin domains.The identification of significant inter-chromosomal interactions must rely on statistical approaches that are specifically designed for this task. Indeed, approaches originally devised for the analysis of ractions . This apulk Hi-C  and singulk Hi-C . StatistAnother promising venue is the modeling and study of 3D genome structure populations, which as discussed in the section “Nuclear Subcompartments” has already provided important insights in inter-chromosomal genome architecture . Neverthtrans-acting factors that contribute to their formation and/or function. For transcription factories, the enrichment of DNA binding motifs within the regulatory regions proximal to the involved loci may provide testable hypotheses as to the transcriptional regulators involved. RNA binding motifs enrichment on pre-mRNAs may similarly help in the context of RNA splicing factories. Further evidence could be gathered by integration of data modalities directly probing for DNA and/or RNA binding of candidate factors. However, these analyses are limited by the availability of well-annotated binding motifs and/or of experimentally determined binding profiles for the cell state of interest.A second key challenge towards clarifying the role of RNA factories is the identification of the in situ proximity labeling, for instance by relying on promiscuous biotinylating enzymes to covalently tag nearby proteins for subsequent streptavidin-based purification and identification through mass spectrometry. These methods have been successfully used to determine protein-protein interactions, and their implementation to study locus-specific regulations are also emerging (reviewed in Robust methods to unbiasedly identify factors that interact with specific nucleic acids are direly needed. Biochemical purification approaches relying on direct capture of nucleic acids have been developed, but so far showed limited sensitivity and specificity (reviewed in Last but not least, the functional characterization of RNA factories is key to determining their physiological relevance. As elaborated in the section “Experimental Testing Strategies and Predicted Outcomes”, mechanistic studies may involve various means of perturbing RNA factories through loss- and/or gain-of-function. While these studies do not pose unsurmountable challenges due to recent advancements in genome editing technologies, they remain time-consuming. Characterization of putative RNA factories may be performed with more throughput by leveraging on functional screening strategies based on the combination of CRISPR/Cas9 perturbations and transcriptional phenotyping through single-cell RNA-sequencing (reviewed in trans-acting regulators onto core nucleic acid targets, leading to their aggregation and to the subsequent recruitment of accessory co-regulated nucleic acids. Future studies will prove, disprove, or modify this model. It will also be key to test the pervasiveness of such regulation to determine whether it involves only a few specific nucleic acids or it is a general property controlling many loci.In summary, emerging evidence suggests a functional bidirectional link between the regulation of RNA biogenesis and inter-chromosomal 3D genome architecture. In other words, the formation of RNA factories organized around TIDs may be not just an emergent property of gene regulation, but also a feature needed to maximize the fidelity and efficiency of such regulations. This is a testable hypothesis based on a clear mechanistic model: the clustered binding of via a combination of specific and non-specific interactions, possibly leading to liquid-liquid phase separation (reviewed in Many key open questions remain in this area. First off, what is the biochemical and/or biophysical mechanism that explains the co-localization of certain nucleic acids from different chromosomes? Besides transcription factors , 2018, sIs dysfunction of RNA factories and/or TIDs involved in the pathogenesis of human disease? Cancer has been linked to alteration of chromosome territories  and of sMoving beyond RNA biogenesis, is proximity between co-regulated nucleic acids a general phenomenon in biology? It is tempting to speculate that other key biochemical reactions such as DNA repair and replication may also benefit from the increased efficiency and fidelity led by local accumulation of relevant regulators. Overall, increasing our spatial awareness of nuclear regulations may prove a worthy and impactful pursuit.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Particularly, the non-vacuum wet-chemical buffer layer formation step has remained a challenge and has acted as a bottleneck in industrial implementations for mass-production. This technical note deals with the comparative analysis of the impact on different methodologies for the buffer layer formation on CIGS solar panels. Cd(1-x)ZnxS S) thin films were prepared by chemical bath deposition (CBD), and chemical surface deposition (CSD) for 24-inch (37 cm × 47 cm) patterned CIGS solar panel applications. Buffer layers deposited by the CBD method showed a higher Zn addition level and transmittance than those prepared by the CSD technique due to the predominant cluster-by-cluster growth mechanism, and this induced a difference in the solar cell performance, consequently. The CIGS panels with S buffer layer formed by the CBD method showed a 0.5% point higher conversion efficiency than that of panels with a conventional CdS buffer layer, owing to the increased current density and open-circuit voltage. The samples with the CSD S buffer layer also increased the conversion efficiency with 0.3% point than conventional panels, but mainly due to the increased fill factor. Scale-up to large-area CuSe The concentrations of CdSOCompositional analysis of the CdS and S layers was performed by scanning electron microscopy combined with energy-dispersive X-ray spectroscopy . X-ray fluorescence spectroscopy  was used for the thickness and compositional analysis of the CIGS layer. Scanning AES  depth profiling was performed on the samples as a supporting tool using a Nanoprobe (PHI 700) system. The formation of CIGS/S/ZnO:Al structure was verified with ex-situ HRTEM  imaging at an accelerating voltage of 300 kV. Both AES and HRTEM measurements were performed at KIST (Korea Institute Science and Technology) Advanced Analysis Center. The transmittance of the buffer layer was determined by ultraviolet-visible (UV/Vis) spectroscopy measurements . The solar cell performance was characterized by current-voltage (J-V) measurements performed by using a solar simulator under AM1.5 equivalent illumination . The external quantum efficiency (QE) was also provided to determine the change in the bandgap of the buffer layers used for the working device by measuring the short-circuit current using spectrally resolved monochromatic light .2+ concentration can inhibit complex formation between the complexing agent NH3 and the Cd2+ ion, thus reducing the growth rate of the film 2+ and S2− in the solution [3)4]2+ with S2−. In this case, the CdS nuclei formation takes place on the substrate under the ion-by-ion growth mechanism instead of adhesion of the particles formed by the cluster-by-cluster mechanism in the solution, such as ZnS particles.Interestingly, the EDX analysis in solution ,33. MeanI–V characteristics of the CIGS PV panels with different buffer layer treatments are shown in 2+ ions in the solution under cluster-by-cluster growth regime and are integrated into the CdS which is formed under a mixed growth regime (cluster-by-cluster and ion-by-ion growth mechanism (OC/cellV) and current density (SCJ). This result well coincides with the previous report that Zn addition increases the ban-gap of the buffer layer. It thus results in lower optical loss from the buffer layer  and a suitable conduction band offset with CIGS light-absorber layer due to a reduced conduction band discontinuity at the S/CIGS junction [OCV) slightly increases by the change on the Zn concentration from 0 to 0.6 (relative to the Cd), above which the presence of spike at the interface hinders a feasible electrons transfer from the CIGS. The echanism b). We emjunction ,35,36. HFF). It is obvious that the increase in FF is primarily attributed to the improved shunt resistance (shR), as the series resistance (sR) remained nearly unchanged S-based buffer layer formed by CSD, the efficiency gain was approximately 0.34 % absolute point, which is mainly resulted from the increased fill factor S formed on a CIGS layer by using the CSD method g confirmeviously ,22,37. FF) as plotted in sR) in FF consequently. As reported previously [As evidenced in external quantum efficiency (EQE) measurements a, an impeviously ,39, the SCJ and OCV. However, the use of CSD with the same solution led to improved cell efficiency because of the increased FF, while the SCJ remained unchanged. EDX and QE results confirmed that the concentration of the Zn component incorporated into the CdS layer differs depending on the surface type. Unlike the CBD buffer layer prepared on the CIGS PV panels, the relatively small quantity of the Zn was found from the S layer prepared by the CSD method. This result could be evidence for the fact that ion-by-ion growth dominant mechanism during the CSD process. Despite the beneficial effect of using the S buffer layer, irrespective of the type of the method, this approach can restrict the use of S for the mass production line due to the reduced deposition rate. This technical note with an equipment-wise approach can be used as a technological guideline for the manufacturers to further develop of wet chemical buffer layer deposition system for the CIGS PV production line. Apart from the statistical and microscopic analysis provided in this technical report, in-depth optoelectronic characteristics must be addressed along with a cost-effectiveness analysis for further development. S layers were grown by chemical deposition using CBD and CSD processes. The transmittance and growth rate of the S buffer layers on the ITO-coated glass were directly and inversely proportional to the Zn addition level, respectively. CIGS solar panels with S buffer layers deposited by both CBD and CSD processes have shown improvement in conversion efficiency. In particular, the conventional CBD process with Cd and Zn components led to increased cell efficiency because of the increase in


The growth plate is cartilage tissue found at the end of long bones in children, responsible for longitudinal bone growth. Injuries to the growth plate cartilage often lead to unwanted bony repair, resulting in growth disturbances such as limb length discrepancy and angulation deformity in children. There is currently no clinical treatment that can fully repair an injured growth plate. Tissue engineering is promising for regeneration of growth plate. Adipose-derived stromal vascular fraction highlight the promising potential as tissue engineering therapy for inducing regeneration of injured growth plate and able to reduce the formation of bony repair that can lead to deformity and limb length discrepancy. Using an animal model of growth plate injury, bone bridge formation is evaluated after 28 days using Enzyme-linked Immunoassay, radiology, histopathology and Immunofloresence examination. Radiological analyses performed by evaluation of grey value using ImageJ software and diameter bone bridge measured from the end to end distance between uninjured growth plate evaluated by histopatology examination. Enzyme-linked Immunoassay and immunofloresence are used to evaluate chondrocyte and chondrogenic marker within the defect. The result shows in group with Adipose-derived stromal vascular fraction have a significant lower bone bridge formation compare to positive control group. This current study represents the first work that has utilized this animal model to investigate whether Adipose-derived stromal vascular fraction can be used to initiate regeneration at the injured growth plate. •Injuries to the growth plate cartilage often lead to unwanted bony repair, resulting in growth disturbances•There is currently no clinical treatment that can fully repair an injured growth plate•Tissue engineering is promising for regeneration of growth plate.•Adipose-derived stromal vascular fraction (AD-SVF) highlight the promising potential as tissue engineering therapy•AD-SVF decrease bone bridge formation on growth plate injuries model on Rat The gA greater understanding of these biological mechanisms could lead to more effective therapeutic interventions for children suffering from growth plate injuries. An earlier study identified four different phases of injury responses in a rat growth plate injury model 28) - the inflammatory, fibro genic, osteogenic and bone bridge maturation remodeling responses occurring during days 1–3, 3–7, 7–14, 10–28, respectively ..12].The advantage of SVF over ADSCs is believed to be in two fundamental areas. Firstly, although similar in properties such as immunomodulation, anti-inflammatory, angiogenesis, and so forth, the distinctive, heterogeneous cellular composition of SVF may be responsible for the better therapeutic outcome observed in comparative animal studies. Secondly, unlike ADSCs, SVF is much more easily acquired, without the need for any cell separation or culturing conditions. Thus, the therapeutic cellular product is instantaneously obtained and has minimal contact with reagents making it comparatively safer and subject to the fulfilment of lesser regulatory criteria.In order to create living tissues, as well as integrate living engineered tissue with native host tissues, cells must be present. The presence of AD-MSC  in SVF will increase the number of progenitor cells in the area of growth plate injury. In addition, some of these cells were found to be MSC like as they expressed the stem cell marker alpha-smooth muscle actin. Furthermore, at the early time point day 4 post-injury, some of these cells already expressed chondrogenic marker collagen-2 and/or osteogenic marker Runx 2, suggesting that this influx of mesenchymal cells may contain a myriad of cells including MSC-like cells, osteoprogenitor cells, pre-osteoblasts, and/or pre-chondroblasts (either pre-existing or newly derived from the infiltrated MSCs). Further indicative of potential multipotency for some of these cells, previous studies have shown that differentiation of these cells and growth plate repair outcomes could be influenced by some growth factors or other signals occurring during this phase , 8]..8].TGFβ is a potential induction factor for chondrogenic differentiation from MSC and other mature tissues derived from MSC. Insulin growth factor I (IGF-1) is one of the growth factors that also has a role in repairing growth plates. Its role in chondrocytes/cartilage includes stimulation of extracellular matrix synthesis and a decrease in matrix catabolism except in OA cartilages. IGF-I induces a large number of anabolic effects and reduces catabolic responses. The chondrogenic differentiation of MSCs is induced by IGF-I but is increased when IGF-I and TGF-B1 are used in combination. Fibroblast growth factors (FGF) involved in the process of repairing growth plates is twofold, namely FGF-2 and FGF-18. In MSC, FGF-2 has the role of increasing proteoglycan synthesis and cell proliferation while FGF-18 has no effect.Stromal Vascular Fraction (SVF) also contains many T regulatory cells (T reg) which are cells in the immunological system. At the growth plate that is injured will release a biomolecule called damage associated molecular pattern (DAMP) which will give effect to the T reg to regulate so that the monocytes turn into macrophages type 2. The more T reg is in the area of the growth plate injury, it is expected there are more and more type 2 macrophages in the area of injury. Type 2 macrophages play a very important role because these type 2 macrophages will initiate a repair process in growth plate injury and have an anti-inflammatory effect that will prevent the occurrence of chronic inflammation which is very detrimental in the ideal healing process. Activation of the pathogenesis pathway through the SoX9 transcription factor will change AD-MSCs to chondrocytes marked by the formation of Col-2a. if this happens it will prevent the occurrence of bone bridge in growth plate injury .From the result we can conclude that AD-SVF prevent the bony bridge formation in injured growth plate in Rat. This study represents the first work that has utilized this rat animal model to investigate whether AD-SVF combine with freeze-dried amniotic membrane can be used to initiate regeneration at the injured growth plate. Questions to be addressed in the future may include analysis of the repair growth plate at different phase of healing process post-operatively to determine the exact mechanism of regeneration process. Second, the potential of MSCs ‘pre-differentiated/committed’ to chondrogenic lineage in vitro prior to transplantation - to improve the possibility of chondrogenic tissue repair. And the last, tracking of the introduced MSCs to determine their role in the repair response - whether MSCs directly contribute to the repair tissue or facilitate the recruitment of endogenous cells. Further study is required to evaluate whether MSC may be a viable therapeutic option for the biological regeneration of the growth plate following injury.


Duchenne muscular dystrophy (DMD/Duchenne) is a progressive X-linked disease and is the most common pediatric-onset form of muscular dystrophy, affecting approximately 1:5000 live male births. DNA testing for mutations in the dystrophin gene confirms the diagnosis of this disorder. This study involves assessment of screening newborns for DMD using an immunoassay for muscle-type (MM) creatine kinase (CK) isoform—the GSP Neonatal CK-MM kit. Comparisons were made with CK activity determination by fluorescence measurement. In addition, the study evaluated the effect of gestational age, age of infant at time of sampling and how stable the CK-MM was over time. This assay discriminates well between normal, unaffected and Duchenne affected populations and is suitable for Duchenne newborn screening. Duchenne muscular dystrophy (Duchenne/DMD) is an X-linked rare disease, affecting approximately 1:5000 live male births worldwide) . DMD occDue to muscle degeneration, individuals with Duchenne have markedly elevated creatine kinase (CK) levels in blood. The elevation of CK in serum of DMD patients was first reported long ago ,9 and deNovel molecular and non-molecular therapies for treating Duchenne are under development . SteroidOne therapy , which targets nonsense mutations, has received conditional approval in Europe and in Australia for use in individuals with Duchenne. The U.S. Food and Drug Administration (FDA), has granted orphan drug designation to Ataluren for the experimental development of Duchenne. In September of 2016, FDA granted approval to the first Duchenne-specific drug therapy in the U.S. via the accelerated approval pathway to Eteplirsen, known commercially as EXONDYS 51, an exon skipping drug .Historically, the primary approach to Duchenne newborn screening (NBS) has been through the detection of CK enzyme activity in serum by fluorescence measurement ,18. CK lThough the CK assay by fluorescence measurement works well with serum samples, it is not well suited for DBS . Due to This paper presents an exploration of the use of a novel immunoassay to quantitatively determine CK-MM concentration in DBS to screen newborns for Duchenne. Comparisons were made with CK activity determination by fluorescence measurement. In addition, the study evaluated the effect of gestational age, age of infant at time of sampling and how stable the CK-MM was over time.Location 1: PerkinElmer, Wallac Oy in Finland using anonymous DBS samples from male and female newborns born in California, USA and obtained from the California Biobank Program;Location 2: Danish Center for Neonatal Screening, Statens Serum Institut (SSI), in Copenhagen, Denmark using anonymized DBS specimens obtained from Danish Neonatal Screening Biobank (DNSB or DNS-Biobank).Studies of the use of an immunoassay to detect Duchenne were performed at two different locations:® 903 filter paper. Archived, leftover specimens that had been stored in −20 °C before inclusion to the study were used. In addition to samples collected from presumed healthy newborns, samples from newborns that had been diagnosed later with Duchenne were included in the study at both locations to enrich the study populations. The Duchenne diagnoses had been confirmed by treating physicians and by molecular genetic testing.The DBS sample collection device was Whatman® Neonatal CK-MM kit.Location 1 studies were devoted to the evaluation of long-term sample stability, determining a preliminary range of CK-MM concentration in a US newborn population and establishing a preliminary cut-off threshold that would be used to categorize the samples as screen positives and negatives. In addition, the GSP CK-MM assay was compared to the fluorescent screening test method traditionally used to measure the CK activity . LocatioThe studies were obtained in accordance with requirements of study location and conducted in accordance with the Declaration of Helsinki, and Institutional Review Board (IRB) or ethic committee approvals or waivers for using anonymous DBS samples and associated data. The Committee for the Protection of Human Subjects, California Health and Human Services Agency (CPHS) had given the favorable opinion for the use of the specimens  at Location 1. In addition, local IRBs at the clinical sites in California had given approvals or waivers to match the Duchenne diagnosis information to the Duchenne affected specimens archived in the biobank. The inclusion of DMD affected specimens required a signed consent by a parent or guardian based on approved protocol by the above-mentioned Institutional Review Boards (IRBs). The California Biobank Program gave a non-patient identifying specimen ID number to all specimens.The specimens were included in the study at location 2 only if the parents/guardians of the newborn had not opted out the further use of the DBS used for NBS. The Danish ethics committee, De Videnskabsetiske Komiteer Region Hovedstaden, reviewed the study protocol and gave a waiver from ethics committee approval . The Statens Serum Institut (SSI) gave a non-patient identifying specimen ID number all specimens.® Inducer (cat. no. 3304-0010), Wash Concentrate (cat. no. 4080-0010), and fully automated GSP instrument (cat. no. 2021-0010) with software version 1.4 . The GSP CK-MM assay principle has been described by Moat et al. [The CK-MM concentration analysis was done using the GSP Neonatal Creatine Kinase -MM kit , DELFIA® inducer solution to the wells dissociates the europium ions from the tracer antibody attached label chelates into solution where they form highly fluorescent chelates. The fluorescence of each sample is proportional to the concentration of CK-MM.The GSP CK–MM assay is a solid phase, 2-site immunofluorometric assay. CK-MM is eluted by the addition of the assay buffer and of the anti-CKMM- Europium tracer solution. CK-MM in the sample reacts with the immobilized mouse monoclonal antibodies and europium chelate–labeled mouse monoclonal antibodies, which recognize 2 separate antigenic sites on the molecular surface of CK-MM. After the incubation step with interval shaking, the paper disc and excess unbound label is washed away from the wells. Addition of the DELFIAN-acetylacetyl-l-cysteine (NAC). CK catalyzes the transphosphorylation of ADP to ATP. Through a series of coupled reactions, the NADPH produced is measured at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The fluorescence is directly proportional to the NADPH concentration. The same calibrator and control material as for CK-MM concentration analysis were used for CK activity analysis . The CK-activities of the calibrators were calculated based on the manufacturer’s reported activity of the pure CK-MM and the dilutions were made for each calibrator level. CK-NAC reagent was obtained from Thermo Scientific (cat. no. TR14010), Di (adenosine-5′) pentaphosphate (DAP) trilithium salt from Merck (cat. no. D6392) and fluorescence was measured with Victor2D Multilabel Counter .The CK activity analysis measures the enzyme activity of CK in the DBS using fluorometry to determine the occurrence of DMD. The assay procedure is based on the reaction principle used by Orfanos and Naylor , employiStatistical analyses were conducted with R, Spotfire S+ 8.1 and Minitab 18 software. The data was visualized with box plots and scatter plots and relevant descriptive statistics  were calculated. In addition, high percentiles  were calculated and used as cut-off values in the study to classify the samples as screening positive and negative. Pearson and Kendall’s Tau correlation co-efficient were calculated and the relationship between the bloodspot CK-MM concentration analysis and CK activity was estimated using Passing–Bablok regression. Long term sample stability was evaluated using storage time specific population medians and visualizations.There were two different sets of DBS samples from a USA population used. The first DBS set included 1200 samples  to determine the long-term stability of CK-MM in DBS when stored in a freezer (−20 °C). The CK-MM concentration across archived samples were comparable to fresh samples up to 7 years of storage time, and that variability within any given storage time point was similar between archived time points and fresh samples. The population median of 10- and 15-year timepoints showed a smaller CK-MM concentration in DBS samples .Second set of DBS samples included 721 anonymous newborn samples of which 19 had been affected by Duchenne, one sample was from a Duchenne carrier and one from a Becker carrier. Samples had been stored for approximately 7–12 years before the bloodspot CK-MM concentration analysis. The storage times of affected and unaffected samples were matched. The age at the time of initial specimen collection varied between 12 and 61 h of age and the birth weight varied between 780 and 5555 g. Three hundred thirty-two (47%) of 700 samples were collected from females and 368 (53%) from males. The CK-MM concentration method was compared to the CK activity method. Same quality controls were run with both methods .p < 0.001).Nineteen of the Duchenne affected samples were from males. In addition, one female Duchenne carrier and one female Becker carrier samples were tested. The CK-MM concentrations measured from the Duchenne affected specimens were within a range  that was fully above the range of the unaffected population. However, the Duchenne carrier result was within the normal range (197 ng/mL) whereas the Becker carrier (2100 ng/mL) separated well from the unaffected population with both cutoff values. All Duchenne affected specimens were detected with the GSP CK-MM kit using the 99th percentile cut-off, whereas the CK enzyme activity method missed one affected Duchenne specimen . The resThe Danish population included data for 3526 anonymous newborn samples of which 16 had been affected by DMD, and three had been affected by BMD. Two thousand ninety-nine of unaffected specimens were used to determine testing site-specific newborn population distribution and higher percentile based cutoff values .The next 1408 unaffected specimens together with 16 retrospective confirmed Duchenne positive samples were assayed and categorized as screening positive or negative based on the cutoff values. The mean value for presumed unaffected specimens was 128 ng/mL and for Duchenne affected 2626 ng/mL. With 99.5th percentile cut-off value, 15 of the Duchenne affected specimens were classified as screening positive and one specimen as screening negative. The specimen had been collected from a premature newborn  when the newborn was 3 days old. The repeat specimen (collected at age of 35 days) was analyzed and CK-MM was higher but not elevated above the unaffected population. Kit discriminated between the normal population and screening positive DMD cases with a positive predictive value (PPV) of 93.8% . In addiThe data from the unaffected samples of US and Danish populations were combined to evaluate the effect of the age of the newborn at the time of sampling to the CK-MM concentration levels. The US population samples had been collected during the first 1–2 days (24–48 h) after birth and most of the Danish population samples were collected at 2–3 days of age or later. The CK-MM concentration was found to inversely correlate with the age of the newborn in unaffected specimens .The data from the unaffected samples of Danish populations was used to evaluate the effect of the gestational age of the newborn to the CK-MM concentration levels. The median CK-MM concentration was increased with the gestational age of the newborn in unaffected specimens .In NBS, determination of presumptive positives for Duchenne would use a cut-off value threshold to distinguish between presumptive negative and presumptive positive values. The cut-off value would be determined based on the distribution of creatine kinase in the newborn population. Blood collected from a heel prick with direct application onto the filter paper is the preferred sample for NBS. The study demonstrated that the novel GSP CK-MM assay discriminates well between normal, unaffected and Duchenne affected populations and is suitable for Duchenne NBS from DBS. In addition, unlike previous methodologies for detecting CK enzymatic activity, the CK-MM concentration analysis shows improved specificity.As reported by Moat et al. , humiditOne of the Duchenne affected newborns that was included in the two studies was collected from a preterm  baby with low birth weight (1200 g) resulting in a false negative result (55.2 ng/mL). The median CK-MM concentration also increased with the gestational age of the newborn in unaffected specimens. Another affected newborn, with a gestational age of 35 weeks and birth weight of 2700 g had a CK-MM concentration of 1100 ng/mL i.e., above the used cutoff values. Previous prenatal studies  have shoDuchenne and Becker muscular dystrophies are considered a spectrum of the same disease, having similar signs and symptoms and being caused by different mutations in the same gene. Both Duchenne and Becker are X-linked recessive diseases, so females are typically asymptomatic carriers of mutations. However, female carriers may manifest symptoms varying from mild muscle weakness to a more severe clinical course and are classified as manifesting or symptomatic carriers . Female In our study, one of the specimens collected was from a female subject, who was a manifesting Duchenne carrier. Both the initial and repeat CK-MM results were screen negative with both cut-offs used. Also, the enzymatic CK activity method result was screen negative. In contrast, a specimen that had been collected from her twin brother, with the same dystrophin gene mutation and the typical Duchenne phenotype, measured as screen positive with both cut-offs and both methods.In addition to Duchenne carriers, Becker muscular dystrophy samples were analyzed. Becker is an X-linked disorder caused by non-truncating dystrophin gene mutations, leading to altered, but detectable dystrophin expression in muscle fibers. Becker is typically characterized by later-onset skeletal muscle symptoms than Duchenne although the phenotype can vary. In our study, one specimen collected from a Becker carrier was analyzed resulting in a screen positive result and four specimens collected from Becker cases had a screen negative result. Some individuals with Becker may have elevated CK-MM concentrations at birth and may screen positive .The CK-MM concentration was found to inversely correlate with the age of the newborn in unaffected specimens. CK-MM is typically highly elevated in Duchenne patients, however during the first few days after birth CK-MM may also be elevated in unaffected newborns due to muscle trauma during birth . After bSince previous pilot Duchenne NBS programs had used creatine kinase enzymatic activity methods, a comparison of the two methods was done. A significant association between the CK-MM concentration and CK enzymatic activity was observed, which further supported the suitability of the CK-MM for Duchenne screening in newborns. The correlation has also been demonstrated previously with 10 blood spot samples from older boys with DMD . However


Differential expressions of estrogen/progesterone receptors (ER/PR) and individual component of extracellular matrices derived from fibroid are reported. Information on the pattern of change in ER/PR expression and amount of tissue fibrosis after hormonal treatment is unclear. We investigated pattern of change in ER/PR expression and percentage of tissue fibrosis in different uterine leiomyomas after gonadotropin-releasing hormone agonist (GnRHa) treatment. Biopsy specimens from fibroids and adjacent myometria were collected after surgery from women with submucosal myoma , intramural myoma  and subserosal myoma . A proportion of women in each group of fibroid underwent treatment with GnRHa for a variable period of 3–6 months. Tissue expression of ER and PR was analyzed by immunohistochemistry. In vitro cell proliferation effect of GnRHa on human umbilical vein endothelial cells (HUVECs) was examined. Distribution of tissue fibrosis was examined by Masson’s trichrome staining with computer-captured image analysis of fibrosis derived from different types of fibroid. PR content was significantly higher than ER in tissues derived from GnRHa-untreated women with SMM and SSM (p = 0.04 for both). Comparing to untreated group, GnRHa-treatment significantly decreased either ER or PR expression in different fibroids. Exogenous treatment with GnRHa dose-dependently decreased proliferation of HUVECs. No significant difference was observed in the percentage of fibrosis in tissues collected from GnRHa-treated and -untreated women with fibroids. The distribution of fibrosis in myoma/myometria and occurrence of fibrosis in perivascular area showed an increasing trend with higher age of the women and with larger size of fibroids. Our findings suggest that despite estrogen dependency, higher PR content in GnRHa-untreated group may indicate a potential role of progesterone in leiomyoma growth. Although GnRHa therapy may shrink fibroids and reduce risk of bleeding during surgery, the occurrence of diffuse tissue fibrosis may impair effective reduction of fibroid size after hormonal treatment. Uterine leiomyomyomas  are the most common benign tumors of female reproductive tract. They are highly prevalent with 70–80% of women burdened by the end of their reproductive years . It is dFibroids are hormone-depended tumors related to an environment of estrogen and progesterone, therefore, in many cases symptoms are relieved at the time of menopause . DiffereExtracellular matrix (ECM) accumulation and remodeling are thought to be crucial for fibrotic disease such as uterine leiomyoma. Fibroids are characterized by elevated levels of collagens, fibronectin, laminins, and proteoglycans –19. ECM At present, several classes of compounds, such as gonadotropin-releasing hormone agonist , GnRH antagonist (cetrorelix acetate), selective progesterone receptor modulators , antiprogestin (mifepristone), and natural compounds such as vitamin D and resveratrol (polyphenol extract) have been studied as medical treatments that target ECM in uterine leiomyoma . DiffereTherefore, we aim to investigate the followings for our current study: (1) immunohistochemical analysis of ER and PR expression in fibroid tissue and myometrium derived from GnRHa-treated and -untreated women with SMM, IMM and SSM, (2) analysis of ER and PR expression pattern in intra-tumor/myometrial vascular cells, (3) protein expression of GnRH receptors in and in vitro effect of GnRHa on vascular endothelial cells collected from human umbilical veins (HUVECs), (4) Masson’s trichrome staining with computer-captured image analysis was performed to examine the distribution of fibrosis in myoma/myometrium and in perivascular area derived from GnRHa-treated and -untreated women with different types of fibroids.The subjects in this study were women of reproductive age. This is a prospective non-randomized cohort study. We prospectively collected biopsy samples from women with fibroids and performed retrospective analysis of biopsy samples derived from GnRHa-treated and -untreated women after surgery. Since this is an observational study to examine the effect of GnRHa on expression profiles of ER/PR/fibrosis, we believe that our samples can be considered representative of a larger population. From April 2014 to December 2016, biopsy specimens were collected from a total of 18 women with submucosal myoma (SMM), 16 women with intramural myoma (IMM) and 17 women with subserosal myoma (SSM) who underwent surgery during this period. The criteria for the classification of SMM, IMM and SSM were based on the classification of the European Society of Hysteroscopy ,38. All The phases of the menstrual cycle in women without hormonal therapy was determined by histological dating of eutopic endometria samples taken simultaneously with removal of myoma nodule. All biopsy specimens were collected in accordance with the guidelines of the Declaration of Helsinki and were approved by the Institutional Review Board of the Research Ethical Committee of Saisaikei Nagasaki Hospital, Nagasaki and Kyoto Prefectural University of Medicine, Kyoto (IRB No. 16005). All patients/samples were recruited/collected in Saiseikai Nagasaki Hospital, Nagasaki and research work was performed in Saiseikai Nagasaki Hospital, Nagasaki/Kyoto Prefectural University of Medicine, Kyoto. All data analysis were performed by an independent investigator (HO) without any prior knowledge of the medical records. All patients in this study received medical treatment during the period of April 2014 and December 2016. All medical records were retrospectively assessed in Saiseikai Nagaskai Hospital during the period of April 2017 and August 2018. A written informed consent was obtained from all women.Nine women with SMM and 6 women each with IMM and SSM underwent hysterectomy. Therefore, we could collect myometrial samples only from these women. Trans-cervical hysteroscopic resection (TCR), laparoscopic myomectomy (LM) or laparoscopy-assisted myomectomy (LAM) were performed in the remaining women. Therefore, instead of studying myometrial samples, we could study only pathologic lesions and endometria derived from these women. Biopsy specimens from the myoma nodule, autologous myometria or endometria were collected from all these women during operation. Biopsy specimens obtained after surgery were analyzed for the histological diagnosis of each fibroid. All collected biopsy specimens were prepared for formalin-fixed paraffin-embedded tissue blocks for subsequent histopathological and immunohistochemical study and also for Masson’s trichrome staining with computer-captured image analysis. In case of multiple myoma, biopsy specimen was collected from the largest myoma and analyzed.We performed immunohistochemical studies to investigate immunoreaction of target antigens in the serial section of biopsies using the following antibodies: ERα ; PR ; anti-human von Willebrand factor (VWF) antibody , a mouse monoclonal antibody, to investigate immunoreaction to HUVECs. GnRHR , a mouse monoclonal antibody against both type I and type II receptor (1:25 dilution), was used to immunolocalize GnRHR expression in HUVECs. Non-immune mouse immunoglobulin (Ig) G1 antibody  was used as a negative control.202/methanol to block endogenous peroxidase activity, sections were pre-incubated with 10% normal goat serum to prevent nonspecific binding and then incubated overnight at 4°C with respective antibody. The slides were subsequently incubated with biotinylated second antibody for 10 minutes, followed by incubation with avidin-peroxidase for 10 minutes and visualized with diaminobenzidine. Finally, the tissue sections were counterstained with Mayer’s hematoxylene, dehydrated with serial alcohols, cleared in xylene, and mounted. We had at least three slides per biopsy for immunohistochemical analysis. The expressions of ER/PR/VWF/GnRHR in fibroid tissue/myometria and in HUVECs were examined. The immunoreaction of ER/PR was expressed as number of ER/PR immunostained cells per high power field (HPF) (x200) and as a mean of five different fields.The details of immunohistochemical staining procedures are described elsewhere ,40. Brie6 cells. An aliquot of 0.5x104 cells was used for our experiments. The detail procedure of HUVECs isolation and preparation is mentioned in a previous report -thymidine-based cell proliferation and carries equal sensitivity and specificity [-9M to 10-5M) of GnRH agonist. The detail procedure of BrdU incorporation assay was described previously [5-Bromo-2-deoxyuridine (BrdU) labeling and detection kit measures cell proliferation by quantitating BrdU incorporated into the newly synthesized DNA of replicating cells . The inccificity . We exameviously ,44. The In order to determine the presence of fibrosis in the biopsy specimens derived from women with SMM, IMM, SSM, and surrounding myometrium, we performed Masson’s trichrome (MT) staining with aniline blue. All reagents were purchased from the Muto Chemical Co. Tokyo, Japan and staining procedures were followed according to the instruction of the manual as supplied by the Muto Chemical Co. Fibrosis in the respective biopsy samples was identified by dense or filamentous (fiber-like) blue staining (aniline blue) instead of green staining (methyl green) as described previously . Distribhttp://fiji.sc). First of all, background correction was done to reduce any color/luminance variations in image. Tissue area  from each stained section was extracted based on luminance information by auto thresholding technique. Automatically calculated threshold was utilized for overcoming non-uniform staining conditions [The details of computer- captured image analysis of MT-stained fibrosis from each sample is described elsewhere ,47. Brienditions . Dense oU-test and Kruskal-Wallis test, respectively. This is an observational study and not a randomized clinical trial. Our main purpose was to examine the effect of GnRHa on the expression profiles of ER/PR/fibrosis in women with uterine myomas comparing to GnRHa-untreated women. Therefore, we did not perform sample size calculation based on pre-specified power in this study. Instead we performed post-hoc power calculation regarding the estimated effect size as the true effect size for Mann-Whitney test in three groups of fibroids. The distribution of ER/PR/fibrosis according to groups was expressed using the box and whisker plots with the medians and IQR. A value of p<0.05 was considered statistically significant. Data analysis was conducted using SAS software version 9.4 .All results are expressed as mean ± SD or mean ± SEM, median and inter-quartile range (IQR). The clinical characteristics of the subjects were compared with one-way analysis of variance. The differences in the ER/PR expression and fibrosis in respective biopsy samples between groups and among groups were analyzed by the non-parametric Mann-Whitney The women with SSM were significantly younger than those of SMM . There was no difference in the mean ages between women with SMM and IMM. The mean sizes of SMM were significantly smaller than those of either IMM or SSM (p<0.0001 for each). The distribution of women with or without GnRHa therapy, phases of menstrual cycle and types of surgery in women with SMM, IMM and SSM are summarized in We analyzed the result of ER and PR expression in respective myoma nodule and myometrium derived from SMM, IMM and SSM regardless of GnRHa treatment . We founIn an attempt to examine the pattern of ER and PR expression in intra-tumor or intra-myometrial vessels, no remarkable change of ER and PR expression was observed in vascular cells in either between groups (myoma and myometrium) or among groups  .We analyzed the results of ER and PR expression in combined myoma and myometrium derived from women with GnRHa-treated and -untreated women with SMM, IMM and SSM. We found that number of PR was significantly higher than ER in myoma/myometria derived from GnRHa-untreated women with SMM (p = 0.04) and SSM (p = 0.04) but not with IMM . CompariWe further analyzed pattern of ER and PR expression in intra-tumor and intra-myometrial vasculatures in GnRHa-treated and -untreated group of these three types of fibroid. There was no difference between ER and PR content in vascular endothelial cells (VECs) of biopsy samples derived from GnRHa-untreated women with either of SMM, IMM or SSM . In contWe found strong immunoreaction of VWF in cultured vascular endothelial cells collected from human umbilical vein (HUVECs) (-8M and 10-5M caused a significant and a dose-dependent inhibition of BrdU incorporation into DNA of HUVECs (p<0.05 for each of 10-8M to 10-5M of GnRHa-treated cells versus GnRHa-untreated cells)  on cell proliferation from day 2 to day 3 without any variation between these days. Therefore, our dose-dependent study was performed with an incubation period of 48 hours. We examined direct cell proliferation effect of a variable dose of GnRHa on HUVECs derived from three cases. GnRHa treatment, at a concentration between 10d cells) . KruskalWe performed Masson’s trichrome (MT) staining to identify aniline blue-stained filamentous collagen fibers (fibrosis) in myoma and myometria derived from women with SMM, IMM, SSM . ComputeThe distribution of fibrosis as measured by Masson’s trichrome-staining in the myoma and myometria derived from GnRHa-treated and -untreated women with SMM, IMM and SSM is shown in The distribution of Masson’s trichrome-stained collagen fibers (fibrosis) was predominant around the small size and large size vessels in the myoma and myometria derived from women with SMM, IMM and SSM . A variath, 40th and 50th years comparing to their 10th and 20th years of age  in their 30s of age . A simils of age .th, 40th, and 50th years of age comparing to 10th and 20th years of age  within these tissues derived from women with different types of fibroids and their response to estrogen suppressing agent. Most of the published reports demonstrated ER/PR expression and individual component of extracellular matrices (ECM) in a specific type of leiomyoma. We came to learn from our study that in GnRHa-untreated women, PR expression was significantly higher than ER in the myoma/myometria derived from women with SMM and SSM. Comparing to GnRHa-untreated women, GnRHa treatment significantly decreased PR expression in biopsy samples collected from SMM and SSM while samples collected from IMM showed a significant decrease of ER expression. We performed post-hoc power calculation regarding the estimated effect size as the true effect size for Mann-Whitney test in three groups of fibroids. We found that the post-hoc power was high for women with SMM (0.908) and relatively low for women with IMM (0.556) and SSM (0.541).There was no remarkable change of ER/PR content in vasculatures between GnRHa (-) and GnRHa (+) women with SMM and SSM while women with IMM showed a significant decrease of ER expression in the vasculatures after GnRHa treatment. The direct anti-proliferative effect of GnRHa on VECs was confirmed in a separate in vitro study using HUVECs. Expression profile of fibrosis in myoma/myometria collected from with these three groups of fibroids failed to show any remarkable change in the accumulation of fibrosis between GnRHa (-) and GnRHa (+) group. The biopsy specimens of myoma and myometria collected from these three groups of women showed a strong perivascular fibrosis with an apparent increasing tendency with higher age of women and larger size of fibroids.One outstanding finding of our study is that PR content was predominant in both SMM and SSM and their adjacent myometria. This finding coincides with the results of Nisolle at al.,  who founWhen we distributed ER- and PR-immunostained SMCs separately in myoma and myometrium derived from women with SMM, IMM and SSM regardless of GnRHa treatment, a predominant expression of PR than ER was observed in the myoma of IMM rather than in adjacent myometrium. There was no difference in the expression profile between ER and PR in either myoma or in myometria collected from women with SMM and SSM. The higher ER content in leiomyomata than in myometrium was reported previously ,55. In tTraditionally medication used to provoke shrinkage of leiomyoma and reduction of blood flow has been based on inducing hypoestrogenism. The only drug that has been shown to be effective is GnRHa (leuprolide acetate). In our previous in vivo and in vitro studies, we demonstrated that in addition to central effect, GnRHa treatment has multiple peripheral effects and was able to significantly decrease tissue inflammation, cell proliferation, angiogenesis, and promotes apoptosis in endometria and myometria derived from women with endometriosis, adenomyosis and uterine leiomyoma ,33. In tWhen we analyzed pattern of ovarian steroid receptors in vascular endothelial cells (VECs) within myoma/myometria, no significant difference between ER and PR expression was found in GnRHa-untreated women with different fibroids. Distribution of ER/PR expression profile separately in the myoma and myometria derived from different fibroids and regardless of GnRHa treatment showed no significant difference between ER and PR in either myoma or in myometrium. Comparing to GnRHa-untreated group, the only significant difference was observed for vascular ER expression in women with IMM after GnRHa treatment. This might to be due to our staining performance with ERα and not with ERβ. Most of the previous studies reported ERβ expression in pregnant and non-pregnant uterine vascular endothelial cells instead of ERα expression ,59. HoweWe previously demonstrated a significant decrease in micro-vessel density as measured by VWF immunostaining in leiomyomata and adjacent myometrium in response to GnRHa treatment . In an aOne of the major disadvantages of using GnRHa in treating uterine leiomyoma is ineffective reduction in size and recurrence of the disease once treatment is stopped. In fact, most leiomyomata return to pretreatment size within 4months following cessation of therapy. This can be explained by withdrawal of HPO-axis down-regulation and stiffness of the uterine wall resulting from dense accumulation of ECM components within myometria and in perivascular area. Fibroids contain substantial amounts of altered and disordered collagens that contribute to their bulk. Rigidity of the uterus is an important factor that may also explain the poor responsiveness to hormonal medication . A variaWe demonstrated in our current study that unlike SMM and SSM, the amount of fibrosis was significantly higher in the myometria than in leiomyomata collected from women with IMM. No significant difference was observed in the percentage of fibrosis in combined myoma/myometria collected from GnRHa-treated and -untreated women with these three types of fibroids. We also found a strong perivascular fibrosis within myoma and myometria in any type of fibroids. The distribution of fibrosis in myoma/myometria and in their perivascular area showed an increasing pattern with higher age of patients and larger size of fibroids. Several lines of evidence supported fibrotic character of uterine leiomyomas by elevated levels of ECM components and myofibroblasts in these tumors ,62. EstrAnother interesting finding in our study is the occurrence of perivascular fibrosis around small size and large size vessels in the myoma and myometrium derived from different types of fibroids. This finding may explain a link in the initiation and extension of fibrosis in the myoma/myometrium of women with uterine leiomyomas. A variable amount of tissue inflammatory reaction in fibroids may produce abundant concentration of TGF-β from infiltrating immune cells and induce a cascade of EMT, FMT, SMM and activation of myofibroblasts ultimately leading to fibrosis ,22,26–28From our findings of perivascular fibrosis in women with different types of fibroid in premenopausal women, we cannot exclude the possibility of sclerotic change in vessels that may increase the risk of thrombotic events as well as hypoxic change of the endometrium. In fact, we found that deposition of collagen fibers in myoma/myometria and in perivascular areas appear to be higher with advancing age of women comparing to younger age groups. Large size fibroid and associated fibrosis can be another predisposing factor. More recently, it has been reported that the uteri of post-menopausal women undergo progressive fibrosis with atherosclerotic changes overtime from the last menstrual period . If simiThere are several limitations in our current study. (1) These findings are limited by the cross-sectional nature of the study and we used only histochemistry and immunohistochemistry. (2) Sample size in each group of GnRHa-treated and -untreated women with fibroid was small. Further investigation is needed with large sample size. (3) Neither specific types of ER (ERα/ERβ) nor PR (PR-A/PR-B) in respective fibroids were analyzed. (4) HUVECs were used in current study instead of uterine arterial endothelial cells in culture system. (5) We examined only the effect of estrogen suppressing agent on E/P receptor expressions and fibrosis. Additional studies on the effect of different progestational compounds are needed to support our current findings.Finally we conclude that our current findings give us some new information on the biological differences in three different types of uterine leiomyoma and on the biological basis of ineffective reduction of fibroid size after GnRHa treatment. Our current findings re-confirmed higher PR content in fibroids and may suggest a potential role of progesterone in leiomyoma growth. GnRHa therapy may transiently shrink fibroids and reduce the risk of bleeding during surgery by decreasing ER/PR expression and proliferation of vascular endothelial cells. The most recent report indicated that after adjusting different confounding factors, size, number of fibroids, and operative time were directly related to risk of bleeding during myoma surgery . These cThe occurrence of myometrial/perivascular fibrosis may impair effective reduction of fibroid size and increase the risk of arteriosclerosis. We need careful follow-up of these women with fibroids even after uterus-sparing surgery such as uterine artery embolization . Based oS1 Figth, 40th and 50th years age comparing to patients in their 10th and 20th years of age. Scale bar = 50μm for each slide.An increasing accumulation of fibrosis was observed in patients in their 30(TIFF)Click here for additional data file.S2 Figth, 40th and 50th years of age comparing to patients in their 10th and 20th years of age. Scale bar = 50μm for each slide.An increasing accumulation of dense perivascular fibrosis was observed in patients in their 30(TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.