Transition immediately after birth is a complex physiological process. The neonate has to establish sufficient ventilation to ensure significant changes from intra-uterine to extra-uterine circulation. If hypoxia or bradycardia or both occur, as commonly happens during immediate transition in preterm neonates, cerebral hypoxia–ischemia may cause perinatal brain injury.2) using specified clinical treatment guidelines during the immediate transition period after birth (the first 15 min) in addition to the routine monitoring of arterial oxygen saturation (SpO2) and heart rate (HR).The primary objective of the COSGOD phase III trial is to investigate whether it is possible to increase survival without cerebral injury in preterm neonates of less than 32 weeks of gestation by targeting cerebral tissue oxygen saturation  defined as any intra-ventricular bleeding or cystic periventricular leukomalacia (or both). Secondary outcomes are neonatal morbidities.The premature infants will be randomly assigned to study or control groups. Both groups will have a near-infrared spectroscopy (NIRS) device , pulse oximeter , and electrocardiogram placed immediately after birth. In the study group, the crSO2 monitoring during immediate transition has been proven to be feasible and improve cerebral oxygenation during immediate transition. The additional monitoring of crSO2 with dedicated interventions may improve outcome of preterm neonates as evidenced by increased survival without cerebral injury.crSONCT03166722. Registered March 5, 2017.ClinicalTrials.gov Identifier:  The transition from the intra-uterine to extra-uterine environment is a complex physiological process characterized by major physiological changes in respiratory and hemodynamic functions, which are predominantly initiated by breathing at birth and clamping of the umbilical cord . If hypo2) and heart rate (HR) measurement with pulse oximetry/electrocardiogram (ECG), blood pressure, and temperature measurements. Special interest has grown in the use of pulse oximetry and ECG to monitor SpO2 and HR during this transitional period [Routine non-invasive monitoring during immediate transition after birth might include arterial oxygen saturation  using near-infrared spectroscopy (NIRS) during immediate fetal-to-neonatal transition [2 is less affected by mode of delivery  [2 and HR. Lower SpO2 and HR values have been reported in infants born via cesarean section compared with infants born vaginally [2 and SpO2 might be due to changes/decreases in cerebral blood flow (CBF) [2 values compared with those who do not [Recently, there has been increasing interest in continuous monitoring of cerebral tissue oxygen saturation crSO using neansition . In 1992ansition . Furthersection) –15 than aginally . The difow (CBF) . Differeow (CBF) , 17. In ow (CBF) –21. It h2 values already during immediate transition compared with neonates without IVH [Therefore, as the brain is the most vulnerable organ of the infant, to monitor cerebral oxygenation in a non-invasive way is a potentially useful to guide supplemental oxygen and respiratory support in neonates. In a two-center prospective observational case control study, we demonstrated that neonates developing an intra-ventricular hemorrhage (IVH) during the first week after birth showed lower crSOhout IVH .2 monitoring in addition to routine monitoring with pulse oximetry and ECG was used to guide respiratory and supplemental oxygen support during the first 15 min after birth. In the NIRS-not-visible group, routine monitoring which consisted of pulse oximetry and ECG was used. The primary outcomes were burden of cerebral hypoxia (<10th centile) or hyperoxia (>90th centile) measured in percentage minutes crSO2 during the first 15 min after birth. In the NIRS-visible group, the burden of cerebral hypoxia was halved with a relative reduction of 55.4%  [Based on the findings in the observational studies –22, pretCerebral ultrasonography is a valuable screening tool to determine significant brain injury like IVH grade 1–3 (+ periventricular hemorrhage), cerebellar hemorrhage, and periventricular leukomalacia (PVL) grade 1–3 when conducted regularly over the first weeks of life in preterm infants . Magneti2 using NIRS INVOS 5100  in addition to routine monitoring of SpO2 and HR to guide supplemental oxygen delivery and respiratory/circulatory support in preterm neonates during the first 15 min after birth.Based on the findings in the observational studies and COSGOD phase I/II trial, the objective of the present clinical trial is to monitor crSOThe primary aim is to increase survival without cerebral injury at discharge or term age. The secondary aim is to assess neonatal morbidity until discharge or term age.2 and SpO2/HR monitoring during the first 15 min after birth will increase survival without cerebral injury and reduce morbidities in preterm neonates of less than 32 weeks of gestation.We hypothesize that supplemental oxygen support and respiratory/circulatory support guided by crSOThe present trial is an investigator-initiated, randomized, multi-center, multi-national, clinical trial that will enroll 362 preterm neonates of less than 32 weeks of gestation in the study group and 362 preterm neonates of less than 32 weeks of gestation in the control group Figs.  and 2. TPreterm neonates of less than 32 weeks of gestation are eligible for the study. Further inclusion criteria are the decision to provide full life support, written informed consent, and the application of NIRS sensors within 3 min after birth. Exclusion criteria are decision not to provide full life support, no written informed consent, or severe congenital malformation of brain, heart, lung, or prenatal cerebral injury or a combination of these.According to data of two European centers  and one Canadian center , the percentage of neonates who survive without cerebral injury ranges from 56% to 77% and the overall percentage is 65%. Given an increase of neonates who survive without cerebral injury from 65% to 75%, 329 neonates in each group are required to detect this difference with a two-group chi-squared test with a 0.05 two-sided significance level and a power of 80%. Given a dropout rate of 10%, a total of 724 neonates will be enrolled.t test or Mann–Whitney U test in the case of continuous variables. To investigate the primary hypothesis whether the frequency of cerebral injury in preterm neonates of less than 32 weeks of gestation will be different in the two groups, a chi-squared test will be performed. Secondary outcome parameters including mortality and neonatal morbidities  will be compared between groups by using the chi-squared test or Fisher’s exact test. Exploratory outcomes  will be compared between groups by using the chi-squared test or Fisher’s exact test; trends of monitoring parameters  during the first 15 min after birth will be analyzed by using linear mixed models. With these linear mixed models, differences in the course of the monitored parameters between groups (study group versus control group) will be analyzed. Baseline characteristics, which will show significant differences between groups, will also be included in the linear mixed models.Demographic and baseline data for the infants will be compared between groups (study group versus control group) using the chi-squared test or Fisher’s exact test in the case of categorical variables and using the https://www.randomizer.at/random/). The ratio of allocation is 1:1. Patients will be stratified according to trial site. In case of multiple births, only the first infant will be randomly assigned.Neonates will be randomly assigned before birth to either the study group or the control group by using the web-based randomization service , developed at the Institute for Medical Informatics, Statistics and Documentation, Medical University of Graz .2 and SpO2/HR monitoring. In the control group, supplemental oxygen support and respiratory/circulatory support are guided by SpO2/HR monitoring according routine resuscitation management; the resuscitation team is blinded to the crSO2 monitoring.In the study group, supplemental oxygen support and respiratory/circulatory support are guided by crSOMonitoring and clinical interventions start within 3 min after birth and last for 15 min after birth.Each neonate will be followed until discharge or until term date (37–42 weeks of gestation).Cerebral ultrasound will be performed at 2–24 h , 2–5 days, 6–8 days, 12–16 days, before discharge/37–42 weeks of gestation and MRI  before discharge, or at 37–42 weeks of gestation.The antepartum medical history and birth history will be collected. Gestational age, birth weight, gender, pH of umbilical artery, and Apgar score will be documented for each neonate. Continuous positive airway pressure and positive pressure ventilation with a face mask, if necessary, will be performed.2 and HR as routine non-invasive monitoring, (ii) ECG for HR as routine non-invasive monitoring, and (iii) NIRS measurements. NIRS measurements will be blinded in the control group.Monitoring during the first 15 min after birth consists of (i) pulse oximetry for SpOFor NIRS measurements, the Invos™ Cerebral/Somatic Oximeter monitor (Medtronic) with the neonatal sensor will be used.2 and HR.Immediately after birth, when the neonate is brought to the resuscitation area, the cerebral NIRS sensor will be placed on the left forehead and fixed with a continuous positive airway pressure cap or elastic bandage on the left forehead. ECG electrodes will be applied to the chest for HR monitoring. One pulse-oximetry sensor will be applied to the right palm or wrist for monitoring pre-ductal SpOResuscitation will be conducted in accordance with the “Consensus Guidelines” on the management of neonatal respiratory distress syndrome , 29, but2 targeting is conducted in accordance with “Local Guidelines” and “Resuscitation Guidelines” [2 remains less than the 10th centile or below the local lower limit, respiratory support will be increased/started or fraction of inspired oxygen (FiO2) will be increased by 10–20% every 60 s. Respiratory support will be reduced/stopped or FiO2 will be reduced by 10–20% if SpO2 remains stable more than the 10th centile [2 is more than the 90th centile [2 monitoring.Depending on the infants’ breathing efforts and HR, the SpOdelines” , 29 but delines”  Fig. 3)2 targeti centile  or above centile  or above2 monitoring is visible to the clinical team . The exploratory outcome measures are need for respiratory support, intubation, and medications during resuscitation; need for mechanical ventilation and treatment with catecholamines on the first day after birth; and monitoring parameters  during the first 15 min after birth.The primary outcome measure is survival without cerebral injury defined as any grade of IVH or cystic PVLAll-cause mortality will be recorded. Cerebral injury will be assessed by cerebral ultrasound at 2–24 h , 2–5 days, 6–8 days, 12–16 days, before discharge/37–42 weeks and optionally cerebral injury will be assessed by cerebral MRI, if performed routinely—mainly without sedation during postprandial sleep—before discharge or term age (37–42 weeks of gestation). Case history will be assessed until discharge or term age, depending on what comes first.Owing to the nature of the trial, the intervention cannot be blinded for the clinical staff and the parents. However, blinding will be used in some other aspect of the trial. The allocation sequence will be concealed.Source data will be registered in the participant’s medical records/case report form (CRF) and the electronic CRF (eCRF). A common web-based eCRF will be used and entered in a central database . Data entry into the central database is the responsibility of the investigators. After establishment of a “clean file”, the database will be locked, and data will be stored for statistical analysis at the Institute for Medical Informatics, Statistics and Documentation, Medical University of Graz.Trial data will be handled in accordance with regulations of the data protection agency in the respective countries. After completion of statistical data analysis, data will be pseudo-anonymized and deposited at the Medical University of Graz. After the end of trial, the data will be archived for 15 years in accordance with GCP guidelines. At each trial site, the data flow will be monitored in accordance with the GCP principles by a locally appointed external monitoring committee.The preterm patient population is a very vulnerable and often seriously ill group. Most adverse events may be of a serious nature with or without the COSGOD trial intervention, and both groups are expected to have a very high proportion of serious adverse events (SAEs). It is therefore not possible, or meaningful, to record and report all adverse events. The SAE to be reported is mortality. Serious adverse reaction and suspected unexpected serious adverse reactions (SUSARs) will be reported to ethic committees and authorities.An independent safety committee will perform a first monitoring after the inclusion of 20% of the neonates to evaluate the risk of SAEs and efficacy (primary and secondary outcome parameters) of the intervention with “certain” or “probably/likely” relationship with the cerebral NIRS oximeter or the application of the treatment guideline. The participants will be insured in accordance with existing legislation of their respective country.The COSGOD trial will start at the different centers randomly assigning participants after approvals from the relevant ethics committees and authorities have been received. All parents will receive written and oral information about the trial before they are asked for their written consent. They will enroll their newborn infant in the trial only by their own free will and can withdraw their consent for participation at any time. If a parent wishes to withdraw the consent for participation, the patient will receive treatment in accordance with the respective hospital’s standard procedures. The trial will be conducted in compliance with the guidelines of the Declaration of Helsinki in its latest form and the ICH GCP guidelines.In case of modifications in the study protocol that are not merely of a formal nature but contain changes pertinent to the study participants, a renewed vote of the ethics committee will be obtained. If applicable, the patients/parents will be informed in the patient information and consent form about changes in the terms and conditions of the trial.2 in addition to routine monitoring of SpO2 and HR to guide supplemental oxygen delivery, respiratory support, or circulatory support (fluid bolus) (or a combination of these) in preterm neonates during the first 15 min after birth. In the pilot study [2 measurement and a treatment guideline could reduce the burden of cerebral hypoxia in preterm neonates with less than 28 weeks of gestation during the first three days after birth [The primary aim of the present clinical trial is to increase survival without cerebral injury in preterm neonates by monitoring crSOot study , we demoer birth . In thiser birth , 32.2 will be kept within recommended/local limits in all neonates (at least between the 10th and 90th centiles of published reference ranges) [2 below the lower limit and hyperoxia defined as SpO2 above the upper limit should be avoided.Since pulse oximetry and ECG are recommended during immediate transition and resuscitation when monitoring is performed , 29, bot ranges) . Therefo2 would suggest increased respiratory support and increased use of supplemental oxygen in case of low cerebral oxygenation. In the COSGOD phase I/II trial, the supplemental oxygen support was lower in the NIRS-visible group in the first minutes [Monitoring of crSO minutes . For COSOwing to the short intervention period, irritation of the skin caused by NIRS sensors will be unlikely. Moreover, this has not been observed in other studies using NIRS during immediate transition , 11. Ris2 and dedicated interventions are feasible during immediate transition and resuscitation and reduce the burden of cerebral hypoxia. The present trial will examine whether monitoring of crSO2 and dedicated interventions will improve survival without cerebral injury.In conclusion, additional monitoring of crSORecruitment started in September 2018 and is expected to be completed at the end of 2020.


Initial reservations regarding spillage and the ability to manage the cyst wall have been allayed by many reports which show satisfying results with minimal complications using laparoscopic techniques. Here, we study the possibility and safety of simultaneous laparoscopic pericystectomy with one of the most common laparoscopic procedure; laparoscopic cholecystectomy.A 37 y/o female from a rural area in Syria was referred to our clinic after one year of total pericystectomy from both right and left lobes of lung. She was scheduled for a second operation to resect a third hydatid cyst from her liver. We noted that she also complained of a cholelithiasis and it would be very helpful to manage both these problems altogether.previously, one manuscript discussed in details the safety of laparoscopic simultaneous partial pericystectomy and total cystectomy for hydatid liver cysts. The anaphylactic shock, surgical site infection (SSI) and recurrence were still challenges in any participation of laparoscopic hydatid pericystectomy with other kind of surgery from the same ports. Here, we also paid an attention for the duration and cost of this practice.The combination of the laparoscopic pericystectomy with other producers may be safe in certain selective cases and does not increase the possibility of postsurgical complications like infections or recurrence. We found this intervention could be effective and useful. It is gSyria lies in the endemic area of the Middle East. We received more than 50 cases of liver hydatid cysts annually in our institute, the main hospital in Damascus. Historically, open surgery has been always in the corner of the management. However, Syrian surgeons, like their colleagues in endemic countries, improved their skills in the laparoscopic management of the liver hydatid cyst. This mini-invasive procedure was firstly described by Saglam et al. . Since t2A 37 y/o female from a rural area in Syria was referred to our clinic after one year of total open thoracic pericystectomy from right and left lobes of lung; the surgical management of hydatid cyst of the liver was delayed till this admission after two shots of albendazole treatment.The patient recently developed also a symptomatic cholelithiasis. There were no other obvious findings in physical examination. Laboratory investigations were in normal limited .Table 1SThe ultrasound showed a cyst in the right lobe of liver and gallbladder stones with no signs of inflammatory. The abdominopelvic computerized tomography (CT) revealed a cystic mass of 5 × 4.5 cm2 with regular borders and minimal calcifications in the 6th segment of liver . It was The plan of the management was discussed and included lagrot partial pericystectomy and cholecystectomy in the same time. The anatomical location of the hydatid liver cyst allowed to use the same ports for both procedures and it alerted, in the same way, to the risk of facing adhesions in the surgical field if we delayed one procedure to another operation which would be scheduled later. This judgment encouraged our team to run both procedures simultaneously.3The laparoscopic surgery was performed. The camera port was applied through the umbilicus following creation of pneumoperitoneum; the hydatid cyst is identified on the lower surface of the liver. Then, the trocar (10 mm) placed in epigastric region and another trocar (5 mm) with cannula is introduced into the peritoneal cavity directly over the hydatid cyst.First of all, we started by introducing into the abdominal cavity a gauze soaked with hypertonic saline solution (10%) which was placed on the diaphragmatic surface of the liver around the cysts to prevent the spillage of fluid. Then we performed the decompression of the cyst by aspiration of the cyst fluid using a wide bore needle through the 5 mm port under laparoscopic guidance, taking care to avoid spillage, and by using a continuous suction cannula around the needle puncture site. We injected the hypertonic saline solution (10%) into the cyst cavity. Ten minutes later, we sucked it and opened the cyst using the electrocautery making a direct inspection of the cavity by introducing the laparoscope into the cyst to seek for remaining cyst elements and biliary leakage, if any, for subsequent attention. We performed then the lagrot partial pericystectomy for the hydatid cyst’s anterior wall, as much as possible, using diathermy and bringing it out of the abdomen. After a clear vision of the gallbladder, a standard cholecystectomy was performed from the same ports, and a drain was inserted under the liver that is removed later after 24 h when no signs of bleeding or biliary leakage were obtained. The patient was discharged in two days. The follow‐up was made as an outpatient at the 30th, 60th and 90th days postoperatively; the patient denied any symptom complaint or sign. see .Fig. 2La4The simultaneous of laparoscopic partial pericystectomy with other producers in the same surgical field may be safe in certain selective cases. Our report shows that these combined surgeries will not increase the risk of intra- or post- operative complications like surgical site infection, spillage or even the recurrence.5In the management of abdominal hydatid cyst disease, several surgical techniques have been advocated, ranging from aspiration to radical and segmental resection. The surgical treatment of liver hydatid disease has evolved dramatically with the improved of the laparoscopy .However, some manuscripts in the first decade of 21 st century reported an anaphylactic shock during the minimally invasive procedure of the hydatid cyst management; this strongly exaggerated the fear which seemed to discourage many surgeons from readily adopting these techniques ,8. LaterIn general, the combined actions in one laparoscopy were discussed separately many times in selective cases . The parIn our case we continue the discussion and questions that others began above; the abdominal hydatid cysts could be present with other pathological problems like a cholelithiasis. The cons and pros of combined surgery should be evaluated every time; the difficulty of techniques, risk of contamination, duration of the procedure and predicting of early or late complications play the main role in the decision of choosing either combined or separated interventions.During the planned procedure; every technique that prevents spillage was done; every instrument that avoids the direct contact between the contents of the cyst and the abdominal cavity on one side; and between these contents and ports in another side were used. Then, the gallbladder was resected classically and the procedure was done, the patient then kept following-up till the complete healing and no residual or new hydatid cyst was reported that go on with perspective exhausted patient from repetition of surgeries.We don't have any conflicts of interest with any organisation, on the contrary our case report was supported by three big educational institution.We haven't received any funding for our manuscript because it is a case report not a research.Our case is exempt from ethical approval in our institute.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request .Aliaa Bakr: corresponding author/ collected the data and wrote the manuscript.Mhd Belal Alsabek: co-author/ the surgeon who prepared the patient, planed the surgery, run the operation and reviewed the PubMed Library.Abdul Rahman Hammadieh: supervisor and guarantor/ reviewed the manuscript.Our manuscript is a case report not a research.Abdul Rahman Hammadieh: MD, AFSA. Assistant professor in the Surgery Department, Damascus University, Faculty of Medicine, Damascus, Syria. The Head of General Surgery Ward, Al-Mouwasat University Hospital, Damascus, Syria.Not commissioned, externally peer-reviewed.


Maternal 25-hydroxyvitamin D (25-OHD) deficiency during pregnancy may increase the risk of preterm and small-for-gestational age (SGA) birth, but studies report conflicting results. We used a multicenter prospective cohort of 2813 pregnant women assessed for 25-OHD levels in the first trimester of pregnancy to investigate the association between maternal 25-OHD concentrations and risks of preterm birth (<37 weeks) and SGA (birthweight <10th percentile). Odds ratios were adjusted (aOR) for potential cofounders overall and among women with light and dark skin separately, based on the Fitzpatrick scale. 25-OHD concentrations were <20 ng/mL for 45.1% of the cohort. A total of 6.7% of women had a preterm birth. The aOR for preterm birth associated with the 1st quartile of 25-OHD concentrations compared to the 4th quartile was 1.53 : 0.97–2.43). In stratified analyses, an association was observed for women with darker skin (aOR = 2.89 (95% CI: 1.02–8.18)), and no association with lighter skin. A total of 11.9% of births were SGA and there was no association overall or by skin color. Our results do not provide support for an association between maternal first trimester 25-OHD deficiency and risk of preterm or SGA birth overall; the association with preterm birth risk among women with darker skin requires further investigation. Vitamin D is generated in the liver and then in the kidneys and is commonly assessed by the measurement of 25-hydroxyvitamin D (25-OHD) levels . VitaminSome research has provided evidence for the existence of an association between vitamin D and preterm and SGA birth, but results have not been consistent. Meta-analyses of randomized controlled trials have reported an association between lower maternal vitamin D concentrations and an increased risk of preterm birth and low birthweight <2500 g , a higheIn this study, our aim was to investigate the association between maternal first trimester vitamin D concentrations and the risks of preterm and SGA birth using a large prospective cohort designed for the assessment of maternal vitamin D during pregnancy which includes 25-OHD concentrations measured between 10 and 14 weeks of gestation as well as data on skin color.The FEPED study is an observational prospective cohort conducted from April 2012 through July 2014 in five French and one Belgium maternity units ,28. The At enrolment, a physician completed a questionnaire to ensure that women met the inclusion criteria. In this questionnaire, skin color was assessed using the Fitzpatrick scale . The FitAfter delivery, sociodemographic and clinical data were abstracted from medical records by personnel trained for the study. Sociodemographic maternal characteristics included parity, maternal age, height, prepregnancy weight, ethnicity and smoking status before and during pregnancy. Information was also collected on medical and obstetrical history , complications of the current pregnancy , the delivery and the infant’s health status at birth.n = 183), miscarriages, abortions and terminations of pregnancy before 22 weeks of gestation (n = 36). Cases with missing data for birthweight (n = 14), gestational age (n = 1), infant sex (n = 5) and 25-OHD maternal concentrations in the first trimester of pregnancy (n = 41) were also excluded. The study population included 2813 women.  of maternal 25-OHD concentrations in the first trimester overall and by maternal characteristics. We then calculated the proportion of women with 25-OHD concentrations <20 ng/mL in the first trimester of pregnancy by selected characteristics. Based on the scientific literature and biological plausibility, we selected co-variables as possible confounders of the association between vitamin D concentrations and preterm birth and SGA, including maternal age, parity, pre-pregnancy body mass index (BMI), smoking, medical and obstetrical history. We also included season at blood draw, skin color and ethnicity, since these were hypothesized to be strongly related to vitamin D status and there is increasing evidence of an association with perinatal outcome. Seasonal variations in respiratory and viral infections, as well as in the temperature, impact on preterm birth ,38,39,40Logistic regression was also used to estimate adjusted odds ratios (aOR) for preterm and SGA birth for the groups, defined by quartile and clinical cutoffs. Based on the hypothesis that there may be an interaction between our outcomes and skin color, we carried out analyses stratified by skin color according to the Fitzpatrick scale . As a se2 (51.8%), Africans and those with a dark skin color (60.4%) , and those with a BMI before pregnancy of ≥30 kg/m (60.4%) . In contp = 0.144), and SGA and non-SGA birth  (results not shown). Compared to the 4th quartile (30 ng/mL+), the adjusted odds ratio associated with 25-OHD concentrations in the 1st quartile was 1.53 (95% CI: 0.97–2.43)) for the risk of preterm birth and 1.07 (95% CI: 0.75–1.54) for the risk of having an SGA infant.Twenty-seven percent of women had 25-OHD concentrations <15 ng/mL and 45.1% had concentrations <20 ng/mL.  There wap value = 0.050) but not for SGA . Analyses stratified by skin color showed that women with light skin had no excess risk of preterm birth, whereas the risk of preterm birth for women with dark skin (type V to VI) was increased for the first quartile of 25-OHD (aOR = 2.89 (95% CI: 1.02–8.18)). Results were similar using clinical cut-offs, with an aOR of 2.63 (95% CI: 0.95–7.27) for 25-OHD concentrations <20 ng/mL compared to those with 25-OH levels >30 ng/mL. No association was observed for SGA using quartiles or clinical cut-offs. Analyses with birthweight and gestational age as continuous variables confirmed the absence of an association between low 25-OHD levels with birthweight in the overall sample and an interaction with skin colour for gestational age (p = 0.05). (The interaction between skin color and vitamin D concentrations was significant for the risk of preterm birth (= 0.05). Restricted cubic splines were used to investigate the association between maternal 25-OHD concentrations and the risk of preterm and SGA birth adjusted for maternal age, parity, BMI before pregnancy, medical or obstetrical history, smoking, ethnicity, skin color and season at blood draw overall. Figures show that the risk of preterm birth and having an SGA infant were not different, regardless of maternal vitamin D concentrations in the overall sample and for women with light skin color.  For womeIn our cohort, almost half of pregnant women had 25-OHD concentrations in the first trimester of pregnancy below the lower clinical cut-off point of <20 ng/mL, one-quarter had 25-OHD concentrations of <15 ng/mL, while only 24% of women had 25-OHD levels above 30 ng/mL. No association was found between low maternal vitamin D concentrations and the risk of preterm and SGA birth in the overall sample and among women with a lighter skin. In contrast, an increased risk of preterm birth, but not SGA, was observed for women with darker skin.Our study has several strengths. We used data from a large multicentre prospective cohort explicitly designed to investigate the association between maternal vitamin D concentrations and perinatal outcomes ,28. All Our aim was to assess the benefit of vitamin D supplementation in early pregnancy on perinatal outcomes and, thus, we did not examine the changes in vitamin D levels between the first and the third trimester of pregnancy. This change was examined in a previous analysis using the FEPED cohort. The increase of 25-OHD levels was estimated to 6.3 ng/mL (SD = 12.6) and 8.5 (SD = 11.5) in women with pre-eclampsia and in those with no pre-eclampsia . In our There was a higher proportion of women with low 25-OHD levels <20 ng/mL in the first trimester of pregnancy among obese women, Africans, darker skin colored and based on season at blood draw. Vitamin D absorption depends on body fat, skin pigmentation and sun exposure and these characteristics are thus well-known risks factors for vitamin D deficiency ,26,48.We investigated two pregnancy outcomes previously associated with vitamin D concentrations and for which plausible biological mechanisms have been suggested. Vitamin D could play a role in the regulation of genes involved in trophoblast invasion and in the angiogenesis for placenta implantation that could impact on fetal growth. However, our results related to SGA do not support an association with maternal vitamin D levels, corroborating a number of previous studies ,23 but cOur results, taken together with the body of conflicting literature on vitamin D and pregnancy outcomes, including the high study heterogeneity in meta-analyses, suggests that the association of vitamin D concentrations and these pregnancy outcomes may depend on the populations studied ,10,11,49In conclusion, our study does not provide support for a common biological pathway linking vitamin D concentrations in the first trimester of pregnancy to risks of preterm or SGA birth. However, further investigation is needed on potential differences in preterm birth risks among women with darker skin color.


Information regarding in vitro fertilization (IVF) as a pregnancy risk factor (yes/ no) is stored in each birth record of the Estonian Medical Birth Registry (EMBR). This study aimed to assess the validity of registration of newborns’ IVF status in the EMBR.To identify the newborns conceived by IVF, the birth records in the EMBR were compared to individual records on the embryo transfer procedures in the Estonian Health Insurance Fund (EHIF) database as a reference. Maternal age was restricted to 40 years, the age limit for IVF treatment covered by the EHIF. The embryo transfer procedures, that dated up to eight weeks before pregnancy, were additionally checked in the infertility treatment clinics. The validity of IVF status was measured by sensitivity, specificity, positive and negative predictive values (PPV and NPV). Relative risk (RR) of unrecorded IVF status among IVF mothers by socio-demographic characteristics and birth plurality was estimated using modified Poisson regression models.There were 3198 newborns identified as conceived by IVF in the EMBR in 2005–2014. Eight of them were incorrectly entered as born after IVF. The record linkage with the EHIF database revealed 1014 newborns with unrecorded IVF status in the EMBR. A total of 4204 newborns were verified as conceived by IVF, 24.1% of them were not categorized as born after IVF. The sensitivity of the IVF status registration was poor (75.9%), specificity (100.0%), PPV (99.8%) and NPV (99.3%) were high. The misclassifications were significantly more common among mothers of younger age or non-Estonians or with singleton birth.Information based on mother’s self-report or her antenatal chart does not accurately identify the newborn’s IVF status. The lack of a specialized country-wide assisted reproductive technology register in Estonia requires routine record linkage of the EMBR, EHIF and the infertility treatment clinics’ databases to obtain adequate information regarding IVF status in the EMBR. Electronic record linkages between databases would help considerably to improve the validity of data to be used in medical decision making, in research and for statistical purposes. Birth records in the Estonian Medical Birth Registry (EMBR) include information on the IVF status of newborns relying on mothers’ self-report or their antenatal charts. As there is no mandate to sanction data exchange between the EMBR and infertility treatment clinics, current study aimed to assess the validity of the IVF status registration in the registry via linkage records of the EMBR and Estonian Health Insurance Fund (EHIF) reimbursement database as a reference. Solidarity based EHIF covers unlimited number of IVF cycles for all women aged 40 years or less. The embryo transfers resulting in birth were identified using the mother’s personal identification number as the key. If necessary, additional verification in the infertility treatment clinics was performed. According to the EMBR data, 146,526 infants with maternal age up to 40 years were born to Estonian residents in 2005–2014. A total of 4204 newborns were verified as conceived by IVF; 1014 newborns had lacked the IVF status on the birth record. The sensitivity of the IVF status registration was poor (75.9%). The IVF status was better recorded for mothers of the older age group (35–40 years) or with a multiple birth, because they were more likely to have IVF pregnancy and were more carefully questioned. As far as Estonia lacks a specialized country-wide assisted reproductive technology register, record linkages of the EMBR, EHIF and the infertility treatment clinics’ databases are requested to obtain adequate information on the IVF status of the newborns in the EMBR.The 2014 Estonian Women’s Health Questionnaire Survey indicated that 15.8% of the sexually active respondents at 18–44 years of age had experienced infertility, and 13.4% of them reported the use of in vitro fertilization (IVF) to have a child [The unique personal identification numbers (PINs) of the newborn and his/ her mother are registered in the EMBR and allow for record linkage . The IVFThe previous study that estimated effectiveness and costs of IVF treatment in Estonia using linkage of the EMBR and EHIF records, reported 15.5% fewer IVF entries in the EMBR in 2006–2011 than were based on the EHIF data. A birth in the EMBR was considered an IVF birth if the embryo transfer procedure was performed within 42 weeks before delivery [Current study aimed to assess the validity of registration of newborns’ IVF status in the EMBR by linking the EMBR and EHIF records.We assessed the validity of registration of newborns’ IVF status in the EMBR by comparing information about it on the records of the EMBR and of the EHIF.The EMBR, currently under the responsibility of the National Institute of Health Development, was established in 1991 and collects data on all infants born in Estonia , 7. The The EHIF, founded in 2001, is the only institution in Estonia that administers the national compulsory solidarity-based health insurance system . The funThe validity of registration of newborns’ IVF status in the EMBR was measured by comparing the birth records in the EMBR and the corresponding embryo transfer records in the EHIF. Thus, the EHIF was treated as a reference. Individual records of the embryo transfers in 2004–2014 (earlier data were not available) including woman’s PIN, date of the procedure and name of the clinic were obtained from the EHIF database  . The beginning of pregnancy after IVF (comparable to that of spontaneously conceived newborns) dates 2–3 weeks prior to the date of the embryo transfer , 12. ThuWhereas the reimbursement claims of the EHIF may contain inaccuracies in documenting the date of the embryo transfer procedure, additional validation of the IVF status was undertaken in the infertility treatment clinics. In site visits, we wished to ascertain that embryo transfers performed during eight weeks before the beginning of pregnancy (by EHIF data), were not related to the pregnancies under study, and these newborns were spontaneously conceived after unsuccessful IVF treatment. Repeated embryo transfers, except the last one before the pregnancy that resulted in birth, were not taken into consideration. Thus, first, a list of eligible mothers  was printed out. Second, in the infertility treatment clinics, the list of these mothers was visually compared with a screen output of their clinical data. As a result, IVF and non-IVF newborns were distinguished Fig. . All birThe validity of the IVF status in the EMBR was evaluated by determining the sensitivity, specificity, positive predictive value and negative predictive value, all often used in validation of health databases –16. The Additionally, we calculated the relative risks  with 95% confidence intervals (CI) of unrecorded IVF status among IVF mothers by available socio-demographic characteristics and birth plurality. Modified Poisson regression models (with a robust error variance)  includedVisual FoxPro 9.0  was used for database management and linkages, and Stata 14  for validity calculations and statistical modeling.According to the EMBR data for 2005–2014, 3198 infants were born to mothers aged 40 years or less and identified as conceived by IVF. The record linkage with the EHIF database resulted in 3912 newborns in the EMBR with the corresponding embryo transfer record (the date of the embryo transfer procedure was within the pregnancy period), whereas 1002 of them were with unrecorded IVF status, i.e., erroneously categorized as conceived spontaneously   Fig. . The resThe 1014 false negative IVF entries resulted in poor sensitivity of the registration of IVF status ; specificity (100.0%), PPV (99.8%) and NPV (99.3%) were high (Table An unrecorded IVF status was significantly more common among mothers of younger age (< 35 years) or with non-Estonian ethnicity or with a singleton birth Table . The misThis study presents the results of the record linkages of the EMBR and the EHIF data to evaluate births following embryo transfer procedures. Although a decade ago the general data quality of the EMBR has been found to be acceptable , the curSimilarly, nearly a quarter of missing IVF entries was found in the Finnish Medical Birth Registry after linkage with insurance reimbursement files, drug prescriptions and IVF statistics . Given tThe IVF status recorded on the antenatal chart is completed by the doctor or midwife at the first antenatal visit. The source of this information can be the electronic health record or self-report of the pregnant woman. In the cases where the IVF status is not mentioned on the antenatal chart, it is based on the mother’s self-report after the delivery as “yes/ no” answer to the question: “Did you have IVF pregnancy?” However, the question is not always asked and the mother can refuse to reveal her IVF status. Additionally, if the abbreviation IVF is used, it may lead to misunderstanding. Expectedly, the IVF status was better registered among mothers aged 35–40 years, and mothers with multiple birth, because they were more likely to have IVF pregnancy and were more carefully questioned. The higher under-recording of IVF status among non-Estonians could be due to language or poor attention to the question in the clinics located in the regions with a high proportion of non-Estonians (northeastern Estonia). We found equally low-quality registration of IVF status when the birth occurred in the same clinic that performed IVF treatment, or when the birth took place in another clinic. This finding indicates that there is limited information exchange even between the departments of the same clinic. Thus, the validity of the IVF entries in the EMBR has so far relied on self-report of mothers and on the accuracy of the medical personnel when completing the antenatal chart.However, there were 266 records of the newborns after IVF in the EMBR with no corresponding embryo transfer record in the EHIF database verifying the IVF status. We had no reason to classify them as non-IVF newborns  as their mothers could have paid for the treatment themselves because they did not have health insurance; or because of a will to have out-of-pocket service; or because they have used IVF services in other countries; or because only the limited number of IVF cycles were covered by the EHIF prior to 2008 . On the The previous linkage study  with somIt is difficult to obtain complete data for newborns’ IVF status in the EMBR by exclusively relying on mother’s self-reported information or her antenatal chart. As far as we do not have a specialized country-wide assisted reproductive technology database in Estonia, the routine linkages between the EMBR, the EHIF database, and data from infertility treatment clinics would be helpful. Furthermore, collecting valid data for IVF pregnancies is important due to the common use of assisted reproductive technology . The inc


These results demonstrated that GKRK-APO is an important potential drug delivery system for glioma-targeted therapy.Therapeutic outcome for the treatment of glioma was often limited due to the non-targeted nature of drugs and the physiological barriers, including the blood-brain barrier (BBB) and the blood-brain tumor barrier (BBTB). An ideal glioma-targeted delivery system must be sufficiently potent to cross the BBB and BBTB and then target glioma cells with adequate optimized physiochemical properties and biocompatibility. However, it is an enormous challenge to the researchers to engineer the above-mentioned features into a single nanocarrier particle. New frontiers in nanomedicine are advancing the research of new biomaterials. In this study, we demonstrate a strategy for glioma targeting by encapsulating vincristine sulfate (VCR) into a naturally available apoferritin nanocage-based drug delivery system with the modification of GKRK peptide ligand (GKRK-APO). Apoferritin (APO), an endogenous nanosize spherical protein, can specifically bind to brain endothelial cells and glioma cells via interacting with the transferrin receptor 1 (TfR1). GKRK is a peptide ligand of heparan sulfate proteoglycan (HSPG) over-expressed on angiogenesis and glioma, presenting excellent glioma-homing property. By combining the dual-targeting delivery effect of GKRK peptide and parent APO, GKRK-APO displayed higher glioma localization than that of parent APO. After loading with VCR, GKRK-APO showed the most favorable antiglioma effect  The efficient treatment for glioma, which is the most frequent primary malignant brain tumor with a median survival less than 2 years after first diagnosis, remains a challenging task. Glioma is difficult to treat because of the special pathological and physiological characteristics. Due to the infiltrate growth of glioma, it is hard to completely remove the glioma through surgery  as the model drug, the anti-tumor efficacy of GKRK-APO was evaluated on in vivo intracranial U87MG tumor-bearing mice. Herein, we report the first study on functional apoferritin nanocage as a glioma-targeted delivery system. This study will help elucidate the functions of apoferritin nanocage and allow for its application as a powerful nanoplatform for brain tumor diagnosis and therapy.However, it is now increasingly acknowledged that glioma is genetically heterogeneous and complex, and shows variations in the expression of the bookmakers postulated to be important for active targeting strategy. For example, the dysregulated TfR expression in glioma cells is often only 3–5-fold higher than that in normal cells  0.2 μm filtered and all chemicals were obtained from Sigma-Aldrich , unless otherwise stated. Sulfate vincristine (VCR) was obtained from Baiyunshan Co. . Maleimided GKRK peptide (GKRK-MAL) was synthesized by Cybertron Medical Technology Co. . All chemicals were of reagent grade and were obtained from Sigma-Aldrich, unless otherwise stated.2.HUVEC cells, U87MG, and bEnd.3 cells were provided by the Cell Resource Centre of IBMS  and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS (Gibco) and 1% antibiotics at 37 °C in humidified atmosphere and 5% COFemale Sprague–Dawley (SD) rats (180–220 g) and Female BALB/c nude mice (weighing 18–22 g) were purchased from Vital River Laboratories . All procedures involving animal housing and treatment were approved by the Animal Care and Use Ethics Committee of the Academy of Military Medical Sciences.GKRK-APO was prepared by coupling GKRK-MAL to APO through sulfhydryl-maleimide coupling reaction  was dissolved in dry DMSO and added to GKRK-APO or APO solution , at a dye to GKRK-APO molar ratio of 10:1. The mixture was gently stirred overnight at 4 °C in the dark and then dialyzed in a dialysis bag to remove free dyes.To monitor the 2, 1 mM CaCl2, 0.5 mM MnCl2) and added to the microtiter wells  by adsorption overnight before blocking with casein blocker . Cy5.5-labeled GKRK-APO were re-suspended in binding buffer  or acetate buffer , respectively. About 0.5 mL of VCR-loaded GKRK-APO or VCR-loaded APO was added to dialysis bag with molecular weight cut-off 12,000–14,000. The dialysis bag was then placed in a flask filled with 30 mL medium at 37 °C. At predetermined intervals, 800 μL of medium was drawn out and replenished with the same volume of fresh medium. The released free VCR at different incubation times was assayed by HPLC, as previously reported . The analysis of stability was carried out by the software of the instrument, as a variation of back-scattering (ΔBS) profiles.The mean diameter and particle distribution of these GKRK-APO carriers were measured by dynamic light scattering . Morphology of the VCR-loaded GKRK-APO was characterized via an atomic force microscopy (AFM)  and transmission electron microscopy (TEM) , respectively. The stability of VCR-loaded GKRK-APO in 10% FBS was evaluated using a Turbiscan LabWtotal drug and Wfree drug represent the total drug in APO and the amount of free drug in the ultrafiltrate, respectively.The VCR encapsulation efficiency (EE) of VCR-loaded GKRK-APO and VCR-loaded APO was calculated using the following equation:To investigate the effect of GKRK peptide density on cellular uptake, Cy5.5-labeled GKRK-APO was prepared at different peptide densities . U87MG cells were seeded into 12-well plates for flow cytometry (FCM) . The concentration of Cy5.5 was 150 ng/mL.In order to assess the binding affinity of GKRK-APO or APO to cells, different 5 μM Cy5.5-labeled GKRK-APO or Cy5.5-labeled APO were incubated with three types of cells  at 37 °C for 2 h, respectively. The cells were washed three times with cold PBS, then centrifuged and re-suspended with PBS for qualitative analysis by confocal laser scanning microscopy (CLMS)  and quantitative analysis by FCM.HUVEC cells or U87 MG cells were seeded into 96-well plates at the density of 5000 cells/well and incubated for 24 h at 37 °C. Then cells were treated with various samples  at a range of concentrations. The cytotoxicity of each sample was determined by MTT method in triplicate.in vitro BBB model, a bEnd.3/U87MG co-culture BBB model was established according to previous reports . U87MG cells were seeded on the basolateral compartment of the insert at 2000 cells/compartment. The cell monolayer integrity was measured by transendothelial electrical resistance (TEER). BBB model was considered constructed successfully when the TEER value reached 200 Ω·cm2. To establish in vitro BBTB model, U87MG cells were plated onto the lower chamber, and HUVEC cells were seeded into the upper inserts of transwell with a density of 1:5 HUVEC/U87MG cells ratio . To confirm that TfR1 and HSPG were expressed in both models, several U87MG cell monolayers in both models were removed, and the expression of TfR1 and HSPG in each model was measured by TfR1 ELISA kit  and HSPG Human ELISA kit , respectively. The measuring processes were performed according to the manufacturer’s instructions. The TfR1 concentration in BBB and BTB models was 43 ± 5 and 46 ± 5 ng/mL, respectively. The HSPG concentration in BBB and BTB models was 210 ± 15 and 220 ± 18 pg/mL, respectively. These results confirmed that TfR1 and HSPG were highly expressed in both models.To establish The culture medium in the upper chambers was changed by 50 μM Cy5.5-labeled GKRK-APO or Cy5.5-labeled APO in 10% FBS containing DMEM. At definite time points, the U87MG cells on the bottom of the cell were immediately visualized by CLSM. To assess the antiproliferative effect of free VCR, VCR-loaded GKRK-APO, and VCR-loaded APO against U87MG cells after penetrating the BBB model or BBTB model, a sulforhodamine-B staining assay was applied. The various samples  were added to the apical compartment of the BBB model or BBTB model, respectively. The final concentration of VCR was 250 ng/mL. After 48 h, the percentage of surviving glioma U87MG cells in the basolateral compartment was determined by the sulforhodamine-B staining assay . Blood was sampled from the retro-orbital sinus at different time points . To prepare samples for analysis, 0.1 mL the internal standard (I.S.) solution  was added to 100 µL plasma sample in a 1.5 mL test tube. The sample mixture was deproteinized with 0.8 mL of methanol and vortex-mixed for approximately 1 min, and the precipitate was removed by centrifugation at 12,000 rpm (revolutions per minute) for 10 min. Then 800 µL of supernatant was transferred to another clean test tube and evaporated to dryness at 37 °C with a CentriVap Concentrator . The dry residue was reconstituted in 100 µL of the mobile phase, vortex-mixed, and centrifuged at 12,000 rpm for another 10 min. Twenty microliters of the clean supernatant was injected into the LC/MS/MS for analysis as described previously .The intracranial glioma-bearing mice were administered Cy5.5-labeled GKRK-APO or Cy5.5-labeled APO  via tail vein injection. Thirty minutes after the i.v. injection, The Cy5.5-labeled GKRK-APO or Cy5.5-labeled APO  was administered via tail vein injection to the mice. After 30 min, the intracranial glioma-bearing mice were anesthetized, and the hearts were perfused with saline, which was followed by 4% paraformaldehyde. The brains were removed for consecutively preparing 5-μm-thick frozen sections. Nuclei were stained with DAPI. The distribution of fluorescence was observed using CLSM.Rv=  × 100%. Where Vdrug is the glioma volume after treatment with drug and Vsaline is the glioma volume after treatment with physiological saline. The remaining six mice in each group were used to monitor survival. The survival time was calculated from day 0 (tumor inoculation) to the day of death. Kaplan–Meier survival curves were plotted for each group. Meanwhile, the body weight of each mouse was measured daily.The intracranial glioma-bearing mice were randomly divided into the following four groups (10 mice per group): Free VCR gourp, VCR-loaded GKRK-APO group, and VCR-loaded APO group and physiological saline group. Eight days after cell injections, each mouse received a dose of 1 mg/kg four times every 2 days. At day 16, four mice from each group were anesthetized and brain cancer was assessed by magnetic resonance imaging (MRI)  with measurement of the tumor diameter. Glioma inhibition was calculated using the formula: After the treatments were finished, the mice were sacrificed to collect the brains. Brain tumors were fixed in 10% buffered formalin, embedded in paraffin, and sectioned at 5 μm thickness. Sections were stained with Hematoxylin and Eosin (HE). The tumor histology was viewed and imaged under optical microscopy .in situ cell death detection kit following the manufacturer’s instructions prior to observation under CLSM.Apoptotic cells in glioma were detected via TUNEL assays using an In order to study the toxicity of VCR-loaded GKRK-APO, the mice were administered with free VCR, VCR-loaded GKRK-APO, and VCR-loaded APO at a single VCR dose of 1 mg/kg body weight via the tail vein, respectively. The mice treated with PBS were investigated as a control group. After mice were sacrificed, samples of brain, heart, liver, kidney, and lung tissue were collected and routinely stained with hematoxylin and eosin (H&E).Cmax and AUC(0-t) were calculated by Data Analysis System 3.16 . A one-way ANOVA was used to analyze the differences among groups. The significance was set at p = .05.Data were expressed as the means ± standard deviation (SD). The Because of the high activity of sulfhydryl group (-SH) in APO, the surface of APO can be easily used for ligand binding by sulfhydryl-maleimide coupling reaction. The GKRK peptide was introduced maleimide group (-MAL), and the peptide was conjugated to APO via the sulfhydryl-maleimide coupling reaction, which enabled GKRK peptide to be conjugated at a specific site (-MAL). To confirm GKRK peptide was functionalized to APO, we analyzed the binding ability of the GKRK-APO to purified HSPG. The GKRK-APO were labeled with the fluorescent agent Cy5.5 and incubated with immobilized HSPG in the wells of a microtiter plate for 2 h. The GKRK-APO strongly bound to the HSPG-coated well , but no p > .05), which showed that the conjugation of GKRK peptide to APO did not affect drug releasing.The physico-chemical properties of the two distinct apoferritin nanocage formulations are summarized in in vivo and in vitro. After an in vitro releasing study, the particle size of GKRK-APO was further analyzed by a laser particle analyzer. The mean particle size of the GKRK-APO was 12.77 ± 0.14 nm, and it had a narrow size distribution (the polydispersity index was 0.10 ± 0.01)  . This pa ± 0.01) . TEM (Fi ± 0.01)  and AFM  ± 0.01)  observatin vivo; therefore, 10% FBS in PBS was employed to mimic the in vivo conditions. The stability of the VCR-loaded GKRK-APO in the 10% FBS was evaluated using Turbiscan Lab® Expert . According to this evaluation . While the cellular uptake of APO was significantly influenced by the increase of peptide/APO molar ratio from 0.5% to 3%. With the further increase of the ratio to 6%, there was no significant difference in uptake compared with the APO with a 3% ratio. This was possibly caused by the saturation phenomenon of HSPG on cells. Limited by the number of receptors and the recycling of endocytosis, receptor-mediated endocytosis is a saturated pathway, which restricts the amount of the GKRK-APO that are available for cellular uptake. Considering the above results, the molar ratio of 3% for GKRK was selected in next experiments.As the density of GKRK peptide in APO was a key factor that will influence the targeting efficiency of GKRK-APO greatly, the cellular uptake of Cy5.5-labled APO with modifications of different densities of GKRK peptide were evaluated in U87MG cells (over-expressed HSPG and TfR1) to guide the formulation optimizing process. As shown in Cellular uptake of Cy5.5-labled GKRK-APO or Cy5.5-labled APO by bEnd.3, HUVEC, and U87MG cells was conducted to assess the targeting property. bEnd.3 cells, the main component of the BBB  when compared with APO . The cytotoxicity studies indicated that the synergistic effect of GKRK peptide and parent APO promoted anti-proliferative activities in U87MG cells that over-expressed TfR1 and HSPG. These results from the cytotoxicities of VCR-loaded GKRK-APO and VCR-loaded APO were consistent with the in vitro cellular uptake results shown in The cytotoxicities of blank APO, blank GKRK-APO, and different VCR formulations were evaluated with the MTT assay after incubation with HUVEC cells and U87MG cells for 72 h. As shown in 74 ng/mL  and 22.874 ng/mL , respectAs we know, the singular properties of proteins are related to their specific structures. The surface modifications of apoferritin in this paper may alter its conformation and affect its activity. As a concept verifying experiment, we did not survey the conformation changes of apoferritin after modifying by GKRK peptide. However, the results of our paper revealed that GKRK-APO had similar drug loading and releasing profiles, TfR1 targeting ability, and cytotoxicity as APO did. For this, the GKRK modified apoferritin did not change its activities that we desired. More studies are needed to explore this problem in the future.in vitro co-culture model of bEnd.3/U87MG cells was constructed to estimate the penetration efficiency of various samples in mimicking conditions in vivo. Cellular uptake of Cy5.5-labeled GKRK-APO or Cy5.5-labeled APO in U87MG cells was investigated by CLSM. As shown in The BBB is a major physiological barrier that prevents drugs or drug delivery systems from entering the brain targeted region. Therefore, an in vitro cytotoxicity assay . It was found that VCR-loaded GKRK-APO and VCR-loaded APO showed similar plasma concentration-time profiles. Both the VCR-loaded GKRK-APO and VCR-loaded APO showed initial high blood circulating levels (0.08 h after injection), while free VCR was quickly cleared from the systemic circulation. The pharmacokinetic parameters are listed in Figure S2(B). VCR-loaded GKRK-APO demonstrated significantly slower clearance rate (CL) and higher AUC when compared with free VCR. Furthermore, there was no remarkable different in CL and AUC between the VCR-loaded GKRK-APO and VCR-loaded APO (p > .05). These results indicate that the conjugation of an adequate amount of GKRK peptide on the surface of APO did not impair the long-circulation characteristic of parent APO.The plasma concentration-time profiles of VCR after i.v. administration of free VCR and VCR-loaded nanocarriers was shown in in vivo. To verify this, tissue distribution was evaluated in nude mice with intracranial U87MG glioma following i.v. administration of free VCR, VCR-loaded APO, and VCR-loaded GKRK-APO, respectively. As shown in Figure S2(C), the concentrations of VCR in the brain of VCR-loaded GKRK-APO was 6.5-fold over that of free VCR, and 1.4-fold over that of VCR-loaded APO, respectively. As shown in Figure S2(D), VCR-loaded GKRK-APO exhibited a similar tissue distribution profile with the VCR-loaded APO in the non-targeted tissue, while a significantly higher level of free VCR was observed accumulated in the liver, heart, spleen, and kidney. Due to the existence of BBB, the levels of VCR in brain are very low, compared with all the other tissues  vs. D: 0.05–0.3% vs. 2.3–6.4%). The circulating time of VCR encapsulated in apoferritin nanocage in blood was prolonged. Hence, it indicated that the body distribution of VCR-loaded APO mainly depended on the distribution behavior of the parent apoferritin in vivo. What is different from VCR-loaded APO was that VCR-loaded GKRK-APO modified with GKRK peptide had significantly enhanced glioma targeting delivery and resulted in higher drug concentration in brain. These results from mice were consistent with the in vitro results shown in in vivo.The selective distribution of drug-loaded nanocarriers in tumors could potentially benefit the antitumor activity of chemotherapy in vivo imaging, the brain accumulation was much higher for the GKRK-APO ).The brain targeting efficiency of GKRK-APO was investigated in nude mice with intracranial U87MG glioma. After the Cy5.5-labeled nanocarriers were given through the tail vein, time-dependent biodistribution of the GKRK-APO and APO were observed using non-invasive NIR fluorescence imaging in live animals. Based on GKRK-APO  and APO GKRK-APO  group. Win vivo glioma targeting capability, immunofluorescence assay was conducted after the treatment of various Cy5.5-labeled samples in mice bearing intracranial glioma. As shown in in vivo imaging results  and VCR-loaded GKRK-APO, overall antiglioma efficacy was observed by MRI for monitoring cancer volume and was confirmed using survival curves. Consistent with the results of glioma distribution . This was mainly attributed to the target systemic delivery of GKRK-APO, which was demonstrated by in vivo imaging  achieving systemic glioma-targeted drug delivery by employing GKRK peptide. GKRK-APO could penetrate BBB and BBTB and target glioma cells. VCR-loaded GKRK-APO could effectively enhance the antiglioma efficacy


This systematic review and meta-analysis aimed to estimate the overall pooled prevalence of preterm birth and the effect of pregnancy induced hypertension (PIH) and multiple pregnancies on preterm birth in Ethiopia.A total of 9 studies with 27,119 participants were included in this systematic review and meta-analysis. The pooled prevalence of preterm birth among mothers who gave births in Ethiopia was found to be 13.32% . Preterm birth was found to be higher among mothers who had pregnancy induced hypertension with odds ratio of 4.69  and multiple pregnancy with odds ratio of 2.40  as compared to the counterparts. In subgroup analysis by region, the prevalence of preterm birth was found to be 12.63%  in Amhara and 10.18%  in Oromia region.The online version of this article (10.1186/s13104-019-4128-0) contains supplementary material, which is available to authorized users. Preterm birth is defined as babies born alive before 37 weeks of pregnancy is completed. Globally, every year, an estimated 15 million babies are born before 37 weeks of gestation of which 85% of the preterm births are concentrated in Africa and Asia . PretermDevelopmental immaturity affects a wide range of organ systems in preterm neonates. Preterm birth results in short term complications, such as respiratory distress syndrome, apnea and brain immaturity . Many ofAround 75% of the preterm births result from spontaneous preterm labour. The remaining 25% are delivered for medical reasons . The cauUse of alternative models of antenatal care , treatinhttps://www.crd.york.ac.uk/PROSPERO and CRD42019118389 respectively.This protocol has been registered with the International Prospective Register of Systematic Reviews (PROSPERO). The web address and the registration number of this systematic review and meta-analysis are The preferred reporting items of systematic reviews and meta-analysis (PRISMA) guideline  was usedIn this study, articles which fulfilled the criteria, (1) observational studies including cohort, cross-sectional, and case–control studies; (2) articles that report the prevalence of preterm birth and/studies that report the association of pregnancy induced hypertension (PIH) and/multiple pregnancy) with preterm birth; (3) studies conducted in Ethiopia; (4) published and unpublished articles at any time; (5) studies which have been written by English, and (6) Studies conducted both at community or institution level were included. Conference papers, editorials, articles without full texts, trials, systematic reviews and meta-analyses, and qualitative studies were excluded.Preterm birth is defined as babies born alive before 37 weeks of pregnancy is completed .We searched all available articles with electronic databases including, PubMed, EMBASE, web of science, and Google scholar. Additionally, we searched using the reference list of included studies and the Ethiopian institutional research repository. Articles were searched using the following search terms: “preterm”, “low gestational age”, “preterm birth”, “preterm delivery”, “early delivery”, “pregnancy induced hypertension”, “hypertension”, “preeclampsia”, “eclampsia”, “pregnancy”, “multiple pregnancy”, “twin pregnancy”, “prevalence”, “incidence”, “predictors”, “factors”, “risk factors”, and “Ethiopia”. A searching string was developed using “AND” and “OR” BOOLEN operators. PubMed searching was done using this searching string  were screened and assessed the title and abstracts followed by full text assessment. Any disagreements between authors were solved by discussion and consensus. Fourth, Two investigators (GM and AW) assessed the quality of studies using the JBI quality appraisal criteria . Any disAfter quality assessment, two independent authors (GM and AW) extracted the data in excel Microsoft spreadsheet. The extracted data items were, first author, year of publication, study area, region, design, population, sample size, prevalence of preterm birth, OR of PIH, and multiple pregnancies. Any disagreements were solved by discussion.2 [2 test statistics results of 25%, 50%, and 75% were declared as low, moderate, and high heterogeneity, respectively [A weighted inverse variance random- effects model  was used2 . I2 testOn the whole, we searched 1435 articles from different data sources of which 1307 articles were from PubMed, 71 from Google scholar, 23 from EMBASE, 11 from web of science, 3 from reference lists of included studies, 7 from institutional research repositories, and 13 from Google. Ninety-seven articles were removed due to duplicates, 1277 due to irrelevant titles and abstracts, 24 due to study area (done in another country), and 10 due to study design. Twenty-seven articles were selected for the full text review of which 18 were excluded after the full text review. Finally, 9 studies were included in this systematic review and meta-analysis to estimate the effect of PIH and multiple pregnancies on preterm birth and pooled prevalence of preterm birth in Ethiopia   for Amhara and 10.18%  for Oromia  among mothers who had pregnancy induced hypertension compared to mothers with no PIH  compared to single pregnancy . This finding is lower than that of Kenya (20.2%)  and . The finding is supported by those of cohort studies, and systematic reviews in China , and KenPreterm birth in Ethiopia is a significant problem. Though the determinants of preterm birth are multifactorial, PIH and multiple pregnancies remained a major contributing factor to the risk of preterm birth. Preconception counseling and antenatal care to facilitate timely management of PIH, more specially controlling hypertension is very important to maintain blood flow to the fetus. Improving health care quality delivered to pregnant women may reduce risk factors for preterm delivery.Strength of this review: Since there has been no similar previous study, this review and meta-analysis showed the national pooled image of preterm birth in Ethiopia and the effect of pregnancy induced hypertension and multiple pregnancy on preterm birth. Strictly following PRISMA guide line and Joanna Briggs Institute Meta-Analysis of Statistics Assessment and Review Instrument (JBI-MAStARI) during critical appraisal is the additional strength of this systemic review and meta-analysis.The search strategy was limited to articles published in English, and this could lead to reporting bias. Relevant predictors might have been missed; hence, future reviews should consider other factors of preterm birth to explore the inquiry more deeply on subject of inquiry. Furthermore, presence of high statistical heterogeneity among studies conducted on preterm birth were considered as limitation of this review.Additional file 1. PRISMA checklist.Additional file 2. Searching strings used for PubMed.Additional file 3. JBI critical appraisal checklist for cross-sectional, case–control and cohort studies.Additional file 4: Fig. S1. Funnel plot for publication bias, log p or LNP (log of proportion in the X-axis and standard error of log proportion in the Y-axis.Additional file 5: Fig. S2. Forest plot of subgroup analysis of the prevalence of preterm birth by region with 95% CI. The midpoint and the length of each segment revealed the prevalence and CI. The diamond shape showed combined prevalence.Additional file 6: Fig. S3. Forest plot of OR of preterm birth among mothers with PIH. The midpoint and the length of each segment indicated OR and 95% CI respectively. The diamond shape indicated the pooled OR.Additional file 7: Fig. S4. Forest plot of OR of preterm birth among multiple pregnancies with 95% CI. The midpoint and the length of each segment indicated OR and 95% CI respectively. The diamond shape indicated the pooled OR.


Perfluoroalkyl substances (PFASs) are widespread industrial pollutants that are extremely persistent in the environment. A previous study in the Danish National Birth Cohort (DNBC) found prenatal perfluorooctanoate (PFOA) exposure was associated with decreased birth weight, but had insufficient statistical power to evaluate adverse birth outcomes. Here, we conducted additional analyses in three samples originating from the DNBC for 3535 mothers and infant pairs to evaluate associations between prenatal PFASs exposures and low birth weight and preterm birth. Maternal plasma concentrations were measured for six types of PFASs in early pregnancy. Several PFASs were associated with a reduction in birth weight and gestational age. We estimated a nearly 2-fold increase in risks of preterm birth for the higher quartiles of PFOA and perflourooctanesulfonate (PFOS) exposure. In spline models, risk of preterm birth was increased for perfluorononanoic acid (PFNA), perfluoroheptane sulfonate (PFHpS) and perfluorodecanoic acid (PFDA) in higher exposure ranges. We also observed some elevated risks for low birth weight but these estimates were less precise. Our findings strengthen the evidence that in-utero PFASs exposures affect fetal growth. Future studies are needed to evaluate whether these associations persist with the decline of PFOA and PFOS in populations and should also investigate newer types of fluorinated compounds introduced more recently. Perfluoroalkyl substances (PFASs) are industrial persistent pollutants that are widespread in the environment . The mosAnimal studies have suggested that prenatal PFASs exposure can affect fetal growth i.e., PFOS and PFOA exposures in-utero can reduce birth weight and gestational age at delivery in rodents ,10,11. SA previous study conducted in the Danish National Birth Cohort (DNBC) found an inverse association between maternal plasma PFOA levels and birth weight , howeverThe DNBC is a nationwide follow-up study of pregnant women and their offspring in Denmark . Pregnann = 6207), non-singleton births (n = 2080), mother who emigrated (n = 51) or died (n = 3), unknown birth outcomes (n = 25), or missing dates of birth (n = 99), and one participant who withdrew from all pregnancies initially enrolled. The source population for this study is further limited to the 83,389 mother-child pairs who completed interview 1 and for whom maternal blood samples were available for PFASs analyses. Previously, three sub-studies have measured PFASs in maternal blood samples in the DNBC [There were 92,576 live-born singletons in the DNBC after excluding unsuccessful pregnancies including abortions and stillbirths  or gestational age <140 days or > 315 days (n = 4). Low birth weight (LBW) was defined as birth weight <2500 g [Birth weight (grams) and gestational age at birth were obtained from the National Hospital Discharge Register at the National Board of Health in Denmark. The information for gestational age recorded in the register was usually based on ultrasound examinations done before 24 weeks of gestation conducted by midwives (~97%) and only few were calculated from the first day of the last menstrual period (LMP). We excluded infants with extreme values of birth weight <500 g or >6800 g  produced by the two laboratories were very high (Pearson correlation r = 0.94 for PFOS and r = 0.95 for PFOA).Comparisons of PFASs measurements in the two laboratories have previously been performed . Althoughttp://www.bsmb.dk) [Information on infant sex, infant birth year (as continuous variable), maternal age , parity  were collected from the medical register, while other potential confounders such as socio-occupational status , pre-pregnancy body mass index , smoking during pregnancy (yes/no) and alcohol intake during pregnancy was recorded in the highly structured questionnaires collected during pregnancy (available at bsmb.dk) . Althougbsmb.dk) ,28, and We used multivariable linear regression to evaluate the expected differences in birth weight (grams) and length of gestation (days) according to maternal plasma PFASs level. In addition, we used multivariable logistic regression to estimate the adjusted odds ratio (OR) and 95% confidence interval (CI) for LBW and preterm birth according to PFASs exposures. The PFASs levels were analyzed as continuous values or categorized in quartiles. For continuous PFASs values, we analyzed log-transformed (base 2) PFASs exposure in the statistical models thus the exposure effect estimate represents an increase per doubling of the PFASs concentration (ng/mL). The PFASs quartile classifications were based on untransformed PFASs values and the lowest quartile was used as the reference. To further evaluate potential non-linear exposure-outcome responses, we fitted restricted cubic spline models with three knots at the 10th, 50th and 90th percentile of the PFASs value on LBW or preterm birth. We also allowed for higher flexibility (4 or 5 knots) in the spline models; but since results did not change substantially, we employed 3 knots to avoid over-fitting. To account for possible laboratory differences or “batch effects”, we added an indicator variable for study sample  when we analyzed PFASs continuously, and we used the study-sample specific cut-off to generate PFASs quartiles.p-values for interaction term between each of the exposure and the potential modifier [All covariates except for infant birth year and gestational age at blood draw (weeks) were introduced into models as categorical variables , and organic food consumption  during pregnancy to evaluate potential confounding by dietary factors . We alsoDemographic and other characteristics of the study participants (unweighted) by study sample are presented in p-value for interaction = 0.05). The negative associations of PFNA or PFDA on gestational age were also stronger among mothers with either lower or higher pre-pregnancy BMI compared with normal weight women .In pooled analyses, we observed that per doubling of exposure in prenatal PFASs (ng/mL) specifically PFOS, PFOA, PFNA and PFHpS were associated with a 45 g, 36 g, 36 g or 39 g decrease in birth weight . A greatSeveral positive associations between prenatal PFASs and preterm birth were observed . The ORsp-value for non-linearity <0.10) for PFOA, PFHxS, PFNA, and PFDA in a spline model of exposure  while PFOS and PFHpS were highly correlated (r = 0.89) (see A non-linear exposure response for LBW was found for PFDA with a slight reduction of odds in the lower exposure range but estimated odds increasing again at higher exposure (>0.2 ng/mL) a. In sen.89) see , even th.89) see .The positive effect estimates between several PFASs and LBW were slightly strengthened using <2650 g as the cut-off while the results became very imprecise with the <2260 g cut-off due to the small number of cases. For preterm birth, the results were also slightly strengthened for PFNA and PFDA using <36 weeks as the cut-off but the effect estimates also became less precise using a <35 weeks cut-off. Our results also remained similar when restricting to samples collected in the first trimester only (data not shown).In this large combined sample from a prospective cohort study in Denmark, prenatal PFASs were generally inversely associated with birth weight and gestational age. Moreover, we found that prenatal exposures to several PFASs may increase the risks for preterm birth, and possibly also LBW but the estimates for LBW were imprecise. Spline models for exposure suggested possible effect on adverse birth outcomes at higher exposure ranges for the less prevalent PFASs in our samples such as PFNA, PFHpS and PFDA. This might be of concerns if these exposures rise in populations with changes in consumer product use of these chemicals ,4,5.2 ng/mL exposure and additionally accounted for possible selection bias due to the sample selection criteria employed.An earlier study in the DNBC  was amonOther cohort studies that investigated the associations regarding prenatal PFASs exposures on fetal growth indicators were mostly small in size and they only assessed PFOS or PFOA exposures ,38. AmonPhysiologically, animal studies have shown the negative effect of prenatal PFASs exposures on birth outcomes and several potential mechanisms have been suggested ,41,42. POur study has several strengths. All three sub-samples were selected from a nationwide well-described cohort of pregnant women and their infants . The PFAThe effect estimates were largely consistent across study samples, but some variations were observed. This could be due to different sampling and selection criteria employed to generate each study sub-sample, or the influence of measurement errors or simply of chance. We adjusted for sampling and selection probabilities using weighted regressions throughout, but some differences in study characteristics across samples may still have remained. A moderate to high correlation between different PFASs make it difficult to disentangle specific exposure effects for each chemical from the effect of the mixtures. Our large sample size allowed us to conduct multi-pollutants analyses, and generally the estimated exposure effects for several PFASs compounds persisted upon co-pollutant adjustments. Some advanced statistical methods for mixture analyses have been proposed ,48, but The observed association may not be causal and can be influenced by bias. There could be unmeasured factors we could not take into considerations leading to residual confounding. When using biomarkers of PFASs, physiological factors that affect accumulation or excretions of PFASs should also be considered. For instance, lower glomerular filtration rate (GFR) in mid- or late-pregnancy has been suggested to be such a possible confounding factor . MothersAny measurement errors in exposures and outcomes are expected to be non-differential and might bias the associations mostly towards the null. Participants were unlikely to be aware of their PFASs levels which limit the possibility of self-selection bias. However, prenatal PFASs exposures may increase risk of miscarriages ,52. Our In conclusion, our analysis using three sub-samples from the DNBC demonstrated that several prenatal PFASs are inversely associated with birth weight and gestational age, and prenatal exposure to several PFASs may increase the risks for preterm birth. Our findings strengthen the evidence that in-utero PFASs exposures might affect fetal growth. Exposure levels of PFOS and PFOA are declining in some western countries but they are still widely detected. Adverse effects on birth outcomes in our samples were also observed in the higher exposure ranges of some of the less commonly detected PFASs such as PFNA and PFDA which might be concerning if exposures to these compounds increase in populations. Considering the ubiquity of PFASs contamination in the environment and in humans, strategies and efforts to prevent PFASs exposures in pregnant women and young infants should continue. Further study is also needed to evaluate whether these associations persist with lower exposure levels for PFOS and PFOA, and substitutes and newer types of fluorinated compounds need to be scrutinized as well.