Currently, there are many studies researched the associations between maternal serum inflammatory indicators  and preterm birth (PTB). The results, however, are inconsistent. Therefore, the aim of this study was to estimate the relationship between maternal serum inflammatory indicators and PTB in a nested case-control (NCC)study.0/7 and 346/7 weeks of gestational age (wGA) from six cities in China between 2015 and 2016. There were 206women who delivered preterm (< 370/7 wGA), and 412 women who delivered term birth, those women were matched by maternal age, birth place, gender of infants, and wGA at blood collection.A NCC study was conducted by Guangxi Birth Cohort Study which enrolled a total of 6203 pregnant women between 5The inflammatory indicators were quantified by immunoturbidimetric methods.p = 0.044) in the first trimester. The association of C3 was extremely related to those who delivered PTB  in the first trimester. Moreover, no significant associations were found in C4 (p = 0.079) and ferritin (p = 0.067) between PTB.Highest quartile concentrations of all inflammatory indicators were determined versus median. After adjusting for maternal age, high levels of CRP (CRP > 16.60 mg/L) are related to the risk of PTB (OR = 2.16, 95% CI: 1.02–4.56, Elevated concentrations of CRP and C3 in the first trimester were associated with increased risk of PTB. Inflammatory indicators may act a pivotal part in early diagnosis and prognosis of PTB. Preterm birth (PTB), an abnormal pregnancy status, defined as delivery before 37 weeks of gestation . It is aSubclinical infections and chronic inflammation may be the main factors of PTB during pregnancy , 4. In aTo better understand the changes in maternal autoimmunity during pregnancy and find appropriate markers in early pregnancy to prevent the occurrence of preterm birth, we measured the concentrations of CRP, C3, C4 and ferritin before 32 wGA, and estimated the relationship between maternal serum inflammatory indicators and PTB in a nested case-control study in Southwest Chinese women.The NCC study was established in 2015 which based on Guangxi Birth Cohort Study. Briefly, participants were enrolled from eight Maternal & Child Health Hospitals in six cities in Guangxi, China. The participants attended obstetric screening during gestation between July and September 2015. Participants from these Maternal & Child Health Hospitals provided blood samples before 32 weeks or less at a time point to test for serum biomarkers. All participants provided written informed consent and the study was approved by the Medical Ethics and Human Subject Committee of First Affiliated Hospital of Guangxi Medical University (ID: 2015(028)).2).The cohort study was conducted among 6203 participants. All participants were followed up until the end of pregnancy. As a result, 5541 participants had pregnancy outcomes and 662 participants lost follow-up. All participants completed a large-scale integrated census through face-to-face interviews and professional maternity checkup by a professional gynecologist. The characteristics of maternal height, pre-pregnancy weight and date of last menstrual period were obtained by self-report. Maternal body mass index (BMI) was calculated. by the pre-pregnancy body weight (kg) divided by the square of height , pregnant women who had medically premature delivery, stillbirth or induced labor, deformity, macrosomia, full term low birth weight neonatal and multiple pregnancy Fig. .Fig. 1FEligibility criteria included women who met all of the criteria listed above and delivered live singleton PTB (< 37 wGA) were selected as PTB. Pregnant women who gave singleton neonate at term (≥ 37 weeks and ≤ 42 weeks). The women were 18–45 years.According to the above criteria, 206 women were selected in the PTB group. These women were matched by maternal age, birth place, gender of infants, and wGA at blood collection in 1:2 ratios and women who delivered term birth infants were selected as control. Finally, 412women were selected as control.About 5 ml overnight fasting venous blood was drawn from the pregnant women when they had a pregnancy test in the morning, and the blood was collected before 32 wGA. Then the blood was centrifuged and stored at − 80 °C until it was sent to the First Affiliated Hospital of Guangxi Medical University for analysis 2 years later. It was the same in the preterm and term group. All samples were analyzed by the same method to ensure reliable results.CRP level (mg/L) was measured on the HITACHI 7600 biochemistry analyzer  by using the immunoturbidimetric methods with CRP assay kits , which intra- and inter-batch CV were 2.37 and 6.02%, respectively. Both C3 and C4 (g/L) levels were measured by immunoturbidimetric technique using the HITACHI 7600 biochemistry analyzer  with an intra-assay CV of less than 6% and inter-assay relative range of less than 8%, which use the Reagent Kits for C3 and C4 Test . Serum ferritin (ng/mL) level was measured with an automatic electrochemical luminescence immunoassay on COBAS 6000 system E601(Elecsys module) analyzer  with an inter-assay coefficients of variation (CV) of 4.5%. The managing obstetricians were blinded to the results of the serum CRP/C3/C4/ferritin concentrations.Laboratory records and analysis of maternal serum CRP, C3, C4 and ferritin biomarkers were obtained from the Laboratory of the First Affiliated Hospital of Guangxi Medical University. The subjects’ data on perinatal and birth outcomes were collected from electronic medical records of 8 Maternal & Child Health Hospitals. The perinatal data included maternal features, pregnancy, parturition and neonate characteristics at birth. All birth reports were diagnosed by professional obstetricians and entered into the maternal and child health care system. All pregnant women’s labor data were exported from this system.All analyses were performed by IBM SPSS 19.0. The continuous variables were performed by Student’s test, and categorical variables were examined by chi-square statistics between two groups.p value < 0.05 was considered statistical significance.We evaluated the associations between maternal serum levels of CRP, C3, C4, ferritin and the risk of PTB by using conditional logistic regression. Concentrations of serum inflammatory indicators were examined continuously and as quartiles. Effect estimates were presented as odds ratios (ORs) with 95% confidence intervals (95%CIs). Initial unadjusted analyses were conducted and subsequently adjusted with covariates showing association with both infant PTB and maternal pathology. Potential confounding factor for inclusion in the final model was maternal age. Statistical test were two-tailed, and Figure p > 0.05). In addition, compared with the TB group, the PTB group had significantly higher levels CRP (p = 0.029). Moreover, no significant difference in ferritin (p = 0.161), C3 (p = 0.255) and C4 (p = 0.451) were found between the two groups (Table The baseline characteristics of the pregnant women at follow-up were shown in Table P p = 0.09. Moreovp < 0.01). The blood biochemical parameters for two groups in the different trimesters were shown in Fig. p < 0.05). By contrast, no significant differences were found in serum C4 and ferritin between PTB and term groups . In PTB group, the inflammation indicators were not significantly different between the second and the third trimester.The result indicated that, with pregnancy progressed, the levels of ferritin dropped while CRP and C3 levels increased among the TB group Fig. . The conp = 0.048). Furthermore, after adjusted age, the OR of PTB in the highest CRP quartile was 2.16 . For C3, the unadjusted OR was 2.28 , while the adjusted OR was 2.53 .The relationship between maternal ferritin, CRP, C3 and C4 levels and the risk of PTB in the first trimester are shown in Table In our study, the levels of inflammation biomarkers, namely, CRP, C3, C4 and ferritin in the peripheral blood serum were investigated during pregnancy. Elevations in the average levels of CRP and C3 in the first trimester were associated with increasing occurrence of PTB.CRP, an acute phase reactant in the innate immune response, is a nonspecific biomarker of inflammation that is usually used as a marker for diagnosis of many inflammatory, infective, and malignant conditions . ElevateIn this study, we found that highly expressed CRP and PTB were remarkably association in the first trimester but no relationship was found in the second trimester Table  and the The pro-inflammatory function of CRP includes the induction of cytokines and tissue factor in the monocytes . The monThe complement, a central part of non-specific and specific immune system, has three renowned physiologic activities, including host defence against infection, bridging interface between non-specific and specific immunity, and phagocytosis cleaning action , 20. AboIn early pregnancy, the elevated serum levels of C3 in pregnant women were positively correlated with PTB. This observation is interesting because infection and inflammation are strongly causal risk factors of PTB . We examAlthough the complement system was activated and involved in inflammation during pregnancy, no significant association was found between C4 and PTB. In addition, the trends of C4 concentrations varied in our study varied, probably because our study was focused on asymptomatic pregnant women, whereas previous studies focused on pregnant women with preeclampsia or systemic lupus erythematosus . In addiFerritin is an acute phase reactant, and elevated level of this molecule has been associated with acute/chronic infection, inflammation, neurodevelopmental disability and malignant diseases . PreviouTo sum up the appeal, in Fig. In a systematic review, similar results were found in populations in the United States, Iran, and Denmark . SpecifiWe chose the primary outcome of pregnancy-associated PTB with evidence of maternal factors, rather than the diagnosis of PTB, to assess whether therapy would prevent serious complications rather than merely modify diagnostic findings.Our study had several strengths. The NCC study design provided an opportunity to include all PTB infants in the study. Importantly, the NCC study design can be clearly illustrated the causal link between serum inflammatory factors and prematurity during the first trimester of pregnancy. To our knowledge, following a MEDLINE search, the current study is the first study to investigate whether inflammatory indicators are associated with Southwest Chinese normal pregnant women. In addition, all participants also adjusted for other potential risk factors, such as maternal age and pre-pregnancy BMI. It is known that the concentration of CRP in the peripheral circulation is associated with high BMI and other obesity markers , 33. ThuThe current study also has some limitations. Firstly, maternal serum samples were collected at one time only and might not accurately reflect the change of CRP and C3 during the whole pregnancy in our study. Secondly, the study was limited by the wide range of wGA of blood drawn (between 6 and 32 wGA). A narrower range of wGA within the data points will help to assess the association of complement markers with more predictive biomarkers.Further predictive analysis requires a large-scale, multicenter, prospective cohort study to investigate the predictive value of these inflammatory markers in maternal blood for preterm delivery in asymptomatic pregnant women and to develop a valuable multivariate predictive model.In summary, a NCC study of 206 cases and 412 controls subjects was performed higher levels of CRP and C3 early in pregnancy were associated with an increased risk of PTB in the first trimester. Our results indicated that a close relationship between early pregnancy inflammatory indicators and PTB in Southwest Chinese women. Further studies are needed to characterize the relationship between inflammatory indicators and long-term outcome using well-defined diagnostic PTB. Additional evaluations are warranted to increase the sample size to target the relationship between high levels of inflammatory indicators and long-term outcome.How to manage these patients to prevent PTB, however, presents several great challenges. Our research merely found that elevated serum CRP and C3 levels increase the risk of preterm birth, and is not a diagnosis in itself. There are many pathways leading to preterm birth and the prevention of each requires different types of scientific inquiry and clinical strategies, which together encompass a wide array of measurement systems and clinical interventions across many health-care disciplines .We need Quantification of the reference range is difficult to predict which will depend on other clinical, cultural, social and economic factors that operate in each environment. Further success is expected in the coming years as other research findings translate into clinical practice, including new ways to treat intrauterine infections, improved maternal nutrition and lifestyle improvements to reduce maternal stress.


Particle radiotherapy has increasingly gained acceptance for locally advanced pancreatic cancers owing to superior tumor conformity and dosimetry compared to conventional photon radiotherapy. However, the close proximity of the pancreas to the stomach and duodenum leads to radiation-induced gastrointestinal toxicities, which hinder the delivery of curative doses to the tumor. To overcome this problem, a surgical spacer was placed between the tumor and gastrointestinal tract, and subsequent proton radiotherapy was performed in this study.Data from 9 patients who underwent surgical spacer placement and subsequent proton radiotherapy were analyzed. The safety and feasibility of the spacer placement surgery were evaluated; the impact of the spacer on dosimetry was also assessed using dose volume histogram (DVH) analyses, before and after surgical spacer placement.Surgical spacer placement and subsequent proton radiotherapy were successfully completed in all cases. Surgical spacer placement significantly improved the dose intensity covering 95%, mean, and minimum doses for the gross tumor volume, and the clinical and planning target volume based on the DVH, while respecting the dose constraints of the gastrointestinal tract. Based on the Common Terminology Criteria for Adverse Events, two patients (22.2%) developed gastrointestinal ulcer (Grade 2) at 1 and 35 months, and one patient (11.1%) developed gastric perforation (Grade 4) at 4 months after proton radiotherapy.Surgical spacer placement in the locally advanced pancreatic body and tail cancers is relatively safe and technically feasible. Comparing radiation plans, surgical spacer placement seems to improve the dose distribution in the locally advanced pancreatic body and tail cancers, which are close to the gastrointestinal tract. Pancreatic cancer is now the fourth leading cause of cancer-related deaths in the United States and is expected to become the second before 2030 . AlthougIn recent years, particle radiotherapy, including proton and carbon-ion radiotherapy, has become increasingly popular worldwide owing to its physical properties, which provide superior dosimetry and conformity to the target. This allows selective tumor irradiation, while minimizing doses to the gastrointestinal tract –11. The The present study evaluated the technical aspects of surgical spacer placement and assessed the dosimetric changes of proton beams before and after surgical spacer placement in patients with locally advanced pancreatic body and tail cancer. In addition, this study investigated the safety, efficacy, and long-term outcomes of surgical spacer placement and subsequent proton radiotherapy in these advanced pancreatic cancers.The medical records of 9 patients who underwent proton radiotherapy at the Hyogo Ion Beam Medical Center after surgical spacer placement at the Kobe University Hospital for locally advanced pancreatic body and tail cancers, between October 2007 and January 2018, were analyzed. In these patients, total doses were restricted by the close proximity of the tumor to the gastrointestinal tract, which necessitated the placement of surgical spacers prior to proton radiotherapy. The general condition of each patient before both surgical spacer placement and proton radiotherapy was evaluated using the Eastern Cooperative Oncologic Group-Performance Status scores; tumor stage was defined according to the Union for International Cancer Control tumor-node-metastasis classification, 8th edition ), D95% (the dose intensity covering 95% of the target volume), Dmean (average dose of the target volume), and Dmin (minimum dose of the target volume) of the GTV, CTV, and PTV; the D0.5 cc (maximum dose received by the highest irradiated 0.5 cc of the organ volume), V48Gy (RBE), V40Gy (RBE), and V30Gy (RBE)  of the stomach and intestinal tract were also assessed. Among these factors, the D0.5 cc and V48Gy (RBE) of the stomach and intestinal tract, and the V95% and V60Gy (RBE) of the GTV and CTV were indicative of adequate dose delivery to the target while respecting the dose constraints of the gastrointestinal tract after surgical spacer placement.After proton radiotherapy, all patients were examined every 3 months with repeated measurements of cancer tumor markers and contrast-enhanced CT and/or FDG-PET scans. As shown in previous reports , 24, locp < 0.05 was considered statistically significant in all the analyses. All statistical analyses were performed using the JMP statistical software package, version 13 .The MST and OS rates were calculated from the date of diagnosis and surgical spacer placement to the date of the event or last follow-up using Kaplan–Meier methods. Freedom from progression and local control rates were calculated from the date of surgical spacer placement to the date of the event or last follow-up using Kaplan–Meier methods. Differences in the survival rates between the groups were compared using the log-rank test. The unpaired Student’s t-test was used to compare the parameters of the dose-volume histograms. A value of The baseline patient and tumor characteristics are shown in Table p = 0.023).Changes in tumor volume and dose intensity based on DVH before and after surgical spacer placement are shown in Table p = 0.0012, p = 0.004, p = 0.005, respectively), V60Gy (RBE) , D95%  and Dmean  while respecting the dose constraints of the gastrointestinal tract. In addition, the D0.5 cc and V48Gy (RBE) of the gastrointestinal tract tended to be reduced.In the GTV, CTV, and PTV, surgical spacer placement significantly improved V95% (The median period of follow-up from spacer surgery was 11 range: 7–47) months; 8 of 9 patients died during this period. The OS rates are shown in Fig. 7 months;In patients with locally advanced pancreatic body and tail cancer, surgical spacer placement was technically feasible and was safely performed; subsequent proton radiotherapy was completed in all cases. The median duration of surgery and intraoperative blood loss was 191 min (median: 122–218 min) and 89 ml (range: 0–388 ml), respectively. Complications related to surgical spacer placement and proton radiotherapy in this group, according to the Common Terminology Criteria for Adverse Events version 4.0, are shown in Table A total of 7 (77.8%) of 9 patients had complications. In the early period, 2 (22.2%), 1 (11.1%), 4 (44.4%), 3 (33.3%), 3 (33.3%), 1 (11.1%), and 1 (11.1%) patients had grade 3–4 leukopenia, grade 1–2 thrombocytopenia, grade 1–2 dermatitis, grade 1–2 abdominal pain (owing to placement of the spacer), grade 1–2 nausea, grade 1–2 anorexia, and grade 3–4 anorexia, respectively. No patients developed gastrointestinal ulcers with hemorrhage.In the late period, 2 (22.2%) patients developed gastrointestinal ulcers at 1 and 35 months after proton radiotherapy. These patients developed grade 1–2 ulcers in the horizontal part of the duodenum, which were healed with conservative management. However, 1 (11.1%) patient developed lower gastric perforation of an unknown etiology 4 months after proton radiotherapy. This patient required removal of the surgical spacer and total gastrectomy to manage the perforation. During the follow-up period, none of the patients with surgical spacers had infections due to long-term retention of a foreign material.A multidisciplinary approach using surgical spacer placement and radiotherapy has been previously employed with conventional photon radiotherapy for other malignancies, such as rectal cancer , 26. HowIn this study, surgical spacer placement was technically feasible and was safely performed; subsequent proton radiotherapy was completed in all cases. Surgical spacer placement significantly improved the dose intensity for the GTV, CTV and PTV based on the DVH, while respecting the dose constraints of the gastrointestinal tract. Although the changes in the volume of CTV and PTV were not examined , the volume of CTV and PTV after spacer placement may have increased because of the absence of proximity to the gastrointestinal tract. There is a limitation to the interpretation of the result that the V95%, V60Gy, and Dmean of CTV and PTV have improved because they are affected by the volume changes. However, the improvement of all parameters of GTV, CTV, and PTV while respecting the dose constraints of the gastrointestinal tract may indicate the effectiveness of the spacer placement.As for radiation-induced gastrointestinal toxicities, considering the beam settings which were planned to deliver maximal doses to be the GTV, CTV, and PTV while keeping the maximum dose to the highest irradiated 0.5 cc of organ volumes in the gastrointestinal tract limited to 48 Gy (RBE), the rate of radiation induced gastrointestinal ulcers is acceptable compared to previous reports , 15, andAs for prognosis, OS, freedom from progression and local control time after spacer surgery in the present study was almost equal to that of previous studies, which included patients with locally advanced unresectable pancreatic cancer treated with curative dose of particle radiotherapy , 13. It While good local control, there were also cases with poor prognosis due to potential micrometastasis. Actually, 3 of our 9 patients died due to peritoneal dissemination within 1 year of treatment despite good local control in this study. Cautious patient selection will be the next agenda for our treatment strategy. As in the NCCN guidelines, now that powerful regimen such as oxaliplatin, irinotecan, fluorouracil, and leucovorin (FOLFIRINOX) or GNP have emerged , 29, 30,Surgical spacer placement and subsequent proton radiotherapy were associated with several problems. The first problem was tumor progression during the interval up to initiation of proton radiotherapy. For our treatment strategy, a median interval of 33 days elapsed between spacer placement and first irradiation. Shortening of this period is an issue; the period from spacer placement to first irradiation is approximately 10 days at present. The surgical limitations of spacer placement posed the second problem. In tumors invading the Treitz ligament, spacer placement is technically difficult due to insufficient space for the spacer material. The motion and range uncertainties during treatment was the third problem. Regarding the motion uncertainties associated with the Gore-Tex sheet, it is impossible to evaluate motion uncertainties during treatment without taking 4DCT. However, Gore-Tex sheets were placed between the pancreas and gastrointestinal tract and sutured tightly to the anterior surface of the pancreas and surrounding tissues. No case required a change in treatment protocol due to the positional disconnection of the spacer maybe due to the tight fixation and postoperative adhesions. Regarding the range uncertainties associated with the Gore-Tex sheet, it is difficult to experimentally determine the stopping depth inside the spacer of the proton beam in the patient’s body, thus, we could not evaluate it. Regarding the Gore-Tex sheet, we adopted conversion from the CT value to the stopping power ratio because the composition of the Gore-Tex sheet is almost the same as that of the human body  . TherefoAlthough there are some problems to be solved, surgical spacer placement and subsequent proton radiotherapy were both technically feasible, and were safely completed in patients with locally advanced pancreatic body and tail cancer. In terms of DVH analysis, the efficacy of irradiation was significantly improved. In view of the better target coverage and improved local control offered by surgical spacer placement and subsequent proton radiotherapy, this may be a promising treatment option for locally advanced pancreatic body and tail cancer. In the future, careful patient selection and effective multimodal therapy may lead to improve prognosis in these patients.Comparing radiation plans, surgical spacer placement seems to improve dose distribution in locally advanced pancreatic body and tail cancer, which are in close proximity to the gastrointestinal tract. Further studies are necessary to assess the prognostic impact of surgical spacer placement and subsequent proton radiotherapy.


P = 6.79 × 10−23, OR = 1.23; Ncases = 4714, Ncontrols = 1,011,520). The variant also confers risk of intervertebral disc disorders  suggesting a common pathogenesis in part or a true pleiotropy.Bell’s palsy is the most common cause of unilateral facial paralysis and is defined as an idiopathic and acute inability to control movements of the facial muscles on the affected side. While the pathogenesis remains unknown, previous studies have implicated post-viral inflammation and resulting compression of the facial nerve. Reported heritability estimates of 4–14% suggest a genetic component in the etiology and an autosomal dominant inheritance has been proposed. Here, we report findings from a meta-analysis of genome-wide association studies uncovering the first unequivocal association with Bell’s palsy (rs9357446-A;  Other symptoms vary from person to person and may include mild facial pain, numbness or stiffness and headache. The annual incidence is about 20–30 cases per 100,000, with about 1 in 60 lifetime risk. The disorder usually occurs in individuals aged 15–40 and affects men and women equally2. Risk factors include hypertension3, diabetes4, immunosuppression5, Lyme disease6, and in rare cases inflammatory demyelinating neuropathy such as Guillain-Barré syndrome7.Bell’s palsy, also known as idiopathic facial paralysis, is an acute or subacute weakness of the facial muscles innervated by the seventh cranial nerve . Symptoms are usually unilateral although both sides can be affected. Although symptoms usually subside within one month, up to 30% of cases show delayed or incomplete recovery with mild to severe residual facial weakness, contracture, and/or hemifacial spasm6. The literature has highlighted several viral illnesses such as herpes simplex type 1, which has been detected in 31–79% of cases9, human immunodeficiency viruses10, varicella-zoster, and Epstein-Barr11. After a primary infection, these latent viruses reside in the geniculate ganglion or in lymphocytes. At times with e.g. stress, the viruses may be reactivated and cause local damage to the myelin8. The two most common treatments are corticosteroids and antivirals. However, studies have found that antivirals alone are not helpful and antiviral treatment in combination with corticosteroids have conflicting results12. Although the heritability has been estimated to be only 4–14%13, numerous familial cases have been reported.While the exact etiology of Bell’s palsy is unknown, it has been suggested that the disorder is caused by inflammation or compression of the seventh cranial nerveHere, we report the first meta-analysis of genome-wide association studies (GWASs) of Bell’s palsy, combining data from Iceland (deCODE genetics), the UK (UK Biobank), Denmark , and Finland (FinnGen), which uncovers an association with a common intergenic variant.Ncases = 290, Ncontrols = 342,122), the UK , Denmark , and Finland . Bell’s palsy cases were assigned ICD-10 (G51.0) diagnoses by a physician in all study cohorts , that confers risk of Bell’s palsy   Table . The fulP-het ≤ 0.05) describing a modest difference in the four datasets. Therefore, we applied a random-effects model which assumes that there may be different underlying true effects estimated in each dataset. The variant maintains a weighted Bonferroni significance (P = 6.83 × 10–23) based on predicted functional impact of the association signal.There is a small evidence of heterogeneity  and of those, eleven are highly correlated  with any known coding or structural variants.Based on the Icelandic data, twenty-two non-coding variants in the region correlate with rs9357446 r > 0.2 ancis-eQTL in blood and adipose tissue in Iceland and did not find an association between rs9357446 and expression of nearby genes (Supplementary Note). The variant did not affect expression in any nearby genes in 18 other databases listed in Supplementary Table N = 1236, P = 0.024). However, this CpG is generally methylated and a slight increase in methylation is not likely to have any biological effect.We examined 15. The Bell’s palsy meta-analysis associates with seven lifestyle traits  and poor health .We performed genetic correlation analyses using the Bell’s palsy meta-analysis and 600 GWASs from the UK Biobank16. This finding replicates in the Icelandic dataset . We did not find association with other lung related phenotypes including asthma, chronic obstructive pulmonary disease, or tuberculosis combining the Icelandic, UK and Danish data in meta-analyses and we did not find associations with other reported risk factors such as hypertension and type 1 diabetes . rs9357446-A is among the top variants at the locus for IDD P = 6.79 × 10–23, OR = 1.23), in a meta-analysis of four study cohorts.The precise pathophysiology of Bell’s palsy is unknown and biologically targeted treatment is lacking. The aim of the study was to search for variations in the human genome affecting risk of Bell’s palsy and thus use genetics to uncover biological underpinnings of this somewhat mysterious disease. Here, we report the first unequivocal association between Bell’s palsy and a sequence variant, rs9357446-A , several sequence variants in the closest gene, cell division cycle 5 like (CDC5L), have been found to associate with related phenotypes, including osteoarthritis17, bone density18, ossification of the spine19, and lung function measured by FVC and forced expiratory volume16. Another potential candidate at the locus, SLC35B2, is a key component of a protein sulfation pathway where it plays a part in modifying C–C Motif Chemokine Receptor 5 (CCR5), that is one of HIV’s host proteins for entry into the cell20. Several other studies point to the importance of CCR5 in determining disease severity of other viral infections in animal models22. In addition, SLC35B2 is involved in proteoglycan synthesis. Cartilage is particularly rich in proteoglycans, and changes in the structure and composition of glycosaminoglycans, forming the sugar chain attached to the protein core of proteoglycans, have been found in osteoarthritis. Mutations in slc35b2 in zebrafish have been shown to cause deformation of the craniofacial skeleton23.While rs9357446-A confers risk of Bell’s palsy, it also confers low risk of IDD as well as reduced lung function measured by decreased FVC24. In Bell’s palsy, a viral infection is considered one of the possible causes8. The main treatment is corticosteroids, which supports the theory of edema induced entrapment neuropathy. The facial nerve traverses through the temporal bone  and branches to innervate the facial muscles. The narrowest part of the canal, the labyrinthine segment, is where most cases of compression occur, which may result in ischemic neuropathy of the facial nerve25.Bell’s palsy is still idiopathic since we do not know what causes it; there are no biomarkers to support the diagnosis and the diagnosis requires exclusion of any other cause of facial paralysisWe were unable to determine a gene at 6p21.1 involved in Bell’s palsy pathology. However, the association of rs9357446-A with IDD brings up the possibility that the variant may confer risk of Bell’s palsy through an effect on cartilage and bone development. Alternatively, the association between Bell’s palsy, IDD, and reduced lung function may be a result of a true pleiotropy. Although more studies are needed, the identification of this variant contributes to the limited understanding of Bell’s palsy.All Icelandic data were collected through studies approved by the National Bioethics Committee  following review by the Icelandic Data Protection Authority. Participants donated blood or buccal samples after signing a broad informed consent allowing the use of their samples and data in all projects at deCODE genetics approved by the NBC. All personal identifiers of the participants' data were encrypted by a third-party system, approved and monitored by the Icelandic Data Protection Authority.The UK Biobank data were obtained under application number 24898. All phenotype and genotype data were collected following an informed consent obtained from all participants. The North West Research Ethics Committee reviewed and approved UK Biobank’s scientific protocol and operational procedures (REC Reference Number: 06/MRE08/65).The Danish Data Protection Agency (2007-58-0015) and the National Committee on Health Research Ethics (NVK-1700407) approved the genetic study on Danish Blood Donor Study (DBDS). The data requested for this study was approved by the DBDS steering committee. Samples from the Copenhagen Hospital Biobank (CHB) were included as part of the study on pain related diseases under the genetics of pain and degenerative musculoskeletal disease protocol (NVK-1803012).Samples and phenotype data were collected from the Finnish biobanks and the national health registers, respectively, for the FinnGen database. The Coordinating Ethics Committee of the Helsinki and Uusimaa Hospital District evaluated and approved the FinnGen research project. The project complies with existing legislation . The official data controller of the study is University of Helsinki.To identify Bell’s palsy cases in Iceland, we searched for patients over the age of 18 with International Classification of Diseases (ICD-10) diagnosis code G51.0 and ICD-9 code 351.0 at the National University Hospital of Iceland and all Icelandic primary and secondary care centers. Records spanned from 1983 to 2019.The UK Biobank study is a large prospective cohort study of ~ 500,000 individuals in the age range of 40–69 from across the UK. Extensive phenotype and genotype data have been collected for participants, including ICD diagnosis codes. Bell’s palsy diagnoses in the UK Biobank were obtained by searching for cases with ICD-10 code G51.0 from UK Biobank General Practice clinical event records  and hospital diagnoses . Only individuals of Caucasian ancestry were included.26. Genetic ancestry quality control was performed in two stages. Firstly, ADMIXTURE v1.2327 was run in supervised mode with 1000G populations CEU, CHB, and YRI28 as training samples and Danish individuals as test samples. Input data for ADMIXTURE had long-range LD regions removed29 and was then LD-pruned with PLINK v.190b3a30 –indep-pairwise 200 25 0.3. Danish samples with less than 0.9 assigned CEU ancestry were excluded. In the second stage, remaining Danish samples were projected onto a principal component analysis (PCA), calculated with in-house tools on an in-house European reference panel. The UMAP package in R31 was used to reduce the coordinates of test samples on 20 principal components to two dimensions. Additional European samples not in the original reference, were also projected onto the PCA and UMAP to help identify the ancestries represented in clusters. A polygon informed by visual inspection was drawn to include all samples with very similar ancestries to the main Danish cluster.The CHB is a research bank, which contains left over samples from diagnostic procedures on in- and outpatients in hospitals in the Danish Capital Region. The DBDS GWAS study is a nationwide prospective cohort study of ~ 100,000 blood donors. We applied for ICD-10 code G51.0 from CHB and DBDShttp://r3.finngen.fi).The phenotype data from the FinnGen study was produced from several national health registries. All Bell’s palsy cases were diagnosed by a physician and categorized using ICD-10 code G51.0, ICD-9 code 351.0, and ICD-8 code 350. The summary statistics for available phenotypes, including Bell’s palsy, were imported on June 16th 2020 from a source available to consortium partners  and 100,000 controls (DBDS) from Denmark, and 1017 cases and 121,360 controls from Finland; in total, 4714 cases and 1,011,520 controls.33. In short, 37.6 million high-quality sequence variants were identified by sequencing 49,962 Icelanders using GAIIx, HiSeq, HiSeqX, and NovaSeq Illumina technology to a mean depth of at least 17.8 × . SNPs and indels were identified and their genotypes called using joint calling with Graphtyper34. Additionally, over 165,000 Icelanders  have been genotyped using various Illumina SNP chips and phased using long-range phasing35, which allows for improving genotype calls using the information about haplotype sharing. The genotypes of the high-quality sequence variants were imputed into the chip-typed Icelanders36. To increase the sample size and power to detect associations, the sequence variants were also imputed into relatives of the chip-typed using genealogic information. All the tested variants had imputation information over 0.8.The preparation of samples and the whole-genome sequencing of 49,962 Icelanders has been described in detail elsewhere37, and the Affymetrix UK Biobank Axiom array38 in the remaining participants. Imputation was performed by the Wellcome Trust Centre for Human Genetics using a combination of the Haplotype Reference Consortium39, UK10K haplotype resources40, and 1000Genomes phase 3 panels28. A total of 96 million variants have been imputed.The UK Biobank samples were genotyped with a custom-made Affymetrix chip, UK BiLEVE Axiom in the first 50,000 individuals41. Samples and variants with less than 98% yield were excluded. We used the same methods as used for the Icelandic data35 to create a haplotype reference panel by phasing the whole-genome sequence genotypes (N = 8635) using the phased chip data , and to impute the genotypes from the haplotype reference panel into the phased chip data. Whole genome sequencing, chip-typing, quality control, and the subsequent imputation from which the data for this analysis were generated was performed at deCODE genetics.Over 332,000 samples from the CHB and DBDS together with ~ 238,000 genotyped samples from Northwestern Europe were long-range phased using Eagle2A custom-made FinnGen ThermoFisher Axiom array  was used to genotype ~ 135,600 FinnGen samples at Thermo Fisher genotyping service facility in San Diego. Imputation was performed using the Finnish population specific WGS backbone. More than 16 million variants have been imputed.32. We used LD score regression to account for distribution inflation due to cryptic relatedness and population stratification in the Icelandic, UK, and Danish data42. In the Icelandic association analysis, we adjusted for sex, county of origin, current age or age at death (first and second order term included), blood sample availability for the individual, and an indicator function for the overlap of the lifetime of the individual with the time span of phenotype collection. In the UK association analysis, we adjusted for sex, age, and the first 40 principal components to adjust for population stratification. In the Danish association analysis, we adjusted for sex, whether the individual had been chip-typed and/or sequenced, and the first 20 principal components. The FinnGen association analysis was adjusted for sex, age, the genotyping batch, and the first 10 principal components.We performed logistic regression using the Icelandic, UK, and Danish data and combined the results with imported association results from Finland to test for association between sequence variants and Bell’s palsy using software developed at deCODE genetics43 to combine the results from the four study groups in which the groups were assumed to have a common OR but allowed to have different population frequencies for alleles and genotypes. Variants with imputation information below 0.8 were excluded from the analysis. Sequence variants were mapped to NCBI Build38 and matched on position and alleles to harmonize the four datasets. We estimated the genome-wide significance threshold and corrected for multiple testing with a Bonferroni procedure weighted for variant classes and predicted functional impact44. A likelihood ratio test was performed for the genome-wide significant variant to test the heterogeneity of the effect estimate in the four datasets. The null hypothesis is that the effects are the same in all datasets and the alternative hypothesis is that the effects differ between datasets.For the meta-analysis, we used a fixed-effects inverse variance methodConditional analysis was performed to identify the most likely causal variant at the locus and to identify any secondary signals. We used the true genotypes of participants in the study from Iceland, the UK, and Denmark. LD between variants was estimated using a set of 8700 WGS Icelanders and was restricted to variants within one Mb from the index variant.P ≤ 0.05/600 = 8.33 × 10–5) from the UK Biobank15 with effective sample size over 5000 were performed using LDSC method45. The LDSC method suggests minimal effective sample size of 5000 for a GWAS trait to get unbiased estimates of genetic correlation and heritability. Since the published GWAS studies used in the analysis have Caucasian ancestry, we used pre-computed LD scores from a 1000 genome panel with r2 from HapMap3 excluding HLA region. The HLA region was excluded for its complex genetic pattern and associations with wide number of traits. Moreover, the default parameters of LDSC method were used to compute genetic correlation and heritability estimates.Genetic correlation analyses between the Bell’s palsy meta-analysis and 600 published GWAS traits .48. MinION and PromethION devices with FLO-106/R9.4 flow cells were used for base calling. Methylation on CpGs was called using Nanopolish (version 0.6.1)49.We performed whole-methylome-sequencing on 4133 samples using ONT Ligation Sequencing Kit (SQK-LSK108) or Rapid Sequencing Kit (SQK-RAD003)Supplementary data.Supplementary information.


The clinical effectiveness of neurally adjusted ventilatory assist (NAVA) has yet to be demonstrated, and preliminary studies are required. The study aim was to assess the feasibility of a randomized controlled trial (RCT) of NAVA versus pressure support ventilation (PSV) in critically ill adults at risk of prolonged mechanical ventilation (MV).n = 78) in four ICUs of one university-affiliated hospital. The primary outcome was mode adherence (percentage of time adherent to assigned mode), and protocol compliance (binary—≥ 65% mode adherence). Secondary exploratory outcomes included ventilator-free days (VFDs), sedation, and mortality.An open-label, parallel, feasibility RCT (p = 0.04) and fewer in-hospital deaths  for NAVA. Although overall sedation was similar, Richmond Agitation and Sedation Scale (RASS) scores were closer to zero in NAVA compared to PSV (p = 0.020). No significant differences were observed in duration of MV, ICU or hospital stay, or ICU, D28, and D90 mortality.In the 72 participants who commenced weaning, median (95% CI) mode adherence was 83.1% (64.0–97.1%) and 100% (100–100%), and protocol compliance was 66.7% (50.3–80.0%) and 100% (89.0–100.0%) in the NAVA and PSV groups respectively. Secondary outcomes indicated more VFDs to D28 (median difference 3.0 days, 95% CI 0.0–11.0; This feasibility trial demonstrated good adherence to assigned ventilation mode and the ability to meet a priori protocol compliance criteria. Exploratory outcomes suggest some clinical benefit for NAVA compared to PSV. Clinical effectiveness trials of NAVA are potentially feasible and warranted.ClinicalTrials.gov, NCT01826890. Registered 9 April 2013. Neurally adjusted ventilatory assist (NAVA) uses electromyographic signals from the diaphragm (Edi) detected via a specialized naso-gastric feeding catheter , as a measure of neural respiratory drive and as a means of controlling the delivery of inspiratory support by a mechanical ventilator . Over thAlthough there is a demand for adequately powered trials of NAVA , uncertaThe primary aim of this study was, therefore, to assess the feasibility of a trial protocol comparing NAVA to PSV, via an assessment of mode adherence. Our primary feasibility objectives were to assess mode adherence (proportion of time adherent to assigned mode) and protocol compliance (binary—≥ 65% mode adherence) during the entire study period. In contrast to previous trials, recruitment was commenced soon after intubation (ability to trigger the ventilator was not a requirement for inclusion), and the intervention was continued to the end of MV support or D28. In addition, reasons for mode cross-over and poor adherence were recorded. Some study results have been previously reported in the form of abstracts , 11.An open-label, parallel-group, randomized feasibility trial was undertaken in four ICUs  comprising 75 beds at a university-affiliated hospital in London, UK. The trial was prospectively registered (NCT01826890) and was approved by the London Westminster ethics committee (13/LO/0012).The inclusion criteria were as follows: adults receiving invasive MV with at least one of the following risk factors for prolonged MV : 1) COP COP1). TResearch staff obtained informed consent from the participant’s legally authorized representative or proxy, and participant consent was sought once capacity was regained as required by the UK law .Using a 1:1 allocation ratio, treatment order was randomized with permuted blocks of random sizes using online randomization software, managed by an independent clinical trial unit . The stuIn the NAVA group, NAVA catheters were inserted within 4 h of randomization. Clinicians were instructed to record the maximum Edi hourly, to use the NAVA mode in place of PSV, and to target an Edi of ≥ 8 μV by adjustment of sedation dose and/or adjustment of MV settings where appropriate. A lower target level of 8 μV was set following basic analysis of existing literature and local audit data to avoid over-ventilation and over-sedation. NAVA parameters were set by clinicians according to a pragmatic protocol  modes and CPAP were permitted in both groups, but no other exclusively spontaneously triggered modes were allowed. A priori, we considered a participant to be protocol compliant if mode adherence was ≥ 65% (binary) and the trial to be feasible if ≥ 65% of participants were compliant. Secondary feasibility outcomes were the proportion of participants with mode cross-over, duration of cross-over, reasons for cross-over, protocol acceptability , and recruitment rates. Secondary exploratory outcomes included ventilator-free days (VFDs) to D28 and D90 and duration of MV from randomization; ICU, hospital, D28, and D90 mortality; ICU and hospital length-of-stay from randomization; mean RASS; and sedation dose and bolus dose frequency per infusion day. Safety outcomes included ventilator-associated pneumonia (VAP), pneumothoraxes, and incidence of unplanned extubation  using the Hodges-Lehmann estimating method [P value of < 0.05 was considered statistically significant. Analysis of clinical outcomes was exploratory and uncorrected for multiple comparisons; therefore, the statistically significant results could be due to chance. Qualitative descriptions of cross-over were categorized using content analysis [Using 65% as the lower bound of the confidence interval (CI)  and an expected attrition of 5% (i.e. no NAVA or PSV weaning), a sample size of 76 patients (38 in each arm) would estimate a protocol compliance of 75% to within a 95% CI of ± 10% . Descripg method  for contg method . Effect g method , 19. Timanalysis . StatistOver a 45-month enrolment period, 774 invasively ventilated patients with COPD, HF, or ARDS were identified, 112 eligible patients were approached, and 78 participants (39 NAVA and 39 PSV) were recruited, a consent rate of 72% (3 patients consented outside the recruitment window and were subsequently excluded)  Fig. .Table 2The main reasons for crossover in the NAVA arm were Edi signal noise or interference causing loss of synchrony 8/28, 22.2%), clinical team preference for use of PSV during deterioration or instability , lack of trial awareness , and clinical inexperience leading to delayed application of NAVA . The three cross-overs in the PSV arm were due to perceived ventilator dyssynchrony , 15.5 VFDs in NAVA (0.0 to 23.0) compared to 0 VFDs (0.0 to 20.5) in PSV (Table p = 0.23). The overall median (IQR) duration of MV was 4.9 days (2.8 to 15.7) (NAVA) versus 9.8 days (3.6 to 25.9) (PSV) . Time to breathing without ventilator assistance and time to alive ICU discharge were shorter in NAVA . No significant difference was observed in ICU mortality , 28-day mortality , and D90 mortality  (Table Median (IQR) VFDs to D28 were greater in NAVA with a median difference of 3.0 VFDs , indicating less sedation while ventilated using NAVA  cross-over from NAVA was observed. Of the reported reasons for cross-over, human factors such as trial awareness, clinician preference, and lack of NAVA experience were most common, implying low confidence in the application of NAVA and need for further training. These issues may have been expected in a large group of clinical staff (over 300) despite attempts to embed NAVA technology into unit practice. This finding also highlights the complexity of NAVA and the likely variation in clinical application caused by ICU contexts and human interactions.While the influence of human factors described above may be modified with increased/improved training and changes to study documents, of more concern for trial feasibility is the difficulty in acquiring and maintaining a satisfactory Edi signal, which occurred in 10 out of 36 (27.8%) NAVA participants. Edi signal problems have been reported previously. Di Mussi et al. reported NAVA mode failure in seven out of 20 patients (35%) due to loss of ‘Edi synchrony’ or low Edi activity, despite having obtained a reliable Edi signal at baseline . DemouleIn contrast to previous studies, the exploratory analysis of ventilation and clinical outcomes found potential benefit in the NAVA arm with increased VFDs to D28, decreased time to breathing without assistance and to alive ICU discharge, and reduced hospital mortality. Acknowledging the low power of these analyses, certain characteristics of our study that are different to previous studies may be relevant to the interpretation of these results. We specifically selected patients with risk factors for prolonged MV  and condp = 0.020), potentially indicating improved sedation management. Decreases in sedation may be driven by the need to optimise the Edi signal and ensure optimal levels of respiratory drive are present prior to the initiation of NAVA [In addition to benefits in ventilation and clinical outcomes and despite no difference in sedation dose and RASS during in all MV modes, RASS scores were closer to zero in the NAVA mode compared to the PSV mode ( of NAVA , 29. Imp of NAVA  and impr of NAVA –33 have  of NAVA  in patie of NAVA  found noA key strength of this study is the description of mode adherence and reasons for poor adherence over prolonged durations. The use of a pragmatic protocol ensures that these adherence data are relevant to the real-world application of NAVA. The study successfully recruited patients at risk of prolonged MV, and treatment separation was improved due to the application of Edi monitoring in the NAVA arm only. While early recruitment during acute critical illness allowed assessment of Edi in CMV modes and NAVA from the very start of spontaneous breathing, it also led to the inclusion of some patients who did not commence weaning and potentially increased the rate of short-term NAVA failure during efforts to commence weaning.Despite conducting the trial in four distinct units, the interpretation of the results and the degree to which the results are generalizable is limited due to its relatively small size and conduct at a single institution. As in the Demoule trial, the study site is an academic centre with relatively high levels of MV experience, which may limit the generalisability of results to less experienced centres. NAVA was introduced 5 years prior to the start of the trial in 2008; approximately 50% of staff were trained and 70% had clinical experience of NAVA. NAVA training and experience was similar across the four ICUs.An analysis of ventilator synchronisation was beyond the scope of this study, and the absence of these data may further limit the interpretation of the exploratory outcomes. Improved synchrony in NAVA compared to PSV has, however, long been established , 35–41, A further potential source of error in the determination of NAVA compliance is the rate of automatic switching to PSV as a NAVA back-up mode, which was not possible to capture during the current trial. Although previous reports suggest levels of automatic switching to PSV of only 0.5 to 2% of total time in NAVA , 33, it As discussed above, NAVA is a complex intervention and its application will vary with human interactions and ICU context. Therefore, to enable interpretation of a multi-site trial a process, a process evaluation will be essential to determine whether the intervention was delivered as intended and to provide descriptive information relating to the research context .This study is the first to our knowledge to report NAVA mode adherence rates beyond 48 h and reasons for cross-over, methodological evidence which is key to the success of future clinical trials. Together with the clinical benefit suggested in secondary outcomes, this study suggests that it is both feasible and justified to conduct a definitive randomized controlled trial to establish the effectiveness of NAVA in patients with a high likelihood of prolonged MV.Additional file 1. Additional methods information.Additional file 2. Supplemental data tables.


REE was lower after simulated early morning shiftwork; however, TEF after breakfast was similar to habitual wake time. Insufficient sleep and caloric intake during a circadian phase of high melatonin levels may contribute to metabolic dysregulation in early morning shift workers.Increased risk of obesity and diabetes in shift workers may be related to food intake at adverse circadian times. Early morning shiftwork represents the largest proportion of shift workers in the United States, yet little is known about the impact of food intake in the early morning on metabolism. Eighteen participants  completed a counterbalanced 16 day design with two conditions separated by ~1 week: 8 h sleep opportunity at habitual time and simulated early morning shiftwork with 6.5 h sleep opportunity starting ~1 h earlier than habitual time. After wake time, resting energy expenditure (REE) was measured and blood was sampled for melatonin and fasting glucose and insulin. Following breakfast, post-prandial blood samples were collected every 40 min for 2 h and the thermic effect of food (TEF) was assessed for 3.25 h. Total sleep time was decreased by ~85 min ( Metabolic diseases, including obesity and type 2 diabetes, continue to increase in prevalence . InsuffiShift workers are chronically exposed to altered behavioral schedules of sleep and wakefulness that result in insufficient sleep and increased food intake during the biological night, as defined by high endogenous melatonin levels. Circadian misalignment, defined as wake–sleep and feeding–fasting behaviors occurring at inappropriate circadian times, has been shown to impair glucose tolerance ,7 and deThe current research literature is comprised predominately of overnight shiftwork studies, yet individuals who begin work in the early morning hours  make upThe purpose of this study was to examine the impact of food intake during a simulated early morning shiftwork protocol on sleep, endogenous melatonin levels, and metabolic outcomes. Specifically, it was hypothesized that simulated early morning shiftwork, including food intake during the biological night, would negatively impact metabolic outcomes as compared to waking and food intake at habitual timing.2) were enrolled into the research protocol. Participants reported being free from any current medical or psychiatric diagnosis, medications, drugs, and were non-smokers. Participants were healthy as assessed by physical, psychological, and sleep disorder screenings and physical exam, blood chemistries, 12 lead clinical electrocardiogram, and urine toxicology. Volunteers had not participated in shiftwork within six months or traveled across more than one time zone within three weeks prior to study. The investigations were carried out following the rules of the Declaration of Helsinki of 1975 , revised in 2013 and approved by the University of Colorado Boulder Institutional Review Board, . Participants gave written informed consent and were compensated for their participation.Twenty-two non-obese, healthy adults  at the medical screening visit and an activity factor that reflected the habitual low level of physical activity (1.5). Timing of sleep and study procedures, including food intake, were scheduled relative to each participant’s habitual sleep timing to maintain relative consistency with individual circadian timing of sleep. Participants were tested under two counterbalanced study conditions in a crossover design : a habit2 in the angle of gaze) for accurate assessment of melatonin levels under controlled conditions. Blood was drawn from an indwelling venous catheter placed after scheduled wake time for melatonin, fasted glucose, and insulin levels. Baseline REE was measured using standard indirect calorimetry with the ventilated hood technique . Respiratory gas exchange was measured for up to 30 min, depending on testing opportunity and values from the first stable 10−20 min were used to determine REE. After the baseline metabolic test, participants were served an identical (within-participant) breakfast 45 min after scheduled wake time in each condition which consisted of 25% of individual daily caloric needs. Participants were given 15 minutes to consume the meal. Blood was then sampled every 40 min for the next 2 h and metabolic testing was conducted every 45 min, beginning 15 min after breakfast, for 3.25 h to measure the thermic effect of food (TEF).After scheduled wake time in both conditions, study procedures were identical with participants remaining seated in a semi-reclined (~35 °) position in dim light  and glucose was assayed using hexokinase, UV . η2G) were calculated using sum of squares from the mixed-effects ANOVA model accounting for variance due to individual differences by including subject as a random factor [η2) were used, even though effects for η2G will be smaller than for η2.Data from one participant were not included due to blood sampling difficulties and three participants completed only one visit. Thus, 18 participants  contributed to the final analysis. Mixed model ANOVA  was used to examine changes in melatonin, insulin, glucose, and metabolic testing outcomes  with condition and sample time as fixed factors and participant as a random factor. Sex and order were initially included in models to test impact on variables of interest and none were significant, thus were removed for final analyses. Insulin and glucose were also analyzed as the homeostatic model assessment of insulin resistance (HOMA-IR) and TEF data were as incremental area under the curve (iAUC) analysis. Planned comparisons using dependent t-tests were used to examine differences between conditions at individual time points. Data are presented as mean ± standard error of the mean (SEM). Effect sizes for condition and condition x time, generalized eta squared , the average age was 23.2 ± 0.9 years, BMI was 23.7 ± 0.6, and body fat percentage was 28.6 ± 0.2%. Upon awakening during both conditions, participants had an average REE of 1.3 ± 0.1 kcal/min, fasting glucose of 85.7 ± 1.2 mg/dL, and fasting insulin of 10.1 ± 0.5 IU/mL.p < 0.0001, medium effect size η2G = 0.33; condition x time interaction, p < 0.0001, small effect size η2G = 0.11; Melatonin levels after waking were higher during early morning shiftwork compared to the habitual sleep condition . This reduction in total sleep time was a result of decreased time spent in Non-Rapid Eye Movement (NREM) Stage 2 and Rapid Eye Movement (REM) sleep . Additionally, participants slept ~85 min less in the simulated early morning shiftwork compared to the habitual sleep condition .Fasting insulin and glucose levels were similar between conditions. Post-prandial glucose levels were significantly elevated after food intake during the simulated early morning shiftwork compared to the habitual sleep condition  for EE, the effect size was less than a small effect size (η2G = 0.01) and none of the planned comparisons for REE or TEF between conditions at individual time points were significant before or after food intake, respectively .Although there was a significant condition x time interaction (Obesity and diabetes are associated with negative health outcomes and are increasingly prevalent among the general population. Shift workers have an increased risk for developing these metabolic diseases, yet the mechanisms underlying this increased risk are unclear. Findings from the current study show that early morning caloric intake after one night of insufficient sleep, when melatonin levels are high, leads to a small increase in glucose levels when compared to the same meal after sleeping and awaking at habitual times. Additionally, EE was similar between conditions. The simulated early morning shiftwork protocol used in the current study was based on observational data from real-world early morning shift workers and, thus, allowed for examination of potential metabolic consequences associated with sleep–wake behaviors that commonly occur in this population. In the early morning shiftwork condition, participants slept ~85 min less and had higher melatonin levels for 2 h after wake time compared to the habitual sleep condition. This is consistent with other studies showing decreased sleep duration in early morning shiftwork , and wasNightshift workers have been shown to have increased risk of cardiovascular and metabolic diseases ,25,26,27In addition to glucose tolerance, the current study sought to determine the impact of one day of simulated early morning shiftwork on components of energy metabolism. Energy expenditure is an important component in weight maintenance, including 24 h energy expenditure, REE, and the TEF. In the current study, REE was lower after waking in the early morning shiftwork condition, then increased after breakfast to become similar to that of the habitual sleep condition. The finding of lower REE is similar to previous work performed by our laboratory. In that study, participants were studied in a simulated overnight shift work schedule in a whole-room indirect calorimeter. Findings demonstrated that total daily EE was decreased on the simulated nightshifts compared to a dayshift, primarily due to lower EE during daytime sleep following simulated night shifts . REE is Another component of total daily EE, the TEF, has previously been shown to vary based on time of day ,36. SpecWe used mixed meal testing to examine the metabolic impacts of simulated early morning shiftwork with induced sleep and circadian misalignment on metabolic and energy expenditure outcomes. Findings from this initial study provide insight into the metabolic challenges that the largest population of shift workers face in real-world settings. However, it is important to consider the limitations and potential confounders of our protocol. First, young and healthy participants were studied and, thus, the generalizability of the findings to shift-working populations who, as mentioned previously, have additional disease burden is not represented in our population. Additionally, we cannot exclude a potential influence of sex differences on these outcomes given that our study was not designed with a sample size to test sex differences. Furthermore, we did not control for menstrual cycle across visits in the females tested. This may be of importance, as Qian and colleagues have recently demonstrated that there may be an impact of sex differences on metabolic outcomes in circadian misalignment . FurtherIn summary, findings from this simulated early morning shiftwork protocol demonstrate possible implications for the metabolic health of early morning shift workers, particularly if these changes were to persist and worsen over time. Moreover, these findings indicate the need for future analysis exploring whether delaying breakfast time after an early morning awakening may be a potential therapeutic target for improving metabolic health in the largest population of shift workers, and perhaps in modern society for people with high morning melatonin levels for hours after awakening ,23.


BRCA1 pathogenic mutation has been well proven to confer an increased risk of breast and ovarian cancer. Despite BRCA1 biallelic pathogenic mutations being extremely rare, they have been reported to be embryonically lethal or to cause Fanconi anemia (FA). Here we describe a patient who was a 48-year-old female identified with biallelic pathogenic mutations of the BRCA1 gene, with no or very subtle FA-features. She was diagnosed with ovarian cancer and breast cancer at the ages of 43 and 44 and had a strong family history of breast and gynecological cancers.The germline carrier of the  BRCA1 or BRCA2 genes. At least one functional allele of BRCA1 is essential for human embryogenesis and development. Biallelic mutations in BRCA1 were considered to be lethal during embryonic development [BRCA1 homozygous knock-out mice models [BRCA1 pathogenic variant have a significant life time risk of breast and ovarian cancers as high as 84% [BRCA mutation carriers [BRCA1 mutations have also been linked to increase risk in cervical, esophagus, liver, stomach, and uterine cancers; however, the increased risks ranged from one to four fold and the exact risk was inconsistent [Hereditary breast and ovarian cancer syndrome is an inherited cancer-predisposition syndrome and is predominantly caused by mutations in the e models ,4,5. BRCe models . Women ch as 84% . Men witcarriers ,10. BRCAnsistent ,12,13,14BRCA1 biallelic pathogenic mutations are extremely rare, which are regarded as embryonically lethal [BRCA1 is considered as FANCS (Fanconi anemia gene), homozygous mutations of the BRCA1 gene contributed to FA group S [BRCA1 mutations.y lethal . FA occuy lethal . It has y lethal . FA is ay lethal . The liky lethal . Mutatio group S . Hence, BRCA1 would have showed an early manifestation of prominent FA features, such as anemia, infection tendency and cancer development [BRCA1 mutations with no or very subtle FA-features.Clinically, we would expect that surviving patients who are compound heterozygous for deleterious mutations in elopment ,22,23. HAt the age of 43, the patient was first diagnosed with poorly differentiated high-grade serous adenocarcinoma of the ovary (stage IIIA), presented with abdominal distension for 1 month and was managed by optimal debulking surgery followed by post-operation paclitaxel and carboplatin (TC) regimen. She had right breast cancer with surgery 7 months later, showing pT1cN3M0 triple-negative invasive ductal carcinoma. She underwent lumpectomy which was followed by simple mastectomy with axillary lymph node dissection. She received adjuvant chemotherapy with 4 cycles of doxorubicin and cyclophosphamide, followed by local-regional radiotherapy up to 66 Gy and docetaxel. The patient developed extensive right chest wall recurrence one year after the mastectomy. She had mixed, but generally poor responses to chemotherapy regimes that escalated from the first line capecitabine. Her condition deteriorated in six months from the time of first recurrence, and progressed to extensive lymphadenopathy, pleural effusion, lung metastasis, liver and urinary bladder metastasis. She was also accidentally found to have an atrophic kidney and renal stone. Second line chemotherapy with 6 cycles of carboplatin (AUC4) and gemcitabine was given, restaging PET with extensive soft tissue metastasis and local recurrence was suggested. Third line chemotherapy with eribulin showed a partial response and eventually changed to fourth line chemotherapy, consisting of weekly doses of vinorelbine and carboplatin (AUC2). She subsequently had a trial of pembrolizumab and paclitaxel, though unfortunately she developed obstructive jaundice and clinically sepsis succumbed at four years after the initial ovarian surgery. No definitive diagnostic test for FA was performed on her due to a lack of clinical indication of FA-like features being observed. Her height was within the average Asian female range  with a BRCA1, BRCA2, TP53 and PTEN. Splicing variant analysis at the transcript level was performed by classic Sanger sequencing on the cDNA. Pathogenic mutations were further reconfirmed by Sanger sequencing. Compound heterozygotes BRCA1 mutations were identified in this proband. One of the mutations was a deleterious deletion in exon 11 , while the other was a splice site mutation in intron 22 . To further confirm the pathogenicity of c.5406+7A>G, we tested the cDNA from the blood of this patient, and confirmed a deletion of 74 nucleotides in her RNA. This deletion resulted in a frameshift termination after an amino acid substitution D1778G   four-gene panel that covered lyfs*27) A,B. ThesBRCA1: c.5406+7A>G; r.5333_5406del74; p.Asp1778Glyfs*27. The unaffected cancer-free daughter aged 19 of the proband carried another mutation, BRCA1: c.4065_4068delTCAA; p.Asn1355Lysfs*10. These findings showed that the two mutations are in trans and the proband has biallelic mutations in BRCA1 . The interpretation of the other mutation is more complex, it is a splice site variant in intron 22, c.5406+7A>G; r.5333_5406del74; p.Asp1778Glyfs*27. The ClinVar database of the National Library of Medicine, USA, shows that this variant as “likely benign” (referenced on 4th Dec 2020), which is supported by four submissions based on germline studies. The associated comments suggest that this variant is a “conservative change”, as the variant occurs at a poorly conserved protein position. The variant has been predicted to be benign by multiple in silico algorithms ). The population frequency quoted in the ClinVar database was “not consistent with disease”. However, these submissions were from privately owned sequencing companies. Unfortunately, no specific details of the clinical evidencFe for these interpretations. In addition, this variant had not been reported in the Genome Aggregation Database  in different populations. No splicing defect was observed in the functional studies [et al., this variant was also reported in Chinese breast and ovarian cancer patients and their protein truncation test has shown an early stop codon after splicing [BRCA1 mutation. In our own Hereditary Breast Cancer Family Registry, we have additional evidence on the disease association of this intron 22 mutation. Two other unrelated families also carried this mutation (unpublished data). One of the families had a female proband with breast cancer diagnosed at the age of 44, suffering from pT1N0M0 ER/PR positive invasive ductal carcinoma, with a significant family history of breast cancers, ovarian cancers, nasopharyngeal cancers and lymphoma in her first and second-degree relatives (Among the two mutations identified in the proband, the deletion in exon 11 mutation,  studies . This vasplicing . By inteelatives A. Anotheelatives B. TherefBRCA1 pathogenic variant. As there are definitive health risks for carriers and their family members, there is a need for better community education on the hereditary cancer risk of BRCA1/2, including for males. Studies showed that males had a low awareness of the BRCA mutation and were less willing to undergo genetic testing until their family members were identified to have a mutation [BRCA1 mutations, their offspring should have a 75% of chance carrying at least one copy of the pathogenic mutation; hence, the clinical burden of developing cancer is predictable. It is still challenging to convey the concept of cancer risk and the prevention strategies of hereditary diseases to the general public. A better community program to educate the community for both males and females is necessary. Usually patients carry biallelic deleterious mutations in cancer-causing genes are expected to exhibit prominent phenotypes and present with malignant diseases at a young age. The ages of onset of malignant diseases in this proband and her sister might have provided proof of this theory. Both proband and her sister had breast and ovarian cancers; however, the proband with two mutations had the conditions occur 10 years earlier than her sister, who carried only one mutation. Unfortunately, there was no genetic information about the four brothers of the proband. We therefore expected that they are likely to be carriers of mutation ,31. Malemutation . As exemBRCA1 mutations exacerbated cancer disease phenotypes. If both parents are carriers of pathogenic BRCA1 mutations, then their offspring should have a 75% chance of carrying at least one copy of the pathogenic mutation. Identification of families that show biallelic inheritance of BRCA1 mutations is important for precise genetic counseling and implementation of disease surveillance or prevention strategies.Compound heterozygosity for deleterious


This could mainly be attributed to an inadequate vegetable intake (OR 0.76 (95% CI 0.59 to 0.96), p = 0.02). In men, smoking was negatively associated with the KIDscore (OR 0.53 (95% CI 0.33 to 0.85), p < 0.01). We conclude that inadequate periconceptional maternal vegetable intake and paternal smoking significantly reduce the implantation potential of embryos after ICSI treatment. Identifying modifiable lifestyle risk factors can contribute to directed, personalized, and individual recommendations that can potentially increase the chance of a healthy pregnancy.Inadequate nutrition and lifestyle behaviors, particularly during the periconception period, are associated with a negative impact on embryonic and subsequent fetal development. We investigated the associations between parental nutritional and lifestyle factors and pre-implantation embryo development. A total of 113 women and 41 partners, with a corresponding 490 embryos, who underwent intracytoplasmic sperm injection (ICSI) treatment subscribed to the mHealth coaching platform “Smarter Pregnancy.” At baseline, nutrition and lifestyle behaviors  were identified and risk scores were calculated. A lower risk score represents healthier behavior. As outcome measure, a time-lapse morphokinetic selection algorithm (KIDScore) was used to rank pre-implantation embryo quality on a scale from 1 (poor) to 5 (good) after being cultured in the Embryoscope™ time-lapse incubator until embryonic day 3. To study the association between the nutritional and lifestyle risk scores and the KIDScore in men and women, we used a proportional odds model. In women, the dietary risk score (DRS), a combination of the risk score of fruits, vegetables, and folic acid, was negatively associated with the KIDScore (OR 0.86 (95% CI 0.76 to 0.98), The online version of this article (10.1007/s43032-020-00220-8) contains supplementary material, which is available to authorized users. Subfertility is still an increasing problem in the Western world, which can be attributed to postponing pregnancy, but also to a decline in sperm count, increasing age of women at the time of conception, obesity, smoking, and other poor lifestyles –3. DespiPre-implantation embryo development can be studied using the EmbryoScope™, which incorporates a specialized built-in microscope designed for automated time-lapse embryo assessment by acquiring images . The EmbMost reproductive challenges, such as fertility problems, miscarriages, congenital malformations, and fetal growth restriction, largely originate in the periconceptional period, which ranges from at least 14 weeks before conception until 10 weeks after conception . InadequNutrition and lifestyle behaviors can be easily assessed using the online mHealth program Smarter Pregnancy, which effectively improves the intake of fruits, vegetables, and folic acid supplements and stop smoking and use of alcoholic drinks . The effIn a prospective cohort study, couples that underwent ICSI treatment were included when embryos were cultured in the EmbryoScope™ time-lapse incubator and baseline data on the five nutrition and lifestyle behaviors of the mHealth program Smarter Pregnancy were available. Couples were included between October 2014 until December 2017 at the Erasmus MC, University Medical Center, Rotterdam, the Netherlands .Patients had to be at least 18 years of age and had to have a good understanding of Dutch speaking and writing. After the introduction of the EmbryoScope™ time-lapse incubator in our clinic, it was mostly used for cycles from patients undergoing ICSI treatment, as in this situation, oocytes can be submitted to time-lapse culture directly after injection and resulting embryos can benefit optimally from undisturbed culture. Only in the more recent years, we also cultured some embryos from IVF treatment cycles in the EmbryoScope™. In this case, fertilized oocytes are submitted to time-lapse culture the day after insemination, after pronuclear inspection. In these cases, the time point of fertilization is less accurate than after ICSI and pronuclear appearance cannot be assessed. We therefore excluded these cycles for the current analysis. Furthermore, we excluded patients with no available data of the Smarter Pregnancy program.This study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving patients were approved by the Medical Ethical Institutional Review Board of the Erasmus, University Medical Center, Rotterdam, the Netherlands. Written informed consent was obtained from all female and male participants at enrolment.Ovarian stimulation, oocyte retrieval, the ICSI procedures, and assessment of embryo morphology were performed as described previously . InseminEmbryo images were automatically recorded in seven focal planes  every 10 min until embryo day 3. Manual annotations were performed by specifically trained members of our team according to the definitions and guidelines by Ciray and colleagues . Time ofTo assess embryo quality and the implantation potential of a pre-implantation embryo, the KIDScore was used . The KIDhttps://www.slimmerzwanger.nl; https://www.smarterpregnancy.co.uk) is a (cost) effective tool to improve nutrition and lifestyle [The mHealth program Smarter Pregnancy , as well as information about diagnosis of subfertility and oocyte retrieval date.As outcome variable, we used the KIDScore as described above. In our hospital, the KIDScore is not used as a decision tool to select embryos for either transfer or cryopreservation. This decision is made by the embryologist based on a single morphological assessment at 66–68 h post fertilization. Only embryos with normal fertilization, as evidenced by the appearance of two pronuclei, that were subsequently transferred or cryopreserved were respectively annotated for research purposes.2) and normal weight (BMI < 25 kg/m2). The effect estimates of the models were transformed into odds ratios using the exponential function on the effect estimate. This odds ratio represents the chance of an individual embryo proceeding to a 1 point higher KIDScore given the associated maternal risk score. In the proportional odds model this odds ratio is assumed to be constant across all levels of the KIDScore.Baseline characteristics of the female and male population in the current study are depicted as median or number with the corresponding interquartile range (IQR) or percentage. All analyses were performed using SPSS package 21.0  and R . To study the association between the nutritional and lifestyle risk scores and the KIDScore in men and women, we used a proportional odds model . This isTo study the association between the fraction of discarded embryos and the nutrition and lifestyle risk scores, we used a generalized linear mixed model (GLMM) approach. Similar to the models discussed above, in the first model no adjustments were made (crude model) where the second model was additionally adjusted for the covariate maternal age. The effect estimates of the models were again transformed into odds ratios using the exponential function on the effect estimate. This odds ratio represents the chance of ICSI treatment resulting in a discarded embryo based on the individual risk score. Since nearly all women used folic acid supplements and nearly none were smokers analyzing these two behaviors was not possible.. From the 41 couples of which the male partner also participated, a total of 185 embryos were cultured in the EmbryoScope™. Of these 185 embryos , 39 embryos were transferred, 100 were frozen, and 46 were of poor quality and thus discarded .Between January 2014 and December 2017, embryos of 544 couples who underwent their first IVF/ICSI cycle were cultured in the EmbryoScope™. Of these couples, 417 (76.5%) were not subscribed to Smarter Pregnancy. In the end, 113 couples were included, of which 41 of the men also participated. In all included couples, fertilization was performed by using intracytoplasmic sperm injection (ICSI), which resulted in a total of 490 embryos that were cultured in the EmbryoScope™. Of these 490 embryos, 104 embryos were transferred, 254 were frozen, and 132 were of poor quality and thus discarded 2, of which 66 (59%) women had a normal BMI and 46 (41%) were overweight/obese (BMI >25 kg/m2). Most women were of Western origin (84%) and were highly educated (51%). The main reason for ICSI treatment was male factor subfertility (81%), of which in 55% of the cases sperm was retrieved surgically. Other indications included unexplained subfertility (2%), female factor subfertility (2%), and combined male-female factor subfertility (15%). The average time between completing the screening of the Smarter Pregnancy program and oocyte retrieval was 49 days (IQR 35–126 days). Most women had inadequate intake of vegetables (n = 79 (72%)) and fruits (n = 63 (58%)). Only two women did not take folic acid supplements (2%). The vast majority of all women did not smoke (n = 107 (98%)) and did not consume alcohol (n = 67 (61%)) (Table The median age and BMI of the women was respectively 32.4 IQR 29.1–35.1) years and 23.2 (IQR 21.7–26.0) kg/m–35.1 yea. In women with a normal BMI, no significant associations were observed between the KIDScore and vegetables and fruit intake and alcohol consumption  Table . After a2) and 17 (41%) were overweight. All men were of Western origin and most were highly educated (59%). The main indication for ICSI treatment was male factor subfertility (78%), of which in 53% of the cases sperm was retrieved surgically. The average time between completing the Smarter Pregnancy screening and oocyte retrieval was 45 days (IQR 34.5–97). The vast majority of men had an inadequate intake of vegetables (n = 27 (66%)) and fruits (n = 23 (56%)). A total of 38 (93%) were non-smokers, while refraining from alcohol was reported by only 13 men (32%) (Table 1).The median age and BMI of the male partner was respectively 34.0 (IQR 29.5–41.5) years and 24.0 (IQR 22.6–27.5). In males, 24 (59%) had a normal BMI  for the crude model and an effect estimate of − 0.63 and an odds ratio of 0.53 (95% CI 0.33 to 0.85) in the adjusted model, respectively Table . Althoug2. In men, we observed that smoking was negatively associated with embryo quality as measured by the KIDScore. Importantly, our results highlight that a majority of subfertile couples undergoing ICSI treatment are not adherent to a healthy diet and lifestyle in the months preceding oocyte retrieval, semen collection, and subsequent fertilization. Despite the fact that many couples score low on a healthy diet and lifestyle, we did not observe associations between these factors and the proportion of underdevelopment of embryos, which were discarded.In this study, we showed that inadequate maternal vegetable and fruit intake, as well as paternal smoking during the periconception period, are associated with the quality and developmental morphokinetics of pre-implantation embryos as outcome of implantation potential. Inadequate periconceptional maternal vegetable intake was negatively associated with the quality of resulting ICSI embryos. Moreover, the effect size was more than doubled in women with a BMI > 25 kg/m, which showed that periconceptional intake of fruits and vegetables significantly improved pre-implantation embryo quality after ICSI treatment, whereas fruit intake was also positively associated with blastocyst formation [Our results are in line with the study of Braga et al.ormation . No effeThe correlation between a maternal healthy diet and periconceptional outcomes has been well established . VujkoviPaternal smoking and high BMI can have a detrimental effect on all semen parameters like volume, density, concentration and morphology , 38, whiA strength of this study is the use of the validated mHealth Smarter pregnancy program using the dietary risk score. Another strength is the use of the KIDScore to evaluate embryo quality, which is generally used and easily applicable. A KIDScore of 5 is associated with high implantation rates, where a low KIDScore of 1 is associated with low implantation rates. Despite its general and easy applicability, the predictive capability of the KIDScore on implantation has an AUC of 0.65, which could be classified as a fair predictor. In addition, we have to consider for inference of our data that developmental kinetics at the cleavage stages are also reflective of genetic integrity and blastocyst quality. Our study was conducted on embryos cultured until day 3 in a time period that culturing until day 3 was routine clinical practice in most hospitals and also in our hospital. From 2019 onwards, we are culturing the embryos until the blastocyst stage (day 5). Therefore, future research should also investigate the associations between periconceptional parental nutrition and lifestyle factors and the outcome after culturing until day 5, i.e., blastocyst quality, pregnancy rates, and outcomes. In our study we did not investigate the association between nutritional and lifestyle scores on clinical pregnancy rate (detectable embryonic heartbeat), which should be done in larger cohorts. Another strength is that using pre-implantation embryo development in vitro allows studying early development outside of the maternal body, allowing contribution and identification of paternal as well as maternal factors from both parental gametes. The uterine environment makes identification of paternal factors impossible, since factors of the uterine environment could bypass and dampen paternal effects, while following early development and implantation in utero is still technically impossible. Importantly, we show that paternal lifestyle significantly impacts on embryo developmental kinetics during the cleavage stages. Since the study population consists of subfertile couples visiting a tertiary university-based hospital, although it does not mean all couples are in need of tertiary referral or care, the results cannot be automatically extrapolated to the general fertile population and this might have consequences for the external validity of our study. All couples underwent ICSI treatment, so little information is available on IVF and female factor subfertility. However, studying the effects of pre-implantation embryo development can only be performed in fertility centres which have availability of time-lapse imaging. Bias to our results cannot be excluded, because only 36% of the male partners were willing to participate. This study revealed the magnitude of effect sizes which will help us to better calculate the sample size for future larger studies or randomized controlled trials.We realized that specific subfertility-related diseases, such as endometriosis and polycystic ovary syndrome (PCOS), are also associated with oocyte and embryo quality. Endometriosis negatively affects the oocyte quality, which could be caused by increased oxidative stress . PCOS isThis study showed that both periconceptional maternal and paternal lifestyle and nutritional factors already have an impact on pre-implantation embryo quality based on morphokinetic evaluation after time-lapse culture. As more than 80% of the reproductive population has one or more inadequate nutrition and lifestyle behaviors, studying and modifying the associations by evidence-based interventions is becoming increasingly important. Moreover, the live birth rate per started IVF/ICSI cycle has remained stable at around 30% per cycle for the last decade. With the increase of subfertility in the Western world, it is essential to determine how to stop the increasing subfertility numbers, improve ART chances and prevent and overcome subfertility causes. Therefore, we conclude that identifying modifiable risk factors, as a first step in the behavioral change pathway to stimulate awareness, can have already a clinical impact by increasing the chance of a healthy pregnancy and having a live-born baby.ESM 12 and BMI<25 kg/m2. (PNG 172 kb)Odds ratios and confidence intervals of the associations between the risk scores for vegetable intake, fruit intake and the dietary risk score and the KIDScore in the female population depicted for the complete group and women with BMI ≥25 kg/mHigh Resolution Image (TIF 19247 kb)ESM 2(DOCX 49 kb)