In a cohort of pregnant women , followed-up between 2012 and 2019 at six sites worldwide4, we studied the associations between ultrasound-derived fetal cranial growth trajectories, measured longitudinally from <14 weeks’ gestation, against international standards6, and growth and neurodevelopment up to 2 years of age8. We identified five trajectories associated with specific neurodevelopmental, behavioral, visual and growth outcomes, independent of fetal abdominal growth, postnatal morbidity and anthropometric measures at birth and age 2. The trajectories, which changed within a 20-25-week gestational age window, were associated with brain development at 2 years of age according to a mirror (positive/negative) pattern, mostly focused on maturation of cognitive, language and visual skills. Further research should explore the potential for preventive interventions in pregnancy to improve infant neurodevelopmental outcomes before the critical window of opportunity that precedes the divergence of growth at 20-25 weeks’ gestation.Many observational studies and some randomized trials demonstrate how fetal growth can be influenced by environmental insults  Similarly, the severity of fetal growth restriction (FGR) cannot be determined unless serial ultrasound scans are performed, and birth weight alone as a proxy measure for the totality of intrauterine growth is insufficient9. In addition, most large-scale pregnancy and newborn epidemiological data are based on retrospective analyses of routinely collected, but rarely standardized, medical records10. In this study, we investigated associations among fetal cranial growth trajectories, measured by serial ultrasound scans from <14 weeks’ gestation, against international standards6 and growth and neurodevelopment up to the key milestone age of 2 years. We sought to identify windows of opportunity for interventions during pregnancy to improve health and neurodevelopmental outcomes.The literature on the association between events during fetal life and postnatal growth and neurodevelopment into childhood is replete with limitations. For example, gestational age is rarely estimated accurately11. More than half  of the fetuses had a steady growth trajectory throughout pregnancy, close to the INTERGROWTH-21st 50th centile5. They comprised the reference group for subsequent analyses (median growth tracking (MGT)). The second trajectory , n = 436) displayed growth faltering throughout pregnancy. The third , n = 268) showed higher (close to +0.5 of s.d.) but parallel growth to the MGT trajectory through the second trimester, with growth starting to falter by the early third trimester. The fourth (accelerating growth (AG), n = 349) showed growth close to the 50th centile through the second trimester and accelerated growth during the third trimester. The fifth (late median growth tracking (LMGT), n = 251) had accelerated growth after 15 weeks’ gestation and then a ‘normalizing’ growth rate in the third trimester  for the Child Behavior Checklist’s (CBCL) attentional problems (16.8%) and emotional reactivity (19.8%) scale14. Scaled total scores were 3.9 ± 2.3 for attentional problems and 5.7 ± 3.6 for emotional reactivity.The INTERGROWTH-21st Neurodevelopmental Assessment (INTER-NDA) cognitive, fine motor, gross motor, negative behavior and positive behavior scores15, 16% of children had visual acuity scores and 11% had contrast sensitivity scores below the norms for 2 year olds , compared to the 10% expected prevalence in the INTERGROWTH-21st population16. The median ages of attaining the World Health Organization (WHO) milestones17 were within the normal ranges: from 6 months for sitting without assistance to 12.8 months for walking without assistance. Overall, children’s height at age 2 was about one third of an s.d. less than the WHO Child Growth Standard (mean z-score = –0.34 ± 1.3) but close to the mean z-score for weight and head circumference (HC)7. The morbidity episodes reported at ages 1 and 2 showed moderate to high rates of clinical conditions, compared to the INTERGROWTH-21st population18, reflecting our strategy of enriching for peri-conceptual risk characteristics at enrollment known to lead to suboptimal size, morbidity and/or neurodevelopment delay , but, from birth onwards, there was a trend to ‘normalization’ with age: the mean infant HC values for the five trajectories were within ± 0.8 z-scores by age 2, with the LF trajectory very close to the MGT trajectory at about the 50th centile , weight  and HC , respectively.In the models adjusted for sex, age at assessment, preterm birth, maternal smoking, age and education, relative to the reference MGT trajectory, the EF trajectory scored poorest on the cognitive  = -6.45, -1.80), language  and fine motor  domains of the INTER-NDA . These cz-scores were slightly higher   on the Child Behavior Questionnaire. Although less affected than children in the EF trajectory, these children had a higher risk of poorer visual acuity  and contrast sensitivity  . Compare6, 0.41) .z-scores for height , weight  and HC  , with a decreased risk of poorer visual acuity  and contrast sensitivity . These children had increased 8, 0.79) .z-scores for height , weight  and HC  . These children were also at marginally lower risk of developing poorer visual acuity  and contrast sensitivity . They also had increased 8, 0.72) .20. We further estimated models that simultaneously included both fetal HC and abdominal circumference (AC) trajectories to evaluate the effect of HC independently of other fetal ultrasound measures. Results were similar to those from the separate HC and AC trajectory models, supporting the hypothesis that associations of HC with growth and development at age 2 are independent of other fetal growth parameters.We evaluated correcting our results for multiple comparisons using the Benjamini-Hochberg false discovery rate correction to control for type 1 error rate at <0.05, and all of our findings with the EF group and all groups related to 2-year growth outcomes remained highly statistically significantn = 5): no relevant changes were observed, except that the HC z-score association with the EF trajectory was weaker. In sensitivity analyses excluding those children missing one or more questions from the INTER-NDA, the associations were similar or slightly stronger for all domains and trajectories, except that the relationship between the AG trajectory and cognitive domain became stronger and statistically significant .We conducted sensitivity analyses excluding preterm births, neonatal intensive care unit (NICU) admissions, serious infant morbidity and frequent infections or hospitalizations, and the results were similar. Sensitivity analyses were also performed including possible outliers of the 2-year anthropometric measures (P < 0.01) for the fine motor, emotional reactivity and contrast sensitivity domains. We adjusted these models for child sex, age at developmental assessment, preterm birth, maternal age and education and smoking during pregnancy. There was no effect on height or HC at age 2, but longer breastfed children in the EF trajectory improved their BMI by that age (P = 0.06)  .21). Although a relationship between behavioral traits and serial measures of HC in healthy infants was described previously22, we have introduced, to our knowledge for the first time, a phenotypic classification of fetal cranial growth with functional relevance to important early childhood outcomes that influence cognition, language, educational achievement and productivity in later life23.We have demonstrated that five fetal trajectories of cranial growth, identified from ultrasound-derived, longitudinal, fetal HC measures, are associated with specific neurodevelopmental, behavioral, visual and growth outcomes at age 2 (ref. 5 and ±1 s.d. of the WHO standard for HC at age 2 (ref. 7), indicating that the association is not the result of extreme growth trajectories or specific study sites contributing the majority of fetuses but, rather, part of the variability among the total study sample. However, we also acknowledge that, as with all biological studies, there was likely random measurement error related to HC, which would likely result in non-differential misclassification of participants into trajectories and bias results toward the null, resulting in an underestimate of the strength of the associations observed24.These effects are independent of 1) other fetal growth measures ; 2) gestational age at birth/preterm status; 3) neonatal and child morbidity; 4) HC growth from birth to age 2; 5) maternal age and level of formal education, smoking and demographic characteristics; 6) gross infant motor markers ; and 7) child sex and exact chronological age at developmental assessment, which strongly suggest a direct effect of differential fetal cranial growth patterns on early child development. Notably, the growth variations identified occur well within the normal range of ±2 s.d. of fetal HC growth25. The diverging patterns are likely related to factors present before this window or even pre-pregnancy.We identified a window between 20 and 25 weeks’ gestation during which cranial growth either tracked the INTERGROWTH-21st 50th centile or its trajectory changed due to growth accelerations/deaccelerations . This isz-score was —0.8 of the WHO standard  is associated with a consistent, mirror (positive/negative) pattern mostly focused on maturation of cognitive, language and visual skills, independent of major confounding variables, such as fetal AC growth, postnatal morbidity and anthropometric measures at birth and age 2.To our knowledge, our study is unique because 1) it was based on following a prospective, multinational cohort exposed to multiple risk factors; 2) pregnancies were accurately dated in the first trimester; and 3) postnatal growth was monitored according to WHO recommendations. Data collection was standardized, which allowed adjustment for possible confounding variables across sites using the same variables and definitions. Hence, the adjusted analyses reflecting independent associations can be considered robust. However, whether the trajectories identified can be used as a screening tool at the population or individual level will require considerably more research.One limitation of our analytical strategy was that we did not explore early pregnancy etiological or risk factors  known to be associated with altered fetal growth. To incorporate such a detailed analysis, given our focus on the association between actual fetal growth trajectories and neurodevelopment, would have required including considerable additional data. We also lacked parental and sibling developmental profiles, which might have helped the adjusted analysis, as well as the family developmental risk that can act as an effect modifier of the observed association.In conclusion, we propose three suggestions for translating the biological evidence presented here into clinical and public health actions. First, it is highly unlikely that a ‘silver bullet’ will ever be identified to prevent or treat FGR because it is such a heterogeneous syndrome. Second, previously tested interventions in pregnancy might have failed to improve neurodevelopmental outcomes because they were implemented after the critical window of opportunity for intervention has passed, or the sample size was too small to show effects on a specific HC trajectory. Lastly, there is no substitute for the strategy of initiating ante-natal care as early as possible for pregnant women worldwide.16.Phase II of the INTERGROWTH-21st Project  was conducted between 2012 and 2019 at six sites: Pelotas (Brazil), Nairobi (Kenya), Karachi (Pakistan), Soweto (South Africa), Mae Sot (Thailand) and Oxford (UK). The sites in Pelotas, Nairobi and Oxford also participated in Phase I of the INTERGROWTH-21st Project, which showed consistent similarities in fetal growth and newborn size at birth and similar patterns of skeletal growth, neurodevelopment and associated behaviors at age 2, in a multinational cohort of healthy, educated and well-nourished women, living in clean environments and at low risk of adverse maternal and perinatal outcomes16. In brief, Pelotas (Brazil) is the third most populous city in the state of Rio Grande do Sul, with 350,000 inhabitants and 4,000 births per year, nearly all of which take place in the city’s four maternity hospitals. Nairobi (Kenya): More than 4,000 births per year in the relatively wealthy Parklands suburb of Nairobi, a geographically delimited urban area, take place in three hospitals, the largest of which, the Aga Khan University Hospital (AKUH), participated in the study. Nairobi is a non-endemic malaria area. The ante-natal HIV prevalence in this hospital is 1%. Karachi (Pakistan): The AKUH, Karachi, is a not-for-profit teaching hospital, serving a range of socio-economic groups in the largest city in Pakistan, with an estimated population of approximtely 20 million people. The hospital has 5,800 births per year; one-third are high risk, referred from four secondary care hospitals . Soweto (South Africa): Chris Hani Baragwanath Academic Hospital, which is attached to the University of the Witwatersrand, Johannesburg, is the only government hospital serving a population of approximately 1.3 million people. It has approximately 17,000 births per year, 75% of which are medium to high risk; this represents 53% of all births in Greater Soweto. The HIV prevalence is 29%. Mae Sot (Thailand): The Shoklo Malaria Research Unit is a Mahidol-Oxford Research Unit field station for studying malaria (including in pregnancy) among the 130,000 people living in refugee camps on the Thai-Myanmar border. Three of its field sites, with a total of approximately 2,000 births per year, participated in the study: two (Wang Pha and Mawker Thai) are clinics for migrants, and the third (Maela camp) is the largest refugee camp along the border. Oxford (UK): The John Radcliffe Hospital covers approximately 75% of more than 8,000 births per year in Oxfordshire, which has a population of more than 650,000 people that includes a large proportion of young, middle-class, well-educated and professional families.Detailed information about each study site was previously published-2 (to facilitate ultrasound scanning of the fetus), natural conception and singleton pregnancy. All other women were eligible to participate after giving written informed consent. Each site’s contribution ranged from 12.4%  to 20.2% . The other sites contributed 17.3% , 15.7% , 16.5%  and 18% , respectively.In the INTERBIO-21st Fetal Study, we enrolled 3,598 women who initiated ante-natal care before 14 weeks’ gestation, irrespective of their risk profile for adverse pregnancy/neonatal outcomes, and monitored their pregnancies to delivery. Thereafter, the health, growth and development of their children were monitored up until age 2. The only inclusion criteria at study entry were maternal age ≥18 years, BMI <35 kg m5. In brief, a comprehensive set of variables was obtained prospectively using data collection forms and an electronic data entry system specifically developed for the study (www.interbio21.org.uk). Baseline information included demographic and nutritional characteristics, medical, gynecological and obstetric history and current pregnancy conditions. Pregnancy follow-up information included routine standard ante-natal care variables, current health, use of supplements or medication and referral to another level of care or hospital. Pregnancy morbidity was also recorded during follow-up, including diagnoses such as gestational diabetes, pregnancy hypertension, pre-eclampsia, vaginal bleeding, preterm premature rupture of membranes, preterm labor, severe vomiting requiring hospitalization, eclampsia/HELLP syndrome , rhesus disease, abruptio placentae, clinical chorioamnionitis, mode of delivery and any pregnancy-related condition or infection requiring treatment .Data collection relating to pregnancy followed the same strategy as in the Fetal Growth Longitudinal Study (FGLS) of Phase I of the INTERGROWTH-21st Project5. In brief, crown-rump length was measured < 14+0 weeks’ gestation, with an angle of insonation as close as possible to 90°, in a mid-sagittal view of the horizontal fetus in a neutral position filling at least 30% of the screen. The calipers were placed on the outer borders of the head and rump, and gestational age was estimated using the INTERGROWTH-21st standards for pregnancy dating6. When the date of the last menstrual period was available, this was also recorded.The ultrasound methods used to estimate gestational age, as well as the training, standardization and quality control processes, were described previouslyAfter the dating ultrasound scan, the women were seen every 5 ± 1 weeks until delivery , as in the FGLS. Identical ultrasound equipment  was used at each site. Of the 3,598 women enrolled, 3,206 had at least three fetal ultrasound scans between <14 and 37 weeks’ gestation.At each visit from 14 weeks’ gestation onwards, the fetal HC was obtained in the transthalamic plane, placing the calipers on the outer border of the skull, using both the ellipse facility and two perpendicular diameters. We selected HC as an ultrasound marker of fetal cranial growth because 1) it is widely used in routine obstetric practice with complementary skeletal growth markers and avoids the measurement errors associated with biometric ratios; 2) measurement was standardized across study sites; 3) it relates directly to brain and cerebellar growth; and 4) importantly for a postnatal follow-up study, HC in childhood is the same measure.The AC was obtained in an axial plane, with the umbilical vein in the anterior third of the fetal abdomen  and the stomach bubble visible. Both the ellipse facility and the two diameters method were used, placing the calipers on the outer border of the body outline (skin covering). All measurements were taken three times from three separately generated ultrasound images. To avoid expected value bias, the ultrasound machines were specially adapted to ensure that the ultrasonographers were ‘blinded’ to the actual values, which were transferred electronically and available only after the examination was completed. In the present analysis, we focused on HC, because it is a reliable proxy for cranial growth; the use of a single measure, rather than a summary measure such as estimated fetal weight, is associated with a lower measurement error, and the same measurement can easily be made longitudinally in postnatal life.The ultrasonographers at each site were selected on the basis of their technical expertise, motivation, reliability and ability to speak the local language(s). Through rigorous training, they gained theoretical knowledge and familiarity with the study protocol, operations manual, data collection and quality control measures. We also conducted centralized, hands-on training, and the Oxford-based Ultrasound Quality Control Unit regularly carried out site-specific standardization procedures to ensure proper use of the ultrasound equipment and protocol adherence. Quality control was maintained throughout the study by taking a random 10% sample of all ultrasound images and assessing their quality using a validated scoring system.19. In brief, newborn measures were ideally obtained within 12 h of birth (and no later than 24 h), using the same equipment at all sites: an electronic scale (Seca) for birth weight (sensitivity of 10 g up to 20 kg) and a specially designed Harpenden Infantometer (Chasmors) for recumbent length. The equipment was selected for accuracy, precision and robustness, as reported in previous studies19, and calibrated twice weekly. HC was measured using a metallic non-extendable tape (Chasmors).The anthropometric measurement protocols and quality control procedures were identical to those used in the FGLS19.All lead anthropometrists were trained centrally to measure newborns according to the study protocol, and, in turn, they trained the local anthropometrists who were standardized at regular intervals; all training materials were based on the original WHO protocols. The Anthropometric Standardisation Unit based in Oxford regularly monitored the performance of all staff. The quality control measures required anthropometrists to take and record all measures twice independently and compare their values with the maximum allowable differences: newborn weight, 50 g; length, 7 mm; and HC, 5 mm. If the difference between the two measures exceeded these values, then both observers independently repeated that measurement a second time and, if necessary, a third timewww.intergrowth21.org.uk). Exclusive breastfeeding up to 6 months and appropriate nutritional support for infants born preterm were promoted during and after pregnancy as recommended26.Across all sites, standardized clinical care and feeding practices were implemented based on protocols developed by the INTERGROWTH-21st Neonatal Group (www.intergrowth21.org.uk). The proportion of infants receiving breast milk and vitamin and mineral supplements and those following a special diet were estimated at age 1 and 2 (refs. 28).Detailed information was obtained from the mother at age 1 and 2 about the infant’s health, severe morbidities, hospitalizations, length of breastfeeding, timing of the introduction of solid food, age at weaning, feeding practices and food intake, using specially produced forms . There were 198 children who attended the 2-year, but not the 1-year, visit. Overall, 2,183 children completed the 2-year follow-up (76% of those eligible) .n = 2,183) was 29.4 ± 5.2 years; their BMI at recruitment was 24.8 kg m-2. Most (88%) were married or co-habiting, 42% were nulliparous and 43% had at least a university education; 71% worked outside the home before pregnancy. Very few mothers smoked or drank alcohol during pregnancy . Fifty-two percent of all newborns were male, and 42% were delivered by Caesarean section; about 9% were preterm, with less than 2% very preterm (<34 weeks’ gestation).The mean age of mothers of children seen at age 2  and the sample participating in the 2-year follow-up component . Women completing the 2-year follow-up were very similar to the full cohort, only they were marginally better educated, and their babies had lower very preterm and NICU admission rates. Infants lost to follow-up were also similar at birth to those completing the 2-year follow-up, except that they were more likely to be preterm and have had a NICU admission , fine and gross motor skills and positive and negative behavior, in an assessment time of 15 min on average. The INTER-NDA has been validated against the Bayley Scales of Infant Development III edition, showing good to moderate agreement13, and has shown good levels of inter-rater  and test/re-test reliability 12.We assessed neurodevelopment at age 2 using the INTER-NDA, an international, multicultural, standardized, psychometric tool, targeted at children aged 22–30 months, which measures multiple dimensions of early development using a combination of directly administered, concurrently observed and caregiver-reported items35; responses were based on caregiver reports. Vision was assessed using the Cardiff Visual Acuity and Contrast Sensitivity tests for binocular vision15. These are indicative of the integrity of the visual pathway and central nervous system and, as directly observed neurodevelopmental markers, are unlikely to be affected by cultural influences.Attentional problems and emotional reactivity were measured on the respective subscales of the Preschool CBCL17. Trained staff collected the data on a form with pictures of the relevant child positions and corresponding definitions. Parents were asked to report the age in months and weeks when they first observed or ‘never observed’ the milestones. We assessed the age (in months) at which WHO gross motor milestones were first achieved.Motor development was assessed against four WHO milestones that are less likely to be affected by recall bias: sitting without support, hands and knees crawling, standing alone and walking aloneAll INTER-NDA assessors were trained centrally according to the study protocol, and, in turn, they trained local assessors who were standardized annually. All assessors were subject to a protocol adherence and reliability assessment after training, and only those with protocol adherence scores in excess of 90% and inter-rater reliability of >0.8 conducted assessments.12. Field staff were unaware of the INTER-NDA domain and total scores for individual children and sites. Data were uploaded onto secure servers as soon as each assessment was completed.The administration of the above tests was supported by a tablet-based data collection and management system, developed for the INTERGROWTH-21st Project16. International normative values have been established by pooling the data from these samples8.The INTER-NDA was used in Phase I of the INTERGROWTH-21st Project. The results clearly demonstrated that the children of healthy, adequately nourished, well-educated women, recruited from five diverse geographical and cultural study sites, who receive recommended ante-natal care, display consistent similarities at age 2 across a comprehensive set of neurodevelopmental outcomesData were managed with a similar system to that used in Phase I of the INTERGROWTH-21st Project. In brief, demographic, medical history and maternal and newborn clinical data were collected initially on paper forms and then entered locally into an online data management system developed for the INTERGROWTH-21st Project (MedSciNet), which sits on a secure MedSciNet server. Blinded data from the ultrasound machines were transferred directly to the database in Oxford. The anonymized databases are accessible only to designated personnel, including the Bill & Melinda Gates Foundation, as part of a data-sharing agreement. Users from each study site have access, at present, only to their own data; a small number of global administrators have access to all the data on a high-security, encrypted server.n = 3,206) with three or more fetal ultrasound scans between 14 and 37 weeks’ gestation (mean = 4.7 scans) to describe fetal growth patterns  with z-scores >5 or <−5 were excluded from the analysis.We included all pregnant women  was blinded at the time of the analysis. The optimal number of groups for each fetal growth measure was selected based on 1) the best model fit using the BIC and 2) the number of participants in the smallest group comprising at least 2.5% of the total sample. Each participant received a posterior probability of being in each group and was then assigned to the definitive group with the highest probability.Posterior group probabilities for models with three to five growth pattern groups were estimated5 was chosen as the reference group; this trajectory is also the one with the largest number of fetuses.Fetal cranial growth trajectories comprised the primary exposures. The trajectory most likely to be adequate (tracking the 50th centile) according to the international INTERGROWTH-21st Fetal Growth Standards12. Behavioral outcomes were based on the CBCL attentional problems and emotional reactivity scales35. Categories of low visual acuity (defined as logMAR >0.4) and high contrast sensitivity (>3%) were based on the Cardiff norms for age 2–3 (ref.15).Outcomes from the neurodevelopmental assessment were based on normative INTER-NDA scores for the six domainsz-scores based on the WHO Child Growth Standards7. Height was considered the primary infant growth outcome. We excluded extreme measurements that were likely to be errors (HC = 1 and height = 7).Height/length, weight, HC and BMI were assessed at age 2; measures were used to estimate age- and sex-standardized As our primary analyses, we used linear regression models to assess the relationships with cognitive, language, fine motor and gross motor domains. The positive and negative affect scores were strongly left- and right-skewed, respectively. The positive affect domain was reverse coded, and both positive and negative scales were modeled as count data using general linear models, using a Poisson distribution and log link function, with a variance correction for over dispersion. Vision outcomes were modeled as binary variables and used Poisson regression as well. The results represent a change in score or RR for each trajectory compared to the reference MGT trajectory. We used linear regression models to assess growth outcomes at age 2 and Cox proportional hazards models to model the age at which gross motor development milestones were first achieved.We selected covariates suspected a priori to be in the causal pathway, using separate directed acyclic graphs for size and neurodevelopmental outcomes. In all adjusted models, we included the child’s sex and age at developmental assessment, preterm birth, maternal age and education  as well as smoking during pregnancy. Age at achieving gross motor development outcome models did not include child age as a covariate.n = 1), height (n = 3) and weight (n = 1) and 2) excluding children missing one or more questions for fine (n = 137) and gross (n = 187) motor domains and those missing more than one question for cognitive (n = 163) and language (n = 147) domains. Finally, we conducted exploratory analyses adjusting by anthropometric measures at birth and age 2.As secondary sensitivity analyses, we evaluated effect modification by duration of breastfeeding (<7 months and ≥7 months) using stratified models. We constructed models that included both fetal HC and AC growth trajectories to explore whether the observed associations with fetal HC were independent effects or partially due to a relationship with the growth trajectory of the abdominal organs, given the value of measuring fetal AC alone to predict birth weight. In sensitivity analyses, we also ran models without including preterm birth as a covariate and excluded (in separate models) children who had 1) any NICU admission; 2) serious morbidity ; 3) at least three infections in a single year requiring antibiotics; and 4) been hospitalized. We also conducted sensitivity analyses 1) including outliers for HC  and STATA .<sec id="S14" sec-type="methods" specific-use="print-only"><title>Online content</title><p id="P59">Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41591-021-01280-2.</p></sec>https://doi.org/10.1038/s41591-021-01280-2.The online version contains supplementary material available at Supplementary InformationSupplementary Materials


Background: The attempt to manage patients with acute myeloid leukemia as outpatients has become increasingly common due to high hospitalization costs, low availability for beds and patient preference. Publications on the subject are scarce, especially in low-income regions and the safety in this population remains to be determined. The present study aims to assess the safety of consolidation with high-dose cytarabine in the outpatient setting. Materials and Methods: We retrospectively analyzed 39 patients who underwent consolidation with high-dose cytarabine, between 2009 and 2018, at Ophir Loyola Hospital, in Belém, Brazil. Patients treated after 2015 were given high-dose cytarabine as outpatients due to the decision of medical staff. Results: Twenty-seven patients received 76 cycles of cytarabine as outpatients; males were 48.14% of the total population, with a median age of approximately 45 years. The occurrence of delay between cycles was significantly lower among outpatients . There was no difference in relapse rates, transfusion requirements and non-relapse mortality between both groups. Hospitalization was required in 40.74% of patients during outpatient cycles and 18.51% of blood cultures were positive for pathogens. Non-relapse mortality was significantly higher among patients above 50 years old and treated on an outpatient basis . Conclusion: High-dose cytarabine administration on an outpatient basis appears to be safe and effective in a low-income population at the Brazilian Amazon region, but toxicity seems to be increased for patients older than 50 years. Multiple cycles of cytarabine at high doses (6000-18000 mg/m²/cycle) have been used as consolidation treatment of patients with acute myeloid leukemia, especially in patients younger than 60 years, with low risk AML 2,3,4. Acute Myeloid Leukemia (AML) is an aggressive disease that requires intensive treatment, usually consisting of induction chemotherapy and consolidation with high-dose chemotherapy or stem cell transplantation 5. In many countries, an average hospitalization period of 3 to 4 weeks for each cycle is adopted 6.Due to the potential risk of complications resulting from prolonged neutropenia, post-induction chemotherapy has traditionally been given on an inpatient basis to patients remaining hospitalized until hematologic recovery 1,7,8. Outpatient treatment can result in a reduction of hospital stay, shorter duration of febrile neutropenia treatment and fewer nosocomial infections9,10,11,12.Prolonged hospitalization, however, increases treatment costs, leads to persistent exposure to hospital-acquired and often multidrug-resistant organisms and impacts on quality of life, increasing rates of depression and sleep disturbances1,13,14,15,16,17,18,19, without significant differences in the incidence of complications 5. None of these reports, however, were performed in low income regions 20,21. The viability, economic impact and safety of the outpatient regimen for high-dose cytarabine consolidation therapy has been previously assessed22. It is important to assure the feasibility of the outpatient strategy in this setting. The benefits of outpatient treatment are even more relevant in developing countries considering costs, access and hospital bed occupancy The present study aims to analyze the safety and feasibility of outpatient consolidation with high-dose cytarabine in a low-income population. This is a retrospective study carried out at Ophir Loyola Hospital . This is the single public health tertiary hospital for treatment of hematologic malignancies in the state.2, twice a day, on days 1, 3 and 5. For patients above 60 years of age, the dose was reduced to 1g/m2 in the same schedule. Data were obtained by chart review of 39 patients diagnosed with AML treated at this hospital from 2009 to 2018, aged ≥ 16 years, who underwent induction therapy, attained complete remission and received consolidation with high-dose cytarabine at a dose of 3g/mPatients treated between 2009 and July 2015 received cytarabine as inpatients. After this date, all patients were treated in an outpatient setting. The protocol shift was adopted by the hematology department, motivated by the high demand and limited bed availability. Registers before 2009 could not be accessed. Patients were clarified and oriented about their disease and care. Medical and transfusion support to outpatients occurred in a day hospital facility, with all patients being advised to return to the hospital in case of fever (> 38ºC) or change in clinical status. Oral antimicrobial prophylaxis consisted of acyclovir 800mg/day, fluconazole 300mg/day and ciprofloxacin 500mg/day. Transfusion requirements were determined by blood counts and clinical status.The studied variables included sex, age, inpatient/outpatient consolidation, date of consolidation cycles, cycle delay, readmissions, complete remission, relapse and death within 30 days after cytarabine cycle (early death). Statistical analysis was performed using SPSS statistics® software and a p value of 0.05 was used as the cut-off for significance.This research complies with the National Health Council's Research Guidelines Involving Human Beings (Res. CNS 466/12) and the precepts of the Declaration of Helsinki. Approval was obtained from the Research Ethics Committee of Ophir Loyola Hospital, CAAE 00675318.0.3001.550 and opinion 3.121.297, on January 24, 2019. Between 2009 and 2018, 12 (30.76%) patients received high-dose cytarabine on an inpatient basis and 27 (69.23%) on an outpatient basis. 58.33% (7/12) of inpatients and 48.14% (13/27) of outpatients were male. Patients receiving outpatient chemotherapy were older than hospitalized patients. Transfusion requirements were slightly higher among outpatients. .Hospitalized patients received 42 cycles of high-dose cytarabine and outpatients received 76 cycles. The occurrence of delay (> 7 days) in at least one cycle was significantly higher among inpatients, with similar relapse rates .Escherichia coli (60% of positive cultures), all resistant to quinolones and sensitive to 4th generation cephalosporins and carbapenems; Pseudomonas aeruginosa (20% of positive cultures), susceptible only to colistin; and Staphylococcus epidermidis (20% of positive cultures), sensitive to all tested antimicrobials. None of the patients with a positive blood culture died.Therefore, 25% of outpatient cycles (19/76) required a short admission period after cytarabine administration. 18.51% (5/27) presented positive bloodstream culture. Agents identified were: The early death rate was similar in both groups, all in remission. No patient had any significant treatment-associated neurotoxicity. However, in outpatients aged 50 years or older, a higher mortality was observed when compared to younger patients. Nonetheless, delays were significantly lower, and all deaths occurred in remission. Transfusion requirements and relapse rates were similar in both groups .19. In Brazil, to our knowledge, this is the first report of high-dose cytarabine consolidation on an outpatient basis. One of the major concerns about implementing this system is the occurrence of delays in administration of cycles, especially due to limitations in access or transportation of patients to the hospital, notably more pronounced in a large country such as Brazil. However, in the present study, significantly lower delays were found among outpatients. This shows that access is probably not a limitation to outpatient consolidation. Other studies also showed a similarity between inpatients and outpatients regarding periodicity 19. There was no difference in relapse rates between groups in our population.In addition, the occurrence of delays or other complications during outpatient administration could impact the incidence of relapse among patients. In previous studies, however, significantly lower rates of relapse were reported among outpatients (32% vs 77%)5. This is probably due to a tendency to adopt early transfusions for outpatients, with less tolerance, while inpatients can be closely watched for longer. It should be noted that there were no cases of significant bleeding in either group.The expressive need for transfusion after chemotherapy is also a concern. In this study, outpatients received a few more transfusions but there were no significant differences compared to inpatients, as shown in previous reports23. In the present study, none of the subjects presented significant neurological signs, reinforcing the findings from previous studies which have already demonstrated lower rates in recent years (0.7%) 23.Administration of outpatient, high-dose cytarabine could also be limited by the classically reported elevated rate of neurotoxicity associated with treatment (8-20%)19. However, transference to the hospital system occurred immediately, reinforcing the safety of this regimen to ensure adequate care for patients’ demands. Nevertheless, considering an estimated 40% to 33% reduction of treatment costs when adopting the outpatient regimen5,24, these results suggest a significant economic impact. It’s also described a better quality of life among patients that experienced a reduction in the number of hospitalized days 25.The number of cycles requiring hospitalization was similar to that previously reported (28%), although the number of patients admitted was higher (20%)26, and was close to rates described for hospitalized patients (14%)13. Corroborating data from the literature, prophylaxis given to outpatients promotes a greater occurrence of resistance to quinolones, especially during the 2nd cycle of consolidation27. However, even with the isolation of a multidrug resistant pathogen, reduction of sepsis occurrence in patients treated in the outpatient setting 10 is reinforced, and there was an absence of deaths among patients with positive culture.The prevalence of positive blood cultures was much lower than previously reported for patients in outpatient consolidation (59%)28. However, there was no significant difference between inpatients or outpatients, possibly reflecting the expected values in the context of the country's population, according to previous surveys  29,30.The early mortality rate was higher than internationally reported for newly diagnosed patients undergoing induction chemotherapy (4%)31,32 using 2 cycles of cytarabine, in our hospital, mortality rates were higher among patients older than 50 years. This is possibly due to a higher prevalence of comorbidities or increased toxicity and infection rates during longer consolidation treatment, evidencing the need for differentiated support strategies and discouraging adoption of this regimen in this specific group of individuals. It is worth mentioning that this research has many limitations. For instance, it is a retrospective analysis based on medical records. In addition, we were unable to directly quantify the economic impact of outpatient treatment. Furthermore, this study represents the situation in a single tertiary hospital, and the results will need to be reproduced in other institutions. Finally, although there are studies showing the safety of outpatient consolidation in elderly patientsTherefore, high-dose cytarabine administration on an outpatient basis appears to be safe and effective in the context of developing regions, possibly due to significantly reducing costs related to treatment and improving patient’s quality of life. However, such a regimen should be used with caution in patients over 50 years due to the high risk of toxicity in this group.


Patients belonged to younger age groups (≤65 years), had a familial status other than married/in couple, and did not present an unfavorable socio-demographic profile. Concerning the pertinence for a psychiatric ED, primary diagnosis of depression/anxiety is the only variable significantly associated with different grade of degree. By the examination of the patients’ trajectory from admission to discharge, the clinical pertinence for a psychiatric ED admission existed for cases assigned to the Echelle Suisse du Tri (EST®) scale degree 1 (corresponding to most urgent and severe conditions), particularly for diagnoses of depression/anxiety associated with SB, SB as primary or comorbid diagnosis, and psychotic and manic/hypomanic episode. However, diagnoses of depression/anxiety without urgent and severe features  constituted the most frequent mode of presentation. Conclusions: Ambulatory and community-integrated settings could be more appropriate for the majority of patients admitted to adult psychiatric EDs. Moreover, the implementation of telepsychiatry strategies represents a very promising opportunity to offer these patients care continuity, reduce costs and filter the demand for psychiatric ED. Background and Objectives: Psychiatric disorders constitute frequent causes of emergency department (ED) admissions and these rates are increasing. However, referring to ED a whole range of conditions that could or should be dealt with elsewhere is imposing itself as a problematic situation. We aimed: (1) to provide a descriptive picture of the socio-demographic and diagnostic characteristics of the visits among adults at the psychiatric ED; (2) to estimate the clinical pertinence of these visits. Materials and Methods: Retrospective analysis of diagnostic/socio-demographic characteristics and clinical trajectories of patients admitted for a psychiatric condition at the adult psychiatric ED of the University Hospital of Geneva (HUG), Switzerland, during a 6-week timespan. Results: In our sample ( In many countries, patients who access emergency departments (EDs) for psychiatric reasons are a concern for health policy leaders and emergency physicians because they present chronic and complex medical issues with high associated costs for health care delivery ,2,3,4,5.Psychiatric disorders requiring ED admissions are considered potentially avoidable when managed through appropriate ambulatory care ,16,17. TCurrently the effectiveness of community- and primary-care-based interventions for limiting ED use for psychiatric illness is not well understood ,30. ThisTo this end, we aimed: (1) to provide a descriptive picture of the socio-demographic and diagnostic characteristics of the visits among adults at the psychiatric ED of the University Hospital of Geneva (HUG), Switzerland; (2) to estimate the clinical pertinence of these visits. We retrospectively examined every admission file of patients registered at the adult division of the psychiatric ED of the HUG, Switzerland, between 1 April 2016 and 15 May 2016, by delineating a descriptive picture of their diagnostic/socio-demographic characteristics and clinical trajectory from admission to discharge. For every visit, diagnoses at admission (primary and comorbid diagnosis performed according DSM 5), demographic data , patient’s access modality to ED , the type of eventual ongoing ambulatory psychiatric treatment , and the decision by the psychiatrist at the discharge from ED ) were determined. ®). The EST® is one of the two screening tools recommended by the Swiss Society for Emergency Medicine and Rescue and is currently used in the three language regions of Switzerland as well as in France and Belgium. With decreasing severity, the EST® scale ranges from degree 1 , to 2 , to 3 , to 4 .Data were also differentiated according to an emergency degree scale used at the HUG ED, which evaluates both the urgency and the severity of a pathological condition: the Echelle Suisse du Tri . Subsequently, differences according to the EST® were performed using Pearson’s Chi-Square and ANOVA test for categorical and continuous variables, respectively. Statistical analyses were based on descriptive statistical methods using SPSS version 25 and the significance was set at This study was conducted in accordance with the Helsinki Declaration as revised in 2013 . It was A total of 763 admissions for psychiatric disorders were made at the adult psychiatric ED of the HUG during the study period. Among them, only admissions that required a psychiatric medical evaluation were included in the study . A final sample of 702 participants were recruited: 383 females (54.6%), 317 males (45.2%), and 2 people (0.2%) with gender disorder. The age ranged from 16 years, the minimal age to be admitted to adult psychiatric ED of the HUG, to 96 years. The mean age was 42.8 ± 18.3 (range 16–96) for females and 38.7 ± 16.5 for males (range 16–88). The differences in the socio-demographic and diagnostic characteristics are shown in ®, 96 cases (13.7%) were classified as degree 1, 312 cases (44.4%) as degree 2, 265 cases (37.8%) as degree 3, and 29 cases (4.1%) as degree 4  and the ≥65 age group the least represented . Half of the patients  were unmarried/non-in a couple. The large majority of patients  lived at their home, did not benefit from a social contribution , and did not have a migrant status   . n = 310, 44.2%), followed by suicidal behavior (SB) , psychotic episode , substance use disorder (SUD) , behavior disorder , somatic problem , behavior disorder in the elderly , manic/hypomanic episode , psychomotor agitation , other reasons , and intellectual disability . Regarding the presence of a comorbid diagnosis, SB occurred in the majority of cases , followed by somatic problem , behavior disorder , SUD , psychomotor agitation , depression/anxiety , behavior disorder in the elderly , psychotic and manic/hypomanic episode , and other reasons . No cases of comorbid diagnosis of intellectual disability was registered  and behavior disorder in the elderly (76.2% vs. 23.8%) while males had significantly more SB (57.3% vs. 42.7%) and behavior disorder (62.7% vs. 37.3). The other not significant differences in terms of the first diagnostic characteristics across gender are displayed in ®, primary diagnosis and no other features  is the only variable significantly associated with a different grade of degree , the diagnoses of psychotic episode  and maniac/hypomanic episode  were the most represented. Psychomotor agitation accounted for one non-voluntary hospitalization. The large majority of patients with SB as principal or accessory diagnosis were hospitalized . For degree 2, 13 (61.9%) women were followed-up by a psychiatrist , 25 (83.3%) returned home, of whom ten (33.3%) were sent to a private psychiatrist. Eight men (32%) came by themselves while undergoing outpatient psychiatric treatment  and eight came by themselves and did not have ongoing outpatient psychiatric treatment. Twenty-two men (88%) returned home, including 14 (56%) with the indication to be monitored in multidisciplinary public structures, and five (20%) were referred to a private psychiatrist. For degree 3, regarding women: 33 (55%) came by themselves and did not a have ongoing psychiatric treatment; and 49 (81.7%) returned home, of whom 22 (44.9%) were referred to private psychiatrists. With regard to men: 20 (58.8%) had no ongoing psychiatric treatment, seven (20.6%) were followed-up in multidisciplinary public structures, and 28 (82.4%) returned home after their ED assessment. For degree 4, 22 cases (75%) did not have an ongoing psychiatric treatment, and 24 cases (82%) returned home  are the most common reasons for referral to the adult psychiatric ED. SB and psychotic episodes accounted for approximately the same proportion of patients. SB was also the most common accessory diagnosis. The distribution of these diagnoses is consistent with the general prevalence of mental disorder within the Swiss population  and, exc® scale degree (degree 1), particularly for diagnoses of depression/anxiety associated with SB, SB as first or accessory diagnosis, and psychotic and manic/hypomanic episodes. However, diagnoses of depression/anxiety without urgent and severe features  constituted the most frequent mode of presentation. First diagnosis of depression/anxiety is the only variable significantly associated with different grade of degree. By the examination of the patients’ trajectory from admission to discharge, clinical pertinence for a psychiatric ED admission existed for cases assigned to the most severe ESTIn detail, among the consultations classified as degree 1 (corresponding to most urgent and severe conditions), the primary diagnosis of depression/anxiety was found in the majority of the cases and associated with SB in one third of the cases. Considering both primary and comorbid diagnoses, SB accounted for the majority of the cases. The psychomotor agitation diagnosis, classified as degree 1 by definition, was surprisingly underrepresented and a small number of psychomotor agitation diagnoses emerged among the non-voluntary hospitalizations. One possible explanation may be that at least some of the agitations were designated as a psychotic and manic/hypomanic episode. With regard to clinical trajectory, a large majority of patients: had a diagnosis of depression/anxiety associated with SB; had SB as a first or accessory diagnosis; had a psychotic and manic/hypomanic episode; arrived by ambulance or police; and were hospitalized after the ED assessment. This may lead to the inference that clinical pertinence with an ED visit exists for degree 1, in particular for these respective diagnoses.Degrees 2 and 3 (corresponding to less urgent and severe conditions than degree 1) accounted for the large majority of the visits. As with degree 1, the most highly represented primary diagnosis was depression/anxiety. However, the proportion of patients with this diagnosis that came by themselves to the ED and having an ongoing outpatient psychiatric treatment was higher compared to degree 1. At the post-ED orientation, the large majority of degree 2 and 3 patients were not hospitalized. Surprisingly, the association of depression/anxiety with a comorbid diagnosis of SB accounted for more cases among degree 2 visits than degree 1, but its clinical severity was lower. Some differences between the patients’ gender with depression/anxiety emerged. Most of these patients were women, had an ongoing outpatient psychiatric treatment, and, after ED assessment, returned home and were referred to a private psychiatrist. Furthermore, they tended to be married/in couple and did not have an unfavorable socio-demographic profile. Men were less likely to have ongoing outpatient psychiatric treatment and were more often referred to public ambulatory multidisciplinary structures offering the possibility of overnight stays, which would suggest clinical diagnoses that were perceived as more severe and/or complicated. In contrast to women, they more frequently had a familial status other than married/in couple and presented a slightly more unfavorable socio-demographic profile. Patients with the described trajectory configure an excessive high percentage for an ED.Degree 4 consultations accounted for a minority of cases, which tended to have mild depression/anxiety diagnoses. Their clinical trajectories were not appropriate for an ED. In summary, visits receiving the diagnosis of depression/anxiety without urgent and severe features  were the most frequent mode of presentation to our adult psychiatric ED. An ambulatory and community-integrated setting could be more appropriate for patients belonging to this group. In particular, they could benefit from access to ambulatory facilities that can provide for a rapid inclusion of unscheduled cases ,17,28,44Another interesting opportunity for patients who inadequately consult psychiatric ED, is the use of telepsychiatry. The latter, also known as telemental health, is commonly defined as “the delivery of mental health care in the form of live and interactive videoconferencing” . The psyThis study had several limitations. First, it was a naturalistic observational study and, consequently, an experimental control was not included, thus precluding inferential conclusions from being drawn. Second, only a short period of observation (six weeks) was monitored, and this limited time span may introduce a potential bias concerning the type of diagnosis that can be more frequent during this specific period. Third, by using the admission files diagnoses, we did not distinguish various types of personality disorders . On the other hand, the strengths of this study were: having a snapshot that included all adult psychiatric ED admissions during the study period ; its location (a large metropolitan area representing the entire Geneva canton); and the concerned population . These latter factors may facilitate the generalizability of the findings to other public psychiatric settings, as well their comparison. The obtained data may inform possible alternative strategies to deal with a whole range of psychiatric patients that can be treated elsewhere than in EDs. Ambulatory and community-integrated settings could be more appropriate for the majority of these patients. Moreover, the implementation of telepsychiatry strategies also represents a very promising opportunity to offer these patients a closer care continuity, reduce costs, and filter the demand for psychiatric ED. The involvement of all key clinicians, along with organizational and technological support, is necessary to guarantee more success to telepsychiatry in ED.


We identified 19 metabolites present in all ejaculate portions and the EE, and reported correlations between the metabolites. Additionally, and for the first time in mammals, we found intra-ejaculate variability in the SP metabolites, observing different relative abundances in choline, glycerophosphocholine and glycine. Regarding their influence in sperm physiology, we hypothesize that these metabolites may explain the specific reproductive characteristics of each ejaculate portion. Finally, the reported SP metabolites could serve as a first steppingstone in the study of quality, functionality, and fertility biomarkers.In pigs, ejaculate is expelled in fractions, mainly the sperm-rich fraction (SRF) and the post-SRF (PSRF), which differ in both sperm content and origin. In addition, intra-ejaculate variability between fractions in terms of sperm reproductive characteristics has been previously reported, the highest sperm quality being observed in the first 10 mL of the SRF (SRF-P1). As seminal plasma (SP) composition has been purported to influence sperm physiology, the aim of this study was to profile pig SP metabolite composition and to find putative differences between the ejaculate portions  and entire ejaculate (EE). To this end, ejaculates ( To date, artificial insemination (AI) remains the most widely used reproductive biotechnology in the swine industry . AlthougAs in other mammalian species, such as humans  and horsIn the last years, the putative impact that SP could exert on pig sperm has acquired greater relevance due to the changes in the ejaculate collection systems in AI-centers. The traditional gloved-hand collection method, which allows the separation and discarding of the PSRF from the SRF ,26, is bThe large-scale study of cellular metabolic products (metabolomics) reflects the downstream events of gene expression ; for thi1H Nuclear Magnetic Resonance (1H RMN) spectroscopy, due to the reproducible and high resolution information that this technique provides regarding the metabolomic composition of biofluids  and Pietrain [n= 3 boars]) belonging to one AI-center . Boars were housed in individual pens in an environmentally controlled building (15–25 °C) with ad libitum access to water and fed with a commercial diet according to the nutritional requirements for an adult boar , Glutamic acid [Glu], Glutamine [Gln], Glycine [Gly], Histidine [His], Lysine [Lys], Phenylalanine [Phe], Tyrosine [Tyr], Valine [Val] and Leucine [Leu]); salts ; nucleosides ; other organic compounds .The To quantitatively compare the SP metabolites of each ejaculate-portion and the EE, 11 of the 24 identified spectral peaks (which corresponded to 11 different metabolites) were included in the statistical analysis. This selection was based on the signal intensity and peak isolation in order to achieve enough signal-to noise and to avoid spectral overlapping (metabolites indicated with an asterisk in p < 0.01) between ejaculate-portions and the EE (p < 0.01) in SP from the SRF-P1  than in SP from the PSRF  and the EE . Similarly, the relative levels of Cho were lower (p < 0.01) in SP from the SRF-P1 (−6.3 ± 4.29) and SRF-P2 (−1.9 ± 5.22) than in SP from the PSRF (4.7 ± 8.03). Moreover, SP from the SRF-P1 (−6.3 ± 4.29) also showed lower Cho levels (p < 0.01) compared to SP from the EE (3.6 ± 6.62). In addition, regardless of the portion of the ejaculate analyzed, our results revealed a strong positive correlation between these three metabolites  differed  score of two PC explained 96.8% of the data variance, achieving more than 99% in the first three PCs A. The PCFinally, to characterize the contribution of each metabolite to the different ejaculate fractions, an abundance plot was created. The abundance plot showed a pattern similar to the PCA distribution, with Gly, Cho and GPC exhibiting the highest abundance in the PSRF and the lowest in SRF-P1 D.Traditionally thought to be a mere vehicle for sperm deposition in the female reproductive tract, studies over the years have shown SP to modulate sperm function, regulate the female reproductive environment, and even affect offspring health ,49,50,511H NMR spectroscopy is emerging as one of the most used, because it provides a uniform metabolite detection of equal sensibility in mixture samples, such as SP [1H NMR spectroscopy.The assessment of male fertility has traditionally focused on the evaluation of sperm attributes, such as motility, morphology and viability ,52. Howech as SP . Therefo1H NMR spectra obtained for pig SP is similar to that of human SP, with all identified metabolites in pig SP also present in human SP [1H NMR spectrometer used [1H NMR spectra of bovine [Our data shows that the pig SP is comprised by 19 metabolites, which include amino acids, salts, nucleosides, and other organic compounds. The human SP ,38,39. Iter used ,38. The ter used ,20. Thesf bovine  and porcf bovine ,54.To the best of our knowledge, this is also the first work that has evaluated the potential relationship between SP metabolites. In general, our results revealed that they were positively correlated. This correlation was strong between Gly, Cho and GPC, and weak between all SP metabolites and Glu. Correlations between metabolites result from the reactions and regulation of networks, and strong positive correlations may indicate a rapid equilibrium, enzyme dominance or similar regulation ,56. Thus1H NMR spectrum of prostatic and seminal vesicle fluids surgically obtained also revealed a higher concentration of GPC, and also lactate, in the seminal vesicle fluid compared to the prostatic fluid. All of the presented data, together with the fact that seminal vesicles represent 80–90% of the total SP protein content [Previous studies suggest that the epididymis mainly contributes to the SP of the SRF-P1, the prostate to the SP of the SRF-P2, and the seminal vesicles to the SP of the PSRF ,16,17,18 content , would s2O2 levels [In pigs, sperm from different ejaculate portions have been shown to differ in their motility and viability , as well2 levels . In effe2 levels . Moreove2 levels . Differe2 levels . Interes2 levels ,67,68.The amino acid Gly has been related to the initiation of the acrosome reaction (AR) in pig, mouse, hamster and human capacitated sperm ,69,70, pDrosophila Melanogaster [2O2 (the main ROS generated by pig spermatozoa) and lipid peroxidation have been found in sperm from the PSRF [On the other hand, both GPC and Cho are metabolites involved in the metabolism of glycerophospholipids. Both of these metabolites are involved in two key aspects of sperm physiology: capacitation and motility. In rats, hydrolysis of GPC by phospholipase C has been shown to occur during capacitation and AR, this lipid being one of those that decrease the most after the onset of capacitation . In addinogaster . Thus, inogaster ,75. Althnogaster , excessinogaster ,78. In tnogaster  and thatthe PSRF . These rthe PSRF ,80. Somethe PSRF ,80. Morethe PSRF , since athe PSRF ,38,79,81To sum up, GPC, Cho and Gly seem to contribute to the regulation of sperm physiology in processes such as motility, capacitation and AR. As aforementioned, SP from PSRF has been extensively suggested to have a negative impact upon sperm reproductive characteristics. Since different relative metabolite abundances between portions were observed, the highest levels for these three metabolites being found in the PSRF, we suggest that GPC, Cho and Gly may contribute towards explaining the sperm function variability between ejaculate portions. What is more, the three metabolites have previously been correlated with different forms of male infertility and suggested as putative fertility biomarkers. Therefore, further studies should elucidate whether GPC, Cho and Gly could also be used as SP biomarkers for pig sperm quality and fertilizing ability.In conclusion, the present study describes the metabolomic composition of pig SP, showing intra-ejaculate variability. The variability in the metabolite abundances of SP between the different portions of the ejaculate suggests that certain metabolites, such as Cho, GPC and Gly, could be linked to sperm fertilizing ability. Due to the processes in which these metabolites are involved, together with previous evidence on specific sperm performance for each portion, SP from PSRF may have a detrimental effect on sperm functionality. On the other hand, this study provides a list of metabolites of pig SP which could serve as a first steppingstone for further studies to uncover the potential of SP metabolites as biomarkers of sperm quality, functionality and fertility in AI-boars. Moreover, because of the high correlations between SP metabolites, it would be possible to assess a reduced number of metabolites as biomarkers of fertility or sperm quality biomarkers instead of performing an in-depth metabolomic analysis of the samples. Progress in this field will likely contribute, not only to the improvement of artificial reproductive technologies in the swine industry, but also to the understanding of sperm physiology. Moreover, based on the similar behavior patterns of human sperm performance between ejaculate fractions and the similar metabolite composition of the SP, the pig could be an appropriate model for the study of the role of SP metabolites in reproductive physiology.


Pseudomonas aeruginosa is an opportunistic pathogenassociated with healthcare-related infections, affecting mainly patientswith underlying diseases and immunosuppression. This microorganism hasseveral virulence mechanisms that favour its pathogenesis, including theproduction of biofilm. This study aimed to analyze the phenotypic productionof biofilms, the occurrence of quorum sensing (QS) genes, and the clonalprofile of clinical isolates of P. aeruginosa fromcolonized/infected patients in a tertiary hospital in Recife-PE.  We obtained 21 isolates that were classified as infection isolates (II), and10 colonization isolates (CI). The phenotypic analysis for biofilmproduction was performed quantitatively. The QS genes were detected byspecific PCRs, and the clonal profile was assessed using ERIC-PCR.  Of the 31 isolates, 58.1 % (18/31) were biofilm producers, of which 70 %(7/10) were CI and classified as weakly adherent; 52.4 % (11/21) of the IIproduced biofilms, and were classified as weak ), moderate), and strongly adherent ). All isolatesharbored the QS genes analyzed. In the clonal analysis, 26 distinct geneticprofiles were identified, highlighting the presence of a clone in foursamples, i.e., one infection isolate, and 3 colonization isolates. P.aeruginosa in addition to the identification of colonizationand infection isolates, especially from complex environments such as ICUs.Further, we define a strategy for monitoring and analyzing P.aeruginosa strains that can potentially cause infections inhospitalized patients.  The detection of biofilm formation is important in  Pseudomonas aeruginosa is a bacillus species found in soil, water,and the digestive tract of some animals. It is grown in the laboratory on cetrimideagar which is used for the selective isolation of bacteria, and it enables theobservation of the large, mucoid, greenish colonies that have a characteristicallysweetish odor and fluoresce in the presence of ultraviolet light due to theproduction of pyoverdin. Additionally, the colonies have been shown to withstandtemperatures of 4 - 42ºC,P. aeruginosa bacterium is an opportunistic pathogen thatcolonizes immunosuppressed individuals that have underlying diseases, and patientswho are admitted to the intensive care units (ICU) in hospitals,,,The P. aeruginosa is an important etiological agent associated withhealthcare-related infections and it has been shown to increase the rate ofmortality and morbidity in patients. It can potentially become multidrug-resistantdue to its ability to acquire different antimicrobial resistance mechanisms,,,,,,,,,P. aeruginosa infections areassociated with biofilm production, such as in patients undergoing mechanicalventilation, or those having cystic fibrosis or burns,,,The pathogen shows the ability to produce biofilms, which are an important factor forvirulence and bacterial resistance, and can have a strong impact on the health ofthe host. The dense polysaccharide matrix of the biofilm contributes to thepersistence of infection, the ineffective action of antimicrobials, and the escapefrom the phagocytic actions of the cells of the immune system of the host, theseeffects result in chronic pathology,,,P. aeruginosa to respond to the self-inductingmolecules called acyl-homoserine lactones (AHLs) that target theLas and Rhl genes. Like mammalian cells, bacteria respond to molecules and interact with the receptorsof other microorganisms to communicatelasI, rhlI, and pqsA genes areresponsible for the biosynthesis of the signaling moleculesN-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), N-Butyrylhomoserine lactone (BHL),and the Pseudomonas quinolone signal (PQS), respectively. Thecorresponding receptors for the signaling molecules are LasR, RhlR, and PqsR,respectively, which are involved in the regulation of bacterial virulence andbiofilm formation,,,Further, the P. aeruginosa isolates fromcolonized and infected patients to verify biofilm production phenotypically, searchfor QS genes , andundertake an analysis of the clonal profile of the isolates.In this study, we aimed at analyzing P. aeruginosa isolates were obtained frompatients, of which 21 were infection isolates (II), and ten were colonizationisolates (CI), based on the criteria adopted by the microbiology laboratory ofthe hospital. The isolates were stored at -20ºC at the Bacteriology andMolecular Biology Laboratory, Universidade Federal de Pernambuco. The frozenisolates were reactivated in Brain Heart Infusion (BHI) medium, incubated for 24h at 37ºC, and were subsequently seeded in cetrimide agar and incubated for anadditional 24 h at 37ºC. These isolates represented diverse clinical andsurveillance samples .A total of 31  samples .P. aeruginosa isolates were grown in BHI broth for 24 h at37ºC. Next, 200 µL of the bacterial suspension was applied to flat bottomed96-well polystyrene plates in triplicate for microtitration. BHI broth was usedas a negative control, and the PA01 strain of P. aeruginosa wasused as a positive control (CP) to test for biofilm production. The plates wereincubated at 37ºC for 24 h, and subsequently, the bacterial suspensions wereremoved, and each well was washed three times with 250 µL of saline (0.9 % NaCl(Dinâmica)). The bacterial samples were fixed using 200 µL of methanol (QuímicaModerna) for 15 min. After methanol removal, the plates were left to dry at roomtemperature and were stained with 200 µL of crystal violet (Newprov) solutionfor 5 min. 570 was measured using an ELISA plate reader, and the samples were classified as described by Stepanovicand collaborators (2000)Next, the plates were washed under running water and dried at room temperature.The absorbance at AlasI, lasR, rhlI, and rhlR,Total DNA extraction was performed using the Brazol kit (LGC-Biotecnologia), asper the protocol provided by the manufacturer, and the DNA was quantified viaspectrophotometry . A simple quantificationwas performed using the absorbance at 260 nm and an assessment of purityperformed using the 260/280 ratio. The genes associated with QS i.e.,2 and 1 U of DNA Taq Polymerase (Promega). The amplificationparameters for ERIC-PCR were initial denaturation at 95ºC for 3 min, followed by30 at 92ºC for 1 min, annealing at 36ºC for 1 min, an extension step at 72ºC for8 min and a final extension at 72ºC for 16 min. The PCR products were stainedwith Blue Green , and analyzed by agarose gelelectrophoresis using a 1.5 % agarose gel. The DNA bands were visualized underUV light and photodocumented (Kasvi) for clonal analysisThe 31 isolates were analyzed via molecular typing by using the enterobacterialrepetitive intergenic consensus-based PCR (ERIC-PCR) technique to determine theclonal profile of the strains. The PCR was performed in a total volume of 25 μLper tube containing 100 ng of DNA, 10 pmol of primers , 1x buffer,200 μM deoxyribonucleotide triphosphate (Ludwig Biotec), 1.5mM ofMgClIn the II samples, the highest prevalence of bacterial isolates was detected inblood 33.3 % (7∕21); 23.8 % (5∕21) of the tracheal secretion and urine samplesshowed the presence of bacteria. In the CI samples, 90 % (9∕10) tested positive,and these were obtained from a rectal swab culture .et al Of the 31 isolates studied, 58.1 % (18/31) were biofilm producers. Afterstaining and analysis using the spectrophotometer, the isolates were classifiedas described in the work by Stepanovic lasI, lasR, rhlI,and rhlR).All 31 isolates had the four QS genes studied P. aeruginosa was significantly responsible for a widerange of infections in critically ill patients that were acquired in the ICU.The II analyzed in this study were obtained from different infection sites, althoughsamples of blood, tracheal secretion, and urine were the most prevalent sources ofP. aeruginosa. Moreover,different studies that used the same technique to analyze biofilm production byP. aeruginosa obtained percentages for biofilm productionranging from 73.7 % to 98.6 % in the isolates analyzed,,,,,We used a quantitative phenotypic test considered to be a gold standard to detectbiofilm production by clinical isolates of The adhesion profile of the biofilm showed a distribution variable of the groups thatwere classified as weak, moderate, to strongly adherent. Similarly, Perez et al.(2013),,P. aeruginosa that were found toinfect cystic fibrosis and non-cystic fibrosis patients and demonstrated that allfour genes were present in the isolates. Lima et al. (2018)lasR, rhlI, and rhlR genes, and a 97.5 %detection rate for the lasI gene. However, these data differ fromthe results obtained by Karatuna and Yagci (2010)lasI and lasR genes, the rhlIgene, and the rhlR gene, respectively. Biofilm formation is a complex virulence mechanism, which requires gene regulation bydifferent components of the QS system. Changes in the expression of the genesrelated to the QS network or the presence of mutations can reduce the production ofthis important bacterial virulence factorlasR, lasI,rhlR, and rhlI genes that prevented theirefficient expression. Lima et al., (2018)P. aeruginosa that were notbiofilm producers. Sequencing of the lasR gene showed theoccurrence of mutations at position 53 of the LasR protein, which is close to thebinding region for its autoinducer, N-(3-oxododecanoyl) homoserine lactone (OdDHL).The authors attributed the non-formation of biofilm by the P.aeruginosa isolates to the mutations observed in thelasR gene.In our study, although all isolates showed the presence of the quorum-sensing genesanalyzed, 41.9 % (13∕31) of the isolates did not produce biofilms in thequantitative analysis. This may occur due to the non-expression of these genes orbecause of the presence of mutations in the genes that regulate the QS system, asfound in a study by Senturk et al., (2012)lasR andlasI genes were unable to form a biofilm, and the resultsindicate that these genes play an important role in QS and the formation ofbiofilms. These findings were similar to those of Persyn et al., (2019)P. aeruginosa isolatesobtained from patients with repeated attacks of pneumonia that were associated withmechanical ventilation. The authors observed that isolates with mutations in thegenes associated with the QS system had a lower degree of virulence.Perez et al., (2013),P. aeruginosa, such asmetalo-β-lactamases, and showed a higher diversity of genetic profiles of P.aeruginosa isolates from public hospitals. Additionally, reports fromstudies in the literature indicate that similar results were obtained using isolatesfrom patients with chronic infections as these individuals are in an environmentfavorable for the appearance of random mutations. Further, antibiotic therapypromotes the artificial selection of bacteria for greater drug resistance, primarilyin cases where P. aeruginosa infection is present, as themucopolysaccharide constituting the biofilm hinders drug diffusion through celllayers,,,The dendrogram in P. aeruginosa in patients admitted to the ICU. The resultssuggest that dissemination between patients plays an important role in theacquisition of P. aeruginosa colonization on the skin and mucousmembranes, and this process precedes the infection that occurs later. Gómez-Zorrillaet al, (2015)P. aeruginosa is a key factor for thedevelopment of infection. Additionally, another studyThe analysis of genetic diversity showed the occurrence of a clone composed of fourisolates, one of them II and three CI. Johnson et al., (2009)P.aeruginosa to survive antibiotic treatment while growing in a biofilmrepresent an important therapeutic challenge. 2016Biofilm production in bacterial isolates is recognized as an important factor forpersistent infectionsP. aeruginosa from patients admitted to a hospital.Additionally, we detected the presence of high genetic diversity between theisolates, including a clone composed of among infection and colonization isolates,indicating that dissemination had occurred in the hospital environment. The resultsindicate the importance of the detection of biofilms in P.aeruginosa in both colonization and infection isolates, especially fromcomplex environments such as ICUs. The strategy outlined in this study can be usedfor monitoring and studying strains that can cause infections in hospitalizedpatients, though the formation of biofilms remains an important therapeuticchallenge. In this study, we described the biofilm production in infection and colonizationisolates of


Cionusolivieri Rosenschoeld, 1838 is described and illustrated in detail for the first time. It is compared with those known from the same genus and other genera in the tribe Cionini and with those of the hypothesized sister tribe Mecinini in the Curculioninae. The larvae of Cionus have three distinctive diagnostic features: the reduced number of setae on the epicranium (only two or three des and one or two fs) and on the epipharyngeal lining ; i.e., distinctly fewer than the most frequent number of setae in weevils, and mandibles dentate or angulate internally near the base. If considered together with Stereonychus Suffrian, 1854, the other genus of Cionini with larvae studied in detail, it is preliminarily suggested that mature larvae of this tribe might be characterized by six main diagnostic features: (1) labial palpi one-segmented, (2) labral rods absent, (3) pedal areas swollen to form large lobes or prolegs, (4) mandible with sharp apical teeth, (5) reduced number of fs on frons, only one or two fs, and (6) reduced number of epipharyngeal setae . It was noticed that C.helleri Reitter, 1904 from Japan, a very distinct species in the genus for some characters of the adult, also possesses distinctive characters in the larva which are uncommon among known cionines. New biological data on C.olivieri with the discovery of its host plant, Verbascumsongaricum (Scrophulariaceae), in central Asia are also reported.The mature larva of  Cionini Schoenherr, 1825 in the subfamily Curculioninae Latreille, 1802 (Curculionidae) currently comprises seven genera occurring predominantly in the Palearctic region . The largest genus is Cionus Clairville, 1798, which is also represented in the Afrotropical and Oriental regions, whereas the other genera include fewer than ten species  or are monotypic . Based on the Palearctic species, Cionus based also on a preliminary study of species of other regions, The tribe Cionusolivieri Rosenschoeld, 1838 are clearly distinguished from other species of Cionus by the following features taken together: body size on average large ; rostrum in lateral view stout, almost evenly curved, approximately of the same width from base to apex, its apical part in dorsal view with parallel sides, not narrower in midlength; antennal insertion in males closer to rostrum midlength; pronotum with almost conically narrowed sides; elytra with sub-rounded sides, short , with the integument concealed by densely distributed scales, without rows of erect setae-like scales; anterior onychia in males of normal length, at most as long as tarsomeres 1 to 3 combined , Afghanistan, and Pakistan. h Africa .Cionus usually include Verbascum, Scrophularia, Buddleja (Scrophulariaceae) and Limosella (Plantaginaceae) .Host plants of Palearctic inaceae) . Larvae inaceae) . It is kium Ten. . In TurkC.olivieri in detail for the first time, in order to provide further characters for the identification of this taxon, and to compare this larva with the larvae of other species of Cionus, Cionini, and the apparent sister tribe Mecinini Gistel, 1848.The aim of the present study is to describe the larva of 42.391391°N; 72.279285°E, 1546 m, Verbascumsongaricum, 2-VII-2018, leg. Chunyan Jiang) and 6 mature instar larvae from Kazakhstan .Fifteen mature larvae from Kyrgyzstan .All described specimens were fixed in 75% ethanol and examined under a Nikon SMZ 1500 optical stereomicroscope with calibrated oculars. To prepare microscope slides, we made dissections as in The observations and measurements were conducted using a compound microscope with calibrated ocular lenses (Leica DM 2500). The following dimensions were measured for each larva and provided in the Description: head width and length, body length (larvae fixed in a C-shape were measured in segments), and body width in the widest place . The relative lengths of all setae can be seen in the figures. Transparent structures were stained with Chlorazol Black E for further examination.Photos of larvae were taken with a Canon-5D camera mounted on the microscope. Images of adults were photographed with a CCD Qimaging MicroPublisher 5.0 RTV mounted on a Zeiss SteREO Discovery. V12 microscope. Images from microscopic slides were taken with a Nikon CoolPix 5400. Photographs in the field were taken with Canon G15 camera.Drawings were made from the original photographs using the software Adobe Illustrator CS6. The numbers of setae in bilateral structures are given for one side only.We used the terms and abbreviations for the setae of the mature larvae found in Taxon classificationAnimaliaColeopteraCurculionidaeRosenschoeld, 1838FF429B55-02FC-52DC-8FBB-A8C9F246892BCionusolivieri Rosenschoeld, 1838: 725. Measurements (in mm). Body length: 9.00–9.75 (N = 18). Body width: 2.50–3.00 . Head width (in front view): 0.68–0.74 (N = 10); length: 0.70–0.78 (N = 10).Figures General. Body subcylindrical, C-shaped, weakly curved, extremely soft, declivous and attenuate posteriorly  present, projecting, anterior one located below stripe at side, externally close to antenna, posterior one located laterally. Setae on head piliform, varying in length, from very long to minute. Dorsal epicranium with three des; des1 and des4 reduced to a basal sensillum; des2 approximately as long as one-third length of des3; very long des3 located anteriorly on epicranium close to frontal suture; des5 located anterolaterally, as long as two-thirds length of des3. Frons only with one fs; fs1, fs2, fs3, and fs5 reduced to basal sensilla; long fs4 located near epistoma. Epicranium with only one les as long as des1. Ventral epicranium with two, minute ves. Posterior epicranium with four, minute pes1–4 and one sensillum. Postoccipital condyles distinct, hypopharyngeal bracon distinct. Tentorial bridge narrow, membranous in middle and half base of sides, strongly sclerotized at sides, with two pairs of acute auricular anterior projections.ule Figs , 6. HeadAntenna  with sclerite distinctly dilated posteriorly and laterally, without posterior extension, cup-like, with one sensillum and one short prms; ligula with one tiny ligs. Postlabium (plb) with M-shaped sclerotization, with three plbs on sclerotized area, all setae separated from each other by about the same distance; short 1plbs and 3plbs as long as one-third length of 2plbs.rts Figs –12. Labrrts Figs  transverynx Fig.  with twoles Fig.  symmetriThorax  by Cleopuspulchellus  by Later, we collected larvae of eri Fig.  from theeri Fig.  in Kazakeri Fig. . This faCionus taxa have been previously described  , and C.scrophulariae  , C.tuberculosus , C.olivieri, and C.thapsus  .The larvae of eight escribed . Unfortuer, 1904 , C.horty, 1785) , C.olens, 1758)  ‒ whereas, 1792)  – were aCionus are probably characterized by three diagnostic features: the reduced number of setae (1) on the epicranium (only two or three des and one or two fs), and (2) on the epipharyngeal lining , i.e., distinctly fewer than the most frequent number of setae in weevils, and (3) mandibles dentate or angulate internally near the base ; and eight epipharyngeal setae . This fact appears particularly interesting. Indeed, C.helleri is unique in Palearctic Cionus in having three tubercles on the pronotum, and mucronate apices of the meso- and metatibiae in males as well as spines on the anterior tarsomere 1. The first two character states are particularly interesting from a taxonomic and phylogenetic point of view because tubercles on the pronotum are possessed only by several species of Afrotropical Cionus and some Oriental Cleopus , whereas the presence of tibial unci in male is a plesiomorphic condition shared in other genera of Cionini, i.e., Nanomicrophyes, Cleopus, and Stereonychidius  in Curculionidae has not been completely resolved, but this has been discussed in previous papers  and Stereonychusfraxini  are problematic due to missing details on chaetotaxy, and it is almost impossible to compare it with Cionus species. Only a detailed larval description, that of Stereonychusthoracicus Faust, 1887 published by fs, and no mes) as Cionus species, but there are some differences: Stereonychus has five des (in Cionus at most three), three als and three ams . It seems that mature larvae of Cionus (excluding C.helleri) + Stereonychus are characterized by six main diagnostic features: (1) labial palpi one-segmented, (2) labral rods absent, (3) pedal areas swollen to form large lobes or prolegs, (4) mandible with sharp apical teeth, (5) reduced number of fs, only one or two fs, on epicranium, and (6) reduced number of epipharyngeal setae , i.e., fewer than the most frequent number of setae recorded in weevils ; (2) dorsum of epicranium with two or three des (vs. five des); (3) frons with one or two fs (vs. three to five fs); (4) epipharynx without mes (vs. with one or two mes); and (5) mala with three to five dms and two to five vms (vs. six or seven dms and four or five vms). The count of palpomeres on the labial palpi was confirmed as one of the most important morphological characters of larvae in the tribe Mecinini (van Cionini have no variability in this character, and the labial palpi have only one palpomere.Since it was hypothesized by a phylogenetic study based on morphological characters of imagos that the tribe Mecinini  we also nini van , but theCionus and related genera and tribes is still impossible because of limited available knowledge. However, it is clear that a detailed description of immature stages is of primary importance for further studies on generic and intergeneric taxonomic relationships within Cionini and/or Curculioninae, as done in other groups of Curculioninae, such as Tychiini (Mecinini (We realize that a thorough study of the immatures of the genus Tychiini  and MeciMecinini .


Key performance indicator (KPI) data provide candidate information required for effective network planning, performance analysis and optimization. However, inadequate KPI data could limit efficient network planning leading to escalating operational cost, and this could adversely affect the subscribers of the network. To this end, this article presents radio frequency (RF) measurements and evaluation of KPIs taken at 1876.6MHz with a bandwidth of 10MHz, for an operational 4G LTE network in Nigeria. The measurements campaign specifically examine the behaviour of the RSRP, RSRQ, RSSI, SINR, PCC PHY DL Throughput, and the PDCP DL Throughput. Huawei Technologies Modem E392 was used for the propagation measurements, and RF measurements cover three evolved node base stations (eNodeBs) with average heights of 25 m. The geographical coordinates of the sites are as follows: Site 1 , Site 2 , and Site 3 . The E392 4G (LTE) Modem is capable of propagation measurements at the various LTE frequency bands, enables LTE download Speed of 100 Mbit/s, supports LTE upload Speed of 50 Mbit/s, utilizes LTE 2x2 MIMO (Multiple Input Multiple Output), and supports 64QAM (Quadrature Amplitude Modulation). The Drive Test (DT) Software version-Genex prove V16, and Genex Assistance V16 were deployed, and the test car carried a test terminal station, a GPS, a Windows supported Computer, and the accompanying drive test system. The test vehicle was driven such that it considered the actual road traffic conditions at a relatively medium speed of up to 30km/h with uniformity thereby reducing possible Doppler effects. Terminal connection was established, and data download services was started . Thereafter, the download simultaneous file downloading limit was set to 5 files . When connection drops, simultaneous connection was re-established using the ftp software, and drive test was carried out within a planned cluster on a bright and sunny day. Statistical descriptions and probability distribution functions of the KPI data is reported and interdependence amongst the KPIs are presented to ease understanding of the interrelationships among the tested KPIs. The data reported would find useful applications in RF planning, radio channel measurements and modelling, feasibility studies and formulation of appropriate regulatory policies for wireless communication systems. Network operators could leverage on the data for appropriate KPI analyses, radio resources management, and research and development. The tested KPIs include the RSRP, RSRQ, RSSI, SINR, PCC PHY DL Throughput, and the PDCP DL Throughput. These KPIs were measured at a 4G LTE frequency of 1876.6MHz with 10MHz bandwidth. The extensive RF measurements span a propagation distance of up to 2km, and measured KPIs were extracted and analysed in IBM SPSS Statistics and MATLAB.The KPIs derived from the experimental data are briefly described as follows. First, the aerial view and the geographical coordinates of the measurements environment are as shown in The statistics of the measured KPIs are given in The probability distribution of the KPIs observed are given in 2The equipment used for measurements is the Huawei Modem E392. The E392 4G (LTE) Modem offers flexibility in RF measurements and post processing of measurements data. The equipment can be used for propagation measurements at various LTE frequency bands, and supports a LTE download Speed of 100 Mbit/s, while the LTE upload Speed supported is up to 50 Mbit/s. Furthermore, the device supports LTE 2x2 MIMO and 64QAM (Quadrature Amplitude Modulation). The Drive Test (DT) Software version-Genex prove V16, and Genex Assistance V16 were selected and carefully connected and assembled in the DT car for seamless propagation measurements. The drive test car carried the test terminal station, the GPS equipment, and a personal computer (PC), and the associated drive test system.In order to achieve quality results, the test vehicle was driven such that it considered the actual road traffic conditions at medium speed of up 30km/h with uniformity. This helps to reduce the possible impacts of Doppler effects. Afterwards, the terminal connection was established, and data download services started using file transfer protocol - ftp, a drive test software, which has the function to download a large file around or up to 20 giga bytes (GB). Thereafter, the download simultaneous file downloading limit was set to 5 files . When connection drops, simultaneous connection was re-established using the ftp software, and drive test was carried out within a planned cluster located in the geographical coordinates of the measurements environment. For data post processing, MATLAB 2018a, a product of Mathworks Incorporated, and the IBM Statistical tool (SPSS) version 24 were used.