While micropropagation systems have been developed for cannabis, little attention has been given to the potential impact of explant source within the plant on subsequent growth and development in vitro. From other species, it is known that the explant source can have a significant impact on future development, presumably due to variations in endogenous phytohormones, sugars, and epigenetic state. This study was performed using a high-cannabinoid cultivar of cannabis, an economically important crop, to compare the growth and development of explants collected from various positions within the plant, ranging from the top to the bottom. Our results show that the multiplication rate , especially with explants derived from middle and apical parts of the mother plant, decreased over multiple subcultures, which can result from epigenetic machinery. In contrast, explants derived from the basal portion of the plant had an acceptable multiplication rate throughout the experiment and produced cannabis plantlets with shorter but wider leaves, indicative of rejuvenation. Hence, these results suggest that basal-derived cannabis explants should be used preferentially as starting material for in vitro propagation.Drug-type cannabis is often multiplied using micropropagation methods to produce genetically uniform and disease/insect-free crops. However, micropropagated plantlets often exhibit phenotypic variation, leading to culture decline over time. In cannabis, the source of these changes remains unknown, though several factors  can result in such phenotypical variations. The study presented herein evaluates the effects of explant sources  over multiple subcultures (4 subcultures during 235 days) on multiplication parameters and leaf morphological traits of in vitro cannabis plantlets. While initial in vitro responses were similar among explants sourced from different regions of the plant, there were significant differences in performance over the course of multiple subcultures. Specifically, explant source and/or the number of subcultures significantly impacted plantlet height, number of nodes, and canopy surface area. The explants derived from the basal and near-basal parts of the plant resulted in the tallest shoots with the greatest number of nodes, while the explants derived from the middle and apical regions led to shorter shoots with fewer nodes. Moreover, the basal-derived explants produced cannabis plantlets with shorter but wider leaves which demonstrated the potential of such explants for in vitro rejuvenation practices with minimal culture decline. This study provides new evidence into the long-term impacts of explant source in cannabis micropropagation. Cannabis sativa L.) is a dioecious, herbaceous crop with a variable growth habit [Cannabis  were selected as explants, using the greenhouse-grown mother plant. Since Honey Banana responds poorly to in vitro culture compared with other cannabis cultivars, it can be considered a potential candidate to study the effect of the explant’s sources on in vitro performance and rejuvenation during prolonged in vitro culture. To study the effect of the explant’s sources, stem segments with two nodes were selected from different parts of the greenhouse-grown mother plant  includin−1 and a PAR photon efficacy of 2.2 μmol J−1.The plant used in this study was cut from a maintained mother plant aged slightly over 2.5 years and was provided with the ideal environment to enable the mother plant to thrive. The mother plant originally came from seed but was renewed from a clone at about the 2-year mark to reinvigorate the plant. The mother plant was transferred to appropriately sized pots as it grew until it was maintained in a 76 L  pot with nutrient-rich soil. A nutrient solution designed for vegetative growth was supplied every 3–5 d to maintain appropriate moisture levels in the soil with an adjusted pH of ~6.5. It contained high levels of nitrogen, with moderate levels of phosphorous, potassium (N-P-K), and micronutrients. Environment conditions consisted of temperatures between 20–25 °C with relative humidity of 55–65%. The mother plant was kept under long-day photoperiods of 18:6 hr light/dark cycles to sustain an indefinite vegetative state. The light source was 300 W broad white-spectrum lights (4.2% red 650–670 nm) with a photosynthesis photon flux of >440 μmol sDriver and Kuniyaki Walnut (DKW) culture medium   (7 drops per 500 mL of solution) for 10 min followed by 3 rinses with sterile distilled water (each time 5 min). Each sterilized explant was cultured in a Magenta GA7 vessel  containing 40 mL of basal DKW medium and kept in the growth chamber at 25  ±  2 °C under a 16 h photoperiod with 40  ±  5 μmol m−2 s−1 light intensity.The decontamination of the explants was performed using 10% commercial bleach with 1.4 mL/L Tween 20  were measured.Several morphological traits of the leaf  were measured using five samples (leaves) from each treatment.p < 0.05. SAS version 9.3 was also used for data analysis. The morphological traits of the leaf were analyzed using ImageJ.The experiment was performed based on a completely randomized design with 2 replicates in a 4 × 4 factorial design (4 sources of the explants and 4 subcultures). The data were analyzed by analysis of variance (ANOVA) followed by Duncan’s multiple range test at In the current investigation, the impacts of the source of explants and the number of subcultures on in vitro multiplication of cannabis were assessed . Based oOur results also showed that the maximum plantlet height was obtained from the explants derived from the basal part (42.34 mm) and the near-basal part (41.72 mm), which was significantly greater than the middle (37.13 mm) and apical (32.97 mm) parts a. SimilaOur results a also shAccording to the correlation coefficient results , a signiIn addition, our results demonstrated that the plantlet’s leaves from all the explant’s sources in subculture I consisted of five leaflets . HoweverProlonged in vitro culture by several subcultures resulted in in vitro grown plantlets with shorter but wider leaves, indicative of rejuvenation . AlthougSalvia tomentosa [Rauvolfia serpentina [Quercus robur [Persea americana [Solanum tuberosum [Ginkgo biloba [Rubus fruticosus [Caralluma lasiantha [Psidium guajava [Hemidesmus indicus [Ficus religiosa [Cedrela montana [Appropriate explant selection is the key initial stage of micropropagation . Althougomentosa , Rauvolfrpentina , Quercusus robur , Persea mericana , Solanumuberosum , Ginkgo o biloba , Rubus futicosus , Caralluasiantha , Psidium guajava , Hemides indicus , Ficus religiosa , and Ced montana  which sh montana ,45. Ther montana ,38,40,44 montana ,46,47. A montana . Thus, cMusa sapientum after eight subcultures. Similar results were also reported in Olea europaea [Coffea arabica [Apart from the source of the explants, the number and duration of subcultures play a fundamental role in the successful in vitro regeneration ,50. Varieuropaea  and Coff arabica . Previou arabica ,55,56,57Prunus sp. plants by studying miRNA172 and miRNA156/157 expression patterns and several of their target genes during rejuvenation. Higher expression of miR156 and miR157 was observed in the leaves of juvenile explants than in mature explants, while the opposite was observed for the expression of miR172 [Pinus radiata plants. The level of DNA methylation in meristematic areas of mature leaves was higher than in juvenile leaves. The modification in the level of DNA methylation during rejuvenation showed that plant rejuvenation can be considered a result of epigenetic changes which are dependent on the explant source [Since explants are typically selected from mature greenhouse-grown mother plants, the explants undergo a rejuvenation process during tissue culture practices . It has f miR172 . These ff miR172 . While tf miR172 ,46,47,63f miR172  reportedt source . Our rest source ,68,69,70This study aimed to evaluate the explant source and number of subcultures on in vitro rejuvenation and phenotypic variations of cannabis plantlets. Our results showed that the number of nodes, especially in the explants derived from middle and apical parts, decreased during prolonged in vitro culture which can be a result of epigenetic somaclonal variation. Moreover, leaf morphology analysis showed that the explants derived from the basal part of the mother plant were better suited to be initial explants for in vitro cannabis rejuvenation practices. Since previous studies on other plant species, such as potatoes , showed


The goal of this study was to characterize the symptoms and outcomes of patients with a symptomatic cervical inlet patch (CIP).Retrospective case series.Tertiary care laryngology clinic in Charlottesville, Virginia.A retrospective chart review of the patient's demographics, comorbidities, prior workup, interventions, and response to treatment was performed. All patients received flexible nasolaryngoscopy and barium swallow study. The analysis was descriptive.Eight patients  were followed for the management of symptoms related to CIP. The mean age at presentation to our clinic was 64.9 (standard deviation = 15.7). Five out of 8 patients presented with a chief complaint of dysphagia, and the remaining 3 with chronic coughs. Five out of 8 patients demonstrated findings of laryngopharyngeal reflux (LPR) including vocal fold edema, mucosal erythema, or postcricoid edema. Swallow study demonstrated hiatal hernia in 3 of 8 patients, and cricopharyngeal (CP) dysfunction  in 3 of 8 patients. One patient presented with a history of Barrett's esophagus. Treatment included increased acid suppression therapy and management of coexisting esophageal pathologies. Ablative procedures were performed in 5 out of 8 cases, with 2 patients requiring repeat procedures. All patients experience subjective symptom improvement.CIP tends to present in complex patients with multifactorial dysphagia, with the most common symptoms being dysphagia and cough. Clinical features of CIP overlap with other more common pathologies encountered by otolaryngologists including LPR and CP dysfunction, and future prospective studies in larger populations should seek to clarify these associations. The gastric inlet patch, also known as a cervical inlet patch (CIP), is an anatomical anomaly wherein secretory gastric mucosa is present in the esophagus, most commonly in the proximal portion. The prevalence of this condition is disputed in the literature, as it is often overlooked in a clinical setting due to difficulty with visualization on routine endoscopy and unclear clinical relevance.Although many cases of CIP are asymptomatic, symptoms of CIP are thought to result from the secretion of acid from gastric parietal cells within the heterotopic mucosa, which can then reflux into the larynx and pharynx causing cough, globus, dysphagia, and excessive throat clearing.Diagnosis can be made fluoroscopically or via direct endoscopic visualization. Endoscopically, CIP appears as a flat, velvety, rose or salmon‐colored patch located between 15 and 21 cm from the central incisors.Treatment for symptomatic cervical inlet patches typically begins with proton pump inhibitor (PPI) therapy. For cases refractory to medical management or showing evidence of dysplasia or neoplasia, endoscopic argon plasma ablation,This study design was reviewed and approved by the University of Virginia Institutional Review Board for Health Science Research (IRB‐HSR) prior to data collection (IRB‐HSR # 22341). Written informed consent for patient information and images to be published was provided by the patients or a legally authorized representative. Records of all patients with a diagnosis of cervical inlet patch up to June 2022 were retrospectively identified with our electronic medical record database using a pre‐existing list of patients seen in the clinic with a diagnosis of CIP or ectopic gastric mucosa. All patients from this cohort were seen in the University of Virginia Voice and Swallowing Clinic during this period. Demographic data, flexible nasolaryngoscopy, esophagoscopy, barium swallow studies, and patient reports of symptoms were obtained. Descriptive statistics were used to summarize baseline characteristics.Eight patients  were identified as having ectopic gastric mucosa in the proximal esophagus with a mean age of 64.9 (standard deviation [SD] = 15.7) at the time of initial evaluation. The average body mass index for this cohort was 31.1 (SD = 10.4). Two patients reported intermittent alcohol use, 1 endorsed a 50‐pack‐year smoking history, and the remaining 5 denied any substance use Table .Five patients presented with a chief complaint of dysphagia and the remaining 3 with chronic coughs. Other symptoms included dysphonia, odynophagia, and globus sensation. Four patients received a diagnosis of cervical inlet patch prior to their presentation to otolaryngology and all patients had been placed on a reflux regimen prior to evaluation by our clinic. Several had multidisciplinary evaluations including pulmonology, allergy, and rhinology.Esophagoscopy performed on each patient provided visualization of the inlet patch in the expected position 15 to 21 cm from the central incisors. Four patients were found to have a single patch, with 3 patients having multifocal patches. The estimated circumference of the patches ranged from <25% to 80%. Biopsies demonstrated either junctional mucosa or gastric epithelium with inflammation with no evidence of malignancy.A fluoroscopic barium swallow study revealed hiatal hernia in 3 of 8 patients. Cricopharyngeal (CP) dysfunction, including CP hypertrophy and Zenker's diverticulum, was found in 3 out of 8 patients. Killian Jamieson diverticulum was present in 1 patient. Flexible nasolaryngoscopy demonstrated findings of LPR, including vocal fold edema, mucosal erythema, or postcricoid edema in 5 out of 8 patients. A validated scoring system assessing LPR symptoms was not used in these patients, and diagnosis of LPR was based on patient‐reported symptoms and clinical findings.2 laser diverticulotomy was performed in 1 patient with CIP and Zenker's diverticulum. One patient was previously diagnosed with Barrett's esophagus. Five patients had their CIP addressed surgically with varying techniques including coblation (2), potassium‐titanyl‐phosphate (KTP) laser ablation (1), hot biopsy forceps ablation (1), or all 3 (1). Two patients required repeat ablative procedures. Variation in the technique depended on the ability to achieve adequate rigid exposure for intervention versus a flexible endoscopic approach.The treatment strategy began with a high‐dose reflux regimen in 6 out of 8 patients. This included increased PPI dosage and/or frequency, and/or addition of alginate or H2 blocker. In addition to CIP management, 5 patients were also managed for coexisting pathologies of the esophagus. Three patients required esophageal dilation due to CP dysfunction. In 1 patient with CIP and a small Killian‐Jamison diverticulum, CP and upper cervical esophagus botulinum toxin injections were performed. COSymptoms recurred in 3 patients following surgical intervention. Upon repeat endoscopy, 1 patient was found to have no evidence of CIP following ablation. The remaining 2 had repeat procedures, with 1 patient experiencing improvement in symptoms and the other with no subjective improvement  receiving KTP laser ablation for CIP treatment. In each case, dysphagia improved following the treatment of CP dysfunction. Dysphagia initially improved in patient 2 following dilation and ablation but soon recurred. Upon repeat esophagoscopy and KTP ablation, the patient experienced no improvement in her dysphagia. Of note, this patient also had the largest  patch in this case series, while the other patients with CP dysfunction had smaller (<30%) patches. Further studies should be performed to better characterize the role of treatment of CIP in patients with CP dysfunction, as these 2 pathologies could be acting synergistically in their contribution to dysphagia.Symptoms of patients with CIP overlap with symptoms of LPR, and the association of these conditions is the subject of active debate. Once thought to be the result of the reflux of gastric contents into the laryngopharynx, the association with CIP is challenging this theory. The proximity of the inlet patch to the laryngopharynx and the superficial location of sensory nerves in the proximal esophagusPatch circumference is related to the severity of symptoms. The 2 patients (#2 and #4) with the largest patches  required repeat ablation, suggesting counseling for this population should include management of expectations following initial ablative treatment.Treatment of patient 2 is ongoing; their symptoms improved after KTP ablation #1, but then recurred with no improvement following repeat KTP ablation. Although the small sample size limited meaningful statistical analysis, there was no apparent relationship between biopsy findings and symptom patterns, severity, or outcomes.All patients in this study received a biopsy of patch mucosa during esophagoscopy, confirming the condition. While definitive conclusions cannot be drawn from this small sample size, there appears to be no association between biopsy findings and symptom severity or response to treatment. Further studies should be carried out to determine if secretory cell types found in inlet patches are associated with an increased incidence of LPR.Incomplete subjective patient data, including Voice Handicap Index‐10 and Eating Assessment Tool‐10 scores, and a small sample size limited a meaningful statistical analysis in this case series. Due to the high prevalence of reflux in CP dysfunction patients, we cannot conclude whether CP dysfunction is a direct result of CIP or a reflux‐related entity. Additionally, direct endoscopic visualization for patients with improved symptoms after the intervention was not obtained routinely, restricting the correlation of symptom resolution with the visual improvement of inlet patches.Clinicians should maintain a high index of suspicion for CIP in patients presenting with dysphagia and/or chronic cough refractory to antisecretory treatment. There is a high prevalence of LPR in this patient population. Ablative procedures may improve symptoms in patients with CIP refractory to medical treatment. Patients with CP dysfunction may have concomitant CIP. The management of these patients can be complicated, and further studies are required to better characterize this relationship and assess the role of ablative procedures in this population.Julian S. De La Chapa, assisted with study design, data collection, data analysis, and manuscript preparation; Christopher J. Harryman, assisted with study design, data collection, data analysis, and manuscript preparation; Patrick O. McGarey, assisted with study design, data analysis, and manuscript preparation; James J. Daniero, assisted with study design, analysis, and manuscript preparationNone.None.


We previously reported the first population‐based study of the epidemiology of microscopic colitis in Northern Ireland. The aim of the current study is to provide updated data on incidence, diagnostic methods and clinicopathological associations, following dissemination of the previous report. A further aim was to compare the findings against relevant recommendations from the 2020 European guidelines.Study cases were identified via the Belfast Health and Social Care Trust pathology laboratory system for new cases of collagenous colitis or lymphocytic colitis diagnosed from 2017 to 2020 inclusive. Demographic and clinical information was collated from electronic healthcare records.p < 0.001). Overall compliance with coeliac serology testing has improved, with 89% tested in 2017–2018 compared with 75% in 2008–2016.Two hundred and seventeen new diagnoses of microscopic colitis were made between 2017 and 2020, comprising 89 (41%) collagenous colitis and 128 (59%) lymphocytic colitis. The overall incidence of microscopic colitis, expressed per 100,000 adult population, ranged from 7.6 to 11.5 . The 2019 peak of 11.5 cases per 100,000 adult population represents a 71.6% increase in incidence compared with the mean incidence of 6.7 per 100,000 adult population from previous data for 2008–2016. There has also been a significant increase in number of cases diagnosed on separate sampling from the right and left colon (85% in 2019–2020 compared with 30% in 2008–2016; Clinicopathological communication has contributed to an increased incidence of microscopic colitis in Northern Ireland through better endoscopic diagnostic sampling and pathology coding practices. Coeliac serology testing has also improved, although continued clinical awareness is required of the need for coeliac serology testing in all patients diagnosed with microscopic colitis. This paper is a follow‐up to the first epidemiological assessment of microscopic colitis in Northern Ireland, providing more complete data on incidence and assessing compliance with endoscopic sampling and coeliac serology testing, both of which are key aspects of the management of microscopic colitis outlined within recent European guidelines.Interest in microscopic colitis (MC), comprising collagenous colitis (CC) and lymphocytic colitis (LC), has increased in recent years. Rising incidence of this disease accompanies improved clinical awareness, particularly in Europe and North America , 2, 3. WWe previously reported the incidence and clinicopathological associations of MC in a population‐based study in Northern Ireland, covering the period 2008–2016 . This stWe therefore conducted a follow‐up audit of the Northern Ireland population, covering the years 2017 to 2020 inclusive. The years 2017 and 2018 account for the interventional period, during which we disseminated the findings from our previous study to clinicians, via presentations at regional gastroenterology and surgical meetings, and to pathologists, by direct e‐mail communication, to flag the publication and specifically to advise on appropriate SNOMED coding for such cases. The years 2019 and 2020 represent a postinterventional period. The primary aim was to provide updated data on the incidence of MC and diagnostic methods. A second aim was to compare selected data items, specifically endoscopic sampling method and performance of coeliac serology testing, against relevant recommendations from the 2020 European guidelines.https://www.nisra.gov.uk/).Study cases were identified through an archival SNOMED search performed within the laboratory information management system of the Belfast Health and Social Care Trust cellular pathology laboratory. This serves two of five trusts within Northern Ireland, providing secondary care to a total population of approximately 725,000 people, including an adult population (aged 18 years or older) of approximately 565,000 . Incidence data were calculated per 100,000 of both the total population and adult population residing in the catchment area during the years 2017 and 2020 , ‘collagenous colitis’ D544130) and ‘lymphocytic–plasmacytic colitis’ (D541710). Note that no specific code is available for ‘lymphocytic colitis’ but, following our previous study, recommended SNOMED codes for MC were disseminated to all pathologists in mid‐2018. No pathology slide review was conducted but all diagnoses were made by one of a team of specialist gastrointestinal pathologists, usually on routine haematoxylin and eosin (H&E)‐stained slides without the use of ancillary stains, such as CD3 immunohistochemistry or collagen stains such as Masson Trichrome or Van Gieson. These stains were typically reserved for borderline cases, reflecting current guidance and routine practice [0 and ‘lyIn order to allow direct comparison with the 2008–2016 cohort, similar patient characteristics were collected as before. Ethical approval was not required for this clinicopathological audit. Information collected included age at diagnosis, sex and endoscopic information regarding the procedure performed (colonoscopy or sigmoidoscopy) and the nature of diagnostic biopsy sampling. For this purpose, the right colon was considered proximal to the splenic flexure and the left colon represented the splenic flexure and descending or sigmoid colon. Incidence data for 2019 and 2020 were analysed separately, given the likely impact on elective endoscopy services of the SARS‐CoV‐2 (COVID‐19) global pandemic . Follow‐A total of 217 new diagnoses of MC were made between 2017 and 2020, comprising 89 diagnoses of CC and 128 of LC. Overall MC incidence, expressed per 100,000 total population, ranged from 5.9 to 9.0, with a peak incidence in 2019 of 9.0, comprising an incidence for CC of 4.2 and for LC of 4.8 during this year  [In the interventional period (2017–2018), 71 of 100 cases (71%) were diagnosed from two or more separately labelled biopsy specimens, usually representing the right and left colon Figure . This up< 0.001) . During Of the 2017–2018 cohort, 89% of patients with MC (95% with CC and 85% with LC) had coeliac serology performed at any time in their clinical history (before or after diagnosis)  the conception and design of the study, or acquisition of data, or analysis and interpretation of data, (2) drafting the article or revising it critically for important intellectual content, (3) final approval of the version to be submitted. Specific contributions are outlined below: MM, acquisition of data and analysis, drafting the article, final approval of the submitted version; HGC, interpretation of data, statistical analysis, article revision, final approval of the submitted version; PBA, interpretation of data, article revision, final approval of the submitted version; MBL, conception and design of the study, interpretation of data, final approval of the submitted version, guarantor.No funding received.None.


Escherichia coli, Pseudomonas aeruginosa, poliovirus type 1 and Cryptosporidium parvum—were used in a human health risk assessment to identify how the storage time of recharged water in the Floridan Aquifer enhances pathogen inactivation, thereby mitigating the human health risks associated with ingestion. We used a quantitative microbial risk assessment to evaluate the risks for a gastrointestinal infection (GI) and the associated disability-adjusted life years (DALYs) per person per year. The risk of developing a GI infection for drinking water no longer exceeded the suggested annual risk threshold (1 × 10−4) by days 31, 1, 52 and 80 for each pathogen, respectively. DALYs per person per year no longer exceeded the World Health Organization threshold (1 × 10−6) by days 27, <1, 43 and 72. In summary, storage time in the aquifer yields a significant reduction in health risk. The findings emphasize that considering microbial inactivation, caused by storage time and geochemical conditions within ASR storage zones, is critical for recharge water treatment processes. Aquifer storage and recovery (ASR) can augment water supplies and hydrologic flows under varying climatic conditions. However, imposing drinking water regulations on ASR practices, including pre-treatment before injection into the aquifer, remains arguable. Microbial inactivation data— The critical need to manage limited and often unpredictable water resources, affected by climate change, population growth and agricultural practices, has resulted in an array of water reuse and recovery applications to address and respond to these increased demands. Aquifer storage and recovery (ASR) is a type of managed aquifer recharge (MAR) technology that injects treated surface water into an aquifer storage zone for later recovery for surface applications . The appThe surface water recharged into aquifer storage zones at ASR facilities must be monitored at the above-ground point of injection for specific constituents, including microbial contaminants, as described in the primary drinking water standards of the Safe Drinking Water Act (SDWA) ,5,6,7,8 Escherichia coli (E. coli), bacteriophages  and eukaryotic viruses  and encysted protozoans  have been shown to be inactivated during the storage of recharged surface water in aquifer zones [By not sampling the recovered and discharged ASR water for the same microbiological constituents as required for the surface water prior to recharge, the influence of time and geochemical conditions of the storage zone on the survival of these microorganisms is overlooked. In fact, fecal coliforms, er zones ,12,13,14er zones ,16. −6). Utilizing a human health risk framework to estimate and interpret health risks associated with environmental hazards and policies has been extensively used for recreational, drinking and groundwater management ,17,18,19In this paper, we describe the application of QMRA as an exercise to translate pathogen reduction during the storage phase in aquifer environments under ASR processes to estimate health risk outputs. The human health risk assessment approach presented in the study assesses the health risks associated with two specific exposure scenarios: ingestion of water from drinking and while swimming. We chose to use both scenarios due to the regulatory requirements for surface water to be treated to drinking water standards at ASR facilities in Florida   and Avon Park Permeable Zone (APPZ) ,16,22. T−1 and 24.4–1524 m day−1 for the wells accessed during the cited studies for the UF and APPZ, respectively [The geologic framework of the Floridan Aquifer System in this region of Florida consists of a thick sequence of predominantly marine carbonates that were deposited during the middle to late Eocene  and Oligocene  . In ordeectively . All ASRThe water quality data summaries from the groundwater wells accessed during the inactivation studies are listed in 3 or NO2 ,16,22.E. coli, Pseudomonas aeruginosa (P. aeruginosa), poliovirus type 1 (PV1) and Cryptosporidium parvum (C. parvum)—followed bi-phasic (E. coli and P. aeruginosa), log-linear (PV1) and Weibull (C. parvum) inactivation models [Previous microbial inactivation studies in the aquifer zones described in n models ,16. The n models ,16,22. −4), given that the water is monitored to meet primary drinking water standards [−6) [The QMRA framework has been extensively applied to evaluate human health risks associated with exposure to microbial contaminants in different water systems  ,19,25,26tandards . The disrds [−6) . DALY esThe study also assumes a closed system for the risk assessment model, in which water that is retrieved from the surface is partially treated via rapid sand filtration and in-line UV disinfection, then injected into the aquifer zone and resides there without assumptions for dilution and no additional water injections or withdrawals. After a specified amount of time, the water is then recovered from the aquifer. The model assesses the human health risks associated with ingestion of that recovered water. E. coli , P. aeruginosa , PV1 (human enterovirus) and C. parvum (encysted eukaryotic protozoan)—are representative of indicators and pathogens that are a significant concern for public health and groundwater. Though PV1 is not as protective as a viral surrogate as other enteric viruses , conservative assumptions are utilized in this work to protectively estimate the overall impact that storage time in the aquifer can have on pathogen concentrations and, therefore, estimated human health risks. Moreover, these four microorganisms have varying survival rates in the anaerobic and reduced groundwater environments [The microorganisms of interest for this human health risk assessment were selected due to their regulatory significance for SDWA standards and treatment ,21,29. Tronments ,16, therGiven that the average storage time for recharged surface water from CERP-related ASR facilities is 3–6 months  and the The microorganism concentrations considered in the exposure assessment were retrieved from environmental studies that enumerated fecal indicator bacteria and pathogen concentrations in Florida surface water targeted for ASR source waters . No diluI) (V) (The dose formula is the product of the concentration of the pathogen in the water (N0) , the patN0) (I)  and the  (I) (V)  in the eE. coli was utilized in a previous microbial inactivation study [E. coli would be pathogenic. A ratio of 1:0.08 of general E. coli to pathogenic E. coli was assumed to adjust the dose of the pathogen [E. coli O157:H7, are best fit by the exponential dose–response function (Equation (2)), which is described by the parameters for survival probability (k) and dose (d) (Equation (2)). E. coli O157:H7 is best fit by the Beta–Poisson dose–response model, which includes the parameters d)  N0 in   infection (under both scenarios) was estimated using Equation (2). An annual risk of infection ,27, assutandards .(4)PannE. coli gastroenteritis, adenovirus gastroenteritis , P. aeruginosa gastroenteritis and cryptosporidiosis (The disability-adjusted life years (DALYs) metric was utilized to estimate the impact of disability (associated with illness) for public health . DALYs pridiosis . DALYs wHealth risks characterized as DALYs  for each pathogen are a product of the probability of illness  on day 0, with estimated infection risks of 2.42 × 10−4. Pathogenic E. coli initially had an elevated health risk comparable to both PV1 and C. parvum, yet the health risks associated with both P. aeruginosa and pathogenic E. coli declined quickly, with pathogenic E. coli experiencing a faster inactivation rate and, therefore, lower health risk than P. aeruginosa. While pathogenic E. coli, PV1 and C. parvum exceeded the risk threshold past day 1, the annual risk of infection for these pathogens decreased by four orders of magnitude by days 31, 52 and 80, respectively. The residual risk for each of the four pathogens eventually decreased to less than 2.01 × 10−8 by day 150. The significant and rapid decrease in the risk of infection associated with P. aeruginosa is likely due to the lower estimated dose of pathogen, resulting in reduced infection risks. Conservatively assuming that recovered water is untreated (except for the ASR storage phase barrier) and used directly for drinking water, the annual risk of infection for each pathogen was estimated . P. aeruE. coli, PV1 and C. parvum—would decrease by four orders of magnitude within 19, 35 and 56 days, respectively. The estimated health risks for P. aeruginosa never exceed the risk threshold. The residual risk for each of the four pathogens is estimated to be below 6.95 × 10−10 by day 150. The human health risks associated with exposure to water while swimming parallel the estimated health risks under the drinking water scenario . The onl−6 DALYs [E. coli, adenovirus (representative for PV1) and Cryptosporidium spp. (representative for C. parvum) were found to exceed the risk threshold on day 0 (E. coli (27 days), adenovirus (43 days) and Cryptosporidium spp. (72 days)) no longer exceeded this benchmark. The DALYs associated with gastroenteritis for P. aeruginosa were found to be lower than the other pathogens and did not exceed the DALYs threshold. Estimated DALYs per person per year associated with gastroenteritis were compared to the acceptable threshold of 1 × 10−6 DALYs . Under ton day 0 . HoweverE. coli, adenovirus and Cryptosporidium spp. slightly exceeded the DALYs threshold, within 49 days, the threshold was no longer exceeded  and WHO DALY (1 × 10−6) recommended thresholds, as demonstrated through QMRA, for all of the pathogens represented in this study. Above-ground pre-treatment requirements for surface water to meet or exceed SDWA regulatory standards at ASR facilities in Florida pose significant increases in economic and financial obligations for the operation and maintenance of those facilities, relative to the existing requirements . However, the results from this study indicate that storage time in the aquifer and geochemical conditions in those storage zones, such as those cited in this study, are effective for the inactivation of pathogens over relatively short storage time periods. Accordingly, the storage time of recharged surface water in selected zones of the Floridan Aquifer System  could be a cost-effective alternative or supplement to the current and proposed above-ground pre-treatments. For example, a storage time of approximately 80 days of storage in the UF or APPZ would result in adequate pathogen inactivation to achieve reduced residual health risks that would meet the suggested annual risk of GI infection  inactivation rate, therefore meeting the Australian health risk guidelines. As shown in the present study, QMRA translates the log reduction of pathogens to reductions in health risks due to different types of treatment zones and can assist in determining the types of treatments that result in acceptable health risks. Additionally, a study evaluating MAR treatment applicability for irrigation through QMRA identified that, for some regions using MAR irrigation management practices, the aquifer treatment zone was the most critical barrier for water quality treatment and pathogen inactivation [Similar studies evaluating MAR systems have also identified that different groundwater characteristics can impact the inactivation rates of pathogens and should be considered in any planned MAR/ASR system ,11,12. Itivation .C. parvum) pathogens had slower inactivation rates than the bacterial microorganisms (E. coli and P. aeruginosa), the health risks posed by potential exposure to these pathogens would still meet the tolerable risks suggested by public health guidelines.QMRA has been identified as a suitable tool to evaluate microbial risks in water systems, such as for MAR/ASR water management practices ,12,13,48The use of deterministic data in the risk assessment likely overestimates the potential human health risks (infection and DALYs), therefore serving as a conservative preliminary approach to evaluate the environmental inactivation of pathogens in the UF and APPZ zones within Floridan Aquifer System in this area of Florida. The availability and use of site-specific pathogen inactivation rates incorporated into this risk assessment framework further emphasize not only the value of QMRA for the water industry but, specifically, that this work can be incorporated into ASR plans and policies by quantifying the effectiveness of aquifer storage in reducing pathogen levels and health risks. Additionally, this risk assessment identified that storage time in the aquifer is a key factor in ensuring effective microbial inactivation and, therefore, mitigated residual health risks, which aligns with findings in other ASR-QMRA work . E. coli (2.98 × 10−3), adenovirus (1.56 × 10−3) and Cryptosporidium spp. (3.22 × 10−3). P. aeruginosa DALYs per case (9.0 × 10−4) was one order of magnitude lower. A previous ASR study determined that a residence time of 70 days was insufficient for adequate pathogen inactivation  [−6) by day 73. When interpreting the DALYs per person per year associated with each of the pathogens, the health risks paralleled the findings from the QMRA. For gastroenteritis, the calculated DALYs per case were comparable for pathogenic tavirus) . Anotheri 2.98 × −3, adenoThe estimated DALYs incorporated into this study utilized the best available information. Metrics regarding gastroenteritis  are limited in the United States due to under-reporting or lack of diagnoses ,49. ManyThe findings presented in this study have potential to inform ASR practices but should be evaluated in the context of their limitations. The QMRA conducted incorporated deterministic values, which inhibits the consideration of the variability in parameter values. This study describes a QMRA model used to assess how pathogen inactivation in the environment correlates to estimated human health risks and, therefore, should be considered risk approximations that can continue to be revised as more relevant data become available. The microbial inactivation rates utilized in this risk assessment are specific to the environmental conditions of the ASR storage zones assessed in this study ,16. HoweThe human health risk outputs estimated in this study can only be assumed to be as good as the variables that were utilized to conduct the risk assessment . AssumptQMRA applications in the water industry translate water quality data to human health impact by quantifying the number of infections that can be prevented due to a treatment process or water management practice. In lieu of costly and logistically challenging epidemiological studies, QMRA measures a body of water as an acceptable source for human use through a range of assumptions related to contaminant type, concentration and virulence based on potential exposure dynamics. The inclusion of QMRA as part of an ASR management plan can contribute to best practices that foster a community’s water and economic sustainability. Under the assumptions utilized in this study, a storage time of at least 80 days in the UF or APPZ storage zones can influence pathogen inactivation at rates that would reduce human health risks (for a GI infection) by at least four orders of magnitude, resulting in estimated risks that no longer exceed suggested annual risk and DALY thresholds. The QMRA framework described in this study emphasizes the critical role that storage time in specific ASR storage zones has on pathogen inactivation, thereby helping to mitigate associated human health risks while complementing current water treatment practices.


We used US nationwide commercial insurance claims data (2011–2015) to study the effect of social deprivation on clinical and demographic risk factors for suicidal ideation (SI) and suicide attempts (SA) among US youth and adults < 65 years, after having a mental health or substance use disorder-related outpatient encounter. Neighborhood social deprivation level was summarized by the quintile of social deprivation index (SDI) at individuals’ zip code level. Cox proportional hazard models were used to evaluate the effect of social deprivation on demographic and clinical risk factors for SI and SA. The study cohort consisted of 317,383 individuals < 65 years, with 124,424 aged < 25 (youth) and 192,959 aged between 25 and 64 (adults). Neighborhood social deprivation impacted risk factors for SI and SA differently for youth and adults. Among youth, SDI interacted with multiple risk factors for both SI and SA. The effects of the risk factors were larger on youth from middle socioeconomic neighborhoods. Among adults, risk of SI was the strongest in the most deprived neighborhoods, but risk of SA did not vary by neighborhood deprivation level. Our findings suggest community-based suicide prevention initiatives should be tailored according to neighborhood deprivation level and the targeted individual’s age to maximize the impact. Despite significant resources allocated towards suicide prevention, the rates of suicidal ideation (SI) and suicide attempts (SA) have increased over the last several years2. Numerous models have been developed to predict the risk of suicide and suicidal behaviors in clinical settings5, focusing on demographic and clinical risk factors associated with SI or SA. A recent study used electronic health records (EHR) data from multiple health systems across the US to identify important clinical risk factors for SA and suicide from over 300 potential predictors, including prior history, diagnoses, medications, healthcare service utilization, and routinely collected depression questionnaires, through developing and validating predictive models4. The most important risk factors identified were long-, mid-, and short-term  mental health and substance use disorder (MH/SUD) related diagnosis, healthcare services utilization, and prescription medications. Using EHR data of almost 20 million visits of individuals aged 13 years or older with an outpatient mental health diagnosis, this study’s models achieved overall accuracy of prediction (c-statistic) over 80%, which significantly outperformed other published models, and thus can be considered as benchmark models for risk prediction.Suicide remains an important public health concern in the US7, it has become possible to study the effects of SDoH on health outcomes. Several studies have found associations between SDoH and suicide, including unemployment, poverty, and divorce rates11. In addition, the independent effects of SDoH on SI or SA persisted even after controlling for the demographic and clinical risk factors of individual participants. In a recent study using the EHR data from the Veterans Health Administration (VHA), adverse SDoH factors, such as housing instability, unemployment, and financial problems, were strongly associated with SI and SA even after adjusting for the presence of major depressive, alcohol use, drug use, anxiety, posttraumatic stress, schizophrenia, and bipolar disorders12. Further, SDoH are associated with risk factors for SI and SA. Living in disadvantaged neighborhoods has also been linked to depression14, substance use disorders in adolescents15, and higher utilization of health services17, all of which are risk factors for suicide and suicidal behaviors4. Therefore, it is important to investigate if SDoH modifies the effects of demographic and clinical risk factors for SI or SA.As many health care systems are moving towards routinely collecting individual-level data on social determinants of health (SDoH) during a clinical encounter and integrating such information within EHRs18. However, this study only considered one dimension of SDoH, neighborhood poverty, which was dichotomized and used as a binary measure. SDoH contains intangible factors from multiple dimensions, including but not limited to economics, food, education, healthcare, social supports, and environment19. Using poverty alone will not be able to effectively identify neighborhoods that require more health care resources. In addition, polarizing neighborhoods into “poor” and “non-poor” oversimplifies reality20; the majority are in the middle of the distribution21. To avoid false dichotomies22, more granular levels of SDoH should be considered in analysis.Few studies have investigated the modifying effect of SDoH on demographic and clinical risk factors for SI or SA. A study based on the Canadian National Longitudinal Survey of Children and Youth data found that youth living in poor neighborhoods were at greater risk of SI and SA, and the effects of psychosocial risk factors were higher in poor neighborhoods, suggesting the need of tailoring community-based youth suicide prevention initiatives to the poverty level of the neighborhoods in order to increase their impact23. The primary objective was to evaluate whether the risks for SI or SA vary across a wide range of neighborhood-level SDoH factors, measured by the social deprivation index (SDI) at each zip code tabulation area (ZCTA). SDI is a population-level composite measure which captures multiple dimensions of SDoH, including income, education, employment, housing, household characteristics, and transportation24. The independent effects of neighborhood social deprivation on SI or SA in addition to individual-level demographic and clinical risk factors were also evaluated. The second objective was to evaluate whether neighborhood social deprivation modifies the effects of clinical and demographic risk factors for SI and SA. Because youth and adults exhibit different patterns for suicidal thoughts and behaviors1, data on youth and on adults were analyzed separately.This study utilized data on commercially insured individuals in the US. Commercial health insurance plans cover over 60% of the US population under age 65, and individuals with commercial insurance coverage tend to have higher income compared to those who have public health plan coverage or are uninsured26. A detailed description of the HCCI database can be found elsewhere27.This nationwide retrospective cohort study was conducted using health insurance claims data from the Health Care Cost Institute (HCCI), a non-profit, nonpartisan, independent research entity. The HCCI holds nationwide deidentified insurance claims data on beneficiaries covered by commercial or Medicare Advantage plans from four major health insurance companies in the US  in a manner that is compliant with the Health Insurance Portability and Accountability Act (HIPAA)28. For individuals with multiple index events in the study window, the first encounter was used as the index event. The three-year continuous enrollment criterion was imposed to fully capture individuals’ recent prior healthcare encounters. Figure We used the HCCI data from 2011 to 2015 to build the youth and adult study cohorts. Specifically, we restricted the cohorts to individuals who were (1) < 65 years of age; (2) subscribed to a commercial insurance plan with mental health coverage; (3) had an MH/SUD related outpatient visit between 1/1/2014 and 6/30/2015 (index event); and (4) at least 3 years of continuous enrollment before the index event. MH/SUD diagnoses were defined by the ICD-9-CM codes lists for mental health, alcohol use, and substance use summarized elsewhereDemographic variables sex , age group , and region  were directly extracted from the membership enrollment file in the month of the index event. Individuals’ five-digit residential zip codes were also extracted. The region  variable was derived from metropolitan statistical areas defined by the US Census core-based statistical areas (CBSAs). Individuals living in a CBSA with a population fewer than 50,000 residents were classified as living in a non-metropolitan setting.24, whereas the SDI version used in this study was updated using the 2011–2015 ACS 5-year estimates and calculated at the ZCTA level. We selected the SDI instead of other measures for two reasons: (1) social deprivation assessed with the SDI is associated with the individuals’ health and health quality29, as well as hospitalization and mortality risks30; and (2) SDI has a version developed at the ZCTA level, making it easier to map to the zip codes recorded in our claims data. Quintiles of SDI were used to allow for deprivation patterns to vary across different characteristics and to preserve the complexity of the effects of disadvantaged neighborhoods. Higher SDI quintile indicates a higher level of social deprivation.Individuals’ neighborhood deprivation level was defined by the quintile of the social deprivation index (SDI) at individuals’ residential zip codes. SDI is a composite measure ranging from 0 to 100 developed to quantify the socio-economic variation at different area levels, i.e., counties, census tracts, Zip Code Tabulation Areas (ZCTA), and Primary Care Service Areas (PCSA). SDI combines seven socio-demographic characteristics from the American Community Survey (ACS) in a particular geographical area: percent living in poverty, percent with less than 12 years of education, percent single-parent household, percent living in rented housing unit, percent living in overcrowded housing unit, percent of households without a car, and percent non-employed adults under 65 years of age. SDI was originally developed using 2005–2009 ACS 5-year estimates and calculated at the PCSA level4, which consisted of a combination of long-, mid-, and short-term diagnoses , medication prescriptions , and healthcare services . Hospitalizations were defined by having claims in the inpatient files. ED visits were identified via claims reporting Revenue Center Codes values for emergency room services: 0450-0459 (emergency room) or 0981 . Diagnoses were defined by ICD-9-CM codes  and time to SA (in days) from the time of the index event. Survival outcomes were chosen because a binary outcome for logistic regression requires insurance beneficiaries to have continuous enrollment of the commercial health insurance plan throughout the entire period of the study, including the follow-up period post index date, which will selectively remove beneficiaries who might have changed or lost employment due to their mental health status and introduce selection bias. SI was defined by ICD-9-CM diagnosis code V62.84 and SA was defined by ICD-9-CM diagnosis codes E950-E958 and E980-E988 (undetermined intent). ICD-9-CM codes E980-E988 were included because using only E95X.X had low sensitivity in identifying a suicide attemptThe distributions of outcome variables (time to SI and SA post index event), demographic characteristics, and risk factors were summarized for the entire cohort, each age-stratified cohort, and each age-stratified cohort by SDI quintile. Chi-square tests were used to compare the distributions of variables between the youth and the adult cohorts, as well as across SDI quintiles within each cohort.4, which contained all the clinical risk factors described in Section "For each outcome variable in each cohort, a sequence of Cox proportional hazards models were fit for various purposes: (1) Model 1 was fit to validate the clinical risk factors identified by othersAll data management and analyses were performed using R statistical software . All methods were carried out in accordance with the HIPAA compliance. This study was reviewed, approved, and informed consent was waived by the Institutional Review Board at Weill Cornell Medicine.Between 1/1/2014 and 6/30/2015, we identified 317,383 individuals younger than 65 years with an index outpatient mental health visit. Among them, 124,424 aged < 25 (defined as youth) and 192,959 were aged 25–64 years (defined as adults). Youth had higher post index event prevalence rates of SI and SA than adults: The post 7-, 30-, 90-, 180-, and 365-day SI rates were 2.98%, 3.76%, 4.76%, 5.65%, and 6.66% among youth, respectively, and 1.16%, 1.50%, 1.91%, 2.36%, and 2.80% among adults, respectively. The post 7-, 30-, 90-, 180-, and 365-day SA rates were 0.40%, 0.61%, 0.92%, 1.25%, and 1.67% among youth, respectively, and 0.16%, 0.24%, 0.38%, 0.53%, and 0.69% among adults, respectively. The SI and SA rates for youths were significantly higher than those of adults at each time period of the outcome . Details of the distributions of the outcome variables , demographic variables, and clinical risk factors for each cohort are summarized in Table Additional main effects of demographic variables to Model 1 significantly improved prediction accuracy : interactions between SDI and age, SDI and alcohol use disorder diagnosis in the past 3 years, SDI and SA in the past 1 year, and SDI and MH emergency department (ED) visits in the past 3 months Fig. . OverallSpecifically, youth under 18 years had higher hazard rates of SI than youth aged between 18 and 24 years; the magnitude of the elevated risk of SI decreased as the neighborhood deprivation level increased (higher SDI quintiles), with the exception of the first two quintiles, where the risks were comparable . Having an alcohol use disorder diagnosis in the past 3 years was associated with an increased risk of SI for individuals from neighborhoods that belonged to the 2nd SDI quintile , but this relationship was not significant for individuals from neighborhoods of other SDI quintiles. Having mental health-related ED visits in the past 3 months was associated with an increased risk of SI for individuals from neighborhoods that belonged to the 2nd  and 4th  SDI quintiles. However, this relationship was not significant for individuals from neighborhoods of other SDI quintiles.Additional main effects of demographic variables to Model 1 were significant : interactions between SDI and SA and schizophrenia in the past 3 years, SDI and SA in the past 3 years, SDI and Benzodiazepine prescription in the past 3 months, and SDI and mental health-related ED visits in the past 1 year. Hazard ratios (HR) of SA by SDI for each risk factor are summarized in Fig. Specifically, having SA in the past 3 years was associated with an increased risk of SA for individuals from neighborhoods that belonged to the 2nd , 3rd , 4th , and 5th  SDI quintiles, but the association was not significant for individuals from neighborhoods that belonged to the first SDI quintile. Taken together, the effect of having SA in the past 3 years increased with neighborhood deprivation level (higher SDI quintiles). Having SA and schizophrenia in the past 3 years was associated with an increased risk of SA for individuals from neighborhoods that belonged to the 2nd SDI quintile , but this relationship was not significant for individuals from neighborhoods of other SDI quintiles. Having benzodiazepine prescriptions in the past 3 months was associated with an increased risk of SA for individuals from neighborhoods that belonged to the 2nd SDI quintile , but this relationship was not significant for individuals from neighborhoods of other SDI quintiles. Having mental health-related ED visits in the past year was associated with an increased risk of SA for individuals from neighborhoods that belonged to the 1st SDI quintile , but this relationship was not significant for individuals from neighborhoods of other SDI quintiles.Additional main effects of demographic variables and SDI were significant  are summarized in Table Additional main effects of demographic variables to Model 1 were significant  are summarized in Table The main finding of this study is that neighborhood level social deprivation impacts the demographic and clinical risk factors of SI and SA differently among commercially insured youth and adults. Among youth, SDI interacted with many risk factors for both SI and SA. The modified effects were usually larger in individuals from less extreme SDI neighborhoods. In adults, SDI was positively associated with SI but not with SA.34. The post 90-day SA rate among youth and adults was 0.92% and 0.38%, respectively. The post 90-day SA rate among adults was within the range of post 90-day SA rates reported by Simon et al., which were 0.26% among primary care outpatient visits and 0.62% among mental health specialty visits4.Among those who had an outpatient mental health or substance use related encounter, SI and SA rates were consistently more prevalent among youth than adults. This finding aligns with a multi-center study of individuals with outpatient mental health conditions conducted during the same study period18. However, with more granular information on neighborhood disadvantage level , we were able to uncover that social deprivation had stronger impact on middle neighborhoods50, which was not apparent in the previous analysis in which a dichotomized measure of neighborhood poverty level was used.We found that, among youth, the impact of social deprivation on the effects of risk factors for SI and SA was usually not the largest in the most deprived or the least deprived neighborhoods. For example, having an alcohol use disorder diagnosis in the past 3 years only impacted youth’s SI among those living in neighborhoods that belonged to the 2nd SDI quintile. A potential explanation would be that parents or educators from other neighborhoods pay more attention to youth’s mental health once they are diagnosed with alcohol use disorder, so that their risk of future SI was more effectively reduced. However, more in-depth studies are needed to understand these magnified effects. Our findings on the youth cohort were consistent with an earlier study on Canadian youth where risk factors for SI and SA varied by neighborhood poverty level36. However, one study found a positive association between relative deprivation in income and SA36, and another study observed a positive association between area deprivation and SA37. Among severely depressed adults, one study reported no association between area-level social deprivation and SI or SA38.Among adults, neighborhood social deprivation level did not modify the effects of clinical and demographic risk factors on SI or SA. However, the main effects of SDI were still significantly associated with adult SI; individuals from more deprived neighborhoods had a higher risk of having SI. The main effects of SDI were not associated with adult SA, after controlling for demographics and clinical risk factors. Several studies also reported a positive association between low income and SI41. However, mental health problems and substance usage such as depression and exposure to drugs and alcohol are rare in very young children42. Therefore, we did not expect a significant portion of children under 12 in our youth cohort. Third, although fitting survival models relaxed the continuous enrollment criterion, we still imposed a three-year before index event continuous enrollment criterion in order to fully capture individuals’ prior healthcare utilization encounters. Individuals with and without continuous insurance plan enrollment might have very different socioeconomic status27. Fourth, since we used claims data for analysis, any encounters that were not billed to the beneficiaries' insurance plan could not be captured. Fifth, analysis in this study was conducted on the commercially insured population. Over 50% of the US population was covered by employer-sponsored health insurance plans43, making commercial insurance the largest insurance coverage type in the US. In our study, the average neighborhood-level percentage of population under 100% Federal Poverty Level (FPL) varied by SDI quintile, from 0.04 to 0.24 in the adults cohort and from 0.04 to 0.23 in the youth cohort (Supplementary Information Figs. 44. Therefore, although the commercially insured population underrepresents people with lower incomes, in our cohorts, there was still enough variability in income to not compromise our findings. Evaluating the impact of social deprivation on risk factors for SI or SA among Medicaid enrollees will be a future direction of our study. Finally, our analysis did not include patients’ race/ethnicity information. It is well documented that suicidality, as well as risk and protective factors for suicidality vary by race and ethnicity47. A recent study also found racial/ethnic disparities in the performance of suicide predictive models48. Racial/ethnic minorities are disproportionately represented in deprived neighborhoods49, therefore, social deprivation might partially explain the racial/ethnic disparities in suicidality. Further studies are needed to evaluate whether SDI mediates the effect of race/ethnicity on suicidality.There are several limitations of our study. First, the deprivation level we investigated was at the individuals’ neighborhood level, which may or may not reflect the individuals’ actual socio-economic status. Second, the HCCI dataset does not provide more detailed brackets for beneficiaries under the age of 18 years. Therefore, we were unable to define the youth cohort as “aged between 12 and 25 years,” which is more commonly reported in the literature50, which is the demographic group sometimes being overlooked by researchers, policy makers, and health professionals planning suicide prevention51.In conclusion, our study provides information on the effects of neighborhood-level social deprivation on suicidal behaviors in youth and in adults. Community-based suicide prevention initiatives may take into considerations the targeted individuals’ age. Our findings suggest that community-based suicide prevention initiatives for adults should target severely deprived neighborhoods. Interventions for youth should focus on those residing in neighborhoods with middle social deprivation levelSupplementary Information.


Taken together, these results indicate that altered amino acid uptake and metabolism could contribute to the acquisition of vinblastine resistance in blood cancer cells. These results will be useful for further research on human cell models.Cancer therapies use different compounds of synthetic and natural origin. However, despite some positive results, relapses are common, as standard chemotherapy regimens are not fully capable of completely eradicating cancer stem cells. While vinblastine is a common chemotherapeutic agent in the treatment of blood cancers, the development of vinblastine resistance is often observed. Here, we performed cell biology and metabolomics studies to investigate the mechanisms of vinblastine resistance in P3X63Ag8.653 murine myeloma cells. Treatment with low doses of vinblastine in cell media led to the selection of vinblastine-resistant cells and the acquisition of such resistance in previously untreated, murine myeloma cells in culture. To determine the mechanistic basis of this observation, we performed metabolomic analyses of resistant cells and resistant drug-induced cells in a steady state, or incubation with stable isotope-labeled tracers, namely,  Current pharmaceutical treatments in anticancer therapies use different compounds of synthetic and natural origin. However, despite some positive results, relapses are common, as standard chemotherapy regimens are not fully capable of completely eradicating cancer stem cells . The pheOf all the mechanisms described above, the failure of chemotherapies is most commonly attributed to two main causes: multidrug resistance (MDR) ,16, causA wide range of natural products (NPs) derived from plants, marine organisms and microorganisms have been introduced into medical treatments and used to optimize the development of new potent and tolerated drugs . These m13C 15N-amino acids. The obtained results clearly show the existence of differences at the metabolic level among the untreated cells (control cells), resistant cells and drug resistant-induced cells. Our results will inform further investigations aimed at testing the impact of vinblastine on human cancer cell lines and primary tumors.In order to improve the knowledge of drug resistance related to vinblastine use, we report here the results of our investigations based on the treatment of P3X63Ag8.653 murine myeloma cells by incubation with low concentrations of vinblastine in culture media . This tr50) [50 value (5 nM) showed a significant increase in cell viability of 86.09 ± 3.08%. Similarly, Ind cells, i.e., those grown for 24 h in culture medium from the resistant cells (IM24), but not for 7 days with low dosages of vinblastine, showed 92.69 ± 2.09% cell viability, thus confirming that drug resistance was induced in P3X63Ag8653 cells.Considering the MTT assay as a reliable tool for measuring cell viability and proliferation, the assays were performed to check for drug resistance in resistant cells (Res) and induced cells (Ind), and the results obtained are shown in 50) ,44. The Metabolomic analyses of cell pellets and culture media were performed for sensitive and resistance cells, before or after induction of resistance. Upon the identification and selection of sensitive and resistant cells, untreated cells underwent metabolic characterization to determine the basal metabolic adaptations that could contribute to resistant mechanisms . The results are tabulated in This reaction is coupled with the synthesis of polyamines, such as spermidine and spermine, both of which are specifically increased in Res and Ind cells . AdditioOf all the pathways differentially regulated in both Res and Ind cells, amino acid metabolism ranked among the most significantly altered factors. Therefore, tracking experiments with stable isotope-labeled amino acids in culture were performed to determine whether the accumulation of these amino acids in Res and Ind cells was attributable to increased uptake or decreased consumption for catabolic or anabolic purposes. The results indicate that none of the amino acids were imported more rapidly by resistant or induced resistance cells than in controls . The obsOver time, the search for molecules capable of interfering with cancer cells has resulted in the concomitant observation of cells that survived the treatments. Such results confirm that despite the promising anticancer properties of many compounds, the incomplete eradication of tumor cells is a common process, which is driven by the development of (or positive selection for) clones that are resistant to/tolerant of the anticancer agent. To shed light on the mechanisms driving such resistance/tolerance, we therefore initiated the investigation of surviving cells, and the results reported here clearly show the possibility of inducing vinblastine-resistant murine myeloma cells, and that previously untreated cells were found to be resistant if grown in IM24 medium. Drug resistance is a complex process that is influenced by several factors. Here, we show that vinblastine-resistant cells are characterized by metabolic remodeling even prior to exposure to the chemical, which could impact the cells’ ability to counteract drug-induced stress and, possibly, promote a possible epigenetic transition, resulting in “Induced” cells. For example, changes in the extent of methionine uptake and metabolism are suggestive of potential alterations in methylation reactions, redox maintenance, polyamine synthesis and coupling to folate metabolism, thereby coordinating nucleotide and redox status ,46. FurtOne of methionine‘s most important functions is its contribution to intracellular methylation by serving as the sole source of the universal methyl donor S-adenosyl-methionine (SAM); SAM is a necessary substrate for all methylation reactions, including those that modulate gene expression , phospholipid integrity, the activity of signaling pathways, the damage repair of isoaspartyl proteins and the biosynthesis of polyamines .Of note, lactate was one of the top 10 most increased metabolites in Res cell media compared to Ctrl, suggesting potential glycolytic reprogramming in this group. Since lactate can function as a signaling molecule, it can regulate gene expression through multiple mechanisms, including the lactoylation of histone proteins . From metabolomics tracing experiments with stable isotope-labeled amino acids, the elevated steady state levels of methionine in resistant cells seem not to be explained by increased uptake. This result is suggestive of the decreased utilization of intracellular methionine in resistant cells, perhaps as a function of decreased de novo protein synthesis  or decreased epigenetic regulation via methylation events . Altogether, the metabolic changes observed could be driven by (i) the law of mass action, as a result of the increased consumption of specific amino acids—perhaps to fuel anaplerotic reactions to sustain viability/proliferation; (ii) the decreased oxidation of specific amino acids at the mitochondrial level, consistent with a metabolic reprogramming towards glycolysis; (iii) epigenetic effects due to a long exposure to the methionine and lactate overproduced by resistance towards induced cells.This study shows several limitations. First of all, validation in human myeloma cell lines and ensuring translatability into pre-clinical animal models in vivo will be necessary, and is currently underway. Like most single omics studies, tentative interpretations of the data presented here will require orthogonal validation with complementary approaches, such as proteomics and transcriptomics, or more targeted analyses . To determine whether the observed metabolic changes are correlative to or causative of the observed resistant/tolerant phenotypes, experiments aimed at mechanistically testing specific hypotheses  will be needed. However, the findings described in this study represent the first step towards understanding the role of metabolic reprogramming in conferring vinblastine resistance, narrowing the scope of future, more focused investigations.2. Upon reaching confluence, the cells were passed into new culture vessels in a 1:10 ratio and the medium was changed every three days.Cells from a murine myeloma P3X63Ag8.653 cell line , derived from the Balb/c strain of mice, were cultured in RPMI-1640 medium supplemented with 10% of heat-inactivated fetal bovine serum and 2 mM glutamine, and incubated at 37 °C in a humidified atmosphere with 5% of COThe use of the activity of the mitochondrial enzyme succinate dehydrogenase as a detector of cell viability is possible through its ability to reduce the tetrazolium dye MTT, or 3--2,5-diphenyltetrazolium bromide, into its insoluble formazan crystals. Since a reduction in MTT is correlated with cell metabolic activity, metabolically active cells are expected to yield high MTT values.4 cells/well in 100 µL of complete RPMI medium were seeded in a 96-well micro plate, and after 24 h of incubation different concentrations of vinblastine  were added. After 24 h, the medium containing the treatment was removed and 100 μL of MTT solution, at the final concentration of 0.5 mg/mL, was added to each well and incubated in the dark at 37 °C for 3 h. The formazan crystals produced were dissolved in 100 μL of DMSO and the optical density (OD) measured at 595 nm by a Tecan Sunrise™  UV-Vis spectrophotometer. The values were expressed as a percentage of cell viability, obtained using the following equation: The MTT assay was performed to evaluate the effect of vinblastine on cell viability . In tota50 Calculator  [50). The values were repeated three times and are reported as mean ± SD.The percent cell viability data, obtained by converting the optical density values as previously described, were processed with an AAT Bioquest ECCA, USA)  to obtai4 cells/mL in a six-well plate and treated for 7 days with low doses of vinblastine , and 5 nM equal to 1/4 of the of EC50 value against P3X63Ag8.653 cells (20 nM). At the end of the treatment period, 1.5 × 104 Res cells/mL were collected and seeded in a six-well plate with fresh culture medium to obtain an Induction Medium (IM24). After 24 h, the IM24 was collected and centrifuged twice at 1500 rpm for 5 min at room temperature. After discarding the pellet (consisting of Res and cell debris), the resulting medium was used to culture P3X63Ag8.653 cells, and after 24 h Ind cells were obtained. P3X63Ag8.653 cells cultured under the same conditions as Res and Ind cells were used as control .In order to induce drug resistance to vinblastine, murine myeloma cells were seeded at a concentration of 1.5 × 104 cells/well of Ctrl, Ctrl Res, Ctrl Ind, Res and Ind were seeded in a 96-well plate, treated for 24 h with 20 nM vinblastine and subjected to MTT assay to evaluate the percentage of cell viability. To evaluate resistance to vinblastine 2 × 106 cells were collected and extracted in 1000 µL of ice cold extraction solution (methanol:acetonitrile:water 5:3:2 v/v/v). Suspensions were vortexed continuously for 30 min at 4 °C. Insoluble material was removed by centrifugation at 18,000× g for 10 min at 4 °C, and supernatants were isolated for metabolomics analysis by UHPLC-MS. Media samples (20 µL) were extracted as described above, in a ratio of 1:25 with the extraction solution.Approximately 2 × 10Analyses were performed using a Vanquish UHPLC coupled online to a Q Exactive mass spectrometer . Ten microliters of sample extracts were loaded onto a Kinetex XB-C18 column . The samples were analyzed using the 3 min isocratic condition or a 5, 9, and 17 min gradient, as described ,52. 13C and 15N isotopes were performed using MAVEN  [Metabolite assignments, amino acids-labeled tracing experiments, isotopologue distributions, and correction for expected natural abundances of NJ, USA) .Data were expressed as mean ± standard deviation (SD) from three independent experiments. Statistical significance between examined samples and control values was determined using one-way analysis of variance. Graphs and statistical analyses  were processed with GENE-E , and MetaboAnalyst 5.0 . Over time, in our search for molecules capable of interfering with cancer cells, we observed cells surviving drug treatment, thus confirming the development of (or positive selection for) clones that are resistant to/tolerant of the anticancer agent. The phenomenon of drug resistance gives cancer resistance and tolerance to anticancer agents. To improve the knowledge about drug resistance linked to the use of vinblastine, a natural product widely used in clinical applications for cancer therapy, as a first step we conducted investigations on murine myeloma cells. We obtained vinblastine-resistant cells and observed the ability of their culture medium to induce vinblastine resistance in myeloma cells never before cultured in the presence of vinblastine. Further, the metabolomic analysis clearly showed that some metabolites produced higher amounts in both resistant and induced cells, and confirmed a process of reprogramming the metabolism of murine myeloma cells kept in culture in the presence of vinblastine (resistant cells), and in murine myeloma cells not treated with vinblastine and cultured in a culture medium taken from resistant cells. Our results will be useful for further investigations of human cancer cells.


Sanitation systems involving onsite storage of faecal matter cause excreta to undergo transformation and primary treatment in-situ. However, little is known about the transformation pathway followed by fresh faeces while contained in situ. The current paper investigated this transformation under ambient conditions during a 16-week in-situ-storage period. Moisture content, drying kinetics, rheological, physicochemical, and thermal properties were analysed to determine the effects of ageing. The faeces experienced dehydration, mainly affecting moisture-dependent characteristics. Moisture content decreased from 79%wt to 26%wt, and water activity of 0.67, which corresponds mainly to the removal of interstitial bound water, reducing mass by 72%. The decreasing moisture content expectantly reduced drying ability, flowability and thermal properties . During this period, negligible biodegradation was recorded (volatile solids reduced by 3%), resulting in consistent chemical oxygen demand, particle size, carbon content and calorific values. Ammonium and nitrates decreased, but total nitrogen remained unchanged. Therefore, ageing affects nitrogen chemical forms and not nutrient composition. The findings demonstrate the benefits of source separation and in particular ventilated storage as a passive way to pre-treat and recover resources from faecal material. During storage, the properties of faeces can undergo modifications in-situ, which can impact the pathogen, nutrient and organic content in the faeces . A substThis study aims to temporally evaluate the properties of human faeces from the time of generation to 16 weeks of storage, replicating dry onsite sanitation systems that have ventilation and employ source separation in their design. The specific objectives are to determine the impact of faecal ageing on the material properties, downstream treatability and possible reuse pathways. The focus of this work was on the change in properties of fresh faeces over time in storage, so urine, anal cleansing material and flush water were excluded. The outcome from this investigation can be applied in conventional UDDTs or other sanitation technologies where the faeces are stored for a given period prior to their treatment or disposal.The faecal samples for this study were obtained from donors from Howard College campus at the University of KwaZulu-Natal (UKZN), Durban, South Africa, and other areas in the college vicinity. An economic incentive was offered to volunteers to donate fresh faeces for the study. They were required to be over 18 years of age and willing to co-operate on the condition of anonymity. Donors received 1 L buckets (pre-weighed and numbered) to facilitate easier collection of faecal samples from the individual donors. The stipulation was to bring the stool in the bucket  to the laboratory within 24 hours. The study received a total of 73 samples from donors where each sample was immediately weighed, sealed, and placed in the cold room at 4 °C until use, to prevent microbial activity and preserve their physicochemical properties. Samples stayed under preservation until enough samples had been collected to cater to all experimental work. Therefore, the cold room storage time ranged from less than a day to one week depending on when the sample was brought in. The Biomedical Research Ethics Committee (BREC) from UKZN approved this study through the ethical clearance with reference number BREC/00000524/2019 before engaging potential volunteers and procuring samples.2) to volume (1133 cm3) ratio of 1:11 . Each bucket contained 900 g of fresh faeces  will be presented in this paper.The analyses of the samples were performed according to the Methods for Faecal Sludge Analysis .Samples of mass 10 – 20 g were dried in the oven at 105 °C for 24 hours to determine moisture content and volatile solids (VS) by igniting the dried residue from the moisture content analysis in a muffle furnace at 550 °C for 2 hours.Radwag Max 50 Thermal Moisture Analyser. The balance in the analyser continuously weighed the sample during drying, and the sample weight was captured every 30 seconds, which allowed determination of the kinetics of the process, i.e. the drying curve (moisture content versus time) and the Krischer curve (drying rate versus moisture content). The Moisture Analyser directly computed the drying curve, whereas the Krischer curve was obtained by the user by calculating the drying rate  at different instants. The drying rate was normalised to g/min moisture removed assuming all samples had the same surface area (6361 mm2).The influence of ageing on the drying behaviour of faecal material was determined from drying tests. A 1.0 – 1.5 g thin film of homogenised faeces spread on a 90 mm diameter aluminium tray was completely dried at 105 °C using a AquaLab Tunable Diode Laser-TDL water activity meter measured the water activity of a 10 ml sample at 25 °C. The instrument maintained this temperature at an accuracy of ±0.005 °C.Water activity estimates the degree to which moisture is bound to the solids in the faeces. An Anton Paar Rheometer MCR72. The flow and deformation of samples were investigated using rotational tests at controlled shear rates. Each test subjected 100 – 150 g of faecal samples to a shear rate that increased from 0.1 to 1 000 s−1 for approximately 15 minutes, using a vane in cup geometry. The RheocompassTM software captured the rheology data, shear strain-shear rate and viscosity-shear rate relationships on a logarithmic scale.Viscosity and shear stress tests on ageing faecal matter were performed at 25 °C using an Yield stress is the lowest shear stress magnitude that needs to be applied to faeces to initiate their flow. The property was determined by plotting viscosity against shear stress . The yieMalvern Mastersizer 3000 particle size analyser. The laser diffraction method used by this analyser assumes particles as spherical. Sample preparation involved dispersing a quarter of a 4 ml-spatula of faecal material in enough water to form a well-mixed slurry in a crucible. Slurry droplets were dispersed in 600 ml of water, and the instrument measured the particle size.The change in particle size of the faecal matter samples at different storage times was analysed using the 4+), nitrate (NO3−), carbon (C) and nitrogen (N) content of samples were analysed to establish how the chemical composition of fresh faeces evolved whilst undergoing ageing. COD, NH4+ and NO3− were measured using spectrophotometry , while C and N were measured using a LECO TruMac CNS analyser using pre-weighed masses of 0.1 – 0.2 g of faeces.The chemical oxygen demand (COD), ammonium , producing gases such as H2 and CO2, which are subsequently converted to CH4 and H2S by hydrogenotrophic bacteria  after achieving the same level of moisture content. For example, the faecal sample at day 0 shown a drying rate of approximately 0.1 g/min at 50%wt moisture content whereas the sample with an age of 8 weeks presented a drying rate of approximately 0.3 g/min at the same moisture content. The ageing process seemed to have allowed moisture redistribution within the samples during the natural drying process. This behaviour is analogous to intermittent drying mechanism wherein materials are rested in between drying periods to allow moisture redistribution within the sample , leadingFrom the results obtained in this section, it is evident that aging passively improves the treatability of the sludge for drying processes, particularly at the first phase of the transformation.The mechanical properties of the faecal samples were measured to determine how sample fluidity evolved during ageing. Three parameters were investigated: viscosity as a function of shear rate (flow curves), yield stress and particle size distribution. The results are displayed in Sample viscosity reduced with increasing shear rate, demonstrating shear-thinning properties a, as alsYield stress increased exponentially from 144 to 1736 Pa during a storage time of 6 weeks and a corresponding solids concentration of 21 to 38%wt total solids b. This eThe fact that increased storage promoted comparatively lower yield stresses compared to faecal sludge and other faeces matrices at the same total solid content implies that this method increased the treatability of faecal material by requiring lower energy for its mechanical handling. The mechanisms causing this phenomenon are unknown and requires further investigation.Particle sizes for all samples ranged between 0.49 and 624 μm . There wThe physiochemical properties of the faeces at different storage times were determined to better understand the changes the material underwent as it aged and to evaluate the potential reuse of the aged faeces as fertilizer and biofuel through the analysis of organic, nitrogenous, and calorific content .Table 1PInitially, the volatile solids content was ∼0.86 g/g dry sample, close to 0.84 g/g dry sample measured for fresh faeces by Fresh faeces exhibited a COD of 1.02±0.03 g/g dry sample, which fell within the range of 0.567−1.45 g/g dry sample found in different studies on fresh faeces . The disCarbon was the primary element in the faecal samples with a content of 0.5 g/g dry sample, representing half the mass of the material, as also found by In summary, minimal degradation was recorded, as the method of ventilated dry storage may slow both anaerobic and aerobic degradation processes, which are usually encountered in non-source separated faecal sludge and centralised sewage sludge respectively. This result could be because material dehydration reduced the microbial activity responsible for degradation, through decreasing water activity.Nitrogen is crucial in the metabolism and growth of plants and usually gets assimilated as ammonium and nitrate. Therefore, it is essential to know the content of nitrogen and nitrogenous molecular nutrients in faeces while ageing to determine their value as an agricultural product. The total nitrogen remained in the range of 0.05-0.08 g/g dry sample, representing 5-8% of the faeces on a dry composition, similar to study findings by When The average calorific value for fresh faeces in this study was ∼ 22 MJ/kg dry sample, lower than that measured by The thermal properties, including the thermal conductivity and heat capacity, were measured for the faecal samples for 16 weeks. The thermal conductivity determines the inherent ability of the samples to conduct heat. The heat capacity indicates the amount of heat required to increase the temperature of the faecal samples.Thermal conductivity varied during storage by decreasing over time a. The inSimilar to the thermal conductivity, the heat capacity experienced a reduction over time, also related to the loss in moisture from the samples during storage a and b. This study has evidenced that ventilated faecal storage passively removes water from faeces without compromising the agricultural or biofuel resource recovery potential. The implications for sustainable non-sewered sanitation are substantial. Firstly, the heat energy saved to reduce 900 g of faeces from a moisture content of 79.05%wt to 25.9%wt is at least 1500 kJ, calculated from the latent heat of pure water vaporisation and without considering the increased energy required for removing bound water at lower moisture contents. This removal of moisture resulted in a mass reduction of 72%, which drastically reduces transport costs. The extent of drying after 16 weeks provided sufficient pre-treatment for the combustion of faeces as autothermal combustion conditions were met  which encourages biochemical reactions, and ventilated conditions which prevent anaerobic digestion. This therefore differentiates the material to other aged faecal sludge types such as septic tank or pit latrine sludge. The faecal material was able to maintain a composition rich in organic matter, nitrogen and a high calorific content, promoting aged faecal material as an attractive alternative for crop-farming products and biofuel, and even construction material .This study showed the benefits of faeces storage under ventilated conditions. The outcomes from this investigation demonstrated that faecal dry storage after source separation is a passive, yet effective method to dry and recover resources from faeces, which is relevant for the global demand for sanitation facilities. However, to enable this in practice, existing source separation systems must incorporate a design which separates fresher faeces from older ones. Further research is warranted to determine the impact of different surface area to volume ratios to understand how to design and optimise passive dehydration chambers, coupled with evaluating safe handling by pathogen inactivation. It would also be interesting to understand the specific mechanisms to why source separated aging improves material treatability in terms of drying rate and rheology and to determine whether it also applies to dewatering and stickiness.10.13039/100000865Bill & Melinda Gates Foundation [Grant number OPP1069575].This research work was supported by the T.M. Chatema: Conceptualization, Methodology, Validation, Formal analysis, Data curation, Writing – original draft, Investigation, Visualization. E. Mercer: Writing – review & editing. S. Septien: Conceptualization, Methodology, Writing – review & editing, Visualization, Supervision, Project administration. J. Pocock: Conceptualization, Methodology, Writing – review & editing, Supervision. C.A. Buckley: Conceptualization, Supervision, Project administration, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


To explore the effects of chemical and physical parameters on embryo developmental competence, we conducted a systematic search on PubMed for peer-reviewed original papers using specific keywords and medical subject heading terms. Studies of interest were selected from an initial cohort of 4141 potentially relevant records retrieved. The most relevant publications were critically evaluated to identify the effect of these parameters on embryo development. Moreover, we generated a literature score (LS) using the following procedure: (i) the number of studies favoring a reference group was expressed as a fraction of all analyzed papers; (ii) the obtained fraction was multiplied by 10 and converted into a decimal number. We identified and discussed six parameters . Moreover, we generated a LS according to five different comparisons . Only two comparisons (37 °C vs. <37 °C and 5% vs. 20% oxygen) yielded a medium-high LS , suggesting a prevalence of studies in favor of the reference group (37 °C and 5% oxygen). In summary, this review and LS methodology offer semi-quantitative information on studies investigating the effects of chemical and physical parameters on embryo developmental competence. In the field of assisted reproductive technology (ART), human embryo culture plays a pivotal role in the success of in vitro fertilization (IVF) treatments. The delicate and intricate nature of preimplantation human development demands a meticulously controlled environment. During human embryo culture, chemical and physical parameters play a crucial role in embryo development and viability ,2,3. TheOil overlay has several important functions and benefits: (i) gas exchange, (ii) temperature stability, (iii) pH regulation, (iv) preventing contamination, and (v) minimizing disturbance. The inherent chemical and physical properties of the oil exert a significant influence on this vital aspect. These properties play a crucial role in shaping and determining the outcome, emphasizing the importance of understanding and considering them when working with human embryo culture . Light eA systematic search was conducted on PubMed to identify peer-reviewed original research articles related to the effects of chemical and physical parameters on development and clinical outcomes. The search strategy involved using relevant keywords and Medical Subject Heading (MeSH) terms. These keywords and Mesh terms were combined in various overlapping combinations to ensure the identification of publications specifically relevant to the topic: (“temperature” OR “oxygen” OR “humidity” OR “oil overlays” OR “light” OR “pH”) AND . Furthermore, additional studies were identified by meticulously examining the reference lists of the selected publications. Full manuscripts were obtained for all the selected papers, and a thorough evaluation of the articles was conducted to make a final decision regarding their inclusion in the review. The most relevant publications, i.e., those concerning the effects of chemical and physical parameters on embryo development, as well as clinical outcome, were critically evaluated and discussed.Two reviewers (AB and FT) independently assessed all studies for inclusion or exclusion. Disagreements were solved in discussion with a last author (LP). During the first screening, titles and abstracts were investigated and studies with a lack of any relevance were excluded; review articles were also excluded . The remThe major limitation of our LS is that it does not take into consideration the variable number of patients/embryos per study to which a different weight should be attributed. Moreover, another limitation is its inability to consider whether or not embryos are taken in and out of the incubator for embryo assessment. It is important to note that our approach does not seek to replace the results yielded using a meta-analysis, but rather serves as a complementary elaboration, enriching the written information presented in the manuscript.Oxygen plays a vital role in supporting embryo metabolism and development. In the female reproductive tract, oxygen concentration is typically around 2–8% . Thus, iInterestingly, recent studies investigated the use of sequential oxygen tension . This is probably to mimic the natural conditions of in vivo embryo development. A sibling zygote randomized control trial showed, although a small sample size, better blastulation rate when oxygen tension is reduced from 5% to 2% on day 3 for extended embryo culture (day 5) , in contWe analyzed 18 studies for the LS calculation, 10 focused on comparing between 5% and a 20% oxygen concentration, resulting in a LS of 7. Additionally, eight studies examined the comparison between monophasic (5%) and biphasic (5–2%) culture oxygen tension, resulting in a LS of 5. These findings suggest there is no evidence that biphasic culture (5–2%) is better than monophasic culture (5%), especially in terms of clinical outcomes .Maintaining the correct temperature is essential for proper gamete function and/or embryo metabolism and development . DeviatiWe analyzed six studies for the LS calculation, obtaining a high LS of 8.3 , suggestHumidity plays a significant role in the incubator environment. Maintaining optimal humidity levels is crucial to prevent excessive evaporation from the culture medium, which can affect embryo development by altering osmolality and pH . HoweverIn conclusion, while basic research studies consistently indicate alterations in pH and osmolality of the culture medium under DC , it is important to note that this consensus does not align with the clinical evidence. For the LS calculation, we analyzed four studies and obtained a LS of 5 , suggestIn human embryo culture, an oil overlay is often used as a covering layer on top of the culture medium to create a specific environment for the embryos. One of the primary purposes of an oil overlay is to facilitate appropriate gas exchange within the embryo culture system, to minimize evaporation, and help maintain a stable environment. Despite this, evaporation could also occur with the use of a mineral oil overlay ,46,73. AIn vivo embryos, which develop inside the female reproductive system, are not directly exposed to light. However, during IVF treatments, embryos may be exposed to light, albeit in a controlled and regulated manner. There is scientific literature available on light exposure and its potential effects on embryo development; nevertheless, contradictory results have been obtained. In a study focusing on pre-implantation rabbit embryos, researchers found that subjecting the embryos to 24 h of visible light exposure did not lead to a significant increase in DNA ploidy abnormalities , in cont2) levels due to the bicarbonate buffering system, in which changes in CO2 concentrations impact the production of carbonic acid, consequently leading to variations in pH. Adjusting the percentage of CO2 gas in the incubator is a fundamental method for precise pH control in the culture medium, which is essential for embryo development [2. Therefore, it is essential to consider altitude and air pressure to maintain a stable and optimal pH for embryo development.Culture media pH is a critical factor in human embryo culture. pH is closely correlated with carbon dioxide  and five different comparisons  . By adopIn summary, this review and proposed LS methodology offer semi-quantitative information on studies investigating the effects of chemical and physical parameters on mammalian embryo culture in order to minimize them in the practice of human IVF. Overall, we identified and critically discussed six parameters . Furthermore, we generated a LS of five different comparisons . Among these, two comparisons (37 °C vs. <37 °C and 5% vs. 20% oxygen) yielded medium-high literature scores, suggesting a prevalence of studies in favor of the reference group (37 °C and 5% oxygen). Conversely, the other three comparisons  produced a low score for 5–2% oxygen, HC, and LE.