P < .05) higher sodium level by biochemistry autoanalyzer, lower albumin, lower protein, and higher maximum percent of physiological weight than the sodium level difference ≤4 mmol/L group. After adjusting for factors showing significant difference between the 2 groups, protein at birth  and percent of maximum weight loss  were factor showing significant associations with sodium level difference >4 mmol/L between 2 methods. Thus, difference in sodium level between blood gas analyzer and biochemistry autoanalyzer in early stages of life could reflect maximum physiology weight loss. Based on this study, if the study to predict the body's composition of extracellular and intracellular fluid is proceeded, it will help neonatologist make clinical decisions at early life of preterm infants.Neonatologists often experience sodium ion level difference between an arterial blood gas analyzer (direct method) and an autoanalyzer (indirect method) in critically ill neonates. We hypothesize that clinical factors besides albumin and protein in the blood that cause laboratory errors might be associated with sodium ion level difference between the 2 methods in very-low-birth-weight infants during early life after birth. Among very-low-birth-weight infants who were admitted to Jeonbuk National Hospital Neonatal Intensive Care Units from October 2013 to December 2016, 106 neonates were included in this study. Arterial blood sample was collected within an hour after birth. Blood gas analyzer and biochemistry autoanalyzer were performed simultaneously. Seventy-six (71.7%) were found to have sodium ion difference exceeding 4 mmol/L between 2 methods. The mean difference of sodium ion level was 5.9 ± 6.1 mmol/L, exceeding 4 mmol/L. Based on sodium ion level difference, patients were divided into >4 and ≤4 mmol/L groups. The sodium level difference >4 mmol/L group showed significantly ( However, neonatologists often experience different sodium ion levels between direct and indirect ion-selective electrode (ISE) methods for preterm infants. In a previous study, we have found that such difference in sodium ion level is associated with serum protein level like adult patients. Most of previous studies have also shown that sodium ion difference can be caused by hypoproteinemia and/or hypoalbuminemia.–7 And sodium ion difference is often associated with critically ill conditions requiring ICU admission.,9Measuring sodium ion level in blood is essential for preterm infants because sodium imbalance can lead to convulsions and brain damage.,7 The United States Clinical Laboratory Improvement Amendments standard for the difference between electrolyte tests recommends that sodium ion level difference between any 2 test methods should not exceed 4 mmol/L. Despite efforts to achieve accurate examination by eliminating difference of sodium ion level, discrepancy remains unsolved, making it difficult for neonatologists to diagnose and treat sodium imbalance, especially in preterm infants during early life after birth.Preterm infants in the early stage of life have physiologic weight loss different from children and/or adults. They may also have unstable vital sign, immature kidney, neonatal morbidities, and death. Moreover, sodium ion level difference exceeding 4 mmol/L between direct and indirect ISE method is often seen in ICU, especially in very-low-birth-weight infants (VLBWIs) during early life after birth.In the current study, in addition to known causes of protein and albumin in blood, clinical factors within the first week of life relevant to sodium ion differences between a blood gas analyzer using direct ISE method and biochemistry autoanalyzer using indirect ISE method in VLBWIs were determined. We hypothesize that clinical factors or characteristics of VLBWIs during early life after birth are associated with sodium ion difference between direct and indirect ISE methods.2The present study included inborn preterm infants who were admitted to Jeonbuk National Hospital NICU from October 2013 to December 2016. Data of VLBWIs (birth weight <1500 g) who underwent arterial blood collection for electrolyte analysis were retrospectively reviewed. Blood test data were collected through electronic medical record chart review. Blood tests were conducted on the first day and the first week. Clinical features of VLBWIs within the first week of life were obtained. First, patients who had hemolytic samples on the first day were excluded from this study. Second, we also excluded patients who used diuretics or steroids that could affect urine amount within a week of life. Third, infants diagnosed as congenital anomalies were excluded. The Jeonbuk National Hospital Institutional Review Board approved this study. The requirement of informed consent was waived due to the retrospective nature of this study.Arterial blood sample was collected within an hour after birth in NICU. Blood tests including blood gas analyzer as a direct ISE method and biochemistry autoanalyzer as an indirect ISE method were performed using the simultaneously collected arterial blood. All samples  of patients were divided into a dry heparin syringe  and a microtube  and sent to the Division of Laboratory Medicine . The sample in the dry heparin syringe was analyzed with a benchtop blood gas analyzer  using the direct ISE method. The blood gas analyzer undergoes an automatic 2-point calibration every 2, 4, or 6 hours and a single-point calibration every 30 minutes or after each sample is input. Another sample placed into the microtube was analyzed with a biochemistry auto-analyzer  using the indirect ISE method. This machine contains an auto-calibration system that maintains the accuracy and precision using a sample buffer. Each sample was tested within 10 minutes after being put in the test container.am by trained nurses. To record the weight exactly, the tube and line were held by nurses and the sensor attached to the patient was removed before the weight of infant was measured. If the patient's weight differed by more than 10% compared to the previous day, it was measured again after zeroing the scale. Humidity and temperature of the incubator were adjusted according to neutral thermal environments. They were adjusted at the discretion of the neonatologist. A patient with less than 25 weeks of gestational age (GA) was cared for with minimal handling to reduce IWL due to immature skin. Phototherapy was applied according to the result of daily total bilirubin.All preterm infants in NICU were managed with the following fluid management protocol. Fluid replacement for patients was commenced at 60 mL/kg for the first day of birth and gradually increased to 120 mL/kg until 10 days after birth. Daily fluid requirements were calculated considering urine output and insensible water loss (IWL). Considering the calculated daily requirement, the input volume was increased within the range of 10 to 15 mL/kg per day. From the first day of birth, tropic feeding (<20 mL/kg) was started. Feeding volume was increased based on feed tolerance. If the amount of feeding did not increase, then parental nutrition was supplemented. All processes of fluid and nutrition management were decided by attending neonatologists. During a week after birth, all VLBWIs were managed in incubators . To control additional IWL from the skin and mucus membrane, babies were wrapped in plastic wraps right after birth. A high humidity was provided to the incubator. Each patient's weight was measured in the incubator at 6  Results of blood tests on the first day and the seventh day included electrolyte, protein, and albumin. Perinatal demographics and maternal factors and preterm infants’ clinical factors within the first week of life were collected. Perinatal demographics characteristics included GA, birth weight, sex, 1-minute Apgar score, 5-minute Apgar score, and small for gestational age (SGA). Maternal factors included maternal age, multiple gestations, completed antenatal steroid, IVF (in vitro fertilization), maternal antibiotics, clinical chorioamnionitis, gestational diabetes mellitus, pregnancy-associated hypertension, and preterm premature rupture of membrane. Vital signs, chemistry, and electrolyte on the first day of birth were collected. Neonatal mortality predicting scores at birth, including clinical risk index of babies II score, SNAP  II score, and SNAP perinatal extension score, were also collected. Preterm infants’ diseases within the first week of life included high grade intraventricular hemorrhage (IVH) (grade 3-4 IVH), respiratory distress syndrome (RDS), hemodynamically significant patent ductus arteriosus (PDA), blood culture proven early sepsis, maximum percentage of weight loss, input volume, amount of urine output, duration of ventilator care, duration of phototherapy, and death within 7 days.VLBWIs were divided into 2 groups based on sodium ion level difference of 4 mmol/L  between blood gas analyzer as a direct ISE method and biochemistry autoanalyzer as an indirect ISE method on the first day after birth.2.1t-test. Deming regression analysis and a Bland–Altman plot were used to evaluate sodium ion level differences between the 2 ISE methods. To find clinical factors associated with sodium ion level difference between the 2 ISE methods, logistic regression analysis was conducted. Univariate logistic regression analysis was performed for clinical factors and blood tests within a week of life. We determined whether clinical factors and blood test variables that showed a difference in univariate analysis at P < .1 might be associated sodium ion level differences by multivariate regression analysis. All analyses were performed using SPSS version 24.0  and MedCalc Statistical Software version 16.0 . A P-value of less than .05 was deemed statistically significant.Two groups categorized by sodium ion level difference of 4 mmol/L of between direct and indirect ISE method were compared with unpaired 3P < .05) higher than 4 mmol/L.Sodium ion levels of a total of 140 VLBWIs were simultaneously analyzed with direct and indirect ISE methods. Among them, 21 VLBWIs with the use of diuretics and/or steroid, 14 with a hemolytic sample, and 9 with congenital anomalies people were excluded from this study. Finally, 106 VLBWIs were included in this study. Of them, 76 (71.7%) had sodium ion level difference between indirect and direct ISE methods exceeding 4 mmol/L at birth. Mean GA and birth weight were 29.4 ± 3.1 weeks and 1138.9 ± 270.6 g, respectively. Sodium ion level at birth measured with the indirect ISE method using a chemistry auto-analyzer was 138.6 ± 4.1 mmol/L. It was 132.6 ± 3.7 mmol/L when the direct ISE method with arterial blood gas analysis was used for measurement. A Bland–Altman comparison of sodium ion level between the 2 methods showed that limits of agreement ranged from −0.2 to 12.0 mmol/L  difference between the 2 groups. Although SGA was high for the sodium ion level difference ≤4 mmol/L group, it was not significantly different between the 2 groups. Except for these factors, other characteristics and maternal factors were not significantly different between 2 groups  in the sodium ion level difference >4 mmol/L group. There was no difference in vital sign or mortality-predicting score such as clinical risk index of babies II, SNAP II, or SNAP perinatal extension score between the 2 groups. The sodium ion level difference >4 mmol/L group showed higher chemistry sodium level by the indirect ISE method, lower albumin level, and lower protein level than the other group (Table Table P < .05). The sodium ion level difference >4 mmol/L group showed a higher maximum percent of physiological weight loss than the other group (P < .05). Except for this, the 2 groups showed no significant difference in neonatal disease . Sodium, potassium, and chloride ion levels on the seventh day did not show a significant difference between the 2 groups either.Neonatal clinical features within the first week of life were compared between the 2 groups in Table P < .001) and percent of maximum weight loss  were found to be factors associated with sodium ion level difference >4 mmol/L between direct and indirect ISE methods  volume through natriuresis and diuresis after birth.In addition to the previously known associated factors such as plasma protein, this study revealed that sodium difference between direct and indirect ISE methods was independently associated with initial maximum weight loss. Since we simultaneously analyzed collected arterial blood samples, sodium difference between 2 ISE methods was not related to difference in a discrepancy between capillary and/or arterial and/or venous blood. Sodium difference was likely to be associated with solid components in the blood such as lipid and other unknown factors. The result of this study showed that the difference in sodium ion level between the 2 groups was mainly due to the chemistry sodium ion level measured with the indirect ISE method. Solid component also makes incorrect sodium ion in indirect ISE method. Unmeasured solid component might make the sodium level difference between direct and indirect ISE methods. These components might also be associated with physiologic weight loss in VLBWIs. Namely, the association could be presented as a way to predict physiologic weight changes.To date, there is no way to predict the amount of physiology weight loss at right after birth. In clinical practice, fluid management is based on subjective indicators including the condition of a patient visible to naked eyes and the neonatologist's experience rather than based on objective data. As a result, the prediction of maximum weight loss at birth could reduce unnecessary fluid intake that might increase mortality and morbidities such as PDA and bronchopulmonary dysplasia. If VLBWIs’ maximum physiology weight loss meaning ECF volume of body water is predictable with sodium ion at birth, it will help fluid management in the first week of life. If there is a substance associated with sodium ion level difference between direct and indirect ISE methods, it is necessary to further study about the association with physiologic weight loss.This retrospective study has some limitations. According to our strict NICU policy, we tried to keep volume appropriate management considering IWL and urine amount. Although, input volume and urine output were not significantly different between the 2 groups, we could not calculate IWL during first week of life. IWL difficult to measure, such as phototherapy, maybe make physiologic weight loss between 2 groups. And antidiuretic hormone and renin-angiotensin-aldosterone system associated hormone those are released with urine amount were not measured. These unmeasured hormonal changes could be an important factor that made sodium level difference. Finally, although this study reflected the condition up to 1 week after birth, it had a limit to reflect overall sodium difference.In conclusion, difference in sodium ion level between direct and indirect ISE methods in the early stages of life might reflect the extent of maximum physiology weight loss in VLBWIs. Based on this study, if the study to predict the body's composition of ECF and intracellular fluid is proceeded, it will help neonatologist make clinical decisions at early life of preterm infants.Conceptualization: Jin Kyu Kim.Data curation: Jin Kyu Kim.Formal analysis: Hyun Ho Kim.Funding acquisition: Jin Kyu Kim.Methodology: Hyun Ho Kim.Writing – original draft: Hyun Ho Kim.Writing – review & editing: Jin Kyu Kim.


The dynamic nature and complexity of the cellular events that take place during the last trimester of pregnancy make the developing cortex particularly vulnerable to perturbations. Abrupt interruption to normal gestation can lead to significant deviations to many of these processes, resulting in atypical trajectory of cortical maturation in preterm birth survivors.invivo MRI in a large sample of healthy term-born infants scanned after birth (n = 259). Then we offer a comprehensive characterization of the cortical consequences of preterm birth in 76 preterm infants scanned at term-equivalent age . We describe the group-average atypicality, the heterogeneity across individual preterm infants, and relate individual deviations from normative development to age at birth and neurodevelopment at 18 months.We sought to first map typical cortical micro- and macrostructure development using In the term-born neonatal brain, we observed heterogeneous and regionally specific associations between age at scan and measures of cortical morphology and microstructure, including rapid surface expansion, greater cortical thickness, lower cortical anisotropy and higher neurite orientation dispersion. By term-equivalent age, preterm infants had on average increased cortical tissue water content and reduced neurite density index in the posterior parts of the cortex, and greater cortical thickness anteriorly compared to term-born infants. While individual preterm infants were more likely to show extreme deviations (over 3.1 standard deviations) from normative cortical maturation compared to term-born infants, these extreme deviations were highly variable and showed very little spatial overlap between individuals. Measures of regional cortical development were associated with age at birth, but not with neurodevelopment at 18 months.We showed that preterm birth alters cortical micro- and macrostructural maturation near the time of full-term birth. Deviations from normative development were highly variable between individual preterm infants. Rapid dendritic arborization, synaptogenesis, ingrowth of thalamocortical afferents combined with the disappearance of radial glia and the dissolution of the subplate drive cortical growth and a significant increase in cytoarchitectural complexity . The corpreterm brain exclusively, with little reference to normative development, or has been limited by small term-born samples. A comprehensive characterization of the cortical sequelae of preterm birth compared to normative development is necessary to better understand the role of cortical (dys)maturation in the encephalopathy of prematurity.Preterm birth, or being born before 37 weeks gestational age (GA), represents a significant risk for adverse neurodevelopment . While win vivo structural and diffusion MRI in a large sample of term-born infants scanned during the neonatal period, to then test how preterm birth shapes the developing cortex. Early brain development is highly variable between individuals, and even those belonging to the same risk or clinical ‘group’ often show heterogeneous pattern of deviations from typical maturation . Infants were scanned at term-equivalent age ) during natural unsedated sleep at the Evelina London Children's Hospital. Infant preparation and imaging have been described previously in detail . Exclusi2.22-weighted scans were acquired with TR/TE of 12 s/156 ms, SENSE=2.11/2.58  with in-plane resolution of 0.8 × 0.8 mm . Images were reconstructed using super-resolution and motion correction resulting in 0.5 mm isotropic grid sizes , TR/TE of 3800/90 ms and 4 phase-encoding directions  and mean diffusivity (MD), in μm2/ms (10−3 mm2/s), were calculated using MRtrix3 .Infants’ dMRI-derived maps were affine registered to native Td in FSL . When prd in FSL   unsatisfactory surface projection or registration to surface template were included . The final sample comprised 259 term-born and 76 preterm infants (Suppl.n = 143 per hemisphere). For all metrics with the exception of SA, where the sum was used, we calculated the mean value per parcel for every infant to be used for subsequent analyses.We used a two-stage quality control (in volume and surface space) to ensure no data affected by (i) motion, or with (ii) artefacts, (iii) poor dMRI to Ts Suppl.. Cortica2.32.3.1pmcfwe < 0.05 .First, we described age-associated changes in cortical microstructure and morphology in the full 259 term-born sample. This was performed with Permutation Analysis of Linear Models (PALM) implemented in FSL using 10,000 permutations and a family-wise error (fwe) correction across modalities (8 metrics) and contrasts (positive/negative association with PMA at scan), including sex in the model . Pearson2.3.2Scikit Learn (Suppl.Scikit Learn Optimize) under 5-fold cross validation and mean absolute error (MAE) as loss function. Optimal hyperparameters were max_depth = 35, max_features = ‘auto’, min_sample_leafs = 2, n_estimators = 1000. The RF predictions were also error-in-variables bias corrected using linear regression  regression in it Learn . The 259n Suppl.. Model hgression . Model p2.42.4.1Prior to testing for group mean differences (‘main effect’ of group) between term-born and preterm infants at TEA, we examined if any of the cortical features showed a significant group * PMA at scan interaction. Analyses were performed in PALM for 259 term-born and 76 preterm infants, including sex as a covariate and correcting across contrasts  and modalities (8 metrics). As a follow-up we also quantified the correlation between PMA at scan and all cortical features in the preterm sample alone (as described in n =259 ) and preterm infants (n = 76). Analyses were performed in PALM, including PMA at scan and sex as covariates in the model and correcting across contrasts  and modalities (8 metrics). We report the t-statistic for parcels indicating a significant group mean difference at pmcfwe < 0.05.If no significant interactions were observed, we then tested for group mean differences between term-born (2.4.2https://sheffieldml.github.io/GPy/). GPR simultaneously provides point estimates and measures of predictive confidence for every observation representing the distance of each individual from the normative mean at that point on the ‘curve’ accounting for modeled covariates. Analogous to the widely employed paediatric height and weight growth charts, this technique allows the local imaging features of individual infants to be referred to typical variation while simultaneously accounting for variables such as age and sex , a Bayesian non-parametric estimation technique, implemented in GPy ( and sex .Suppl.p < 0.001)  .2.4.3Z| > 3.1 divided by the total number of infants), individually for every cortical feature.The spatial prevalence of extreme negative/positive deviations across the preterm sample was examined by calculating a parcel-based percentage map of extreme deviations (number of infants with |2.4.4Z < |3.1|) or atypical (Z > |3.1|) to then calculated a positive and a negative whole-cortex atypicality index. These indices capture the extreme positive/negative deviations for every infant and represent the ‘overall burden’ as a percentage of cortical parcels with extreme deviations (Z > |3.1|) relative to the total number of parcels. Thus, if an infant had 29 parcels with extreme negative deviations in cortical FA (Z < −3.1), the FA- atypicality index for this infant will be 10% (29 out of 286).For every cortical feature and infant, we classified each parcel as either normative (2.4.5U (for group differences) for the atypicality indices. Analyses were corrected for multiple comparison correction (FDR).To test if age at birth influences cortical development, we examined the association between GA at birth and GPR derived Z-scores; and between GA at birth and the whole-cortex atypicality indices . To assess if deviations from normative cortical development relate to later behavior, we looked at the correlation between Bayley III Scales of Infant and Toddler Development (BSID-III)  composit2.5http://www.developingconnectome.org/. dHCP surface template is available at https://brain-development.org/brain-atlases/atlases-from-the-dhcp-project/cortical-surface-atlas-bozek/. Code for MSM surface registration can be found at https://github.com/ecr05/MSM_HOCR, partial volume correction code at https://git.fmrib.ox.ac.uk/seanf/dhcp-neonatal-fmri-pipeline/-/blob/90f2f7317fff0cd809d0b1b4a400d970c1ea115c/doc/surface.md. Python code and metadata to perform the main analyses in this work are available from https://github.com/ralidimitrova/neo_cortexDev .The MRI data for the dHCP are freely available at 3p = 0.90), nor in sex distribution (p = 0.89) (Suppl.p<0.05), no infants had severe WM injury such as periventricular leuomalacia and only one preterm infant had more than 20 PWMLs. One preterm infant had cystic lesions in both thalami extending to internal capsule and left putamen, one had cerebellar hemispheric parenchymal loss accompanied by brainstem atrophy, and one had middle cerebral artery infarct . No group differences were observed at the 18 month follow-up . All, but two , preterm infants scored within 2 standard deviations from the population mean.Term and preterm infants did not differ in PMA at scan ( = 0.89) . Distrib3.1Suppl.We observed heterogeneous and regionally specific associations between PMA at scan and MRI-derived features of cortical micro- and macrostructure during the neonatal period . With in2 = 0.74 . Overall, SA was the ‘best’ predictor of PMA at scan, with 47% (108/229 parcels) of the 10% most important features. ODI followed with 37 parcels (16%), cortical thickness with 29 (13%), fICVF with 21 (9%), FA with 14 (6%), MD with 11 (5%), sulcation with 6 (3%) and curvature with 3 parcels (1%) .RF regression predicted PMA at scan in the hold-out term-born sample with MAE = 0.60, MSE = 0.56 (weeks) with a correlation between observed and predicted age of ρ = 0.85, R3.23.2.1Suppl. Fig. 7) consistent with the associations observed in the term-born sample . Indeed, preterm infants showed correlations between PMA at scan and cortical micro- and macrostructure . Model MAE maps are shown in Suppl. Fig. 9.We observed widespread extreme positive deviations in MD and thickness, and extreme negative deviations in fICVF in preterm infants B. While 3.2.3Suppl. Fig. 10). We observed no association between Z-scores and BSID-III neurodevelopmental scores at 18 months.Lower GA at birth related to higher MD and lower fICVF in the parietal cortex . Term-born infants did not have higher atypicality index in any of the cortical features we studied.As a group, preterm infants had higher atypicality indices for most metrics except FA and SA . On averFDR < 0.05). Being born earlier was also associated with a higher proportion of extreme positive deviations in MD (τ = −0.24), thickness (τ = −0.20) and sulcation .Lower GA at birth was related to a higher proportion of extreme negative deviations in ODI (τ = −0.15), fICVF (τ = −0.26), curvature (τ = −0.24) and sulcation  and reduced neurite density index (fICVF↓) in the occipital, parietal, temporal and the somatosensory cortex compared to term-born peers. Group differences in cortical microstructural ‘geometry’ were evident in a few parcels, mainly in the insular region, suggesting reduced neurite orientation complexity (FA↑ and ODI↓). GA at birth was negatively associated with MD and positively with fICVF in the parietal cortex, while in the insular cortex increased GA related to lower FA and higher ODI. The exact cellular mechanisms underlying this altered microstructure development are unknown. Dysmaturation of the neurons in the subplate, that offer initial targets and guidance of thalamocortical afferents before they reach the cortical plate may have a contributing role  ODI, fICVF, curvature and sulcation, and with more parcels with extreme high (Z > 3.1) MD, thickness and sulcation. This argues that when reaching the time of full-term birth, the cortex is more atypical in those born at lower GA. While we did not detect mean differences in curvature and sulcation between the two groups, preterm infants were more likely to show higher loading of extreme deviations from the normative curvature and sulcation compared to term-born infants. Atypical folding pattern has been previously reported in preterm infants  or very  specific aspect(s) of the underlying architecture  Biomedical Research Centre at Guy's and St Thomas’ Hospitals NHS Foundation Trust. The study was supported in part by the Wellcome Engineering and Physical Sciences Research Council Centre for Medical Engineering at King's College London  and the Medical Research Council (UK) [grant MR/K006355/1]. D.C. is supported by the Flemish Research Foundation . J.O. is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society . G.M. received support from the Sackler Institute for Translational Neurodevelopment at King's College London and from National Institute for Health Research (NIHR) Maudsley Biomedical Research Centre (BRC). J.O., A.D.E. and G.M. received support from the Medical Research Council Centre for Neurodevelopmental Disorders, King's College London [grant MR/N026063/1]. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, the Department of Health and IMI 2JU.The dHCP project was funded by the European Research Council (ERC) under the European Union Ralica Dimitrova: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. Maximilian Pietsch: Conceptualization, Formal analysis, Methodology, Software, Validation, Visualization. Judit Ciarrusta: Conceptualization, Methodology, Investigation, Writing – original draft. Sean P. Fitzgibbon: Investigation, Methodology, Resources, Software, Validation. Logan Z.J. Williams: Investigation, Methodology, Resources, Validation. Daan Christiaens: Data curation, Investigation, Resources, Software. Lucilio Cordero-Grande: Data curation, Investigation, Resources, Software. Dafnis Batalle: Methodology, Resources, Software. Antonios Makropoulos: Methodology, Resources, Software. Andreas Schuh: Methodology, Resources, Software. Anthony N. Price: Data curation, Methodology, Investigation, Resources. Jana Hutter: Methodology, Investigation, Resources. Rui PAG Teixeira: Methodology, Investigation, Resources. Emer Hughes: Data curation, Methodology, Investigation, Resources. Andrew Chew: Data curation, Investigation, Resources. Shona Falconer: Data curation, Investigation, Resources. Olivia Carney: Data curation, Investigation, Resources. Alexia Egloff: Data curation, Investigation, Resources. J-Donald Tournier: Investigation, Methodology, Resources. Grainne McAlonan: Funding acquisition, Investigation, Supervision, Writing – review & editing. Mary A. Rutherford: Data curation, Resources, Supervision, Writing – review & editing. Serena J. Counsell: Data curation, Resources, Supervision, Writing – review & editing. Emma C. Robinson: Methodology, Software, Supervision, Validation. Joseph V. Hajnal: Funding acquisition, Methodology, Resources. Daniel Rueckert: Funding acquisition, Methodology, Resources. A. David Edwards: Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing. Jonathan O'Muircheartaigh: Conceptualization, Methodology, Resources, Software, Supervision, Validation, Writing – original draft, Writing – review & editing.


States which reduce fetal oxygen delivery are associated with impaired intrauterine growth. Hypoxia results when barometric pressure falls with ascent to altitude, and with it the partial pressure of inspired oxygen (‘hypobaric hypoxia’). Birth weight is reduced when native lowlanders gestate at such high altitude (HA)- an effect mitigated in native (millennia) high altitude populations. Studying HA populations offer a route to explore the mechanisms by which hypoxia impacts fetal growth.Between February 2017 and January 2019, we prospectively studied 316 pregnant women, in Leh, Ladakh  and 101 pregnant women living in Delhi .th birth weight centile) vs 19% of newborn at low altitude. At HA, increased maternal body mass index, age and uterine artery diameter were positively associated with growth >10th weight centile.Of Ladakhi HA newborns, 14% were small for gestational age (<10This study showed that Ladakhi offspring birth weight is relatively spared from the expected adverse HA effects. Furthermore, maternal body composition and greater uterine artery size may be physiological HA adaptations and warrant further study as they offer potential mechanisms to overcome hypoxia related growth issues. Birth weight is an important determinant of fetal and infant survival –4. Smallrd trimester UtA flow at high altitude (HA) is greater in Tibetans than Han Chinese supplying blood to the growing pregnant womb increases in diameter as gestation advances . At 20 wn Chinese. Increasn Chinese.Fetal genotype influences birth weight at low altitude, independent of maternal genotype . HoweverThe Ladakhi people of the Indian Jammu and Kashmir regions represent an ancestral HA population, dwelling between the Karakoram and Himalayan mountain ranges at ≥3400m. They share some elements of ancestral history with neighbouring Tibetans , but havi)Report the association of maternal and offspring anthropomorphic characteristics with birth weight impacts, and specifically that of increased UtA diameterii)Collect biological samples to investigate genetic and metabolic mechanisms that might help explain the phenotypes identifiedWe thus set out to perform a larger prospective observational study in order to elucidate the effects of HA hypoxia on birth weight in a single native HA Ladakhi population. Specifically, we sought to: th September 2016, the Office of the Chief Medical Officer Leh (3rd August 2016), the All India Institute for Medical Sciences and the University College London research ethics committee (3634/002).Two linked prospective observational cohort studies were carried out. Pregnant women were recruited from Sonam Norboo Memorial (SNM) Hospital, Leh, Ladakh  from the antenatal clinics and antenatal ultrasound departments over a 2-year period (Feb 2017-Jan 2019). Infants born at the All India Institute for Medical Sciences, New Delhi (216m above sea level) were studied in parallel over a 1-year period (Jan 2018-December 2018). Ethical permissions were granted for the study from the Indian Health Ministry’s Screening Committee (HMSC) on the 7Pregnant women were eligible if they were aged over 18; not known to be genetically related to the father (First cousin or closer); having a singleton pregnancy; planning to deliver at the hospital; < 28 weeks gestation using last menstrual period (LMP) and in whom an estimated due date (EDD) could be confirmed by ultrasound. Where LMP was uncertain or where there was a difference of more than 5 days by an appropriate early ultrasound, the ultrasound determined EDD was used. Excluded were those pregnancies with obvious fetal structural or chromosomal abnormalities.Pregnant women completed a questionnaire documenting their dietary patterns (meat eater/ vegetarian); and family, obstetric and medical histories . Geographical ancestry was recorded with, where possible, birth locations for more than five generations. Blood pressure and anthropomorphic measures (height and weight pre-pregnancy and at attendance) were recorded using standard anthropometric techniques at the enrolment, booking appointment. UtA diameters were measured in longitudinal section at its crossover with the external iliac artery using transcutaneous ultrasound, between 18-24 weeks gestation  by a sinInformation concerning the birth process (mode of delivery) and the neonatal characteristics  were recorded. Arterial transdermal oxygen saturation was recorded by pulse oximeter within first 15 minutes of birth from both a finger and toe with babies lying, resting and not crying and in the absence of supplemental oxygen delivery.th centile according to the Intergrowth software package (https://intergrowth21.tghn.org).Deliveries were classified preterm if <37 weeks gestation. All deliveries up to 42 weeks’ gestation were included in the analysis. Small for gestational age (SGA) infants were defined by a birth weight of <10Outcome measures were defined by cases- pregnancies affected by SGA versus controls  offspring). Analysis was first undertaken to compare maternal characteristics between SGA and AGA pregnancies in the HA and LA locations. Comparisons between HA and LA locations were also undertaken for the entire cohorts and then stratified by SGA and AGA outcome.Statistical analysis was performed using the STATA 16 package. Normality was confirmed through visual representation of birth weight distribution by histogram plot. Maternal baseline characteristics are reported as means [standard deviation SD] unless otherwise stated. Unpaired t-test were performed and reported for baseline characteristics between women who delivered AGA babies and those that delivered SGA babies.3. Logistic regression was used to identify factors contributing to the probability of SGA delivery in the HA population. Logistic regression results are reported as odds ratios with 95% confidence limits. Explanatory variables are assessed as significant at the 5% level. Univariable logistical analysis of maternal variables was first undertaken from the entire HA cohort. Maternal variables that retained significance in the first model were included in the final multivariable model to identify the association of UtA diameter and being an SGA case. Data were initially analysed by univariable logistic regression and those with the lowest P value used to generate the final multivariable model. Sensitivity analysis using forward stepwise regression confirmed the validity of this approach.Birth weight centiles and birth weight centile ratios were calculated using Intergrowth standards as previously documented. Ponderal index was calculated as infant birth weight (g)*100/ birth length (cm)Sample size was calculated prior to commencement of the study using the STATA statistical software package. At 90% power with a 0.05 significance level, 184 subjects would allow for detection of a 300g birth weight difference between SGA and AGA cases. This assumes a SD of 540g, which is based on previous studies , 22 withOverall, 316 pregnant women were recruited in Leh (HA), and 101 in Delhi (LA) (1 year due to later start of enrolment). Over the two-year period, all women where gestational age could be ascertained and thought to be <28 weeks, were approached to be in the study. Maternal characteristics are presented in In Leh, more than 96% of women considered themselves of Ladakhi descent , whilst 10 (3.2%) described themselves as Tibetan. Birth weight was greatest in the Tibetan ancestry group (3.62kg) vs Ladakhi (3.14kg), p 0.0009.Of AIIMS participants, the majority of women had lived in Delhi for more than 5 years, but more than half came originally from outside of Delhi area. Overall, 94/101 (93.1%) described themselves as Indoaryan ethnicity, 2 as Indo-European and 5 as Dravidian.2, p=0.002) and age  differed. Compared to women who delivered an AGA baby, those who delivered an SGA baby were statistically older and lighter.Characteristics of HA women who delivered SGA verses AGA babies were similar. Maternal ethnicity, parity, smoking status (no subjects disclosed being a smoker at HA), chronic illness and socio-economic  did not differ . No sign2 [p=0.03]). Hypothyroidism was reported once in each group with one report of maternal hypertension in the SGA group.Baseline maternal characteristics of women who delivered at LA revealed a similar pattern. Ethnicity, parity, age and smoking (one woman with an SGA pregnancy reported current smoking) were not found to be different between groups. Maternal BMI was different between groups . Missing UtA data was due to the sonologist not being available to scan (n=29), women being outside of 18-24-week gestation period (n=34) or if the blinded analysis disagreed (n=32). Data were not used where the second operator disagreed on blinded analysis by >0.1cm.At HA, mean UtA diameter was 56.5 [10.7] mm in AGA pregnancies vs 51.5 [9.3] mm in SGA pregnancies (p=0.009). Birth weights in the 187 AGA neonates where UtA data was available were similar to that in the overall cohort recruited (3.23kg). SGA pregnancies with UtA data reported (n=34) were also found to have similar birth weight (2.51kg) to overall study population  and SGA was reported at a similar rate (15%). In the SGA group there was no statistical difference in birth weight between those with UtA measurements (2.51kg) and those without (2.54kg), p= 0.69.Univariable logistical regression analysis of maternal characteristics between AGA and SGA babies at HA revealed body mass index (BMI) and age to be significantly different between groups. Therefore, a final multivariable logistical regression model was run incorporating maternal BMI, age and UtA diameter in babies born AGA versus SGA at HA. Maternal UtA diameter remained statistically different between women who had an SGA baby versus those who had an AGA baby (OR 1.06 p=0.0001) with increased maternal UtA positively associated with having an AGA baby. UtA diameter was statistically smaller in the LA group vs HA groups 47.3 [4.65] vs 55.5 [11.2] mm respectively, p=0.0001). In LA babies, UtA diameter was appropriately recorded in 61 (60.3%). In this group the average birth weight was 2.99kg in AGA pregnancies (n=47) and 2.43kg (n=14) in SGA pregnancies. There was no difference in size in the UtA diameter between AGA and SGA pregnancies  in the LA cohort.Neonatal data for HA and LA sites are presented in The mean birth weight in the LA cohort was 2.89 [0.4] kg, significantly less than the HA cohort (p=0.0001) with 19 (19%) infants identified as SGA (mean 2.43 [0.22] kg). Birth length was 48.1 [1.7] cm and SGA babies were again shorter at 47.1 [1.44] vs AGA babies 48.3 [0.19] cm, p= 0.0067. Babies were born earlier than HA counterparts . No significant difference was noted between gestational age that AGA vs SGA babies born .Birthweight was greater in male vs female babies at both locations. Males at HA were heavier. than females weighing 3.21 [0.45] kg vs females at 3.1 [0.45] kg, p= 0.03. At LA, male vs female weight difference was not found to be statistically significant (p=0.19). Male/female numbers were similar at both HA and LA sites . Overall babies born at HA of Ladakhi descent were heavier  than their LA counterparts. Comparing just SGA pregnancies at HA and LA, significant differences between birth weights and length were not maintained.2 shortly after birth was lower at HA than LA (88.9 [8.32] % vs 92.5 [13.2] % respectively), p= 0.005. SaO2 did not differ between SGA and AGA babies at HA (88.7%), but initial SaO2 was higher in AGA than SGA babies born at LA  respectively, although not reaching significance.At birth there was no statistically significant difference between baby APGAR scores at 1 minute between HA 8.26 [1.17] vs LA 8.47 [1.45], p=0.11 . At HA AWe performed a comprehensive prospective study of a HA native population in relation to reproductive outcomes and compared this with a LA non-native population.Individuals gestated and born at HA had a significantly higher birth weight than expected for an Indian population. The only other published study specifically of birth weight in the Ladakhi population reported significantly lower birth weights than those recorded in this study as well as shorter birth lengths with a much higher rate of low birth weight <2500g (27% versus 7.8%) . It is dBirth weights in Ladakhi subjects were on average 3.15kg, up to a maximum birthweight of 4.4kg. Within this population those individuals who identified as Tibetan still showed the greatest adaptation with an average birth weight of 3.62 kg. These figures correspond to other adapted high-altitude populations. A retrospective review of a cohort of South American birth weights revealed an average birthweight for high altitude Andean (longest adapted) subjects of 3.15kg (n=728), compared with their European (shortest) counterparts at high altitude at 2.96kg (n=167) (https://dhsprogram.com/pubs/pdf/FR339/FR339.pdf) for term low birthweight (LBW) individuals of 16.6% (versus 7.8% in our Leh population). The proportion of babies defined as small for gestational age (according to birth weight centile) at HA (14%) was lower than at LA (19%). Birth weight centiles take into account gestational age and this finding suggests adaptation in HA cohort is not purely a result of gestational age differences.Our data also support the idea that rates of poor fetal growth (as defined by Intergrowth reference standards) were lower than expected in the Ladakh population with significantly lower rates than documented by the recent Indian National family health survey 4 2015-16 . AMPK was found to be present in utero-placental tissue and influenced vasodilation in the presence of chronic hypoxia  and PPAR γ expression differences associate with both birth weight and maternal UtA diameter, physiological mechanisms through which they may be acting warrant further investigation as they offer the potential to overcome hypoxia related growth issues.In the Ladakhi population, HA adaptation is represented through enhanced birth weight protection seen by lower than expected rates of SGA. Adaptation in pregnancy may be mediated through increased UtA diameter with resultant improved oxygenation to the placenta and fetus.


Background and Aims: Declining fertility is a key driver behind the rapid aging of populations worldwide. Finland has experienced a 25% decline in fertility from 2010 to date and ranks low even on the European and Nordic scales. This study aimed to address the association between sociodemographic indicators and birth rate  in Finland.Methods: Open data on 310 Finnish municipalities were retrieved from the public database of Statistics Finland. Several sociodemographic subdimensions , each converted to standard deviation units, were modeled against birth rate at the municipality level using generalized estimating equations.Results: In this dataset, average annual birth rate was 8.8 per 1,000 individuals. Birth rate was positively associated with change in population size , percentage of <15-year-olds , percentage of individuals living in their birth municipality , and percentage of foreign language speakers . In contrast, birth rate was negatively associated with percentage of ≥65-year-olds , percentage of unemployed individuals (0.98. 0.95−0.99), income , and number of individuals living in the same household unit .Conclusion: The present findings are expected to advance the allocation of resources to areas and subpopulations that have high or low birth rate, and thus contribute to the development of a more family-friendly society. Future studies are encouraged to evaluate the sociodemographic indicators of birth rate in other low fertility countries, and to address the individual-level mechanisms behind the municipality-level associations identified in this study. While the average 1950s woman had five children, the current global average is around 2.5 children per woman . It is eThere is regional variability in birth rate  and fertility rate  across the globe . While tThe social determinants of health  include In the context of a low fertility country, this Finnish study aimed to identify sociodemographic predictors of birth rate at the municipality level. The fundamental aims were to advance the detection of areas and subpopulations that are inclined to high or low birth rate, and thus provide tools for an efficient resource allocation and development of a more family-friendly society.The material for this study was retrieved from the StatFin registry  which isFor this study, the StatFin database was queried for sociodemographic indicators available at the municipality level. The variables of interest were retrieved between October 2020 and January 2021, when the database followed Finland's current regional division into 310 municipalities. Data of all the 310 municipalities were retrieved from 2011 to 2018, i.e., the earliest and latest years when all the variables of interest were available, respectively. While The data collections were based on Finnish citizens and foreigners legally residing in Finland for at least 12 months. The number of live births within a calendar year and total population size at the end of each year were obtained from the official Finnish population register maintained by the Digital and Population Data Services Agency (Digi- ja väestötietovirasto). Change in population size was calculated as the difference between two consequent years in percentages, with positive and negative percentages indicating increase and decrease in population, respectively. Population structure was further estimated by calculating the percentages of females, <15 and ≥65-year-olds relative to the total population of each municipality, on the basis of the population register. Here, the general birth rate was chosen as the outcome instead of fertility rate as the study aimed to identify sociodemographic predictors of birth count across Finnish municipalities, in contrast to studying the predictors of female fertility.Data on completed educational qualifications and degrees in Finland and abroad were obtained from several databases which are mainly updated by educational institutions. Individuals are classified according to highest completed education into primary, secondary  and tertiary levels . Here, low education was estimated by calculating the percentage of individuals with only primary education relative to all ≥15-year-olds.Employment and occupational statistics are constructed on the basis of several data sources such as Finnish Tax Administration , Social Insurance Institution of Finland (Kela), employment registers, and student and conscript registers. For this study, municipalities' unemployment rates were calculated as the percentage of unemployed relative to the labor force . Data on income were obtained from the registry of Finnish Tax Administration. The registry includes all individuals who receive taxable incomes within a year. Here, the median annual gross income (in euros) of each municipality was used in the analyses.2) of each municipality was calculated as total population size divided by land area. Land areas were obtained from the official records of National Land Survey of Finland (Maanmittauslaitos). Division between individuals living urban and rural was made according to urban-rural classification of Finnish Environment Institute (Suomen ympäristökeskus). The classification is geographical information system-based and allocates 250 × 250-meter map grids as urban areas  or rural areas .The location of individuals, i.e. address, was obtained from the population register. Population density  was calculated relative to all household units. The percentage of individuals whose current municipality of recidence was the same as their municipality of birth was also calculated.The percentage of foreign language speakers  was calculated using records from the official population register. Divorce rate  was based on data reported by courts of law to the population register. Car ownership rate was calculated as the total number of registered passenger cars relative to total population. Car registration data were obtained from the traffic affairs register of Finnish Transport and Communications Agency (Liikenne- ja viestintävirasto). Finally, crime rate  was constructed using data from the Ministry of Interior's police information system.The characteristics of the municipalities were first explored using descriptive statistics. Means with standard deviations or medians with interquartile ranges were presented, depending on the distribution of data. Characteristics were tabulated according to rough population size tertiles , and differences between the tertiles were analyzed using one-way analysis of variance or Kruskal–Wallis test, depending on the distribution of data.P-values were extracted from the GEE output.After considering previous literature , the assP-values < 0.05 were considered statistically significant.As a sensitivity analysis, the models were re-run after excluding the 1 and 99% percentiles of each variable and including interaction terms between time and each predictor in the models. These approaches did not essentially alter the results of the initial models. Statistical analysis was performed using SPSS Statistics version 26 . The analysis was based on 310 Finnish municipalities with population sizes ranging from 91 to 648,042. Characteristics of the municipalities are presented in Initial univariate and final multivariable GEE models for the association between sociodemographic indicators and birth rate are presented in According to the final multivariable GEE model, birth rate was positively associated with change in population size , percentage of <15-year-olds , percentage of individuals living in their birth municipality , and percentage of foreign language speakers . In contrast, birth rate was negatively associated with percentage of ≥65-year-olds , percentage of unemployed individuals (0.98. 0.95−0.99), income , and number of individuals living in the same household unit . The remaining indicators  showed no statistically significant independent association with birth rate.This nationwide study utilized open data from 310 Finnish municipalities in an attempt to reveal municipality-level sociodemographic indicators of birth rate. Indeed, the analysis identified several independent indicators of higher birth rate  as well as lower birth rate . In addition to confirming associations that were previously known, this study also revealed novel information on the set of indicators that have value in estimating birth rate in a low fertility country.Population structure and birth rate are at a constant interplay. It is clear that birth rate is one of the key factors contributing to the increase or decrease in a municipality's population size, and that municipalities with a higher proportion of fertile-aged individuals also tend to have a higher number of young families . These cA novel finding of this study was the positive association between individuals currently living in their municipality of birth and birth rate. It may be that individuals who do not need to relocate are able to settle down and consider starting a family earlier. Alternatively, the association may reflect the possibility of certain municipalities having low move-out rates due to, e.g., central location which simultaneously attracts young families to move in. A previous Austrian study  found thOf direct socioeconomic indicators, unemployment and median income had an independent association with birth rate, while low education did not. A closer look at RRs indicates, firstly, that within the pool of socioeconomic indicators, mean income exceeds both low education and unemployment rates in predictive value at the municipality level. Secondly, the findings suggest that areas of higher wealth have lower reproduction rates and vice versa. This phenomenon has been widely recognized and discussed , 12–14, Foreign language speakers were associated with higher birth rate at the municipality level. This finding is likely to be mediated by foreign background and cultural traditions in starting a family. Recent papers , 18 repoThe number of individuals in a household unit, a proxy for average family size, was negatively associated with birth rate in the multivariable models. This may be explained by residual confounding, as the percentage of <15-year-olds had a strong positive association with birth rate in the dataset. Alternatively, large family size may indicate lower desire for a new child. Correspondingly, household overcrowdedness was not associated with birth rate in the full model; its potential effect is likely to be mediated by geographical factors , socioeconomic status , and age distribution .Divorce rate had no association with birth rate in this dataset. Despite a Nordic report  which coThis study had several strengths. First, the dataset was comprised of nationwide data from a country with a generally low birth rate. The datased covered all municipalities and included data from 2011 to 2018 for maximal representativeness. There were no missing data. Second, all data were based on official statistics recorded and distributed by Statistics Finland. Thus, the data were reliable and accompanied by detailed descriptions regarding the construction of each variable. Third, the present analysis was based on a wide range of sociodemographic indicators including population structure, education and income, location and living, divorces, car ownership rate, and crime rate. The analysis was performed using GEE which accounted for the internal correlations within the data. A multivariable approach was considered highly valuable in order to reveal the independent effect of each predictor relative to the outcome. Finally, all data used in this study are public and available for further scrutiny under the CC BY 4.0 license.There were also several limitations to this study. First, only municipal-level data on birth rate and the socioeconomic parameters were available. However, similar apporaches have been used elsewhere , and theThe public health implications of this study are manifold. In a low fertility country such as Finland, the study of birth rate indicators is of high importance as they may advance the detection of areas and subpopulations that are inclined to high or low birth rate. Evidence-based data on birth rate indicators may be used to guide resource allocation in order to develop a more family-friendly society and, ultimately, influence fertility. Firstly, efforts to reduce unemployment and poverty seem advisable also from the viewpoint of fertility. Secondly, resources could be directed to education- and career-oriented individuals (proxied by high income) to better facilitate parenthood via, e.g., parental leaves, daycare services or other means of municipal support. Thirdly, it seems advisable for municipalities to attract young citizens to reside in the municipality instead of relocating. Fourthly, the present findings underline the importance of robust and well-organized maternity and child health clinics in municipalities that meet the characteristics associated with higher birth rate. In particular, municipalities should aim to ensure that all services are readily available and accessible for foreign-language speakers. Lastly, despite these remarks, the author wishes to emphasize that an individual's voluntary choice not to have children should always be respected.This Finnish nationwide study identified municipality-level sociodemographic indicators of higher birth rate  and lower birth rate . In a low fertility country, the present findings are expected to advance the allocation of resources to areas and subpopulations that have high or low birth rate, and thus contribute to the development of a more family-friendly society. Future studies are encouraged to evaluate the sociodemographic indicators of birth rate in other low fertility countries, and to address the individual-level mechanisms behind the municipality-level associations identified in this study.https://www.stat.fi/tup/tilastotietokannat/index_en.html .Publicly available datasets were analyzed in this study. This data can be found here: Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.PO is the sole author of the manuscript and worked on the conception of the project, data curation, analysis and interpretation of the data, and writing the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of this study was to investigate the prevalence of depressive symptoms and associated factors in women who underwent treatments for fear of birth; internet-based cognitive therapy, counseling with midwives, continuity with a known midwife or standard care. A secondary analysis was performed using data collected from four samples of women identified with fear of birth and receiving treatment with different methods. A questionnaire was used to collect data in mid-pregnancy and at follow-up 2 months after birth. Depressive symptoms were assessed using the Edinburgh Postnatal Depressive Scale. In mid-pregnancy, 32% of the 422 women with fear of birth also reported a co-morbidity with depressive symptoms. At postpartum follow-up, 19% reported depressive symptoms 2 months after birth, and 12% showed continued or recurrent depressive symptoms identified both during pregnancy and postpartum. A history of mental health problems was the strongest risk factor for presenting with depressive symptoms. None of the treatment options in this study was superior in reducing depressive symptoms. This study showed a significant co-morbidity and overlap between fear of birth and depressive symptoms. Screening for depressive symptoms and fear of birth during pregnancy is important to identify women at risk and offer specific treatment. Fear of birth and depressive symptoms each affect 10–20% of all pregnant women. There is, however, sparse knowledge about the impact of different treatment options for fear of birth when depressive symptoms also are prevalent during pregnancy and after birth.There is no universal definition of fear of birth and several methods are used to identify women with fear of birth during pregnancy  in a national sample of pregnant women , four medium-sized cities (projects 1 and 2), and one small town (project 3) in the middle and northern parts of the country. All women were enrolled for antenatal care in their community, following the national program for antenatal care (SFOG & SBF p < 0.001). The area under the ROC was 0.89 indicating high sensitivity with a FOBS cut-off point of 54. Sensitivity was 89%, specificity 79%, and Youden index 0.68. Positive predictive value was 85% and negative predictive value 79%. For practical reasons, after discussion with counseling midwives and obstetricians, a cutoff point of FOBS 60 or more is now used in clinical practice. For the combined present sample Cronbach’s alpha was 0.85. The mean FOBS was 73.82 (SD 17.57), range 0–100. An overview of the projects, the treatments, the grouping for the present study, and the context of care is presented in Table All participants included in the projects presented with fear of birth. The level of fear of birth was assessed using the Fear of Birth Scale (FOBS). The FOBS scale consists of two 100 mm Visual Analog Scales that are summed and averaged to get a score. When filling out the scale study, participants are asked to respond to the question “How do you feel right now about the approaching birth?” and are instructed to place a mark on the two scales which have the anchor words calm/worried and no fear/strong fear. The cut-off point of 60 or more was used to classify women with fear of birth. FOBS has been psychometrically tested and validated against the Wijma Delivery Expectancy Questionnaire (WDEQ), in a large Australian sample  and one follow-up questionnaire 2 months after birth. Women in project 1 completed all questionnaires online, whereas women in projects 2–4 received the printed questionnaire by post at their home address. The follow-up questionnaire was distributed in the same way, except women in project 3 also had the opportunity to access a web-based questionnaire if they preferred.Background data in the first questionnaire included self-reported information about age (years), parity (primiparas vs multiparas), marital status (cohabiting with partner vs single), level of education (high school or lower vs university education), country of birth (Sweden vs other countries), infertility treatment (yes/no), history of any previous mental health problems (yes/no), birth preference , and tobacco use (yes no).Depressive symptoms were assessed using the Edinburgh Postnatal Depression Scale (EDPS)  after birth.Descriptive statistics were used to present the background characteristics. Differences between women’s background characteristics and treatment for fear of birth was explored using chi-square test. The continuous EPDS scores over time and between treatment methods were analyzed using a mixed between-within subjects analysis of variance (ANOVA) test, with and without covariates included, and with calculation of mean scores and reduction in mean scores over time Pallant .p-value < 0.05 was regarded statistically significant. The odds ratios were calculated using a multi-nominal regression analysis in the statistical program SPSS.To study co-morbidity, a composite variable was created with the score 0 = no depressive symptoms during pregnancy or after birth (reference category); 1 = depressive symptoms during pregnancy but not after birth; 2 = depressive symptoms after birth but not during pregnancy; and 3 = depressive symptoms on both occasions. Differences between the groups in the composite variable, background factors, and treatment options were calculated using odds ratios with a 95% confidence interval or Fisher’s exact test. A The total sample consisted of 422 women with fear of birth. Table Figure Table p < 0.000).Almost similar proportions of multiparous and primiparous women had had counseling (49% vs 51%). Around 60% of women who received iCBT and continuity of midwifery care were primiparas, and so were 91% of the women who received standard care. These differences were statistically significant (p 0.017). There were also some differences in previous mental health problems (p 0.005). The highest proportion of mental health problems was found in women who had counseling (52%), followed by women who had had iCBT (42%), continuity (38%), and the lowest proportion in women who received standard care (24%).In the four treatment groups 50–57% had a university education. The exception was women who had standard care, where only one in three had a high level of education (p 0.010). More than one in three women in the iCBT group preferred a cesarean Section (33%) and so did a quarter of women who received counseling (25%). Among women who had continuity of midwifery care, 18% preferred a cesarean section and so did 11% of women who had standard care. There were no differences between the groups in EPDS-scores, or in the sub scores of anxiety in EPDS.There were also statistically significant differences in birth preferences (p 0.027) and were more likely to have had EPDS-scores above 13 in mid pregnancy (p 0.000). The largest drop-out rate was found in women randomized to iCBT (76%). In total, 19.1% scored 12 or more on EPDS 2 months after birth.The follow-up questionnaire was completed by 260 women , but only 256 completed EPDS after birth , with moderate effect size , but no difference between the treatment methods (p 0.424). The result remained statistically significant when adjusting for covariates , but the effect size was low and there was still no difference between the treatment methods (p 0.873).In the next step of the analysis, a mixed between-within subjects’ ANOVA was conducted to assess the impact of the four treatment options on participants’ EPDS scores across time. There was a statistically significant main effect for time, with a reduction in the mean scores on EPDS for the whole sample , and a preference for cesarean section . Depressive symptoms only after birth were associated with a low level of education . Depressive symptoms both during and after pregnancy were associated with single status , and a history of mental health problems . No other associations were found, either in background characteristics or in treatment for fear of birth.Of the 251 women who completed both questionnaires, a composite variable was created. The composite variable was built on the prevalence figures of depressive symptoms from the different time points to get a picture of the trajectories. Altogether, 169 (67.3%) women with fear of birth did not have depressive symptoms at all (reference group), 34 women (13.5%) reported depressive symptoms during pregnancy but not after birth, 18 women had EPDS scores of 12 or more after birth (7.2%); these women did not have depressive symptoms in mid-pregnancy. Finally, 30 women (12.0%) presented with depressive symptoms in pregnancy and after birth. The multi-nominal regression analysis showed that depressive symptoms The main findings of this study were that women with fear of birth presented with fairly high co-morbidity with depressive symptoms during pregnancy. A large proportion had a history of mental health problems, which was the strongest explanatory factor for the composite variable of xx.High levels of depressive symptoms were found in women with fear of birth, with more than 30% during pregnancy and in nearly 20% of women 2 months after birth. These figures are higher compared to the 10–20% of women in general childbearing populations with depressive symptoms , the mixed between-within subjects’ ANOVA showed no statistically significant differences between the treatment groups in terms of reduction in EPDS scores. Time seemed to be the factor that reduced depressive symptoms, not treatment for fear of birth. We do not know if women in these treatment groups had received any help for depressive symptoms or if women with fear of birth in the four studies that form the basis of this secondary analysis, presented with totally different reasons behind their fears. Women in the continuity group and those who received standard care comprised a large proportion of women living in a rural area with a long distance to hospital, which might be the main reason for expressing fear of birth . These figures could partly be explained by the co-morbidity with fear of childbirth and the previous suggestion that women with fear of birth carry a high degree of psychological strain (Rondung et al. What is most noteworthy is the proportion of women classified as having depressive symptoms n = 50, the standard deviations of the EPDS scores vary considerably in some comparisons.This study is compromised by its observational design when conducting a secondary analysis with a mix of women in the treatment groups. Another important issue to consider is the large drop-out rate, mainly in the iCBT group. iCBT has been recommended as a valuable treatment for depressive symptoms in general populations (Swedish National Board of Health and Welfare This study showed a significant co-morbidity between fear of birth and depressive symptoms, with previous mental health problems as the most important explanatory factor. When identified with fear of birth, an additional anamnesis or screening for depressive symptoms are suggested. Screening for depressive symptoms and fear of birth during pregnancy is important to identify women at risk and offer specific treatment. None of the treatment options for fear of birth in this study was superior in reducing depressive symptoms, but more research is needed to develop models that consider the physical, emotional, and social aspects of pregnancy and childbirth.


KNAT7 gene expression on SCW formation in transgenic poplars, we prepared poplar KNAT7 (PtKNAT7) overexpression (PtKNAT7-OE) and antisense suppression (PtKNAT7-AS) vector constructs for the generation of transgenic poplar lines via Agrobacterium-mediated transformation. Since the overexpression of homologous genes can sometimes result in co-suppression, we also overexpressed Arabidopsis KNAT7 (AtKNAT7-OE) in transgenic poplars. In all these constructs, the expression of KNAT7 transgenes was driven by developing xylem (DX)-specific promoter, DX15. Compared to wild-type (WT) controls, many SCW biosynthesis genes downstream of KNAT7 were highly expressed in poplar PtKNAT7-OE and AtKNAT7-OE lines. Yet, no significant increase in lignin content of woody biomass of these transgenic lines was observed. PtKNAT7-AS lines, however, showed reduced expression of many SCW biosynthesis genes downstream of KNAT7 accompanied by a reduction in lignin content of wood compared to WT controls. Syringyl to Guaiacyl lignin (S/G) ratios were significantly increased in all three KNAT7 knockdown and overexpression transgenic lines than WT controls. These transgenic lines were essentially indistinguishable from WT controls in terms of their growth phenotype. Saccharification efficiency of woody biomass was significantly increased in all transgenic lines than WT controls. Overall, our results demonstrated that developing xylem-specific alteration of KNAT7 expression affects the expression of SCW biosynthesis genes, impacting at least the lignification process and improving saccharification efficiency, hence providing one of the powerful tools for improving bioethanol production from woody biomass of bioenergy crops and trees.The precise role of KNAT7 transcription factors (TFs) in regulating secondary cell wall (SCW) biosynthesis in poplars has remained unknown, while our understanding of KNAT7 functions in other plants is continuously evolving. To study the impact of genetic modifications of homologous and heterologous  KNAT7 gene expression in poplars has not yet been reported, but such knowledge will assist in using transgenic woody biomass for bioethanol production in the future.Plant cell walls serve as significant sinks for the irreversible sequestering of fixed atmospheric carbon . LignoceArabidopsis, AtKNAT7 (KNOTTED ARABIDOPSIS THALIANA) is one of the KNOX (KNOTTED1-like homeodomain) TF gene family members, which is highly conserved across the angiosperms  or collapsed xylem phenomenon due to weak SCWs. Thus, AtKNAT7 appears to be differentially regulating SCW formation in different cells/tissues of the same plant. AtKNAT7 was also shown to be a functional ortholog of poplar KNAT7 (PtKNAT7) because PtKNAT7 complemented the Atknat7 mutant  experiments. The NbKNAT7 overexpression lines had thicker cell walls, suggesting that KNAT7 TF is a positive regulator of the SCW biosynthesis in tobacco. Also, the saccharification from SCW biomass of the VIGS NbKNAT7 lines was 40% higher than the controls. It was recently reported that KNAT7 also positively controls the xylan biosynthesis pathway by binding to the promoter of the IRX9 gene, and expression studies showed that the transcript levels of IRX9, IRX10, and FRA8 genes were lower in the Atknat7 mutant  and suppression (antisense) vector constructs were designated as PtKNAT7-OE and PtKNAT7-AS. The full-length KNAT7 cDNA from poplar stems was amplified using gene-specific primers . The PtKNAT7 insert was flipped over in the opposite direction for the antisense construct from the sense constructs. For the second part of this study, we overexpressed AtKNAT7 in poplars using the same DX15 promoter and designated this construct as AtKNAT7-OE. For this vector construct, AtKNAT7 cDNA was amplified using gene-specific primers and ligated into XbaI/SacI digested DX15pBI101 vector to replace the GUS gene. All the three constructs, PtKNAT7-OE, PtKNAT7-AS, and AtKNAT7-OE, were used for the transformation of hybrid poplar  explants using Agrobacterium C58 strain by leaf disc infiltration method (The mers see  with intn method .KNAT7 and Arabidopsis KNAT7 using Integrated DNA Technologies (IDT) software. The total reaction was 12 μl containing 6 μl of SYBR green, 1 μl of each primer (1 μM), 1 μl of cDNA template, and 3 μl of RNase-free water. The reaction for each gene was performed in triplicates with thermocycler conditions as follows: 95°C for 10 min followed by 45 cycles for 95°C for 30 s, and 60°C for 30 s. Relative gene expression was calculated by the ΔCT method. Actin7 was used as an internal control  scraped from 20 to 50 aerial internodes of hybrid poplar plants using the TRIZOL  method as described earlier . Extract control .For histology, sections were cut at the seventh internode from the apex of poplar plants (with about 50 internodes above the soil) and preserved in ice-cold FAA . The internodes were infiltrated under a vacuum for about 10–15 min and kept overnight at 4°C. Dehydration and embedding in wax were done for fixing the sections on slides. Dewaxing was performed using two washes of xylene and decreasing concentrations of ethanol followed by washing with water. Autofluorescence was observed from dewaxed sections using fluorescent light imaging.The growth characteristics of the greenhouse-grown plants were measured every week. All the independent lines for each gene construct were grown under identical conditions in the greenhouse as described by After scraping the developing xylem from transgenic plants, the wood was dried at room temperature for 2 weeks and then milled through a 20-gage mesh filter. One gram of wood powder was stored in falcon tubes, destarched with amylase and ethanol extracted (described in the following section), and 4 mg of destarched-extracted material was used for lignin analysis. Three samples from each gene construct were sent to National Renewable Energy Laboratory (NREL) for analysis by Pyrolysis-Molecular Mass Beam Spectrometry (Py-MBMS) to estimate lignin content and for S/G ratio measurement . DuplicaPretreatment and enzymatic hydrolysis of biomass was carried out as described previously with some modifications , 2018. BKNAT7 gene in wild-type control (WT) and transgenic KNAT7 poplars were quantified using qRT-PCR. The PtKNAT7 gene transcripts were significantly increased in PtKNAT7-OE lines compared to the WT plants  genes involved in the SCW formation (irx phenomenon) suggesting a positive regulation of SCW biosynthesis by KNAT7 TF. Overexpression of the KNAT7 gene did not change the SCW thickness of vessels and fibers in transgenics compared to wild-type control plants (NbKNAT7 in tobacco resulted in thickening of the SCW in the xylem while suppression of NbKNAT7 in tobacco VIGS and RNAi plants resulted in thinner xylem SCWs coupled with increased xylem area. Furthermore, the positive regulation of xylan biosynthesis by KNAT7 TF has been recently reported in Arabidopsis inflorescence stems (KNAT7 transgenes in this work. In the present study, we overexpressed poplar and Arabidopsis KNAT7 genes in transgenic poplars under the regulation of DX15 promoters and our observations suggest that KNAT7 acts like a positive regulator in poplars. It is also possible that there are species-specific differences in KNAT7 functions in SCW biosynthesis. Overall, the xylem area in PtKNAT7-AS lines increased by 26% while the xylem area was decreased in PtKNAT7-OE lines by 15% as compared to WT stems confirming the earlier observations in NbKNAT7 transgenic lines of tobacco (PtKNAT7 transgenic lines. It has not escaped our attention that AtKNAT7-OE transgenics showed xylem area phenotype similar to PtKNAT7-AS and not like PtKNAT7-OE. We are still far away from understanding the impact of such manipulations on plant phenotypes and it appears that at least, in this case, specific-specific variations in the KNAT7 gene source might be responsible for this phenotype. But further investigations are warranted into this phenomenon in the future.Investigations into the role of KNAT7, one of the members of the plant KNOX II TF family, began with the ormation . To undel plants . Similarce stems  and seedce stems . The usece stems . We have tobacco , where tAtknat7 mutants had thicker interfascicular fibers but curiously also had irx phenotype in the vessels. However, these studies could not explain the irx phenotype in vessels when interfascicular fiber cells displayed SCW thickening at the same time. Recently, On the contrary, PtKNAT7 and AtKNAT7 genes did not significantly increase the total lignin content although some monolignol biosynthesis genes, as well as some other representative genes involved in SCW formation, were upregulated. This observation is similar to AtKNAT7 gene was overexpressed. However, antisense PtKNAT7 suppressed lines in poplars had lower lignin content and suppressed expression of SCW genes. This observation is again different than the earlier findings by knat7 mutants. We also observed that S/G lignin ratios were significantly increased in both PtKNAT7-OE, AtKNAT7-OE, and PtKNAT7-AS lines. Considering the potential importance of KNAT7 TF in syringyl lignin biosynthesis (KNAT7 may increase S lignin synthesis but it is puzzling why antisense suppression of KNAT7 also increased S lignin content. It is possible that all the components of the TF network that interact with KNAT7 are not yet completely identified and there might be some complex regulation of S lignin synthesis or suppression of G lignin production that might be occurring in such transgenic lines.We observed that overexpression of the ynthesis , it is eKNAT7 poplar lines have been examined for their lignin quantities and content as well as saccharification efficiencies. We have observed increased saccharification efficiencies in all the transgenic lines of poplar altered in KNAT7 gene expression compared to WT controls. The decrease in lignin content in PtKNAT7-AS lines and an increase in S/G ratios in all transgenic lines are positively associated with increased saccharification efficiencies. Similar to present findings, NbKNAT7 VIGS lines had increased saccharification efficiency than control plants.Recently, KNAT7 transgenic poplar lines show almost similar or slightly better growth phenotypes compared to WT control plants. These observations are similar to an earlier report in tobacco KNAT7 transgenic plants where normal-looking transgenic KNAT7 plants were produced (KNAT7 transgenic poplar lines from a bioenergy production perspective. In conclusion, the genetic alteration of KNAT7 could be one of the powerful strategies for increasing saccharification efficiency in transgenic bioenergy plants and has a great potential for improving bioethanol production.While the focus of this study is on the biotechnological application of genetic manipulation of KNAT7, it is a common observation that in transgenic plants manipulated in lignin biosynthesis genes show abnormal growth phenotypes e.g., . We obseproduced . An incrThe original contributions presented in the study are included in the article/KNAT7 experiments in poplars. AB performed saccharification assays. AH-W and CD did lignin analysis. All authors contributed to manuscript preparation.YA and CJ planned the experiments and interpreted the results. AN initiated the The views expressed in the article do not necessarily represent the views of the DOE or the United States Government. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work or allow others to do so, for the United States Government purposes.AN is currently employed by Kaveri Seeds. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


IntroductionAutism spectrum disorder (ASD) is a set of neurodevelopmental disorders characterized by deficits in social behaviors and nonverbal interactions. The disorder is believed to be multifactorial regarding etiopathology. This study aimed to investigate the possible risk factors associated with the development of autism in the prenatal and postnatal periods.MethodsWe conducted an unmatched case-control study composed of 56 autistic cases and 85 control children in North Cyprus. Cases recruited were previously diagnosed by a pediatric psychiatrist as being on the autistic spectrum. Parental questionnaires were distributed, and the collected data were analysed using Statistical Package for the Social Sciences (SPSS) version 24 . Binary logistic regression was used to compute the adjusted odds ratios (aOR), adjusted for possible confounders.ResultsOur results showed increased odds of developing ASD in mothers with mental disorders such as depression and anxiety , mothers with medical conditions such as Hashimoto’s thyroiditis , mothers using aluminum-containing anti-acids , mothers exposed to loud noises during pregnancy , mothers with ≥ two previous miscarriages , neonates with birth weight <2500 grams , male gender neonates , and neonates exposed to MRI or CT scan during the first year of life . Decreased odds of ASD development were observed in mothers using multivitamins during pregnancy , mothers consuming slight amounts of baking powder during pregnancy , mothers with threatened abortion , and neonates taking iron supplementation during the first six months of life .ConclusionThere were various maternal and neonatal factors associated with ASD development in North Cyprus. Although there is some evidence to suggest that exposure to specific factors during prenatal or postnatal periods may increase the risk of ASD, there is insufficient evidence that implicates a specific factor for autism etiology. Future studies are recommended to be performed on larger scales to support further the factors associated with ASD development. The world health organization estimates that the prevalence of autism spectrum disorder (ASD) is one per 160 children worldwide . ASD encDespite being a common condition with a lifelong course, ASD showed no apparent connection with a single specific risk factor . InsteadMany researchers in the literature tried to discover the relationship between ASD and possible risk factors, together with potential protective factors. One meta-analysis study linked ASD development with abnormal fetal presentations, umbilical cord complications, fetal distresses, birth injury or trauma, multiple births, maternal hemorrhage, summer birth, low birth weight, small for gestational age, and congenital malformations . AnotherOur study investigated the relationship between autism and prenatal, perinatal, and postnatal conditions to suggest possible risk factors and protective factors for ASD. We conducted an unmatched case-control study among children with and without autism in North Cyprus. Because there is no autism registry in North Cyprus, we obtained cases from schools of children with developmental disabilities.Study sampleWe conducted a case-control study in the Turkish Republic of Northern Cyprus in 2017 among children under the age of 18. According to the Statistical Yearbook of Northern Cyprus published in February 2016, the total number of children in North Cyprus under 18 years was 73,500, with an average of 4000 new births per year . ApproxiProcedureWe obtained ethical approval from the Ethics Committee of Eastern Mediterranean University. In addition, informed consent was obtained from the parents of autistic children by visiting eight special education centers in North Cyprus and calling the parents of autistic children registered in those centers. We then distributed hard-copy questionnaires to be filled by the mothers of autistic children and collected the questionnaires one month later. We also obtained informed consent from the mothers of controlled children by reaching out to two public schools in North Cyprus and distributing the questionnaires to the heads of those public schools. The questionnaire was distributed in the Turkish language and was developed based on a thorough literature review of similar studies. The questionnaire asked yes-no questions about the possible exposure to each variable during the pregnancy period.VariablesThe demographic variables in our study include gender, child’s age, mother’s and father’s ages at the birth of their child, child’s order, birth weight, birth month, mother’s country of birth, living in an old house, mother having medical conditions, and previous miscarriages. The other part of the survey focused on the possible risk factors or protective factors associated with the development of autism during the prenatal, perinatal, and postnatal periods. The odds ratio was adjusted for potential confounders, including mother’s age, father’s age, child’s gender, birth weight, previous miscarriages, mother’s medical conditions, and pregnancy-induced medical conditions. These confounders were chosen based on data from previous literature studies.Statistical analysisData analysis was done through the Statistical Package for the Social Sciences (SPSS) version 24 software  and statistical significance was set at a two-sided P-value of <0.05. Descriptive statistics were computed on all variables as the primary method of data evaluation. We compared the distribution of demographics and general characteristics between autistic and non-autistic children by performing Pearson’s chi-square test, as well as an independent sample t-test for quantitative variables including child’s age, mother’s age, father’s age, and order of the child. We also used the binary logistic regression analysis with a 95% Confidence Interval (CI) to calculate the unadjusted odds ratios (OR) and the adjusted odds ratios (aOR) for potential confounders. The statistical analysis on SPSS was done using the number of responses received for each variable, and the missing responses were not included in our statistical calculations.Demographical characteristicsThe total sample size included in our study comprised 141 subjects, 56 cases and 85 controls. The potentially eligible number of autistic cases in North Cyprus was estimated to be 100; out of these cases, we managed to collect responses from the mothers of 56 autistic children, which was the number of cases suggested by “openepi website”. We also distributed 100 questionnaires for the control group, and we managed to collect responses from the mothers of 85 controlled children. Reasons for non-participation in this study included parental preferences, as some parents did not want to feel guilty for potentially causing the disease to their children. The means of ages in cases and controls were 9.6±5.3 and 6.3±2.8, respectively (P<0.0001). In the cases recruited, 78.6% were males, and 21.4% were females, while in the controls recruited, 56.5% were males, and 43.5% were females (P=0.007). These findings reveal the unmatching of ages and genders between the cases and controls in our study. The mean of mothers’ ages at birth was also significantly different between cases (27.8±4.9) and controls (30.9±4.4) with P<0.0001. However, the mean of fathers’ ages showed no significant difference between both groups (P=0.213). The mean order of a child was slightly higher in the cases (1.5±0.7) compared to controls (1.3±0.5) with P<0.049. The birth weight and birth month showed no differences between both groups (P >0.05). Table Continuous variables that were categorized in our study included birth weight, which was categorized into <2500g and >2500g; mother’s and father’s ages at delivery, which were categorized into <30 years and >30 years of age; and birth month, which was categorized into winter months  and non-winter months. Potential confounders were chosen based on data from previous literature studies. Adjusted confounders in our study included mother’s age, father’s age, child’s gender, birth weight, previous miscarriages, mother’s medical conditions, and pregnancy-induced medical conditions.Maternal factors during pregnancySeveral maternal factors have shown statistically significant results concerning the development of autism. Among these maternal factors, increased risk of autism was seen in mothers with mental disorders during pregnancy, such as anxiety and depression , mothers with medical conditions during pregnancy, such as Hashimoto’s thyroiditis and anemia , mothers using aluminum-containing antacids during pregnancy , and mothers exposed to prolonged periods of noise pollution, i.e., loud noises during pregnancy . Decreased risk of autism was seen in mothers taking multivitamins during pregnancy , mothers using baking powder twice a month while cooking , and mothers with threatened abortion . The following maternal factors showed no statistical significance in autism development: maternal smoking during pregnancy, maternal second-hand smoke exposure, pregnancy-induced conditions, such as gestational diabetes and hypertension, maternal vitamin D deficiency, hyperemesis, insecticide exposure, hair dying, consuming seafood and caffeine, heavy metal exposure, such as mercury, maternal vaccination, microwave usage, owning a pet, and medication use during pregnancy (P ≥ 0.05). Table Neonatal factors Several neonatal factors during and after delivery have shown statistically significant results related to the development of autism. Among these neonatal factors, an increased risk of autism was seen in neonates whose birth weight was less than 2500g , male neonates , and neonates exposed to CT or MRI during the first year of life . Neonates taking iron supplements during the first six months of life demonstrated a decreased risk of autism . The following factors showed no statistical significance in autism development: delivery during winter months, difficulties during delivery, such as oxygen deprivation, neonatal jaundice, taking vitamin D and anti-reflux medications during the first six months of life, and having lactose intolerance or colic pain during the first six months of life (P ≥ 0.05). Table Parental factors Some parental factors were related to the development of autism in their offspring. An increased risk of autism was seen in mothers with ≥ two previous miscarriages . Fathers’ ages and mothers giving birth in a country other than North Cyprus revealed no statistically significant results (P ≥ 0.05). Table Our case-control study was able to establish various neonatal and maternal risk factors for the development of ASD. When considering maternal factors, compared to their control counterparts, significant risk factors for autism were observed in mothers with mental disorders, mothers with medical conditions, mothers using aluminum-containing antacids, and mothers exposed to loud noises while pregnant. On the other hand, protective factors were multivitamins usage during pregnancy, small amounts of baking powder usage during pregnancy, and mothers with threatened abortion. Neonatal factors implicated in the development of ASD were male gender, birth weight below 2500g, and exposure to MRI or CT in the first year of life.Several meta-analyses and review articles were found in the literature combining multiple case-control studies related to ASD development. Studies that showed similar findings to our results include a meta-analysis done by Chen et al. 2016), which revealed that maternal autoimmune disease is a significant risk factor for ASD development with a pooled OR of 1.34 (95% CI 1.23 - 1.46) . Materna, which rWang et al. 2017) revealed that cigarette smoking was a non-significant factor  revealed. These eThe meta-analysis done by Wu et al. 2016) focused solely on finding a connection between ASD and parental age. The findings differed from our study, as they showed a significant risk of developing ASD with increased parental ages . A 10-ye16 focuseLimitationsThe differences in the results found in our study compared to literature might be attributed to the smaller sample size used, as opposed to the wider sample sizes used in the meta-analyses. Additionally, most of the meta-analyses included multiple pieces of research from different geographical areas, allowing the inclusion of various factors related to the studied population. A major limitation of our study is its retrospective design, predisposing it to recall bias. Prenatal, obstetric, and postnatal characteristics were evaluated based on surveys answered by mothers depending on their ability to remember past events. Furthermore, matching was not accomplished in our study due to the small sample size of the Northern Cyprus population, making it difficult to find many autistic children of similar ages. The ages of autistic children included in our study were increased to include children up to 18 years of age, aiming to reach larger numbers of autistic children. One difficulty that may also arise when exploring this topic is that many of these risk factors are connected, causing a possible confounder effect; mothers with medical conditions usually give birth to underweight neonates, pinpointing which risk factor is the most significant remains a challenge.Autism is one of the leading mental causes of disability in children worldwide. Looking at potential risk factors might give us insight into the possible preventative measures. Our study among children in Northern Cyprus showed evidence suggesting several maternal and neonatal factors associated with autism. The results showed increased odds of developing ASD in mothers with mental disorders such as depression and anxiety, mothers with medical conditions such as Hashimoto’s thyroiditis, mothers using aluminum-containing anti-acids, mothers exposed to loud noises during pregnancy, mothers with ≥ two previous miscarriages, neonates with birth weight <2500 grams, male gender neonates, and neonates exposed to MRI or CT during their first year of life. The factors that demonstrated decreased odds of ASD development were observed in mothers using multivitamins during pregnancy, mothers consuming slight amounts of baking powder during pregnancy, mothers with threatened abortion, and neonates taking iron supplementation during the first six months of life. Although there is some evidence to suggest that exposure to specific factors during the prenatal or postnatal periods may increase or decrease ASD risk, there is insufficient evidence that implicates a specific aetiological factor for autism. It is recommended that future studies be performed on a larger scale to support further the factors associated with ASD development.