GABRA2 rs279858-linked variants might be a key genetic modulator for heroin vulnerability by affecting the connections of reward network and cognition. The role of the reward network connections that mediates the effects of rs279858 on cognition would be disrupted by heroin addiction. These findings provide new insights into the neurocircuitry and genetic mechanisms of addiction.The reward system plays a vital role in drug addiction. The purpose of this study is to investigate the structural connectivity characteristics and driving-control subnetwork patterns of reward circuits in heroin abusers and assess the genetic modulation on the reward network. We first defined the reward network based on systematic literature review, and built the reward network based on diffusion tensor imaging data of 78 heroin abusers (HAs) and 79 healthy controls (HCs) using structural connectomics. Then we assessed genetic factors that might modulate changes in the reward network by performing imaging-genetic screening for 22 addiction-related polymorphisms. The genetic association was validated by performing genetic associations (1032 HAs and 2863 HCs) and expanded-variant analysis. Finally, we estimated the association between these genetic variations, reward network, and clinical performance. We found that HAs had widespread deficiencies in the structural connectivity of the reward circuit (center in VTA-linked connections), which correlated with cognition deficiency. The disruptions synchronously were shown on the reward driving system and reward control system.  In recent decades, the prevalence of heroin abuse and on-medical prescription opioid use sharply increased in the USA and Europe3. The resurgence of heroin abuse has triggered dramatic increase in heroin overdose deaths4 and the spread of human immunodeficiency virus and hepatitis C virus infections5. Heroin misuse makes more attacks on the vulnerable population, such as adolescents and young adults, and can be life-threatening with significantly high morbidity and mortality. Adding new insight into the biological mechanism and identifying precise targets for neuromodulation are vital for the prevention and treatment of this devastating disease6.Heroin is one of the major addictive drugs that generates considerable public health concerns both in China and worldwide8. Despite the diversity in chemical structure and molecular targets, addictive drugs mediate their reinforcing properties by increasing dopamine (DA) concentrations in reward circuits, primarily in the ventral tegmental area (VTA) and its major projections  and medial prefrontal cortex (mPFC))9. Drug addiction is hypothesized to develop from an imbalanced dual system in these reward circuits, the hyperactive reward motivation (mainly involves VTA and NAc) and hypoactive reward control  and anterior cingulate cortex (ACC))11. This hypothesis has been validated in electrochemical and behavioral studies13. Recent advances in neuroimaging technology and brain network analysis allow us to investigate the reward circuit from a connectome perspective.Drug addiction including heroin addiction is seen as a disease of aberrant neuroadaptation in the brain reward system15 (see Method section). Although the resting-state imaging studies have revealed that drug abusers exhibit functional connectivity deficiencies in the executive control system and impulsivity system17, it remains largely unknown how the reward driving-control system is affected by drug addiction.The reward processing in addicted individuals has been extensively studied by measuring brain reactivity to drug cues and non-drug rewards (mostly monetary). According to the systematic review we conducted to identify the disrupted reward circuit of substance abusers  may also be involved in this pharmacological processing19. Genetic factors contribute roughly 50% to the etiologies of addiction20. Since variants within the opioid-related genes may influence the various stages and endophenotypes of heroin addiction, it is important to understand how these genetic factors may modulate changes in the reward network of heroin addiction.Heroin primarily acts on endogenous opioid receptors and then triggers DA release in VTA and NAc, which are primarily modulated by GABA and the glutamate system22. These SNPs were in genes involved in the opioid system, DA system, GABA system, glutamate system, 5-hydroxytryptamine system, and other key polymorphisms. The supporting reasons for the selection of the 22 candidate SNPs are summarized in Table We evaluated heroin addiction-related genes and chose 22 variations as candidate genetic markers, which overlapped with SNPs reported in literature reviews associating genetic risk variants to opioid dependenceThe present study aims to investigate the structural connectivity and driving-control subnetwork patterns of the reward circuit, and subsequently investigate the potential genetic modulation responsible for these changes. We also estimated the association between these genetic variations, reward network, and clinical performance.Step 1: We first investigated the reward network changes in heroin abusers (HAs). To better define the network nodes, we conducted systematic literature review and identified the most disrupted brain areas for rewarding stimuli in substance abusers. Then the reward network was built by probabilistic fiber tracking the diffusion tensor imaging (DTI) data from 78 HAs and 79 healthy controls (HCs). The changes in network characteristics and driving-control subnetworks were estimated.Step 2: We then assessed genetic factors that may modulate changes in the reward network in HAs by performing imaging-genetic screening for 22 addiction-related polymorphisms. The genetic association was validated by performing genetic associations (1032 HAs and 2863 HCs) and expanded-variant analysis.Step 3: Finally, the association between the genetic variation, changes in reward network, and clinical performance was analyzed by mediation analysis.The systematic literature review was conducted to help identify disrupted reward-related brain regions which responses to rewarding stimuli  in substance abusers. The search terms included neuroimaging terms, substance addiction-related terms, and stimulus-related terms. The focus of the studies selected for the systematic review was on the comparisons between substance abusers and HCs and whole-brain analysis, which was published online before 30 November 2016. We identified a total of 65 studies and organized the findings by specific brain regions Table . The seaDiagnostic and Statistical Manual of Mental Disorders, 4th edition, but not for other substances . Alcohol abusers were excluded from the study based on the Michigan Alcoholism Screening Test (score ≤ 4). In addition, we recruited 79 matched male HCs from the local community through newspaper advertisements. All of the participants were ethnic Han Chinese, and native to southern China. The HAs self-reported that they had no past or current major medical conditions and no personal or family history of major psychiatric disorders other than their current addiction. The exclusion criteria included (1) level of education < 9 years, (2) left-handed, and (3) contraindications for magnetic resonance imaging (MRI).A cohort of 78 male HAs was recruited from drug addiction treatment centers and the local community in Zhongshan city, Guangdong province, China. They all met the criteria for heroin dependence based on the The genetic validation dataset included 1032 HAs , who were recruited from multiple drug addiction treatment centers in Guangdong and Hubei provinces and 2863 HCs  from the respective local communities. The MRI group was a subsample group derived from the genetic sample group (only from Zhongshan city). The characteristics of the subjects are summarized in Table The study was approved by the Peking University Institutional Review Board. All subjects were informed of the entire procedure and potential risks before being requested to sign a written informed consent form. All participants received monetary compensation for contributing to the study. None of HAs received systemic pharmacological substitution treatments during this study.MRI subjects underwent a detailed series of standardized neuropsychological tests, either on the same day or within 7 days of MRI scans. This included (1) the Montreal Cognitive Assessment (MoCA) to assess global cognitive ability, (2) the Barratt Impulsiveness Scale (BIS-11) to determine the level of impulsivity, (3) the Iowa Gambling Task (IGT) to evaluate decision-making ability, and (4) a visual analog scale to assess self-reported average heroin craving during the past week. Detailed descriptions of these assessments are available in the b0 images without diffusion weighting; 25 non-collinear diffusion-weighting gradients with b = 1000 s/mm2; acquisition matrix: 128 × 128; field of view: 256 × 256 mm2).MRI was performed using a 1.5-T MR Signa HDxt imaging system  with a standard eight-channel head coil. Two experienced radiologists examined the T2 images, and no abnormalities were observed in the subjects. T1-weighted sagittal three-dimensional images were acquired with a spoiled gradient recalled echo sequence with coverage of the entire brain. For DTI, a total of 28 image sets was acquired with 56 axial slices  using the following steps: (1) Eddy-current and head motion correction, estimation of the diffusion tensors, and calculation of functional anisotropy (FA); (2) co-registration of T1 scans with their corresponding DTI b0 image so that ROIs that were defined in the structural MRI space could be transformed into native diffusion space; (3) the FA images were spatially normalized to the Montreal Neurological Institute (MNI) space using DARTEL of SPM8, resampled to 3 × 3 × 3 mm3 during the normalization, and smoothed with an 6-mm full-width at half-maximum Gaussian kernel.The DTI data were preprocessed using FSL5.0.7 , and the reward-control subnetwork comprised the dlPFC, ACC together with OFC and INS that mainly exerted inhibitory control for substance reward.We defined the addiction reward-related brain regions (12 regions in each hemisphere), which were both highly reported in our systematic literature review and involved in biological mechanism of heroin addiction, as the nodes of reward network. These involved the dlPFC, ACC, VTA, orbitofrontal cortex (OFC), insula (INS), ventral striatum (VStr), caudate, amygdala (AMY), hippocampus (HIP), thalamus (THA), putamen, and pallidum . Probabilistic fiber tracking was repeatedly sampled from the distributions of voxel-wise principal diffusion directions to generate a probabilistic streamline on the location of the true streamline and thereby build a connective value map in diffusion space. All the fibers were tracked with a curvature threshold of 0.2. Then, both the FA-value images and the probabilistic streamline images were warped from diffusion space into MNI space. The one-sample The average connective strength of each edge was measured for each subject independently, including connections within the reward-driving subnetwork (Driving) and reward-control subnetwork (Control), connections between driving/control subnetworks (BTN-connections), and connections with significant differences between HAs and HCs (Diff-connections).Genomic DNA was isolated from peripheral blood lymphocytes using the QIAamp DNA Mini Kit . MassArray  was used to genotype all markers using allele-specific Matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Primers and multiplex reactions were designed using RealSNP.com website. None of the individual proportions significantly deviated from Hardy–Weinberg equilibrium (HWE) among HCs and HAs. The genotype call rate was >98% for each SNP.http://biostats.usc.edu/Quanto.html) was higher than 0.9. The variances were similar between the groups that were being statistically compared. Two-sample t-test was used to determine differences in demographic and clinical characteristics using SPSS 21.0 software. Each structural connection was compared between the HA and HC groups using 10,000 non-parametric permutation tests based on two-sample t-test scores, with “cigarettes smoked per day” and age as covariates. The threshold of Diff-connections was set at p < 0.00018116 (0.05/276) with multiple-comparison correction to identify statistically significant differences. The assessment of HWE was performed using the χ2 goodness-of-fit test. Additive linear regression was used for the association analysis of SNPs with structural connection, with “cigarettes smoked per day” and age as covariates using PLINK23. Linkage disequilibrium (LD) analyses were performed using Haploview 4.1 (http://www.broad.mit.edu/mpg/haploview/). The mediation analysis was performed using the model 4 in PROCESS24 to bootstrap the sampling distribution of the indirect effect.The statistical power of sample size computed by the Quanto soft  and neurocognitive performances are shown in Table p < 0.00018116 (0.05/276) with multiple-comparison correction; Fig. t-value were selected out   with rs279858, had similar significant consequences on the mean connective strength of the reward network . Significant differences in the allele and genotype frequencies of GABRA2 rs279858 were observed between HAs and HCs. The rs279858*G allele frequency was significantly higher in HAs than HCs . However, this association for rs279858, reward network, and cognition was not observed in HAs  mainly in default-mode, attentional and visual systems26. However, the convergent changes in the reward circuit, the most critical system for addiction, were insufficient for analysis. The reward network has a significant amount of neural projections. Hence, we adopted the probabilistic fiber tracking method, which is more effective at determining the crossing fibers and intricate branching configurations than the deterministic approach27. By adopting the FA-weight probabilistic tracking approach, we observed the widespread decrease and no increase in connections in the reward network of HAs. These results were rather unexpected since it is generally considered that in addiction the reward-driving network was increased, whereas the reward-control network was decreased. Heroin use may trigger numerous forms of synaptic plasticity in the brain reward regions, and reduce inefficient and redundant neurons for acquiring sensitization toward addiction-related cues28. The neurotoxic effects of heroin are involved in loss of gray matter and white matter, cognition, neuronal apoptosis, synaptic defects, depression of neurogenesis, and so on34. The number of synapses, dendritic spines density, and membrane resistance of VTA dopaminergic neurons could be profoundly affected by chronic morphine use36. Consequently, HAs could have deficiency in myelination integrity and dendritic spine density of neural fibers within the reward network37, which reflected by the deficiency in DTI-based connectivities. The dynamic effects of heroin on reward network could be further investigated using HAs which had abused heroin for different time.The advances in DTI tractography method allows us to delineate the neural axon fibers non-invasively based on the diffusion patterns of water molecules38. We attempted to understand the neurobiological meaning of these changes through concerning the top and bilateral changes, which mainly presented at the VTA-centered core projections of the reward circuit. The VTA is considered as an essential center for reward and motivation39. The main reciprocal projections of the VTA are with NAc, mPFC , and AMY, and these connections form the core of the reward-loop7. Our finding suggests the main structural connections that are damaged in heroin addiction are presented at the core projections of the reward circuit, which parallel the reward driving system and reward control system. A possible explanation is that the initial drug-induced plasticity in the DAergic midbrain and subsequently in the VStr would recruit more dorsal striatal regions during chronic drug use and reshape the connectivity within these projections. This leads to compulsive drug dependence at the late stages of addiction40. Hence, the driving and control subnetworks of the reward circuit would have compact interactions and operate together to monitor reward processing.By systematic review of the literature for imaging studies, we found the highly reported brain areas of rewarding stimuli in substance addiction were mainly in the neurobiology of addictionA α2 receptor which encoded by GABRA2 located on chr4p1241 was highly expressed in the mesolimbic DA reward pathway, including HIP and dopaminergic neurons in the substantia nigra and VTA42 47. In line with previous studies, we provided evidence of the association between GABRA2 rs279858 and variants in the rs279858-linked low-expression haplotype block with heroin addiction vulnerability49. GABAA receptors may influence the opiates-reduced excitability of DA neurons within the reward pathway51 (GABAA α2 receptors are highly expressed). The rs279858 variant may affect the distribution pattern of GABAA α2 receptors52 that may be implicated in weaker neural connectivity in reward-loop and less inhibition of DA neurons, which results in the increased risk for addiction via developmental mechanisms53. However, addictive drug use could alter the expression of GABAA subunits genes54. We have also speculated and assessed the epigenetic modifications of GABRA2 may be involved in the gene–heroin interaction. However, we have not found significant group difference for the epigenetic rate of CpG island in part of GABRA2 promoter, as well as its correlation with rs279858 and addiction characteristics. It was needed to detect more regions and pattern of epigenetic modulation of GABRA2 in the future. So, the overall interactive effects of heroin-by-rs279858 throughout the reward circuitry may be dynamic.GABA55. Here, we only observed general cognition abnormality in sober HAs associated with the strength of reward network, and not in decision making and impulsivity. A hypothesis to the deficiency of decision making and impulsivity in HAs would be reversible to some degree after abstinence, as we had demonstrated in our previous study56. However, damage of addiction on general cognition would be long-lasting and simultaneously occur with connectivity changes. Furthermore, GABRA2 rs279858 accounted for significant changes to cognition, in part through affecting the mean strength of the reward network in healthy individuals. However, this association was indistinct in heroin users, which may be due to the detrimental effects of heroin after long-term use.Cognitive impairment is a common risk associated with heroin addiction, which is aggravated by heroin useGABRA2 variants on the reward circuit could be performed using optogenetics and in vivo microscopy in preclinical models. Fourth, the study used a 1.5T MRI system, which was considered suboptimal by modern imaging standards. Other possible influence factors, such as the IQ and Socioeconomic Status, should be counter for in our future studies. Prospective and/or follow-up studies are needed to determine the causal role of GABRA2 rs279858 in the reward circuit in heroin addiction.The present study does have some limitations. First, our imaging and behavioral data focused only on Han male subjects. However, according to the genetic data, there was much more men than women in the HA group, while much women than men in control group. This disequilibrium in gender distribution between HA and HC group is the limitation of this study. Second, we did not utilize whole-genome SNP analysis  in our imaging-genetics screening. Hence, we could have missed critical SNP associations with heroin addiction. Third, the validation of the neurobiological mechanism underlying the consequences of GABRA2 rs279858-linked SNPs were associated with susceptibility to heroin addiction by affecting the connections of the reward circuit and cognition. These findings provide new insights on how genetic variants influence the neurocircuitry that underlies individual heterogeneity to heroin addiction vulnerability. Our results provide evidence to garner interest in the scientific community to incorporate a circuit-level understanding of molecular and morphology changes that underlie drug responses.HAs have widespread deficiencies in the VTA-centered structural connectivity of the reward network, both in the reward-driving and reward-control systems. The Supplemental material


Background and objective: Endometrial angiogenesis is a prerequisite for successful pregnancy. Optical coherence tomography (OCT) is a non-invasive physically optical imaging technique widely used in ophthalmology and cardiology. However, there is no study using OCT to evaluate endometrium. The aim of this study was to use OCT and traditionally histological methods to investigate endometrial vascularization in women undergoing in vitro fertilization-embryo transfer (IVF-ET) treatment and to determine the association with the pregnancy outcome. Methods: A total of 47 women were included in this study. OCT was used to assess endometrial vascularization by determining the high signal areas precisely on the seventh day after luteinizing hormone surge in non-conception natural cycles. Endometrial biopsies were obtained following OCT and immunohistochemistry was used to determine micro vessel and expression of vascular endothelial growth factor-A (VEGF-A) in the luminal epithelium, glandular epithelium and stroma, separately. Micro vessel counting was performed and the result was expressed as micro vessel density (MVD). A semi-quantitative H-score was used to determine the staining intensity of VEGF-A. Results: In women who successfully conceived after embryo transfer, the proportion of extensive high signal area in the uterine body detected by OCT , MVD  and stromal expression of VEGF-A  were found to be significantly higher than those of women who did not conceive after embryo transfer in the subsequent IVF-ET treatment . In addition, a significantly higher stromal expression of VEGF-A  and MVD  was found in women with extensive high signal area in uterine body, compared to those with focal or no high signal area . Conclusions: Both immunohistochemistry and OCT demonstrated significant difference in vascularization of the peri-implantation endometrium between subjects who did and did not conceive after IVF-ET treatment. Our findings also suggest OCT appears to be a promising non-invasive or minimally invasive alternative to study endometrial vascularity in women with reproductive failure. A timely arrival of a viable embryo and receptive endometrium are two pre-requisites for successful pregnancy. In in vitro fertilization-embryo transfer (IVF-ET) treatment, implantation is considered to be successful when there is evidence of intrauterine gestational sac. There are several causes accounting for failed implantation, mainly including gamete/embryo factors and uterine factor. Angiogenesis is the formation of new blood vessels from existing vascular structures  and endoOptical coherence tomography (OCT) is a physically optical imaging technique widely used in ophthalmology  and cardTraditionally histological measurement of micro vessel density (MVD) has been thought to be an accurate method to examine endometrial vascularization in several previous studies. One study has examined MVD in women with intrauterine adhesion and found that endometrial MVD in women recovering from transcervical resection of adhesions treatment was significantly higher than those non-responding to the treatment, which indicates that adequate angiogenesis is important for endometrial repair and function . AnotherVascular endothelial growth factor (VEGF)-A is one of the most important factors in angiogenesis regulation and it is synthesized in a phase-depending manner in human endometrium. It has been shown a three-fold increase of VEGF in the secretory phase, when compared to the proliferative phase , which sIn this study, our aim was to use OCT and immunohistochemistry to measure endometrial vascularization in the peri-implantation period in women undergoing IVF-ET treatment. We also investigated whether endometrial vascularization was associated with IVF pregnancy outcome.This study was approved by Joint Chinese University of Hong Kong - New Territories East Cluster Clinical Research Ethics Committee (CREC-2014.575 and CREC-2016.160). All the endometrial biopsies were collected with written informed consent of the participants. A total of 47 women undergoing IVF-ET treatment were included in this study. All of them had regular menstrual cycle and natural ovulation. Women who had uterine structural abnormalities or abnormal hystersalpingogram result were excluded in this study.In this study, although we have recruited 47 subjects, only 20 of them underwent examination by an optical coherence tomography imaging system (OCTIS) prior to the endometrial biopsy in this study, which was partly because of the unavailability of the OCTIS machine during a program update and machine maintenance and partly because of the unavailability of the person performing OCTIS. Nevertheless, the 20 women who underwent OCTIS represented a consecutive series of subjects, without bias in patient selection. OCTIS consists of an imaging computing system and a sterile detachable probe. The OCT probe is a catheter measures 2.5 mm in diameter and 15 cm in length which is sealed with a transparent outer sheath with a 1 mm long window near its tip for image scanning and capturing. Side-viewing probe was chosen as it is suitable for the imaging of tube-like hollow organs such as uterine cavity. There is a flexible fiber optical shaft for rotational imaging at 600–1200 rpm inside the sheath. The optical fiber is illuminated by a broad wavelength-range light source operating at 1300 nm and a sweeping rate of 50 kHz. The images acquisition speed is at 20 frames per seconds with 15 μm axial and 25 μm lateral resolution and depth up to 3 mm in both grey and color modes. The scanning window at the distal tip of the catheter is brought to the area of interest to capture OCT image. The OCTIS is United State Food and Drug Administration (K102599) approved for medical imaging. To obtain OCT images of endometrium, speculum examination was performed and OCT probe was inserted into the women’s uterus via cervix. The OCT probe was placed at the fundus of uterine cavity while simultaneously operating a pull-back along the uterine cavity to cervix to perform a scan of the whole uterine cavity. The OCTIS then created cross-sectional plane of endometrium which allowed real-time and off-time analysis of each section. Endometrial biopsy was performed afterwards. Red-yellow area on OCT image represents high backscattering signal and was used to assess endometrial vascularization. Three random OCT images were selected from uterine fundus, uterine body and isthmus region, respectively, for each woman and all images were mixed before assessment. The image was divided into four quadrants  and the density of high signal area was scored with (i) score 0 if no high signal identified, (ii) score 1 if focal high signal area (<50% in a quadrant) observed (iii) score 2 if extensive high signal area (>50% in a quadrant) observed . Each quAll participants in this study performed daily urine dipstick test from day 9 of the menstrual cycle onwards to detect the luteinizing hormone (LH) surge and endometrial biopsies were precisely obtained on day LH+7 in the non-conception cycles. A Pipet Curet  was used to obtain endometrial specimens, which were then immediately put into 10% neutral buffered formalin for over-night fixation. After embedded into paraffin wax, endometrial specimens were firstly examined with routine H&E staining. For the immunohistochemistry staining, as stated in our earlier study , endomet2.As stated in our previous work , systemai (i+1), in which i represented the intensity of staining  and Pi represented the percentage of cells at each intensity (0–100%). H-scores were calculated in different endometrial compartments, namely luminal epithelium, glandular epithelium, and stroma, separately. H-score was used to grade and calculate the intensity of VEGF-A expression, based on the equation: H-score = ∑PIn natural cycle, daily hormonal monitoring of estrodial/LH/progesterone was performed four days prior to the expected day of ovulation or if estrodial ≥400 pmol/L until evidence of LH surge. Ultrasound was performed in arranged in the peri-ovulatory period to measure endometrial thickness and follicle diameter. Ovulation was assumed to occur 36 hours after LH surge. Day-three embryos and blastocycsts were thawed and transferred three days and five days following the estimated day of ovulation, respectively.In hormonal replacement treatment cycle, 6 mg oestradiol was given daily orally from day 2 of the menstrual cycle for 14 days. Transvaginal ultrasound was performed on day 14 of the estrogen therapy to assess endometrial thickness and to exclude ovarian activity. If endometrial thickness was ≥8 mm, plasma progesterone level was <4 nmol/L, progesterone was commenced using either Endometrin  100 mg TDS vaginally. Day-three embryos and blastocycsts were thawed and transferred three days and five days following progesterone administration, respectively.’st test and Mann–Whitney U test were applied to compare normally distributed data and nonparametric data, respectively. The difference of qualitative data was compared by chi-square test. p < 0.05 was considered significant. All of the statistical analysis was performed using SPSS software .A Shapiro–Wilk test was used to check the data distribution. The mean ± SD was used to present the normally distributed data while median (range) was used for skewed data. The StudentIn this study, 25 women conceived, and 22 women did not conceive in the subsequent IVF-ET cycle, out of which 42 women had single embryo transferred and the other five women had double embryos transferred; in those with two embryos transferred, one woman conceived twins and four women did not conceive. A total of 38 women were transferred day-five blastocysts (21 in the pregnant group and 17 in the non-pregnant group) and nine women were transferred day-three embryos (four in the pregnant group and five in the non-pregnant group). None of the participants underwent pre-implantation genetic test (PGT) treatment. The demographic details and embryo information of these participants were summarized in The immunostaining of endometrial vWF expression from pregnant women and non-pregnant women was shown in Representative images of H and E staining are shown in An image of endometrium demonstrated by parallel histology and OCT image was shown in Among the 20 women undergoing OCT imaging, a significantly higher stromal expression of VEGF-A and MVD was found in women with extensive high signal area in uterine body, compared to those with focal or no high signal area. There was no significant difference in MVD, luminal epithelial expression of VEGF-A, and glandular epithelial expression of VEGF-A between women with extensive high signal area and those with focal or no high signal area in the uterine fundus and isthmus regions .2; VEGF-A, vascular endothelial growth factor-A; OCT, optical coherence tomography.MVD, micro vessel density, calculated as number of micro blood vessels/mmIn this study, we have used a new method, namely OCT, to evaluate endometrial vascularization and subsequently observed a significantly increased proportion of an extensive high signal area in women who were pregnant. Using immunohistochemistry, we have also found an increased MVD and higher stromal expression of VEGF-A in the peri-implantation endometrium in women with successful subsequent pregnancy during IVF-ET treatment. In addition, there was an association between the two methods of measuring endometrial vascularity.Embryo implantation remains one of the most critical steps in determining the success of IVF treatment despite the significant advances in this field in recent years. A prerequisite for successful implantation requires vascular adaptation in the endometrium and endometrial angiogenesis has been therefore thought to be a marker of the receptive endometrium . An earlUsing non-invasive ultrasonography combined with color Doppler, women with detectable endometrial blood flow were found to have better clinical outcomes, suggesting an adequate blood flow to endometrium is essential for successful pregnancy . MoreoveOCT combined with angiography has been widely used in ophthalmology to provide retinal and choroidal vasculature and structure ,18. HoweOne previous study has examined the predictive value of endometrial micro vessel for IVF treatment outcome . The autEmbryo implantation is a dynamic process, which consists of apposition, adhesion, and invasion . In the Whilst we have demonstrated altered endometrial vascularization in women undergoing IVF-ET treatment, there are still several unanswered questions yet. Firstly, given that it has been considered that early embryo development requires a relatively low oxygen environment , it is uThere are several limitations of this study. One possible criticism of our study was that observations in the non-conception endometrial biopsy cycle (four weeks prior to embryo replacement) may not be exactly the same as the endometrium at the time of embryo transfer, given that there some degree of cycle to cycle variation may occur . MoreoveTo conclude, both immunohistochemistry and OCT demonstrated significant difference in vascularization of the peri-implantation endometrium between subjects who did and did not conceive after IVF-ET treatment. Our findings also suggest OCT appears to be a promising non-invasive or minimally invasive alternative to study endometrial vascularity in women with reproductive failure.


Chemical synapses are specialized interfaces between neurons in the brain that transmit and modulate information, thereby integrating cells into multiplicity of interacting neural circuits. Cell adhesion molecules (CAMs) might form trans-synaptic complexes that are crucial for the appropriate identification of synaptic partners and further for the establishment, properties, and dynamics of synapses. When affected, trans-synaptic adhesion mechanisms play a role in synaptopathies in a variety of neuropsychiatric disorders including epilepsy. This review recapitulates current understanding of trans-synaptic interactions in pathophysiology of interneuronal connections. In particular, we discuss here the possible implications of trans-synaptic adhesion dysfunction for epilepsy. Synaptic connections are important defining features of neurons. They continue to be formed and eliminated throughout the development and postnatal period allowing neurons spreading and processing complex patterns of impulses that underlie cognition. Synapses must maintain and strictly regulate the positioning of two opposite molecular architectures – their receptor and channel specification at the postsynaptic site, and the highly dynamic system of neurotransmitter-containing vesicles at the presynaptic site. Moreover, the alignment of both synaptic constituents must be coordinated in time during junction formation, thus neurotransmitters released from the pre-synaptic membrane could act on receptors located at the post-synaptic site in order to ensure fine flow of information between neurons .The biochemical and ultrastructural studies of a synapse suggest that adhesion might be critical for the proper assembly and molecular composition of pre- and postsynaptic specializations . In factThe crucial role of trans-synaptic CAMs in shaping synapses is also reflected by the fact that many of these molecules have been linked to synaptopathies in neuropsychiatric disorders, including epilepsy. Epilepsy is a condition that has an impact on more than 60 million people throughout the world . The disAt least to some extent, plasticity of interneuronal connections seems to be based on synaptically implemented and strictly controlled enzymatic digestion of trans-synaptic CAMs and their adhesive contacts by extracellular proteases . The bioEach chemical synapse is composed of a patch of presynaptic cell membrane from one neuron facing a patch of postsynaptic cell membrane from a second neuron. The space between them is called a synaptic cleft. The presynaptic side of the synapse harbors the molecular machinery essential for a turnover of neurotransmitter-containing vesicles. Its role is to initiate synaptic responses. The postsynaptic side of the synapse contains receptors and ion channels and triggers downstream cellular signals. During synaptic activity presynaptic neuron executes the release of neurotransmitter molecules into the synaptic cleft, then their binding to the postsynaptic receptors activates channels and evokes molecular responses in the postsynaptic neuron. Apart from signal transmission a role of synapses is to wire neurons into specific functional circuits. Synaptic connections are very dynamic and as a result of synaptic activity they undergo structural and functional changes – a process that is called synaptic plasticity. Synaptic plasticity involves alterations of molecular components present at a synapse, which leads to changes in synaptic geometry and communication efficacy. The phenomenon of synaptic plasticity is widely involved in synapse formation, maintenance and elimination and underlies learning, memory and regenerative processes . UnderstThe trans-synaptic CAMs play particularly important role in regulation of pre- and postsynaptic molecular assembly during diverse steps of synapse lifespan. During contact establishment at the site of future synapse, the trans-synaptic CAMs mediate recognition of partner cells. Then, they ensure functional specification of developing synaptic contact by the recruitment of molecular components to active zones, and postsynaptic densities, which eventually leads to the different types of functional synapses with distinct physiological properties .The use of trans-synaptic adhesion term here might be, however, misleading because these molecules are not only static structural elements of adhesion, but also dynamic modulators of continual morphological and functional synaptic plasticity . Trans-sFigure 1).There are several classes of extracellularly acting proteases that are modulated by synaptic activity and are well established to be engaged in synaptic plasticity . The mosApart from metaloproteinases, trypsin-type serine proteases are also well-evidenced to be involved in synaptic plasticity, with a particular involvement of neuropsin (KLK8) in modulation of trans-synaptic complexes .in vivo animal models have identified several potentially important trans-synaptic CAMs and their associated proteases and linked them to synaptic dysfunctions. Due to the large number of molecules and considering the fact that experimental evidence supporting the involvement of some direct trans-synaptic interactions and their digestion in synapse function is sometimes compelling, we wish to acknowledge here only the most exemplary ones (summarized in Table 1).One can easily notice that any alterations in this complex and proteolytically modulated trans-synaptic engineering might result in serious neuronal dysfunctions in developing or mature synapses. Indeed, scientific investigations on neuronal cultures and Figure 2).Trans-synaptic adhesion system is based on either a homo- or a heterophilic interactions between CAMs over the width of synaptic cleft . A canonin vivo studies on knockout (KO) animals demonstrated that neuroligins as well as neurexins are crucial for the formation of synaptic contacts and their function . The above notions have been supported by many studies on neuronal cultures. For instance, cells lacking either N-cadherin or β-catenin showed multiple synaptic malformations such as more filopodia-like thinner spines with parallel decrease of total spine number  is one of the most abundant cadherins in synapses. It belongs to the classic cadherins – a single-pass transmembrane proteins containing five extracellular cadherin domains, a transmembrane motif, and a cytoplasmic domain. The protein homophilic adhesion is calcium-dependent . Molecule number .Alteration of N-cadherin function induces detectable synaptic deficits that clearly demonstrate structural and functional association of N-cadherin with the processes of synapse stabilization, synaptic transmission and plasticity at both sides of the synapses. For example conditional KO mice are characterized by reduced LTP and impaired spine enlargement as demonstrated by in vitro that EphB2 receptor can also be cleaved by MMP-2 and MMP-9 in dissociated hippocampal neuronal cultures  is a glycoprotein which expression was reported at both pre- and postsynaptic constituents. The N-terminal region of NCAM binds to the partner NCAM molecule and contains five Ig domains followed by a region containing two fibronectin type III-like repeats . NumerouContactins are Ig-CAMs that contain six N-terminal Ig-like domains . There aSeveral types of LRR (leucine-rich repeats) proteins were identified at synapses, in particular NGLs (netrin-G ligands), SALMs (synaptic adhesion-like molecules), and LRRTM (leucine rich repeat transmembrane) proteins. The three NGLs expressed in vertebrates (NGL1-3) are type I membrane proteins localized to postsynaptic sites. Their name comes from the NGLs characteristic potential to bind to presynaptic netrins (netrin-G1 and -G2) . CrucialSynapse-organizing functions were also reported for SALMs . Like NGin vitro and in vivo is associated with decreased AMPAR trafficking . Dystroglycan is a duplex of α- and β-subunits. The α-dystroglycan is a highly glycosylated extracellular component, whereas β-dystroglycan intermediates in interaction between α-subunit and the cytoskeleton. Dystroglycan, like neuroligin, also binds specifically to a subset of the LNS domains of neurexins (Dystroglycan is an ubiquitous membrane protein and part of dystrophin-glycoprotein complex. It links the cytoskeleton to the extracellular matrix and stabilizes the dystrophin-glycoprotein complex at the membrane (eurexins . The dyseurexins .The epilepsy comprises a very heterogeneous group of neuropsychiatric disorders. They are defined by the occurrence of recurrent seizure episodes . In addistatus epilepticus) that leads to a latent period and eventually to the occurrence of chronic manifestation of unprovoked tonic-clonic seizures associated with histopathological changes that are characteristic for TLE  the most common form of epilepsy in adults. Rodents with recurrent TLE seizures are typically generated with use of chemoconvulsants such as pilocarpine, or kainate . Animals for TLE . Like stactivity . KindlinPost-traumatic epilepsy (PTE) is one of the leading causes for acquired epilepsies in adults. PTE develops often as a result of traumatic brain injury (TBI) caused by external mechanical force. This initial brain insult triggers cascade of molecular changes that leads to the appearance of spontaneous recurrent seizures . ConsideOver the last years, many genes with effect on human epilepsy have been reported providing important evidence for heritability of the condition . There aDistinct origins of the condition and associated seizure types are reflected by the very diverse and complex molecular mechanisms underlying epilepsies. Given the accumulating research evidence based on animal models epilepsy is now clearly recognized also as a synaptopathy. Synaptic alterations contribute to the epilepsy development, i.e., epileptogenesis, recurrent seizure initiation and propagation, and associated cognitive deficits. This notion is further supported by clinical studies suggesting that several of these synaptic mechanisms might involve dysfunctions in trans-synaptic adhesion.Since neuroligin/neurexin interaction regulates the proper balance between the number of excitatory and inhibitory synapses, it is not surprising that their alterations are important epileptic hallmarks. For instance, it was shown that neuroligin-1 and its binding partner neurexin-1β are intensively expressed in epileptic foci of human patients with TLE . FurtherThe involvement of classical cadherins in epileptogenesis and epilepsy has not apparently been examined experimentally. Nevertheless, studies on animal models revealed a potentially pathogenic mechanism related to N-cadherin. N-cadherin expression was reported to be increased in hippocampus up to 1 month following pilocarpine-induced seizure and was found consistent with the induction of mossy fiber sprouting .Most of the epilepsy incidents are idiopathic or acquired, however, there is a strong evidence for heritability of the condition. Important non-classic cadherin implicated in genetic cases of epilepsy is protocadherin-19 (PCDH19). Protocadherins possess strong structural similarity to classic cadherins particularly, they contain extracellular motifs that are homologous to those of classic cadherins. The name protocadherin suggest an evolutionary old group of molecules, however, protocadherins have very late origin and for the first time appear in primitive vertebrates . The PCDThe strong association of PCDH19 with epilepsy inheritance is also stressed by genetic screening demonstrating that 11 out of 45 patients suffering from Dravet syndrome (severe myoclonic epilepsy of infancy) carried mutations in PCDH19 . MoreoveThe expression pattern and cellular distribution of EphB receptor/ephrinB complex was investigated in patients with intractable TLE and correlated with data obtained from lithium-pilocarpine-treated rats . In compNCAM-1 concentration in cerebrospinal fluid was found elevated in epilepsy patients in comparison to healthy individuals. Intensive NCAM-1 content was also detected in drug-resistant epilepsy patients when compared to the drug-effective patients . TherefoTo explore the role of polysialylated-NCAM in epilepsy, endoneuraminidase-based inactivation of PSA-NCAM was performed by administration of the drug into the contralateral ventricle of mice with chronic seizure. That action induced neurodegeneration of cells in dentate gyrus and CA3 region of hippocampus and promoted the early onset of focal seizures . AnotherContactin-6 deletions in humans were reported to be associated with various neurological syndromes including seizures as a comorbidity . This ststatus epilepticus in the acute phase and decreased spontaneous recurrent seizures evoked by pilocarpine. Inhibition of SALM3 also prolonged the latent period in the PTZ kindling model. The aforementioned results suggest that SALM3 plays a role in epileptogenesis and that its suppression might exert therapeutic effect.SALM3 has been lately implicated in epileptogenesis and epilepsy. The study by in vitro that abnormal epileptiform activation of neurons with prolonged zero magnesium conditions decreased the expression of β-dystroglycan in mice cortical brain slices. The authors also showed that the β-dystroglycan deficit was associated with the dysfunctions of astrocytic endfeet which suggests epileptogenic downregulation of β-dystroglycan at blood-brain interface. Interestingly, clinical studies have identified two girls with alpha-dystroglycanopathy who presented severe and farmaco-resistant epilepsy characterized with electrographic myoclonic seizures (Initially, seizures .Among extracellular proteases the most intensively studied in epilepsy pathogenesis are metzincin metalloproteinases. Some of them have already had an identified trans-synaptic target which makes metzincins potential contributors to aberrant synaptic plasticity that occurs in epilepsy development.The best examined metzincin that is associated with trans-synaptic CAM and plays the significant role in different models and types of epilepsy is MMP-9 . The verSubsequently, the presumed role of MMP-9 in epileptogenesis was confirmed with use of other models namely treatment with either pilocarpine or 4-aminopyridine . RecentlThe role of other MMPs in epilepsy development has been so far poorly investigated. Experimental data indicates the significant increase of MMP-2 activity in animal models of TLE after systemic injection of pilocarpine  or kainaApart from MMPs, other proteases targeting CAMs were also implicated in epileptogenesis and epilepsy. For instance, ADAM9 and ADAM10 expression were reported to be induced in dentate gyrus of hippocampus following kainite-induced status epilepticus . MoreoveThere are also other types of proteases definitely implicated in cleavage of trans-synaptic CAMs, and with strong relation to epileptogenesis and epilepsy. Particularly worth to mention is for instance calpain -a protein that belongs to the family of calcium-dependent cysteine proteases  that wasAlthough evidence implicating different trans-synaptic complexes in the formation and maintenance of synaptic structure is considerable and their role in synaptic dysfunction convincing, further experimental data is required in order to understand the biological consequences and significance of the proteolytic trans-synaptic CAMs cleavage in modulation of synaptic function. Initial studies have already started to shed light on the dynamics of the key trans-synaptic adhesion molecules cleavage products, in response to synaptic activity. Such experimental data will be essential for an in-depth understanding of the function of adhesion molecules in synaptopathies. Particularly in epilepsy where the trans-synaptic CAMs seem to be crucial for many epileptogenic mechanisms and therefore comprise promising targets for the future pharmacological therapeutic interventions.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This retrospective analysis compared the efficiency of the gonadotropin- releasing hormone (GnRH) antagonist (GnRH-ant) protocol and the GnRH agonist long (GnRH-a) protocol for patients with diminished ovarian reserve (DOR). A total of 1,233 patients with DOR (anti-Mullerian hormone <1.1 ng/mL) were recruited for this retrospective case-control study. They were divided into two groups according to female age. Younger patients were assigned to POSEIDON group3 (PG3: age ≤35 years); older patients were assigned to POSEIDON group 4 (PG4: age >35 years). All patients with DOR underwent controlled ovarian stimulation and fresh embryo transfer (ET) on day 3. We recruited 283 GnRH-a and 54 GnRH-ant cycles for PG3, and 663 GnRH-a and 233 GnRH-ant cycles for PG4. In PG3, the GnRH-a protocol was associated with a lower ET cancellation rate  and a higher live birth rate  than the GnRH-ant protocol for the initiated cycles. Furthermore, the GnRH-a protocol was correlated with a higher implantation rate than the GnRH-ant protocol for ET cycles . No differences in the ET cancellation rate, live birth rate and implantation rate between GnRH-a and GnRH-ant groups were observed among PG4 patients. In conclusion, the GnRH-a protocol was more effective than the GnRH-ant protocol for young patients with DOR. The low ET cancellation rate and high implantation rate may be related to embryo quality or endometrial receptivity, which warrant further investigation. Clinical management of patients with poor ovarian responses in assisted reproduction technology (ART) cycles is challenging. The rates of clinical pregnancy and live birth are lower in poor ovarian responders than in age-matched normal responders –3. SelecThe gonadotropin-releasing hormone (GnRH) antagonist (GnRH-ant) protocol is associated with less gonadotropin and fewer injections than those in the GnRH agonist long (GnRH-a) protocol for normal responders –7. ThereAnti-Müllerian hormone (AMH) and antral follicle count (AFC) are the most reliable ovarian reserve markers for predicting the ovarian response in clinical settings –14. Low The definition of poor ovarian response by AMH or AFC is inconsistent in the literature. In 2011, to reach a consensus and to benefit future clinical trial and meta-analyses, Bologna criteria were proposed for poor ovarian responders . The BolThe POSEIDON group further stratified poor or suboptimal ovarian responders to more effectively guide their clinical management . One of This retrospective case-control study evaluated the efficiency of the GnRH-ant and GnRH-a protocols for patients with DOR in ART cycles. Medical records related to ART cycles were analyzed from the database of a previous prospective cohort study for the effect of genetic polymorphism in ART cycles. All the data were fully anonymized before we accessed them. Approval from the Institutional Ethics Review Board of Chung Shan Medical University Hospital was obtained for the prospective cohort study (CS-13194 and CS2-14033), which including ~95% of the ART cycles in Lee Women’s Hospital from 2013 to 2015. All the participates signed informed consent for the subsequent analysis of medical records for other studies. We have two physicians, one favors using GnRH-a long protocol for the first or second IVF and the other favors using GnRH-anta protocol for the first or second IVF cycles for most patients. Both physicians are well experienced and achieve similar live birth rates according to the records of our infertility center. The following procedure of oocyte retrieval and in vitro embryo culture is shared by embryologists with fixed rotating schedule in our hospital and thus the following treatment will not influence the comparison of these two protocols.The recruited patients were divided into three groups according to their age on the day of entry into COS cycles. ART cycles combined with fresh embryo transfer (ET) were included. For patients with diminished ovarian reserve, day 3 ET is our routine practice. If the patients requested blastocyst transfer, we would collect embryos from 2 or more oocyte retrieval. Then we would perform blastocyst transfer in frozen embryo transfer cycles. Such cycles are not included in the present study. Women with uterine factor infertility, severe endometriosis, cycles for gamete donation program, and cycles for preimplantation genetic testing for aneuploidy were excluded.The GnRH-ant (cetrorelix) protocol was performed as described previously . The decThe women participating in this study followed the GnRH-a protocol as described previously . In brieVarious biological parameters related to ART cycles were compared between the two treatment groups (GnRH-a vs. GnRH-ant) through Student’s t test. The rates of fertilization, ongoing pregnancy and live birth for the three groups were compared using the chi-squared test or Fisher’s exact test, as applicable. All analyses were performed using the Statistical Package for Social Sciences . P < .05 was considered to indicate statistical significance.We recruited 1,233 women with AMH <1.2 ng/mL; they underwent first or second ART cycles, 946 GnRH-a protocols and 287 GnRH-ant protocols, at Lee Women’s Hospital, Taichung, Taiwan. These cycles were divided into three groups by age . The youThe ovarian reserve markers  were similar between the patients in the GnRH-a and GnRH-ant protocols in each group . As expeWe further analyzed the clinical outcome of ART cycles for patients with DOR after ET . The dayWe explored the outcome of COS and ET for each initiated cycle . In addiThe present study demonstrated that the GnRH-a protocol was more effective than the GnRH-ant protocol for PG3 patients in terms of lower ET cancellation rates, higher implantation rates, and higher live birth rates. Nonetheless, the efficiency of the two protocols was similar with each other for PG4 patients or poor ovarian responders satisfying the Bologna criteria. The total gonadotropin amount was less when the GnRH-ant protocol was applied than when the GnRH-a protocol was applied for PG3 and PG4B patients.Clinicians initially recommend the patient-friendly GnRH-ant protocol in COS for patients with poor ovarian response  , 8. One The reason for a higher ET cancellation rate for the GnRH-ant protocol than that for the GnRH-a protocol for PG3 patients is unclear. A large retrospective analysis for young patients (< 35 years of age) with good prognosis also demonstrated a higher cancellation rate prior to ET for the GnRH-ant protocol than the GnRH-a protocol . In our Our results indicated low implantation rates for the GnRH-ant protocol for ET cycles in PG3, possibly because of differences in endometrial receptivity. Researchers have suggested that various ovarian stimulation protocols in IVF are linked to endometrial receptivity , 21. An By contrast, a study using microarray data revealed that the gene expression profiles of endometrial cells following GnRH-ant treatment are more similar to those in natural cycles compared with those after GnRH-a treatment . OverallEmbryo quality or euploid rates are another possible cause of impaired implantation rates in the GnRH-ant protocol. The good embryo rates in the present study did not significantly differ between the two protocols among PG3 patients. Although the oocyte maturation rates were lower in PG4B patients using the GnRH-ant protocol, no difference in the oocyte maturation rates was not observed in PG3. By contrast, a borderline higher abortion rate was observed among PG3 patients treated using the GnRH-ant regimen. The ET cancellation rates for those treated using the GnRH-ant protocol were similar in the three age groups; whereas rates for those treated using the GnRH-a protocol were correlated with age. Zygote score was reported to be associated with pregnancy outcome when the GnRH-ant protocol is applied but not when the GnRH-a protocol was applied . On the In conclusion, the advantage of the GnRH-a protocol in the present study was evident only among the PG3 patients; the protocol exhibited no advantages for PG4 patients or true Bologna poor ovarian responders. The GnRH-a protocol was associated with a low ET cancellation rate, high implantation rate and high live birth rate. Embryo euploid rate or endometrial receptivity might explain the difference in implantation rate between the GnRH-a and GnRH-ant protocols for young patients with DOR (PG3). Additional large scale randomized control trials are required to confirm the findings of this study.S1 Dataset(XLS)Click here for additional data file.


Multi-modal interventions are effective in increasing hand hygiene (HH) compliance among healthcare workers, but it is not known whether such interventions are cost-effective outside high-income countries.To evaluate the cost-effectiveness of multi-modal hospital interventions to improve HH compliance in a middle-income country.Staphylococcus aureus bloodstream infections (MRSA-BSIs) alone would make HH interventions cost-effective in intensive care units (ICUs). Transmission dynamic and decision analytic models were combined to determine the expected impact of HH interventions on MRSA-BSI incidence and evaluate their cost-effectiveness. A series of sensitivity analyses and hypothetical scenarios making different assumptions about transmissibility were explored to generalize the findings.Using a conservative approach, a model was developed to determine whether reductions in meticillin-resistant Interventions increasing HH compliance from a 10% baseline to ≥20% are likely to be cost-effective solely through reduced MRSA-BSI. Increasing compliance from 10% to 40% was estimated to cost US$2515 per 10,000 bed-days with 3.8 quality-adjusted life-years (QALYs) gained in a paediatric ICU (PICU) and US$1743 per 10,000 bed-days with 3.7 QALYs gained in an adult ICU. If baseline compliance is not >20%, the intervention is always cost-effective even with only a 10% compliance improvement.Effective multi-modal HH interventions are likely to be cost-effective due to preventing MRSA-BSI alone in ICU settings in middle-income countries where baseline compliance is typically low. Where compliance is higher, the cost-effectiveness of interventions to improve it further will depend on the impact on hospital-acquired infections other than MRSA-BSI. Hospital-acquired infections (HAIs) are a major cause of morbidity and mortality among hospitalized patients Direct patient contact with healthcare workers (HCWs) transiently contaminated with nosocomial pathogens is believed to be the primary route of transmission. Improving HCW hand hygiene compliance can minimize the impact of this transmission route and reduce the incidence of nosocomial infection Transmission dynamic models are useful tools to help understand the likely impact of interventions to control communicable diseases. Moreover, their use in health-economic evaluations of interventions that reduce transmission is essential to fully capture intervention benefits. However, whereas several studies have used dynamic models to consider hospital infections The aims of this study are to develop a dynamic model-based framework for evaluating the cost-effectiveness of hand hygiene promotion interventions and use it to evaluate the cost-effectiveness of such interventions in a middle-income country. Our analysis is informed by data from a typical regional hospital in Thailand, a middle-income country with a gross domestic product (GDP) per capita approximately equal to the world median. We focus on MRSA-BSI as this is one of the most serious and best-studied types of infection in ICU patients, there is clear evidence of frequent patient-to-patient transmission of MRSA, and evidence that such transmission can be interrupted by improved hand hygiene Transmission dynamic and decision analytic models were combined to simulate the transmission dynamics and evaluate the impact and cost-effectiveness of hand hygiene interventions. Two ICU settings were considered: a paediatric intensive care unit (PICU) and an adult intensive care unit (adult ICU). Epidemiological and economic parameters were derived from detailed local data from a typical tertiary hospital in North-east Thailand. Information about catchment area, staff:patient ratios, and further details about the ICUs have been described elsewhere A previously described deterministic host–vector model was constructed to simulate MRSA transmission dynamics in an ICU  [24]. PaModel parameters were obtained from various sources . MRSA caCost-utility analysis was performed from a healthcare provider's perspective. The cost of the hand hygiene intervention was estimated over a period of one year. Health benefits were measured with a lifetime horizon and a 3% discounting rate. Costs were adjusted to 2016 values with the exchange rate of 33 Thai Baht per US$1 There were two main cost components: cost of hand hygiene promotion; and costs associated with MRSA-BSI. The latter includes costs of additional hospital stay and antibiotic treatment . The cosCosts associated with MRSA-BSI were estimated from additional stay and treatment. Hospitalization cost was calculated as the excess length of stay due to MRSA-BSI multiplied by the cost per bed-day. Retrospective data from routine clinical and microbiological laboratory databases at the local hospital (2003–2010) were used to identify MRSA-BSI cases. Additional stay due to infection was estimated with a multi-state model accounting for time-dependent bias using the ‘etm’ package within R Treatment for MRSA-BSI was assumed to require a 14-day course of vancomycin with dose regimens following treatment guidelines for hospital-acquired MRSA-BSI. Drug costs were obtained from the Drug Medical Supply Information Center Estimates of life expectancy among post-ICU patients were taken from a previous study in North-east Thailand Four scenarios with different baseline versus post-intervention hand hygiene compliance values were considered: (a) 10% vs 20%; (b) 10% vs 40%; (c) 10% vs 60%; and (d) 40% vs 60%. These are consistent with results from a systematic review where odds ratios were estimated to be 6.5 and 11.8 for WHO-5 and WHO-5 plus other interventions among studies using an interrupted time-series design  In each comparison, point estimates of incremental costs (ΔC) and QALYs gained (ΔQ) due to the intervention and the incremental cost-effectiveness ratio  were calculated. The threshold willingness to pay per QALY gained (λ) was taken as GDP per capita (US$4848), and a threshold value of three times GDP per capita was considered in a scenario analysis RA), the expected number of MRSA cross-transmissions resulting from a single colonized patient during a single ward stay, assuming all other patients on the ward are susceptible, was varied between 0.5 and 5, while prevalence of MRSA colonization on ICU admission was varied between 1% and 15%. Changes in costs and health outcomes under different baseline compliance and improvement levels were calculated and combined to evaluate the cost-effectiveness of such interventions in terms of the IMNB. The maximum level of investment at which the intervention would still be cost-effective, the prevalence reduction, and final prevalence in all scenarios were also determined.A series of hypothetical scenarios with different assumptions about the transmissibility and prevalence of MRSA colonization at admission were considered. The ward reproduction number (Under base case assumptions (with a pre-intervention hand hygiene compliance of 10%), a multi-modal hand intervention (WHO-5) is highly likely to be cost-effective in both PICU and adult ICU settings if it increases hand hygiene compliance to ≥20% . ConversRisk of death due to MRSA-BSI in our study hospital was estimated to be between two- and three-fold higher than in high-income countries A = 0.5), where prevalence of MRSA colonization at admission is 5%, and where baseline compliance is ≤20% in both the PICU and the adult ICU, the intervention is always cost-effective even with only a 10% compliance improvement using the cost estimates in In hypothetical scenario analyses, the hand hygiene intervention was found to be cost-effective in most scenarios, especially when there was high transmissibility and a high prevalence of MRSA colonization on admission . In the Hand hygiene promotion using the WHO multi-modal campaign is likely to be highly cost-effective for ICU settings in Thailand where baseline compliance is low (≤20%) solely as a result of preventing MRSA-BSI. Factors that tended to make the intervention more cost-effective were low baseline compliance, high prevalence of colonization at admission, and high rates of transmission. With higher baseline compliance, the intervention may often still be highly cost-effective as a result of reduced MRSA-BSI alone if rates of MRSA carriage on ICU admission or ICU transmission are sufficiently high.Acinetobacter spp., where a segmented regression analysis found that a hand hygiene intervention was associated with a substantial change in incidence of infections with extensively drug-resistant Acinetobacter spp. in Taiwan Acinetobacter spp. has also been reported to be a frequent contaminant of the hands of HCWs in endemic settings in South-east Asia, strengthening the evidence for a causal link between increased hand hygiene and reduced infections Acinetobacter spp. are the largest single cause of hospital-acquired BSI in North-east Thailand, if infections with these organisms can be reduced substantially by improved hand hygiene, the implications for our analysis could be substantial Acinetobacter spp. and the effects of hand hygiene are needed to inform it.Because we ignored impacts of the intervention on other types of HAI (other MRSA infections and infections with other pathogens) our analysis is conservative and likely to underestimate health benefits. MRSA-BSIs represent only 5.1% of hospital-acquired BSIs in North-east Thailand, whereas Gram-negatives account for 67.6% et al. (in Switzerland), and Chen et al. (in Taiwan) used data from observational studies to estimate reductions in infections due to hand hygiene interventions. Pittet et al. concluded that if only 1% of the observed reduction was due to the intervention it would have been cost-saving. Chen et al. also concluded that their intervention was likely to be cost-saving. Huis et al. (in the Netherlands) used trial data to inform a cost-effectiveness analysis, assuming a linear relationship between hand hygiene compliance and reduced infections, concluding that the intervention was likely to be cost-effective if the willingness to pay for a 1% reduction in the HAI rate was about US$6000. A study in the UK also concluded that hand hygiene interventions were likely to be cost-saving even if the reduction in rates of HAI were as low as 0.1%. As in our study, this report explicitly calculated QALY gains. However, unlike the other studies, staff time was not accounted for when costing the intervention. A previous study in a middle-income country (Vietnam) concluded that a hospital-wide hand hygiene promotion was cost-saving To the best of our knowledge, our study is the first economic evaluation of a hand hygiene intervention to make use of a dynamic model in a developing country Direct comparison of these findings with ours is difficult for three reasons. First, only one of the previous studies quantified benefits in terms of final health outcomes (QALYs) Our study has some limitations, the most important of which is that data are not yet available that allow us to include other pathogens in the model. In addition, since local information on the resources used for the hand hygiene intervention is limited, we were dependent on estimates of resource used for hand hygiene interventions from studies outside Thailand. A further limitation is that we evaluated the intervention over only a one-year post-intervention period where the initial investment would be most. Additional costs to maintain the compliance in the later years are difficult to quantify but likely to be less costly than the first year.In conclusion, effective multi-modal hand hygiene interventions are likely to be cost-effective in ICU settings in typical middle-income countries due to reduced incidence of MRSA-BSI alone under a wide range of circumstances. When this is not so, the cost-effectiveness of interventions to further improve hand hygiene will depend on the impact on other infections and other pathogens. Further work is needed to quantify this.


As optoacoustic tomography (OT) emerges as a mainstream pre-clinical imaging modality, understanding the relationship between optoacoustic and other imaging biomarkers in the context of the underlying tissue biology becomes vitally important. Complementary insight into tumour vasculature and hypoxia can be gained using OT alongside magnetic resonance imaging (MRI)-based techniques. To evaluate the relationship between these metrics and the relative performance of the two modalities in assessment of tumour physiology, co-registration of their output imaging data is required. Unfortunately, this poses a significant challenge due to differences in animal positioning during imaging. Here, we present an integrated framework for registration of OT and MR image data in mice. Our framework combines a novel MR animal holder, to improve animal positioning during imaging, and a landmark-based software co-registration algorithm. We demonstrate that our protocol significantly improves registration of both body and tumour contours between these modalities, enabling more precise multi-modal tumour characterisation. Taking the example of cancer imaging, dynamic contrast enhanced (DCE) MRI 2 biomarkers have been shown to indicate tumour vascular maturity in vivo cancer imaging.MSOT-measured THb and SOThe correct combination of spatial information from different imaging modalities requires careful alignment of the images and hence an efficient co-registration algorithm. This is usually achieved in both patient and small-animal imaging by careful body positioning and scanning process optimisation, aided by software-based alignment. Well-established, clinically used solutions are available Here, we present a new integrated framework for registration of MSOT and MR image data in pre-clinical studies of mice, which can be applied to soft, deformable tissues such as tumours. The method combines a novel animal holder design and a robust co-registration algorithm. We first describe the method and show its performance for co-localization of the internal tumour structures between the modalities. We then demonstrate the improvement in co-registration achieved by the combination of hardware and software-based solutions, compared to the manual overlay of the tumour regions with standard animal holders used for MSOT and MRI. Finally, we demonstrate the application of the co-registration framework for comparison of perfusion-based data recorded using MSOT and MRI.22.16 LNCaP prostate adenocarcinoma cells, n = 3 mice; 1.5 × 106 PC3 prostate adenocarcinoma cells, n = 3 mice; 1 × 106 mouse K8484 pancreatic adenocarcinoma cells, n = 3 mice) in 100 μL phosphate buffered saline (PBS). Using three different cell lines allowed us to investigate the co-registration procedure across a range of morphological and functional characteristics.All animal procedures were conducted in accordance with project (70-8214) and personal license (IDCC385D3) issued under the United Kingdom Animals (Scientific Procedures) Act, 1986 and were approved locally under compliance form number CFSB0671. Subcutaneous tumours were established in male BALB/c nude mice  by inoculation of cells from one of three different cancer cell lines in both flanks  was used. Briefly, a tunable optical parametric oscillator (OPO) pumped by an Nd:YAG laser provides excitation pulses with a duration of 9 ns at wavelengths from 660 nm to 1200 nm at a repetition rate of 10 Hz with a wavelength tuning time of 10 ms and a peak pulse energy of 90 mJ at 720 nm. Ten arms of a fibre bundle provide uniform illumination of a ring-shaped light strip of approximately 8 mm width. For ultrasound detection, 256 toroidally focused ultrasound transducers with a centre frequency of 5 MHz (60% bandwidth) are organized in a concave array of 270 degree angular coverage and a radius of curvature of 4 cm.Mice were prepared according to our standard operating procedure 2.3A 9.4 T Agilent MRI system  running VnmrJ 3.1, using an Agilent quadrature transmit/receive millipede volume coil of 38 mm inner diameter was used. The same anaesthesia protocol as for optoacoustic imaging experiments was maintained. A physiological monitoring system was used for observing mouse status and for sequence triggering . The core temperature of the mouse was monitored using a rectal probe, and stabilized to 37 Dynamic contrast enhanced (DCE)-MRI data were acquired using a spoiled gradient echo sequence  were scanned in the custom silicone holder . Transfe2.5MI with the fixed (or reference) image FI. The transformation matrix The main objective of any general co-registration software is to merge the coordinate system of the moving image MI and the matching positions in the MSOT image as FI. These vectors were then used to calculate the transformation matrix and transform both modalities into the same coordinate space, minimising the euclidean distance between the landmarks. A landmark-based approach was chosen to maximise robustness. High structural homogeneity of xenograft tumours and their regular shape can limit the performance of other, internal information or shape based A landmark-based co-registration approach fitgeotrans.The registration procedure was implemented in two steps, each constituting a landmark-based co-registration process. The first step ensured proper alignment between the body contours in MSOT and MRI, which provides constrained initial conditions to the tumour alignment, while the second step provided further alignment of the tumours. Landmarks for the first step were the spine and characteristic anatomical features visible in MRI and MSOT, such as contact points between tumours and body . Second 2.6All image analysis was performed in MATLAB 2016a (Mathworks) using the Image Processing Toolbox, the Computer Vision Toolbox and custom scripts unless otherwise stated. All image data and custom analysis codes will be made openly available at doi: 10.17863/CAM.39741.pinv function in MATLAB 2016a) was applied to the measured optoacoustic spectrum in each pixel to calculate the relative oxy- [Hb] signal. The presented images illustrate the total haemoglobin signal [Image reconstruction was performed using an acoustic backprojection algorithm  with an electrical impulse response correction, to account for the frequency dependent sensitivity profile of the transducers. Images were reconstructed with a pixel size of 100 All MR images were flipped horizontally prior to image registration. The position of the slice analysed was chosen by the operator to best match the location of the imaging slice in OT, acquired directly before the MRI.The analysis of registration accuracy of body and tumour contours was performed by calculating the Dice Similarity Coefficient (DSC). This coefficient is defined as:The results were compared on a per-tumour basis. Differences in DSCs between conventional and novel holder geometries as well as before and after landmark-based registrations (for body and tumour) were statistically tested with two-tailed paired DCE-MRI signal area under the curve (AuC) 1 min after contrast administration was compared to the change in blood oxygen saturation  protocols, which have been previously shown to relate to tumour perfusion and vascular function 4Co-registration of images between modalities enables the combination of complementary information provided by different imaging methods. Due to deformation of the animal or patient between scans, correct alignment of images can pose a significant challenge and require both hardware and software-based optimisation approaches. In this work, we describe an integrated hardware and software framework for co-registration of mouse MSOT and MR imaging data. Without co-registration, these modalities produce very different images of the sample, due to different animal positioning and stress distribution.On the hardware side, a novel silicone MRI animal holder was developed, which was designed to mimic the external stresses acting on the mouse body in the MSOT. Introducing the new holder alone already significantly improved the similarity in the shape of the entire mouse body contour as well as the individual tumour contour, contributing to more accurate co-registration. Importantly, the use of the holder did not increase animal preparation time or cause any side effects for animal welfare during imaging. Fabrication of the holder is simple and inexpensive, as soft two-component silicone is poured over a 3D printed mouse mold. The protocol offers a simple solution to improve MSOT/MR image co-registration.A software tool for landmark-based image co-registration was then established to further improve the co-registration and enable per-pixel analysis of the combined multi-modal images. The transformation matrix for the MSOT images was calculated to maximise similarity between body and tumour outlines in both modalities as well as to minimise the distances between anatomical landmarks. The result of applying this software tool was a co-localisation error up to several 100 s of microns, comparable to the typical resolution of both modalities. The robustness of the landmark-based co-registration approach was verified by comparing the positioning of landmarks by several operators, yielding a close agreement. This framework also enabled per-pixel combination and comparison of the insight offered by MSOT and MRI in functional imaging. The relationship between tumour perfusion, provided by early DCE-MRI enhancement Despite the clear improvements in image co-registration achieved, there remain some limitations to our study. Firstly, the described two-step hardware and software framework is designed to aid with 2D co-registration, which assumes already the correct, manual choice of matching imaging slice between the modalities. The use of the silicone holder can help in this task, as the similar cross-sectional shape of the tumour in the MRI can help match it qualitatively to the geometry in the MSOT. Slice misalignment, although minimised with the use of the holder and an optimised slice registration protocol, can introduce additional error in the co-registration procedure.A second limitation arises in the design of the silicone bed, which aimed to mimic the effects of the polyethylene film holder used in the MSOT, as well as the stresses due to water submersion during MSOT imaging. In order to support the weight of the animal, the silicone had to be stiffer than optimal, causing some discrepancy in MRI/MSOT mouse positioning. Further optimisation using silicones of different elastic properties could better match the distribution of forces and should be investigated in future experiments.Finally, the silicone bed was created for a specific mouse size based on the typical usage in our experiments. If needed, additional silicone beds could be created to account for different mouse sizes, across strain and age for instance, and taking the individual tumour position into consideration. The optimal approach would utilize 3D modeling to create mouse-specific holders and require standardisation of the modelling, printing and casting workflow.4.1We have demonstrated the feasibility of a hardware- and software-based image registration framework for MRI and MSOT images. We use a novel silicone MRI holder, as well as a software tool to perform landmark-based co-registration of the images. Both steps led to a significant improvement in the registration of the tumour outlines and internal structure between the modalities. This simple, inexpensive approach can be readily implemented for multi-modal MSOT/MRI studies of mice, which will help to provide valuable insight into relative performance of these two modalities in revealing vascular architecture and function in cancer.