It is known that the rate of caesarean section (C-section) has been increasing among preterm births. However, the relationship between C-section and long-term neurological outcomes is unclear. In this study, we utilized diffusion tensor imaging (DTI) to characterize the association of delivery method with brain white matter (WM) microstructural integrity in preterm infants. We retrospectively analyzed the DTI scans and health records of preterm infants without neuroimaging abnormality on pre-discharge term-equivalent MRI. We applied both voxel-wise and tract-based analyses to evaluate the association between delivery method and DTI metrics across WM tracts while controlling for numerous covariates. We included 68 preterm infants in this study . Voxel-wise and tract-based analyses revealed significantly lower fractional anisotropy values and significantly higher diffusivity values across major WM tracts in preterm infants delivered via C-section when compared to those delivered vaginally. These results may be partially, but not entirely, mediated by lower birth weight among infants delivered by C-section. Nevertheless, these infants may be at risk for delayed neurodevelopment and could benefit from close neurological follow up for early intervention and mitigation of adverse long-term outcomes. Preterm birth (< 37 weeks gestation) accounts for more than 10% of live births worldwide., 3 TheseMultiple studies have found significant associations of C-section delivery with adverse cognitive outcomes, emotional problems, sleep problems, and increased risk of developing autism spectrum disorder and attention deficit hyperactivity disorder later in life.-15 Some Although conventional brain MRIs can identify drastic structural and pathologic abnormalities, advanced MR techniques such as diffusion tensor imaging (DTI) can further characterize brain development by evaluating WM microstructural integrity. Prior studies have utilized DTI to characterize WM microstructure in infants across a range of gestational ages, identifying specific WM regions that are microstructurally delayed in infants of lower gestational age., 24 We hIn this study, we compared the early WM microstructural integrity of radiologically normal singleton preterm infants born via C-section versus vaginal delivery after correction for gestational age at birth and time of scan, weight z-scores at birth, and other pertinent clinical variables. Our results elucidate neurobiological correlates of delivery method and explain the possible microstructural underpinnings of prior studies reporting adverse neurological outcomes in association with C-section delivery.We reviewed the imaging and electronic health records of all infants who had brain MRI performed within 3 months of birth between January 2013 and March 2021 at our center. Data access and retrieval were performed on March 31, 2021. The authors had access to information that could identify individual participants during and after data collection. Preterm infants at our institution routinely undergo brain MRI at term equivalent age or prior to discharge. This predischarge MRI scan includes DTI series at the end, if tolerated by the infant. We included infants who (1) were born between 24- and 37-weeks gestational age at birth, (2) had no structural or signal abnormalities on conventional brain MRI series, (3) had DTI performed as part of their MRI study, and (4) had sufficient information in the health record regarding: delivery methods, indication of early delivery, gestational age at birth and time of scan, birth weight, and 1- and 5-minute APGAR scores. All birth weights were converted to Z-scores using the 2013 Fenton growth chart for preterm infants. In additAs prior studies have identified low birth weight as a risk factor for delayed neurodevelopment and adverse neurological outcomes, a sub-cohort excluding infants born small for gestational age  was identified to conduct additional analyses., 28 All 2 acquisitions. Motion artifact for neonatal MRI is reduced at our institution preferentially using the “feed-and-swaddle” technique with anesthetic sedation being used as a second line option. atlas. For everThe data are presented as mean ± standard deviation, median (interquartile range) or frequency (percentage) as appropriate. The “stats” package in R was used to determine if significant differences existed with regards to clinical variables between infants delivered vaginally and via C-section. A two-sample t-test was used for continuous variables, a Wilcoxon rank-sum test was used for APGAR scores, and a chi-squared test was used for binary variables. R was also used to conduct tract-specific linear regression analyses.Despite differing significantly between the two experimental groups, neither birth weight Z-score nor presence of maternal preeclampsia were significantly associated with any DTI metrics on voxel-wise GLM analysis or tract-specific regression after adjusting for delivery mode and other covariates listed above. Use of general anesthesia during delivery was also not significantly associated with any DTI metrics on voxel-wise GLM analysis or tract-specific regression after adjusting for delivery mode and additional covariates.Nine infants born SGA  were excluded from the sub-cohort. Supplemental Table 1 summarizes the clinical demographics of the infants included in the sub-cohort. Of note, birth weight Z-score differed significantly between infants delivered vaginally and via C-section in the sub-cohort. The excluded infant delivered vaginally required preterm delivery due to preterm labor. Indications for preterm C-section delivery among the excluded infants included severe or worsening preeclampsia or HELLP syndrome (n = 5), fetal heart rate abnormalities (n = 2), and breech presentation with failed external version (n = 1). Of the excluded C-section deliveries, one required the use of general anesthesia.Supplemental Fig. 1 depicts the results of the voxel-wise GLM analyses for the association of delivery method with FA and RD values in the sub-cohort after controlling for gestational age at birth, gestational age at scan, 5-minute APGAR, birth weight Z-score, presence of preeclampsia, and presence of chorioamnionitis as covariates. These results were further confirmed by tract-specific analyses . FA values in the body of the corpus callosum, corona radiata, and right tapetum were found to be significantly lower in infants delivered via C-section compared to those delivered vaginally . RD values in the pontine crossing tract, corpus callosum, cerebellar peduncle, right posterior corona radiata, fornix, and stria terminalis were significantly higher in infants delivered via C-section than those delivered vaginally . GLM analysis did not reveal any significant association between MD values and delivery method but MD values in pontine crossing tract, cerebellar peduncle, left retrolenticular internal capsule, right posterior corona radiata, fornix, stria terminalis, and right superior longitudinal fasciculus were found to be significantly higher in infants delivered via C-section than those delivered vaginally on tract-specific analysis . There was no significant association of delivery method with AD values on voxel-wise or tract-specific analyses. Birth weight z-score, maternal preeclampsia, and use of general anesthesia during delivery were not significantly associated with any DTI metrics in the sub-cohort after adjusting for delivery method and additional covariates.In both voxel-wise GLM and tract-based linear regression analyses, preterm infants requiring C-section delivery had reduced WM microstructural integrity compared to those delivered vaginally, most prominently in the corpus callosum, internal capsule, and corona radiata. Notably, the difference between delivery mode of preterm infants was found after adjusting for gestational age at birth, gestational age at scan, 5-minute APGAR, birth weight Z-score, presence of preeclampsia, and presence of chorioamnionitis as covariates – and after excluding infants from multiple gestations and those with any maternal history of drug use. Although birthweight Z-score and presence of maternal preeclampsia differed significantly between infants delivered vaginally and via C-section, additional voxel-wise GLM analyses found no significant association between these risk factors and DTI metrics after controlling for delivery method and other clinical variables. In the sub-cohort excluding SGA infants, areas of significant association between delivery method and DTI metrics were limited compared to the primary cohort but still included pertinent WM tracts such as the corpus callosum, internal capsule, and corona radiata.Our results provide the neurobiological indications and early WM microstructural correlates for clinical studies reporting that preterm infants who require C-section are at risk for adverse neurodevelopmental outcome. The difference in early WM microstructural development may be a consequence of multiple factors necessitating C-section delivery; nevertheless, our findings highlight the higher risks of neurodevelopmental delay among preterm infants delivered via C-section and the need for close follow-up and potential intervention to mitigate these risks in this vulnerable cohort.The myelination of WM tracts as infants age reduces the random diffusion of water molecules and results in alignment of diffusion along WM tracts. Thus, changes in DTI metrics such as increased FA and reduced MD and RD are corollaries for WM maturation. DTI metrThe relationship between delivery method and infant brain development has not been extensively studied. Deoni et. al have compared the DTI metrics between term infants delivered via C-section (n = 11) versus vaginally n = 32). Similar . SimilarAs we demonstrate the relationship between delivery method and WM maturation on neuroimaging, the question of how delivery method may influence early brain development arises. It has been suggested that vaginal delivery is an important contributor to the normal development of infants’ gut microbiome and that alterations in this process (due to C-section) can disturb normal brain development., 41 The Exclusion of infants born SGA in the sub-cohort analyses limited the regions of apparent significant association between delivery method and DTI metrics when compared to primary cohort analyses. This suggests that the results of our primary analyses could be partially mediated by the lower birth weight and birth weight z-score among neonates born by C-section versus vaginally in the primary cohort. Indeed, prior reports have described the association between birth weight and DTI metrics as well as long-term neurological outcomes.-47 HowevOur study is limited by its retrospective, single center design and small sample size. Our small sample size also limits the power of our study in including multiple covariates, which may complicate the interpretations of our analyses on the independent associations of birthweight z-score, maternal preeclampsia, and general anesthesia use during delivery with DTI metrics. The small sample size especially limited the sub-cohort analysis after exclusion of 9 SGA preterm infants. DTI metric analysis can be influenced by acquisition protocol and timing, as well as processing methods used to generate DTI metric maps. Although we controlled for several demographic and clinical variables, including those that differed significantly between experimental groups, we could not account for all possible covariates that may influence delivery method and explain observed differences in DTI metrics between the experimental groups in this study. Of note, recent work has suggested that intrauterine inflammation impacts fetal and infant neurodevelopment and although we controlled for the presence of chorioamnionitis as a covariate, we did not collect placental pathology data to assess for other causes of intrauterine inflammation., 49 We aPreterm infants requiring C-section section delivery, compared to those delivered vaginally, have neuroimaging markers of delayed WM maturation on predischarge MRI scans, most prominently in the corpus callosum, internal capsule, and corona radiata independent of gestational age at birth, gestational age at scan, 5-minute APGAR, birth weight Z-score, maternal preeclampsia, and chorioamnionitis. Possible confounders of birth weight z-score, rates of maternal preeclampsia, and use of general anesthesia during C-section delivery are not independently associated with WM maturation in this cohort. Lastly, our primary results may be partially, but not entirely, mediated by lower birth weights among infants delivered by C-section versus vaginally. These findings suggest that preterm infants requiring C-section are at risk for delays in WM maturation and thus may require close neurologic follow up to identify and mitigate adverse long-term neurological outcomes.


Although BW correlation with CdB was not statistically significant, hierarchical linear regression  suggested a fetotoxic effect. These results confirmed the threat PTMs may represent and the need for more extensive research to elucidate their role in inadequate FG in developing countries.Prenatal exposure to potentially toxic metals (PTM) may impair fetal growth (FG). We investigated the relationship between maternal exposure to lead (Pb), cadmium (Cd) and manganese (Mn) and birth weight (BW) of 74 newborns. Blood was collected during the second trimester of pregnancy to determine Pb (PbB) and Cd (CdB), while hair (MnH) and toenails (MnTn) were used for Mn. Samples were analyzed by graphite furnace atomic absorption spectrophotometry (GFAAS). Sociodemographic and BW data were collected from questionnaires and maternity records, respectively. The medians (P25th–P75th) of PbB, CdB, MnH, and MnTn were, respectively, 0.9 (0.5–1.8) µg/dL; 0.54 (0.1–0.8) µg/L; 0.18 (0.1–0.4) µg/g; and 0.65 (0.37–1.22) µg/g. The means (standard deviation) of birth weight according to sex were 3067 (426.3) and 3442 (431) grams, respectively, for girls and boys. MnTn presented an inverse correlation with the BW/gestational age ratio for girls (rho = −0.478;  Low birth weight (LBW), defined as birth weight less than 2500 g , represeAs a preventable event, LBW highlights a severe issue of health inequity, and it is, therefore, urgent to investigate possible approaches to improving this situation . LBW hasHumans are commonly exposed to different types of pollutants simultaneously. There is growing concern about the consequences of multiple exposures to PTMs. Indeed, some studies have already demonstrated the possibility of combined effects of PTMs as a mechanism of toxicity ,22,23,24Given the above, this study aimed to (i) investigate the association between exposure to these metals and children’s birth weight; and (ii) investigate the possibility of a combined influence of these metals on birth weight, taking into account the social context of the study population.This study population derived from the project “Socioenvironmental Determinants of Child Neurodevelopment” (DSAN-12M), which examined a prospective birth cohort in the municipalities of Nazaré das Farinhas and Aratuípe in the Recôncavo Baiano region of Brazil. More detailed information is presented elsewhere . The recruitment was based on the network of primary care units (PCU) of the National Health System (SUS is its acronym in Portuguese), including 11 units in Nazaré and 4 units in Aratuípe. During prenatal consultations, the pregnant women were invited to receive the research team at their homes for explanations about the project goals and methods. If they accepted, adult participants or legal guardians (of pregnant adolescents) signed the consent form, while minors were asked to sign an assent form. The enrollment of 164 participants was carried out in two phases due to the COVID-19 pandemic: from July 2019 to March 2020, and from July 2021 to September 2022. This prospective study considered pregnant women’s exposure from insertion until delivery and the outcome of the newborn’s weight. The maternity wards of Hospital Luís Argolo  and the Santa Casa da Misericórdia (in Nazaré das Farinhas) were included in the survey to collect information from medical records after delivery. For the inclusion criteria, we selected pregnant women with a gestational age (GA) of fewer than 24 weeks who started their prenatal consultation at one of the PCUs of the municipalities and had lived in the region for at least one year before the pregnancy. Women with twin pregnancies, who had prescriptions for medications potentially neurotoxic for the fetus or pregnancy complications (or were classified as high-risk pregnancies) and women who had undergone difficult childbirths were excluded from the study. Ethical issues: This project was approved by the research ethics committee of the Faculty of Pharmacy-UFBA through Resolution 466-CNS/2012, with approval No. 3246555.Sociodemographic DataTrained interviewers administered semi-structured questionnaires to participants to collect data on daily habits, education level, occupation, and probable sources of PTM exposure. Socioeconomic status (SES) was stratified into five categories from A to E, based on the criteria of the Brazilian Association of Population Studies . ConsideFG was evaluated based on anthropometric data and GA collected from the medical records of the two maternity hospitals where the deliveries occurred. The data collected were: birth weight (BW), GA, length, and head circumference. When available, GA was primarily based on a first- or second-trimester ultrasound examination (best obstetric estimate). When ultrasound was unavailable, the estimate was based on the last menstrual period, provided by the pregnant woman. Low birth weight (LBW) if weight < 2500 g; normal weight if weight ≥ 2500 g, andPremature delivery if gestational age < 37 weeks; delivery at term if gestational age ≥ 37 weeks.For the analyses, we mainly considered birth weight as the outcome variable. The other variables helped to assess the condition of the newborn. BW and GA were dichotomized as follows ,28: Low Information on the number of prenatal consultations and the baby’ sex was also collected from the medical records. The number of prenatal consultations was dichotomized into “adequate prenatal care” (≥6 visits) and “inadequate prenatal care” (<6 visits) according to the recommendations of the Ministry of Health . RegardiThe intrauterine exposure of newborns was estimated based on the assessment of maternal biomarkers of exposure, that is, the concentrations of the PTMs in pregnant women’s biological matrices , in addition to environmental indicators (domestic settled dust). 2/30 days. Sample collection and processing details are described in Bah et al. . For bioConsidering the available data and variables, we established the conceptual model for this analysis based on the DSAN-12M project model presented by Bah et al. . The framework took a holistic approach to contaminant toxicity study in environmental science based on PTMs. Taking a multilevel perspective, the participants’ situation was divided into the four contexts of the bioecological model of Bronfenbrenner  (1979): We considered the dyad’s sociodemographic variables and exposure biomarkers in the microsystem. Arrows B, C and D represent how variables from other contexts influence pregnant women and the outcome in the microsystem.This conceptual model was used as a criterion, followed by statistical aspects, to analyze the data, especially in multivariate regression. To support this, we used the statistical analysis process suggested by Leal et al.  and VictConsidering that the inclusion criterion was filling in the birth weight availability from hospital records, we compared the sociodemographic characteristics of the pregnant women included or not in the study population in this preliminary presentation.A descriptive analysis of the children’s sociodemographic data, exposure, and outcome variables was presented. The Chi-squared test helped to assess possible differences. The distributions of continuous variables were evaluated using the Kolmogorov–Smirnov (KS) or Shapiro–Wilk (SW) test, and the central measures of tendency were presented as mean ± standard deviation and median (P25th–P75th). Student’s t-test was used to compare the means of birth weight according to sociodemographic variables and degrees of exposure to the PTMs.Raw, orTransformed into another variable: weight (in grams)/GA (weeks) ratio, to control for the influence of GA on birth weight.Using Spearman’s correlation coefficient, possible relations were estimated between birth weight, other anthropometric parameters, and metal exposure biomarkers. Birth weight was used in the following ways to analyze its relationship with exposure biomarkers:p ≤ 0.05), the variables were included in the model.The exposure biomarker distributions, being non-parametric, were log10-transformed. Hierarchical linear regression analysis (HLR) was used to assess the association between biomarkers and FG variables (birth weight and weight/GA ratio). Following the conceptual model  or basedp < 0.05 was applied.The SPSS software version 23 for Windows was used in the statistical analysis, and a significance level of From 164 pregnant women followed up until delivery, data on 74 births (44.5%) were collected based on completion of the birth weight data in the medical records and the baby health booklet. Depending on the variables collected in the questionnaire or medical records and the availability of biological samples, we had additional data losses.Almost all pregnant women included in this analysis (93.1%) declared themselves as black or mixed race, with a median age of 27 ± 6.1 years and a pre-pregnancy BMI of 25.1 ± 3.8 kg/m². Nearly half (47.9%) were from low SES and lower D/E, and over two-thirds (69.2%) received government assistance. The majority (73.8%) reported being able to count on family and friends to care for their children. Almost 30% reported being passively exposed to cigarette smoke .Regarding FG parameters, only birth weight and gestational age showed a normal distribution. Considering the birth weight and GA references, few births presented a worrying situation; only 4.2 (7.7%) of the NBs were classified as LBW or were born preterm. Means ± SD for GA at delivery and birth weight were 39 ± 1.7 weeks and 3275 ± 463 g, while medians (P25th–P75th) for length and head circumference of newborns were 48 (47–50) and 34 (33–35) cm, respectively. p = 0.029), being higher in SES with lower D/E.Regarding exposure biomarkers, all exposure markers and biomarkers showed a non-parametric distribution by the KS test. Overall, the medians (P25th–P75th) of RtPb, PbB, MnH, MnTn, and CdB were, respectively, 13.0 (3.7–34.6) μg Pb/m²/30 days; 0.9 (0.5–1.8) µg/dL; 0.18 (0.1–0.4) µg/g; 0.65 (0.37–1.22) µg/g; and 0.54 (0.1–0.8) µg/L. Of the sociodemographic characteristics, only SES showed a significant association with MnTn  and medians , respectively, in boys compared to girls. Mothers’ sociodemographic characteristics and babies’ anthropometric data at birth  showed nt-test (p = 0.018) with the BW/GA ratio. This influence of sex can be seen in p = 0.400) with the BW/GA ratio, but only for boys. Regarding biomarkers, bivariate analysis demonstrated no association with birth weight or BW/GA ratio in either Student’s t-test  or Speart-test . Howeverp = 0.099) with the BW/GA ratio.HLR  confirmeIn this study, we evaluated the impact of prenatal exposure to PTM on birth weight. In general, only Mn exposure stood out, having an inverse correlation between MnTn levels and BW/GA ratio only in girls, which suggests the effect modification of sex. Despite the worrisome exposure of the participants to Cd, we did not detect a statistically significant effect on birth weight, as suggested in other studies. The CdB showed a negative correlation only in boys, which, although statistically insignificant, also suggested a possible fetotoxicity modified by sex. It was impossible to investigate the probable combined effects of the three PTMs on birth weight due to the small sample size. Missing anthropometric measurements or other FG parameters in hospital records led to losing an essential part of the study population outcome data. The main inclusion criterion of this study being the availability of information on the outcome, we evaluated the possibility of extending these preliminary findings to participant groups that were not included. Despite the similarity of exposure to PTMs in the two groups, considering the importance of waste-burning and house renovation on MnTn and CdB levels , the lowRegarding exposure to PTMs, the pregnant women had low levels of exposure to Mn and Pb, while in the case of Cd, a worrying situation was observed. Considering the references recommended in the literature ,35, 1.9 The average birth weight in our study was similar to that reported by Silveira et al.  and BarrOf the sociodemographic characteristics (of the mother or the newborn), only the newborn’s sex, GA, length, and head circumference were associated with the outcome (birth weight and BW/GA ratio). This finding is not consistent with other reports in the literature; for example, cigarette smoking , alcoholHair and nails are keratinized tissues and are therefore rich in cysteine and are capable of chelating metals in sulfhydryl bonds throughout their growth. These matrices showed better performance when used to investigate exposure to Mn ,35 and tAnother fact that must be emphasized when comparing our findings with studies carried out with other matrices was the role of sex. Although the association between MnTn and birth weight was not maintained in HLR , the negative correlation with birth weight only among girls is consistent with other studies that show evidence of the sex-specific toxicity of PTMs ,49,50,51p = 0.149) with MnH but a negative correlation with MnTn. This difference may be due to the micronutrient status of Mn and the fact that the hair matrix indicates low recent Mn exposure, which is consistent with this tendency [Considering only the suggested biological effects on girls , contrary trends can be seen from the observed correlations and the graph in tendency . We did not find a significant association between the endpoints and exposure to Pb and Cd in this study. However, the literature presents sufficient evidence ,52,53,54p = 0.099) between LogCdB and the BW/GA ratio  found in the HLR analysis. In the case of Cd, we found exposure levels similar to those of workers and populations living close to industrial dump sites ,56. LeveConcerning Pb, it is crucial to consider the level found in this work with caution, as it is not consistent with other studies carried out ,59 in thConsidering the outcome of this work, some studies have already demonstrated the adverse effects of Pb on birth weight at levels below the CDC reference value ,20. EvenThe prospective design of this analysis and the biological plausibility of our findings meet the two main principles of causality established by Hill ). In theNevertheless, in addition to the COVID-19 pandemic, some contextual realities of the study area  hindered our data collection and may have influenced our final results, such as lack of statistical significance between PTM exposure and FG outcome. We found some resistance  to participating or assisting in field data collection. Due to logistical and resource limitations, we could not locate (due to change of address or inaccessible zone) some of the pregnant women. These realities contributed to the losses observed, culminating in the small sample size. That may have led to a selection bias and the inclusion of more pregnant women with low exposure to the three PTMs because of their easier geographical accessibility, for example. Also, more than half of the medical records in the maternity hospitals or the health booklet (NB and pregnant women) were not filled out, greatly hindering data collection, and this may explain the low proportion of LBW we found. Another limitation of this study may be that it included deliveries performed by cesarean section (37.8%) and premature births, which represent a risk factor for low birth weight. Although we included them in the models as adjustments to the HLR models, their impact may persist.Since pregnant women, fetuses, and children are more sensitive to PTMs, there is a need for information on their exposure and the extent to which they are affected in the southern hemisphere. Despite the limitations of this study, we detected an inverse sex-dependent relationship between low birth weight and exposure to Mn or Cd. Although we did not find any deleterious effects of Pb, it is vital to consider any level of exposure as a health risk. It is necessary to deepen this investigation, considering the population’s perception of the risk these metals pose, to reduce further exposures.


It is known that the rate of caesarean section (C-section) has been increasing among preterm births. However, the relationship between C-section and long-term neurological outcomes is unclear. In this study, we utilized diffusion tensor imaging (DTI) to characterize the association of delivery method with brain white matter (WM) microstructural integrity in preterm infants. We retrospectively analyzed the DTI scans and health records of preterm infants without neuroimaging abnormality on pre-discharge term-equivalent MRI. We applied both voxel-wise and tract-based analyses to evaluate the association between delivery method and DTI metrics across WM tracts while controlling for numerous covariates. We included 68 preterm infants in this study . Voxel-wise and tract-based analyses revealed significantly lower fractional anisotropy values and significantly higher diffusivity values across major WM tracts in preterm infants delivered via C-section when compared to those delivered vaginally. These results may be partially, but not entirely, mediated by lower birth weight among infants delivered by C-section. Nevertheless, these infants may be at risk for delayed neurodevelopment and could benefit from close neurological follow up for early intervention and mitigation of adverse long-term outcomes. Prematurity is also the leading cause of infant mortality, contributing to approximately one million infant deaths each year3. These infants suffer from a range of pathologies in the neonatal period including respiratory distress, intraventricular hemorrhage, and necrotizing enterocolitis among others4. Numerous studies have also linked prematurity to adverse long-term neurodevelopmental outcomes, including increased rates of cerebral palsy, cognitive impairment, and behavioral disorders in preterm infants when compared to term births7. The degree of prematurity has also been associated with increased risk of such adverse neurological outcomes9. Notably, while C-sections account for more than a fifth of all live births globally, the rate of C-section in preterm births has risen from around 25% to over 35% in the past three decades11. Thus given the increasing prevalence of C-section delivery, particularly in prematurity, a particular area of recent interest has been the relationship between delivery method and infant outcomes.Preterm birth (< 37 weeks gestation) accounts for more than 10% of live births worldwide15. Some of these adverse outcomes may—at least partially—be mediated by confounding maternal and infant factors such as birthweight, maternal age, maternal substance use and infant sex17. In contrast, some studies report no association between delivery method and infant mortality, neurodevelopmental delay, or rate of neurological disorder development among term, extremely preterm or very low birth weight infants22. Given these conflicting reports, we aimed to examine the association of delivery method with preterm infants’ brain white matter (WM) microstructure, a biomarker of developmental disorders.Multiple studies have found significant associations of C-section delivery with adverse cognitive outcomes, emotional problems, sleep problems, and increased risk of developing autism spectrum disorder and attention deficit hyperactivity disorder later in life24. We have recently shown the associations of gestational age at birth and corrected age at the time of scan on infants’ brain WM microstructural development23. Deoni et al. have also utilized DTI to study how delivery method is associated with infant brain development in full-term, healthy infants, finding that those born via C-section displayed signs of lower WM development at two weeks. However, they noted that such differences seemed to be less apparent later in life25.Although conventional brain MRIs can identify drastic structural and pathologic abnormalities, advanced MR techniques such as diffusion tensor imaging (DTI) can further characterize brain development by evaluating WM microstructural integrity. Prior studies have utilized DTI to characterize WM microstructure in infants across a range of gestational ages, identifying specific WM regions that are microstructurally delayed in infants of lower gestational ageIn this study, we compared the early WM microstructural integrity of radiologically normal singleton preterm infants born via C-section versus vaginal delivery after correction for gestational age at birth and time of scan, weight z-scores at birth, and other pertinent clinical variables. Our results elucidate neurobiological correlates of delivery method and explain the possible microstructural underpinnings of prior studies reporting adverse neurological outcomes in association with C-section delivery.26. In addition, we excluded infants whose mothers engaged in documented substance use during pregnancy and those who were the product of a multiple gestation. We recorded the incidence of maternal preeclampsia, chorioamnionitis, premature rupture of membranes, need for infant intubation, need for infant therapeutic hypothermia, pertinent postnatal pathologies, and indications for premature delivery for our included subjects.We reviewed the imaging and electronic health records of all infants who had brain MRI performed within 3 months of birth between January 2013 and March 2021 at our center. Data access and retrieval were performed on March 31, 2021. The authors had access to information that could identify individual participants during and after data collection. Preterm infants at our institution routinely undergo brain MRI at term equivalent age or prior to discharge. This predischarge MRI scan includes DTI series at the end, if tolerated by the infant. We included infants who (1) were born between 24- and 37-weeks gestational age at birth, (2) had no structural or signal abnormalities (gross distortion or severe motion artifact) on conventional brain MRI series, (3) had DTI performed as part of their MRI study, and (4) had sufficient information in the health record regarding: delivery methods, indication of early delivery, gestational age at birth and time of scan, birth weight, and 1- and 5-min APGAR scores. All birth weights were converted to Z-scores using the 2013 Fenton growth chart for preterm infants 28. This study was approved by our institutional review board and the need for consent was waived by the Yale Human Research Protection Program Institutional Review Boards due to the retrospective nature of the study. Additionally, all research was conducted in accordance with the Declaration of Helsinki to ensure ethical respect to human subjects.As prior studies have identified low birth weight as a risk factor for delayed neurodevelopment and adverse neurological outcomes, a sub-cohort excluding infants born small for gestational age  was identified to conduct additional analyses2 acquisitions. Motion artifact for neonatal MRI is reduced at our institution preferentially using the “feed-and-swaddle” technique with anesthetic sedation being used as a second line option29. Preprocessing of DTI scans was performed using FMRIB’s Diffusion Toolbox (FDT) in FMRIB’s Software Library (FSL). This including corrections for susceptibility distortions using the TOPUP tool, and correction for eddy currents and subject movement using the EDDY tool. Then, the DTIFIT tool was used to fit diffusion tensors and generate fractional anisotropy (FA), mean diffusivity (MD), radial diffusivity (RD), and axial diffusivity (AD) maps. All DTI metric maps were visually inspected for quality control prior to further analysis.MRI scans were performed on a Siemens 3 T Skyra scanner. DTI series were obtained using a single-shot echoplanar image sequence with the following parameters: repeat time = 10,200 ms, echo time = 94 ms, flip angle = 90°, field of view = 18 × 18 cm, slice thickness = 2.5 mm, matrix = 128 × 128, including a single b = 0 and 30 noncolinear direction b = 1000 s/mm30. As birth weight Z-score and presence of preeclampsia differed significantly between infants delivered vaginally and via C-section  onto the MNI-152 brain space template. Similar TBSS and GLM analyses were repeated for the sub-cohort excluding infants born small for gestational age.The TBSS toolbox in FSL was used for voxel-wise statistical analysis of DTI metrics along WM tracts. As a part of this process, the most representative FA map of the cohort was first identified by non-linearly co-registering each FA map to all other FA maps. The TBSS script then identifies the most “typical” or representative FA map among the cohort. This representative FA map was then co-registered using affine alignment registration to the MNI-152 standard brain space. The FA maps of all other subjects were then similarly co-registered to MNI-152 by combining the non-linear co-registration to the representative FA map and the affine alignment co-registration of the representative map to MNI-152. This process was repeated to bring MD, RD, and AD maps to the MNI-152 space. A skeletonized WM tract map across all subjects was generated by averaging the aligned FA maps using a threshold value of 0.1. Then, we used the “randomise” tool in FSL for GLM analyses to examine the association of delivery method with DTI metric values across WM tracts while controlling for gestational age at birth, gestational age at scan, 5-min APGAR, birth weight Z-score, and presence of preeclampsia as covariates. Supplemental Fig. 23. For every subject, the means of non-zero DTI metric values were calculated in each of the 48 WM tracts from JHU atlas using FSL. Then, we applied linear regression analyses to determine the association of delivery method with mean DTI metric values in each WM tract while controlling for gestational age at birth, gestational age at scan, 5-min APGAR, birth weight Z-score, and presence of preeclampsia as covariates. Similar tract-specific linear regression analyses were repeated for the sub-cohort excluding infants born small for gestational age. All p-values were also adjusted for multiple comparisons using the Benjamini–Hochberg procedure33.The results of our GLM analyses were further verified by conducting additional linear regression analyses across 48 WM tracts based on the John’s Hopkins University (JHU) atlasThe data are presented as mean ± standard deviation, median (interquartile range) or frequency (percentage) as appropriate. The “stats” package in R was used to determine if significant differences existed with regards to clinical variables between infants delivered vaginally and via C-section. A two-sample t-test was used for continuous variables, a Wilcoxon rank-sum test was used for APGAR scores, and a chi-squared test was used for binary variables. R was also used to conduct tract-specific linear regression analyses.Figure Figure Despite differing significantly between the two experimental groups, neither birth weight Z-score nor presence of maternal preeclampsia were significantly associated with any DTI metrics on voxel-wise GLM analysis or tract-specific regression after adjusting for delivery mode and other covariates listed above. Use of general anesthesia during delivery was also not significantly associated with any DTI metrics on voxel-wise GLM analysis or tract-specific regression after adjusting for delivery mode and additional covariates.Nine infants born SGA  were excluded from the sub-cohort. Table Figure In voxel-wise GLM analyses, preterm infants requiring C-section delivery had reduced WM microstructural integrity compared to those delivered vaginally, most prominently in the corpus callosum, internal capsule, and corona radiata. Notably, the difference between delivery mode of preterm infants was found after adjusting for gestational age at birth, gestational age at scan, 5-min APGAR, birth weight Z-score, and presence of preeclampsia as covariates—and after excluding infants from multiple gestations and those with any maternal history of drug use. While tract-specific linear regression analyses supported the results of our voxel-wise GLM analyses, significant associations between delivery method and DTI metrics were not observed after correcting for multiple corrections in these analyses. Although birthweight Z-score and presence of maternal preeclampsia differed significantly between infants delivered vaginally and via C-section, additional voxel-wise GLM analyses found no significant association between these risk factors and DTI metrics after controlling for delivery method and other clinical variables. In the sub-cohort excluding SGA infants, significant association between delivery method and DTI metrics was noted in similar regions to the overall cohort, including the the corpus callosum, internal capsule, and corona radiata.Our results provide the neurobiological indications and early WM microstructural correlates for clinical studies reporting that preterm infants who require C-section are at risk for adverse neurodevelopmental outcome. The difference in early WM microstructural development may be a consequence of multiple factors necessitating C-section delivery; nevertheless, our findings highlight the higher risks of neurodevelopmental delay among preterm infants delivered via C-section and the need for close follow-up and potential intervention to mitigate these risks in this vulnerable cohort.34. DTI metrics have also been shown to predict neurological outcome, with studies reporting associations between DTI metrics measured during term-equivalent scans and cognitive, motor, and visual outcomes in childhood and adolescence36. Of note, some have reported associations between the development of cerebral palsy and other motor abnormalities and term equivalent DTI metrics in the same WM regions where we identified significant association between delivery method and DTI metrics such as the corpus callosum and internal capsule38. Thus, term-equivalent DTI can potentially help identify infants at risk for adverse neurological outcomes with regards to their method of delivery, especially when neurological exam and standard neuroimaging might otherwise be unremarkable.The myelination of WM tracts as infants age reduces the random diffusion of water molecules and results in alignment of diffusion along WM tracts. Thus, changes in DTI metrics such as increased FA and reduced MD and RD are corollaries for WM maturation25. Similar to our results, they reported higher FA values in WM tracts of term infants delivered vaginally compared to those delivered via C-section. We found higher MD and RD in addition to reduced FA in preterm infants delivered via C-section. Deoni et al. also noted slight variations in results with the inclusion of additional covariates, suggesting that factors beyond delivery method could be contributing to perceived differences in WM maturation. We attempted to address this by controlling for clinical variables known to be independently associated with WM maturation in infants  in addition to those we found differed significantly between our two experimental groups , among others23. As prior reports have raised concern that exposure to general anesthesia, particularly inhaled agents such as sevoflurane, may contribute to delayed neurodevelopment and adverse long-term neurological outcomes, we also confirmed that exposure to general anesthesia during delivery was not independently associated with WM maturation in our cohort40. Of note, Deoni et. al found no difference in WM tract DTI metrics when comparing separate cohorts of eight-year-old children delivered via C-section (n = 23) versus vaginally (n = 37). Thus, they proposed that WM microstructural differences in association with delivery mode may only be present in early (pre-discharge) MRIs and not resolve at older age—although they analyzed different children’s cohorts from infants.The relationship between delivery method and infant brain development has not been extensively studied. Deoni et al. have compared the DTI metrics between term infants delivered via C-section (n = 11) versus vaginally (n = 32)42. The impact of differential infant hormonal expression between vaginal and C-section deliveries on early brain development has also been proposed43. However, these explanations are complicated by studies reporting contrasting results regarding the association of delivery method with neurodevelopmental disorders45. Even among the numerous studies suggesting that delivery method may be associated with neurological outcomes, differences in methodology  and characteristics of experimental cohorts among prior reports complicate interpretation. Additionally, some investigators report that delivery method may be associated with neurological outcome only in the context of additional factors such as infant gender, APGAR scores, multiple gestations, and use of general anesthesia during C-section32. Given the many maternal and infant factors that may influence need for C-section versus vaginal delivery, it is challenging to control for all covariates that may influence brain development independently or cooperatively with delivery method. Nevertheless, our study provides WM microstructural correlates for prior clinical studies reporting that preterm infants requiring C-section are at higher risk of neurodevelopmental delay. While our findings may be related to multifactorial circumstances necessitating C-section delivery, they highlight the need for close neurological follow up and timely interventions in these vulnerable children.As we demonstrate the relationship between delivery method and WM maturation on neuroimaging, the question of how delivery method may influence early brain development arises. It has been suggested that vaginal delivery is an important contributor to the normal development of infants’ gut microbiome and that alterations in this process (due to C-section) can disturb normal brain development48. However, we address this by controlling for birth weight z-score as a covariate in analyses of the primary and sub-cohorts and also demonstrating a lack of significant independent association between birth-weight Z-score and DTI metrics in the primary and sub-cohorts.Exclusion of infants born SGA revealed a significant difference in birth weight z-score between infants delivered by C-section and those delivered vaginally. Of note, prior reports have described the association between birth weight and DTI metrics as well as long-term neurological outcomes50. Additional postnatal pathologies such as bronchopulmonary dysplasia, necrotizing enterocolitis and sepsis have also been described to influence neurodevelopmental outcome in preterm and low birth weight infants53. However, given that the rate of such pathologies did not differ significantly between infants born by C-section or vaginally in our primary or sub-cohort, we elected not to include these pathologies as covariates in our analyses. We also described various indications for respective delivery method but could not control for all such indications given the widely varying delivery indications in our cohort. Finally, long-term neurological outcomes were not available in our dataset to corroborate clinical correlates of DTI findings.Our study is limited by its retrospective, single center design and small sample size. Our small sample size also limits the power of our study in including multiple covariates, which may complicate the interpretations of our analyses on the independent associations of birthweight z-score, maternal preeclampsia, and general anesthesia use during delivery with DTI metrics. DTI metric analysis can be influenced by acquisition protocol and timing, as well as processing methods used to generate DTI metric maps. Although we controlled for several demographic and clinical variables, including those that differed significantly between experimental groups, we could not account for all possible covariates that may influence delivery method and explain observed differences in DTI metrics between the experimental groups in this study. Of note, recent work has suggested that intrauterine inflammation due to chorioamnionitis and other pathologies impacts fetal and infant neurodevelopmentPreterm infants requiring C-section section delivery, compared to those delivered vaginally, have neuroimaging markers of delayed WM maturation on predischarge MRI scans, most prominently in the corpus callosum, internal capsule, and corona radiata independent of gestational age at birth, gestational age at scan, 5-min APGAR, birth weight Z-score, maternal preeclampsia, and chorioamnionitis. Possible confounders of birth weight z-score, rates of maternal preeclampsia, and use of general anesthesia during C-section delivery are not independently associated with WM maturation in this cohort. These findings suggest that preterm infants requiring C-section are at risk for delays in WM maturation and thus may require close neurologic follow up to identify and mitigate adverse long-term neurological outcomes.Supplementary Information.


Presuming that the incidence of cerebral palsy (CP) in Korea is decreasing due to medical advances, we analyzed the trends and risk factors of CP in changing circumstances. We identified all women who delivered a singleton between 2007 and 2015 using the Korea National Health Insurance (KNHI). Information on pregnancy and birth was obtained by linking the KNHI claims database and data from the national health-screening program for infants and children. The 4-years incidence of CP decreased significantly from 4.77 to 2.52 per 1000 babies during the study period. The multivariate analysis revealed that the risk of developing CP was 29.5 times higher in preterm infants born before 28 weeks of gestational age, 24.5 times higher in infants born between 28 and 34 weeks, and 4.5 times higher in infants born between 34 and 36 weeks, compared to full-term appropriate for age (2.5 ~ 4 kg of body weight) infants. 5.6 times higher in those with birth weight < 2500 g, and 3.8 times higher in pregnancies with polyhydramnios. Additionally, respiratory distress syndrome increased the risk of developing CP by 2.04 times, while necrotizing enterocolitis was associated with a 2.80-fold increased risk of CP. In Korea, the incidence of CP in singleton decreased from 2007 to 2015. We need to continue to focus on developing medical technologies for the early detection of high-risk neonates and minimizing brain damage to reduce the incidence rate of CP effectively. In addition to physical disability, CP can be accompanied by cognitive impairment, communication problems, and epilepsy, which cause considerable personal and socioeconomic burdens. The medical community’s ongoing efforts to reduce this burden have significantly improved healthcare technology over the past few decades. The early identification of women at risk of preterm birth and protective methods for reducing perinatal complications may decrease the incidence of preterm birth and fetal mortality2. Moreover, asphyxia is suspected, brain cooling has been widely used to protect the neonatal brain. Presuming that the incidence of CP in Korea is decreasing due to medical advances, we attempted to analyze the latest trends and risk factors for CP in changing circumstances.Cerebral palsy (CP) is a group of permanent disorders of the development of movement and posture causing activity limitation attributed to non-progressive disturbances in the developing fetal or infant brainAlmost all Koreans are covered by the health insurance policies of the Korea National Health Insurance (KNHI) of the Health Insurance Review and Assessment Service, except for 3% of the population that the Medical Aid Program covers. Thus, the KNHI claims database contains claims information for most Koreans except for uninsured procedures, such as plastic surgery. As a part of the National Health Insurance Corporation healthcare system, the national health-screening program for infants and children, for which children aged 4–80 months are eligible, began in 2007 and is composed of seven consecutive health examinations according to age . The national health-screening program consists of history taking, physical examination, developmental screening, visual acuity, and a dental examination.Information on pregnancy and birth was obtained by linking the KNHI claims database and national health screening program data for infants and children. Using the KNHI claims database, we identified all women who delivered babies between 2007 and 2015. A total of 3,778,561 women who had deliveries were identified. Excluding 56,925 deliveries of multiple births, a total of 3,278,916 single births were included in our study. To ensure the reliability and validity of our analysis, we excluded twin babies from the study sample as they could result in duplicate data from the same mothers. Moreover, since twin births represent only a small proportion of all births, their exclusion allowed for a more representative sample of the general population. Detailed information, such as medical illness, delivery mode, pregnancy complications, and single or multiple pregnancies, was confirmed using data from the KNHI claims and the national health-screening program for infants and children.We assumed an initial diagnosis of CP for early treatment if a diagnostic code had been registered. In addition, we defined cases of cerebral palsy using the Tenth Revision of the International Classification of Diseases: spastic quadriplegic CP (G80.0), spastic diplegic CP (G80.1), spastic hemiplegic CP (G80.2), dyskinetic CP (G80.3), ataxic CP (G80.4), other CP (G80.8), CP, unspecified (G80.9). Hereditary spastic paraplegia (G11.4) was excluded. Upon diagnosis of CP, the number of people was summed based on the child's birth year. The observational period was at least four years after birth.The yearly incidence of CP per 1000 live births was calculated using the total number of infants born during the study year as the denominator and the number of infants diagnosed with CP during subsequent follow-up as the numerator.Pre-pregnancy health status: “hypertension (I10)”, “diabetes mellitus (E11)”, and “advanced maternal age (> 35 years)”.Pregnancy complications: “gestational hypertension (O13)”, “preeclampsia (O14)”, “gestational diabetes (O24)”, “chorioamnionitis (O41.1)”, “premature rupture of membrane (O42)”, “oligohydramnios (O41.0)”, “polyhydramnios (O40)”.Conditions during delivery: “preterm birth (< 37 weeks of gestation)”, “mode of delivery”, “low birth weight (< 2500 g)”, and “large for gestational age (≥ 4000 g)”.Conditions after delivery: “respiratory distress syndrome (P22.0)”, “Bronchopulmonary dysplasia (P27.1)”, “Necrotizing enterocolitis (K55.3)”.Statistical analysis was performed using SPSS software version 12.0 . The Student's t-test was used to compare continuous variables between groups. The categorical variables were compared using the χ2 test. We compared temporal trends by using the χ2 test. Multivariate logistic regression analysis was used to estimate the adjusted odds ratio (OR) and 95% confidence interval (CI). All P-values were two-sided and were considered statistically significant if 0.050 or less.The institutional review board of the Korea University Guro Hospital approved the study (2020GR0468) and granted a waiver for informed consent because of its retrospective nature.As shown in Fig. p < 0.001 for vaginal births; p = 0.001 for total live births; p = 0.021 for cesarean births).As shown in Fig. According to a combined analysis of maternal and their babies' records, the frequency of advanced maternal age, hypertension, and diabetes before and during pregnancy, diabetes mellitus before pregnancy, cesarean delivery, gestational hypertension, gestational diabetes mellitus requiring insulin treatment, chorioamnionitis, premature rupture of membrane, oligo- and polyhydramnios, preterm birth, low birth weight, male sex, and large for gestational age was higher in the group diagnosed with CP than in the controls Table .Table 1CIn Table p  = 0.015), as well as a slight increase in the proportion of full-term small-for-gestational-age, from 1.85 to 1.91% (p  = 0.032). On the other hand, we observed no significant variation in the occurrence rate of polyhydramnios over the study period.Figure Our data revealed that the incidence of CP per 1000 live births declined significantly from 4.77 to 2.52 babies among all live single births between 2007 and 2015. The likelihood of developing CP is 4.54–29.49 times higher in preterm babies than in full-term babies of the appropriate weight. Additionally, full-term babies who are small-for-gestational-age (body weight <2.5kg) have a 5.39 times higher risk of developing CP, and pregnancies with polyhydramnios have a 3.81 times higher risk of CP. Neonatal conditions, such as respiratory distress syndrome and necrotizing enterocolitis, have increased the risk of developing CP by 2.04-fold and 2.80-fold, respectively.3. According to a meta-analysis analyzing 19 previous reports using live births as the denominator, the overall prevalence of CP is 2.11 per 1000 births4. The included studies were from the USA, Canada, Australia, or Europe, except one from China. Nationwide population studies in other Asian countries have recently reported a prevalence of CP of 1.09 per 1000 children under 20 years of age in Taiwan5, 1.19 in children aged 0–6 years in China6, and 2.26 in children aged 0–4 years in Japan7. Our study findings indicate that the incidence of CP in Korea experienced a significant decline from 4.77 to 2.52 per 1000 live births from 2007 to 2015. This decrease was similar to the degree observed in other Asian countries.A previous study in South Korea found that the incidence of CP increased from 2.2 to 3.2 per 1000 children born between 1999 and 20039. The immature brain cannot effectively maintain a blood supply because there are fewer collateral vessels or anastomoses around the peripheral blood vessels and immature walls. The vessels cannot compensate for hypoxic-ischemic damage to limited vasodilation capacity10. Thus, various attempts have been made to prevent preterm birth; 17α-hydroxyprogesterone caproate may help prevent recurrent preterm birth13. Furthermore, vaginal progesterone prevents premature birth of mothers with short cervixes and improves neonatal outcomes16. Overall, progesterone administration lowered the rate of preterm births by approximately 50%17. Cervical pessary use or cervical cerclage prevents repeated preterm births in high-risk women with a short cervix18. Despite medical advances, the rate of preterm births increased from 2.9% (1997–1999) to 4.5% (2011–2013)19 and from 3.31% (1997–1998) to 6.44% (2013–2014)20 in Korea. The increase in twin births is thought to have contributed to the rise in preterm births. As shown in Fig. p = 0.015) in our study.Aside from our study results, preterm birth is already well-known as a risk factor for CP21. Fetal growth restriction has been reported as a more critical risk factor for CP than fetal inflammation and birth asphyxia combined22. In our study, based on the limitations of the available data, an alternative approach was taken to analyze the risk of cerebral palsy associated with growth abnormalities. Since accurate gestational age data was unavailable from the claim data, term babies were categorized based on their birth weight into three groups: those weighing less than 2.5kg, those weighing between 2.5kg and 4kg, and those weighing 4kg or more. Despite the limitations, this method allowed for a more detailed investigation of the potential risk factors linked to cerebral palsy in infants with growth abnormalities. Our results showed that full-term babies who are small-for-gestational-age (body weight <2.5kg) have a 5.39 times higher risk of developing CP compared to full-term babies of appropriate weight, concordant with the previous studies.The association between the development of CP and fetal growth restriction is well known24. Even when the results of a detailed ultrasound examination of the fetus were normal, polyhydramnios doubled the risk of genetic syndromes, neurologic disorders, and fetal malformations diagnosed after birth25. The incidence of polyhydramnios did not change over the study period .In our study, polyhydramnios increased the risk of CP by 3.81 times. Polyhydramnios has also been associated with increased perinatal morbidity and mortality risk, such as preterm birth, aneuploidy, cesarean section, fetal anomalies, and perinatal and postnatal mortality26. Moreover, a meta-analysis demonstrated a 1.59-fold increased risk of CP in neonates with necrotizing enterocolitis, possibly due to heightened exposure to proinflammatory cytokines and the associated risk of sepsis27.Our study found that respiratory distress syndrome and necrotizing enterocolitis were associated with a 2.04-fold and 2.80-fold increased risk of developing CP, respectively. Our study's results align with the prior studies, as it reported that moderately late and late preterm infants (32–36 weeks) who experienced respiratory distress syndrome had a two times higher incidence of CP than those without respiratory distress syndrome at the same gestational weeks4) stabilizes blood pressure, reduces vasoconstriction in the cerebral arteries, and restores circulation in preterm neonates29. Treatment with MgSO4 in preterm labor may lower the risk of CP32. The proportion of moderate to severe CP decreased significantly in babies born in women with preterm birth who were treated with magnesium 31; several meta-analyses also support this result35. Second, the prenatal administration of corticosteroids for fetal lung maturity may reduce the occurrence of CP36. Finally, brain or whole-body cooling has become standard management for neuroprotection in newborns with birth asphyxia38.In addition, it is important to note that although not included in our study data, the advancements in various medical technologies aimed at protecting the neonatal brain may have contributed to a reduction in the incidence rate of CP. First, magnesium sulfate (MgSO39. Moreover, our study revealed a continued decline in the incidence of CP in Korea.Through various efforts, including improvements in prenatal and neonatal care, Korea has decreased the infant mortality rate from 4.7 in 2004 to 3.0 in 2014In this study, we assumed that the registration of a diagnostic code indicates an initial diagnosis of CP. However, it is important to acknowledge that diagnosing CP can be challenging and may change as more information is gathered or the child develops. Children with mild CP may not be diagnosed until later in life, as their symptoms may not be as apparent during early childhood. Therefore, our study may include cases where a tentative diagnosis was made to receive early physical therapy. In some healthcare settings, it is common practice for healthcare professionals to diagnose a child with CP for this purpose tentatively. However, it is important to note that including such cases may have contributed to potential inaccuracies in our findings, which should be considered when interpreting our results. We also acknowledge the limitations of relying solely on diagnostic codes for diagnosing CP in our study. Using administrative data in research may result in potential inaccuracies due to the lack of clinical data in the analysis, a recognized limitation commonly encountered in studies utilizing administrative data. Given the absence of accurate gestational age data in the medical claim database, we categorized term babies based on birth weight and analyzed them to alternatively assess the risk of cerebral palsy associated with growth abnormalities.While our study successfully linked birth records of infants born in the hospital to their respective mothers, it is essential to note that our study's scope is limited to infants born within the hospital. Thus, our study did not include roughly 1.5% of births outside the hospital. Furthermore, a small number of individuals who may have emigrated or died at some point after birth cannot verify their status within our research data.1, our study was unable to identify perinatal risk factors of CP such as birth asphyxia, neonatal sepsis, or respiratory distress syndrome, as well as early infantile risks such as encephalitis or head trauma.Finally, while a child can be diagnosed with CP if a triggering event occurs before the affected function fully developsIn Korea, the incidence of CP decreased from 2007 to 2015. Based on our findings and analysis, we can infer that the efforts to protect the brain of neonates before and after birth through advanced technologies have effectively reduced the incidence rate of CP. Therefore, we need to continue to focus on developing medical technologies for the early detection of high-risk neonates and minimizing brain damage to reduce the incidence rate of CP effectively.


This study confirmed breech presentation risk factors maternal age, nulliparity, tobacco smoke, a previous cesarean delivery (CD), neonatal female sex, gestational age, and birth weight. Black race and Hispanic origin were verified to be protective factors. We found breech prevalence among ART and OST to be stable during the study period. Meanwhile, newborn birth weight was increased, and the gap between breech and other presentations in ART was reduced. Our results indicate that singleton pregnancies conceived by ART or OST were associated with a higher risk of breech at delivery. Well-known risk factors for the breech presentation were also confirmed. Some of these factors can be modified by implementing interventions to reduce their prevalence .Since essential factors have changed in recent years in assisted reproduction technologies (ARTs), this study reassessed the association between ART and breech presentation. We primarily aimed to estimate the correlation between ART and breech at delivery. Secondary purposes were to evaluate the correlation between other subfertility treatments (OSTs) and breech and to assess possible confounding factors and temporal trends. This study investigated the 31,692,729 live birth certificates from US states and territories in the 2009–2020 period. The inclusion criteria were singleton births reporting the method of conception and the presentation at delivery. The outcome was the breech presentation at delivery, while the primary exposure was ART, the secondary exposure was OST, and the potential confounding factors from the literature were considered. ART (OR 2.32 CI.95 2.23–2.41) and OST (OR 1.79 CI.95 1.71–1.87) were independent and significant risk factors for breech at delivery ( Since the introduction in the 1970s of assisted reproduction technology (ART) in the human species, many improvements have been implemented . In receAlthough in the past, singleton infants conceived with ART showed a higher hazard for low birth weight and prematurity, the introduction of the freeze-all concept decreased some negative perinatal outcomes such as the low birth weight ,6. HowevThe breech presentation is a familiar condition in obstetrical practice, accounting for approximately 3–8% of singleton fetuses at delivery ,10,11. MART was previously associated with breech presentation ,15,16. HIn this cross-sectional retrospective study, we used the US National Center for Health Statistics birth certificate data that are part of the National Vital Statistics System . We consPregnancies were selected according to the subsequent inclusion and exclusion criteria. We included all consecutive singleton pregnancies with information about the mode of conception and maternal age at delivery between 18 and 49 years old. We considered the following as exclusion criteria: women older than 49 years old or younger than 18 years old, records with imputed values for sex or multiple pregnancies, multiple pregnancies, gestational age below 22 or above 49 weeks of gestation, no data about the mode of conception, and chromosomal anomalies. In http://www.strobe-statement.org/, accessed on 1st May 2023) [While preparing the manuscript, we observed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement guidelines for observational studies (ay 2023) . All dathttps://www.cdc.gov/nchs/data_access/Vitalstatsonline.htm#Tools (accessed on 2 December 2021).From the original datasets, the following variables were extracted: maternal age, parity, race, pre-pregnancy body mass index (BMI), fetal presentation at delivery, mode of delivery, previous CD, multiple pregnancies, multiple pregnancies imputation label, neonatal sex, neonatal sex imputation label, gestational age at delivery, neonatal weight, chromosomal anomalies, and tobacco smoke. The primary outcome was the presentation at birth. Type of conception was used as the primary explanatory variable . The typp-value < 0.05 to be statistically significant. The normality of continuous variables was evaluated using the Kolmogorov–Smirnov test. We show parametric continuous variables as mean ± standard deviation and non-parametric ones as the median and interquartile range (IQR). We display dichotomous or polychotomous variables as percentages and absolute values, excluding missing values (“NA”) from the denominator (unless otherwise defined). We show the logistic regression results as odds ratio (OR) and 95% confidence interval (CI.95). We analyzed categorical variables (dichotomous or polychotomous) employing the chi-square test or the Fisher exact test. Meanwhile, we analyzed continuous variables using the Wilcoxon test (non-parametric variables) or the t-test (parametric variables). We also conducted a logistic regression analysis considering fetal presentation as a dependent variable and mode of conception  as the primary explanatory variable (independent). We also considered all possible confounding factors derived from the literature and available in the dataset as independent variables. We carried all potential predictive factors (p < 0.05) from univariate analysis in the multivariate model. The initial multivariate model incorporated all variables and their interactions, and when interactions turned out to be non-significant, the estimation without interaction model was employed.All investigations were performed using R software (version 4.1.3) . The pro2 (IQR 22–30), and 31.24% of women were nulliparous. The majority were white only (74.73%), 14.92% were black only, and 25.55% were of Hispanic origin. A positive history of tobacco smoke was present in 7.35% of cases, and the history of a previous CD was present in 15.12% of cases. In the majority of cases, an SC of 98.82% was recorded ; in 0.49%, an OST of 154,445; and in 0.69%, an ART of 218,212.The total number of singleton pregnancies to comply with the inclusion criteria was 31,692,729. The median maternal age was 29 years (IQR 24–33). The median pre-gestational BMI was 25 kg/mThe median gestational age at delivery was 39 weeks (IQR 38–40), the median birth weight was 3320 g (IQR 3005–3647), and the median birth weight MoM was 0.99 (IQR 0.90–1.08). Most (94.17%) fetuses at delivery were in cephalic presentation, and 3.00% were in breech presentation. Spontaneous vaginal birth delivery was observed in 65.67% of cases and CD in 31.04%.p < 0.05). p < 0.001) (p < 0.001) (p < 0.001) (ART was a significant risk factor for breech presentation at delivery OR 2.2 (CI.95 2.16–2.23) . Other r< 0.001) . Figure < 0.001) . Meanwhi< 0.001) . The mul< 0.001) .We found ART and OST to be independent and significant risk factors for breech presentation at delivery. Our analysis confirmed risk factors for breech presentation to be maternal age, nulliparity, tobacco smoke, previous CD, neonatal female sex, gestational age, and neonatal birth weight. Black race and Hispanic origin were confirmed to be protective factors. We further found breech prevalence among ART and OST to be stable during the study period. Meanwhile, newborn birth weight MoM was increased during the study period, and the gap between breech and other presentations in ART was reduced to a non-significant difference.The literature is controversial about the association between ART and breech presentation at delivery. Previously published small observational studies have documented a raised risk of the breech presentation associated with ART ,16,42,43Multiple hypotheses can explain our findings. First, as both OST and ART are associated with breech presentation, some factors related to subfertility are probably associated with the risk of breech presentation at delivery independently of gestational age, parity, and the other factors accounted for in this study. This hypothesis is corroborated by previous data where adverse pregnancy outcomes were attributed to factors associated with infertility rather than to elements linked to ART . Second,https://www.sartcorsonline.com—accessed on 12 April 2022). The neonatal birthweights also increased, coherently with the literature linking FET to large for gestational age newborns [In the US population, the number of FETs on the total number of embryo transfers (ET) increased from 31.5% in 2014 to 44.1% in 2019 in cycles with the patient’s own oocytes alone  also found in different settings. These points mean that these results are readily applicable also in other contexts. However, not all the results are equally generalizable. For example, the CD rate and the use of ART and OST change according to local management and the policies of the different health systems, and this issue limits the generalizability of our findings.These data suggest that there is still space to reduce some of the risk factors associated with breech presentation and thus reduce the CD rate. For example, interventions aimed at reducing tobacco smoke in pregnancy or preventing primary CD can also favor the reduction in breech presentation. Furthermore, there is probably room for a further decrease in CDs favoring a greater diffusion for the version for external maneuvers from breech to cephalic.However, studies are needed to demonstrate whether these interventions can actually be cost-effective. Furthermore, the connection between breech presentation and ART is not yet evident; further studies to better understand these mechanisms would lead to benefits. Again, additional development of ART and OST techniques could further reduce the differences observed between pregnancies obtained through these routes and those conceived spontaneously.In summary, our results reveal that singleton pregnancies conceived by ART or OST were associated with a higher risk of breech presentation at delivery. We confirmed well-known risk factors for breech presentation at delivery, such as maternal age, nulliparity, tobacco smoke, previous CD, neonatal female sex, gestational age, neonatal birth weight, and protective factors such as Black race and Hispanic origin. Some of these factors can be modified by implementing further interventions to reduce their prevalence . Furthermore, the CD rate is at the highest level among ART and OST singleton pregnancies, and there is probably a margin to lower it.


Scedosporium spp. and Lomentospora prolificans have become a serious threat in clinical settings. The high mortality rates associated with these infections can be correlated with their multidrug resistance. The development of alternative treatment strategies has become crucial. Here, we investigate the in vitro and in vivo activity of luliconazole (LLCZ) against Scedosporium apiospermum  and Lomentospora prolificans. The LLCZ MICs were determined for a total of 37 isolates  according to EUCAST. Furthermore, the LLCZ antifungal activity was tested in vitro, using an XTT  growth kinetics assay and biofilm assays . In addition, a Galleria mellonella infection model was used for in vivo treatment assays. The MIC90 of LLCZ was determined to be 0.25 mg/L for all tested pathogens. Growth was inhibited within 6 to 48 h of the start of incubation. LLCZ inhibited biofilm formation in both preadhesion stages and late-stage adhesion. In vivo, a single dose of LLCZ increased the survival rate of the larvae by 40% and 20% for L. prolificans and Scedosporium spp., respectively. This is the first study demonstrating LLCZ activity against Lomentospora prolificans in vitro and in vivo and the first study showing the antibiofilm effect of LLCZ in Scedosporium spp.Infections with IMPORTANCELomentospora prolificans and S. apiospermum/P. boydii are opportunistic, multidrug-resistant pathogens causing invasive infections in immunosuppressed patients and sometimes in healthy persons. Lomentospora prolificans is panresistant against the currently available antifungals, and both species are associated with high mortality rates. Thus, the discovery of novel antifungal drugs exhibiting an effect against these resistant fungi is crucial. Our study shows the effect of luliconazole (LLCZ) against L. prolificans and Scedosporium spp. in vitro, as well as in an in vivo infection model. These data reveal the previously unknown inhibitory effect of LLCZ against L. prolificans and its antibiofilm effect in Scedosporium spp. It represents an extension of the literature regarding azole-resistant fungi and could potentially lead to the development of future treatment strategies against these opportunistic fungal pathogens. Lomentospora prolificans is a fungal pathogen known for its resistance against multiple anti-infective agents belonging to different substance classes, such as echinocandins, pyrimidines, allylamines, polyenes, and azoles (to a limited extent) , mostly in combination therapy, as suggested on the basis of both in vitro data and clinical studies  . Scedospericin B , 5. For ericin B . In the  studies , 8. Howe studies –11. The  studies , 13. Amo studies . Biofilm studies . Like ottructure . For thiL. prolificans and Scedosporium spp. is luliconazole (LLCZ). Clinically, LLCZ is well known for its effect against dermatophytes  and is currently approved only for topical use in the treatment of superficial mycoses such as tinea pedis, tinea cruris, tinea corporis, and onychomycosis  has also been documented   approved LLCZ for topical use in the United States . FurtherL. prolificans and S. apiospermum/P. boydii in vitro and in vivo.The aim of this study was to investigate the effect of LLCZ against the planktonic growth and biofilm formation of L. prolificans ranged between 0.004 and >0.25 mg/L, for S. apiospermum/P. boydii, the obtained values were between 0.008 and 0.06 mg/L. The overall MIC90 and MIC50 values were 0.25 mg/L and 0.06 mg/L, with no significant differences between the distinct organisms -2H-tetrazolium-5-carboxanilide salt] growth kinetics assay, revealing that various concentrations of the antifungal agent had a significant effect  assays at different formation stages. While the effect of the drug was similar for all three organisms, the results of the two assays differed. The XTT assay  showed aL. prolificans isolate 2229 was treated with an LLCZ concentration of 0.03 mg/L . .03 mg/L  and half.03 mg/L . In bothGalleria mellonella . No difference was recorded in metabolic activity when the drug was added at 0 h or 2 h after incubation. It is possible that LLCZ effectively inhibits the metabolic activity and development of L. prolificans and Scedosporium spp., despite their high biofilm production. This may be the explanation for the significant effect of LLCZ treatment against mature biofilms (48 h after incubation). Comparable to LLCZ, the novel antifungal drug olorofim also showed a partial (40%) inhibition of L. prolificans and Scedosporium spp., but it has been described as ineffective against late-stage biofilm of azole-resistant A. fumigatus isolates  were used. Due to the nature of the CV assay (staining biomass without distinction between dead or living cells), dead hyphae could be detected. However, according to the XTT assay, these hyphae were metabolically inactive.The biofilm formation capacity of LLCZ-treated fungi was assessed via two separate assays. While the XTT assay showed a significant decrease in metabolic activity (75%) in all organisms when the drug was directly incubated with the fungal suspension (0 h), this could not be correlated with a decrease in biomass, since the CV revealed a decrease of only 22% of the total generated biofilm mass . When the drug was added at a later time point (2 h after incubation), the amount of produced biomass in biofilm was reduced by approximately 20%. This decrease could be attributed to the washing step prior to the addition of the drug , an inoculum of 2 × 107 CFU/mL was chosen for all three organisms, due to their rather reduced virulence  twice a day for two doses, followed by 4 mg/kg twice a day /P. boydii (n = 3) isolates was relatively low due to a lack of availability. Similarly, for the in vivo experiments, the larvae were infected with only one isolate of each organism. Further, the invertebrate animal model may also have been impacted by several external factors, such as light sensitivity and weather conditions, which may have impacted the viability of certain larval batches and influenced the reproducibility of the experiment . After washing once with 1× phosphate-buffered saline (PBS), the inoculum was adjusted to an appropriate concentration in RPMI 1640 medium.The fungal suspensions were prepared as described elsewhere . In brieLLCZ was purchased from Sigma-Aldrich . Dimethyl sulfoxide (DMSO) was used for dilution of the drug into stock solutions, which were stored at −20°C until further use. The stock was diluted to various working concentrations, followed by a 1:100 dilution in RPMI 1640 medium. A DMSO concentration of 1% remained in the aliquots. For further information, refer to the corresponding sections below.(i) Broth microdilution. The isolate-specific MIC of LLCZ was determined via broth microdilution according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) ((EUCAST) . LLCZ wa(ii) Growth kinetics. To assess the effect of LLCZ against the planktonic growth of L. prolificans (8 isolates) and S. apiospermum/P. boydii (6 isolates), an XTT-based colorimetric microbroth assay was used . The growth was assessed after 0 h, 6 h, 12 h, 24 h, and 48 h of incubation with the drug. Two hours before the end of incubation, 50 μL of 2.5 mg/mL XTT  plus 125 μM menadione (Sigma) solution was added to each well. Aliquots (150 μL) of the suspensions were transferred from each well onto new 96-well plates with a U-shaped bottom. The OD492 was measured using a microplate reader .was used . The iso(iii) Biofilm assays. The biofilm analysis methods were previously described  of the suspensions were incubated in 24-well plates at 35°C. LLCZ was added at different time points after the initial incubation: 0 h (t0), 2 h (t2) and 48 h (t48). The t0 group was directly incubated with LLCZ for 48 h, while for the t2 and t48 groups, the biofilms were washed with 1× PBS and incubated with LLCZ after initial incubation times of 2 h and 48 h, respectively. Afterward, the biofilms were washed with 1× PBS and analyzed via XTT and crystal violet (CV) assays. For the XTT assay, 500 μL of a solution of XTT (0.5 mg/mL) plus 25 μM menadione was pipetted into each well of the plates, which were then incubated at 36°C for 3 h in the dark. For the crystal violet assay, the biofilms were stained with a 0.01% CV solution for 20 min, air-dried overnight at room temperature, and then incubated for 30 min in a 30% acetic acid solution. Aliquots (100 μL) from each well were transferred into a U-shaped 96-well plate, and the optical density was measured in a plate reader at 492 nm (XTT assay) and 620 nm (CV).escribed , 32, 34.(iv) Confocal laser scanning microscopy. The effect of LLCZ against biofilm formation was visualized via confocal laser scanning microscopy (CLSM) using the Elyra LSM 710 instrument  with a laser at 405 nm and a 20-fold magnification objective. Suspensions were incubated in μ-Slide 8-well glass-bottom plates , as described above. The biofilms were fixed with 100% methanol for 1 min. Images were processed using ZEN black software (Zeiss).Galleria mellonella larva handling.(i) Galleria mellonella  was used as the in vivo model. The standardized protocol for handling of the larvae was previously published (ublished . Briefly(ii) Infection treatment. The treatment of the larvae infected with L. prolificans, S. apiospermum, and P. boydii with a single dose of LLCZ was tested. Based on a previous infection assay , the caterpillars were injected in the prolegs with an inoculum of 2 × 107 CFU/mL from each pathogenic organism. LLCZ was injected at a concentration of 5 mg/kg body weight, equaling 152 mg/L (152 mg/L . In para152 mg/L . The lar(iii) Recultivation of the organisms. Recultivation of the pathogens from dead larvae was performed as previously described (escribed . The deaIn vitro.(i)  All experiments were performed in triplicate. The growth percentage of each treated well was calculated in comparison to that of the untreated control. A statistical analysis of the growth percentage was carried out using the program GraphPad Prism 9 . The statistical significance level was determined using Dunnett’s multiple-comparison test; significance was determined at P < 0.05 .In vivo.(ii)  All experiments were performed in triplicate. The survival data were analyzed using the program GraphPad Prism 9 (GraphPad Software Inc.). Survival curves were plotted using the Kaplan-Meier estimator. To compile the results from multiple repetitions, the raw data were calculated as a single experiment on the datasheet. The statistical significance of the survival curves was determined with a log-rank (Mantel-Cox) test; significance was set at P < 0.05 .