Preterm birth has been related to inequalities in maternal educational level, but the causal mechanism is not entirely known. Some factors associated with preterm birth and low educational level such as chronic medical conditions, pregnancy complications and related-health behaviours could have a mediation role in the pathway. This study aimed to evaluate the association between maternal educational level and preterm birth, analysing the mediation role of these factors. We performed a retrospective cohort study based on hospital electronic records of 10467 deliveries that took place in the Hospital Clínic of Barcelona between 2011 and 2017. Poisson regression was used to obtain crude and adjusted relative risk of preterm birth in women with different educational level and the percentage of change in relative risk was calculated when mediation variables were included in the model. Women with a lower educational level had a higher risk of preterm birth . The loss of association after the inclusion of body mass index in the model suggests an important mediation role of maternal overweight. Other variables such as smoking, drug use, preeclampsia and genitourinary infections also appear to play a role in the observed inequality between women with different levels of education. Efforts to promote health literacy and to improve preventive interventions, before and during pregnancy, could decrease preterm birth rates and perinatal health inequalities Preterm birth is defined as all birth occurring before 37 completed gestational week and it is the leading cause of neonatal mortality worldwide  and of iHeterogeneity in preterm birth rates have been found according to socioeconomic status in several studies and different countries –18. In BMany variables related to higher risk of preterm birth such as substance abuse , overweiWe carried out a retrospective cohort study using the maternity hospital database of the Hospital Clínic of Barcelona. We included women who were followed up during their pregnancy and gave birth at our hospital between January 2011 and December 2017. The Hospital Clínic of Barcelona is a public high complexity hospital that provides care to two different populations. On the one hand, all women living in four districts of Barcelona city are assigned to the Hospital Clínic for their pregnancy follow-up, regardless of their obstetric risk. On the other hand, Hospital Clínic assists women referred due to high-risk pregnancies from other hospitals in semi-urban or rural areas. In order to minimise the selection bias inherent in hospital-based studies, for this study we only included women of the first group, who lived in Barcelona and were a priori assigned to our hospital. We excluded pregnancies terminated before 22 completed gestational weeks, multiple pregnancies and legal terminations of pregnancy after 22 gestational weeks but we included stillbirths as they share causal mechanisms with preterm birth .This study was approved by the Clinical Research Ethical Committee of the Hospital Clínic (HCB/2019/0145).Maternal educational level was collected in the first pregnancy visit and considered in 3 categories: low (without studies and primary studies), medium (secondary level) and high (university or more). Preterm birth was defined as any birth after 22 and before 37 completed gestational weeks. Gestational age, collected from hospital database, was calculated based on the crown-to-rump length at first trimester ultrasound or a combination of last menstrual period and the fetal biometry when first trimester ultrasound was not available. Preterm birth was classified according to its etiopathogenesis in spontaneous or iatrogenic  and according to gestational age thresholds in two groups; less than 34 weeks (moderate and very preterm birth) or 34 weeks or more (late preterm birth).th gestational week. BMI was categorized into underweight (<20kg/m2), normal weight (20–24.9 kg/m2), overweight (25–29.9 kg/m2) and obesity (>30 kg/m2). Pregnant women were asked about smoking, alcohol consumption and drug use at first follow-up visit and any level of consumption was considered. Hypertension and diabetes included the pre-existing conditions and their onset during pregnancy, considering all the ICD-9-CM diagnosis documented. Genitourinary infection included diagnostics of chorioamnionitis, urinary tract infection and vaginitis. Preeclampsia and anemia were considered when appeared during pregnancy. Poor prenatal care was recoded when codified as an ICD-9-CM diagnosis.Information about maternal age at the beginning of pregnancy, mothers’ height and weight, country of birth, neighbourhood of residence and smoking, alcohol and drug use were obtained from electronic medical records. Maternal chronic health conditions and complications during pregnancy were obtained from the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes present in the Minimum Basic Data Set  of SpaniA directed acyclic graph (DAG) , was creor)x100] . FurtherA total of 21437 deliveries took place at our hospital between January 2011 and December 2017. Among these, 11357 belonged to women who lived in the hospital’s area. We excluded 261 multiple pregnancies, 49 legal terminations of pregnancy and 580 women with no information in the exposure variable, leaving a study sample of 10467 singleton pregnancies.Confounder variables identified through the DAG were maternal age at the beginning of pregnancy, maternal region of birth and deprivation index of the neighbourhood as they are related to the exposure and the outcome, but they were not in the causal pathway. We considered BMI, smoking during pregnancy, alcohol consumption, drug use, pregnancy complications such as genitourinary infection, preeclampsia, diabetes, anemia and hypertension and inadequate prenatal care as intermediate variables.The overall rate of preterm birth was 3.5%, which corresponds to 370 preterm deliveries during the study period. Among those, 231 (62.4%) had a spontaneous etiology and 139 (37.6%) were iatrogenic, 121 (32.7%) were infants born before 34 weeks of gestational age and 249 (67.3%) were born at 34 complete weeks or after. The incidence of preterm birth among the 580 women excluded due to missing data on the exposure variable was 17.1%. One thousand eight hundred twenty-six women (17.4%) had a low educational level, 3607 (34.5%) had a medium educational level and 5034 (48.1%) had a high educational level. The prematurity rate was 4.7% among women with a low educational level, 3.7% among those with a medium level and 3.0% among women with higher education. Less than 20% of women with a high educational level were from low-income countries .Women with low educational level showed an increase of 60% in the risk of preterm birth whereas women with medium educational level appeared to have a non-significant increased risk. The increased RR remained when adjusted for confounder variables . In the Regarding the association between educational level and possible mediators adjusting for confounders , we founThe detailed results of the subanalyses by etiology and gestational age are available as supporting information. Briefly, women with a low educational level were at higher risk of spontaneous preterm birth and before the 34 weeks of gestation, with greater risks than those observed in the main analysis. The risk of preterm birth after the 34 weeks was similar than the observed for the whole group of preterm infants. For the iatrogenic etiology, a gradient of inequality was also observed, with an increased risk in less educated women that did not reach statistical significance . The disFirst, several factors may explain the small preterm birth incidence observed in our cohort, which, although low, shows the usual proportion in terms of etiology and gestational age thresholds . On the Women with low educational level have a significant increased risk of preterm birth in comparison to women with a high educational level. Instead, women with medium educational level have a non-statistically significant increased risk. The increased risk seems to be explained by intermediate variables related to both exposure and outcome. The BMI, smoking, drug use, genitourinary infections and preeclampsia were the variables that reduced the RR of preterm birth the most when included in the model.Women with low and medium educational level are more likely to have overweight and obesity in our population, even when adjusted for maternal age, region of birth and deprivation index of the neighbourhood. These inequalities in the social distribution of overweight and obesity have been observed in many studies performed in middle and high-income countries, especially in women , 25. At Smoking was unequally distributed among participants in this study. Women with low and medium educational level had a higher PR of smoking and at the same time, women who smoked during pregnancy showed an increased risk of preterm birth. Drug use followed the same pattern, although its prevalence was lower. Both harmful health behaviours partially explained the RR inequalities observed. Our findings conform with results in other European cohort studies , 39, whiThe onset of preeclampsia and genitourinary infection during pregnancy produced the greatest increase in preterm birth RR in this study. Both factors had a higher incidence in women with a low educational level. In contrast, women with a medium educational level had a higher risk of preeclampsia without an increase of genitourinary infection when compared to women with high educational level.A cohort-study conducted in California observed a mediation role of preeclampsia for the association between socioeconomic level, based on educational level and insurance status, and preterm birth: socioeconomically advantaged white women had a longer gestational period partly explained by a reduction in the risk of preeclampsia , but thiUnlike other studies , 44, 45,Regarding the subanalyses results, statistically significant educational disparities were found in spontaneous preterm birth and both gestational age groups. Although we observed a gradient of inequality for iatrogenic preterm birth, it did not reach statistical significance, consistently with prior findings , even whThe main strengths of this study are the large population and the availability of individual-level data about educational level, used as a proxy indicator of socioeconomic level. Data related to anthropometric measures, substance use, chronic health conditions and pregnancy complications was available and used in the analysis to assess their role in preterm birth inequalities. In order to reduce the possible selection bias in hospital-based studies, women with a different reference health facility who were referred to our high complexity hospital due to high obstetric risk were identified and excluded.Despite data was collected prospectively, cross-sectional data was also included: maternal educational level, confounder variables and some intermediate variables such as BMI, smoking, alcohol consumption and drug use were collected once, during the first follow-up visit. This may limit the interpretation of the results as causality cannot not be established between educational level and these factors. Although maternal region of birth was considered a confounder variable in the analysis, we did not have available data about ethnicity nor about the time since their arrival to the country. Maternal occupation and household income may play a confounder role in the association between maternal education and preterm birth, but they were not available for analysis. Some intermediate variables were considered only when the ICD-9-CM diagnose was assigned by a medical practitioner. Therefore, overrepresentation of most severe cases of some conditions such as preeclampsia or genitourinary infection should be contemplated. Finally, due to the lack of this variable in the database, it has not been possible to assess the mediator role of assisted reproductive techniques, which are more prevalent in women with a high socioeconomic status  and haveFindings of this study suggest that low educational level increases the risk of preterm birth and that some medical conditions and harmful health behaviours more prevalent in social disadvantage women could explain this inequality. The health literacy level, which has been found to be insufficient in almost 60% of Spanish population , may expFirstly, the prevalence of maternal smoking is high in our population, around 13% in women with low and medium educational level, although it does not reach the 20% observed at national level in Spain . MoreoveFurthermore, high risk of preeclampsia and genitourinary infection in women with a lower educational level could be decreased through interventions that promote early detection. Whitney et al  demonstrRegarding the role of BMI, we observed a concerning prevalence of high BMI in women with a low and medium levels of education. Although regional and national clinical guidelines include nutritional counselling for pregnant women, there are no available information about the coverage and outcomes of this health promotion activity . On one In conclusion, we found maternal educational inequalities in preterm birth risk in the study population. These inequalities are explained by factors such as overweight and obesity, smoking, drug use, preeclampsia and genitourinary infections. A better understanding about preterm birth inequalities’ etiology may help clinical practitioners, public health officers and policy makers to find strategies for prevention of preterm deliveries and reduction of perinatal health inequalities.S1 TableRR: Relative Risk; adj: Adjusted for maternal age, maternal region of birth and neighbourhood deprivation index; 95%CI: 95% confidence interval.(PDF)Click here for additional data file.S2 TableRR: Relative Risk; adj: Adjusted for maternal age, maternal region of birth and neighbourhood deprivation index; BMI: body mass index; % change = [(RR without the mediator–RR with the mediator) / (1- RR without the mediator) x 100].(PDF)Click here for additional data file. 12 Dec 2022
 PONE-D-22-22954

 Maternal educational level and preterm birth: Exploring inequalities in a hospital-based cohort study

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Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors demonstrated that educational status may be indeed work as risk factor of preterm birth. This is due to the effects over different covariates including BMI, smoking drugs and genitourinary infections. This is not novel nor surpising but the methods is valid and results are sound. I have a good impression of this work and I'm providing some suggestions for improving the paper before publication:A recent study established a generalized methodology with machine learning for building-up an evidence-based holistic risk assessment for PTB to be used in clinical practice. This evidence should be cited (ref 1) along with the concept that selection of predictive risk facctors is essential along with a hierarchical procedure.A further limitation is lack of etiological differentiation of PTB: iatrogenic vs spontaneous. This etiological dichotomy is particularly prominent in IVF pregnancies which are indeed a high-risk group for preterm birth (ref 2). I reccomend citing this concept and considering this variable for their study. If this is not possible it should be mentioned as a limitation along with the appropriate referencing.recent work showed that phenotypic classification of PTB may provide a better understanding of the etiologic factors and mechanisms associated with preterm birth than continuing to consider it an exclusively time-based entity. (ref 3). This fits with my comment number 2 related to etiology. Do they believe that the model would work better truing to predict different and specific PTB phenotypes/etiology? add a comment on that.The same comments related to etiology (dichotomic) or phenotype (multiple classes) may be applied to gestational age tresholds. Did they try to assess PTB<34 or 28 weeks? What are the results? And the implications? The study would be much stronger if they try to look at different GA tresholds.References1. Della Rosa PA, et al. A hierarchical procedure to select intrauterine and extrauterine factors for methodological validation of preterm birth risk estimation. BMC Pregnancy Childbirth. 2021 Apr 16;21(1):306. doi: 10.1186/s12884-021-03654-3. PMID: 33863296; PMCID: PMC8052693.2. Cavoretto P, et al. Risk of spontaneous preterm birth in singleton pregnancies conceived after IVF/ICSI treatment: meta-analysis of cohort studies. Ultrasound Obstet Gynecol. 2018 Jan;51(1):43-53. doi: 10.1002/uog.18930. PMID: 29114987.3. Villar J, et al. JAMA Pediatr. 2021 May 1;175(5):483-493. doi: 10.1001/jamapediatrics.2020.6087. PMID: 33646288; PMCID: PMC7922239.Reviewer #2: Thank you for this interesting manuscript that provides insight how the effect of sociodemographic factors influence birth outcomes. I have some minor questions1) Could you elaborate on the fact of low incidence of preterm birth in your cohort i.e are results applicable in another population?2) The authors state that all types of preterm birth was included and no further distinction on onset or type was performed. I think it could actually differ between spontaneous and induced onset how much educational level matters as a factor and also how the mediating factors differ. Could you elaborate on this in analysis or if no data is available mention as a limitation if you agree.3) Different approaches  of mediation could be used. I understand that this is based on logistic regression and a sort of attributable factor calculation. Did the authors consider to conduct other type of mediation calculation for example SEM analysis, and how does this analysis differ in the statistical epidemiological approach of the method used here.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: NoReviewer #2: No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  14 Feb 2023Reviewer #1The authors demonstrated that educational status may be indeed work as risk factor of preterm birth. This is due to the effects over different covariates including BMI, smoking drugs and genitourinary infections. This is not novel nor surprising but the methods is valid and results are sound. I have a good impression of this work and I'm providing some suggestions for improving the paper before publication:First of all, the research team would like to sincerely thank you for your time and effort reading and evaluating our work, for your positive assessment and your very interesting suggestions. Your review has allowed us to get to knowledge of recent and relevant bibliography, to add important limitations, to include two subanalyses of the data considering the preterm birth etiology and gestational age thresholds. We consider that all these changes have definitely improved the manuscript. *All line numbers refer to the document “Revised Manuscript Track Changes”1. A recent study established a generalized methodology with machine learning for building-up an evidence-based holistic risk assessment for PTB to be used in clinical practice. This evidence should be cited (ref 1) along with the concept that selection of predictive risk factors is essential along with a hierarchical procedure.Thank you very much for suggesting this study. We found its results very relevant. It has allowed us to reflect in depth on the usefulness of identifying risk factors in clinical practice. We added a sentence about the study in the discussion section.Discussion section, line 359: “Moreover, the identification of most relevant preterm birth risk factors for specific populations may help clinicians when addressing individual patient risk [Ref].”Ref: Della Rosa PA, Miglioli C, Caglioni M, Tiberio F, Mosser KHH, Vignotto E, et al. A hierarchical procedure to select intrauterine and extrauterine factors for methodological validation of preterm birth risk estimation. BMC Pregnancy Childbirth. 2021;21(1):1–17. doi: 10.1186/S12884-021-03654-3.2. A further limitation is lack of etiological differentiation of PTB: iatrogenic vs spontaneous. This etiological dichotomy is particularly prominent in IVF pregnancies which are indeed a high-risk group for preterm birth (ref 2). I recommend citing this concept and considering this variable for their study. If this is not possible it should be mentioned as a limitation along with the appropriate referencing.Regarding the etiological differentiation, we address this issue in the following points (3&4). Regarding the IVF, we fully agree that this is a relevant issue as IVF are more prevalent in women with high socioeconomic status and they are a risk factor of preterm birth. Unfortunately, we do not have the variable informing about “IVF” or “assisted reproductive technique” in our database. We have added the limitation with two relevant citations in “Strengths and limitations” section. Strengths and limitations section, line 347: “Finally, due to the lack of this variable in the database, it has not been possible to assess the mediator role of assisted reproductive techniques, which are more prevalent in women with a high socioeconomic status [Ref1] and have been established as a risk factor of preterm birth [Ref2].”Ref1: Sauer M V. Reproduction at an advanced maternal age and maternal health. Fertil Steril.2015;103(5):1136–43. doi: 10.1016/J.FERTNSTERT.2015.03.004.Ref2: Pandey S, Shetty A, Hamilton M, Bhattacharya S, Maheshwari A. Obstetric and perinataloutcomes in singleton pregnancies resulting from IVF/ICSI: a systematic review and metaanalysis. Hum Reprod Update. 2012;18(5):485–503. doi:3. Recent work showed that phenotypic classification of PTB may provide a better understanding of the etiologic factors and mechanisms associated with preterm birth than continuing to consider it an exclusively time-based entity. (ref 3). This fits with my comment number 2 related to etiology. Do they believe that the model would work better trying to predict different and specific PTB phenotypes/etiology? add a comment on that.4. The same comments related to etiology (dichotomic) or phenotype (multiple classes) may be applied to gestational age thresholds. Did they try to assess PTB<34 or 28 weeks? What are the results? And the implications? The study would be much stronger if they try to look at different GA thresholds.Answers to points 3 & 4: Initially, we aimed to use the etiology of preterm birth (spontaneous vs. iatrogenic) in our analysis, but considering some experts opinion regarding the limitations of the dichotomous classification of preterm birth  we decided not to use it. Even so, we have this variable available, so after reading your comments and considering that both reviewers agreed that it would improve de manuscript, we considered appropriate to perform a subanalysis according to the dichotomic etiology and to point out that recent literature indicates that this classification is somewhat obsolete and citing the work in relation to the identification of more than two phenotypes of preterm birth. Further, we have added a subanalysis according to gestational age thresholds <34 weeks and 34 weeks or more . We have added the complete results of the subanalyses as a supplementary material S1 & S2 Tables, and the following comments in the main article: Methods• Line 97 “Preterm birth was classified according to its etiopathogenesis in spontaneous or iatrogenic  and according to gestational age thresholds in two groups; less than 34 weeks (moderate and very preterm birth) or 34 weeks or more (late preterm birth).”• Line 135 “Further, we performed two subanalyses considering the aetiology of preterm birth, spontaneous or iatrogenic, and the gestational age threshold.”Results• Line 162 “Among those, 231 (62.4%) had a spontaneous etiology and 139 (37.6%) were iatrogenic and 121 (32.7%) were infants born before 34 weeks of gestational age and 249 (67.3%) were born at 34 complete weeks or after.”• Line 220 “The detailed results of the subanalyses by etiology and gestational age are available as supporting information. Briefly, women with a low educational level were at higher risk of spontaneous preterm birth and before the 34 weeks of gestation, with greater risks than those observed in the main analysis. The risk of preterm birth after the 34 week was similar than the observed for the whole group of preterm infants. For the iatrogenic etiology, a gradient of inequality was also observed, with an increased risk in less educated women that did not reach the statistical significance (S1 Table). The distribution and magnitude of risk factors diverged between the subgroups. Overweight and obesity increased the risk of iatrogenic and late preterm birth. Preeclampsia and genitourinary infection were relevant risk factors in all groups, but while preeclampsia was especially prominent in iatrogenic preterm births, genitourinary infection was particularly important in spontaneous preterm birth and preterm infants born before 34 weeks (S1 Table). The mediating role of BMI, smoking, drug use, genitourinary infections and pre-eclampsia in the association between educational level and preterm birth was also observed in the different subgroups, although with different magnitudes. In summary, the factors that accounted for the higher percentage of inequality in women with a low educational level were: infection and smoking in spontaneous preterm births; BMI and preeclampsia in iatrogenic preterm deliveries; the BMI and infection among those born before the 34 weeks of gestational age and the BMI, smoking and preeclampsia among those born after 34 weeks (S2 Table).”Discussion• Line 310 “Regarding the subanalyses results, statistically significant educational disparities were found in spontaneous preterm birth and both gestational age groups. Although we observed a gradient of inequality for iatrogenic preterm birth, it did not reach the statistical significance consistently with prior findings [Ref1], even when adjusted by maternal age, which has been pointed as a possible explanation of the increase in preterm birth rates among socioeconomically advantaged women [Ref2]. The BMI accounted for an important percentage of inequality in infants born before and after the 34 weeks, as well as it reduced the RR observed in iatrogenic preterm birth, but their role was less important in spontaneous preterm birth. This result is in consonance with the findings of Brink et al [Ref1], who observed a lower prevalence of obesity in women who had spontaneous preterm births compared with iatrogenic. Infection was especially relevant in inequalities observed in spontaneous preterm birth and infants born before the 34 weeks, consistently with prior results regarding the role of infection in deprivation and spontaneous and very preterm birth [Ref3]. Finally, it is worth mentioning that the etiopathogenesis classification of preterm birth (spontaneous vs iatrogenic) has proven to be insufficient to explain the complexity of preterm birth syndrome  and a new classification has been proposed, based on twelve preterm birth phenotypes[Ref6] Using this classification in future research could lead to a better understanding of disparities in preterm birth according to its etiology mechanisms.Ref1: Brink LT, Nel DG, Hall DR, Odendaal HJ. Association of socioeconomic status and clinical and demographic conditions with the prevalence of preterm birth. Int J Gynaecol Obstet. 2020;149(3):359. doi: 10.1002/IJGO.13143.Ref2: Joseph K, Fahey J, Shankardass K, Allen VM, O’campo P, Dodds L, et al. Effects of socioeconomic position and clinical risk factors on spontaneous and iatrogenic preterm birth. BMC Pregnancy Childbirth. 2014; 14(117). doi: 10.1186/1471-2393-14-117Ref3: Smith LK, Draper ES, Manktelow BN, Field DJ. Deprivation and infection among spontaneous very preterm births. Obstet Gynecol. 2007;110(2 I):325–9. doi: 10.1097/01.AOG.0000270158.57566.2F.Ref4: Kramer MS, Papageorghiou A, Culhane J, Bhutta Z, Goldenberg RL, Gravett M, et al. Challenges in defining and classifying the preterm birth syndrome. Am J Obstet Gynecol. 2012;206(2):108–12. doi: 10.1016/j.ajog.2011.10.864.Ref5: Goldenberg RL, Gravett MG, Iams J, Papageorghiou AT, Waller SA, Kramer M, et al. The preterm birth syndrome: issues to consider in creating a classification system. Am J Obstet Gynecol. 2012;206(2):113–8. doi: 10.1016/j.ajog.2011.10.865Ref6. Barros FC, Papageorghiou AT, Victora CG, Noble JA, Pang R, Iams J, et al. The distribution of clinical phenotypes of preterm birth syndrome: implications for prevention. JAMA Pediatr. 2015;169(3):220–9. doi: 10.1001/JAMAPEDIATRICS.2014.3040.Reviewer #2Thank you for this interesting manuscript that provides insight how the effect of sociodemographic factors influences birth outcomes. I have some minor questions. First of all, the research team would like to sincerely thank you for your time and effort reading and evaluating our work, for your positive assessment and your very interesting suggestions that have improved the manuscript. Your review has allowed us to think more deeply about some questions, such as the low prevalence of preterm birth in our cohort (which is definitely an issue we must have addressed) and the need to include a subanalyses considering the etiology of preterm birth. *All line numbers refer to the document “Revised Manuscript Track Changes”1. Could you elaborate on the fact of low incidence of preterm birth in your cohort i.e are results applicable in another population? Thank you for this first comment, it is a very relevant point that we had missed. The overall preterm birth rate in our cohort is 3.5%, which is indeed lower than the rate in Barcelona (6.5%) and in Spain (7%). There are several factors that could explain this low prevalence. Although tertiary hospitals usually have higher rates of preterm birth, we excluded from our analysis all these women that have been referred to our hospital because of their high obstetric risk: The Hospital Clinic of Barcelona is a public high complexity hospital that provides care to two different populations. On the one hand, all women living in four districts of Barcelona city are assigned to the Hospital Clínic for their pregnancy follow-up, regardless their obstetric risk. On the other hand, Hospital Clínic assists women referred due to high-risk pregnancies from other hospitals in semi-urban or rural areas. In order to minimise the selection bias inherent in hospital-based studies, for this study we only included women of the first group, who lived in Barcelona and were a priori assigned to our hospital. We have added this explanation in the methods section for a better understanding of the participants’ selection. Considering the above, this low incidence of preterm birth is probably explained by the fact that women without a specific high risk are followed in a high complexity centre where early detection and prevention practices are included in the usual clinical management of pregnancy. Moreover, we have to add that the prevalence of preterm birth would be higher (4.25%) if we had included those women without information in the exposure variable. Among the 580 women who did not have information about the educational level, the rate of preterm birth was 17.1%. Our interpretation is that these women did not attend regular follow-up visits , which is a sign of reduced accessibility to the health system, poor health literacy and low socioeconomic level. We have also included this important information in the manuscript. So, despite of the high standards in clinical practices, socioeconomic disparities are still present in our hospital (and probably they are higher than reported here) and, therefore, we consider that these results are applicable to other populations with similar social disparities, even though they have higher rates of preterm birth. We have included these explanations and interpretations in the following sections: Methods, line 74 “The Hospital Clinic of Barcelona is a public high complexity hospital that provides care to two different populations. On the one hand, all women living in four districts of Barcelona city are assigned to the Hospital Clínic for their pregnancy follow-up, regardless of their obstetric risk. On the other hand, Hospital Clínic assists women referred due to high-risk pregnancies from other hospitals in semi-urban or rural areas. In order to minimise the selection bias inherent in hospital-based studies, for this study we only included women of the first group, who lived in Barcelona and were a priori assigned to our hospital.” Results, line 164 “The incidence of preterm birth among the 580 women excluded due to missing data on the exposure variable was 17.1%.”Discussion, line 241: “First, several factors may explain the small preterm birth incidence observed in our cohort, which, although low, shows the usual proportion in terms of etiology and gestational age thresholds [Ref]. On the one hand, although preterm birth rates in high complexity hospitals are usually above population rates, the selection of participants in this study have attempt to represent general pregnant women population. On the other hand, those women with missing data on the educational level, which were excluded from the analysis, represented a high preterm birth burden. Our interpretation is that these women did not have this information collected because they did not attend the regular follow-up visits, which could be related to reduced accessibility to the health system, poor health literacy level and low socioeconomic status. Therefore, inequalities in our preterm birth rates could be greater than those reported in this study. Finally, the Hospital Clínic is a high complexity centre where early detection and prevention practices are part of the standard of care clinical management of pregnancy, regardless of their obstetric risk. In spite of this, educational disparities are still present in Hospital Clinic’s perinatal outcomes and we consider these results could be applied to other populations with similar social disparities and healthcare resources.”Ref: Goldenberg RL, Culhane JF, Iams JD, Romero R. Epidemiology and causes of preterm birth. Lancet. 2008;371(9606):75–84. doi: 10.1016/S0140-6736(08)60074-42. The authors state that all types of preterm birth were included and no further distinction on onset or type was performed. I think it could actually differ between spontaneous and induced onset how much educational level matters as a factor and also how the mediating factors differ. Could you elaborate on this in analysis or if no data is available mention as a limitation if you agree.Initially, we aimed to use the etiology of preterm birth (spontaneous vs. iatrogenic) in our analysis, but considering some experts opinion regarding the limitations of the dichotomous classification of preterm birth  we decided not to use it. Even so, we have this variable available, so after reading your comments and considering that both reviewers agreed that it would improve de manuscript, we considered appropriate to perform a subanalysis according to the dichotomic etiology and to point out that recent literature indicates that this classification is somewhat obsolete and citing the work in relation to the identification of more than two phenotypes of preterm birth. Further, we have added a subanalysis according to gestational age thresholds <34 weeks and 34 weeks or more . We have added the complete results of the subanalyses as a supplementary material S1 & S2 Tables, and the following comments in the main article: Methods• Line 97 “Preterm birth was classified according to its etiopathogenesis in spontaneous or iatrogenic  and according to gestational age thresholds in two groups; less than 34 weeks (moderate and very preterm birth) or 34 weeks or more (late preterm birth).”• Line 135 “Further, we performed two subanalyses considering the aetiology of preterm birth, spontaneous or iatrogenic, and the gestational age threshold.”Results• Line 162 “Among those, 231 (62.4%) had a spontaneous etiology and 139 (37.6%) were iatrogenic and 121 (32.7%) were infants born before 34 weeks of gestational age and 249 (67.3%) were born at 34 complete weeks or after.”• Line 220 “The detailed results of the subanalyses by etiology and gestational age are available as supporting information. Briefly, women with a low educational level were at higher risk of spontaneous preterm birth and before the 34 weeks of gestation, with greater risks than those observed in the main analysis. The risk of preterm birth after the 34 week was similar than the observed for the whole group of preterm infants. For the iatrogenic etiology, a gradient of inequality was also observed, with an increased risk in less educated women that did not reach the statistical significance (S1 Table). The distribution and magnitude of risk factors diverged between the subgroups. Overweight and obesity increased the risk of iatrogenic and late preterm birth. Preeclampsia and genitourinary infection were relevant risk factors in all groups, but while preeclampsia was especially prominent in iatrogenic preterm births, genitourinary infection was particularly important in spontaneous preterm birth and preterm infants born before 34 weeks (S1 Table). The mediating role of BMI, smoking, drug use, genitourinary infections and pre-eclampsia in the association between educational level and preterm birth was also observed in the different subgroups, although with different magnitudes. In summary, the factors that accounted for the higher percentage of inequality in women with a low educational level were: infection and smoking in spontaneous preterm births; BMI and preeclampsia in iatrogenic preterm deliveries; the BMI and infection among those born before the 34 weeks of gestational age and the BMI, smoking and preeclampsia among those born after 34 weeks (S2 Table).”Discussion• Line 310 “Regarding the subanalyses results, statistically significant educational disparities were found in spontaneous preterm birth and both gestational age groups. Although we observed a gradient of inequality for iatrogenic preterm birth, it did not reach the statistical significance consistently with prior findings [Ref1], even when adjusted by maternal age, which has been pointed as a possible explanation of the increase in preterm birth rates among socioeconomically advantaged women [Ref2]. The BMI accounted for an important percentage of inequality in infants born before and after the 34 weeks, as well as it reduced the RR observed in iatrogenic preterm birth, but their role was less important in spontaneous preterm birth. This result is in consonance with the findings of Brink et al [Ref1], who observed a lower prevalence of obesity in women who had spontaneous preterm births compared with iatrogenic. Infection was especially relevant in inequalities observed in spontaneous preterm birth and infants born before the 34 weeks, consistently with prior results regarding the role of infection in deprivation and spontaneous and very preterm birth [Ref3]. Finally, it is worth mentioning that the etiopathogenesis classification of preterm birth (spontaneous vs iatrogenic) has proven to be insufficient to explain the complexity of preterm birth syndrome  and a new classification has been proposed, based on twelve preterm birth phenotypes [Ref6]. Using this classification in future research could lead to a better understanding of disparities in preterm birth according to its etiology mechanisms.Ref1: Brink LT, Nel DG, Hall DR, Odendaal HJ. Association of socioeconomic status and clinical and demographic conditions with the prevalence of preterm birth. Int J Gynaecol Obstet. 2020;149(3):359. doi: 10.1002/IJGO.13143.Ref2: Joseph K, Fahey J, Shankardass K, Allen VM, O’campo P, Dodds L, et al. Effects of socioeconomic position and clinical risk factors on spontaneous and iatrogenic preterm birth. BMC Pregnancy Childbirth. 2014; 14(117). doi: 10.1186/1471-2393-14-117Ref3: Smith LK, Draper ES, Manktelow BN, Field DJ. Deprivation and infection among spontaneous very preterm births. Obstet Gynecol. 2007;110(2 I):325–9. doi: 10.1097/01.AOG.0000270158.57566.2F.Ref4: Kramer MS, Papageorghiou A, Culhane J, Bhutta Z, Goldenberg RL, Gravett M, et al. Challenges in defining and classifying the preterm birth syndrome. Am J Obstet Gynecol. 2012;206(2):108–12. doi: 10.1016/j.ajog.2011.10.864.Ref5: Goldenberg RL, Gravett MG, Iams J, Papageorghiou AT, Waller SA, Kramer M, et al. The preterm birth syndrome: issues to consider in creating a classification system. Am J Obstet Gynecol. 2012;206(2):113–8. doi: 10.1016/j.ajog.2011.10.865Ref6. Barros FC, Papageorghiou AT, Victora CG, Noble JA, Pang R, Iams J, et al. The distribution of clinical phenotypes of preterm birth syndrome: implications for prevention. JAMA Pediatr. 2015;169(3):220–9. doi: 10.1001/JAMAPEDIATRICS.2014.3040.3. Different approaches  of mediation could be used. I understand that this is based on logistic regression and a sort of attributable factor calculation. Did the authors consider to conduct other type of mediation calculation for example SEM analysis, and how does this analysis differ in the statistical epidemiological approach of the method used here.Thank you for this comment. From the beginning we proposed this type of analysis, inspired by other articles on social inequalities that used this method, which we find easy to interpret, in the sense of being able to observe how the relative risk changes when adding the different risk factors and to understand the underlying process. Regarding the SEM-based mediation analysis, we could certainly have used it to compare the proportion of mediated effect obtained through SEM and the % of change obtained through this method. We decided to use the percentage of reduction in de RR in order to make our results more comparable with other studies in the field. However, we are now aware of the increasing use of SEM in the field and therefore will definitely consider this approach in future projects.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 20 Mar 2023Maternal educational level and preterm birth: Exploring inequalities in a hospital-based cohort studyPONE-D-22-22954R1Dear Dr. Granés,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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Electric injuries (in the form of lightning or electric shock) may lead to various implications in the human body, the most important of which include neurological insults. The damage caused is influenced by the route of its entry into the body, its strength, and the duration of exposure. The muscles of the larynx receive motor supply from the recurrent laryngeal nerve (RLN) . Recurrent laryngeal nerve (RLN) palsy leading to vocal cord palsy is seen in several pathologies, but after thorough research of existing literature, we could only find a single case of vocal cord palsy following electric injuries, which was also lost in follow-up. In this report, we present a case of unilateral vocal cord palsy following an electric injury on the ipsilateral arm of a young male. He presented to the emergency department of our center soon after the accident. A multidisciplinary team was engaged in the overall management of the patient . He was started on steroids, speech therapy, and other supportive management. On follow-up, his condition improved, and laryngeal endoscopy showed positive signs. This case highlights a unique but rare possibility of vocal cord palsy following electric injuries and may help in the prompt diagnosis and management of the same. Electric current, when passing through the body, may interfere with the normal functioning of the internal organs and may burn susceptible tissues. The severity of the injury is influenced by the pathway through which the current enters the body, the duration of its exposure, and the resistance faced by tissues . Such elWe hereby present a unique case report from an accident in hilly and remote areas of Uttarakhand, India, where a high-voltage wire  fell on an under-construction metallic bridge. Several people were struck, and a few lost their lives or got severely injured. A 35-year-old, previously healthy male also got trapped on the bridge, and his left arm got scorched by the electric current. After the accident, he fell unconscious and was brought to the emergency room of our institute, where he was initially resuscitated and treated for burn injuries. He acquired a third-degree asymmetrical burn on the lateral aspect of his left arm, accounting for approximately 2% of his body surface area. He subsequently developed acute kidney injury, which was probably the consequence of degraded blood products in his urine. He developed mild difficulty breathing, which was evaluated, and he was found to have bilateral pleural effusion along with ascites. The nephrology, neurosurgery, and burns and plastic surgery teams were involved in the multidisciplinary management of the patient. Meanwhile, he started complaining of a mild sore throat and a change in his voice (hoarseness) the next day. He was thoroughly examined, and on laryngeal endoscopy , which supplies all the muscles of the larynx (except the cricothyroid), is a branch of the 10th cranial nerve, the vagus. On the right side, it arcs around the right subclavian artery, and on the left, around the aorta, making it more susceptible to injury owing to its long course. Insults to the vagus or RLN during its course can lead to vocal cord paralysis. Patients with a single-sided immobile vocal cord may be asymptomatic in as many as 30%-50% of cases. The most frequent causes of RLN palsy are tumors, surgeries, idiopathy, and post-intubation [Injuries due to electric current can be explained in three different ways. The first probable mechanism is the tissue destruction and coagulative necrosis caused by the conversion of electric energy into thermal energy. The second mechanism proposed is the alteration of cell membrane potential and tetanization caused by direct damage due to current. The last is the indirect injuries by trauma as a result of falls of surrounding objects/violent contraction of muscles .Neurological deficits are not uncommon following electrocution and manifest in different forms. After extensive research of previously available literature, only a single case of bilateral vocal cord palsy was found. The unique case of unilateral vocal cord palsy after electric shock injury thus opens the gateway for unexplored, but further intertwined loops in the medical sciences.The patient reported here sustained an electric injury that entered his body through his left upper arm, and the electric current, in a very possible way, traveled in a retrograde fashion to his upper torso, including the head and neck region. As the nervous system has low resistance to the flow of external electric current, it is more susceptible to electrothermal injuries. This exposure to an unnatural and aberrant electric current leads to hypoxic and vascular compromise in the neurons, which leads to cell death. Another postulated mechanism of nerve injury is the demyelinating injury caused by free radical injury, which leads to delayed impairment .For further investigations, laryngeal electromyography (LEMG) could be deployed to distinguish the cause of palsy to be hidden in upper/lower motor neurons, peripheral nerves, neuromuscular junctions, muscle fibers, cartilages, or joints , althougAny case of hoarseness of voice or sore throat in a case of electrocution needs to be evaluated with a detailed history and laryngeal examination. After ruling out all possible anatomical/organic causes for recurrent laryngeal nerve palsy, a course of systemic steroids can be started along with speech therapy and other supportive management. Surgical intervention (thyroplasty) may be advocated in cases that do not respond adequately to medical management. Unilateral vocal cord palsy following electrocution may add a unique entity to the existing literature and needs to be further explored with the help of technological advancements.


Bolsa Família cash transfer program with birth weight–related outcomes.This cohort study investigates the association of participation in the  Bolsa Família Program (BFP) associated with improved birth weight–related outcomes?Is the This cohort study of 4 277 523 live births found that BFP participation was associated with a reduction in low birth weight and an increase in birth weight. Outcomes were more pronounced among groups at increased risk, with a greater decrease in odds of low birth weight found among mothers with fewer years of education and indigenous mothers.These findings suggest that BFP may be associated with improved birth weight–related outcomes and decreased birth weight inequalities. There is limited evidence of the association of conditional cash transfers, an important strategy to reduce poverty, with prevention of adverse birth-related outcomes.Bolsa Família Program (BFP) and birth weight indicators.To investigate the association between receiving benefits from the This cohort study used a linked data resource, the Centro de Integracao de Dados e Conhecimentos Para Saude (CIDACS) birth cohort. All live-born singleton infants born to mothers registered in the cohort between January 2012 and December 2015 were included. Each analysis was conducted for the overall population and separately by level of education, self-reported maternal race, and number of prenatal appointments. Data were analyzed from January 3 to April 24, 2023.Live births of mothers who had received BFP until delivery (for a minimum of 9 months) were classified as exposed and compared with live births from mothers who did not receive the benefit prior to delivery.Low birth weight (LBW), birth weight in grams, and small for gestational age (SGA) were evaluated. Analytical methods used included propensity score estimation, kernel matching, and weighted logistic and linear regressions. Race categories included Parda, which translates from Portuguese as “brown” and is used to denote individuals whose racial background is predominantly Black and those with multiracial or multiethnic ancestry, including European, African, and Indigenous origins.A total of 4 277 523 live births  were assessed. BFP was associated with an increase of 17.76 g  in birth weight. Beneficiaries had an 11% lower chance of LBW . BFP was associated with a greater decrease in odds of LBW among subgroups of mothers who attended fewer than 7 appointments , were Indigenous , and had 3 or less years of education . There was no association between BFP and SGA, except among less educated mothers, who had a reduced risk of SGA .This study found that BFP was associated with increased birth weight and reduced odds of LBW, with a greater decrease in odds of LBW among higher-risk groups. These findings suggest the importance of maintaining financial support for mothers at increased risk of birth weight–related outcomes. It has more than 13 million beneficiary families per year.31 Although Brazil was one of the pioneers in implementing CCTs in Latin America and there has been some evaluation of the association of this program with child health,26 there is still a lack of evidence to support an association of the BFP with birth weight indicators.The Our objective was to estimate the effectiveness of PBF, focusing on its potential association with a decreased likelihood of LBW and SGA, as well as improved birth weight (in grams). It is recognized that the association of BFP with birth weight indicators may vary by population subgroup.The Research Ethics Committee of the Institute of Collective Health, Federal University of Bahia approved the protocol for this cohort study and waived informed consent because this study uses electronic data without any personally identifiable information. The Reporting of Studies Conducted Using Observational Routinely Collected Health Data (RECORD) statement has been followed.32 from 2012 to 2015 among mothers aged 10 to 49 years who were registered on CadÚnico  at any time from 2004 to 2015  and (2) multiple births and newborns with congenital anomalies (given that these conditions are associated with adverse birth weight indicators35).The eligible study population consisted of children from live births in the Centro de Integracao de Dados e Conhecimentos Para Saude (CIDACS) Birth Cohort to 2015 . Births  to 2015 . Our sel36 Low-income families  are eligible for the BFP if they include at least 1 individual from a priority group, such as pregnant individuals, breastfeeding mothers, children, or children or adolescents aged 0 to 17 years.36 Ideally, cash payments are directed toward women, contingent on the fulfillment of specific program requirements .36 These criteria encompass the necessity for consistent school attendance and use of health care services throughout childhood (including maintaining an up-to-date vaccination schedule), during pregnancy , and in the postpartum period.37 Further details on eligibility criteria and program characteristics are described in eAppendix 2 in Live births were classified as being exposed to BFP if the mother started receiving BFP at any time during the cohort period, considering an exposure window of at least the estimated period of a complete pregnancy (9 months), without interruptions. Mothers who discontinued receipt were not considered in the analysis. Newborns of mothers who did not receive BFP at any time before delivery were classified as unexposed. BFP eligibility criteria are CadÚnico registered family per capita income and family composition . Families with a monthly per capita income of up to R $89.00  are considered extremely low income and eligible, independent of their composition.38 linked to the Live Birth Information System (SINASC) from January 1, 2004, to December 31, 2015. The cohort database contains records of 114 001 661 individuals (40 542 929 families) with low income eligible for social assistance programs via CadÚnico. This linkage constitutes the CIDACS Birth Cohort (a subset of the 100 Million Brazilian Cohort).32 All data sets were evaluated with deidentified, linked data .Our outcomes were birth weight categorized as LBW (<2500 g) and non-LBW , birth weight in grams, and SGA  and appropriate for gestational age . Newborn size was defined by sex-specific curves corresponding to single live births as established by the International Fetal and Newborn Growth Consortium for the 21st Century (INTERGROWTH-21st) Consortium40 PS estimation was performed using complete data . The descriptive analysis based on missing data is available in eTables 1-4 and eAppendix 4 in 41 A 2-sided P value < .05 indicated statistical significance.We estimated the association of BFP with birth-related indicators using propensity score (PS)–based methods. Analyses were described in detail in the research protocol.42 as an alternative approach : gestational age, sex of live-born infant, maternal age at birth, and type of delivery. Adjusted risk ratio was calculated using the δ method to verify discrepancies in the odds ratio (OR) , educational level , and attendance at prenatal appointments (<7 and ≥7 appointments). All PSs were estimated separately for each population subgroup, with the variable defining the subgroup excluded from the calculation. Similarly, kernel-weighted logistic and linear models were calculated overall and separately within each population subgroup  were to nonbeneficiary mothers, and 2 799 824 births (65.4%) were to mothers who received BFP. Mean (SD) and median (IQR) birth weight among mothers who received BFP  were higher than among mothers who did not receive BFP  . OccurreAccording to PS variables, differences between beneficiaries and nonbeneficiaries were minimized after weighting  . BFP wasConsidering the frequency of prenatal care appointments attended, BFP participation was associated with a greater reduction in odds of LBW  and greater increase in birth weight  among mothers who attended fewer than 7 appointments . EstimatIn this cohort study, we found that BFP participation was associated with reduced chances of LBW and an increase in birth weight in grams. BFP participation was associated with a greater decrease in odds of LBW and increase in birth weight in grams among higher-risk population subgroups classified in our study: mothers who attended fewer than 7 antenatal care appointments; were Black, Indigenous, or Parda; and less educated . An association between BFP participation and decreased odds of SGA was found among Indigenous mothers and those with less education.47 However, we also explored the association of BFP with outcomes in pregnant individuals from different social and ethnic subgroups, showing greater changes in outcomes among the highest-risk groups. In addition, use of information on previous childbirths enabled adjustment for birth intervals, previous LBW, and previous prematurity.48 Although CCTs have an association with an increased interval between births,49 the association with beneficiary fertility among mothers is controversial.50 The first pregnancy and grand multiparity are risk factors for LBW and SGA.19Our findings are consistent with those of a previous study that examined 100 Million Brazilian Cohort data and other studies that evaluated the effect of CCTs on birth weight.45 A randomized study conducted in rural villages in Togo, West Africa, found that receiving a UCT reduced the chance of having a baby with LBW .46 In Colombia, a study on the Familias en Acción program showed a 578-g increase in birth weight in urban treatment locations.47 Increased birth weight in the US Food Stamp Program  provides further evidence that prenatal nutritional intake may play a role in child birth outcomes.51 In the US, Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) services have also been associated with reduced LBW and increased birth weight in grams, especially among subgroups of Black women and those with late prenatal care or no prenatal consultation.52The magnitude of outcomes associated with other CCTs and UCTs has varied by program characteristic. A study of the effectiveness of the Oportunidades program, a CCT implemented in Mexico, demonstrated a 127-g increase in mean weight at birth among beneficiary children and a 4.6% lower prevalence of LBW in this group.54 In the US, poverty relief during the prenatal period (an income tax credit) was associated with an increase in birth weight of 15.7g (12.5 g when adjusted for smoking).53 In retrospective cohort studies, cash transfers during the prenatal period provided to women with lower incomes who were residents in a municipality of Canada  were associated with a 26%55 and 29%56 lower risk of LBW.Cash transfer strategies are also implemented in high-income countries.55To the best of our knowledge, this is the first study to evaluate the association of CCT with weight standards at birth by gestational age in a high-risk Brazilian population. In a study in Canada, the only study found that assessed the association of a CT  with SGA, an association was found with a decrease in SGA births .56 psychosocial health,57 increased social capital, and female decision-making power.58 The second is the association of conditionalities with outcomes and the benefits provided by integrated, health-related actions.61The difference between CT designs may explain the variability reported in estimates (heterogeneity of findings). We may consider 2 hypotheses for the mechanisms behind the association of CCTs with birth weight outcomes. First, CTs enable the family to diversify the food purchased , which is associated with family food security indicators,62 These groups include individuals experiencing more deprivation with greater difficulty in accessing this service. Our investigation found associations between BFP and increased birth weight and decreased odds of LBW within specific subgroups. These subgroups included mothers who attended fewer prenatal appointments; those who were Black, of mixed race, and Indigenous; and those with lower levels of education. Furthermore, our study found that BFP beneficiaries had lower odds of SGA only in subgroups of Indigenous mothers and those with lower education levels. These results suggest that beneficiaries and nonbeneficiaries may be more homogeneous in relation to characteristics not observed in subgroups of mothers at higher risk. The group with a lower number of prenatal consultations likely consisted of individuals who faced greater challenges in accessing this service, particularly those with lower socioeconomic status. Education was the only variable that was not well-balanced between groups. Therefore, disparities persisted, with a higher percentage of mothers in the group with fewer consultations having low levels of education. These findings are consistent with those of a recent review on CCTs and child health in low- and middle-income countries showing that these programs exhibited considerable heterogeneity among subgroups by socioeconomic status indicator.30Despite the significant increase in attending prenatal appointments in Brazil between 2000 and 2015, inequality remains pronounced, particularly among Black and Indigenous women and those with a lower level of education.53 The difficulty of reducing birth weight–related outcomes indicates the need to intensify policies with this focus.6 Thus, there is a need to strengthen social, redistributive, and health policies that act on the negative consequences of inequalities, seeking to minimize their effects on health, striving for food and nutritional security, prenatal care, and assistance during labor.12LBW, as a result of poverty, can contribute to worse health status over time and consequently maintain inequality from generation to generation.40 providing data analysis transparency and greater result comparability. Several strengths can be highlighted in this study. The population-level database encompasses a wide range of socioeconomic variables at family and personal levels and a variety of risk factors, which are rarely available in administrative data. A robust analytical approach using kernel-based PS weighting and IPTW was used to account for observed confounding factors in the study. Beneficiary and nonbeneficiary groups were well-balanced for covariate distributions.This study used PS-based approaches to evaluate the association of BFP with maternal-child health results in a population of low-income and extremely low-income Brazilian families. The study followed a previously defined and published research protocol,26 which dealt with the issue in a similar way to our study, by following a kernel matching approach to select a set of nonbeneficiary BFP observations within the CIDACS 100 Million Brazilian Cohort. This method enabled us to balance groups by observable characteristics. The external validity of the study was affected by the population choice given that we considered only 1 child per mother. BFP is a binary variable in our study, and this proposal did not investigate nuances related to the value received and poverty levels. Another limitation of this study is the bias related to unmeasured confounding. Important unmeasured factors should be considered, particularly family income, which could not be included in this study. Moreover, we were unable to investigate the distribution of some established biological risk factors associated with LBW and SGA, including chronic diseases, gestational weight gain, prepregnancy body mass index, smoking, and drug use among BFP and non-BFP groups. Another important limitation of our study is that we exclusively focused on live births. Consequently, stillbirths and spontaneous abortions were not taken into consideration. Nevertheless, it is plausible that outcomes associated with these factors are attenuated when analyzing the association of BFP with birth weight indicators in more homogeneous subgroups.Several limitations should also be considered. Receiving BFP is not a random attribution but the result of a self-selection process by families. A BFP selection bias was reported in another study,This cohort study found that BFP participation was associated with improved birth weight indicators. The magnitude of the improvement was greater in higher-risk groups. These findings contribute to the scope of literature evaluating integrative policies and highlighting the importance of maintaining financial support for high-risk mothers. We emphasize the importance of reducing barriers to access and use of health services. Future studies may also assess the quality of prenatal care provided to socioeconomically high-risk populations. We also highlight the importance of evaluating the association between BFP participation and the occurrence of stillbirths, abortions, and infant survival. We highlight that our evidence is associative. However, our contribution is robust and adds data to literature on the association of CCTs with maternal-child health.


MT2A), using RNA sequencing. We found that silencing MT2A partially reversed the inhibitory effect of IPFSC-EVs on fibroblast proliferation in the inflammatory environment. In conclusion, IPFSC-EVs inhibit the progression of knee arthrofibrosis by regulating MT2A, which inhibits fibroblast proliferation in the inflammatory environment.Knee arthrofibrosis is one of the most serious complications of knee surgery; however, its pathogenesis is unclear, and current treatment methods have not achieved satisfactory results. Mesenchymal stem cells (MSCs) have good anti-inflammatory and antifibrotic properties, and studies have reported that human infrapatellar fat pad-derived MSCs (IPFSCs) have the advantages of strong proliferative and differentiating ability, ease of acquisition, and minimal harm to the donor. Increasing evidence has shown that MSCs function through their paracrine extracellular vesicles (EVs). Our study is aimed at exploring the effects of human IPFSC-derived EVs (IPFSC-EVs) on knee arthrofibrosis and the underlying mechanisms in vivo and in vitro. In the in vivo study, injecting IPFSC-EVs into the knee joint cavity effectively reduced surgery-induced knee arthrofibrosis in rats. In the in vitro study, IPFSC-EVs were found to inhibit the proliferation of fibroblasts in the inflammatory environment. Additionally, we screened a potential IPFSC-EV molecular target, metallothionein 2A ( Knee arthrofibrosis following knee surgery is a major challenge for most orthopedic surgeons, manifesting primarily as recurrent joint pain, knee contracture, and limitations in flexion and extension functions, all of which substantially deteriorate patients' postoperative quality of life . DespiteAt present, the mechanism of arthrofibrosis following knee surgery is unclear. It is believed that the main causes of knee arthrofibrosis are excessive fibroblast proliferation and extracellular matrix production in response to various inflammatory cytokines and growth factors , 4. TherMany strategies for preventing arthrofibrosis in animals have been developed in recent years, including topical applications of hydroxycamptothecin, mitomycin C, and artesunate –9. HowevStudies have reported that mesenchymal stem cells (MSCs) have positive therapeutic effects on fibrotic diseases, such as pulmonary, hepatic, and renal fibrosis –13. Howeα) and inhibited the degree of postoperative knee arthrofibrosis. Using transcriptomic sequencing, we screened metallothionein 2A (MT2A), a potential target gene of IPFSC-EVs, for its ability to inhibit knee arthrofibrosis. Studies have shown that MT2A plays an important role in regulating cell proliferation and inflammatory responses [MT2A in this process is expected to provide new intervention targets for treating fibrotic diseases in clinical practice.In this study, we established a rat model of surgery-induced knee arthrofibrosis, injected IPFSC-EVs into the knee joint cavity, and collected specimens for histological preparation 4 weeks after surgery. Using hematoxylin and eosin staining, Masson staining, and immunohistochemistry, we found that IPFSC-EVs reduced the expression of proinflammatory factors (IL-6 and TNF-esponses –27. Thern = 12 per group): a phosphate-buffered saline (PBS) control group and three IPFSC-EV intervention groups . The IPFSC-EV concentrations used were selected based on a previous study [The study was approved by the Animal Ethics Committee of Yangzhou University. A total of 48 male Sprague–Dawley rats weighing 250–300 g were purchased from the Experimental Animal Center of Yangzhou University . Before the experiment, all rats were carefully housed in the animal room at a constant temperature (24 ± 0.5°C) and humidity (50%–60%). The rats were randomly divided into four groups  was administered for 3 consecutive days after surgery to prevent infection. From the first postoperative week, 10 μL of IPFSC-EVs at various concentrations  was injected into the knee joint cavity under ultrasound guidance. The control group was injected the same dose of PBS twice a week for 4 weeks.The knee arthrofibrosis model was established as described in a previous study . Brieflyα) and fibrosis marker (α-SMA) in the fibrotic tissues were also observed via immunohistochemistry.Rats were euthanized 4 weeks after establishing the model, following which the knee joint specimens were collected for histological analysis. The specimens were fixed in 4% paraformaldehyde for 1 week, fully decalcified in ethylenediaminetetraacetic acid, and embedded in paraffin. Subsequently, the four groups of specimens were cut into sections and stained with hematoxylin and eosin to observe the degree of fibrosis. The density and content of collagen (collagen I and III) in the fibrotic tissues were observed via Masson staining and immunohistochemistry, respectively. The content of proinflammatory cytokines  at 37°C for 10 h. The cell suspension was then filtered through a 40 μM cell strainer and resuspended in DMEM supplemented with 10% FBS and 1% PS.The human fibroblast cell line was purchased from ScienCell Research Laboratories . Fibroblasts were cultured in Dulbecco's Modified Eagle Medium  supplemented with 10% fetal bovine serum  and 1% penicillin–streptomycin  in a humidified atmosphere with 5% CO7 cells/mL. Anti-CD90, anti-CD44, anti-CD105, and anti-CD73 antibodies were then added separately to the cell suspension. After incubation at room temperature for 30 min in the dark, the cells were detected using flow cytometry.IPFSC surface antigens were detected using the Human MSC Analysis Kit  according to the manufacturer's instructions. After reaching 90% confluency, the cells were digested with trypsin, and the cell concentration was adjusted to 10This experiment was performed using the human adipose MSC induction and differentiation kit  strictly according to the manufacturer's instructions. The osteogenic, adipogenic, and chondrogenic induction media were prepared in advance, and the induction was performed for up to 3 weeks. Mineral content of the cultures was determined using Alizarin Red staining, endoacidic mucopolysaccharide content in cartilage tissue was determined using Alician Blue staining, and lipid accumulation was determined using Oil Red O staining.After reaching 60% confluency, the IPFSCs were washed twice with PBS. The cell culture medium was then replaced with serum-free medium for EVs . After culturing for 48 h, the conditioned IPFSC medium was collected to isolate the EVs, which were extracted using ultracentrifugation. The morphology of IPFSC-EVs was observed using transmission electron microscopy. The particle size distribution and concentration of IPFSC-EVs were determined using the NanoSight LM10 instrument . Protein markers of EVs were detected by western blot, including CD63 and CD81. Fibroblasts were incubated with PKH26- (red) labeled IPFSC-EVs, and the uptake of IPFSC-EVs by fibroblasts was observed under a confocal laser scanning microscope.β. The fibroblasts were treated with 10 ng/mL of IL-6 and IPFSC-EVs at various concentrations  for 24 h. Subsequently, 10 μL of Cell Counting Kit-8 (CCK-8) buffer was added to each well, and the plate was incubated at 37°C for 2 h. The absorbance of cells was measured at 450 nm using a microplate reader.The fibroblast suspension was added to a 96-well plate at a density of 5000 cells/well. To simulate the cellular inflammatory environment, the inflammatory fibroblast model was established using interleukin (IL)-19 particles/mL) for 24 h, and total RNA was extracted using TRIzol . After the RNA samples had passed the quality control, mRNAs with polyA tails were enriched using Oligo (dT) magnetic beads. Next, a cDNA library was created, and after it was qualified, on-machine sequencing was performed, with the DESeq algorithm used to calculate differentially expressed genes. Using |log2(FoldChange)| > 0 and Padj < 0.05 as the screening conditions, a volcano plot of genes differentially expressed between the IPFSC-EV and control groups was constructed. Subsequently, Gene Ontology (GO) analysis was performed to help elucidate the biological implications of the differentially expressed genes, including biological processes, cellular composition, and molecular functions, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to identify significantly enriched pathways affected by the differentially expressed genes. Combined with the specific functions of these genes, the molecular target of IPFSC-EVs was identified.Fibroblasts in the inflammatory environment were treated with IPFSC-EVs (109 particles/mL) for 24 h in an inflammatory environment, washed with PBS, and then fixed in 70% ice-cold ethanol overnight. The fibroblasts were then rewashed with PBS, and each sample was resuspended in 0.5 mL of propidium iodide and allowed to stand for 30 min in the dark. The samples were then assessed using flow cytomtery, and the data were analyzed using FlowJo software.This experiment was performed using the Cell Cycle Detection kit . Fibroblasts were treated with IPFSC-EVs (109 particles/mL) for 24 h, 50 μM of EdU working fluid was added to each well, following which the wells were allowed to stand for 2 h. The cells were fixed with 4% paraformaldehyde for 30 min and then permeabilized with PBS containing 0.5% Triton X-100. After counterstaining with Hoechst 33342 reaction solution, EdU-positive cells and nuclear staining were observed under a fluorescence microscope.This assay was performed using the EdU Cell Proliferation Kit (keyGEN BioTECH). Fibroblasts in the inflammatory environment were treated with IPFSC-EVs  overnight at 4°C, followed by incubation with the corresponding secondary antibodies for 2 h at room temperature. Finally, protein signals were detected using chemiluminescence by visualizing the membranes under the BIO-RAD ChemiDoc XRS imaging system.A western blot was performed as previously described . Brieflyβ (10 ng/mL) and IPFSC-EVs (109 particles/mL) for 24 h, and total RNA was extracted using TRIzol , after which cDNA was synthesized using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit . Subsequently, RT–PCR was performed using the AceQ Universal SYBR qPCR Master Mix kit . Gene expression levels were determined using the 2−∆∆CT method. The primer sequences used for RT–PCR are listed in Fibroblasts were treated with IL-1MT2A. Fibroblasts were incubated with the lentiviral vector for RNA interference (RNAi) of MT2A (LVMT2A) and negative control RNAi  at a multiplicity of infection of 20 for 16 h according to the manufacturer's instructions. After transfection for 72 h, the transfection efficiency was determined using fluorescence microscopy. Lentiviral-transfected fibroblasts were cultured with 2 μg/mL puromycin  for 48 h. RT–PCR and western blot analyses were performed to determine the gene and protein expression levels of MT2A, respectively. Stably transfected fibroblasts with silenced MT2A were used for subsequent experiments.Lentiviral-mediated permanent transfection was performed to silence t-test, whereas multiple-group comparisons were performed using one-way analysis of variance. A P value of <0.05 was considered statistically significant.The data for this study are presented as mean ± standard deviation (SD). The statistical analysis was performed using the SPSS 19.0 statistical software. Pairwise comparisons were performed using Student's 10 particles/mL  into the knee joint cavity to observe whether IPFSC-EVs could delay the progression of knee arthrofibrosis. Hematoxylin and eosin staining revealed that the control group had dense fibrotic tissues, and the IPFSC-EV groups exhibited significantly less degree of fibrosis than the control group. With increasing IPFSC-EV concentration, the degree of fibrosis in the knee joint cavity was gradually reduced. Similarly, Masson staining revealed that the collagen content in the knee joint cavity gradually decreased with the increase in IPFSC-EV concentration (α) and fibrosis marker (α-SMA) in a concentration-dependent manner  to create a cellular inflammatory model. The model's reliability was validated using RT–PCR, and results showed that the cellular inflammatory model could promote the expression of inflammatory cytokines (IL-6 and TNF-α) (β inflammatory environment could promote the expression of fibroblast proliferation-related genes (CCND1 and PCNA)  . We alsond PCNA) . In othey effect . Therefo9 particles/mL) in the inflammatory environment for 24 h and then performed transcriptomic sequencing (RNA-seq). The volcano plot revealed differentially expressed genes between the IPFSC-EV and control groups  were partially reversed , which supported the hypothesis that high MT2A expression could promote breast cancer cell proliferation [MT2A expression differs significantly among cells derived from tissues of various diseases, and its effect on the proliferation of tissue-derived cells also differs considerably. Therefore, our findings might thus provide a foundation for future research.Human metallothioneins (MTs) are a class of low-molecular-weight proteins that are widely present in mammals, higher plants, and certain prokaryotes and are a large family of cysteine-rich molecules . MTs havr pylori –52. MTs feration , 54. MT2feration . These rα) and fibrosis marker (α-SMA). The above results suggested that IPFSC-EVs had the potential to inhibit the progression of knee arthrofibrosis.In the in vivo study, we established a model of surgery-induced knee arthrofibrosis in rats. The histological analysis of knee fibrotic tissues stained with hematoxylin and eosin indicated that IPFSC-EVs could significantly reduce fibrosis and the number of fibroblasts. It is well known that excess collagen deposition is a key factor in the formation of fibrosis. Masson staining was performed to detect the collagen content in fibrotic tissues, which revealed that the collagen content was significantly reduced in the IPFSC-EVs groups compared with the control group. In addition, the expression of collagens I and III was detected using immunohistochemistry. Our findings indicated that IPFSC-EVs could significantly suppress the expression of collagens I and III. Meanwhile, immunohistochemical analysis also revealed that IPFSC-EVs could reduce the expression of proinflammatory cytokines (IL-6 and TNF-CCDN1 and PCNA) was decreased after treatment with IPFSC-EVs, which was in agreement with the aforementioned results. These results indicate that IPFSC-EVs could inhibit fibroblast proliferation in the inflammatory environment. Intriguingly, when MT2A was silenced in fibroblasts, the IPFSC-EV-induced inhibitory effect on cell proliferation was reversed.In the in vitro study, the results of CCK8 and EdU cell proliferation assays demonstrated that IPFSC-EVs inhibited the viability and proliferative ability of fibroblasts in the inflammatory environment. Moreover, RT–PCR and western blot analyses confirmed that the expression of proliferation-related genes (This study was aimed at preliminarily exploring the inhibitory effect of IPFSC-EVs as a cell-free therapy on knee arthrofibrosis and screening and verifying the molecular targets of IPFSC-EVs to inhibit fibroblast proliferation in the inflammatory environment. However, this study is only a preliminary study and presents several limitations. First, IPFSC-EVs were administered via local injection, and although no adverse complications occurred in any animal, the safety and long-term effects of IPFSC-EVs need further experimental verification. Second, we did not explore which components in the EVs are involved in regulating cell proliferation. Third, because the situation in patients with severe knee arthrofibrosis is much more complex than in animal models, it remains unknown whether IPFSC-EVs can inhibit severe knee arthrofibrosis in clinical practice. Therefore, follow-up studies will be conducted in the future.MT2A expression, which inhibits fibroblast proliferation in the inflammatory environment. Thus, IPFSC-EVs could be used as a novel therapeutic strategy to prevent knee arthrofibrosis following joint surgery.The findings of our study demonstrated that IPFSC-EVs can inhibit the progression of knee arthrofibrosis. This effect may be mediated by upregulating