The objective of this study was to evaluate the relationship between random capillary glucose levels in healthy pregnant women and infant size at birth and childhood growth to the age of five years. This population-based cohort study comprised 10,937 healthy mother–child dyads. Data on highest maternal random capillary glucose level during pregnancy and sequential anthropometric data on their children during the first five years of life were gathered from the Uppsala County Mother and Child Cohort. Statistical analyses were performed with linear regression and linear mixed effect regression models. We found that higher glucose level during pregnancy was associated with higher weight z-score  0.08–0.11), length z-score  and BMI z-score  at birth, adjusted for maternal BMI and country of birth, smoking during pregnancy and parity. The association did not remain at 1½, 3, 4 and 5 years of age. There was a positive relationship between higher glucose level during pregnancy and a decrease in weight z-score, height z-score and BMI z-score from birth to 5 years of age. In conclusion, higher random capillary glucose levels in pregnant healthy women were associated with greater infant size at birth, as well as decreased growth velocity in early childhood. Obesity is a great contributor to the global disease burden and causes morbidity and premature death in cardiovascular disease. The risk of obesity-associated morbidity and mortality increases for every year lived with obesity4. Children with rapid weight gain and accelerated BMI in early childhood run increased risk of sustained obesity in adult life5. Furthermore, children with accelerated height development during the first years of life are more prone to develop obesity in childhood6. Once obesity is established, it is difficult to lose weight due to homeostatic adaptations in the body7.The prevalence of childhood obesity is increasing worldwide9. Good pre-gestational and gestational glycaemic control in diabetic mothers decrease the risk of macrosomia and large for gestational age (LGA) fetuses12. Further, infants to mothers with untreated mild hyperglycaemia during pregnancy have higher birth weight than infants to mothers with normal blood glucose levels during pregnancy, independent of maternal BMI, parity and maternal educational level13.Diabetes mellitus during pregnancy is strongly associated with greater size at birth16. So far there are conflicting results about the relationship between maternal glucose levels within normal range in pregnancy and risk of obesity in early childhood. One study investigated the growth in children from birth until 2 years of age in non-diabetic mothers. In this study, they examined the relationship between the level of randomly collected venous glucose, within normal range, in gestational week 28 and childhood growth. They observed a catch-down growth in children from birth to 24–36 weeks of post-natal age in children with mothers having a glucose level within the highest third of normal range17. Another study provided some evidence that maternal glucose levels within normal ranges during pregnancy correlate positively to adiposity in 10–14 year old children18.In addition, even infants with mothers who have not developed gestational diabetes are at higher risk of being born LGA or with macrosomia the higher maternal glucose levels during pregnancy19. The screening procedure for gestational diabetes are either based on offering all pregnant women OGTT or a risk factor-based screening, where only those with risk factors are offered OGTT. One risk factor is a high non-fasting random capillary blood glucose level, a test that is repeatedly offered during pregnancy in all women in Swedish maternal health care22.Different diagnostic and screening methods are utilised for gestational diabetes (GDM). The diagnosis is based on an Oral Glucose Tolerance Test (OGTT), although cut-offs for the diagnosis differ between, and sometimes, within countriesUntil now, most studies that have investigated how glucose levels during pregnancy affect size at birth and childhood growth have been using fasting blood glucose or blood samples collected under oral glucose tolerance testing. Thus, it is not known how randomly collected capillary blood glucose levels are associated with infant size at birth or with early childhood growth until the age of 5 years. Hence, the aim of this study was to investigate the relationship between the highest level of randomly collected capillary blood glucose in healthy non-diabetic pregnant women and infant size at birth as well as growth trajectories from birth to 5 years of age.2. A majority of the mothers, 62.6% (N = 4971), had an educational level corresponding to post-secondary or university degree. Furthermore, most mothers, 84.9% (N = 6748), did not smoke at first maternal health care visit and/or in pregnancy week 30–32. Mean level of highest measured randomly collected capillary blood glucose during pregnancy was 6.2 mmol/L. Among the children, 4.4% (N = 348) were born LGA and 1.1% (N = 87) were born SGA.Of the 10,937 mother–child dyads in Uppsala County Mother and Child Cohort born between 2007 and 2015 we finally included 7,945 mother–child dyads  g and mean birth length was 50.9 (± 2.0 SD) cm. A decrease in weight, height and BMI z-score was seen from birth to five years of age. Mean weight z-score was 0.12 (± 0.9 SD) at birth and −0.08 at 5 years of age. Length z-score at birth was 0.08 (± 0.9 SD) whilst we observed the lowest level −0.02 (± 1.0 SD) of height z-score at 3 years of age. BMI z-score was 0.13 (± 0.9 SD) at birth and −0.11 (± 0.9 SD) at 5 years of age. Of all 5-year-old children, 10.9% (N = 362) were overweight and 2.2% (N = 74) were obese , Table . For eveThe range of weight z-score, height z-score and BMI z-score, between lowest and highest glucose quartile, were larger at birth than at 1½, 3, 4 and 5 years of age Fig. . We showWe found an inverse relationship between maternal glucose quartiles and weight z-score growth trajectory from birth to 1½, 3, 4 and 5 years of age. The decrease in weight z-score were least in children to mothers with glucose level within Q2 and greatest for Q4 compared with children to mothers with glucose level within Q1  of the highest random capillary blood glucose values measured in our population were collected in pregnancy week 25 or later. Nevertheless, a fifth (19.8%) of the random capillary blood glucose levels were collected before pregnancy week 13. Our data suggests that it may not matter when in pregnancy you measure a high glucose value within normal range; a higher glucose level measured at any time in pregnancy, in a non-diabetic mother, is associated with a greater size at birth and a decreased growth velocity in early childhood. Since blood glucose samples were random collected and not fasting samples there could be non-differential misclassification. Another limitation of the study is the limited information on potential confounders. We have used educational level as a proxy for socioeconomic status. Unfortunately we do not have any information on maternal or child diet and behavioural factors, such as physical activity, which might have an impact on fetal and childhood growth. Further we do not have information on anthropometry at age 2 years.A limitation of this study was that we did not have information on in which gestational week the highest glucose level is noted in each individual. This makes it hard to draw any conclusions about the importance of the timing of the glucose sample when estimating the relationship between maternal glucose level and size and growth of the child. The insulin sensitivity changes during pregnancy and decrease when pregnancy is proceeding. Secretion of insulin increases from second trimester until delivery along with the decreasing insulin sensitivityWe saw a growth pattern with a greater size at birth and a decrease in weight and BMI z-score until 5 years of age. Based on our data we cannot know the reason for this. Though we think that the greater size at birth and the decreased size at 5 years can be a result of our selected population of healthy children born at term to healthy mothers. Diseases that may affect the growth of the offspring were excluded, e.g. preeclampsia and children with malformations and chromosome aberrations. Another contributing factor may be our relatively highly educated population, Uppsala County Mother and Child Cohort, because Uppsala is a University City and the educational level is higher compared with the national average. In general, high educational level is associated with a healthier lifestyle and the growth from birth until the age of five is highly influenced by the lifestyle in the family.Randomly collected capillary glucose levels during pregnancy in non-diabetic mothers correlate with infant size at birth and a decrease in weight, height and BMI z-scores in early childhood. However, there were no associations between the maternal glucose levels during pregnancy and anthropometry at 1½, 3, 4 and 5 years of age. Factors others than glucose levels during pregnancy may be of greater importance for size in early childhood.Database of statistics of the Child Health Care Unit in Uppsala County, which holds information on repeated anthropometric measurements  and health data . Children living in Uppsala County have an attendance in child health care of 97%44.This study is based on data from The Uppsala County Mother and Child (UCMC) Cohort, a population-based cohort originally based on a local register for the Child Health Care in Uppsala County. The database comprise nearly 60,000 children born at Uppsala University Hospital, between 2000 and 2015. The UCMC Cohort use data from the Medical Birth Register (SMBR) and the Registry of Education 45, the Registry of Total Population , the Causes of Death Registry . Finally, the UCMC Cohort holds data from the Inpatient Care Registry, containing parental and child discharge diagnoses, covering all private and public hospitalizations, and the Prescribed Drug Registry with information on all prescribed drugs dispatched in Swedish pharmacies. All Swedish residents allocate a unique national registration number. With use of these national registration numbers, data from the different registers were linked.Further, the UCMC Cohort database incorporates data from a number of the Swedish health registries, including the Swedish Medical Birth Register is a nationwide register that comprises prospectively collected data of about 99% of all pregnancies in Sweden. The maternal, delivery and child health care services in Sweden provide standardized schedules and are free of charge. Every health care provider is compelled to report information about all pregnancies, with a duration of 22 + 0 weeks or longer, to SMBR36. Complications and diseases are classified and coded according to the International Classification of Diseases (ICD). SMBR applies Marsal’s intrauterine growth curves as reference standards for growth data for new-borns46. Children with weight or length + /− 2 standard deviations from mean of reference population for new-borns are defined as large for gestational age (LGA) and small for gestational age (SGA), respectively48.The Swedish 49. In pregnancy week 30–32, mothers are asked a second time about smoking habits. Randomly collected capillary blood glucose is analysed at maternal health care visits in pregnancy weeks 25, 28–29, 33 and 37.The first antenatal visit in maternal health care usually takes place before gestational week 13 and includes an interview with questions about the medical and obstetric history as well as social situation including smoking habits. Weight and height are measured and randomly collected capillary blood glucose is analysed. Gestational age is predominantly estimated with ultrasonography in first or second trimester of pregnancyThis study includes information from the UCMC Cohort of 10,937 mother–child dyads with infants born between 2007 and 2015. Children born preterm  and postterm  were excluded . We excluded children born after multiple pregnancies (n = 855), and children lacking data on birth weight z-score and SGA or LGA status . To decrease the risk of impaired growth caused by diseases in the children, we excluded children with chromosome aberrations and congenital malformations (n = 59). Individuals with data on size  at birth, 1½, 3, 4 and 5 years of age less than −5 or more than + 5 standard deviations (SD) from mean were regarded as misclassified and excluded (n = 16). Furthermore, children of mothers with inflammatory or autoimmune disease, essential and gestational hypertension, preeclampsia, cardiovascular disease, hyperthyroidism and epilepsy were excluded (n = 901). Children with mothers diagnosed with pregestational diabetes and gestational diabetes were excluded (n = 125). Mothers with missing data for highest glucose level (n = 39) and mothers with a highest value of randomly collected capillary blood glucose > 12.1 mmol/L and their children were excluded due to potential risk of maternal diabetes mellitus (n = 2). The final population consisted of 7945 mother–child dyads.2, mothers with a previous history of gestational diabetes or having a child born LGA or mothers with parents or siblings with diabetes mellitus type 2 underwent OGTT. Furthermore, OGTT was also carried out in mothers with suspected increased fetal growth based on symphyseal fundal height, weight estimated to >  + 22% or polyhydramnios at ultrasonography. Pregnant women that underwent OGTT with a fasting plasma glucose level ≥ 7.0 mmol/L or a plasma glucose level ≥ 9.0 mmol/L 120 min after glucose intake were diagnosed with gestational diabetes (GDM), and excluded from this study. Data about maternal glucose levels were gathered from maternal records and linked with register data.The highest measured randomly collected non-fasting capillary blood glucose level in mmol/l obtained at any time throughout the pregnancy was the exposure in the study. The value of the first glucose sample, collected before pregnancy week 13, were included in the data collection for statistical analyses. Glucose samples were collected capillary by midwives, as a part of routine maternal health care and analysed on point-of-care testing instruments at the maternal health unit. The instruments are provided and validated by Uppsala University Hospital laboratory and are routinely checked and calibrated to secure quality of analyses. The pregnant women were not told to be fasting before sampling. The blood samples were collected at any given point in the day and at any time in relation to meals which is the clinical practise in Sweden. In Uppsala screening with OGTT for gestational diabetes is based on risk factors, not offered to all women. Mothers with a randomly collected capillary blood glucose level ≥ 8.8 mmol/L, mothers with a BMI ≥ 35 kg/m50. The child’s BMI was calculated with weight in kg with two decimals at birth and at 1½ years and weight in kg with one decimal at 3, 4 and 5 years of age and height in metres with two decimals for all ages. Only children with anthropometric values collected two months before or after 1½, 3, 4 and 5 years of age were included.Offspring growth expressed as weight z-score, height z-score and BMI z-score at birth, 1½, 3, 4 and 5 years were the outcomes. Swedish reference curves are used for documentation of anthropometric data in child health care, and standard deviation scores (z-scores) were calculated using the Swedish reference populationTo select covariates for making adjustments for potential confounders in analyses we drew a directed acyclic graph (DAG)  of study population, and categorical variables as proportions (percentages). We made separate univariate and multivariate linear regression analyses for highest random capillary glucose level during pregnancy and the outcomes child weight z-score, height z-score and BMI z-score for ages 0 (birth), 1½, 3, 4 and 5 years, respectively. Results were expressed as beta coefficients with 95% CI.In order to take repeated measurements into account and to examine the growth trajectories of the children, we performed linear mixed effect regression model analyses. We divided the children into quartiles according to the highest level of their mothers’ random capillary blood glucose during pregnancy. The first glucose quartile (Q1) included children to mothers with random capillary blood glucose level < 5.6 mmol/L, the second quartile (Q2) 5.6–6.0 mmol/L, the third quartile (Q3) 6.1–6.7 mmol/L and the fourth quartile (Q4) ≥ 6.8 mmol/L. We performed separate linear mixed regression model analyses where we compared the weight z-score, height z-score and BMI z-score trajectories of the glucose quartile groups from birth to the follow-up ages 1½, 3, 4 and 5 years. In the analyses, we handled each child (e.g. subject) as random effect to allow the intercept to vary by child. Age and glucose quartiles were handled as fixed effects as well as the covariates parity, maternal BMI, maternal country of birth and smoking. For all interactions between maternal glucose quartiles and age of the children, we used glucose Q1 as reference value. Growth trajectories from birth to the four follow-up points  for children to mothers with glucose levels within Q2, Q3 and Q4 were compared with the growth trajectory of children to mothers with glucose level within Q1. We presented the results as beta coefficients with 95% CI for fixed effects.Due to a large proportion of missing values for smoking at first maternal health care visit and/or in pregnancy week 30–32 (11.3%) we conducted supplementary analyses after multiple imputation by chained equations. We imputed data for smoking at first maternal health care visit and/or in pregnancy week 30–32 by using the child’s birth weight, highest maternal level of education and highest measured glucose level during pregnancy.p < 0.05. We conducted all statistical analyses with R version 3.5.3 51 packages lme452, tidyr53, mice54 and figures were produced using package ggplot255.Statistical significance for all analyses were set to 56.The Regional Ethical Review Board in Uppsala attested ethic permission of the project (no. 2014/353). This study was performed in accordance with national and international guidelines for medical research. Statistics Sweden merged and de-identified the registry data of Uppsala County Mother and Child Database before delivery to the research group. Since participants were neither identified nor contacted, informed consent was not required. The need of informed consent was waived by the Regional Ethical Review Board in Uppsala. When a study has been approved by a relevant ethics committee, informed consent is not requisite in Sweden and other Nordic countries for large registry-based studies in generalSupplementary Information.


Birth tourism refers to non-resident women giving birth in a country outside of their own in order to obtain citizenship and/or healthcare for their newborns. We undertook a study to determine the extent of birth tourism in Calgary, the characteristics and rationale of this population, and the financial impact on the healthcare system.A retrospective analysis of 102 women identified through a Central Triage system as birth tourists who delivered in Calgary between July 2019 and November 2020 was performed. Primary outcome measures were mode of delivery, length of hospital stay, complications or readmissions within 6 weeks for mother or baby, and NICU stay for baby.Birth Tourists were most commonly from Nigeria (24.5%). 77% of Birth Tourists stated that their primary reason to deliver their baby in Canada was for newborn Canadian citizenship. The average time from arrival in Calgary to the EDD was 87 days. Nine babies required stay in the neonatal intensive care unit (NICU) and 3 required admission to a non NICU hospital ward in first 6 weeks of life, including 2 sets of twins. The overall amount owed to Alberta Health Services for hospital fees for this time period is approximately $694 000.00.Birth Tourists remain a complex and poorly studied group. The process of Central Triage did help support providers in standardizing process and documentation while ensuring that communication was consistent. These findings provide preliminary data to guide targeted public health and policy interventions for this population.The online version contains supplementary material available at 10.1186/s12913-022-07522-4. Birth Tourism: the practice of non-residents of a country traveling to a new country with the intention to give birth in the new country.Birth Tourist: an Uninsured Prenatal Patient who is a non-resident who travels to a new country with the intention of giving birth there. In Canada, Birth Tourists do not qualify for publicly funded health care coverage, even if they are Canadian citizens, because they do not reside in Canada. Occasionally Birth Tourists have private health insurance that partially or entirely covers their medical bills.Uninsured Prenatal Patient: a patient who does not have provincial healthcare but who resides in Canada. They may not have Alberta Healthcare Coverage (AHC) for various other reasons, including.Convention Refugee: a person who meets the refugee definition in the 1951 Geneva Convention relating to the Status of Refugees . This definition is used in Canadian law and is widely accepted internationally. Health Care costs for Convention Refugees are covered by the federal government.Refugee Claimant or Asylum Seeker: a person seeking refugee status whose case has not been decided.Other: A pregnant patient who is neither a Convention Refugee nor Birth Tourist, but resides in Alberta. These patients may have an expired Canadian Work Permit, Visitor Visa, or Student Visa. They may also be undocumented meaning residing in Canada without legal documentation.Jus soli: the principle of jus soli allows for a child born on Canadian soil to a visiting foreign national to obtain Canadian citizenship .jus soli); the notion that the costs of medical care are lower in that country compared to the individual’s home country; the perception that medical care in that country is safer; and potential access to public schooling, healthcare, and sponsorship of other family members in the future. In recent years, discussions regarding Birth Tourism have been more prominent globally [The term Birth Tourism refers to the practice of a country traveling to a new country to give birth there for a variety of personal reasons. Such reasons may include: to obtain Citizenship for the infant in that country , which is current standard of data extraction, does not separate birth tourists from non-birth tourists. Subsequently, we hoped these changes would help to better support Uninsured Prenatal patients. However, that process has not been streamlined to the same degree at this time.Our primary objective was to collect detailed information on pregnant persons without Alberta Health Care (AHC) coverage in order to identify Birth Tourists in Calgary, evaluate their clinical outcomes, and characterize their financial and resource burden on the healthcare system. Our secondary objective was to collate the information collected to contribute to advancing the literature on Birth Tourism as this topic is relevant across the globe.We developed a centralized intake process for prenatal patients without AHC delivering in Calgary. Starting in July 2019, all prenatal referrals in Calgary for patients without AHC coverage were redirected to a Central Triage (CT) office. This office was consistently run by one administrator with the support of a three member physician team. Patients were administered a questionnaire and subsequently triaged to a care provider based on patient preference, risk profile and city quadrant of residence. Care providers, with the exception of midwifery who had a separate process for billing of patients, then invoiced CT directly. Invoices were required to be consistent with what would have been billed to Alberta Health and were reviewed by the physician leads for the same e.g. excess modifier codes could not be billed. In addition to ensuring standardized payments for services, CT ensured that the Canadian Medical Protective Association’s Governing Law and Jurisdiction form for all providers and Alberta Health Services was reviewed and completed as well as consent for information sharing among providers as well as Alberta Health (AH). Patients stayed in contact with CT with respect to when they would be leaving the country and timing of refund. In situations where English was not the primary language, language line or other translation services (through family or physician) was used. Refunds were issued once all invoices were received (approximately eight weeks postpartum). If a patient refused CT, we endeavored to follow up with how and where the patient received care.A ‘Central Triage package’ was reviewed and provided to all patients deemed to be Birth Tourists in person, via email, or via registered mail. Within this package, there was full disclosure of the concern regarding potential cost burden of Birth Tourism within Calgary and the rationale for the creation of CT – particularly to deter the process of Birth Tourism. A deposit of $15,000 was collected and held in trust by CT from each Birth Tourist to cover cost of physician service fees. In alignment with both the Alberta Medical Association (AMA) and the College of Physicians and Surgeons of Alberta (CPSA), patients were also able to pay for their fees individually following the service and care was never refused. The physician service fees for Birth Tourists were billed at a rate of five times the rate determined by AH in accordance with the Uninsured Services guidance document provided by the Alberta Medical Association (AMA) . ServiceThe CT system also sought to create a system by which uninsured patients could be identified quickly and thus be provided support letters and advocacy for more timely access to AHC given the impending medical care required. These scenarios were also assessed on a case by case basis allowing for provider discretion. Scenarios involving Uninsured Prenatal patients were found to be more complex with more individuation required which was another reason to not formally include them in the analysis. Our past experience with Uninsured Prenatal patients showed often face significant economic barriers that would make it difficult to pay an advance deposit for medical services. These patients were referred to an appropriate obstetrical provider with full understanding that appropriate medical care would be provided regardless of ability to pay. Physician fees for services provided to Convention Refugees were billed directly to the federal government.Although a deposit was collected by CT to cover the cost of physician services, a deposit was not collected to cover Alberta Health Services (AHS) site fees for hospital stay which are different for each hospital. AHS site fees are charged based on a daily rate. As part of the CT package, all Birth Tourists were given a copy of the current AHS site fee schedule and were made aware that the AHS finance office would bill them directly at the time of hospital admission.The questionnaire was developed by the study authors (see Appendix The CT team identified Birth Tourists by questionnaire responses in the following ways: (1) patient self-identifying as a Birth Tourist (2) the Birth Tourist arrived in Canada while pregnant, on a visitor visa, and had no intention to live permanently in Canada immediately following the delivery. Women who were classified as Uninsured Prenatal Patients were excluded. If there was a case that was unclear, the case was brought to the three-member physician team and reviewed sometimes with the support of hospital social work. There were no cases where consensus was not achieved through this method. Most patients were administered the questionnaire during the antenatal period, although, some presented for the first time at the hospital during the time of delivery. In this circumstance, the delivering physician would refer the patient to CT and attempts were made to administer the questionnaire postpartum. Occasionally a deposit was also collected post- partum to streamline invoice payment.Physicians sent invoices for services provided directly to CT, who then paid physicians from the deposit. An itemized receipt for all physician services was provided to each Birth Tourist and a refund was issued to the Birth Tourist where applicable. Any billing codes used were done so in the standard of the Alberta SOMB and were required to match the service provided. The physician invoices including the billing codes were also used to determine mode of delivery or any additional care required. Comments provided on these invoices or from the patients following delivery to administration at CT were also collected.We completed a qualitative descriptive analysis using information obtained from the CT patient questionnaires and physician invoices from July 1, 2019 to November 1, 2020. Only questionnaires completed by persons identified to be Birth Tourists were included in the review, with all identifying information removed. A data dictionary was used to collect the variables analyzed . This study was unfunded.In total, 102/227 patients captured by CT from July 15, 2019 to November 1, 2020 were identified as Birth Tourists. The 125 patients who were not Birth Tourists but instead Uninsured Prenatal patients were not included in the analysis. 89/102 (87%) patients were identified by direct referral to CT. The remaining 14 (14%) Birth Tourists were identified after delivery by invoices from physicians. The average and median maternal age was 32 years . Of these women, 42% had not had a previous live birth, 42% were multiparous, and parity of 16% was unknown. There were 5/102 (5%) Birth Tourists who self-referred to midwives for home birth or birth center delivery. 83/102 (81%) had an encounter with an AHS Calgary zone hospital. Of the remaining 19 patients (19%), 8 were lost to follow up, 2 delivered elsewhere in Alberta, 3 went back to their home country for delivery , 3 stated they would seek Midwifery care for home birth within Calgary, and 3 went to Ontario. There were 17 (17%) Birth Tourists that did not have a Regional Health Number.83% of patients stated they came to Canada with a Visitor Visa. The type of visa for 14% of patients was unknown, 2% had possessed a Student Visa, and 1 patient was a Canadian Citizen who had never lived in Canada. The date of arrival in Canada was known for 77/102 patients; the average time from arrival in Calgary to the expected due date was 87 days (SD 76) with a range of 7 to 502 days. Exact date of departure from Calgary is known for 34/102 patients; for these patients, the average length of stay in Calgary after delivery was 49 (range 18–80) days;Birth Tourists were most commonly from Nigeria (25%), Middle East (18%) China (11%), and India (8%) and Mexico (6%). There were no birth tourists from Western Europe or Australia. 77% of Birth Tourists stated that their primary reason to deliver their baby in Canada was for the desire for a Canadian baby who would be eligible for Canadian citizenship. 8% stated their reason to deliver in Canada was to access better health care. 40% of questionnaire respondents chose Calgary specifically because they had family and/or friends in the city. We were unable to expand further beyond this on other reasons for delivery in Canada or outside their home countries.38% of patients delivered vaginally and 35% delivered by cesarean Sect. 29% of deliveries were of unknown type. Two mothers required readmission to hospital within 6 weeks postpartum; one was admitted to ICU for 5 days for cardiac reasons, and another was admitted for severe preeclampsia and stroke. Another patient had severe postpartum preeclampsia but discharged herself against medical advice. Her subsequent outcome is not known. Nine babies required stay in the neonatal intensive care unit (NICU) and 3 required admission to a non NICU hospital ward in first 6 weeks of life, including 2 sets of twins. The average length of stay was 18 days with a range of 1–63 days.With respect to maternal health history prior to arrival in Canada: 9 had a history of previous cesarean section, 6 patients reported having some form of Diabetes Mellitus, 3 arrived with a cerclage in place, 2 arrived acutely hypertensive, 2 had history of myomectomy, 2 had threatened preterm labour (one actively in preterm labour), 2 had blood borne infection, and 2 had known twin pregnancies. There was one new diagnosis of HIV following arrival in Canada.There was AHS billing information captured for 83/102 (81%) Birth Tourists  paid the $15 000 deposit for physician services Appendix . Of thesOf the 227 patients captured by CT, 125 (55%) were deemed to not be Birth Tourists and were classified as Uninsured Prenatal patients. As stated earlier, this group was not included in the analysis. However, we do feel it prudent to state that we feel a process is urgently required to support this population. Since the initiation of this project, the CT team has been able to directly liaise with AHS to help expedite AHC insurance for those that qualify. We would advocate for a similar process that is available to all eligible women across the province. Having a system in place to distinguish Uninsured Prenatal patients is of benefit to the patient and the provider. It provides a means to better identify and support those for whom health care coverage can be optimized and also allows an opportunity to better support the needs of the patient. The additional support required can be difficult for individual community providers to provide in isolation. Our process did not specifically have a role for social work and additional community supports. However, we do feel it would be beneficial to create a dedicated process to support Uninsured Prenatal patients not only in the acquisition of AHC but also the opportunity for the provision of community and social supports that may be required. Women with an undocumented status may delay prenatal care due to concerns of being deported, and as a result increase adverse outcomes. We feel this population would benefit from additional community support and discussion of ensuring safe treatment without the threat of deportation and also creation of a system where cost is not a barrier to accessing care.Birth Tourists represented 44% of patients seen by CT. Of these, 87% of Birth Tourists were identified by direct referral to CT. There may be a few explanations for non-referral to CT. There is a complex network of care providers in Calgary which likely led to slow dissemination of the information regarding CT. There was some hesitancy from obstetrical providers to refer to CT perhaps due to pre-existing relationships or the feeling of obligation within communities with higher rates of Birth Tourism, the concern that there would not be a distinction between Birth Tourists and other Uninsured Prenatal patients, and the concern that patients may avoid care altogether. Through standardized communication we were able to address a common issue identified by providers: the fact that Birth Tourists were receiving incorrect and often varied messaging regarding costs associated with delivery, not only from their friends and online forums, but also from different obstetric providers in Calgary. Previously, the practice of different providers charging different fees for the same service led to ‘deal making’ between providers and Birth Tourists. Clear communication and collection of a standardized deposit in advance for physician services mitigated some of this problem. Many potential patients contacted the CT office from overseas prior to their arrival in Canada. Physicians did not have to collect payment in hospital or postpartum, which is often very difficult. It is concerning that 39% of patients did not pay the deposit. The reasons for this remain unclear and were not explored in the breadth of this study. Though some of these patients did ultimately pay for the physician service provided following the delivery, it was not clear to CT if all providers were paid and what amount. 83/102 (81%) of Birth Tourists had an encounter with an AHS Calgary Zone hospital. Of the remaining 19 patients (19%), 8 were lost to follow up. That is not insignificant and speaks to the need for greater administrative support to ensure that both mother and baby had access to care in a timely fashion. 5% of Birth Tourists identified that they would be self-referring to midwifery specifically for home birth or birth center delivery. Unfortunately, we were not able to capture the financial data with respect to cost of home birth versus birth center in such scenarios. Midwifery care is unique funding model in that there is a direct contract between AHS and the Alberta Association of Midwifery [The majority 76/102 (75%) of visas granted to patients seen through CT were Visitor Visas. Some patients did state they had taken out private insurance. Our process did not have consistent collection of private insurance information or the extent of coverage. This is a limitation of our study and examination of this in further studies may help to better understand the financial options available to patients.The average time from arrival in Calgary to delivery/EDD was 87 days with a range of 7–502 days (average 87 days). The 502 days reflects an individual who ultimately left and returned to their home country. This is a limitation of our data in that we are not able to capture exactly when all individuals left. This also underscores the difficulty in capturing a subset population of uninsured and undocumented pregnant women. When the time of stay surpasses 365 days, the individual can no longer be considered as a tourist . The indThe average of 87 days arrival in advance of EDD or delivery demonstrates potentially significant implications in care provision and supports the evidence that many women in this population seek care late in pregnancy. Women who arrive very close to the end of pregnancy or postdates may be in a rush to obtain care. Potential for complications including elevation in maternal blood pressure and stillbirth rise as women approach and pass their due date. This rush for care puts an increased stress of immediate care provision in an already stressed system. Improved education with VISA issuance would be helpful to inform women of the risks they may be taking in traveling to deliver overseas late in pregnancy.Almost a third of women presenting to CT had a known preexisting medical condition. This included one person actively experiencing preterm labour and three with a cerclage in the current pregnancy. Many Birth Tourists were concerned about hospital stay > 24 h due to the additional daily AHS fee. In some cases this lead to Birth Tourists discharging themselves Against Medical Advice. The potential for increased morbidity is significant. A limitation to our study is that we were not able to assess if advance knowledge of the CT process may have changed the decision to undertake Birth Tourism.In comparison to the 35% of Birth Tourists delivered cesarean section, the general cesarean section rate in Alberta in 2018 was 31% . The NICThe time from delivery to departure was 49 days with a range of 18–80 days. Approximately one third of patients did not specify a date of departure but stated they would be leaving ‘after delivery. Though the data is limited by the fact that 1/3 did not specify an actual date, the average of 49 days to departure is consistent with the definition of a Birth Tourist not permanently staying Canada at that given time. This does have implications for the Canadian born child’s ability to access Alberta Health care. Specifically, a person must make Alberta his/her home and be physically present in Alberta at least 183 days in a 12-month period . 17% of children born to mothers identified as ‘birth tourists’ received AHC with a third of those mothers still having unpaid bills for maternal care. This poses the question as to whether or not the criteria for obtaining Alberta Health Care for neonates born to mothers without Canadian Health Care is rigorous enough.The most common reason stated by the patients in our study for travel to Canada to give birth for ‘a Canadian baby’. A Canadian citizenship is perceived as a valuable item not only in the short term but also long term for health care and education. The long term implications to this are complex and multifaceted and should not be simplified. However, our data does show that the Canadian Citizenship is held in value and is provided as reason for Birth Tourism.The overall amount owed to Alberta Health Services during the study period is almost $700,000 Table . There wOur AHS financial data was not clearly outlined in terms of what costs were paid. We do feel that moving forward a clearly itemized invoice for hospital services should be provided to patients as well as CT. There were a number of people who were issued refunds from CT for physician service fees yet still had outstanding AHS fees owing. We propose to only issue CT refunds once a fully paid hospital invoice is shown. A further limitation of our financial analysis was that we did not include foreign parents hiring Canadian surrogates to carry a child who will leave the country.Another limitation of our study was that all out of country patients were not identified through the CT. These cases were often recognized through communication from ancillary care providers such as pediatrics asking for clarification on billing. Some out of country patients presented for the first time at delivery. There were also some out of country patients making individual arrangements with maternity care providers that also came to light when additional care providers asked for clarification regarding billing. Given the lack of a robust system of data collection, it is very possible that Birth Tourists and Uninsured Prenatal patients were missed. Unfortunately, we were also unable to assess which women remained in Canada after expiration of their visitor visa. There were many scenarios in the financial data were there were outstanding maternal charges, AHC was obtained for the infant and there was no referral to CT. It is possible that some of these women were not truly Birth Tourists but rather Uninsured Prenatal patients that overstayed a visa and we were not able to capture the specifics of their scenario.Our analysis overlapped with the Covid-19 pandemic. We had anticipated birth tourism to drop to almost zero with the lack of air travel. However, that was not the case, suggesting that at a time of reduced international travel, birth tourism continued. It would be important to collect data on an ongoing basis as we suspect that rates of birth tourism in past and future years may be higher.The process of Central Triage did help support providers in standardizing process and documentation while ensuring that communication was consistent.The process 1) enabled us to differentiate Birth Tourists from non-Birth Tourists, which is something that DIMR data has been previously unable to, and thus identify populations that may need additional support 2) ensure that physicians are more likely to get paid for services rendered 3) standardize a fee schedule across the city for Birth Tourists. We do feel that an area of further investigation that is required is better understanding the non-Birth Tourist population. This group seems to have greater complexity in terms of background and potential need for support.With increasing strain on healthcare budgets, outstanding patient invoices warrant attention. In a publicly funded system that is often under strain, we do feel that our process of CT did help to improve efficiency and provide more timely access to appropriate care for Birth Tourists. We do believe that having a clearly delineated policy for Birth Tourists would allow for better collection of data, consistency of messaging and communication between all parties involved in health care from the patient to the frontline worker to the administrator. There may be unintended consequences of such policy on the non-Birth Tourist group, in particular the undocumented patient. It would be imperative that the determination of Birth Tourists was very clear and not reliant on self –identification alone.Additional file 1. Additional file 2: Additional file 3.


The first study of the flexo-ionic effect, i.e., mechanical deformation-induced electric signal, of the recently discovered ionic liquid crystal elastomers (iLCEs) is reported. The measured flexo-ionic coefficients were found to strongly depend on the director alignment of the iLCE films and can be over 200 µC/m. This value is orders of magnitude higher than the flexo-electric coefficient found in insulating liquid crystals and is comparable to the well-developed ionic polymers (iEAPs). The shortest response times, i.e., the largest bandwidth of the flexo-ionic responses, is achieved in planar alignment, when the director is uniformly parallel to the substrates. These results render high potential for iLCE-based devices for applications in sensors and wearable micropower generators. The past few decades have witnessed a flourishing development of electro-mechanical transducers for use in soft robotics ,2,3, sen−2–10−3 Flexoelectricity exists even in centrosymmetric materials by coupling electric polarization with strain gradient. It has been studied in both solid crystals and liquid crystals since the 1960s . In crysceramics ,13, wher0−3 μC/m ,15. In l0−3 μC/m . The fle/m -2-methylbenzene) were purchased from Synthon chemicals. IL (1-Hexyl-3-methylimidazolium hexafluorophosphate (HMIM-PFuctivity .6) was added to the LCE precursor solution and mechanically stirred for 15 min after heating to 80 °C to achieve complete mixing. A re-determined weight percentage (25%) of ionic liquid was used as per our previous experience on this system for electric actuation . I,9. I~2000.33TiO3 ), crysta0.33TiO3 , and ben0.33TiO3 ,19 as in


The fistulization of a hepatic hydatid cyst to the duodenum is an extremely rare case demonstrated in computed tomography and confirmed surgically. We reported a case of 56 years-old woman representing this unusual complication.We show, through this work, the importance of radiological signs that can help to make the diagnosis before surgery. Hepatic hydatid cyst is a zoonotic disease commonly caused by Echinococcusgranulosus in cattle and sheep rearing regions.It is endemic in countries around the Mediterranean, such as Morocco, and constitutes a real public health problem due to its frequency, morbidity and potential mortality.Numerous complications can occur, including compression, infection, or rupture.We see various types of ruptures  but into the gut, especially the duodenum, still extremely rare and challenging for the surgeon.A 56-years-old woman with a history of asymptomatic hepatic hydatid cyst came to the emergency with fever, sweating and right upper quadrant pain that increased with inspiration for 2 days.Laboratory studies revealed a white blood cell count of 13 300/μl, with 37% eosinophils. Biochemistry revealed a creatinine level of 140 μmol/l.CT scan showed a hepatic cavity occupying segment 6 at his lower pole; we observed a fistula communicating with the duodenum. A second sign that testifies this communication was the air-liquid level.Emergency laparotomy was carried out under the diagnosis of an infected hydatid cyst. The patient was operated on through a bilateral subcostal laparotomy and finding 1 hydatid cyst that is close to the pylori but with direct contact with the first duodenum knee.Exploring this cystic cavity revealed communication with the first duodenum affecting its anterior wall, which was repaired and the hydatid cyst removed A and B.FHydatidosis is a parasitic disease considered benign but can become dangerous because of its complications. It is a real public health problem in the world by its frequency, morbidity, and its potential mortality. It is caused by infection with the larval stage of Echinococcusgranulosus.Many serious complications dominated by rupture and secondary infection We consider that the rupture is the most notable complication of hepatic hydatid cyst  The rupture is caused by 2 major factors the inflammation, the diameter of the cyst, the permanent contact with is and the delay of the treatment lead to erosion of the wall of the organ adjacent to the cyst.It usually occurs into biliary ducts (9%-30% in large series), causing jaundice and acute cholangitis, or into the peritoneal cavity (1%-12.5%), which might lead to a life-threatening anaphylactic reaction Only a few cases in literature talk about the rupture or fistulisation of the hydatid cyst into the duodenum (0.15%), and its diagnosis is usually after surgery.The presence of air in the cyst was observed in most of the cases in the literature, which may alert the physician to fistulisation formation, but is not a specific sign.However, in our case and without barium opacification, we can identify the fistula target communicating with the first knee of the duodenum, and at the same time, the cyst contains an air-fluid level (Nowadays, interventional radiological treatment takes place, especially for stable patients that cannot support surgery. However, we can propose conservative surgical treatments such as partial cystectomy or drainage.The surgical management depends essentially on the view of hollow viscus damage, in other words, the status of the defect if it is a small gap or not and if it is revamped by the local inflammatory condition.In terms of what is present, the surgical procedure to log out the cysto-digestive fistula by simple suture associated with gastric aspiration or other options such as duodenostomy or duodenal diverticulization if the duodenal gap was important and would not lend itself to reparation.Radical surgical procedures like hepatic resection or pericystectomy can also be performed in selected complicated cases Treatment of the parasite by preoperative and postoperative albendazole effectively prevents recurrence, and the recurrence rate after all surgical treatment alternatives is less than 10% The lesson learned from this case study is to look for the clue findings in order to make the diagnosis of the fistulisation of the hepatic hydatid cyst which is extremely rare and always challenging for the surgeon.


The use of RNA interference has become increasingly popular for investigating insect physiology, testing the functionality of insect genes and as a potential control strategy. Hemiptera include many vectors for destructive plant diseases. A major characteristic of the order of Hemiptera is feeding on the phloem sap of their plant hosts. Phloem feeders face high osmotic stress between the gut lumen and hemolymph due to the high level of sucrose in phloem sap. Targeting the osmoregulation mechanisms in Diaphorina citri Kuwayama, which transmits ‘Candidatus Liberibacter asiaticus’, the putative causal agent of Huanglongbing in citrus may lead to an effective control strategy. Herein we downregulate the expression of aquaporin, representing a major mechanism of osmoregulation, by RNA interference., Diaphorina citri Kuwayama is devastating the citrus industry worldwide. It transmits ‘Candidatus Liberibacter asiaticus’, the pathogen of Huanglongbing in citrus. RNA interference is an excellent tool for functional genomics and for screening target genes for pest control. Herein, we silenced the aquaporin (AQP) gene (DcAQP) homologue in D. citri to study its functionality and whether it could be a good target for a control strategy. AQP is an integral membrane channel protein that aids in the rapid flux of water and other small solutes that move across the lipid membrane. In Hemiptera, it is well established that AQP plays important roles in adjusting to physiological challenges including (1) regulating osmotic stress between the gut lumen and hemolymph after imbibing large quantities of a low nitrogen, sugar-rich liquid diet; (2) avoiding or preventing dehydration and desiccation; and (3) surviving at elevated temperatures. The dsRNA-DcAQP was applied twice to nymphs of the 4th and 5th instars through a soaking technique. Silencing AQP caused a significant increase in nymph mortality. Emerged adults showed malformations and a shorter lifespan. Silencing DcAQP provoked alterations in some metabolites and increased the uric acid content in emerged adults. DcAQP could be a useful target to control D. citri.The Asian citrus psyllid Aquaporins (AQPs), as the name denotes, are specialized integral membrane proteins that form pores through which water and small neutral molecules are selectively channeled across cellular membrane . UbiquitThe AQPs form water channels through the plasma membrane of the cell in order to passively transport water in response to osmotic gradients, and they have an important role in cell volume regulation and excretion . SpecifiD. citri and other hemipterans depends on their ability to osmoregulate after feeding on plant phloem sap, which is known for its rich sucrose content, but such feeding may increase the osmotic pressure up to 4–5 times [D. citri is by sucrose hydrolase activity in the gut [Bemisia tabaci and Acyrthosiphon pisum mentioned that the silencing of AQP1 provoked disruption of osmoregulation due to a significant elevation in the osmotic pressure [Insects belonging to the order Hemiptera ingest large quantities of plant sap to acquire sufficient levels of nitrogen and other nutrients. Plant sap contains primarily water and sugars as main components, which provoke the increase of osmotic pressure in the insect gut. In order to overcome this challenge, the insects quickly break down sucrose into fructose and glucose, which are transported to the interior of the cells, and the remaining sugars in the gut are oligomerized and eliminated as honeydew ,14,15. A–5 times . In fact the gut . Other wpressure ,18.Cicadela viridis and in the anterior ileum of the hindgut of B. tabaci [Some Hemipterans such as leafhoppers, froghoppers, whiteflies, and psyllids have a characteristic water-conducting anatomical complex called the filter chamber that bypasses part of the digestive tract and functions to shunt rapidly and directly large amounts of fluids from the foregut to the midgut . This st. tabaci ,20 where. tabaci . In othe. tabaci . AQP1 wa. tabaci .ClAQP in the human bed bug Cimex lectularius, which transmits Trypanosoma cruzi, severely affects its excretion, water homeostasis and reproduction [Rhipicephalus (Boophilus) microplus, which transmits the protozoan Babesia bovis, was decreased by silencing of RmAQP2 [ApAQP1 caused significant elevation in osmotic pressure of the hemolymph [Bactericera cockerelli, Ibanez, Hancock and Tamborindeguy [Owing to their high specificity, RNA interference (RNAi) techniques to silence genes have been widely accepted as an alternative method to control insect pests of importance to human health and agriculture ,23. Theroduction . The fitf RmAQP2 . In the emolymph . Recentlrindeguy  found thDiaphorina citri Kuwayama (Hemiptera: Liviidae) is one of the most notorious agricultural pests because it transmits and spreads the bacterium ‘Candidatus Liberibacter asiaticus’ that causes huanglongbing (HLB) or citrus greening disease [D. citri. However, despite efforts to reduce the population of insects with insecticides and biological controls, these measures have not been sufficient [D. citri, RNAi techniques have proven to be a powerful tool to assess the efficacy of genes targeted to reduce psyllid survival and fecundity, increase susceptibility to insecticides, increase mortality, cause abnormal conditions, or interfere with homeostasis [ disease ,26. HLB fficient ,28. In Deostasis ,31,32,33DcSuh) in D. citri using RNAi, which resulted in disruption of the osmotic homeostasis in D. citri [D. citri using in silico analysis and silence the AQP gene in D. citri by means of RNAi technique. We hypothesize that silencing DcAQP using dsRNA will result in knockdown of its transcript expression, which will cause increased mortality in adults, owing to their inability to maintain internal water homeostasis.AQP is one of three mechanisms insects use to regulate osmotic pressure. In addition to AQP, sucrose hydrolase acts to break down sucrose into fructose and glucose, while sucrose transglucosidase oligomerizes glucose into long-chain oligosaccharides, primarily trehalose, which is excreted as honeydew . These tD. citri . This leD. citri were continuously reared on 6-month-old Citrus macrophylla (Alemow) plants with new flush and enclosed in insect rearing cages . The D. citri colonies were maintained in temperature-controlled growth rooms set at 25 ± 3 °C, 60 ± 5% relative humidity (RH) and with a 16:8 h (light:dark) photoperiod. The citrus and insect cultures were located at the Citrus Research and Education Center (CREC), University of Florida, Lake Alfred, FL, USA.Laboratory colonies of D. citri Genome and Transcriptome https://citrusgreening.org/organism/Diaphorina_citri/genome (accessed on 10 September 2021). Several AQPs sequences from the databases were compared with the information in NCBI and UniProt through BLAST with aquaporins well described in other insects. These protein sequences included AQP of A. pisum (accession no. NP_001139376.1), B. tabaci  and Myzus persicae (accession no. A0A0H3XRM3) from Hemiptera and Aedes aegypti (accession no. Q9NHW7.2) and D. melanogaster (accession no. NP_001286316.1) from Diptera. Multiple amino acids sequence alignment was performed using Clustal W and Boxshade 3.21. to visualize conserved regions.The aquaporin sequences were queried from DcAQP protein sequence were examined with TMHMM and SMART. The theoretical isoelectric points (pI) and molecular weights (MW) of protein were computed using the Compute pI/MWTool . The SWISS-MODEL and GENO 3D servers were used to predict the secondary structure to validate template selection, and it was visualized using the PyMOL . The phylogenetic relationship was performed by MEGA7 using the neighbor-joining method with 1000 bootstrap replicates to evaluate the significance of the nodes.Membrane topology and domain structure predictions of the D. citri predicted aquaporin AQPA (DcAQP), mRNA is 815 bp in length (NCBI-GenBank: XM_008486010.2). Primers were designed from the open reading frame sequence (ORF) using the Primer Plus 3 program, which considers several parameters including CG content (50–55%), melting temperature (59.95–60.01 °C), product length (300–401 bp) and undesirable alignments. A primer pair DcAQP-F and DcAQP-R was generated to obtain a 306 bp DNA fragment through gBlocks® Gene Fragments , and all primer sequences used in this work are presented in DcAQPT7 F-R) and used as the template to generate dsRNA with the MEGAscript RNAi kit (Ambion). We synthesized the dsRNA-GFP (primers: gfpT7 F-R) following the same procedure mentioned above for use as a negative control. The quantity and integrity of synthesized dsRNA was evaluated by a NanoDrop 1000 spectrophotometer  at 260 nm and by agarose gel electrophoresis.The D. citri were removed from the leaves and branches of C. macrophylla plants with a #2 camel hairbrush. Then, nymphs were examined under a stereomicroscope and classified into 1st through 5th instars based on morphological features described in [DcAQP were prepared in RNase-free water as follows: (ng·µL−1) 25, 50, 75, 100, 200, 500 and 1000. In addition, RNase-free water and dsRNA-GFP were used as negative controls. Third through fifth instar nymphs were starved for 2 h and then soaked with a 10 μL dsRNA droplet, and the application was repeated after 24 h. The dsRNA droplet was withdrawn by a pipette after soaking for about 10 min. After dsRNA applications, treated nymphs were placed on filter paper to dry, and then were transferred onto small C. macrophylla plants housed in modified Berlese funnel traps (funnel removed).The nymphs of ribed in . CombiniDcAQP transcript fragment evaluation by semi-quantitative RT-PCR, 2 h after the second dsRNA application, the nymphs were washed with RNase-free water to removed dsRNA-DcAQP residues and were processed. A primer pair (DcAQPSRT F-R) was designed to be inside the dsRNA-DcAQP trigger sequence for semi-quantitative RT-PCR analysis, which produced an amplimer of 234 nucleotides. Then, total RNAs were extracted in TRIzol® Reagent (Invitrogen) from untreated eggs, nymphs in first, second, third, combined fourth-fifth stages, and adults using a Direct-zolTM RNA MiniPrep kit . Total RNA (100 ng) from each sample was used to amplify the transcript fragments of DcAQP, and actin was used as an internal control  with the Affinity Script One-Step RT-PCR kit (Agilent). Reactions were subjected to the thermal program: 45 °C for 5 min, 92 °C for 1 min; 30 cycles of 92 °C for 20 s, 57 °C for 20 s and 72 °C for 30 s; followed by maintenance at 72 °C for 3 min. PCR products were analyzed on a 1.5% agarose gel (Thermo Fisher Scientific) and visualized by GelRed staining .For DcAQP on aquaporin gene expression, we treated nymphs with 200 and 500 ng/µL dsRNA such as mentioned above using the same soaking protocol, and the application was repeated after 24 h. After adults emerged, total RNA was isolated from the whole bodies of adult D. citri. The primers used for qRT-PCR analysis (DcAQPRT F-R) were used at 125 nM. First-strand cDNA synthesis was initiated with 0.6 µg of purified RNA using SuperScript IV VILO Master Mix . The synthesized cDNA was diluted 1:5 (10 ng/µL working concentration) and stored at −20 °C until use. The quantitative RT-PCR reactions were carried out in a 20 µL mixture consisting of 10 µL of Power Up SYBR Green master mix (2×) , 1 µL of primer pair mix, 2 µL of cDNA and 7 µL of RNase-free water. All the samples were placed in QuantStudioTM 3 Real Time PCR detection system . One reference gene, actin (Genbank accession number DQ675553) was used to normalize the amount of cDNA added to the PCR reactions.To evaluate the effect of dsRNA-D. citri nymphs each (from 4th and 5th instars) were treated with dsRNA-DcAQP at the concentrations mentioned above. The mortality rate was calculated based on the numbers of emerged adults 7 d after treatment. Experimental data from mortality assays were carried out with JMP software, using one-way ANOVA, and multiple comparisons between treatments were performed with the Tukey’s Honestly Significant Difference (HSD) test. For the survival assay, we recorded the numbers of adults dead daily, and the analysis was performed with JMP software using the Kaplan–Meier method. Statistical significance was established as p < 0.05. During the experiment, the dead or dying D. citri were examined morphologically under a stereomicroscope. The psyllids with malformations were photographed using a Canon Power Shot S3IS digital camera coupled to a M3Z stereomicroscope (Leica).For mortality studies, three replicates per treatment consisting of 20 −1) dsRNA-DcAQP. The adult body length measurements were taken after 10 days with the emerged adults. We made two measurements: (A) from the head to the end of the wings, and (B) from the head to where the body ends. We calculated the average body weight of the emerged adults by dividing the total weight of living adults by the number of adults. We statistically analyzed the experimental data from weight and body length measurements with SigmaPlot software, one-way ANOVA, and Tukey’s HSD test for multiple comparisons between treatments.Three biological replicates (20 nymphs per treatment) were treated with 0, 25, 100, 200, 500 and 1000  trifluoracetamide (MSTFA) [D. citri, respectively, after two applications (24 h apart) of dsRNA-DcAQP at 200 ng/µL as nymphs and immediately placed them into 1.5 mL microcentrifuge tubes with the appropriate extraction solvent for each derivatization , and kept them at −20 °C until analysis. A glass bead was added to each tube, and the tubes were cooled in liquid nitrogen for 10 min. Thereafter, the insects were macerated by homogenizing in a TissueLyser II  in the extraction solution for 2 min. Samples were then placed on an Enviro-Genie  at rotate/rock 20/40 overnight at 5 °C. After the overnight extraction, the tubes of insect homogenate were vortexed briefly and centrifuged at 5 °C for 20 min at 12,000 rpm in a refrigerated centrifuge  to remove solid debris. The supernatant was collected from each tube and placed into new 1.5 mL tubes. Two biological replicates were made per methodology.To determine the effects of silencing  (MSTFA)  and meth (MSTFA) . We use D. citri supernatant into silanized GC vials with 200 µL fused-inserts  along with 10 µL of internal standard . As a blank reference sample, we derivatized 100 μL extraction solvent plus 10 μL ribitol IS added. The extracted samples and blank samples were dried under a nitrogen stream before adding the derivatization reagents. Methoxyamine hydrochloride solution (MOX) in pyridine (2%) and N-methyl-(N-trimethylsilyl) trifluoracetamide (MSTFA) were purchased from ThermoFisher Scientific. To each of the dried samples, 30 µL MOX was added and incubated at 60 °C for 1 h. Finally, 80 µL MSTFA was added and incubated an additional 1 h at 60 °C. The derivatized samples were injected splitlessly into the GC injector (0.5 µL) for analysis using the same GC-MS column and conditions previously described by Killiny [For the first GC-MS analysis using MSTFA derivatization, we placed 100 µL aliquots of adult  Killiny .D. citri supernatant to 1-mL silanized GC-MS inserts and concentrated them to approximately 20 µL under a nitrogen stream. To the concentrate, we added 180 µL of NaOH (1 M). The alkaline sample was mixed with 167 µL of methanol and 34 µL of pyridine, followed by the addition of 20 µL of MCF. The sample was vigorously mixed for 30 s. An additional 20 µL of MCF was added, and the sample was mixed for another 30 s. A 100 µL aliquot of chloroform was added with vigorous mixing for 10 s, followed by 200 µL of sodium bicarbonate (50 mM) with vigorous mixing for 10 s. The upper layer was discarded, and approximately 100 µL of the organic layer was transferred to a new insert. A few milligrams of sodium sulfate were added to dry the organic layer, and 1 µL was injected into the GC-MS. The GC-MS conditions for MCF derivatizations were as reported previously [For the second GC-MS analysis using methylchloroformate, we transferred aliquots (100 µL) of adult eviously .D. citri genome possesses several sequences with significant similarity to the aquaporin  from human, Homo sapiens  as a candidate gene to be targeted by RNAi based on the position of hallmarks of the aquaporin family such as the domains that provide the functionality as a selective water channel, as well as the score of the alignments with HsAQP1. The multiple sequence alignment of the AA sequence of DcAQP protein with other insects such as A. pisum, B. tabaci and M. persicae (from Hemiptera) and A. aegypti and D. melanogaster (from Diptera) revealed a high similarity between them   B.DcAQP consists of 270 AA residues and has a theoretical isoelectric point (pI) of 8.73 and a predicted molecular mass of 28.85 kDa as obtained by pI/MW Tool. The sequence showed two main regions, the canonical signature Asn-Pro-Ala (NPA) localized in B  and E  loops which are intracellular and extracellular, respectively. Furthermore, the bioinformatic analysis of AA sequences using the TMHMM Server v. 2.0 to predict the transmembrane helices of DcAQP and using the Simple Modular Architecture Research Tool (SMART) to predict the domain architecture suggests a high topological similarity between DcAQP from psyllid and HsAQP1 from human. DcAQP protein has several key features that are present in a member of the aquaporin superfamily such as the presence of six membrane-spanning helices (TM1 to TM6) separated by five loops with intracellular N- and C-termini  (−1), and the similarity between both sequences was 47.5%. The Ramachandra plot indicated that 82.4% of amino acid residues were in the favorable region, 14.3 in the allowed region, 2.7% in the generously allowed region and 0.5% in the disallowed region. Taken together, these findings suggest that the DcAQP belongs to the traditional Class 1 of aquaporin, which is mostly water selective.Although the three-dimensional (3D) structures for arthropod aquaporins are not available yet, we were able to predict the 3D secondary structure of D: 2D57) C. In GenD. citri life cycle to target DcAQP, we investigated the transcript levels of DcAQP throughout the whole life cycle  and in adults, but not the early stages  (DcActin.To determine the best stage(s) of the fe cycle . We founinstars) . All stuDcAQP and the application method, we studied the effect of dsRNA-DcAQP on the transcript levels of DcAQP in the 4th–5th instars in comparison with H2O as a control. RNA was extracted from nymphs treated twice with dsRNA-DcAQP. DcAQP-silenced nymphs, indicating the knockdown of gene expression. Furthermore, the effect of DcAQP silencing on the nymph mortality (%) of 4th–5th instars of D. citri carried out using seven concentrations of dsRNA-DcAQP revealed that the nymph mortality (%) increased after the application of dsRNA-DcAQP compared to H2O and dsRNA-GFP  and maintained them on citrus to obtain the emerged adults. The Kaplan–Meier analysis of cumulative survival up to 40 days showed that the survival of the emerged adults from dsRNA-DcAQP-treated nymphs was significantly lower at all concentrations compared to the controls (H2O and dsRNA-GFP), and it was not dose-dependent   but not the low concentrations  significantly decreased the survival and shortened the lifespan of emerged adults, compared to the mock- and dsRNA-GFP-treated controls suggesting a threshold response  A. Instearesponse B.DcAQP approximately 12 days post-treatment (dpt) in the emerged adults from 4th–5th nymph instars treated with the most effective concentrations (200 and 500 ng·µL−1). The DcAQP gene expression decreased slightly in the emerged adults from nymphs treated with 200 ng·µL−1 dsRNA-DcAQP (DcAQP silencing on the uric acid content of the whole body of emerged adults. Although there were no significant differences between the two studied concentrations (200 and 500 ng·µL−1 dsRNA-DcAQP), both concentrations increased the endogenous levels of uric acid in comparison with the mock control  had some malformations (reduced body size and incompletely molted) (DcAQP silencing on body measurements (weight and length). Interestingly, all studied concentrations of dsRNA-DcAQP  significantly reduced the body fresh weight (μg·insect−1) 10 dpt in comparison with both mock- and dsRNA-GFP-treated controls (DcAQP silencing reduced the body length of emerged adults from treated nymphs (−1 dsRNA-DcAQP), but not when nymphs were treated with 25 ng·µL−1 (−1 dsRNA-DcAQP or higher (−1 but without affecting the length of the wings.We observed that  molted) A–D. Thercontrols E. Furthecontrols F. Generad nymphs G,H. The  ng·µL−1 G. Likewir higher H. These D. citri adults treated by dsRNA-DcAQP. We detected 41 compounds by gas chromatography-mass spectrometry (GC-MS) through MSTFA derivatization, and 17 compounds through MCF derivatization , glucose (two peaks), chiro-inositol, an unknown sugar alcohol 5, glucuronic acid, myo-inositol and oleic acid. Interestingly, all compounds that were significantly different increased in the range of 2–3 fold with respect to the control, and none were decreased (except peak 2 of the unknown sugar eluting before glucose). Otherwise, through MCF derivatization, we found that L-phenylalanine and heptadecanoic acid (C17:0) were increased, but L-proline, L-glutamic acid, L-histidine and maleic acid were significantly diminished with respect to the control.The level of sugars, organic acids, and amino acids were affected in tization . By MSTFSince the earliest identification of the molecular water channel (aquaporin-1 (AQP1)) in red blood cells in the early 1990s, a number of genomic and transcriptomic projects have identified numerous homologous water channel proteins in bacteria, plants, and animals ,8. Most DcAQP sequences of the six transmembrane helices (TM1-6) and five loops (A-E) typical of other aquaporins. Loops B and E are highly symmetrical and contain the canonical Asn-Pro-Ala (NPA) region. Both the N- and C-terminus of the protein are intracellular. These characteristic features comprise the classic ‘hourglass’-shaped model for AQP1 [DcAQP were matched with sequences of B. tabaci, A. pisum, M. persicae, A. aegypti and D. melanogaster that have been well-characterized as functional water channels. Additionally, the results of the alignment and secondary structure prediction show that the DcAQP sequence could belong to class 1 of AQPs subfamily. AQP1 belongs to class 1 and is expressed in the alimentary tracts and Malpighian tubules (insect kidney) in several insects, where it plays an important role for osmoregulation in sap feeders like leafhopper [In this work, the results from bioinformatic analysis clearly demonstrated the presence in for AQP1 ,9,12,41.afhopper , pea aphafhopper ; and whiafhopper .DcAQP transcript in D. citri eggs was undetectable in agarose gel after semi-quantitative RT-PCR analysis. This result is possible if the eggs were diapaused or because the AQPs are expressed at different levels in different tissues during insect development, depending on the physiological necessity of the development stage [Recently, several roles for AQPs were described in silk moth egg development, including hydration and osmotic swelling of oocytes, and water transport during vitellogenesis . Howevernt stage ,18.D. citri life stages and have allowed the identification of target genes that could be beneficial for controlling this vector. Specifically, these methods include the use of an artificial diet, soaking, and topical feeding [D. citri fitness only when we applied the dsRNA twice in a 24 h interval through the soaking method. It could be that we obtained these results because successful RNAi depends on several factors including species, the gene targeted, its expression profile, as well as intracellular transport and degradation which is more susceptible when is applied orally or by soaking instead of injection [Actin and coat protein II (Sec23) while cell lines of Coleoptera produced siRNA after soaking in the same dsRNA [The silencing of target genes through RNAi technique using dsRNA is becoming a powerful tool for insect pest management ,44,45,46 feeding ,32,33,47njection ,49. For DcAQP was high and showed a tendency to decrease the survival probability even at low concentrations. However, by the end of the experiment, we considered that there was no impact on the survival parameter. Additionally, the results of the gene expression indicated a total knockdown only 24 h after application of the dsRNA-DcAQP in nymphs, and the effect was dramatically reduced on DcAQP gene expression when the adults emerged approximately 15 days after treatment. These results indicated that the dsRNA was effective only during the first few days after application. The loss of effect on the DcAQP expression could be because the dsRNA is lost or consumed during nymph development, leaving little for suppression in the adults. In our earlier work with CYP4 genes [D. citri, gene expression was suppressed significantly for 8 dpt [It is interesting to note that the mortality measured on day 7 after the last application of dsRNA-P4 genes , we alsoor 8 dpt . This tror 8 dpt . It is rDcAQP was transient, it was enough to cause a reduction in body size, likely due to dehydration from the effects of high osmotic pressure and the inability to move water through the cell membranes. However, because we did not measure the insect dry weight in our experiments, we cannot confirm dehydration as the cause of reduced body size. We hypothesize that this mass reduction is due to water loss because we silenced AQP gene, which is one of the important mechanisms controlling insect osmotic pressure. The nymphal stage is characterized by voracious feeding behavior [DcAQP in nymphs disrupts the transport of water, nutrients, metabolites, hormones and body wastes more in nymphs than in adults, which feed less. Furthermore, in the silk moth, Hyalophora cecropia, it is essential to keep the osmotic pressure constant in order to allow the diffusion between molting fluid and hemolymph during the molting process [DcAQP.Homeostasis of water and energy is one of the many critical roles of cellular membranes. Several physiological processes that are dependent on osmotic equilibrium start to fail when the internal steady state is lost ,11,51. Tbehavior . Therefo process . It is pD. citri emerged from treated nymphs confirms the function of AQPs in D. citri. Recent studies revealed that insects store uric acid in the fat body and utilize it in diverse manners such as supplying the nitrogen requirement for egg production in females or as a physiological antioxidant [DcAQP could have provoked poor distribution of oxygen due to osmoregulation disruption and then caused ROS damage in some tissues [DcAQP may have assisted in the recovery of nucleotides produced by damage tissues and incorporated them into the nucleicolytic pathway with the purpose of storing uric acid for later use. Additionally, in bed bugs, the silencing of ClAQP1 and ClGLp1 significantly reduced water and urea excretion after blood feeding [DcAQP in 4th–5th instar nymphs reduced nitrogen excretion resulting in uric acid accumulationAdditionally, in newly emerged adults we found an increase of uric acid in comparison with the controls. The accumulation of uric acid in adult ioxidant ,55. One ioxidant . It is aioxidant . The sil tissues , but thi feeding , so it iDcAQP. We observed no effect on sucrose metabolism, indicating that sucrose hydrolase was breaking down the sucrose into glucose and fructose. However, glucose accumulated significantly, which suggests that glucose was not totally integrated into the metabolic pathway of glycolysis and the tricarboxylic acid cycle. The alterations in primary and secondary metabolites in D. citri adults emerged from RNAi-treated nymphs demonstrate the impact of silencing of DcAQP during the metamorphosis.Normal excretion of amino acids in some insects occurs through the Malpighian tubules and through the rectum . We founD. citri, we found significant increases in the nymph mortality, reduction in body fresh weight and length and increases in the uric acid content associated with RNAi-treated D. citri. Some of these effects were transient, as was found in our previous work with CYP4. The silencing of DcAQP could have a stronger, more durable effect in the control of D. citri if applied in conjunction with other RNAi gene targets such as DcSuh to further enhance disruption of D. citri osmoregulation. Future research efforts in this area should include evaluation of ROS damage, measurements of hemolymph osmotic pressure or water content, and DcAQP localization to specific tissues to further support the functional roles of aquaporin in D. citri. In addition, delivery of DcAQP as a plant-incorporated protectant via transgenic citrus or as a field-applied or injected product should be investigated as the current method is limited to laboratory investigations.In this first study of silencing aquaporin in


Prematurity and perinatal stress, such as intrauterine growth restriction (IUGR) and chorioamnionitis, are pathological processes creating an impaired intrauterine environment. These intrauterine factors are associated with the development of proteinuria, hypertension, and chronic kidney disease (CKD) later in life. Initially, this was thought to be secondary to oligonephropathy, subsequent glomerular hypertrophy, and hyperfiltration, leading to glomerulosclerosis, a further decrease in nephron number, and finally CKD. Nowadays, there is increasing evidence that prematurity and perinatal stress affect not only nephron endowment but also the maturation of podocytes and vasculogenesis. IUGR is associated with podocyte damage and an aggravated course of nephrotic syndrome. Moreover, preterm birth and IUGR are known to cause upregulation of the postnatal renin-angiotensin system, resulting in hypertension. Chorioamnionitis causes damage to the glomeruli, thereby predisposing to the development of glomerulosclerosis. This review aims to summarize current knowledge on the influence of prematurity, IUGR, and chorioamnionitis on the development of different glomerular structures. After summarizing human and experimental data on low nephron number in general, a specific focus on the current understanding of podocyte and glomerular capillary formation in relation to prematurity and different causes of perinatal stress is presented. Developments in perinatal and postnatal care have increased the survival of neonates at the expense of increased morbidity later in life, with report of neurologic and pulmonary complications in up to 50% of extreme preterm or extreme low birth weight neonates. Kidney development continues until 34–36 weeks of gestation and may be hampered by preterm birth and/or perinatal stress, such as intrauterine growth restriction (IUGR) and chorioamnionitis. These constraints on kidney development are associated with hypertension, proteinuria, focal segmental glomerulosclerosis (FSGS), and chronic kidney disease (CKD) later in life. Low birth weight (LBW) is associated with a 70% increased risk and prematurity (< 28 weeks gestation) with a threefold increase in risk of CKD , 2. TherThe pathophysiological mechanisms underlying the development of proteinuria, hypertension, and CKD in the context of perinatal stress or preterm birth are not fully elucidated. Much research has been conducted into the field of reduced nephron endowment and the glomerular hyperfiltration theory as described by Brenner and colleagues in 1981, which explains how a reduced nephron number could lead to glomerular sclerosis and kidney failure . HoweverThis raises the question of whether different pathophysiological pathways are affected by different causes of perinatal stress and the effect of their timing during gestation. There is growing evidence that in addition to the glomerular number, development of other structures such as podocytes and the vascular system are also affected. This review aims to summarize current knowledge on the influence of prematurity, IUGR, and chorioamnionitis on the development of different glomerular structures Fig. . After sEvidence is accumulating that prematurity and perinatal stress significantly increase the risk of postnatal kidney pathologies . A meta-In the presence of comorbidities, perinatal stress and prematurity impose an additional risk of developing CKD. In an adult population of patients with hypertension and/or diabetes, Fan et al. showed that patients with LBW were 56% more likely to develop CKD than those with a normal birth weight . The aveMicroalbuminuria is an early marker of glomerular hyperfiltration, and several studies report on the correlation with birth weight and prematurity. A meta-analysis by White et al. reports an 80% increased risk of developing microalbuminuria with LBW . KeijzerPrematurity and perinatal stress are associated with hypertension both in childhood and in adulthood. In children at the age of 30 months and born preterm (< 30 weeks), 30.9% were found to have hypertension compared with 3.9% in term born children . At the During normal development, nephrons are formed until 36 weeks of gestation, after which no new nephrons develop. At term birth, each kidney holds approximately 900,000 nephrons but a wide range in numbers has been described . After tThe magnitude of reduction in nephron numbers upon preterm birth is dependent on the timing of delivery. Nephron formation starts at 15 weeks of gestation, with an exponential increase in nephrons in the third trimester, leading to a plateau around 36 weeks of gestation . Before There are several human studies showing that glomerulogenesis can continue after preterm birth . NephronAccording to the hyperfiltration theory, in oligonephropathy, a reduced nephron number leads to hyperfiltration of the remaining glomeruli, resulting in glomerular hypertrophy with secondary damage to the GBM and podocytes, leading to proteinuria, glomerular sclerosis, and eventually kidney failure. Both Sutherland and Rodriguez found an increased renal corpuscle cross-sectional area in the first couple of months after birth as a morphological indication of glomerular hypertrophy , 28. BreIUGR is strongly correlated with reduced nephron endowment , 32. HowTiming of IUGR during organogenesis is associated with risk profiles later in life, as shown by Roseboom and colleagues using the Dutch Famine Cohort . PregnanGlomerular maturation in neonates born after IUGR with ongoing nephrogenesis may be altered more severely, compared with prematurity alone. Rodriguez reported human autopsy data of neonates after preterm birth with birth weight < 1000 g. Overall, the authors concluded there is no postnatal ongoing glomerular maturation beyond 40 days after birth as ongoing subcortical nephrogenesis and S-shaped glomeruli were only found in the preterm group autopsied within 40 days after birth . HoweverChorioamnionitis is a bacterial intrauterine infection characterized by acute granulocyte infiltration in the fetal-maternal or fetal tissues. Chorioamnionitis is the most frequent cause of preterm delivery. Up to 50% of preterm birth is associated with chorioamnionitis , with reResearch data on the effect of intrauterine inflammation on nephron development is currently only available from pre-clinical models, in which intra-amniotic (IA) lipopolysaccharide (LPS) delivery induces chorioamnionitis and FIRS. The first published study regarding nephron development shows that a single IA high-dose injection of LPS given at the equivalent of 33 weeks human gestation leads to a 23% reduction in nephron number and increased renal corpuscle volume in lambs . On the Podocytes form an important part of the capillary filter in the glomerulus and are important in selective resistance to passage of larger molecules . The celPodocyte development starts in the comma-shaped stage of glomerular development with mesenchymal cells differentiating into primitive podocytes . In the As podocytes are highly differentiated cells with limited capacity for replacement, their number per glomerulus is mainly determined during development. Kikuchi showed that the podocyte number increases until the capillary loop stage, after which the number remains static until full development . HoweverThe effect of IUGR on podocytes in an animal model of protein restriction has been studied by Menendez-Castro and Chen , 52. In In human cohort studies of patients with nephrotic syndrome, IUGR and prematurity have been found to be a risk factor for the development and aggravated course of nephrotic syndrome (NS). Several studies have shown that LBW and IUGR are associated with a steroid-dependent and steroid-resistant course of nephrotic syndrome , 54. ComAdditional to the effect IUGR has on the course of NS, there is evidence that it also aggravates the course of other glomerular diseases that potentially progress to glomerulosclerosis. Plank et al. studied two animal models of IUGR, by uterine artery ligation and maternal protein restriction, and induced glomerulonephritis , 59. In Glomerular vasculogenesis is regulated through the renin-angiotensin system (RAS) and vascular endothelial growth factor (VEGF) cascade. Vascular development within the glomerulus starts in the S-shaped phase with podocytes excreting VEGF-A leading to the infiltration of endothelial cells, which subsequently excrete PDGF-β causing the migration of mesangial cells into the glomerulus. Mesangial cells are essential for the vascular loop formation and therefore increase the glomerular surface area . On the The renal microvascular development encompasses not only glomerular vasculogenesis but also the development of peritubular capillaries. The development of these peritubular capillaries is also regulated by VEGF and RAS . StudiesSutherland and Staub both used preterm sheep models with sheep born at 130 days of gestation (comparable to 35 weeks gestation in humans) , 64. BotThe correlation between IUGR and glomerulovascular development has been studied in animal models. Baserga is the only one to report on the VEGF cascade and found a downregulation of VEGF in the kidney during nephrogenesis in a rat model with growth restriction secondary to uteroplacental insufficiency . At 21 dCompared with the aforementioned studies on prematurity and IUGR and their effect on glomerular vasculogenesis, research involving chorioamnionitis has focused on the postnatal effect on RAS and the subsequent development of hypertension—one of the known long-term consequences of perinatal stress. Ding et al. and Hao et al. showed in rodent models that chorioamnionitis increased the intrarenal expression of renin and angiotensin II in the weeks after birth without elevated levels of plasma renin activity or angiotensin II , 69. HypAnother effect of chorioamnionitis on the glomerular capillaries is the correlation between chorioamnionitis and glomerulovascular damage rendering the glomeruli increasingly susceptible for glomerulosclerosis . KidneysThe predisposition to develop CKD after preterm birth and/or perinatal stress, such as IUGR and chorioamnionitis, has long been acknowledged. Since Brenner’s hyperfiltration theory supported the correlation between oligonephropathy and proteinuria, hypertension, and CKD, initial research mainly focused on the correlation between prematurity, perinatal stress, and oligonephropathy. However, ongoing research has shown that the described predisposition for CKD involves pathways beyond oligonephropathy. Research has shown that IUGR is strongly associated with the risk of proteinuria  and podoAll three aforementioned factors individually increase the risk of kidney disease later in life Fig. , and the