The application of nanomedicine to antiretroviral drug delivery holds promise in reducing the comorbidities related to long-term systemic exposure to highly active antiretroviral therapy (HAART). However, the safety of drugs loaded with silver nanoparticles has been debatable. This study is aimed at evaluating the effects of HAART-loaded silver nanoparticles (HAART-AgNPs) on the behavioural assessment, biochemical indices, morphological, and morphometric of the hippocampus in diabetic Sprague-Dawley rats. n = 8) A (nondiabetic+vehicle), B (nondiabetic+HAART), C (nondiabetic+HAART-AgNPs), D (diabetic+vehicle), E (diabetic+HAART), and F (diabetic+HAART-AgNPs). Morris water maze, Y-maze test, and weekly blood glucose levels were carried out. Following the last dose of 8-week treatment, the rats were anaesthetized and euthanized. Brain tissues were carefully removed and postfixed for Nissl staining histology.  Conjugated HAART-AgNPs were characterized using FTIR spectroscopy, UV spectrophotometer, HR-TEM, SEM, and EDX for absorbance peaks, size and morphology, and elemental components. Forty-eight male SD rats (250 ± 13 g) were divided into nondiabetic and diabetic groups. Each group was subdivided into  alleviates diabetes-induced neurotoxicity. Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycaemia. The long-term effects of diabetes can lead to conditions such as neuropathy, nephropathy, and cardiovascular complications , 2. TypeMany drugs cause insulin resistance or impaired insulin secretion, leading to diabetes in individuals with risk factors. The association between highly active antiretroviral therapy (HAART) and the development of T2DM has been reported , 4.Diabetes mellitus has been implicated in various neurological diseases such as neuropathy and neuroinflammation. Its long-term effects on the brain can manifest at physiological and structural levels . In diabThe standard HAART comes in a combination form of two nucleoside reverse transcriptase inhibitors  and with one of the nonnucleoside reverse transcriptase inhibitors (efavirenz) or protease inhibitors (ritonavir/atazanavir) or integrase inhibitor  .The HAART, a combination of tenofovir (TDF), emtricitabine (FTC), and efavirenz (EFV) as a fixed-dose therapy for the treatment of Human Immunodeficiency Virus (HIV), has proven to be more effective when compared to other fixed-dose combinations . While HThe mechanism through which HAART causes diabetes is not fully understood. However, a study by Avari and Devendra  has attrThe application of nanobased drug delivery systems in the treatment of diseases including HIV/AIDS is relatively new but rapidly developing, where nanoparticles are employed as a vehicle to deliver the therapeutic agent to specifically targeted cells in a controlled manner .The emergence of the drug nanodelivery system has tremendous improvement on HIV/AIDS treatment because of its ability to achieve drug efficacy, crossing biological barriers, targeting specific cell populations, and long-term drug release , 26. NevSilver nanoparticles (AgNPs) have been used for a wide range of applications in the biomedical field due to their novel property and simple synthesis method . AgNPs hHence, this study is aimed at evaluating the effects of HAART-loaded silver nanoparticles (HAART-AgNPs) on the behavioural assessment, biochemical indices, and morphological and morphometric of the hippocampus in STZ-induced diabetic Sprague-Dawley rats.3), trisodium citrate, streptozotocin (STZ), and sodium hydroxide of analytical grade were bought from Sigma-Aldrich Company, Johannesburg, South Africa. All the chemicals were of analytical quality. Sterile distilled water was used in all the stages of this experiment throughout.The highly active antiretroviral therapy (HAART), a fixed-dose combination of efavirenz , emtricitabine , and tenofovir disoproxil fumarate , was bought from Dis-Chem Pharmacy Ballito, South Africa. The other chemicals such as silver nitrate (AgN03) was used as a template, trisodium citrate (TSC) was used as a reducing and stabilizing agent, and distilled deionized water was used as a dissolving agent. An aqueous solution of 0.30 M AgNO3 was prepared by drying the AgNO3 crystal in an oven at 100°C, weighing 5.10 g of the dried crystals in a 100 mL volumetric flask, and dissolving double-distilled water. A series of TSC solutions  was prepared from an aqueous stock solution of 2 M (147 g in 250 mL of double-distilled water). Four TSC solutions of varying concentrations  were used for the study. The AgNPs formed were under continuous stirring at 90°C for 5 minutes at a pH of 10.5. After that, the solution changed in colour from colourless to amber, signifying the formation of AgNPs.Silver nanoparticles were synthesized according to Turkevich et al. . Silver 15 g of the HAART was weighed out and dissolved in 10 mL concentrated sodium hydroxide, put in a 50 mL volumetric flask, and added distilled water. The 50 mL HAART solution was mixed with 100 mL AgNP aqueous solution. The concentration of HAART used was 1.05 M. The ultrasonicator was used to ensure the proper blending of the constituents, the AgNPs, and HAART.The resultant nanoformulations, HAART-AgNPs, were centrifuged at 4,500 rpm at 40°C, at 40 minutes to separate the unincorporated drug. The supernatant was analyzed using a UV spectrophotometer at a wavelength of 285-315 nm to calculate the quantity of unincorporated drug (W1) from the total amount of drug coupled with silver nanoparticle (W2).The percentage incorporated efficiency for the formulated HAART-AgNPs was estimated usingThe formulated nanoconjugates (AgNPs and HAART-AgNPs) were confirmed by ultraviolet-visible (UV-Vis) spectroscopy  and Fourier transform infrared (FTIR) spectroscopy . The morphology and size of the nanoparticles were evaluated by a high-resolution transmission electron microscope  operated at a voltage of 200 Kv. Field emission scanning electron microscope  performed at a voltage of 5 keV with energy dispersive X-ray .Forty-eight  adult maThe animals were handled in accordance with the standard guides for the animal laboratory, and the University of KwaZulu-Natal Animal Ethics Committee approved the protocol (AREC/044/019D).ad libitum) for two weeks followed by an overnight fasting and single intraperitoneal injection of STZ (40 mg/kg B.W) in 0.9% NaCl with 100 mM sodium citrate buffer (pH 4.5). The control rats received the vehicle (citrate buffer).Type 2 diabetes mellitus was induced in the experimental animals according to Wilson and Islam . BrieflyThe diabetic condition was confirmed five days after STZ injection by blood glucose level ≥ 200 mg/dL.Drug administration began after the rats were confirmed diabetic. The rats were either administered vehicle, HAART, or HAART-AgNPs once daily using oral cannula except for HAART-AgNPs, which was administered intraperitoneally (5 days/week).The animals were grouped and treated for eight  weeks asGroup A: nondiabetic control+vehicle (0.5 mL/100 g)Group B: nondiabetic+HAART Group C: nondiabetic+HAART-AgNPs (24.5 mg/kg b.w i. p)Group D: diabetic control+vehicle (0.5 mL/100 g)Group E: diabetic+HAART Group F: diabetic+HAART-AgNPs (24.5 mg/kg b.w i. p)Everson et al. .The rats' spatial learning and memory performance were evaluated using a Moris water maze, as previously described by Greish et al. and Morris , 35. TheThe latency to reach the hidden platform submerged 2 cm below the water level was recorded.Two days later, the probe trial was performed on each animal, following the same procedure as the training trial. In the probe trial, the hidden platform was removed from the pool, but other cues remained in the same place for both training trials and probe trials, and animals were given 120 seconds of swimming in searching for the platform. The starting point for both the training trial and probe trial is the marked point in the quadrant opposite the platform quadrant, and this was used for the probe trial recording. The percentage of time spent swimming in the quadrant previously contained platform was recorded and calculated. The longer the time spent in the quadrant that previously had the platform was considered as a measure of learning and memory retention.The spontaneous alternation was used to measure the spatial working memory and locomotion activity according to Hughes and Typlt et al. , 37. BriEach arm is identified by letters A, B, and C. The rats were placed at the centre of the maze and allowed to move freely for 5 mins, and their tails must completely enter into the arm from the centre before recording as one entry.The number of entries was recorded using a video camera, and two independent observers recorded the data from the computer system. Between each experiment session, the apparatus was cleaned with a tissue soaked with alcohol and, after that, dry with tissue paper.Percent alternation was calculated as follows:by Typlt et al. The brain tissue was harvested and rinsed in PBL. 0.5 g of the harvested brain tissue was homogenized in 5 mL sodium phosphate buffer with 1% triton with 1% triton X-100 . The homogenates were centrifuged for 10 mins at 20,000 g (4°C). The supernatants were decanted into 2 mL Eppendorf tubes, labelled, and preserved at -80°C until further analysis.The brain homogenates were used to measure the concentration of reduced glutathione (GSH), antioxidant enzymes (SOD), and lipid peroxidation (MDA) using a spectrophotometric assay.Glutathione (GSH) level was assessed according to Ellman's  protocol2.All the rats from each group were anaesthetized and euthanized at the end of the experiment, followed by the total body perfusion with 0.1 M phosphate buffer saline (pH 7.4) transcardially. The brain was carefully removed and weighed, then postfixed in 4% paraformaldehyde for 48 h. The hippocampus of the rat was dissected from the brain and processed for paraffin embedding. Following this, the brain tissues were infiltrated with 30% sucrose for three days at 4°C. The paraffin-embedded brain tissues were sectioned at 5 𝜇m using Leica RM 2255 microtome, and these tissues were stained according to the method of Gu et al. . The corp < 0.05 was considered statistically significant.Most measurements are continuous variables and are presented graphically by descriptive statistics. ANOVA was used to compare more than two groups to determine the statistical significance. Where one-way ANOVA was used, Dunnett's multiple comparison post hoc test was carried out. All analyses were done using GraphPad Prism 9 for Windows , and values were expressed as mean ± SEM. + to Ag0 and stabilizing the AgNPs. The IR spectrum of AgNPs shows absorption bands at 3227.27 cm−1 .As presented in .27 cm−1  while HAcm−1  with a total weight of 55.2 g percentage of silver nitrate. In addition, a concentration of 1.5 M contained 16.89% of Ag, and 23.34% of Ag was discovered in 0.5 M, while 2 M concentration possessed the least Ag percentage with 8.38%.p < 0.05) increase in blood glucose level in diabetic groups compared to their respective control in pre- and postadministration. Interestingly, after 8 weeks of treatment, group F (diabetic+HAART-AgNPs) shows a significant (p < 0.05) reduction in blood glucose level compared to group E (diabetic+HAART), as shown in There was a significant (p < 0.05. Group F (diabetic+HAART-AgNPs) shows a significant reduction in the latency to reach the hidden platform between the first and third-day trials. Interestingly, diabetic rats treated with HAART-AgNPs significantly enhanced learning by reducing the latency compared to diabetic control group D on the last day trails, as shown in Spatial learning was assessed in nondiabetic and diabetic groups treated with vehicle, HAART, and HAART-AgNPs using Morris water maze (latency to reach the hidden platform 2 cm below water level). The control group A shows learning behaviour by significantly reducing the latency to reach the submerged platform in the pool in four trials for three consecutive days p < 0.05) improved memory by increasing the time spent in the quadrant previously contained platform compared to diabetic control (group D), as shown in The percentage of time spent swimming in the quadrant previously contained platform was significantly reduced in group D (diabetic control) compared to group A (nondiabetic control). Administration of HAART-AgNPs to the diabetic rats significantly (p < 0.05) reduction in spontaneous alternation compared to nondiabetic control (group A). There was a significant improvement in the spatial working memory of diabetic rats treated with HAART-AgNPs (group F) compared to diabetic groups treated with HAART (group E) and diabetic control (group D), as shown in Working memory was assessed on nondiabetic and diabetic groups treated with vehicle, HAART, and HAART-AgNPs using Y-maze. Diabetic control (group D) shows a significant (p < 0.05) in diabetic control compared to nondiabetic control group A. Interestingly, group F (diabetic+HAART-AgNPs) improved locomotion compared to group D (diabetic control) and group E (diabetic+HAART), as shown in Locomotion was evaluated using a total number of arm entries/5 mins in the Y-maze. Locomotion activity was significantly reduced (p < 0.05) in the SOD and GSH levels of diabetic control (group D) compared to nondiabetic control (group A). MDA also significantly increased in the diabetic control compared to nondiabetic control. Notably, there was a significant (p < 0.05) improvement in GSH, and MDA levels of the diabetic rats treated with HAART-AgNPs (group F) compared to diabetic rats treated with HAART only (group E), as presented in There was a significant decrease  shows poorly Nissl staining compared with the nondiabetic control group. Interestingly, the Nissl staining for the diabetic group treated with HAART-AgNPs (group F) shows improved Nissl staining of the pyramidal cells, as shown in p < 0.05) decrease in positive Nissl-stained neurons in diabetic control (18.62 ± 0.866) compared to nondiabetic control (35.08 ± 2.377). Notably, diabetic rats treated with HAART-AgNPs (31.30 ± 2.024) showed a significant increase p < 0.05 in the number of surviving positively stained neurons compared to diabetic control and diabetic-treated HAART (21.40 ± 2.494) as shown in There was a significant   was loadIn the present study, the formulated HAART-AgNPs showed a percentage incorporation efficiency of 90.52 ± 0.5%. This indicated that a more significant amount of the HAART was incorporated with silver nanoparticles.−1 which was within the range of our observations at different concentrations of the stabilizing agent. In addition, this result indicated that the structural components of HAART were not disorganized despite silver nanoparticle interaction.The IR spectra for the incorporated HAART-AgNPs revealed different absorbances at the O-H functional group after the incorporation. These changes in the stretching vibrations of the O-H group at various concentrations indicate stabilizing agents on the nanoparticles. A study by  observedOur formulated HAART-AgNPs revealed a spherical shape and mean nanoparticle sizes between 19 and 32 nm in all concentrations. Spherical shapes with nanoparticle size between 20 and 50 nm of nanoparticles have been described as not toxic to the biological tissue –46. The The SEM displayed a nanorod covered with nanosphere structure for 0.5 M, 1 M, and 1.5 M. This finding agrees with a study by  that repFinally, the elemental constituents of the formulated HAART-AgNPs were confirmed by EDX, the result in this study indicated that the concentration of 1.5 M formulated HAART-AgNPs has moderate silver (16.89%), and the presence of elemental compositions such as oxygen (O), carbon (C), fluorine (F), and chlorine (Cl) indicates that HAART was fully incorporated with AgNPs. Studies have shown that the elemental compositions of the drugs must be shown in the conjugated nanodrug to establish the proper incorporation , 48. TheBased on the toxicity criteria as reported in the literature –46) such such46])Diabetes mellitus in people living with HIV is associated with various neurological complications , 8, 31. In the present study, daily oral administration of HAART to diabetic animals further increases the blood glucose levels in diabetic rats. This observation shows that HAART has metabolic effects by increasing the blood glucose level in experimental rats, supporting the findings of Maganga et al. , who repIn this study, rats administered with HAART failed to show differences between day one and day three latency trials to reach the submerged platform. This result suggests impairment in spatial learning and memory in the diabetic group treated with HAART. The evidence regarding the potential neurotoxicity of HAART related to cognitive impairment has been reported . HAART wInterestingly, HAART-coupled silver nanoparticle conjugate (HAART-AgNPs) showed a consistent spatial learning pattern by significantly reducing the latency to reach the hidden platform in the three-day trials.The HAART-AgNPs have high CNS penetration-effectiveness due to their side (10-100 nm), enhanced bioavailability, and reduced CSF viral loads while reducing toxicity profile .An investigation on the spatial working memory of the diabetic rat treated with HAART showed significant learning and memory deficit. The memory deficits observed in this study may result from the diabetogenic effects of HAART on the experimental rats by increasing the blood glucose level in diabetic rats. Despite the benefit of HAART, changes in the brain cytoarchitecture and related impairments on spatial memory and learning have been observed in experimental animals and normal HIV-positive individuals , 58. HowDrosophila melanogaster treated with EFVb-HAART and possible neurotoxic consequences.HAART-AgNPs significantly improved locomotion activity in diabetic rats compared to HAART-treated only. This reduction in locomotion seen in diabetic rats treated HAART may be related to anxiety-like behaviour. This finding corroborates the report of Gyang et al. , who obsOxidative stress (OS) forms the biological basis for many diseases, and it plays a vital role in neuronal injury . In thisThis current study showed that HAART-loaded silver nanoparticles showed an improvement with antioxidant enzymes. The relatively small size of silver nanoparticles has a prospect in drug delivery by reducing the dosing frequency .Hippocampal CA1 pyramidal cells are thought to be involved in memory and spatial learning . The redThe HAART-AgNPs improved hippocampal pyramidal cell Nissl staining and a significant increase in the number of positive cells for Nissl staining compared to the diabetic group and diabetic rats treated with HAART. Silver nanoparticles are known for their antioxidant, antidiabetic, antiviral, and antifungal properties . An imprIn this study, the administration of HAART to diabetic rats aggravates the complications of diabetes and promotes neurotoxic effects on the experimental rats, while HAART-loaded silver nanoparticle (nHAART) alleviates diabetes-induced neurotoxicity.


Morphostenophanes is a small arboreal group of darkling beetles that are endemic to the Oriental region. The complete mitochondrial genome of the M. sinicus population from the Manwan Town was first characterized. The mitogenome consisted of a circular DNA molecule of 15,662 bp with a 66.352% AT content. It comprises 13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes. The PCGs have a typical ATN (Met) start codon, except for nad1 (TTG as a start codon), and are terminated by typical TAN stop codons.The genus  Morphostenophanes, which is a small arboreal group of darkling beetles endemic to the Oriental region  was collected from Yunnan, China . The specimen was deposited by the corresponding author in the insect specimen room of the Institute of Plant Protection, Guangxi Academy of Agricultural Sciences and given a specimen accession number (GIPP-20200924-002). Genomic DNA was isolated and fragmented to generate a genomic library, using the VAHTS® Universal DNA Library Prep Kit for MGI . Sequencing was subsequently performed on the MGI-SEQ 2000 platform , using an insert size of 400 bp. We obtained approximately 9.179 Gb raw data (2 × 150 bp paired-end reads), of which 9.046 Gb (98.548%) was high-quality, clean data. The genome was assembled de novo using SOAPdenovo v2.04 (https://github.com/aquaskyline/SOAPdenovo2/)   consists of a circular DNA molecule that is 15,662 bp . Using Perna and Kocher’s formula (Perna and Kocher The mitogenome of M. sinicus contains 13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes, which were annotated using the MITOS Web Server (http://mitos.bioinf.uni-leipzig.de/)  had an ATA start codon, one (atp8) had an ATC start codon, six  had an ATG start codon, and one (cox2) had an ATT start codon. All 13 PCGs had typical TAN stop codons: six genes  had TAA stop codons, three  had TAG stop codons, and four  had an incomplete stop codon, consisting of a codon that was completed by the addition of A nucleotides at the 3′ end of the encoded mRNA. The 22 tRNA encoding genes were interspersed throughout the coding region and ranged from 60 bp (tRNA-Cys and tRNA-Tyr) to 70 bp (tRNA-Lys). The genes encoding lrRNA and srRNA were 1214 and 750 bp, respectively. A maximum overlap length of 7 bp was located between nad4 and nad4l, atp8, and atp6. The maximum length of the intergenic region was 32 bp, located between lrRNA and tRNA-Val.The mitogenome of M. sinicus, its mitochondrial PCGs, and those of 14 other species of Insecta, were used to construct a maximum-likelihood phylogenetic tree using the MEGA X software  is shown. The percentage of trees in which the associated taxa were clustered together is shown below the branches. The initial tree(s) for the heuristic search were obtained automatically by applying Neighbour-Join, and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with a superior log likelihood value. A discrete gamma distribution was used to model evolutionary rate differences among sites ). The rate variation model allowed for some sites to be evolutionarily invariable ((+I), 23.53% sites). The tree is drawn to scale, with branch lengths representing the number of substitutions per site (above the branches). The percentage of trees in which the associated taxa were clustered together is shown in red. This analysis involved 15 amino acid sequences. The complete mitogenome of


SNCA mutations, there are no data for long‐term outcome.Although the short‐term efficacy of bilateral subthalamic deep brain stimulation (DBS) has been reported in a limited number of Parkinson's disease (PD) patients with SNCA mutations, implanted with bilateral subthalamic DBS. We compared demographic and clinical data at baseline and last follow‐up. Clinical data of motor and nonmotor symptoms and motor fluctuation were collected up to 10 years from DBS surgery.This multicenter retrospective study investigated previously reported PD patients with SNCA duplication and one had c.158C.A (p.A53E) mutation. The mean post‐implantation follow‐up duration was 5.4 ± 3.7 years. All patients with SNCA duplication showed favorable outcome, although one died from breast cancer 1.5 years after DBS. The patient with the missense mutation became wheelchair‐bound due to progressed axial, cognitive and psychiatric symptoms after 3.5 years from DBS despite the benefit on motor fluctuation.Among four subjects, three had SNCA mutations, especially those with SNCA duplication, and cognitive and psychiatric symptoms are important for the long‐term outcome of subthalamic DBS.Based on findings in our small cohort, subthalamic DBS could be beneficial for motor fluctuation in PD patients with  This study assesses the long‐term outcome of bilateral subthalamic deep brain stimulation (DBS) in patients with Parkinson's disease (PD) related with SNCA mutations.Based on our results, we suggest bilateral subthalamic DBS could be effective treatment option in PD with SNCA mutations if the candidates are well‐selected.Cognitive, psychiatric symptoms and axial motor symptoms are important for the long‐term outcome of subthalamic DBS in PD with SNCA mutation. PD pati2SNCA mutations  studies with PD patients with nongenetic causes or monogenic etiology but not SNCA mutation, (2) studies with DBS on other targets, (3) review or other studies without original data, or (4) animal study. Initial screening was independently done by 2 researchers (J Youn and JW Cho), and disagreements were resolved after discussion. Fourteen duplicated studies and 14 nonrelevant studies  were excluded. Finally, three patients from three case reports were selected we contacted three centers with previous relevant studies to participate in this retrospective study , and the last follow‐up after DBS (long‐term outcome) were collected from February 2019 to February 2020. Demographic data including age, age at the onset of PD, age at DBS, family history and follow‐up duration, and clinical outcome about motor and nonmotor symptoms and fluctuation were collected using the standardized data collection form in all enrolled subjects. The clinical outcome from DBS was assessed based on the three categories; (1) motor symptoms using Unified Parkinson's disease rating scale (UPDRS) part 3 and Hoehn and Yahr stage, (2) nonmotor symptoms with mini‐mental status exam (MMSE) and Beck's depression inventory, and (3) fluctuation/dyskinesia with the item #32 and #39 of UPDRS part 4. We added other clinical scales used at each center.33.1SNCA mutations, duplication was identified in three (case #1–3)  were followed up to 6.5 and 10 years from DBS and revealed excellent motor outcome from bilateral STN DBS (Table SNCA duplication (case #1) died from breast cancer 1.5 years after DBS, but her parkinsonism was well‐controlled till her death.Two subjects with In the subject with missense mutation (case #4), disease itself progressed with levodopa‐nonresponsive or ‐induced symptoms such as depression, perceptional problem, and cognitive decline. However, motor benefit from STN DBS was still evident, considering she still took PD medications less frequently with less dyskinesia compared to the baseline.4SNCA mutations. Despite small number of patients, our results suggest a good outcome in the three cases with duplications whereas outcome in the patient with the missense mutation was not robust because of axial symptoms and cognitive and psychiatric problems.This is the first study to investigate the outcome of STN DBS up to 10 years in the PD patients with SNCA duplication (case #2 and #3) 10 and 6.5 years after DBS, and in another (case #1) until she died 1.5 years after DBS. Although one with missense mutation (case #4) had unfavorable outcome, subthalamic stimulation was still effective for motor fluctuation and dyskinesia till the last follow‐up.For the motor fluctuation/dyskinesia, STN DBS showed favorable outcome in all cases regardless of genotypes. Fluctuation/dyskinesia was dramatically controlled in two patients with SNCA mutations  but deteriorated only in one subject with missense mutation (case #4) after DBS. Additionally, even with STN DBS, many studies reported preserved or improved cognition or psychiatric symptoms  Grant ARS01_01081, Cariparo Foundation; He serves as consultant for Boehringer–Ingelheim for legal cases on pathological gambling. J.I. Lee received research support from Medtronic and Boston Scientific. J.W. Cho, T. Kuusimäki, D. Kim, K.R. Cho have no conflict of interest to declare.JY: Research project conception, organization, execution; statistical analysis design, execution; manuscript preparation writing of the first draft, review and critique. GO: Research project conception, execution; statistical analysis execution, review and critique; manuscript preparation writing of the first draft, review and critique. NH: Statistical analysis execution, review and critique; manuscript review and critique. YS: Research project organization, execution; statistical analysis review and critique; manuscript preparation review and critique. TK: Research project conception, organization, execution, statistical analysis execution, manuscript preparation review and critique. VK: Research project organization, execution, statistical analysis review and critique, manuscript preparation review and critique. AA: Research project organization, execution, statistical analysis review and critique, manuscript preparation review and critique. JIL: Research project organization, execution, statistical analysis review and critique, manuscript preparation review and critique. DK: Research project organization, statistical analysis execution; manuscript preparation review and critique. KRC: Research project Organization, execution, statistical analysis review and critique; manuscript preparation review and critiquehttps://publons.com/publon/10.1002/brb3.2503The peer review history for this article is available at


Intestinal disorder is an important factor contributing to growth lag and high rates of morbidity and mortality of piglets with intrauterine growth retardation (IUGR). Resveratrol (RSV) and its derivative pterostilbene (PT) are natural stilbenes possessing various bioactivities, such as antioxidative and anti-inflammatory effects. This study compared the protective potential of RSV and PT on the intestinal redox status and gut microbiota in weanling piglets with IUGR.Eighteen male piglets of normal body weight (NBW) and 54 same-sex IUGR piglets were chosen according to their birth and weaning weights. The NBW piglets accepted a basal diet, while the IUGR piglets were allotted to one of three groups according to their body weight at weaning and received a basal diet, an RSV-supplemented diet (300 mg/kg), or a PT-supplemented diet (300 mg/kg), respectively.P < 0.05). Administering RSV and PT also enhanced jejunal superoxide dismutase activity and the mRNA and protein expression of superoxide dismutase 2 of IUGR piglets by promoting nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation (P < 0.05). Comparatively, PT was more effective than RSV in elevating the villus height/crypt depth ratio and occludin mRNA and protein levels in the jejunum of IUGR piglets (P < 0.05). PT was also superior to RSV in increasing Nrf2 nuclear translocation and inhibiting malondialdehyde accumulation in the jejunum of IUGR piglets (P < 0.05). Additionally, RSV modulated the composition of cecal microbiota of IUGR piglets, as evidenced by increasing the prevalence of the phylum Bacteroidetes and the genera Prevotella, Faecalibacterium, and Parabacteroides and inhibiting the growth of the phylum Proteobacteria and its genera Escherichia and Actinobacillus (P < 0.05). Moreover, RSV significantly increased the butyrate concentration in the cecum of IUGR piglets (P < 0.05).Compared with IUGR piglets, both RSV and PT improved the IUGR-associated decrease in jejunal villus height and increases in plasma diamine oxidase activity and D-lactate level and jejunal apoptosis of piglets (PT is more potent than RSV to prevent intestinal oxidative stress, while RSV has a stronger capacity to regulate gut microbiota compared to PT.The online version contains supplementary material available at 10.1186/s40104-021-00589-9. Maternal malnutrition and placental insufficiency perturb the growth and development of the embryo/fetus and its organs during pregnancy and induce a low birth weight, which is defined as intrauterine growth retardation (IUGR) . IUGR noOxidative stress is considered to be a trigger for the onset and development of intestinal diseases and it is implicated in the pathophysiology of IUGR-associated intestinal injury , 8. At bin vivo [Resveratrol (RSV) is a natural stilbene, mainly present in red wine, grapes, rhubarb, and blueberries, conferring multiple biological benefits, including anti-inflammation, anticancer, and lipid/glucose metabolism-regulating effects . Recent in vivo . In thisin vivo is still scant. Also, there remains a crucial gap in understanding the protective effects of PT on intestinal oxidative stress and subsequent intestinal dysfunction. Therefore, the present study compared the in vivo protective potential of RSV and PT in intestinal redox status and gut microbiota using a porcine model of naturally occurring IUGR. Given the omnivorous nature and the similarities in intestinal anatomy and physiology between humans and pigs [PT is a naturally occurring methoxylated analog of RSV, sharing similar biological functions to those of RSV. Owing to the presence of methoxy groups, PT exhibits superior lipophilicity and metabolic stability to RSV, contributing to better cellular uptake and biological activities , 19. Preand pigs , this stA total of 72 healthy pregnant multiparous sows (Landrace × Yorkshire) with similar expected farrowing dates (less than 3 d) and parity (second or third) were preselected during gestation. At farrowing, the sows that had 11–13 live-born piglets and met the selection criteria for IUGR were chosen. Piglets were defined as intrauterine growth-retarded (IUGR) piglets when their birth weight were 2 standard deviations below the average birth weight of the total population, while those have a birth weight within 0.5 standard deviations of the average birth weight of the total population were deemed to be the normal body weight (NBW) piglets , 10. Aftg for 15 min at 4 °C and then stored at − 80 °C until analysis. After blood sampling, the piglets were anaesthetised via electrical stunning and sacrificed by exsanguination. Subsequently, the abdominal cavity was opened and the jejunal segment was harvested immediately according to a previous study [At 35 days of age, all piglets were weighed, and one piglet having the nearest average weight of its replicate was selected for sampling. The blood samples were withdrawn into heparin-coated tubes from the precaval vein. The plasma samples were separated by centrifugation at 3,500×us study . ApproxiPlasma DAO activity was determined using a colorimetric assay kit  according to the method of Hosoda et al. . BrieflyAfter fixing in paraformaldehyde solution overnight, the jejunal samples were embedded and stained based on the method of Luna . BrieflyThe apoptotic index of the jejunum was determined using a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) BrightRed Apoptosis Detection Kit  in accordance with the manufacturer’s guidance. After deparaffinization and aquation, the jejunal specimens were dipped into proteinase K (20 μg/mL) for 20 min at room temperature, followed by incubation with the TUNEL mixture (deoxynucleotidyl transferase enzyme and BrightRed Labeling Mix) at 37 °C for 1 h in a dark and humidified chamber. Subsequently, the slices were counterstained with the 4,6-diamidino-2-phenylindole solution  and the images were captured using a fluorescence microscope . The percentage of TUNEL-positive cells in the jejunum was quantified from 15 well-oriented villi each section by an independent and blinded observer who was not aware of the treatment procedures, using Image Pro Plus 6.0 software.The jejunal mucosa tissues were homogenized in saline solution  and centrifuged at 3,500 r/min for 10 min. The supernatants were then diluted into the optimal content for detecting the activities of total superoxide dismutase , glutathione reductase  and glutathione peroxidase , and the concentration of malondialdehyde  using the reagent kits . SOD activity was measured at 550 nm according to the method of Sun et al. , which dg for 10 min at 4 °C, the supernatants were obtained to detect GST activity by the procedure of Saydam et al. [g for 10 min at 4 °C for determination of GSH and GSSG levels according to the method of Teare et al. [The activities of glutathione S-transferase  and the concentrations of reduced glutathione  and oxidized glutathione  in the jejunal mucosa, were determined using commercial kits . After homogenization of jejunum tissues in 0.1 M potassium phosphate buffer  and centrifugation at 8,000 m et al.  using che et al. . A chromoccludin (OCLN),  claudin-1 (CLDN-1), zonula occludens-1 (ZO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), heme oxygenase 1 (HO1), superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), and beta-actin (ACTB) using a TB Green® Premix Ex TaqTM kit  with the QuantStudio 5 Real-time PCR System . The relative expression levels of the target genes were calculated using a quantification approach (2-ΔΔCt method) and normalized against the reference gene (ACTB) expression level. A complete list of qRT-PCR primers is presented in Table The RNAiso Plus Reagent  was used to isolate the total RNA of the jejunal mucosa. The quality and concentration of extracted RNA were detected using NanoDrop 1,000 , and the RNA integrity was evaluated with 2.0% agarose gel electrophoresis. Subsequently, the complementary DNA was produced using a PrimeScript™ RT Master Mix kit . The qRT-PCR assay was conducted to determine the mRNA expression levels of Total protein isolation from the jejunal mucosa was carried out with a radioimmunoprecipitation assay lysis buffer containing protease inhibitor cocktail . The nuclear protein in the jejunal mucosa was prepared using a Nuclear Protein Extraction Kit . The concentrations of total cellular protein and nuclear protein in the jejunal mucosa were measured by the bicinchoninic acid protein assay kit . Thereafter, equal quantities of protein were resolved by SDS-PAGE gel and then transferred onto polyvinylidene difluoride membranes. After that, the membranes were incubated with blocking buffer  for 1 h at room temperature and probed with the primary antibody against OCLN , CLDN-1 , ZO-1 , nuclear factor erythroid-2-related factor 2 , Kelchlike ECH-associated protein-1 , NQO1 , HO1 , SOD1 , SOD2 , Histone H3  and ACTB  overnight at 4 °C. Then, the membranes were washed by Tris-buffered saline with 0.05% Tween-20 and incubated with a suitable secondary antibody for 1 h at room temperature. Finally, these target bands were visualised using the Tanon 5200 Multi-Imaging System  and analysed by Image Pro Plus 6.0 software.Total bacterial DNA was extracted from cecal digesta samples using the TIANamp Stool DNA Kit  according to the manufacturer’s guidance. The V3-V4 region of the bacteria 16S rDNA genes was amplified by PCR procedure with the specific primers (338F: 5′-ACTCCTACGGGAGGCAGCAG-3′ and 806R: 5′-GGACTACCVGGGTATCTAAT-3′) . After cAfter sequencing, the raw fastq files were preprocessed to remove the adapter pollution and the reads of low quality. The paired-end clean reads were merged to tags using FLASH (V 1.2.11). Subsequently, the clean tags were assigned into operational taxonomic units (OTUs) with a threshold of 97% identity by UPARSE (V 7.0.1090), and the chimeric sequences were identified and eliminated using UCHIME (V 4.2.40). Species annotation analysis was performed by an RDP Classifier (V 2.2) based on the Greengene database (V 201305) with a confidence threshold of 80%. The rarefaction curve, species accumulation curve, Shannon, Simpson, and Chao1 indices were calculated using RStudio software (V 3.5.3) with the vegan package. To estimate the beta diversity, principal coordinates analysis (PCoA) based on Bray-Curtis distance and the analysis of similarities (ANOSIM) were conducted to compare the differences between the treatments using RStudio software (V 3.5.3).The concentrations of cecal short-chain fatty acid, including acetate, propionate, and butyrate, were quantified using gas chromatography  according to the method reported by Yu et al. .P-value smaller than 0.05 was identified as statistically significant.The SPSS for Windows version 22.0 statistical package  was performed for statistical analysis. Dataset normality was determined by the Shapiro-Wilk test. When appropriate, one-way ANOVA and Tukey’s post hoc test were used to assess the significant difference among groups. Otherwise, the statistical significance of differences was analysed by the Kruskal–Wallis non-parametric test followed by pairwise differences in rank sums. The results were expressed as means with their standard errors. A P < 0.05). Neither RSV nor PT supplementation significantly affected the growth performance of IUGR piglets (P > 0.05). There were no remarkable differences in initial and final body weight, ADG, ADFI, and FCR between the two supplemented groups (P > 0.05).In comparison with the NBW group, the IUGR piglets had lower initial and final body weight, ADG and ADFI Table  during tP < 0.05). In contrast, both RSV and PT administration effectively increased jejunal VH and reduced jejunal apoptotic index (P < 0.05). Moreover, a PT-supplemented diet also significantly increased the jejunal VH/CD ratio . The VH, CD .As revealed by Fig. tio Fig. d of IUGR CD Fig. c, VH/CD P < 0.05). In addition, western blot analysis revealed that the IUGR piglets showed decreased protein levels of jejunal OCLN . Consistently, IUGR downregulated the gene expression of ZO-1 in the jejunum compared with the NBW controls (P < 0.05). Conversely, both RSV and PT administration to IUGR piglets increased jejunal ZO-1 expression at transcriptional and translational levels compared with the IUGR subjects (P < 0.05). Moreover, PT markedly upregulated the mRNA and protein levels of jejunal OCLN of IUGR piglets and induced a higher OCLN mRNA abundance of IUGR+PT piglets than that in the IUGR-RSV group (P < 0.05). No significant differences were observed in jejunal CLDN-1 mRNA or its protein levels among groups (P > 0.05).Compared with the NBW group, plasma DAO activity Fig. a and D-lCLN Fig. c compareP < 0.05). Nevertheless, the decreased jejunal SOD activity in the IUGR group was reversed to normal values by both RSV and PT treatment (P < 0.05). PT also obviously increased GST activity . Furthermore, we observed a higher jejunal GSH level in the IUGR+PT group when compared with the IUGR+RSV group (P < 0.05). No significant alterations were found in jejunal GPx .The IUGR piglets displayed decreased SOD activity Fig. a, GSH coity Fig. d, the coity Fig. e and GSHGPx Fig. b, GR , but the protein level of Keap1, a repressor of Nrf2, was not altered by IUGR (P > 0.05). In parallel with the lower nuclear Nrf2 protein level, IUGR downregulated jejunal protein levels of NQO1 and SOD2 . Moreover, the lower jejunal SOD2 mRNA abundance . In contrast, both RSV and PT supplementation dramatically stimulated the nuclear Nrf2 accumulation in the jejunum of IUGR piglets (P < 0.05); PT exhibited a more remarkable effect than RSV in this process (P < 0.05). Meanwhile, piglets in two supplemented groups displayed higher mRNA and protein levels of jejunal SOD2 relative to the IUGR piglets (P < 0.05). PT treatment to IUGR piglets also increased the mRNA and protein levels of NQO1 and the protein level of SOD1 in the jejunum, compared with those that received a control diet (P < 0.05).Compared with the normal controls, the nuclear translocation level of jejunal Nrf2 Fig. a of IUGROD2 Fig. b, two imnce Fig. c was alsP < 0.05) and tended to decrease the Chao1 index (P = 0.054) in comparison with the NBW piglets, which were not improved by RSV or PT (P > 0.05). We also determined the beta diversity of the cecal microbiota by the PCoA analysis , IUGR and IUGR+RSV , and IUGR+RSV and IUGR+PT  groups. However, the cecal bacterial communities in the IUGR and IUGR+PT groups were similar .To investigate the impact of RSV and PT on the gut microbial environment, we profiled the cecal bacterial community composition of each treatment using 16S rDNA gene sequencing. The rarefaction analysis and species accumulation curve Fig.  illustraP < 0.05). Instead, these alterations of IUGR piglets were reversed by an RSV diet (P < 0.05). Moreover, PT intervention also tended to reduce the relative abundance of Proteobacteria (P = 0.055). At the genus level, a total of 79 bacterial genera were annotated and the top 35 of which were depicted in Fig. Oscillospira and Parabacteroides of IUGR piglets were decreased (P < 0.05). After 2-weeks RSV supplementation, the relative abundance of Prevotella, Faecalibacterium, and Parabacteroides were significantly increased, and the relative abundance of Actinobacillus and Escherichia were remarkably decreased (P < 0.05). These results collectively demonstrated that both RSV and PT modulated the structure of cecal microbes of IUGR piglet.In order to further identify the changes in cecal microbiota composition, the dominant phyla and genera in all the groups were analysed. At the phylum level Fig. b, FirmicP = 0.081), and no significant differences were observed in the concentrations of total short-chain fatty acid, acetate, or propionate between the NBW and the IUGR groups (P > 0.05). In contrast, RSV intervention significantly increased the butyrate concentration (P < 0.05) and tended to enhance the acetate concentration in cecal digesta (P = 0.068).As illustrated in Fig. IUGR animals often exhibit growth lag in their early growth stage , 7, 33. Intestinal integrity plays a fundamental role in intestinal physiological functions, which can be evaluated by the circulating DAO activity and D-lactate concentration . In thisOxidative stress has been recognized as a crucial mechanism in intestinal disorders of IUGR neonates , 10. SODOn the other hand, the potent potential of PT in the activation of Nrf2 pathway was also found in this study, which is consistent with a previous study suggesting that PT administration induced an approximately four-fold increase in Nrf2 protein levels in the colon compared with RSV at an equivalent dose . Nrf2 isEscherichia and Actinobacillus [Escherichia and Actinobacillus, indicating the potential of RSV and PT in suppressing the proliferation of harmful bacteria. Bacteroidetes is the most predominant anaerobe producing short-chain fatty acid in the gut, which can be modulated by RSV to maintain energy metabolism homeostasis in high-fructose or high-lipid models [Prevotella and Parabacteroides in the cecum was enhanced by RSV. Faecalibacterium is one of the main butyrate producers, which provides energy for the gut epithelial cells and confers anti-inflammatory properties [Faecalibacterium prausnitzii mitigates colorectal colitis through production of butyrate and modulation of the Th17/Treg balance. Here, we observed apparent increases in the prevalence of Faecalibacterium and butyrate levels in the cecum after RSV treatment, suggesting that RSV may avail the intestinal health by regulating potential probiotics and their metabolites in the gut.Intestinal flora is the crucial determinant of intestinal development and immune system maturation. Previous studies revealed that low birth weight can disturb gut microbiota , 34, whibacillus , 51. It bacillus , 53. Aftd models , 53. In operties . Zhou etoperties  illustrain vivo, such as increasing the Bacteroidetes-to-Firmicutes ratios and the growth of Bacteroides, Lactobacillus, and Bifidobacterium [It is worth noting that the ability of RSV to regulate gut microbiota was higher than that of PT, which seems in striking contrast to its insufficient antioxidative activity of IUGR piglets mentioned above. Actually, given the low bioavailability/high bioactivity paradox of RSV, growing evidence supports the hypothesis that RSV is possibly acting primarily through remodeling the gut microbiota , 57. Altacterium , 53, 57.acterium , 59, whiacterium . Also, racterium , 60. ThuBoth RSV and PT protect against the IUGR-associated intestinal morphological changes, increased intestinal permeability, redox imbalance, and gut dysbiosis of weanling piglets. PT shows more potency than RSV in alleviating oxidative stress and activating the Nrf2-associated antioxidant system in the jejunum of IUGR piglets. RSV is superior to PT in regulating the cecal microbiota of IUGR piglets.Additional file 1: Table S1. Composition and nutrient levels of the basal diet.Additional file 2: Table S2. Primer sequences for quantitative real-time PCR.Additional file 3: Figure S1. Rarefaction curve and species accumulation curve of cecal microbiota based on operational taxonomic units.Additional file 4: Figure S2. Unique and shared OTUs analysis and the alpha diversity of the cecal microbiota.


The aims of this study were to clarify the following: (1) how often does prolonged pregnancy ≥34 weeks occur in patients with emergent cerclage without progesterone and (2) the risk factors preventing such pregnancy continuation.  This retrospective observational study was performed using medical records of patients for whom emergent cerclage had been performed between April 2006 and December 2018 in our institute.  Emergent cerclage was performed in 123 patients . Primiparous patients numbered 44 (36%). A history of spontaneous preterm birth (SPTB) was present in 30 (24%). The median presurgical cervical length (CL) was 16 (8–21) mm at surgery. Of the 123, 20 (16%) were delivered at 33 + 6 weeks or less (<34 weeks). We conducted logistic regression analysis of the risk factors of SPTBs <34 weeks after cerclage. Three risk factors were identified that increased the risk of SPTB <34 weeks: presurgical CL 0 mm (odds ratio (OR): 5.30; 95% confidence interval (CI): 1.58–17.7), a history of SPTB , and the presence of sludge .  Three risk factors predicted SPTB <34 weeks after emergency cerclage without progesterone administration: unmeasurable CL (CL 0 mm), a history of SPTB, and the presence of sludge on ultrasound. SPTB <34 weeks occurred after emergency cerclage in 16% of patients, being comparable with the recent data with progesterone. Treatment and prophylaxis for spontaneous preterm birth (SPTB) are challenging: administration of progesterone , tocolytProgesterone has been widely recommended as a treatment for threatened PTB in global guidelines –8. HowevThis study was approved by the ethics committee of our institute (20–070). We retrieved medical charts of patients for whom cervical cerclage was performed between April 2006 and December 2018 in our institute, in which approximately 1,000 high-risk deliveries are managed annually. Our treatment strategy for SPTB fundamentally adhered to the Japanese guidelines –11. The μL, high-grade fever, and fetal tachycardia), (3) prolapse of the membrane (bag) into the vagina, (4) rupture of the membrane, (5) continuous preterm labor pains despite administration of tocolytic agents, (6) lethal major fetal anomalies, and (7) those not consenting to the cerclage. We excluded iatrogenic preterm births including preeclampsia, medical complications, and non-reassuring fetal status before labor pains from this study. We also excluded placenta previa, fetal abnormalities, and multiple deliveries. Amniocentesis to measure subclinical infection was not performed. Although emergent cerclage was performed from 15 to 24 weeks' gestation, it was exceptionally performed at 24 to 26 weeks in a few cases based on patients' wishes and obstetricians' decisions.We performed emergent cerclage in patients with CL <25 mm at less than 24 weeks of gestation irrespective of the presence or absence of a history of SPTB with patients' consent. Transvaginal ultrasound was routinely performed between 19 and 23 weeks of gestation. In high-risk SPTB patients, we measured CL weekly or biweekly from 15 to 24 weeks. Patients referred from other institutes due to shortened CL were also included. We included patients with shortened CL regardless of the presence or absence of preterm labor pains. If CL could not be measured because of cervical effacement and cervical shorting, we defined the condition as CL 0 mm. We did not employ cerclage in the following patients (exclusion criteria): those with (1) active vaginal bleeding, (2) evident chorioamnionitis  in some patients using a wet gauze and/or small balloon . After the operation, systemic antibiotics  were administered intravenously for more than 3 days. After the operation, the patients were hospitalized for more than one week. Ritodrine hydrochloride, magnesium sulfate, or their combination was administered as tocolysis in all patients. The attending obstetricians decided on the periods of these administrations and hospitalizations. Vaginal or intramuscular progesterone was not administered because it is not covered by national insurance. Vaginal ulinastatin, which is a granulocyte elastase inhibitor, was administered every day based on patients' wishes during hospitalization and also based on the Japanese guideline –11. BlooU-test and Fisher's exact test (two-tailed) were used to compare characteristics, maternal backgrounds, and outcomes between deliveries at less than 34 weeks' gestation (+) vs. (−). Parameters significant (P < 0.15) on univariate analysis were used for logistic regression analysis. All analyses were performed using JMP Pro version 15 , with P < 0.05 considered significant.From electronic medical records, we retrieved maternal backgrounds , and a history of SPTB), CL, presence/absence of sludge on ultrasound, vaginal bacterial culture, and the presence/absence of cervical elastase , gestatiLactobacillus and ureaplasma were present in 89 (72%) and 42 (34%), respectively. The prolapsed membrane was replaced by pushing-back at surgery in 22 (18%). No uterine malformation was noted. Premature rupture of the membrane at surgery did not occur in any patients.P=0.003) and (ii) significantly more frequent ultrasound-detectable sludge (8 (40%) vs. 16 (16%), respectively (P=0.029). Patients with SPTB <34 weeks frequently had a history of SPTB, although it was not significant (8 (40%) vs. 22 (21%), respectively (P=0.091). The median duration between the cerclage and discharge showed a shorter tendency in patients with SPTB <34 weeks than those with ≥34 weeks (35 (IQR: 21–84) vs. 76 (IQR: 13–98) days, respectively (P=0.057).P value of the duration was 0.057. Three risk factors were identified that increased the risk of SPTB <34 weeks: presurgical CL 0 mm (odds ratio (OR): 5.30; 95% confidence interval (CI): 1.58–17.7), a history of SPTB , and the presence of sludge .We made a few important observations in patients necessitating emergent cerclage without progesterone administration. SPTB <34 weeks occurred after emergency cerclage in 16% of patients. Three risk factors predicted SPTB <34 weeks: unmeasurable CL (CL 0 mm), a history of SPTB, and the presence of sludge on ultrasound. These results may be similar to those of patients managed with progesterone administration.3 or CRP >15 mg/L), are associated with PTB <32 weeks. There are similarities and differences between Fuchs's study and our study. The similarities were that a history of PTB and cervical dilation (“CL = 0 mm”) was associated with PTB. Contrarily, there were some differences in inclusion criteria and risk factors to be examined. He included patients with bulging bag into vagina, whereas we excluded them. Regarding infection, he took WBC and CRP into consideration; however, we evaluated sludge and cervical elastase. Another study concluded that, of patients receiving cerclage during 10–24 weeks, emergent cerclage, a history of conization, and CL <25 mm were risk factors for PTB <32 weeks [A few studies have been reported regarding risk factors for SPTB after cerclage under different pre- or postoperative managements, including no routine tocolysis and progesterone administration. Fuchs et al.  reported32 weeks . Inclusi32 weeks .A history of SPTB was a significant risk for SPTB <34 weeks in this study. Numerous studies showed that a history of SPTB is a risk factor for SPTB. Of those, a systematic review and meta-analysis showed that the rate of SPTB (<37 weeks) was high as 30% in 55,197 patients with a history of a preterm singleton livebirth . In addiAn ultrasound-detectable sludge was shown to be a risk for SPTB in this study. A systematic review showed that patients with sludge had higher incidences of SPTB , 17. RecUnmeasurable CL (CL 0 mm) was also a risk factor for SPTB in this study. Shortened CL is a well-known risk factor for SPTB, and emergent cerclage shows the preventive effect for SPTB in patients with shortened CL. Berghella et al.  showed tP=0.04) in a randomized study [n = 106) did not decrease SPTB without progesterone administration [Comparing the effectiveness of cerclage or any other treatment with the endpoint being the incidence often causes some difficulty. Our protocol for cerclage may not be completely consistent with the global standard regarding the following two points: (1) Progesterone was not administered, and (2) cerclage was performed for a patient with CL <25 mm even without a history of SPTB. A study involving a large number of showed that cerclage decreased the SPTB incidence for a singleton pregnancy in the presence of a history of SPTB with CL <25 mm, but only in those with a history of SPTB . It remaed study . On the stration . A few astration , 30. Regstration . As descThis study had some limitations. First, this was a retrospective analysis, and the study number was relatively small. Second, confounding risk factors could not be completely excluded. For example, intra-amniotic infection can cause SPTB; however, we only checked for the presence or absence of sludge, serum CRP, and cervical elastase. Although interleukin-6 and -8 in amniotic fluid can indicate intra-amniotic infection , we did Three risk factors predicted SPTB <34 weeks after emergency cerclage: unmeasurable CL (CL 0 mm), a history of SPTB, and the presence of sludge on ultrasound. SPTB <34 weeks occurred after emergency cerclage in 16% of patients, being comparable with the recent data. This study may have indicated the data of cerclage effectiveness without progesterone, data excluding the effect of progesterone.


P < 0.05). EV-associated gastrointestinal infections were more common in patients with a transplant (children [71%] and adults [46%]), compared to nontransplant patients (P < 0.05). Additionally, nontransplant patients had a higher number of hospital stays (P < 0.05), potentially reflecting more severe disease. However, transplant patients were more likely to have symptom persistence after discharge (P < 0.05). Finally, children and adults with a transplant were more likely to have additional enterovirus infection episodes (P < 0.05). In our cohort, enterovirus infections did not seem to be more severe after transplantation; however, patients tended to present with different clinical symptoms and had genotypes rarely found in nontransplant recipients.Enterovirus infections are known to cause a diverse range of illnesses, even in healthy individuals. However, information detailing enterovirus infections and their severity in immunocompromised patients, such as transplant recipients, is limited. We compared enterovirus infections in terms of genotypes, clinical presentation, and severity between transplant and nontransplant patients. A total of 264 patients (38 transplant recipients) with 283 enterovirus infection episodes were identified in our hospital between 2014 and 2018. We explored the following factors associated with enterovirus infections: clinical presentation and diagnosis on discharge, length of hospital stay, symptom persistence, and infection episodes in both children and adults. We observed some differences in genotypes between patients, with enterovirus group C occurring mainly in transplant recipients (IMPORTANCE Despite the high prevalence of enteroviruses in the community and the increasing demand for transplants from an aging population, knowledge on enteroviruses in solid organ transplant recipients is currently limited. Transplant recipients represent a significant patient population and require additional considerations in patient management, particularly as they have an increased risk of disease severity. Enteroviruses are known to cause significant morbidity, with a diverse range of clinical presentation from over 100 different genotypes. In this study, we aimed to provide a more comprehensive overview of enteroviral infections in transplant recipients, compared to nontransplant patients, and to bridge some gaps in our current knowledge. Identifying potential clinical manifestation patterns can help improve patient management following enterovirus infections. In recent years, improved antirejection treatment for solid organ transplantation has improved the outcome for transplant recipients . HoweverEnteroviruses are ubiquitous in the community, with studies indicating an 11–13% prevalence even among healthy individuals . In the Enteroviruses have vast variations in their genomes and can be categorized into four different family groups, A, B, C and D, each with their own subspecies, resulting in over 100 different genotypes . Each geEnterovirus infections following transplantation are neither fully understood nor studied. Indeed, literature on enterovirus infections in transplant recipients consists mainly of case studies and small patient cohorts, involving either one type of organ transplant or specific enterovirus genotypes , 11. It A more comprehensive overview of enterovirus infections in solid organ transplant recipients, both in children and adults, may be required to bridge some gaps in our current knowledge. In this study, we compared transplant patients with nontransplant patients by (i) investigating the distribution of genotypes, (ii) exploring clinical manifestations, and (iii) determining potential differences in severity.A total of 264 patients with 283 enterovirus infection episodes, covering 33 genotypes, were included in the study. Overall, 23 transplant recipients (26 samples) and 128 nontransplant recipients (148 samples) were excluded due to a poor sequencing result . For the(i) Patient characteristics. Most patients included in our study were children younger than 16 years (174 children versus 90 adults), with an average age of 5 years in children and 45 years in adults (n = 5) were identified and in older children, six liver and one kidney transplant recipients were included. Meanwhile, in adults, lung transplant recipients  represented the largest group, followed by kidney transplant recipients . No significant differences were found between males and females (P = 0.79).n adults  and 2. Wn adults . In smal(ii) Sample characteristics. In small children, enteroviruses were most frequently found in feces (P = 0.009). In adults, enteroviruses were most commonly detected in fecal samples from transplant recipients and respiratory samples from nontransplant recipients, with 63.3% and 42.4%, respectively. In comparison, only 16.7% of enteroviruses were detected in feces in nontransplant adults. Interestingly, of the 16 respiratory samples collected from transplant patients, 10 detections were from lung transplant recipients. Most strikingly, enteroviruses were only detected in cerebrospinal fluid (CSF) from nontransplant patients.in feces  and 2. Hn = 3, 17.5%) was the most frequently detected genotype in children with a transplant and occurred solely in liver transplant recipients (Table S2). Interestingly, EV-C were most commonly detected in adults with a transplant, comprising of 57% (n = 17) of detections. Coxsackievirus A22 (CV-A22) was subsequently identified as the most frequently detected EV-C genotype in transplanted adults , occurring in all solid organ transplants included in the study (Table S2 and S3). In comparison, there were only 13 EV-C detections, including one CV-A22 detection, in adults without a transplant (20% of adult detections). EV-D (in which the only genotype detected was EV-D68) were found in all ages in the nontransplant population and exclusively in adults in the transplant population (P < 0.05). A complete breakdown of the number of genotypes found in each sample material and transplant type can be found in Tables S2, S3 and S4.Although EV-A and EV-B were most commonly detected in children in both populations, EV-D68 was the most frequently identified genotype in children without a transplant Table S2). Meanwhile echovirus 11 (pulation  and 2. E. Meanwhi(i) Co-detections. An enterovirus was the only pathogen identified in 21.3% (n = 10) of detections in transplant patients and 46.6% (n = 110) of detections in nontransplant patients. Rhinoviruses were found to be the most commonly identified co-detection (29.85%) associated with infections in the nontransplant population. Furthermore, co-detections with adenovirus (10.6%), along with other viruses associated with gastroenteritis, such as rotavirus (4.3%) and norovirus (4.3%), were additionally found in the nontransplant population. Similarly, co-detections with adenovirus were observed in the transplant population, along with norovirus (20%) and human parainfluenza type 3 (13.3%). A detailed overview of the co-detections found at the same time as the detected enterovirus are shown in Tables S5 and S6.Based on the documented clinical symptoms at presentation, patients were divided into four categories : respiratory, gastroenteritis, neurological or other  . As age (i) Clinical manifestations in children. Respiratory symptoms and subsequent respiratory infections were most commonly associated with enteroviruses detected in children without a transplant, with EV-D68 causing the majority of infections  (P < 0.05). Interestingly, other than EV-D68, no other genotype was found to have caused more than one respiratory infection each, in the nontransplant population. In comparison, gastrointestinal symptoms  were most commonly found in children with a transplant (P < 0.05) , of which three infections were EV-A, one EV-B and one EV-C (Table S8).24, 80%)  and S8. (ii) Clinical manifestations in adults. Notably, enteroviruses in adults without a transplant were most frequently detected in cases of neurological symptoms and infections (49%)  were found to result in the majority of neurological infections, with echovirus 30 (E-30)  and echovirus 16  observed to have the highest frequency (Table S8). In contrast, no neurological infections from enteroviruses were found in adults or children with a transplant. Meanwhile, enteroviruses found in adults with a transplant were most commonly associated with either respiratory infections (four EV-D and two EV-C) or gastrointestinal infections (five EV-C and one EV-D) (Table S8). Gastrointestinal infections were also more likely (P < 0.05) to occur in adult transplant patients, compared to nontransplant adults. Interestingly, CV-A22 (EV-C) was found to be the causative agent in 3/6 gastrointestinal infections in adult transplant patients (Table S8).ns (49%) . EV-B  and adults .(i) Length of hospital stay. The majority of enterovirus infections in children without a transplant required hospital admission (83%) (n = 42) of children without a transplant had a short-term stay of 2–6 days and 30% (n = 23) had a stay longer than 7 days. On average, these children were likely to have a hospital stay of 6.5 days, with infections most commonly caused by EV-B  and EV-D  (Table S9). Longer admission periods were strongly associated with neurological infections. Hospital stays of >30 days were reported from an enterovirus A71 infection causing meningitis and two EV-D68 infections, one causing acute flaccid myelitis (AFM) and the other causing pneumonia. In addition, 70% (n = 5) of infections in children with a transplant led to hospital admission of two or more days. In these five cases, enteroviruses were found to have caused gastrointestinal infections. Meanwhile, 50% (n = 5) of gastrointestinal infections in nontransplant children led to a hospital stay of two or more days. However, children with a transplant did not have a significantly longer length of hospital stay (LOS) than children without a transplant (P = 0.58).on (83%) . A totaln = 30) . EV-D68 infections tended to be more severe, with six infections in children and three infections in adults resulting in a LOS of 7–30 days. Adults with a transplant were more likely to be seen in an outpatient setting with only 31% of enterovirus infections leading to hospital admission (average of 2.8 days). As there were only two group C infections in nontransplant adults, statistical power was not sufficient to determine differences in severity. However, it could be noted that three out of the four infections in adults with a transplant that caused a hospital stay of more than 2 days were caused by EV-C (Table S9). Overall, nontransplant patients tended to have a longer LOS compared to transplant patients, with an average of 5.2 days  and 3.3 days (most commonly from gastroenteritis or respiratory infections), respectively (P = 0.014).The majority of adults without a transplant were also admitted to the hospital (70%) (n = 30)  and tend(ii) Patient recovery. Most patients with an enterovirus infection made a full recovery (82.3% infections) (P < 0.05). However, this was not found to be significant between children (P = 0.084) or adults (P = 0.104). Of note, adults with a transplant had symptom persistence from four EV-C infections: two cases of gastrointestinal symptoms from coxsackievirus A1 and two cases of respiratory symptoms from enterovirus C109 and EV-C105 (Table S10). An additional adult with a transplant had persistent respiratory symptoms from an EV-D68 infection (Table S10). Meanwhile, adults without a transplant had no symptom persistence from EV-C infections, but persistent neurological  and respiratory  symptoms, from EV-B and EV-D (Table S10). An additional adult had lingering symptoms from a coxsackievirus B5 infection causing myocarditis. Children without a transplant had symptom persistence following EV-A , EV-B , and EV-D (n = 6 infections [54.5%] including a child with AFM).ections) . Overall(iii) Enterovirus recurrence. Although the majority of patients in this study had only one infection episode, patients with a transplant were more likely to have a second and third enterovirus infection (P = 0.002) and adults (P = 0.020). Although multiple infection episodes were rare, five patients were found to have had three separate infections with the same/different enterovirus genotype. Of note, in the transplant population, one child had three separate E-11 infection episodes, while another had a CV-A4 infection followed by a co-enterovirus infection from EV-C104 (respiratory sample) and CV-A6 (plasma sample). Finally, an adult with a transplant had three separate CV-A22 infection episodes, highlighting the possibility for chronic enterovirus infections.nfection . Indeed,nfection . This waEnterovirus infections can cause a diverse range of illnesses, even in healthy individuals –15. Howe–Children (<16 years) without a transplant had the highest number of enterovirus detections, particularly in small children (0–5 years) . This isn = 4, 80%). As our hospital performs approximately 30 pediatric liver transplants annually, it could account for the high number of liver transplants in this age group . Additionally, transplanted individuals tended to have more symptom persistence resulting from EV-C infections.Generally, the frequency of EV-A and EV-B detections, which include the majority of the coxsackieviruses and exclusively the echoviruses, declined with increasing age  and 2. T22–n = 24) of detections in children and 67% (n = 10) of detections in adults presenting with respiratory illness, reiterating EV-D68 as a respiratory disease pathogen. However, in adults with a transplant, we found EV-D68 to have caused the respiratory infection in 40% (n = 4) of detections. Nevertheless, it must be noted that transplant recipients tended to have more co-detections and could account for the lower number of detections found to have solely caused the infection (Table S7). Overall, children had a more severe EV-D68 infection than adults in our study, with a higher number of hospital stays (P < 0.05).The most detected enterovirus in this study was EV-D68 and was primarily associated with respiratory infections. We found EV-D68 to have caused the respiratory infection in 72.7% , only 10 were determined to have caused the infection (11.6%). This suggests that while enteroviruses may cause gastrointestinal symptoms in nontransplant children, the vast majority of detections are not associated with clinical illness. In transplanted children enteroviruses were found to cause gastrointestinal infections in a third of cases. Although the number of patients in this category is small to draw a definite conclusion, it does appear that children with a transplant tend to experience more gastroenteritis associated with enteroviruses than their nontransplant counterparts. Furthermore, while we only detected seven enterovirus gastroenteritis cases, five required hospital admission, suggesting gastrointestinal infections can lead to considerable morbidity, which has been observed previously . Additionally, they were also more likely to have a single outpatient appointment at the time of sampling and a shorter LOS, compared to nontransplant patients. This is not surprising as transplant patients are more likely to have a higher number of check-up appointments and any new symptom will be treated with urgency with the patient being tested, regardless of hospital admission. Nevertheless, the longer recovery time and enterovirus recurrence could suggest patients could benefit from additional care. More follow-up studies monitoring the prognosis of enterovirus infections could provide additional information for clinicians for patient management, particularly as studies investigating gastroenteritis in transplant recipients are currently limited , and are associated with various clinical manifestations, it can render definitive conclusions challenging, due to the multiple comparisons required. Furthermore, as this study was carried out in a tertiary hospital, the overrepresentation of patients with complex medical histories or severe illness could imply that the nontransplant population were not necessarily previously healthy or immunocompetent. A future study could include non-university hospitals and primary care to provide a clearer picture of the clinical burden of enterovirus associated diseases. Finally, it should be noted that the enteroviruses described in this study are reflective of the circulation in the Netherlands during this 4-year period, including three outbreaks of EV-D68. As a result, fluctuations in enterovirus prevalence are to be expected outside this time frame and region.In spite of these limitations, it is possible to draw a few careful conclusions from this study. First, we have found that enteroviruses are not necessarily more severe in transplant patients. This is especially the case for neurological infections, which we did not detect in the transplant population. Second, it may be the case that some enterovirus genotypes are more pathogenic in transplant recipients. We showed that nearly all EV-C detections occurred in the transplanted population. In contrast, EV-A and EV-B infections were more likely to affect younger children, with the transplant population not appearing to be especially vulnerable. Third, although the question of enteroviruses being regarded as a true gastrointestinal pathogen is not entirely solved, according to the clinician’s opinion, enteroviruses were more commonly thought to cause gastrointestinal infections in transplant individuals.The University Medical Center Groningen (UMCG) is the largest transplant center in the Netherlands. The UMCG performs approximately 200 kidney (adults only), 60 liver, 35 lung and 12 heart transplants annually. Approximately 3,000 transplant recipients are being followed-up. All patient information gathered during admission or outpatient visits were recorded and extracted from the electronic patient database system.An enterovirus was initially detected through reverse transcriptase real-time PCR, followed by genotyping using Sanger sequencing by targeting the viral protein 1 gene . SequencPatients were included in the study if they had been admitted to the UMCG for an overnight stay (≥ one night) or had symptoms during an outpatient appointment. Only patients with enterovirus detection after transplantation were included. An enterovirus infection episode was defined as a single clinical period, including clinical symptoms and viral detection. Repeated samples that yielded the same genotype from the same patient within a 3-week period were removed . If a paA retrospective examination of all medical records from selected patients was performed. The following clinical information was collected: age, gender, comorbidities, enterovirus clinical presentation, final diagnosis on discharge, length of hospital stay co-detections (microorganisms detected at the same time as the enterovirus), recovery and frequency of all enterovirus detections between January 2014 and December 2018. The following sample information was collected from BioNumerics: enterovirus genotype, sample material and viral load. Samples were collected depending on the clinical symptoms of the patients and tested in our syndromic panels for respiratory, gastrointestinal and neurological infections. If an enterovirus was detected in more than one sample, only the material with the highest viral load, i.e., the lowest cycle threshold value was used for sequencing.To investigate whether enteroviruses found in patients were thought to be the cause of the illness, we examined both the recorded clinical symptoms (which prompted the sampling) and the final diagnosis of the patient following a clinical investigation. The subsequent documented diagnosis was used to assess if the treating physician considered the detected enterovirus as incidental, or a contributing cause of the illness (either as a causative agent or part of an infection caused by more than one microorganism). Only detections where an enterovirus was deemed to be the causative agent were counted as an infection, leaving out the detections which were judged to be incidental findings.P < 0.05).Qualitative and quantitative data were analyzed using IBM SPSS (v23) and compared using chi-square tests, multinomial regression analysis and Mann-Whitney U tests to determine significant differences between transplant and nontransplant patients (This study was evaluated by the local UMCG Ethics Committee and a waiver “METc-2021/143” was obtained. All samples and patient data were pseudonymized before analysis according to local guidelines.The data that support the findings of this study are available from the corresponding author upon reasonable request.