Cases of Type 2 Diabetes Mellitus (T2DM) are increasing at an alarming rate due to the rise in obesity, sedentary lifestyles, glucose-rich diets and other factors. Numerous studies have increasingly illustrated the pivotal role that human islet amyloid polypeptide (hIAPP) plays in the pathology of T2DM through damage and subsequent loss of pancreatic β-cell mass. HIAPP can misfold and form amyloid fibrils which are preceded by pre-fibrillar oligomers and monomers, all of which have been linked, to a certain extent, to β-cell cytotoxicity through a range of proposed mechanisms. This review provides an up-to-date summary of recent progress in the field, highlighting factors that contribute to hIAPP misfolding and aggregation such as hIAPP protein concentration, cell stress, molecular chaperones, the immune system response and cross-seeding with other amyloidogenic proteins. Understanding the structure of hIAPP and how these factors affect amyloid formation will help us better understand how hIAPP misfolds and aggregates and, importantly, help identify potential therapeutic targets for inhibiting amyloidosis so alternate and more effective treatments for T2DM can be developed. Diabetes mellitus is a complex condition linked to increased blood glucose levels, compromised insulin production or action, that can be subdivided into five major types: monogenic diabetes, Type 1 Diabetes mellitus (T1DM), Type 2 Diabetes Mellitus (T2DM), gestational diabetes and, more recently, Type 3 Diabetes (T3DM) . In addiT2DM is a complex metabolic condition characterised by hyperglycaemia and peripheral insulin resistance, leading to depletion and dysfunction of pancreatic islet β-cells . A rangeT2DM has been associated with several risk factors that are genetic, epigenetic and environmental . More thSocial-economic factors are also a risk factor linked to T2DM. Individuals from low-socioeconomic communities are more likely to suffer from chronic stress which is associated with increased blood pressure and elevated blood glucose levels . Those fFusobacterium and Ruminococcus have been detected in T2DM patients compared to non-diabetic patients. These bacteria form part of the microflora of the intestines that are thought to regulate levels of lipopolysaccharides (LPS), which have been linked to the advancement and development of T2DM [Microorganisms have also been associated with T2DM, as highlighted in recent studies looking at whether the gut microbiome is linked to T2DM . Elevate of T2DM . Levels  of T2DM .Prescribed medications to manage T2DM include antihyperglycemic drugs such as metformin and sulfonylureas. Although these have proven to reduce mortality and assist in the control of blood glucose levels, metformin has associated side effects such as nausea and diarrhoea in 50% of patients. This is thought to be as a result of metformin increasing levels of glucagon-like peptide 1 (GLP-1) and increasing intestinal glucose turnover, as well as influencing the microflora of the gut . SulfonyIn order to progress with therapeutic interventions and help prevent disease onset, we urgently need a better understanding of T2DM at the molecular level. Despite a plethora of scientific literature on diabetes, exactly what initiates the disease and what factors are responsible for accelerating the condition are still unclear. In this review, we have focused on the role of hIAPP and its tendency to misfold and aggregate in the molecular pathology of T2DM, since accumulating evidence suggests that different conformations of the protein are associated with the initiation and/or acceleration of the disease.Deposits of incorrectly folded (“misfolded”) hIAPP in the pancreas of people with T2DM have been reported in up to 90% of all T2DM patients . The chahIAPP is a 37 amino acid protein with an element of intrinsically disordered structure and is secreted from β-cells together with insulin . It is cThe gene that encodes hIAPP is found on chromosome 12, includes 3 exons  and is iSeveral functions have been ascribed to IAPP, including suppression of glucagon release, control of gastric emptying and regulation of satiety , all of Understanding the structure of hIAPP and its various conformations is important for understanding how it contributes to T2DM and hence for designing effective therapeutics. In T2DM, evidence suggests that hIAPP can adopt different conformations, including pre-IAPP (an un-cleaved form), monomers, dimers, oligomers and fibrils . Like otIn vitro, synthetic hIAPP monomers dissolved in phosphate buffer or diluted hexafluoro-2-isopropanol form random coil conformations, which misfold into β-sheet structures and fibrils . AnalysiRecently, several cryo-EM structures of recombinant hIAPP have been produced ,41,42,43The most detailed, and biologically relevant structures of hIAPP fibrils was published recently by Cao et al., 2021 , showingSince membranes have been suggested as crucial to hIAPP misfolding and aggregation , it is eImportantly, decreased insulin degrading enzyme (IDE) levels can lead to hyperinsulinemia, which may contribute to insulin resistance . IAPP isDifferent mechanisms have been proposed explaining how monomers stack together and assemble into oligomers. Identifying exactly how the different conformations form and the key residues involved is important for developing therapeutics to inhibit aggregate formation, especially at the early stages of the process before extensive cellular damage occurs. NMR experiments using hIAPP peptides in solution suggest the initial events may involve adjacent monomers binding via noncovalent interactions involving residues 26–32 before assembling into oligomers . AnotherSeveral studies indicate that the amyloidogenic properties of human IAPP are linked to residues 20–29, one of the regions previously identified as key to oligomer assembly . ResidueA Ser20Gly mutation found in Chinese and Japanese populations has been linked to an increased rate of amyloid aggregation, and an increased propensity to T2DM, further supporting the importance of the 20–29 region in amyloidosis . The quaA conserved disulphide bond found outside the cross β-core is located between residues Cys 2 and Cys 7 of hIAPP. Removal of this bond through mutation or Cys-Cys disulphide reduction was shown to increase hIAPP amyloidosis while also decreasing hIAPP-induced membrane leakage, highlighting the importance of this region in fibril formation and related cytotoxicity .Accumulating evidence suggests that hIAPP oligomers play a crucial role in cytotoxicity. Due to their soluble nature and short lifespan, it has been extremely difficult to isolate them to characterise their structure. Altamirano-Bustamante et al.  used varHIAPP has been linked to cytotoxicity of pancreatic β-cells, but more recently other cells such as neuronal cells . Whilst A crucial question in the understanding of hIAPP aggregate formation is what initiates and accelerates the formation of the fibrils that have been linked to T2DM. If we knew what triggered the misfolding cascade in the first place, then T2DM (and possibly other PMDs) may be preventable. Distinguishing between what initiates and what accelerates the process of amyloidosis is key but may involve the same factors. Several factors may contribute to the formation of these misfolded aggregates, and we have summarised these in There is an important relationship between the concentration of hIAPP and the rate of aggregation . When a A proposed accelerator of hIAPP aggregation is cell stress. Endoplasmic reticulum (ER) stress is a known factor in T2DM pathology . An ER sAnother form of cell stress found in T2DM is oxidative stress that can be caused by chronic hyperglycaemia. As evidenced by methods such as mass spectrometry and high-performance liquid chromatography, markers of oxidative tissue damage have been identified in plasma taken from individuals with T2DM ,83. OxidMedicago sativa administered to people with insulin resistance has shown promising results [Chaperones are proteins required for the correct folding of other proteins and, thus, play an important role in preventing protein misfolding and aggregation. Molecular chaperones are important in response to ER stress  in which results .ER stress triggers the activation of signalling pathways, which lead to a process known as the unfolded protein response (UPR) in an attempt to restore ER homeostasis. Mechanisms of UPR include the increase in ER size, decrease in protein synthesis, increase in protein folding capacity and an increase in the removal of protein aggregates. If the UPR is unable to cope with increased amounts of aggregates forming in conditions such as T2DM, the accumulation of aggregates in the cells activates apoptotic pathways, leading to cell death and a decrease in β cell mass .Heparan sulphate proteoglycans (HSPGs) vary in subcellular localisation and can be divided into one of four HSPG subtypes, depending on their location . The latter have been identified as having an essential modulatory function in multiple developmental signalling processes through the regulation of molecules, including WNT transforming growth factor and fibroblast growth factor . A type Interleukin-1β (IL-1β) is an inflammatory cytokine that plays a role in immune responses to injuries and infections  and is aProteins involved in other protein misfolding diseases such as Aβ in Alzheimer’s disease have been observed directly interacting with hIAPP. Monomers are thought to interact via their β-sheet structures (found in both Aβ and hIAPP), leading to cross-seeding and the formation of hybrid fibrils in the brain . In vitrhIAPP aggregates have been observed in organs such as the brain, but it is unknown whether hIAPP is produced in those areas or it migrates there via the peripheral circulation . Recent Accumulating evidence suggests a link between Parkinson’s Disease (PD) and T2DM through protein interactions between alpha-synuclein (known to misfold in PD) and hIAPP. Co-aggregation of the two proteins has been linked to an increased risk of PD in T2DM patients . Hybrid 2+, which also controls the equilibrium between insulin monomers, dimers and hexamers. A decrease in Zn2+ ions shifts the equilibrium of insulin oligomers in the direction of zinc-free monomers and dimers, which bind to IAPP monomers more efficiently than insulin hexamers. This may explain the observed link between loss of function mutations in the ZnT8 zinc ion transporter expressed on secretory granules with a decrease in hIAPP aggregation [The concentration of zinc in the body is thought to contribute to T2DM. Zinc concentrations of 10–25 µM, which is the range found extracellularly, can decelerate the aggregation process by elongating the lag phase and decreasing the elongation rate of the fibrils, as shown in vitro, whilst the opposite is true for higher zinc concentrations . Perhapsregation .There are currently several treatments being commonly used to treat those with T2DM, mostly insulin and metformin. However, since this disorder is closely linked to obesity, preventative methods such as changes in diet and fitness are usually suggested as the best preventative method for people at high risk of T2DM . Results from a randomised controlled trial using 5238 participants demonstrated that without additional standard medical treatment, diet change and increased activity does not necessarily reduce the risk of T2DM pathology; however, it does reduce the risk of T2DM in people with increased glucose impairment .For those with T2DM, the most common drug prescribed for the management of T2DM is metformin, part of the biguanide class of medications. It is thought to operate by minimising glucose production by hepatic cells and suppresses cell inflammation via interaction with metabolic organs , immune cells, as well as influencing the activity of the glucose-lowering hormone glucagon-like peptide 1 . The mosThe different protein conformations that IAPP can misfold into have been proposed as viable treatment targets, since the oligomeric and fibrillar forms have been linked with pathology . VariousManganese-salen chloride derivatives EUK-8 and EUK-134 that have been shown to possess antioxidant properties can also inhibit hIAPP amyloidosis . The antAn alternative treatment option proposes utilisation of the hIAPP oligomers for active immunisation. Studies in vivo by Bram et al.  showed tT2DM will soon be a medical emergency as levels of obesity increase and our populations live longer . Since aThe observation of polymorphic structures with varying residue orientations also implies that there may be variations between the structures of different fibrils, which could affect the formation of fibrils as well as their cytotoxicity ,65. One Cytotoxicity of hIAPP is an area of important research that needs to be investigated further to determine the precise molecular events involved. An essential part of understanding hIAPP aggregation is knowing what accelerates the process, as this also assists in identifying viable treatment options.There is additionally a need to develop alternative treatments with minimal side effects . In thisPart of the ER stress response involves molecular chaperones ,95. ReduOther amyloidogenic proteins can accelerate the misfolding of hIAPP through cross-seeding and co-aggregate with hIAPP. Targeting other proteins may provide a good therapeutic target for T2DM, and vice versa, which has already been suggested in the literature .Additionally, as more biochemical and structural information becomes available regarding the different forms of hIAPP, specific parts of these structures can be targeted when developing therapeutics. These include molecules to stabilise monomeric hIAPP to prevent formation of misfolded hIAPP, or those that bind to misfolded forms to block binding of additional hIAPP molecules or target misfolded forms for removal. Treatments can also target molecules in pathways associated with accelerating the misfolding and aggregation of hIAPP, as summarised in T2DM is a condition that is increasing at an alarming rate worldwide. In this review, we have highlighted a plethora of studies that link the protein hIAPP with T2DM. There are several factors linked to T2DM that have been shown to accelerate hIAPP amyloid formation by directly or indirectly interacting with hIAPP oligomers, monomers, and fibrils under hyperglycaemic conditions. Hyperglycaemia causes an environment which increases hIAPP aggregation. HIAPP aggregates and oligomeric species have been shown to be crucial for β-cell apoptosis and for T2DM development. Precisely what initiates these reactions is unclear, in addition to the precise molecular mechanisms involved in these pathways; however, amyloidosis and interactions with membranes appear to be essential factors linking hIAPP to T2DM.The propensity to aggregate lies in the primary structure of hIAPP, as seen in studies comparing rat and human IAPP . Underst


The contamination rate was associated with the mode of delivery and showed a significantly higher contamination rate of 9.7% when compared with cesarean deliveries (1.4%). The 10-year period consistently shows an average contamination rate between 4% and 6% per year. It is conceivable that the inverse relationship between volume and contamination rate might be related to thinner veins although no scientific evidence has been provided so far. The lower contamination rate in cesarean sections appears to be related to the sterile operating setting. Overall, the rate of bacterial contamination varies and depends on the type of birth, the way of delivery, and probably the experience of the staff.Cord blood (CB) collected at birth has become a valuable stem cell source for hematopoietic stem cell transplantation (HSCT). However, the collection of umbilical cord blood always bears a risk of microbiological contamination, both in vaginal birth and in cesarean section. A total of 10 054 umbilical cord stem cell samples were successfully cryopreserved between 2010 and 2020, of which 783 (8%) samples were tested positive for bacterial contamination. Umbilical CB with a volume of less than 60 mL showed a bacterial contamination rate of 12%, and above 60 mL volume a rate of 6% was found demonstrating an inverse relationship between sample volume and contamination rate (correlation coefficient   This is of particular importance for patients from racial and ethnic minorities, as for those it can be difficult to find a donor.4.Umbilical cord blood (UCB) stem cells have been established as an alternative stem cell source for pediatric and adult patients with various oncologic, hematologic, immunologic, and inherited metabolic disorders, especially by lacking a related or unrelated donor.6 In addition, few studies have confirmed that the relapse rate after UCB transplantation is low compared to unrelated donor transplants, indicating that UCB may be the preferred source for patients at high risk of relapse.7 UCB transplantation has the advantage of a lower rate of chronic graft-versus-host disease (GvHD) compared to hematopoietic stem cells from peripheral blood, which is reflected in lower long-term morbidity and mortality.7 Research studies have shown that cord blood transplants can also be performed in cases even when the donor and the recipient are partially matched, so cord blood increases the patient’s chance to find a more suitable donor.9 However, with increasing differences in the donor and recipient HLA systems, the risk of delayed or absent hematopoietic reconstitution also increases in UCB transplantation.10 In contrast, the greater availability of high-quality and high-cell-content UCB units has resulted in increasingly improved engraftment and survival outcomes after UCB transplantation.12Numerous retrospective studies from recent years have shown that UCB transplantation in patients with hematological malignancies can result in disease-free survival (DFS) comparable to that of adult donor transplants.13 Cord blood preparations with latent virus and environmental toxin contaminations are lower than those with a bone marrow transplant, but appear to be more highly contaminated by bacteria than those from bone marrow and peripheral blood are, which may be due to the collection technique.14-16 Bacterial contamination of human blood products is another challenge as it occurs quite frequently and it is observed that bacteria developed resistance, which can be life-threatening, especially in immune-compromised patients.18 Therefore, sterility testing is mandatory according to the guideline standards to prevent transplantation transmitted bacterial infections causing severe transfusion-associated sepsis in immune-suppressed patients.20There are various providers worldwide, which accompany or sometimes even offer the process from collection to storage. Importantly, the collection of UCB always bears a risk of microbiological contamination, both in spontaneous/vaginal birth and in cesarean section.21 In case of contamination, a bacterial determination should be carried out again from a suitable reference sample to exclude second contamination or higher risk of unsuccessful clinical outcome, so high quality and safety of hematopoietic stem cells are required for any successful transplantation.16 Using sterile techniques and a closed system during the whole preparation process is usually used to reduce the risk of any bacterial contamination.16 Freezing bags or tubes must comply with the current state of technology and exclude cross-contamination between different samples during storage.16Moreover, bacterial contamination of hematopoietic stem cells obtained from UCB may cause a rejection of a stem cell graft.The aim of this study was to investigate the bacterial contamination rate related to the delivery mode including collection experiences of the staff and collection sample volume. In addition, the most commonly detected bacterial strains were determined.A total of 10 054 UCB samples for potential autologous use was cryopreserved in the transfusion medicine and hemostasis department from September 2010 to September 2020 in cooperation with the Erlangen University Hospital (Germany).Institutions that manufacture, test, process, store, or market stem cell products in Germany must ensure compliance with legal requirements, maintain a quality management system in accordance with Section 3 AMWHV (“Arzneimittel- und Wirkstoffherstellungs-verordnung”) and the principles of “good manufacturing practice” (EU GMP Guideline). The UCB collection was carried out in all maternity clinics in Germany that had signed a contract with the Eticur GmbH Germany company in accordance with the current SOP “Guidelines for Transplantation of Stem Cells Part III, Umbilical Cord Stem Cells, 3.1 Collection” and the individual preparations were carried out by the transfusion medicine and hemostasis department of the University Hospital Erlangen in accordance with the SOP “Guidelines for Transplantation of Stem Cells, Part III Umbilical Cord Stem Cells, 4th CB Stem Cell Preparation.” The University Hospital of Erlangen is licensed by the Federal Agency PEI (Paul Ehrlich Institute). Eticur GmbH Germany is a 100% subsidiary of Famicord Europe and has established collaboration with the University Hospital Erlangen. In this regard, the applied methods for cryopreservation and microbiology testing were identified and described in SOPs .Informed consent from the parents, a prescribed anamnesis questionnaire, and a production protocol were mandatory for the collection of UCB. All clinics and the University Hospital Erlangen provided a valid manufacturing permit. Approval for the pre-drug stage was carried out by the “Qualified Person” in accordance with § 14 AMG .+ cells, total nucleated cell  count, and colony-forming units (CFU). The CFU method was performed according to the protocol described by Dempke et al,22 which shows the in vitro capacity of the proliferation of hematopoietic stem cells.Cord blood samples were transported immediately after birth to the stem cell laboratory were analyzed and cryopreserved (10% v/v DMSO) within 48 h from the delivery time. Every sample was analyzed in terms of total blood volume, a total count of CD34+ and CD45+ cells were analyzed by immunofluorescence on FACSCalibur  using anti-CD34-PE and anti-CD45-FITC  according to the protocol described by Cassens et al.23CD34To check the quality parameters such as the microbiological control, the purity, and the vitality of the product as well as to control the manufacturing process or cryopreservation, sufficient preparation samples according to Section 18 (1) AMWHV must be ensured until clinical use. These samples must be stored under conditions comparable to those of the umbilical cord blood stem cells.In an attempt to determine the contamination with bacteria samples were taken from the incoming cord blood bag as well as the processed stem cell preparation followed by microbiological cultures were performed by using BD Bactec Standard Anaerobic/F 40 mL culture vials (REF 442024) and BD Bactec Standard/10 Aerobic/F 40 mL culture vials (REF442027)  to detect anaerobic and aerobic bacteria after 7 incubation days. The sample volume was identical for all samples (percentage volume). The method validation included a slow grower and all standard bacteria and fungi. Each bag was tested twice and both assays (primary and secondary contamination rates) were comparable (data not shown).A total of 10 054 UCB stem cell samples were successfully cryopreserved between September 2010 and September 2020. Most cord blood samples had volumes between 50 and 100 mL. The overall distribution of all cord blood samples taken shows a Gaussian-like distribution with the maximum at less than or equal to 70 mL volume .r = −-0.9)  samples were tested positive for bacterial contamination. UCB with a volume of less than 60 mL showed a contamination rate of 12%, and above 60 mL volume, a rate of 6% was found demonstrating an inverse relationship between sample volume and contamination rate (correlation coefficient = −-0.9) .).Considering all samples with a volume of 50–100 mL, an inverse correlation between sample volume and contamination rate is clearly shown, the higher the volume collected, the lower the contamination rate .Furthermore, the results show that vaginal birth is much more frequently performed than cesarean section, and a Gaussian-like distribution can also be seen for both birth modes, where a maximum is seen with the highest number of collections is at a sample volume of 60–90 mL .Regarding the contamination rate related to collection sample volume as well as delivery mode, both birth modes showed the inverse correlation between sample volume and contamination rate . For spoIn addition, the contamination rate associated with the mode of delivery showed a significantly higher average contamination rate of 8.4% compared with cesarean deliveries with only 1.5% ; Fig. 2DStaphylococcus- (37.7%), Bacteroides- (22.1%), and Enterococcus-strains (21.8%), as well as Escherichia coli species (13.5%) .Considering all 783 non-sterile samples the most detected bacterial strains were  (13.5%) . All detacopoeia i–iii.staphylococcus species, 6% of Enterococcus species, and E. coli species with 6% were detected . The quality of the umbilical cord stem cell preparation is also determined using the cell number of TNC (CD45+) as well as the CD34+ stem cells that are included in the TNC population. In addition, the viability of both cell populations is indicative of the quality of the stem cells. It is clearly shown that the higher the volume of the umbilical cord blood, the higher is the TNC cell count .At cesarean section, the most frequent strains in collected UCB is the propionibacterium acnes with 40% as well as 34% of detected ; Fig. 3Bll count . Regardi+ stem cells, which (with a few exceptions) display a very good vitality of over 90% independent of the sample volume .Regarding the TNC, it became apparent that a vitality of 70%-80% was tendentially achieved with smaller volumes, in contrast to the CD34e volume , 4D. Cone volume . AlthougThe main objective of this study was the investigation of bacterial contamination rate under a routine condition in the cord blood stem cell products after vaginal delivery and cesarean section related to sample volume and bacterial strains. Additionally, the correlation between the cell numbers, sample volume, and delivery mode has been established.24 This is probably one reason for fewer cases of acute or chronic GvHD. It has also been reported that not all HLA characteristics need to be matched in allogeneic hematopoietic stem cell transplantation, making cord blood an alternative to bone marrow or peripheral blood stem cell transplantation.24-27The quality and associated safety of hematopoietic stem cells are one of the most important requirements for successful transplantation. Another important factor is considered the high concentration of adult pluripotent progenitor stem cells with increased proliferative potency, which are usually not yet affected by environmental factors and are young and vital. It is possible that due to the relative immaturity of the T cells in the CB, greater immunological tolerance can be expected after transplantation.+ stem cells .39-44Some reports also described that vaginal delivery was associated with higher TNC counts than at cesarean section.The inverse relationship between volume and contamination rate might be related to small veins, especially for smaller volumes. Small veins may result in a longer collection time, which may have some  impact on the complete sterility of the cord throughout the collection process. Furthermore, it should be noted that inexperienced staff may also contribute to higher contamination rates. Although this was not systematically evaluated in our study, there was a trend to a higher contamination rate when blood samples were drawn by less experienced staff and a separate study is underway to further address this observation.16 Moreover, it is evident that the preparation of CB for clinical use is another challenge due to the small sample volume and special sterility requirements that must be met. Studies are currently ongoing to optimize UCB sample collection with a focus on diluting the starting material, determining the best time for sample collection, and collecting test samples from UCB residues, ie, red blood cells.45 However, it should be noted that any sampling for testing would reduce the quantity of stem cells available for transplantation.Also, re-attempting collection, especially if the umbilical cord collapses quickly, can sometimes occur and sterility may no longer be optimal. Beside the vaginal or as perianal microbiomes source for contamination, kit contamination as well as bacteria from skin cannot be excluded as source during collection preparation and cryopreservation.Further results regarding the contamination rate related to collection sample volume as well as delivery mode, both birth modes showed also the inverse correlation between sample volume and contamination rate . For spo39 The lower contamination rate in cesarean sections is caused by the environmental conditions of an operating room.21The dependence of contamination rate on the mode of delivery is even more evident in the significantly higher average contamination rate of 8.4% for vaginal deliveries compared to 1.5% for cesarean deliveries ; Fig. 2Dn = 783) the most detected bacterial strains were Staphylococcus with 37.7% and Bacteroides species with 22.1% as well as E. coli species with 13.5%  and Bacteroides vulgatus  were found (i and ii). The third most common bacterial strain identified was Enterococcus , especially Enterococcus faecalis as the most common with 81.8%  in cesarean sections (The most frequently described bacterial strain sections ; Table 347 Even if the average bacterial contamination rates is not higher than those of other studies, the analysis of the last 10 years shows an average contamination rate between 6% and 9.5% (16Other authors also confirm the identification of relatively high concentrations of propionibacterium acnes in cord blood samples.and 9.5% , which a44 it is clear the processes starting with the collection of the umbilical cord blood, through the processing to the stem cell preparation to the cryopreservation according to the national guidelines and laws must be trained and optimized repeatedly to be able to fulfill the required parameters.14 Furthermore, it must be ensured that multi-resistant bacteria, one of the great challenges of our time, do not continue to advance.Even if not all bacteria survive the process of cryopreservation,The contamination rate of cryopreserved UCB is dependent on delivery mode and is inversely correlated with the volume of sample collected at birth. Based on the study presented here several lines of evidence indicate that bacterial contamination rates of UCB collected at birth for transplantation purposes may be reduced by the collection of samples after cesarean delivery together with a high sample volume. In addition, utilizing experienced and well-trained collection staff is able to successfully complete the collection in a sterile manner even in the presence of smaller veins, which can successfully reduce insufficient puncture maneuver, which can clearly increase the contamination rate.


Central nervous system (CNS) disorders are a therapeutic area in drug discovery where demand for new treatments greatly exceeds approved treatment options. This is complicated by the high failure rate in late-stage clinical trials, resulting in exorbitant costs associated with bringing new CNS drugs to market. Computer-aided drug design (CADD) techniques minimise the time and cost burdens associated with drug research and development by ensuring an advantageous starting point for pre-clinical and clinical assessments. The key elements of CADD are divided into ligand-based and structure-based methods. Ligand-based methods encompass techniques including pharmacophore modelling and quantitative structure activity relationships (QSARs), which use the relationship between biological activity and chemical structure to ascertain suitable lead molecules. In contrast, structure-based methods use information about the binding site architecture from an established protein structure to select suitable molecules for further investigation. In recent years, deep learning techniques have been applied in drug design and present an exciting addition to CADD workflows. Despite the difficulties associated with CNS drug discovery, advances towards new pharmaceutical treatments continue to be made, and CADD has supported these findings. This review explores various CADD techniques and discusses applications in CNS drug discovery from 2018 to November 2022.  Mental disorders including neurological and psychiatric disorders represent an area of medicine where there is a considerable unmet need for new and more advanced treatments. Diseases such as Alzheimer’s disease and Parkinson’s disease only have treatments available that provide symptomatic relief ,2. As thThe discovery of new drugs targeting mental disorders is associated with some of the highest fail-rates in drug discovery, with 85% of drugs failing in phase II and III clinical trials . This maCADD is typically classified into ligand-based and structure-based methods . Ligand-In this review, we summarise the most common CADD methods, including homology modelling, molecular docking, molecular dynamics simulations, pharmacophore modelling, quantitative-structure-activity relationship (QSAR) methods, and the more recent deep learning (DL) applications that have proven their efficacy in CADD. Furthermore, we provide examples of their application in drug discovery projects for psychiatric and neurological conditions. The selected examples are from 2018 to January 2023 and focus on applications that are supported by experimental data to demonstrate the validity and value of the in-silico methods applied. The examples cover a wide range of biological targets including cannabinoid receptor 1, acetylcholinesterase and the α7 nicotinic acetylcholine receptor. We demonstrate that CADD is an important tool in the discovery of CNS drugs, especially during the early stages of the drug discovery pipeline. Ligand-based drug design (LBDD) uses data from a known ligand or set of ligands to anticipate the activity of other small molecules based on the commonality with the molecules being investigated. This works on the principle that compounds with a similar chemical structure will exhibit similar binding properties.In 2D based methods, the chemical structure is represented as a set of descriptors, which are then used to screen for molecules possessing similar properties. A range of physicochemical properties and molecular features are calculated from a 2D molecular graph . These pA pharmacophore model is a collection of chemical features that defines the interactions of a ligand with a biological target that elucidates a physiological response . Pharmac3D-pharmacophore models are comprised of molecular features, such as the steric and electrostatic properties that allow the ligand to bind to the target receptor to produce a pharmacological response. Functional groups represented as hydrogen bond donors or acceptors, charges, hydrophobic and aromatic are identified in the pharmacophore . In esseTo develop a robust model that is representative of all known active molecules, pharmacophore generation must be an iterative process whereby models are scored and refined until the most suitable solution is ascertained. Typically, the scoring and generation of pharmacophore models can be categorized into either overlay or root mean square derivative (RMSD) scoring . OverlayD) or functional potency (EC50 or IC50) values. Data may be obtained in-house , or, more commonly, chemical databases  is underpinned by the correlation between the physicochemical properties and topological features of a molecule and the biological activity they exert on a target . QSAR statabases  are usedIdeally, the molecules used in model generation should be split into approximately 80% for training and 20% for testing in addition to an external validation set ,27. It iThe dimensionality of molecular descriptors in QSAR may range from zero-dimensional to six-dimensional (0D to 6D). Increasing dimensions of chemical representation will increase the level of detail about molecules in the QSAR model and, subsequently, the complexity and computing power needed. The examples in this study are limited to 3D methods, and therefore dimensions 4D–6D will not be discussed. All dimensions of QSAR are explained and reviewed in detail by Manoj et al. . Table 1Once a dataset has been curated and descriptors generated, an algorithm must be selected to complete the regression task. Broadly speaking, the algorithms used in QSAR can be categorised into linear and non-linear methods . Popular2 (1–(RSS/TSS), where RSS is the residual sum of squares and TSS is the total sum of squares) and q2 are the most popular parameters to check the goodness of fit for QSAR models [2 value is obtained by calculating R2 using leave-one-out cross validation [2 should be as close to 1 as possible for goodness of fit [2 > 0.5 and R2 > 0.6 is sufficient [2 does not always guarantee good external validity [To ensure the QSAR model being used has robust predictive abilities, validation studies must be undertaken. This is another recommendation from the OECD for the development of a credible QSAR model . It has R models ,40. The lidation . Ideallys of fit ; howeverfficient . It is ivalidity . Alternavalidity . Structure-based drug design (SBDD) is a branch of CADD and is utilised when the 3D-structure of a biological target is available. The insight into the composition of a ligand binding site allows for the screening of compound libraries and the specific design of molecules to fit optimally to the ligand binding site. Recent advances in crystallisation techniques and technical advances in cryogenic electron microscopy (cryo-EM) means that the pool of available 3D-structures of important drug targets is rapidly expanding. Furthermore, the completion of the Human Genome Project and use of artificial intelligence-based structure prediction tools such as AlphaFold2  and RoseThe first step in structure-based drug design (SBDD) is selecting a target structure and identifying the binding site of interest. The target structure can be determined experimentally by nuclear magnetic resonance (NMR), X-ray crystallography or cryo-EM. The Protein Data Bank (PDB)  is a repMolecular docking is a SBDD technique that predicts the preferred orientation and conformation of a ligand in a binding site. It can be used to screen databases of ligands, provide scores for the predicted binding energy and rank the ligands to assist in finding potential hit molecules ,64. In mv1.7 towards new therapies for neuropathic pain, used docking studies in BioSolveIT to refine a chemical library of 1.5 million ligands down to nine leads with demonstrated efficacy in a mouse model of neuropathic pain [v1.7 inhibitors at the active site was noted with the new ligand. Generally, scoring functions can be divided into three different categories: (1) empirical [The scoring of ligands determines which ligand pose is the most energetically favourable and ranks the library of screened ligands to indicate which compounds are most likely to be active and suitable for further research . This ishic pain . An impompirical ,71,72,  can account for this as a technique either before or after docking to study protein conformation, flexibility, stability and ligand sampling . Due to MD aims to simulate the time-based change in atom positions, using Newton’s equation of motion (F = ma) to offer a relationship between the force and acceleration of the atom dependent on atomic mass over time. These algorithms are implemented as force fields, with common examples including CHARMM , GROMOS MD simulations require a starting point, typically experimental models based on X-ray crystallography, cryo-EM or NMR spectroscopy. Computational models such as homology modelling, described in + or Cl−. Simulation systems require several steps of processing before they can be simulated for production runs, with this including minimisation and equilibration, where the MD system is simulated for a short period to reduce the overall system energy and to stabilise the system for production simulations. Post-simulation analysis metrics include measures such as the root mean square deviation (RMSD), a measure to determine the overall structural deviation from the initial starting pose, and root mean square fluctuations (RMSF), a measure to determine the movement of individual residues in the protein to determine the flexibility of the protein and the ligand of interest. Further analysis including ligand-to-residue contact analysis, ligand environment analysis and binding pose clustering is also performed to determine potential ligand binding poses and intermediate binding sites.In MD simulation preparation, membrane proteins are placed within a membrane model consisting of one or more lipid species such as phosphatidylcholine (POPC), phosphatidylethanolamine (POPE) and cholesterol. In an explicit water simulation system, individual water molecules are placed to fill the simulation box and random water molecules are removed and replaced with ions such as NaA key challenge in all drug discovery endeavours is ensuring that new leads have acceptable pharmacokinetic parameters of absorption, distribution, metabolism and excretion whilst also minimising toxic effects (ADMET). There exists further difficulty for CNS pharmaceuticals that must cross the blood–brain barrier (BBB), as this is a complex process that involves both the passive and active diffusion mechanisms . The incThe prediction of BBB permeability by passive diffusion can largely be predicted from physicochemical properties including lipophilicity, polarity and ionisation at physiological pH. However, several mechanisms of active transport into and efflux out of the CNS must also be accounted for in these predictions . AdditioWhile deep learning (DL) is not a new methodology, with it having been applied to language and image processing for several decades now, its applications to drug discovery efforts have only emerged in the last few years . This haThe most widely used application of CNNs are in image recognition, as the architecture mimics that of the visual cortex . CNNs coRecurrent neural networks can process sequential data by linking information from one time point back to an earlier time point. This property means they require a memory buffer to store information from previous states . These pIn computational drug discovery, as demonstrated above, deep learning applications are producing advances over more traditional techniques. Novel algorithms which combine both CNNs and LSTM for de novo design, such as the RELATION model , show prComputational methodologies have become an essential part of drug design. Recent examples have demonstrated the potential of these techniques in terms of accelerating the drug discovery pipeline and reducing the time and money spent on laboratory testing and clinical trials. 50 data from ChEMBL [1, A multi-target activity 3D-QSAR model against acetylcholinesterase (AChE), serotonin transporter (SERT), beta-secretase 1 (BACE1) and glycogen synthase kinase-3 (GSK3β) was built to towards new therapeutics for Alzheimer’s disease . The QSAm ChEMBL , with bo2, A structure-based application of the multi-target approach was also used to identify lead compounds with dual activity against AChE and α7 nAChR. The ZINC15 database , consist−1). This model was selected to screen a dataset of 2 million compounds, of which 6 were identified as suitable for docking with AutoDock Vina, MD simulations using GROMACS and in vitro assessment. Benzyl trifluoromethyl ketone , with an accuracy of 91.57%. In addition, the CNN model outperformed traditional machine learning algorithms. The top 10 ranked compounds from the unknown samples underwent molecular docking against the 5-hydroxytryptamine receptor 2A (5HTR2A) to ascertain favourable interactions between these ligands and the target proteins. Pimvanserin was used as a positive control, for which three of the ten ligands had comparable binding energy, of which the topoisomerase inhibitor topotecan 4, B was the5, Another deep learning approach, using deep neural network architecture was built to identify piperine-like compounds and drugs against these targets. The model demonstrated an accuracy 87.5%. A total of 57,423 compounds from the ZINC and PubChem databases underwent a similarity search based on piperine to find similar structures. In all, 101 compounds were selected for further investigation through docking in AutoDock 4.0, of which 5 were suitable for MD simulations on the AMBER platofrm. The docking and MD studies revealed that an additional ring in top performing compounds 5, B is like50 values for both the wild-type and G2019S mutant enzyme, with LY2019-005  antagonists was curated from the literature and split into training and test sets in a 4:1 ratio, with the randomization of this process repeated 50 times to avoid bias. MOE software was used to generate 206 molecular descriptors, which then underwent dimensionality reduction using principal component analysis (PCA). An atom-based 3D-QSAR model was developed using the partial least squares technique, with the final model having an RMSE of 0.29 and Ramine 7, C can be 50 values were identified. Compound 8 (A highlight on target sequence (HoTS) deep learning model was used to scan the purinergic P2X3 protein sequence to identify new binding sites in search of treatments for neuropathic pain . Once thmpound 8 C was the2 of 0.56, and was selected for screening. A pharmacophore model was also generated using Glide for use in virtual screening. Over 2000 small molecules from the DrugBank database were screened against the QSAR model, which had a pKi cut-off of 5.5. A total of 823 ligands were then screened against the pharmacophore model, before the best 120 underwent shape-based screening. Ligands that shared similarities to siramesine and ligands with a piperazine-containing scaffold or tetrahydroisoquinolinyl structures were kept for in vitro testing. These scaffolds are known for high S2R binding affinity. A total of 30 compounds possessed this scaffold and were identified as promising leads. Six molecules underwent biological testing, which revealed three FDA approved drugs, nefazodone, cinacalcet and pimozide, had nanomolar binding affinity values, with nefazodone  reported in the literature. MOE software was used to generate molecular descriptors of each ligand for QSAR model generation. Four algorithms were generated, namely stepwise regression, Lasso, genetic algorithm (GA) and an algorithm, GreedGene, which was developed by the authors. GreedGene had the best performance, with an Rodone 9, D being t50 value of 5.4 µM [A pharmacophore was generated using 11 α7 nAChR agonists from the literature. The pharmacophore consisted of a hydrogen bonding region, a hydrophobic centre and one positively ionised group. To reduce the number of false positives, a recursive partitioning model was also used. A virtual screening of the ChemDiv database against these two models was performed. After filtering to ensure no Lipinski parameter violations, 13 ligands were selected for in vitro assessment, 10 of which had demonstrated inhibitory effects. T761-0184 was selected for further investigation due to its high potency. This ligand underwent induced fit docking to a homology model of α7 nAChR to ascertain the binding mode for structural optimisations. Of the 51 optimised structures, B10 10, D exhibitf 5.4 µM .Computational drug design is a powerful tool in the pursuit to discover new therapies for neurological and psychiatric conditions. This literature review presents a distillation of the computational approaches to drug design from multi-disciplinary research articles to highlight the importance of CADD in finding new CNS drugs. The exemplars provided demonstrate how CADD methodologies have been utilised to present a basis for future drug development. It is apparent from the literature presented that progress towards novel therapies in CNS drug discovery is being made, in particular for drug targets where there are no therapeutics available. It is hoped that by creating novel medications towards these new targets, the standard of patient care will improve by both increased treatment efficacy and reducing side effects. In the context of CNS computational drug discovery, there is a clear trend that the majority of research is focused on investigating new treatments for neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease. However, psychiatric conditions such as schizophrenia and substance abuse disorder, as well as neurological conditions such as brain injuries and neuropathic pain, are other research foci, all of which present significant disease burdens for both the patient and society as a whole. Although it is evident there is still a long way to go for new lead molecules to become approved drugs, CADD is helping to hasten this process. As newer techniques such as deep learning become more mainstream within academic drug design research, it is expected that the efficiency with which studies can be carried out will greatly increase.


Chronic obstructive pulmonary disease (COPD) is associated with significant morbidity, mortality and healthcare costs. Beta blockers are well-established drugs widely used to treat cardiovascular conditions. Observational studies consistently report that beta blocker use in people with COPD is associated with a reduced risk of COPD exacerbations. The bisoprolol in COPD study (BICS) investigates whether adding bisoprolol to routine COPD treatment has clinical and cost-effective benefits. A sub-study will risk stratify participants for heart failure to investigate whether any beneficial effect of bisoprolol is restricted to those with unrecognised heart disease.1 < 80% predicted, FEV1/FVC < 0.7), a self-reported history of ≥ 2 exacerbations requiring treatment with antibiotics and/or oral corticosteroids in a 12-month period since March 2019, age ≥ 40 years and a smoking history ≥ 10 pack years. A computerised randomisation system will allocate 1574 participants with equal probability to intervention or control groups, stratified by centre and recruitment in primary/secondary care. The intervention is bisoprolol (1.25 mg tablets) or identical placebo. The dose of bisoprolol/placebo is titrated up to a maximum of 4 tablets a day (5 mg bisoprolol) over 4–7 weeks depending on tolerance to up-dosing of bisoprolol/placebo—these titration assessments are completed by telephone or video call. Participants complete the remainder of the 52-week treatment period on the final titrated dose  and during that time are followed up at 26 and 52 weeks by telephone or video call. The primary outcome is the total number of participant reported COPD exacerbations requiring oral corticosteroids and/or antibiotics during the 52-week treatment period. A sub-study will risk stratify participants for heart failure by echocardiography and measurement of blood biomarkers.BICS is a pragmatic randomised parallel group double-blind placebo-controlled trial conducted in UK primary and secondary care sites. The major inclusion criteria are an established predominant respiratory diagnosis of COPD (post-bronchodilator FEVThe demonstration that bisoprolol reduces the incidence of exacerbations would be relevant not only to patients and clinicians but also to healthcare providers, in the UK and globally.ISRCTN10497306. Registered on 16 August 2018Current controlled trials The online version contains supplementary material available at 10.1186/s13063-022-06226-8. The order of the items has been modified to group similar items (see Chronic obstructive pulmonary disease (COPD) is a lung disease characterised by progressive airflow obstruction . GloballAcute deteriorations in symptoms known as exacerbations are an important clinical feature of COPD. They are associated with accelerated lung function decline, reduced physical activity, reduced quality of life and increased mortality –16. COPDBeta blockers are a class of drug with proven benefit in people with heart failure or ischemic heart disease, particularly those with left ventricular impairment. The rationale for repurposing beta blockers for use in COPD comes from the findings of observational cohort studies that beta blocker use in people with COPD is associated with a reduced risk of exacerbation –24. A syBeta blockers competitively antagonise the effects of catecholamines on beta-adrenoreceptors. Beta1-adrenoreceptors are found only in the heart, whereas beta2-adrenoreceptors are more ubiquitous, being found in the heart and lungs. Bronchodilating beta2-agonists are the mainstay of COPD and asthma treatment and beta blockers that antagonise beta2-agonists could have adverse respiratory effects, and for asthma, a condition with reversible airflow limitation, beta blockers are usually avoided . A systeWe describe here the bisoprolol in COPD study (BICS) which tests the hypothesis that adding bisoprolol to routine COPD treatment reduces the rate of exacerbation. A sub-study will test the hypothesis that any beneficial effect of bisoprolol is restricted to those with unrecognised heart disease. The full protocol is available as a The primary objective is to determine the clinical and cost-effectiveness of adding bisoprolol to usual COPD treatment in patients with COPD at high risk of exacerbation as evidenced by a history of at least two COPD exacerbations in a previous year.Hospital admissions with a primary diagnosis of COPD exacerbationTime to first exacerbation of COPDTotal number of emergency hospital admissionsTotal number of major adverse cardiovascular events (MACE)Lung functionChanges in breathlessness during treatmentAll-cause, respiratory and cardiac mortalityDrug reactions and serious adverse eventsHealth-related quality of lifeDisease specific health statusHealth care utilisationIncremental cost-per-exacerbation avoidedCosts to the NHS and patients and lifetime cost-effectiveness based on extrapolation modellingModelled lifetime incremental cost per quality-adjusted life yearThe secondary objectives are to compare the following outcomes between participants treated with bisoprolol and those treated with placebo:o.Treatment effects in participants with and without unrecognised heart diseaseThe sub-study will risk stratify participants to investigateBICS is a pragmatic randomised, double-blind, parallel group, placebo-controlled, multicentre clinical trial investigating whether bisoprolol is superior to placebo when added to current COPD therapy for 52 weeks in patients with COPD who have had two or more exacerbations of COPD in a previous year treated with oral corticosteroids (OCS) and/or antibiotics. Figure There is a sub-study to the main BICS study—that stratifies BICS participants into those with and without unrecognised heart disease based on blood biomarkers and echocardiogram to investigate whether any beneficial effect of bisoprolol is restricted to those with previously unrecognised heart disease.https://w3.abdn.ac.uk/hsru/BICS/Public/Public/index.cshtml.Participants are being recruited from primary and secondary care sites across the UK. In primary care some General Practices are acting as recruitment sites, whereas others act as participant identification centres (PICs) with identified participants being evaluated in other primary or secondary care recruitment sites. A list of the study sites can be found at Aged ≥ 40 yearsA smoking history of at least 10 pack years ([average number of cigarettes/day x years smoked]/20)1 < 80% predicted, FEV1/FVC < 0.7) .The full protocol is included as The immediate trial team based in the coordinating centre  meet weekly initially. The frequency of these meetings is likely to reduce as the trial progresses, but can revert to weekly meetings as and when required. On a monthly basis, the immediate team is joined by a wider team . A Project Management Group (PMG) and Trial Steering Group (TSC) oversee the project. The PMG meets approximately every three months and comprises the chief investigators, grant holders  and the trial office staff. The TSC meet approximately every 6 months and includes an independent chair, clinical and methodological expertise and lay representatives.The independent Data Monitoring Committee (DMC) comprises an independent chair and independent members with clinical, statistical and methodological expertise. The DMC meets every 6 months or more frequently if required. The DMC is independent of the sponsor and competing interests and reports to the chair of the Trial Steering Committee.The trial complies with the UK National Health Service National Research Ethics Service guidelines for reporting adverse events . AdverseThe trial office monitor aspects of the study on an ongoing basis as described in the study monitoring plan prepared at the outset of the study. The trial is also monitored and audited by the Sponsor. Individual sites may be monitored by their local Research and Development department.Changes to the protocol require the trial office to seek permission from the funder, sponsor, REC and NHS R&D offices.A summary of the study findings will be sent to surviving trial participants, their GPs and investigators. The clinical study report will be used for publication and presentation at scientific meetings and at patient and clinical interest group events. The publication policy is included in the protocol There is a pressing need to identify interventions that reduce the impact of COPD, a common disease that continues to be associated with high morbidity, mortality and healthcare costs. BICS is a pragmatic randomised double-blind placebo-controlled trial that tests the hypothesis that adding the beta blocker bisoprolol to routine COPD treatment reduces the rate of exacerbation. The cardiac sub-study tests the hypothesis that any beneficial effect of bisoprolol is restricted to those with unrecognised heart disease.Recruitment to the BICS trial was paused in March 2020 due to the COVID-19 pandemic and remained paused until July 2021 when the sponsor and funder agreed that the study could re-open to recruitment. As part of the plans for re-opening, a number of revisions were made to the protocol.Firstly, we reduced the number of face to face assessments. When the study first opened to recruitment in 2018, there were up to 7 face-to-face assessments . In response to COVID-19, the number of face-to-face assessments for the main BICS study was reduced to one baseline assessment; although if the site or participant prefer to carry out the baseline assessments remotely , this is permitted. Those participants who wish to take part in the sub-study will have two additional face-to-face assessments, one soon after recruitment for the blood samples and echocardiogram, and one around the 52-week follow-up for blood samples.1/FVC < 0.7 was permitted but FEV1 < 80% predicted needed to be demonstrated at baseline. The inclusion criteria were amended so that both FEV1/FVC < 0.7 and FEV1 < 80% predicted are based on historical readings recorded in the medical records.Secondly, we amended the inclusion criteria. When the study first opened to recruitment, one of the inclusion criteria was “at least two exacerbations requiring treatment with antibiotics and/or oral corticosteroid use in the previous year, based on patient report,” and this was revised so that the two exacerbations could have occurred since March 2019, providing they were within 12 months of each other. The number of exacerbations experienced by people with COPD declined during the COVID-19 pandemic probably because of COVID-19 mitigation measures, e.g. shielding, social distancing measures and face coverings . The ECL1. Again, because of the limitations on spirometry in the NHS, the titration schedule was revised so that FEV1 was replaced with participant self-report of their breathing—with a particular focus on deterioration since the study medication was started or the dose of study medication was increased.Thirdly, we amended the titration schedule within the study. When the study first opened to recruitment, the titration schedule included FEVBeta blockers are predominantly used in the management of cardiovascular conditions e.g. hypertension, heart failure; however, it has been observed that beta blocker use in people with COPD is associated with a reduced risk of exacerbation –24. A syThe sample size for current study was based on the ECLIPSE study reporting that for people with COPD with two or more exacerbations in a year, the mean (SD) number of COPD exacerbations within the subsequent one year was 2.22 (1.86). In our trial of low-dose theophylline in COPD (TWICS), for people with COPD with two or more exacerbations in the previous year, the mean (95% CI) number of exacerbations in the subsequent year was 2.23 . The TWICS study also confirmed reports that patient recall of COPD exacerbations is reliable over a year with 79% concordance between participant and GP clinical records of exacerbation in a validation exercise , 50.1 < 80% predicted) conducted in the USA (protocol NCT02587351) [p = 0.004). The BICS trial differs from BLOCK COPD in several important respects. In BICS the beta blocker bisoprolol is being tested because, unlike metoprolol, it is licensed for use in heart failure in the UK and has a higher beta1:beta2 receptor selectivity ratio (14:1) than metoprolol (2:1) [1 < 70% predicted) with ≥ 1 exacerbation in the previous year will be randomised 1:1 to bisoprolol or placebo, the treatment period is 2 years and the doses of bisoprolol will be 1.25 mg, 2.5 mg or 5 mg od. The primary outcomes include all-cause mortality, hospitalisation for COPD exacerbation, hospitalisation for primary cardiac cause and major adverse cardiovascular events. A single-arm open label trial of bisoprolol in people with moderate/severe COPD (FEV1 < 80% predicted) and hypertension conducted in Japan (protocol UMIN000024712) appears to have ceased recruitment [BICS is one of several trials investigating the role of beta blockers in people with COPD. BLOCK COPD was a multicentre randomised double blind placebo controlled trial of metoprolol in people with moderate/severe COPD (FEVol (2:1) . In BLOCruitment . A totalruitment . All parIt is almost certain that a substantial proportion of the participants in BICS will have severe lung disease and will have limited exercise tolerance. When the study first opened to recruitment, allowances were made in the trial design to facilitate participation by this group of patients: at site discretion, participants could be recruited and dose titrated home using portable spirometers and sphygmomanometers, and those unable to attend for follow-up assessment visits would be assessed by telephone review and postal collection of quality of life questionnaires. We hope that the revisions made to the protocol to allow restart have made the study even more accessible to this group of patients. Study medication is to be couriered directly to the homes of participants, thus avoiding travel to study centres to collect supplies.The dose each participant takes for the duration of the study is determined by titrating the dose of study drug during the first 4 weeks of the treatment period; however, to increase flexibility and to replicate ‘real life’, there is sufficient medication in the first batch of medication for the dose titration to be conducted over 7 weeks. The study drug is provided as 1.25 mg bisoprolol tablets; the dose titration results in final doses of 1, 2, 3 or 4 tablets a day. A single tablet was chosen to avoid the complications of different tablet strengths and doses. The dose titration schedule is a conservative interpretation of the ‘start low, go slow’ advice provided in heart failure guidelines designed for use by appropriately trained nurses in primary care settings , 30. DecBisoprolol is a relatively cheap drug familiar to many clinicians whose use is established in people with heart failure and COPD. The demonstration that the addition of bisoprolol to routine COPD treatment reduces the likelihood of exacerbation will be relevant not only to patients and clinicians but also to healthcare providers, both in the UK, and globally.The current protocol is version 7, 14 May 2021. The first participant was recruited on 16 October 2018. The trial was paused to recruitment in March 2020 due to the COVID-19 pandemic. It remained paused to recruitment until July 2021 when the sponsor and funder agreed that the study could re-open to recruitment. BICS is currently recruiting patients until 31 March 2022 by which point discussions with the funder regarding study viability in relation to COVID-19 and recruitment will have been completed.Additional file 1.


It remains unclear whether the rate of fetal death has changed during the COVID-19 pandemic. We assessed the impact of COVID-19 mitigation measures on fetal death in Sweden , Denmark  and Norway  using robust population-based registry data. We used Cox regression to assess the impact of the implementation of pandemic mitigation measures on March 12th, 2020, on miscarriage  and stillbirth . A total of 11% of 551,914 pregnancies in Denmark and Norway ended in miscarriage, while the proportion of stillbirths among 937,174 births across the three countries was 0.3%. There was no difference in the risk of fetal death during the year following pandemic mitigation measures. For miscarriage, the combined hazard ratio (HR) for Norway and Denmark was 1.01 , and for stillbirth, the combined HR for all three countries was 0.99 . We observed a slightly decreased risk of miscarriage during the first 4 months, with an HR of 0.94  after lockdown. In conclusion, the risk of fetal death did not change after the implementation of COVID-19 pandemic mitigation measures in the three Scandinavian countries. It was suggested early on that pandemic mitigation measures could influence the rate of pregnancy outcomes; however, whether this is the case is controversial due to inconsistent findings across countries3. Some studies report evidence of an increase in stillbirths during the first wave of the pandemic9, while other studies found no evidence of a difference17. A meta-analysis concluded that there was significant evidence of an increase in stillbirths after pandemic lockdown18. The majority of the existing studies had a limited sample size, with fewer than 30 stillbirth cases after the onset of the pandemic12. The largest study to date used information on more than 400,000 births during the pandemic identified through National Health Service hospital admissions in England and indicated no difference in the rate of stillbirth after the start of the pandemic19. There are no existing studies that compare the rate of miscarriage following pandemic lockdown to rates during the prepandemic period.The COVID-19 pandemic has had broad social, economic and health-related impacts22. There were clear changes in the behaviour of the populations in all three countries from mid-March, resulting in a decreased use of public transportation, less workplace commuting and more time spent at home23. Behavioural measures indicated that the strict lockdowns of Denmark and Norway resulted in more drastic behavioural changes than in Sweden24. We hypothesized that the pandemic mitigation measures could plausibly decrease the risk of fetal death, as pregnant women were likely to have less exposure to infections26.The Scandinavian countries, Sweden, Denmark and Norway, are similar with regard to their universal healthcare, levels of income inequality, and fertility patterns. When the World Health Organization declared that COVID-19 had reached a pandemic level , the number of infected persons was relatively low in all three Scandinavian countries. Denmark and Norway initiated strict pandemic mitigation measures in mid-March in an attempt to avoid the high burdens on the health-care systems observed across several other European countries, while Sweden followed a less strict approachThe objective of this study was to assess the impact of COVID-19 mitigation measures on the risk of fetal death using national registry-based data from Sweden, Denmark and Norway.We extracted information on pregnancies registered with a live birth, stillbirth or miscarriage between January 2017 and March 2021. In Denmark and Norway, information was available from national Medical Birth Registers and National Patient Registers and included all pregnancies resulting in any contact with specialist health-care services . The data from Denmark and Norway therefore included all births (live and stillbirths), in addition to all recognized miscarriages resulting in contact with specialist health-care services. This is likely to include the overwhelming majority of recognized first-trimester miscarriages and all second trimester miscarriages. In Norway, the exact gestational age of the first-trimester miscarriages occurring before 12 completed gestational weeks was not available. Based on registrations in Denmark, the median gestational week of the registered first-trimester miscarriages was 8 weeks . From the Swedish Pregnancy Register, we retrieved information on all births at ≥ 22 gestational weeks during the same time period; in Sweden, 92% of births are included in the national register (18 of 21 Swedish regions). We therefore had information on all registered pregnancies for Denmark and Norway, while we only had information on births for Sweden. Further details of the data sources are listed in the appendix (supplementary online methods). This study was approved by the Regional Committee for Medical and Health Research Ethics of South/East Norway (#141135) and the Swedish Ethical Review Authority . Denmark, the study was registered with the Danish Data Protection Agency via the University of Southern Denmark (reg. no. 364 20/17416) and via Statistics Denmark. Each committee provided a waiver of consent for participants. The ethical committees mentioned above in the three Nordic countries are grounded in foundational ethical principles embodied in the Declaration of Helsinki of 1964 and its subsequent revisions and the Belmont Report.Although the intensity and timing of COVID-19 mitigation measures differed between the three countries, the majority of measures were introduced around March 12th, 2020  and the national Patient Registry . For Norway, the Medical Birth Registry included all pregnancies ending after 12 completed gestational weeks, while information on pregnancies ending prior to this time was obtained through the National Patient Registry. In Sweden, we obtained information on deliveries after 22 completed gestational weeks from the Pregnancy Register. Further details on the identification of pregnancies across the three different countries are available in the supplemental methods. We examined the risk of miscarriage only in Denmark and Norway, while the risk of stillbirth was examined in all three countries.28. The Danish Medical Birth Register includes all births up to December 31, 2018, but only a proportion of births from the first quarter of 2019. Therefore, from January 1, 2019, we identified live births based on registrations of International Classification of Disease version 10 (ICD-10) codes Z38 and O80-84, and stillbirths based on registrations of ICD-10 code P95 in the National Patient Registry. Any registration of stillbirths with a birthweight of < 500 g and where gestational age was missing or less than 22 weeks was reclassified as miscarriages. Miscarriages were further identified using ICD-10 codes O02- “spontaneous abortion” and O03- “other abnormal products of conception”. In Norway, information on all pregnancies ending after 12 completed gestational weeks was available from the Medical Birth Registry of Norway29. All pregnancies recorded in the birth registry are designated as either having resulted in a live birth or a fetal death. We distinguished fetal deaths according to whether they were a miscarriage or a stillbirth based on information on birthweight and gestational age. We defined a fetal death as a miscarriage if the gestational age was < 22 completed weeks and the birthweight was < 500 g, while a fetal death with a gestational age ≥ 22 completed gestational weeks or a birthweight ≥ 500 g was classified as a stillbirth. Information on miscarriages prior to 12 completed gestational weeks was available from the patient registry30. As in Denmark, we used the ICD-10 codes O02 and O03 to identify miscarriages. In Sweden, we had information on all deliveries (live and stillbirths) after 22 completed gestational weeks from the Swedish Pregnancy Register. This quality register was initiated in 2013 and includes 92% of all births in Sweden (18 of 21 regions).In Denmark, information on all pregnancies was available from the National Patient Registry and the Danish Medical Birth RegisterI2 statistic, calculated as 100% × (Q–df)/Q, where Q is Cochrane's heterogeneity statistic and df denotes degrees of freedom31. The main analysis examined whether the implementation of pandemic mitigation measures on March 12th, 2020, influenced the risk of fetal death during a 12-month follow-up period. Secondary analyses examined more immediate effects of the implementation of the pandemic mitigation measures during the 2, 4 and 6 months following March 12th, 2020. Analyses were performed using Stata version 16 .Individual-level data were analysed for each country separately. We examined differences in the risk of miscarriage and stillbirth using Cox proportional hazards regression with the gestational day as the time metric. We chose to use a Cox regression to distinguish between the time of the pregnancy that was pre-pandemic as opposed to during the pandemic. To avoid oversampling of short pregnancies and fetal losses towards the end of the study period, we excluded all pregnancies that did not have the opportunity to reach 42 completed gestational weeks by the end of follow-up based on the best estimate of the start of pregnancy, prioritising ultrasound measurements where this was available. Exposure to pandemic mitigation measures  was used as a time-varying exposure, derived using gestational day on the implementation of pandemic mitigation measures . Women could therefore contribute both exposed and unexposed follow-up time if their pregnancy started before and ended after March 12th, 2020. We adjusted for calendar week as a discrete variable to account for seasonal variation in the risk of the outcomes of interest. We examined the validity of the proportional hazards assumption by visually inspecting the Schoenfeld residuals. The results from each country were subsequently combined using a random effects meta-analysis. As the individual-level datasets were housed at independent institutions in the three countries, it was not possible to conduct a pooled analysis of the individual-level data and test differences between the countries using an interaction term. Heterogeneity was instead assessed using the A total of 449,347 births were recorded in Sweden during the study period, while 290,857 pregnancies  were recorded in Denmark, and 261,057 pregnancies  were recorded in Norway. The overall proportion of miscarriage among all pregnancies during the follow-up period was 11.4% in Denmark and 11.9% in Norway. Among all births, the proportion ending in stillbirth was 0.3% in all three countries. The proportion of stillbirth and miscarriage remained relatively stable in all three countries during the prepandemic study period.2 0%, p value 0.39)   Fig. . SimilarUsing national registries from Sweden, Denmark and Norway, we found no increase in fetal death after the implementation of COVID-19 pandemic mitigation measures across three Scandinavian countries. We observed a modest decreased risk of miscarriage during the first 4 months following March 12th, 2020.9, but the evidence is inconsistent17. As the majority of the existing studies have a very small sample size (with between 20 and 140 cases of stillbirth after lockdown), there is a high degree of uncertainty in the estimates. A meta-analysis of 6 studies suggested an increased risk of stillbirth during the first wave of the pandemic, with a combined incidence rate ratio of 1.33 18. Differences in the findings across studies could be due to variations in the sample size, the country income level, financing of the health-care system, and the extensiveness of pandemic mitigation measures. Two of the previous studies included in the meta-analysis had ~ 140 cases of stillbirth after lockdown7, while the rest of the studies had only approximately 20 cases of stillbirth after lockdown or less12. The largest study to date  from the UK had a clear null finding, similar to that observed across the three countries in our study, with a risk of 0.36% during the pandemic versus 0.37% during the prepandemic period (p = 0.16)19.Some existing studies suggest an increase in stillbirths following COVID-19 pandemic mitigation measuresTo our knowledge, studies examining the change in the risk of miscarriage after the implementation of pandemic mitigation measures are lacking.35 and stillbirth38, although understanding of the biological mechanisms remains inadequate. Our findings are reassuring given that general access to health care among pregnant women was reduced during the early stages of the pandemic. There are a number of potential explanations for how pandemic mitigation measures could plausibly contribute to a modest decreased risk of miscarriage. For example, certain infectious diseases are associated with an increased risk of miscarriage26, and a reduced number of social contacts (as a result of pandemic restrictions) may have led to fewer infections (not only COVID-19) among pregnant women. It is also possible that pandemic restriction measures resulted in less physical stress, which again could have reduced women’s risk of miscarriage, although the impact on work-related physical strain on the risk of miscarriage is likely to be modest25. However, the fact that we observed this modest decreased risk of miscarriage only during the first months after implementation of pandemic mitigation measures might also indicate that this modest reduction is unlikely to be causal. For example, if a larger proportion of miscarriages was seen only in primary care  during this rather chaotic time, this could also explain this finding. The lack of a causal relationship between pandemic mitigation measures and risk of fetal death is further substantiated by the fact that we did not see any changes in stillbirth.There are several well-known risk factors for miscarriage39. The missing registrations did not depend on birth outcomes and would not bias associations. Information on early miscarriages registered in specialist health-care services in the Danish and Norwegian data at the population level is unique from an international perspective. For the Danish data, the introduction of new registration procedures during the study period may have led to inconsistencies in registration practices over time. Notably, we were only able to capture miscarriages resulting in contact with specialist health-care services. Very early miscarriages for which the woman did not seek health care or only contacted primary care services are therefore not captured. Based on previous estimates from the Norwegian general practitioner database, approximately ¼ of first-trimester miscarriages are only seen in primary care40. Therefore, our study will have underestimated the number of miscarriages. This could have contributed to our observation of a modest reduction in the number of miscarriages during the first months after the pandemic lockdown if a greater proportion of miscarriages were seen in the primary care during this time period because of a higher threshold for referring early miscarriages to specialist care during this early period.We studied more than one million pregnancies in the three Scandinavian countries from January 2017 through March 2021. The universal (free) health-care system in the Nordic countries and the mandatory registration of all contacts with the health-care system in the national registries ensured that we captured the majority of pregnancies during this period. For Norway and Denmark, all live and stillbirths (including home births) during the study period were included. For Sweden, we captured 92% of registered births (live and stillbirths), as information on approximately 8% of births is missing due to incomplete electronic data transfer in 3 of Sweden’s 21 counties44 or any evidence to support an increased risk of fetal death after vaccination47.The aim of our study was to assess the overall impact of pandemic mitigation measures on the rate of fetal death. We therefore did not include information on infection or vaccination for SARS-CoV-2. However, the current limited evidence indicates no strong evidence of an increased risk of fetal death among women who experienced SARS-CoV-2 infection during pregnancyIn conclusion, we observed no overall evidence of a change in the risk of miscarriage or stillbirth in the year following the implementation of pandemic mitigation measures across three Scandinavian countries.Supplementary Information.