There is limited research into how midwives use social media within their professional role. Small pilot studies have explored the introduction of social media into maternity practice and teaching but there is little evidence around how midwives use social media professionally. This is important as 89% of pregnant women turn to social media for advice during pregnancy, and how midwives use social media could be influencing women, their perception of birth and their decision making.To analyse how popular midwives portray birth on the social media platform Instagram.This is an observational mixed methods study using content analysis. Five ‘popular’ midwives from each country  were identified and their posts about birth collated from a one-year period (2020–21). Images/videos were then coded. Descriptive statistics enabled comparison of the posts by country. Categorisation was used to analyse and understand the content.n = 466), and UK (n = 239), Australia (n = 205) and New Zealand (n = 7) respectively. Images/videos were categorised into ‘Birth Positivity’, ‘Humour’, ‘Education’, ‘Birth Story’ and ‘Advertisement’. Midwives’ portrayals of birth represented a greater proportion of vaginal births, waterbirths and homebirths than known national birth statistics.The study identified 917 posts from the 20 midwives’ accounts, containing 1216 images/videos, with most coming from USA (n = 17). Both the midwives and women portrayed in images were primarily white, demonstrating a disproportionate representation.The most popular midwives identified mainly had private businesses (There is a small midwifery presence on Instagram that is not representative of the broader profession, or the current picture of midwifery care. This paper is the first study to explore how midwives are using the popular social media platform Instagram to portray birth. It provides insight into how midwives post an un-medicalised, low risk representation of birth. Further research is recommended to explore midwives’ motivation behind their posts, and how pregnant and postnatal women engage with social media. Fear and anxiety related to birth has increased on a global scale , and matWomen are using social media as a source of advice and support during childbirth , 5, withEvidence suggests that health discussion and promotion on social media contain high levels of incorrect information . DespiteIn 2020, 3.6 billion people used social media globally, with the most popular platform being Facebook with 2.9 billion active users . However‘prominent social media users who accumulated a dedicated following by crafting an authentic online persona’ [Whilst anyone can have an account, Instagram has facilitated the uprising of ‘Instagram Influencers’ who are a group of popular account holders who use their status and engagement with their followers to market products and achieve celebrity status , 23. Thepersona’ . Howeverpersona’ , 26, sugIf social media can strongly influence an individual’s health and their choices, then it could be argued that there is a role for the midwife in steering individuals’ understanding, beliefs and choices around birth. This study aims to analyse how popular, or ‘influencer’ midwives portray birth on the social media platform Instagram.This study was an observational mixed methods study using media content analysis to analyse data from Instagram. The primary objective was to analyse how midwives post about birth on the social media platform Instagram. As healthcare systems vary significantly across the world, influences from different systems could be reaching pregnant women via global platforms. Therefore, this study looks to explore how midwives post about birth, with comparisons between the UK, New Zealand, USA and Australia.The study used media content analysis, a method frequently used within media and communication studies . Health Prior to commencement of this study, ethics approval was achieved through Bournemouth University Ethics Committee.Social media content is already freely available for use within the public domain, however consideration was taken to anonymise Instagram account information as best practice.In consideration of the concept of ‘Instagram influencers’ , 23, a s- Reviewing blogs and websites of ‘the best midwives to follow’- Using a generic search engine to search for ‘ < country > midwife Instagram’ and reviewing the first 20 results- Identifying accounts mentioned in the last 20 posts by each country’s professional body’s Instagram account or events pages.A pilot study was undertaken to ensure the strategy collected midwives’ accounts that would provide sufficient data. This led to a purposive strategy of selecting midwives identified as ‘Influencers’ by:- ‘Midwife’ of ‘Labour RN’ declared on account- Recent posts (5 or more posts within the last two years)- Accounts not ‘Private’, and therefore publicly availableInclusion criteria:- Nurses, doulas or other allied professionals- Not active on account in two yearsExclusion criteriaInclusion and exclusion criteria are shown below:Accounts were then reviewed to ensure that they met inclusion criteria and collated. If more than five accounts qualified per country, purposeful selection was made of the accounts with the most Followers.Data were collected using the datascraping tool PhantomBuster, which was chosen due to its ease of use and the applicability of the information it scrapes. All posts related to birth were identified by a researcher throughout the period 1/9/2020 to 31/08/2021 to accommodate for fluctuations throughout the year, and the URLs collated. URLs were then inputted into PhantomBuster to produce databases of raw data on each post including post URL, account holder, captions, number of likes and comments and date published.Due to the pioneering nature of this study, no coding sheets or data collection tools existed related to Instagram and birth. Therefore a manual data collection tool was developed including codes based on De Benedictis, Johnson  and elemAn element of the coding including Categorisation. Categorisation is an element of Discourse Analysis often used within Journalism and Communication research. Categories were made by considering Potter and Wetherell  as a metn = 60), randomly selected using an online random number generator. In line with Miles and Huberman [Intercoder reliability was ensured by one researcher separately coding 5% of posts  were identified Table . A totaln = 130,836) and post numbers (n = 1731) on average (Table Midwives from the USA had the most followers (ge Table . The majn = 17). Of those businesses, 100% (n = 17) provided Antenatal and/or Postnatal Education, 24% (n = 4) sold a book and 24% (n = 4) offered direct midwifery care. Two businesses (12%) offered resources for healthcare professionals.Most midwives had private businesses associated with their accounts (n = 3721), with UK second (n = 1369), Australia third (n = 580) and NZ fourth (n = 323). The USA received the highest mean number of ‘Comments’ (n = 123), with the UK and Australia receiving similar numbers at n = 60 and n = 58 respectively. Posts from New Zealand received the lowest number of ‘Comments’ with an average of 13 per post.Table n = 466), followed by the UK (n = 239), Australia (n = 206) and New Zealand (n = 7) (Table n = 667), UK (n = 263), Australia (n = 281) and New Zealand (n = 7). In Australia, New Zealand and the UK, images outweighed videos significantly, but in the USA videos represented nearly a third of all content.Midwives from the USA posted the most posts about birth  and Australia (61%) Table . A furthOf the total images/videos about birth, New Zealand posted the largest proportion of photographic representations (100%), followed by the USA (70%), Australia 38%) and finally the UK (33%) .In total, 42% of images and videos showed women using a pool for analgesia, with numbers as high as 48% in the USA , whereas fewer from Australia (17%), USA (15%) and New Zealand (0%) featured a healthcare professional.In all countries, the majority of images/videos of birth were of white women. The USA (26%) and UK (9%) were the only countries where black women were represented.The representations largely depict an un-medicalised portrayal of birth, with the majority of births across all countries (where mode of birth was clear) being spontaneous vaginal births 75%) (Table % (Table Intercoder reliability tests were undertaken in collaboration with another researcher (VH) Table . All fieOverall, the midwifery influencers, or ‘most popular’ midwives on Instagram were not representative of the profession and this study suggests that the profession does not have a strong presence on the social media platform Instagram. How they posted about birth, including mode of birth, location of birth and ethnicity of the birthing person, was not representative of known birth statistics in each country, portraying a much lower intervention version of birth.Unless midwives had a business or something to market then they appear less likely to engage with Instagram, or with considerably less outreach. Even where midwives did engage, their following was small. Some lifestyle Instagram influencers have several million followers , yet theThe popular midwives included in this study were predominantly white, with only two out of the twenty midwives being from other ethnic backgrounds. Whilst this may be an artefact of the sampling strategy, it is relevant as these midwives had the highest follower count and are therefore likely to reach the most women. This high disproportion is mirrored in their posts of women in labour, of whom 69% were white. Considering that in each country, maternal morbidity and mortality are highest for women from black or minority ethnic backgrounds (including indigenous women) –47, cleaRepresentation was not only skewed in terms of ethnicity, but also in the picture of labour and birth depicted. When midwives posted photographic images and videos of women, the focus was on an unmedicalized labour and birth with high rates of vaginal birth and homebirth. Whilst there are risks and benefits for both caesareans and vaginal births and a personalised approach should be taken, it is largely accepted that morbidity and mortality are overall lower for low risk mothers and babies after a vaginal birth . SimilarWhilst the focus on more physiological birth may be positive for a midwifery model of care, thought needs to be directed towards how women receive this information and the effects that it has on them. How women receive information is largely unknown within the research. Audience theories, from Media and Communications fields, explore how people respond to information within the media, proposing that this is linked to an individuals’ background, motivation, their passive or active consumption of information and age amongst other factors . Birth in = 17). Whilst this may not be surprising in countries where maternity care is largely private, such as the USA, in countries like the UK and Australia where most women can access free care this is clearly disproportionate. All businesses sold Antenatal Education, which is in line with findings that 77% of women turned to their smartphone for antenatal education [It is also noteworthy that most influencer midwives had businesses associated with their Instagram accounts (ducation . With liducation  exploredIt appears that within the field, research around social media is rarely undertaken by midwives. Of the small amount of published material available around pregnancy or birth on Instagram, none to date is authored by midwives. Most studies were by individuals working in the field of journalism, communication or digital or social media studies –65 or otEarly research to explore why midwives don’t engage with social media or the broader media has found a common theme of fear of professional retribution for saying or doing the wrong thing , 16. ForThere are many limitations of this research. First, only midwives who posted using English language were included due to limited resources. It is also relevant that the data scraping and data analysis involved in analysing social media data is extremely time consuming. The use of dominant coding may unintentionally have reinforced existing mainstream discourse, however every attempt was made to provide a balanced narrative. Although only 20 accounts were chosen, the volume of data was significant, and reducing this to the content relevant to this study took a significant amount of time. This limited the sample size. Furthermore, considering that only seven posts were included from New Zealand, it is likely that the selection strategy did not identify some significant midwifery users on Instagram. It is recognised that whilst influencers were chosen to gain insight into the content to which women were most likely to be exposed, it does have potential to limit their representation of the broader profession. Whilst this research does explore the content that midwives posted, it does not explore their intention or motivation when posting, or how women have received the information.This is the first study to explore how popular midwives are using the social media platform Instagram. The findings indicate a small midwifery presence that is unlikely to be representative of the broader profession, or the current picture of midwifery care. Midwives posted a largely un-medicalised portray of birth, and the influence of this on women and their expectations of birth needs further exploration. Given the potential for social media to influence individuals’ understanding, beliefs and choices around birth, this is an area that the midwifery profession needs to develop. Further research is recommended to explore midwives’ motivation behind their posts, as well as how pregnant or postnatal women receive information through social media. Tailored training packages for midwives and student midwives to empower them to use social media and refine their current usage may be helpful.


To what extent does prenatal folate status modify associations between early pregnancy perfluoroalkyl and polyfluoroalkyl substance (PFAS) exposure and birth outcomes?In this cohort study of 1400 mother-singleton pairs in the US, higher early pregnancy plasma PFAS concentrations were associated with lower birth weight and gestational age and a higher risk of low birth weight. These findings were observed only among mothers in the lowest quartile of prenatal folate status measured by dietary intake or plasma biomarker.The findings of this study suggest that PFAS exposure may be associated with adverse birth outcomes only among mothers with low folate status in early pregnancy. Prenatal perfluoroalkyl and polyfluoroalkyl substances (PFAS) have been linked to adverse birth outcomes. Previous research showed that higher folate concentrations are associated with lower blood PFAS concentrations in adolescents and adults. Further studies are needed to explore whether prenatal folate status mitigates PFAS-related adverse birth outcomes.To examine whether prenatal folate status modifies the negative associations between pregnancy PFAS concentrations, birth weight, and gestational age previously observed in a US cohort.In a prospective design, a prebirth cohort of mothers or pregnant women was recruited between April 1999 and November 2002, in Project Viva, a study conducted in eastern Massachusetts. Statistical analyses were performed from May 24 and October 25, 2022.Plasma concentrations of 6 PFAS compounds were measured in early pregnancy . Folate status was assessed through a food frequency questionnaire and measured in plasma samples collected in early pregnancy.z score, standardized by gestational age and infant sex; low birth weight, defined as birth weight less than 2500 g; and preterm birth, defined as birth at less than 37 completed gestational weeks.Birth weight and gestational age, abstracted from delivery records; birth weight z score only among mothers whose dietary folate intake  or plasma folate concentration  were below the 25th percentile . No associations were found among mothers in the higher folate level groups, although the tests for heterogeneity did not reject the null. Associations between plasma perfluorooctane sulfonic acid and perfluorononanoate (PFNA) concentrations and lower birth weight, and between PFNA and earlier gestational age were noted only among mothers whose prenatal dietary folate intake or plasma folate concentration was in the lowest quartile range. No associations were found among mothers in higher folate status quartile groups.The cohort included a total of 1400 mother-singleton pairs. The mean (SD) age of the mothers was 32.21 (4.89) years. Most of the mothers were White (73.2%) and had a college degree or higher (69.1%). Early pregnancy plasma perfluorooctanoic acid concentration was associated with lower birth weight and birth weight In this large, US prebirth cohort, early pregnancy exposure to select PFAS compounds was associated with adverse birth outcomes only among mothers below the 25th percentile of prenatal dietary or plasma folate levels. This cohort study examines the outcomes associated with folate status and exposure to perfluoroalkyl and polyfluoroalkyl substances in women who are pregnant. Previous studies reported inverse associations between folate and PFAS concentrations in a representative US population,16 and protective effects of folate on PFAS–lower antibody associations in children.17 Folic acid supplementation has been universally recommended to prevent neural tube defects.18 Folate plays a critical role in DNA synthesis and methylation.19 Folate and PFAS may share common transporters in the human body,28 thus resulting in potential biological interactions between folate and PFAS. This study aimed to examine whether prenatal folate status modifies the association between PFAS exposure and singleton birth outcomes.Prenatal PFAS exposure has been linked to lower birth weight, shorter gestational age, and preterm birth in epidemiological studies,29 The institutional review board of Harvard Pilgrim Health Care approved the study protocol. Participants provided written informed consent. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for cohort studies.Mother-singleton pairs were recruited at their first prenatal visit between April 1999 and November 2002, in Project Viva, a large prebirth cohort in the US. Data analysis was performed from May 24 to October 25, 2022. Details on the Project Viva study design and population are described elsewhere.In brief, sociodemographic characteristics, reproductive history, early pregnancy diet, and nutrient supplementation were assessed by questionnaires and interviews. Participants provided a nonfasting blood sample at recruitment .There were 1400 mother-singleton pairs with plasma PFAS measurement, dietary folate intake, and birth outcome data. Among these participants, a subpopulation of 898 mothers had plasma folate concentrations quantified. We compared the study population with the original Project Viva cohort. The participant inclusion diagram can be found in eFigure 1 in 31 Limits of detection were 0.2 ng/mL for PFOS and 0.1 ng/mL for all other PFAS compounds. Concentrations below the limit of detection were imputed with the limit of detection divided by the square root of 2.33 Detection rates for PFDeA (47.52%) and PFOSA (10.93%) were low and were excluded because imputing values may incur bias.33 All other PFAS were detected in more than 99% of the samples.The nonfasting blood samples obtained in early pregnancy were centrifuged and stored in non–PFAS-containing cryovial tubes in liquid nitrogen freezers (≤−130 °C). Plasma samples were sent to the Division of Laboratory Sciences at the Centers for Disease Control and Prevention for quantification of 8 PFAS compounds, including perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), perfluorohexane sulfonic acid, perfluorononanoate (PFNA), 2-(N-ethyl-perfluorooctane sulfonamido) acetate (EtFOSAA), 2-(N-methyl-perfluorooctane sulfonamido) acetate (MeFOSAA), perfluorodecanoic acid (PFDeA), and perfluorooctane sulfonamide (PFOSA), using online solid-phase extraction coupled to high-performance liquid chromatography isotope dilution tandem mass spectrometry.35 An interview was conducted at recruitment to assess supplement intake for the 3-month period before the date of the index pregnancy detection. Pregnancy dietary folate equivalent (DFE) intake (micrograms per day) included both natural food folate and folic acid from fortification and supplement intake and was estimated by multiplying the frequency of intake of standard portion sizes of each food item or supplement times its folate or folic acid content, according to the Harvard nutrient composition database.35 To account for the variations in folate intake by total energy consumption, we adjusted the DFE intake for total energy intake via the residual method.36A semiquantitative food frequency questionnaire was used to assess diet from the last menstrual period of the index pregnancy to the time of study recruitment.Baseline plasma samples for a subpopulation were sent to Boston Children’s Hospital’s Clinical and Epidemiological Research Laboratory for folate quantification  by an electrochemiluminescence binding assay . The assay is approved by the US Food and Drug Administration and has high reliability for plasma folate measurement (day-to-day imprecision <3%).11 We computed birth weight z score standardized by gestational age and infant sex, using a US reference.37 Preterm birth was defined as birth less than 37 completed gestational weeks (259 days) and low birth weight as less than 2500 g.Date of birth and birth weight in grams were abstracted from medical records. Gestational age was calculated using the date of the last menstrual period and corrected by ultrasonography (16-20 weeks) if the 2 estimates differed by more than 10 days (9%).39 Index pregnancy was self-reported as planned or unplanned. Breastfeeding history was estimated using parity and breastfeeding of the index birth as previously described.11 We calculated the Alternative Healthy Eating Index for Pregnancy score as a measure of diet quality in early pregnancy.41Data on maternal characteristics were collected by questionnaires and interviews at recruitment. Demographic and socioeconomic characteristics included maternal age, maternal and paternal educational level, self-reported maternal race and ethnicity, and annual household income. Maternal prepregnancy body mass index was calculated by self-reported weight in kilograms divided by the square of self-reported height in meters. Smoking status was grouped as ever smokers, former smokers, and early pregnancy smokers. Reproductive history included parity and infertility history defined as having tried for more than 12 months (if age <35 years) or 6 months (if age ≥35 years) to achieve the index pregnancy or having a diagnosis of infertility or claims for infertility treatments and/or prescriptions in medical records.The study population was grouped by folate quartiles of DFE intake or plasma folate concentrations. We conducted descriptive analyses on population characteristics and PFAS distributions by folate groups. Spearman correlation coefficients of 6 PFAS concentrations, DFE intake, and plasma folate concentration were calculated.42 Multivariable linear regression models were used to assess differences in the continuous birth outcomes—gestational age (days), birth weight (grams), and birth weight z score, per doubling of plasma PFAS concentrations—by DFE intake or plasma folate concentration groups. Adjusted covariates were selected a priori using a directed acyclic graph and included maternal age, educational level, race and ethnicity, body mass index, smoking history, parity, breastfeeding history, infertility, planned pregnancy, infant sex, gestational age at recruitment, and Alternative Healthy Eating Index for Pregnancy score as well as paternal educational level and annual household income , adjusting for the above-mentioned covariates.Quantile-based g computation (QGC) was used to assess the joint association of the total PFAS mixture with birth outcomes by folate groups.The associational estimates in each folate group were obtained from regression models with the interaction term of folate group × individual PFAS concentration. Likelihood ratio tests were used to test statistical significance of interaction terms in models with meaningful associations.46 Generalized additive models were used to explore nonlinear modification by folate (as DFE or plasma folate concentration) in individual PFAS and birth outcome associations (eMethods in The 2 binary outcomes—preterm birth and low birth weight—were examined using logistic regressions (eMethods in P < .20). All analyses were conducted in R, version 4.0.3 . In the linear and logistic regressions, we used multiple imputation with chained equations (MICE) to impute missing covariate values   age was 32.21 (4.89) years. Most mothers identified as White (1025 [73.2%]), had a college degree or higher (968 [69.1%]), had an annual household income greater than $70 000 (820 [58.6%]), planned for the pregnancy (879 [62.8%]), and reported periconception folic acid supplement use (957 [68.4%]) . The incAcross DFE groups, mothers in the lowest quartile were more racially and ethnically diverse, less educated, reported lower annual household income, were more likely to smoke during pregnancy, and were less likely to be nulliparous, plan for this pregnancy, have infertility, and use periconception folic acid supplements, compared with the other 3 DFE quartile groups . HoweverThe 6 PFAS compounds were detected in more than 99% of the samples. Distributions of plasma concentrations of the 6 PFAS compounds were similar in the study population and the subpopulation with plasma folate measurements and were similar across folate groups (DFE or plasma) (eTable 2 and eTable 3 in r = 0.24) with plasma folate  and EtFOSAA (r = −0.12), and no other correlations were found  and 0.74 (PFOA and PFOS). Dietary folate intake had a low correlation  (P = .49 for heterogeneity) , and no associations were found for the other plasma folate quartile groups (PFOA: geneity) . Higher geneity) . No assogeneity) .z score among the lowest DFE quartile  and the lowest plasma folate quartile groups , but not in the higher quartile groups   . The remP = .13 for heterogeneity) , while no associations were found for higher quartiles (geneity) . No assogeneity) .P = .15 for heterogeneity) , but not among mothers with higher dietary folate intake or plasma folate concentrations in early pregnancy. No consistent associations were found between PFAS exposure and risk of preterm birth or between PFAS mixture exposure and any of the birth outcomes in any of the folate groups.In this large, prospective prebirth cohort, we found that early pregnancy exposure to select PFAS compounds was associated with lower birth weight, birth weight 52 A previous study with 1645 mothers from Project Viva reported inverse associations between early pregnancy plasma PFNA concentration and birth weight z score and gestational age in the total population, but the study did not examine birth weight as an outcome.11 In the present study, the associations between PFNA and lower gestational age were only seen in the lowest quartile group of plasma folate concentrations. A lower birth weight z score was associated with PFOA instead of PFNA only among mothers whose early pregnancy folate status was in the lowest quartile range. We additionally found associations of prenatal PFOA, PFOS, and PFNA concentrations with lower birth weight, and of PFOS with higher risk of low birth weight only in mothers with the lowest folate status. The early pregnancy plasma PFAS concentrations in our study population were similar to those in women of reproductive age in the general US population during similar time periods.53Our findings of early pregnancy PFAS exposure and lower birth weight, lower gestational age, and higher risk of low birth weight among women with the lowest folate status are consistent with findings from several birth cohorts.54 which may not be representative of folate status over the entire pregnancy due to changes in food preferences in very early pregnancy. In contrast, plasma folate concentrations measure the internal dose of folate in circulation within a short time frame (days) before the time of sample collection in early pregnancy,55 when 86% of the Project Viva population reported using prenatal vitamin or folic acid supplements. Mothers in the lowest dietary folate quartile generally had lower socioeconomic status (SES) compared with mothers with higher folate status. However, SES in general was similar across quartile groups by plasma folate concentrations. Drawing similar conclusions from the 2 different folate measurements showing the PFAS associations with adverse birth outcomes were only found in mothers whose dietary folate intake or plasma folate levels were lower than the 25th percentiles supported our interpretation that these findings were not primarily due to residual confounding by SES. Furthermore, models were adjusted for parental educational level and household income. These results support our hypothesis that higher prenatal folate status could reduce or counteract PFAS exposure in birth outcomes. A recent study reported that higher PFAS concentrations were associated with lower antibody levels to rubella and mumps among adolescents with lower red blood cell folate levels but not in adolescents with higher folate levels in the US general population.17 In addition to neural tube defect prevention, these cumulative findings suggest that adequate folate status may counteract PFAS adverse effects and possibly the adverse effects associated with other environmental pollutants, as reported in experimental research.58To our knowledge, this is the first study to examine prenatal folate status as a modifiable factor for the associations between prenatal PFAS exposure and birth outcomes. We examined folate as both dietary intake and as a plasma biomarker, representing complementary folate assessments. The early pregnancy dietary folate intake crudely reflected folate intake from the last menstrual period up to the first trimester,60 Sufficient folate supply in pregnancy has been associated with a decreased risk of low birth weight and small for gestational age.62 Evidence also shows that both folate and PFAS are substrates for several shared transporters in the human placenta, specifically the organic anion transporter family63; solute carrier family, such as folate receptor α; and the adenosine triphosphate–binding cassette family, including the breast cancer resistance protein and P-glycoprotein.66 Folate might compete with PFAS on shared transporters and thus reduce the placental transport of PFAS to the fetus. Furthermore, prenatal PFAS exposure is related to epigenetic changes in cord blood.67 Sufficient folate status might counteract PFAS-related epigenetic changes through its critical methyl-donor role in DNA methylation, thus mitigating the deleterious health effects.70The mechanisms by which folate interacts with PFAS in birth outcomes are unclear and need further experimental investigation. Folate plays an important role in DNA synthesis and methylation, both critical to fetal growth.11 Third, dietary intake and plasma folate concentrations were both used to facilitate comparison and interpretation.This study examined prenatal folate as a modifiable factor for the associations between PFAS exposure and birth outcomes. The first strength of the study is its relatively large sample size, permitting stratified analyses by folate status. Second, PFAS concentrations were measured in plasma samples collected in early pregnancy, precluding potential confounding by hemodynamic changes occurring in late pregnancy.55 Mothers in Project Viva were folate replete; the 25th percentiles of the sample had dietary folate intake and plasma folate concentration above the recommended levels for neural tube defect prevention .71 Further studies are needed to replicate the present findings in other populations with lower prenatal folate intake. Multiple comparisons were conducted that could result in inflated type I error. However, tests were based on prior hypotheses and consistent patterns of heterogeneity by folate status were identified that were unlikely due to chance. Dietary folate intake measured by food frequency questionnaires could have nondifferential measurement errors. However, food frequency questionnaires perform well in measuring the relative dietary intake  in the population.54 While covariate adjustment included overall diet quality, residual confounding by diet cannot be ruled out. Because of the observational nature of this cohort study, causal effects of folate status on PFAS-related birth outcomes cannot be inferred. Selection bias is also possible given the differences in SES factors in the study sample with the original population in the Project Viva cohort. However, differences were subtle and several SES factors were included as covariates in the models. In addition, because of the complexity of the data analyses, we did not further examine sex-specific heterogeneities in the infants or nonlinear associations.The study has limitations. Although both dietary and plasma folate status were evaluated, data on red blood cell folate, which reflects medium- to long-term folate intake, were not available.z score, and gestational age, and higher risk of low birth weight only among mothers whose early pregnancy dietary folate intake or plasma folate levels were below the 25th percentiles. Findings suggest that mothers with the lowest folate status were more susceptible to PFAS-related adverse birth outcomes. Folate might be used as a PFAS prevention measure during preconception and pregnancy. If confirmed in other settings, the findings may have important implications for identifying vulnerable populations and implementing intervention studies.In this prebirth cohort study, higher early pregnancy PFAS concentrations were associated with lower birth weight, birth weight


Acanthamoeba castellanii genotype T4 is a clinically significant free-living amoeba that causes granulomatous amoebic encephalitis and amoebic keratitis in human beings. During the initial stages of infection, trophozoites interact with various host immune responses, such as lactoferrin (Lf), in the corneal epithelium, nasal mucosa, and blood. Lf plays an important role in the elimination of pathogenic microorganisms, and evasion of the innate immune response is crucial in the colonization process. In this study, we describe the resistance of A. castellanii to the microbicidal effect of bovine apo-lactoferrin (apo-bLf) at different concentrations . Acanthamoeba castellanii trophozoites incubated with apo-bLf at 500 µM for 12 h maintained 98% viability. Interestingly, despite this lack of effect on viability, our results showed that the apo-bLf inhibited the cytopathic effect of A. castellanii in MDCK cells culture, and analysis of amoebic proteases by zymography showed significant inhibition of cysteine and serine proteases by interaction with the apo-bLf. From these results, we conclude that bovine apo-Lf influences the activity of A. castellanii secretion proteases, which in turn decreases amoebic cytopathic activity. Acanthamoeba is a genus in the FLA group and is responsible for opportunistic and non-opportunistic infections in humans and other animals [Acanthamoeba castellanii is a member of this genus, which promotes granulomatous amoebic encephalitis (GAE), cutaneous lesions, nasopharyngeal, and pulmonary infections in immunocompromised patients, whereas, in healthy people, it can cause amoebic keratitis (AK) [Free-living amoebae (FLA) are protozoa with a cosmopolitan distribution. This group of microorganisms can be isolated from soil, dust, air, drinking water, swimming pools, eyewash solutions, and contact lenses ,2. Acant animals . Acanthatis (AK) . During tis (AK) ,5,6. Thetis (AK) . Lf is atis (AK) ,9. The ptis (AK)  and 80–9tis (AK) ,12. The tis (AK) .A. castellanii. We found that A. castellanii possesses the ability to evade the amoebicidal and amoebostatic effects induced by bovine apo-Lf (apo-bLf). We also found that, despite this lack of effect, apo-bLf inhibits the cytopathic effect of A. castellanii on MDCK cells, possibly due to inhibition of the proteolytic activity of amoebic serine and cysteine proteases.In 2012 it was demonstrated the protective effect of oral administration of Lf in the “dry eye syndrome” caused by an age-induced decrease in lacrimal gland secretory function . It has Acanthamoeba castellanii genotype T4 (ATCC 50370) trophozoites were kindly provided by Dr. Simon Kilvington . These amoebae were obtained from human cases of Acanthamoeba Keratitis and were cultured at 25 °C in Chang’s medium supplemented with 10% (v/v) of fetal bovine serum . The identification and characterization were performed as previously described [A. castellanii were harvested during the logarithmic growth phase (48–72 h).6 trophozoites were incubated with different concentrations  of apo-bLf  for 1, 3, 6, and 12 h. The apo-bLf was diluted in the respective amoebic culture medium without FBS. Apo-bLf-free and serum-free cultures of A. castellanii were used as the experimental controls.To assess the viability of the amoebic cultures, trypan blue and FACs assays were performed. Briefly, 5 × 10p > 0.05) .For the trypan blue technique, trophozoites were recovered after each apo-bLf treatment. The amoebae were then incubated with 0.5% trypan blue, and at least 100 cells from five different fields were analyzed by light microscopy . The results are reported as the percentage of live amoebae. In addition, a quantitative analysis of viability was performed using a fluorescent SYTOX Green assay  according to the supplier’s protocol. Serum-free cultures and trophozoites fixed with 2% paraformaldehyde  were used as experimental controls. The data were obtained using a FACS Calibur flow cytometer  and analyzed using Summit 5.1 software . Three independent assays were performed for both viability assays. The statistical analysis was performed by one-way ANOVA using GraphPad Prism 5.0  and incubated with apo-bLf at the concentrations previously mentioned for 1, 3, 6, or 12 h. At each time interval, trophozoites were removed from the culture tube surface by chilling for 30 min. To determine the number of trophozoites, the samples were directly counted by light microscopy using a Bright-Line Hemacytometer . As a control, the trophozoites were incubated in a serum-free medium. The statistical analysis of the three independent assays was performed by one-way ANOVA (p > 0.05) using GraphPad Prism 5.0 .For the ultrastructural analysis, the trophozoites were processed for transmission electron microscopy (TEM). The trophozoites were incubated with 500 µM of apo-bLf and fixed with 2.5% glutaraldehyde in 0.1 M of sodium cacodylate buffer (pH 7.2). Next, the samples were embedded in epoxy resin, and semi-thin sections were stained with toluidine blue (0.5%). The thin sections were contrasted with uranyl acetate and lead citrate and examined using an EM-910 Zeiss transmission electron microscope.A. castellanii of different concentrations of apo-bLf was analyzed. Madin-Darby canine kidney (MDCK) cells were cultured in minimal essential medium (MEM)  supplemented with 10% FBS in an atmosphere of 5% CO2 and at 37 °C for 48 h until they reached 95% confluence (monolayer). Subsequently, the trophozoites (1 × 106) were co-cultured with MDCK cells in serum-free MEM at 37 °C in a 1:1 ratio in 24-well culture plates  in the presence of 500-µM apo-bLf. After 12 h, the trophozoites were removed by chilling for 30 min, and the integrity of the cell monolayers was observed by light microscopy. The MDCK cells without trophozoites, the MDCK cells incubated with apo-bLf (500 µM), and the MDCK cells incubated with the trophozoites without apo-bLf were used as experimental controls. Three independent assays were performed under each condition.The cytopathic effect on g for 10 min, and washed with PBS 1X (pH 7.2). Subsequently, the trophozoites were lysed in PBS 1X (pH 7.2) by six freeze–thaw cycles with hot water and liquid nitrogen. Conditioned medium (CM) was prepared according to a previously-described protocol [g for 10 min, and finally passed through a 0.22 µm Durapore membrane . Next, the samples were precipitated with absolute ethanol at a 3:1 ratio and stored at −20 °C for 2 h. The CM was centrifuged at 6000× g for 30 min. The protein concentrations of the TCEs and CM were quantified using the Bradford method [v/v) Triton X-100 solution  for 30 min and incubated overnight with 100 mM of Tris-HCl (pH 7.0) and 2 mM of CaCl2. Finally, the gels were stained overnight with 0.5% (w/v) Coomassie Brilliant Blue R-250 . The protease activity was identified as clear bands on the blue background. Three independent assays were performed for each sample.The protease activity was determined by 10% SDS-PAGE co-polymerized with 0.1% porcine gelatin , bovine , and human apo-lactoferrin . Total crude extracts (TCEs) of trophozoites incubated with or without 50, 100, and 500 µM apo-bLf were obtained as previously described , with soprotocol . Brieflyd method . Fourteev/v) Triton X-100 solution  for 30 min and incubated overnight with 100 mM of Tris-HCl (pH 7.0) and 2 mM of CaCl2. The gels were then stained overnight with 0.5% (w/v) Coomassie Brilliant Blue R-250 . Three independent assays were performed for each sample (26).For the proteinase inhibition assays, the TCEs and CM were preincubated for 1 h at 37 °C with different protease inhibitors under constant agitation. The concentrations of inhibitors were as follows: for the cysteine proteases, 10 mM of p-hydroxymercuribenzoate (pHMB), and 10 µM of E-64; for the serine and cysteine proteases, 5 mM of phenylmethylsulfonyl fluoride (PMSF), and 1 mM of aprotinin . We loaded the samples onto 10% SDS-PAGE co-polymerized with 0.1% bovine apo-lactoferrin , and then performed electrophoresis at 4 °C in an ice bath at 80 V for 3 h. The gels were then washed twice with 2.5%  of apo-bLf, as compared to that of the untreated trophozoites . The TEM analysis did not show any effect of the apo-bLf on the trophozoites. In the case of the apo-bLf-treated and non-treated A. castellanii, the amoebae showed typical morphological features of contractile vacuoles and nuclei (N) with a prominent nucleolus. The cytoplasmic membrane showed complete integrity, but with the presence of electron-dense material and an increase in the number and size of mitochondria  , partial (PMSF), and non-specific (aprotinin) inhibitors of cysteine proteases. The pHMB inhibitor completely decreased the proteases detected in TCEs  compared with our study (5 × 106). Another important difference may be related to the isolation method or strain of A. castellanii employed in each study (we employed A. castellanii ATCC 50370), which means that, in terms of specific Acanthamoeba strains, the susceptibility or resistance to Lf needs to be explored. In this context, a study of the incubation of A. castellanii with 1.48 mg/mL (19.24 µM) of Lf found that it does not cause alteration in the adhesion of A. castellanii on the SV40-human cornea epithelial cell line; however, the authors did not report the death of trophozoites under this condition [During an om tears . The intof 10 µM . Initialondition .Candida species), and protozoans such as Toxoplasma gondii, Trypanosoma brucei, T. cruzi, Giardia lamblia and Babesia caballi [A. castellanii cultures, even at higher concentrations and after 12 h of incubation. However, in the cultures of A. castellanii incubated with apo-bLf after 6 h, we observed some morphological changes by light microscopy, such as the amoebae becoming larger and slightly rounded. Moreover, in the TEM study, a thickening of the plasma membrane and an accumulation of electron-dense material in the cytoplasm were observed. We also observed an increase in the number of mitochondria, with an increase in their size with respect to the amoebae that were not treated with apo-bLf, which could be explained by a possible interaction between the apo-bLf and the A. castellanii trophozoites at the membrane level. The binding of Lf to the surface of E. histolytica trophozoites has also been reported [Candida species, where cell wall changes induced by apo-bLf were observed, causing eventual cell death [A. castellanii trophozoites, the antimicrobial activity of apo-bLf was not observed at higher concentrations. Even though there is no apparent effect on amoebic viability at high concentrations of apo-bLf, interestingly, when exploring the cytopathic effect of these amoebae on MDCK cells, inhibition of cell monolayer destruction was observed. This protective effect is consistent with a study reported by Coronado-Velazquez et al., where MDCK cells were incubated with A. mauritaniensis cells that were previously preincubated with serine protease inhibitors, resulting in an abolition of the cytopathic effect and a reduction in the degradation of epithelial tight junction proteins, demonstrating that Acanthameoba proteases are an important virulence factor for this pathogen [A. castellanii in both TCEs and CM. Protease inhibition revealed that cysteine proteases present in TCEs from A. castellanii trophozoites were involved in apo-bLf degradation. The proteases present in CM without apo-bLf revealed that cysteine and serine proteases could degrade the apo-bLf and holo-bLf. We previously reported that the secretion of cysteine proteases from A. castellanii is capable of degrading holo-Lf [E. histolytica correlated with our results, where cysteine activities at 250, 100, 40, and 22 kDa cleaved holo-bLf at pH 7.0 and pH 4.0 [E. histolytica [Another mechanism related to apo-bLf is the microbiostatic effect, which has been correlated with the ability of Lf to chelate iron and render it unavailable to microorganisms ,16,20. T caballi ,19,37. Ireported . A similll death . Howeverpathogen . The cytpathogen ,26,27. Td pH 4.0 . Lf coultolytica .A. castellanii trophozoites were incubated with apo-bLf, the protease secretion was inhibited and no proteolytic degradation was observed. Similar results were observed in zymograms co-polymerized with apo-bLf. These results could be due to apo-bLf functioning as a protease inhibitor or somehow inhibiting their secretion. These results are similar to those found in other microorganisms, such as Actinobacillus pleuropneumoniae and Mannheimia haemolytica [Escherichia coli (EPEC), which causes disease in several species, it has been reported that Lf damages the type III secretion system, the main secretion system of virulence factors in this bacterium, blocking actin polymerization and the degradation of EspB [A. castellanii and apo-bLf and peptides derived from bLf .However, when molytica ,39. In t of EspB . In our  of EspB . HoweverA. castellanii is one of the few microorganisms that can resist the amoebecidal or amoebestatic effects of apo-bLf. However, the apo-bLf inhibited the protease activity of A. castellanii and drastically reduced the cytopathic effects of the trophozoites on MDCK cells. Therefore, we believe that amoebic proteases are a key virulence factor during pathogenesis, and apo-bLf could be considered as a possible therapeutic alternative against A. castellanii.Our results demonstrated that


Formation of the functional connectome in early life underpins future learning and behavior. However, our understanding of how the functional organization of brain regions into interconnected hubs  matures in the early postnatal period is limited, especially in response to factors associated with adverse neurodevelopmental outcomes such as preterm birth. We characterized voxel-wise functional centrality (weighted degree) in 366 neonates from the Developing Human Connectome Project. We tested the hypothesis that functional centrality matures with age at scan in term-born babies and is disrupted by preterm birth. Finally, we asked whether neonatal functional centrality predicts general neurodevelopmental outcomes at 18 months. We report an age-related increase in functional centrality predominantly within visual regions and a decrease within the motor and auditory regions in term-born infants. Preterm-born infants scanned at term equivalent age had higher functional centrality predominantly within visual regions and lower measures in motor regions. Functional centrality was not related to outcome at 18 months old. Thus, preterm birth appears to affect functional centrality in regions undergoing substantial development during the perinatal period. Our work raises the question of whether these alterations are adaptive or disruptive and whether they predict neurodevelopmental characteristics that are more subtle or emerge later in life. The brain is continuously changing across the lifespan but is most plastic and modifiable during early life . FollowiPreterm birth (<37 weeks’ gestation) most commonly happens during the third trimester and is estimated to affect around 11% of all live births around the world . AlthougThe graph-theoretical modeling framework has provided an important insight into the topological architecture of the neonatal brain. Both the structural and functional brain networks show highly efficient connectivity patterns, with structural network development preceding the development of functional network architecture. The networks develop in a primary-to-higher-order ontogenetic sequence and establish a pattern of functional connectivity characterized by both segregation and integration . For a cInitial work in infants has shown that the neonatal functional connectome shows a heavy-tailed degree distribution , with thThe Developing Human Connectome Project (dHCP) represents the largest open-source sample of rs-fMRI neonatal data with high spatial and temporal resolution , thus prhttp://www.developingconnectome.org/). Ethical approval was given by the UK National Research Ethics Authority (14/LO/1169), and written consent was obtained from all participating families prior to data collection. For the purpose of this study, we included subjects without visible abnormality on magnetic resonance imaging of possible/likely clinical significance following reporting by an experienced neonatal neuroradiologist. Findings leading to exclusion were major lesions within white matter, cortex, cerebellum and/or basal ganglia, small head/brain <first centile, and isolated nonbrain anomalies with possible clinical significance. Incidental findings with unlikely significance for clinical outcome were not excluded, such as subdural hemorrhages, isolated subependymal cysts, and punctate white matter lesions. In the case of a multiple pregnancy, only 1 sibling was included in the subsequent analysis. Subjects were also excluded due to excessive in-scanner motion (>10% framewise displacement [FD] motion outliers [FD > 1.5IQR + 75th centile]). The resulting population was 366 neonates. Of these, 300 were born at term and 66 were born preterm, i.e. before 37 weeks’ gestation. Very preterm-born (<32 weeks GA at birth) and extremely preterm-born (<28 weeks GA at birth) neonates represented 17/66 (26%) and 8/66 (12%) of the total preterm population. Scans were acquired at term or term-equivalent age, respectively. See the demographic and clinical details in n = 300). The effect of preterm birth was assessed by treating GA at birth as a binary variable, with comparisons carried out between the neonates born <37 weeks  and those born at >37 weeks . As the <37 week cut-off for preterm-birth is somewhat arbitrary (n = 366), and GA at birth was treated as a continuous variable.Research participants were recruited as part of the dHCP  and T2-weighted images  were acquired for registration and clinical reporting  and a neonatal 32 channel receive head coil and imaging system  . Images eporting .The data were preprocessed using a pipeline optimized for neonatal imaging as part of the dHCP . In brieGraphs were constructed from the data at a voxel level. The BOLD time series for each voxel within the predefined gray matter mask was extracted, and a correlation matrix was made using the temporal Pearson’s correlation of time series between each pair of voxels. A threshold of 0.20 was used to avoid including spurious connections in the correlation matrix  and onlyThe network analysis was repeated using no threshold for positive connections (i.e. >0.0) and a proportional threshold of 10% to determine whether the results were affected by threshold choice.z-scores to allow comparisons across subjects and were used as the inputs of the subsequent group-level analysis . Hence, these DC values are relative and reflect centrality relative to the rest of the brain rather than absolute centrality. In order to account for the differences in network cost influencing network topology . A groupwise comparison was conducted between preterm and term infants in a GLM, including PMA at scan, sex, network density, total number of FD outliers, and tSNR as covariates (y ~ β0 + β1Birth Status + β2PMA + β3Sex + β4Density + β5FD + β6tSNR). The effect of GA at birth as a continuous variable was also explored in a GLM with the same covariates as in the groupwise analysis (y ~ β0 + β1GA + β2PMA + β3Sex + β4Density + β5FD + β6tSNR). A GLM assessing the interaction between group (term vs. preterm) and PMA was also run to assess whether the effect of PMA at scan on DC varies as a function of preterm birth (y ~ β0 + β1Birth Status + β2PMA + β1Birth Status* β2PMA + β3Sex + β4Density + β5FD β6tSNR). Network density and tSNR were included as covariates because subjects have varying network density (0.58 ± 0.15 [mean ± standard deviation {SD}]), and the within-subject distribution of tSNR in the neonatal fMRI scans is not uniformly distributed. FD outliers were included as a covariate as head motion during fMRI data acquisition is known to cause signal artifact and affect the estimates of functional connectivity , including PND, sex, network density, total number of FD outliers, and voxel-wise temporal signal-to-noise ratio (tSNR) as covariates (ectivity . A versiectivity  adapted ectivity  was usedectivity . Threshoectivity .z-score) was extracted, and partial correlations were conducted between median DC and PMA at scan . In a multivariate model, the relationship between birth status (preterm vs. term-born neonates) and median DC in the RSNs was assessed with PMA at scan, sex, FD outliers, and network density as covariates (y ~ β0 + β1Birth Status + β2PMA + β3Sex + β4Density + β5FD). Partial correlations were also carried out between the median DC and GA at birth as a continuous variable . Reported P-values were uncorrected, but false discovery rate procedure  previously identified in the neonatal brain . The RSNrocedure  was apply ~ β0 + β1PMA + β2Sex + β3Density + β4FD + β5Outcome Measure + β6IMD). To explore the relationship between DC and outcome at the RSN level, univariable analyses were conducted with partial correlations between median DC for each RSN and each outcome measure, controlling for PMA at scan, sex, network density, total number of FD outliers, IMD, and age at follow-up assessment as covariates. Multiple linear regression was also used to test if the median DC in the 11 RSNs significantly predicted outcome, again controlling for PMA at scan, sex, network density, total number of FD outliers, and IMD in the model. To assess whether the relationship between median DC and outcome differs with birth status (preterm vs. term), univariate analyses were also conducted in the term-born cohort and the preterm-born cohort separately, both at the voxel- and RSN level, and the interaction between median DC in each RSN and birth status was included in a multivariable model.The relationship between relative DC and outcome at 18 months was assessed both at the voxel and RSN levels. The relationships between voxel-wise DC and each outcome measure  were explored in GLMs, including PMA at scan, sex, network density, total number of FD outliers, tSNR, and IMD as covariates in the combined cohort of preterm- and term-born infants (http://data.developingconnectome.org.The dHCP is an open-access project. The imaging data used in this study were included in the 2021 (third) dHCP data release, which can be downloaded by completing a data usage agreement and by registering at z-scored values, respectively.Analysis of the average brain connectivity matrices from term-born infants showed that the highest raw DC is evident within the left and right supramarginal gyri, the left and right precentral gyri, and bilaterally in the cuneus and is lowest in the inferior temporal regions . Panels In term-born infants, increasing PMA at scan was associated with an increase in voxel-wise relative DC within the cuneus and calcarine fissure and small regions within the lingual gyrus and superior occipital gyrus. PMA at scan was also associated with a decrease in relative DC in the bilateral precentral and postcentral gyrus in addition to the rolandic operculum, insula, and small regions within the transverse temporal gyrus, the right angular gyrus, and the right inferior parietal gyrus . A negative relationship was also observed in the auditory RSN  and the lateral motor RSN .A similar pattern of results was observed when exploring median DC in 11 established neonatal functional RSNs . After cAt the voxel-level, preterm-born infants showed a higher relative DC in the right and left cunei and the left and right calcarine fissures. Higher DC was also observed in very limited regions within the left middle and inferior frontal gyri. Preterm-born infants showed lower centrality predominantly within the right cingulum and precunueus in addition to the left paracentral lobule and the right and left precentral gyrus  and increased centrality in the visual RSN (P = 0.009) when compared with term-born controls , and median DC in the visual RSN was negatively associated with GA at birth , significant after multiple comparison correction  and on networks thresholded to only include the strongest 10% of connections . Our results were largely replicated see –6, suggeThere were no voxel-wise relationships between relative DC and neurodevelopmental outcomes at 18 months significant after multiple comparison correction in the term- and preterm-born infants combined, in the term-born infants only, or in the preterm-born infants only. There were also no relationships that survived multiple comparison correction between DC and outcome at 18 months at the RSN level. Importantly, this was also the case for the regions affected by preterm birth at term-equivalent age.P < 0.05, some significant relationships were present. As this may be informative for future studies, these results have been included in the r = −0.155, P = 0.009), and CBCL “other” was associated with median DC in the auditory network  in the term- and preterm-born infants combined , and CBCL Internalizing was associated with median DC in the somatosensory RSN , Bayley Motor was associated with median DC in the prefrontal RSN , and CBCL Externalizing was associated with the Visual Association RSN  in the neonatal brain and explore how it is associated with age at scan in term-born infants and GA at birth in a cohort of term-born and preterm-born infants. In the early postnatal period of term-born infants, our results suggest that the typical maturational trajectory involves an increase in relative functional centrality predominantly within the visual cortex and a decrease in relative functional centrality within the primary motor and auditory cortices. Further, preterm birth appears to affect centrality largely in the same brain areas, although alterations in functional centrality associated with preterm birth in the motor cortex correspond to largely medial and association RSNs compared to the lateral motor RSN being associated with age at scan in term infants. Comparing median values corresponding to previously defined neonatal RSNs , pretermThe changes in relative functional centrality associated with age at scan in term-born infants are located within primary RSNs. The maturation of RSNs shows a primary-to-higher-order ontogenetic sequence, with RSNs encompassing primary motor, visual, and auditory cortices already showing adult-like topology in term-born infants . This isWhile spontaneous retinal activity allows the visual pathways to start to function early in the third trimester, following birth, there is a large increase in visual stimuli, inducing increased cerebral activity and maturational mechanisms . The facSimilar to the visual network, the primary motor and auditory networks show adult-like topology at birth . In contWith the brain undergoing dramatic changes in functional activity during the third trimester, infants born prematurely during this sensitive period are vulnerable to the influence of developmentally unexpected sensory stimuli which is not age-appropriate and significantly different from the womb in duration, complexity, and intensity . Such anNotably, the areas that appear to be most affected by preterm birth in the present study are also predominantly located within the motor and visual RSNs. This suggests that preterm birth affects the regions which are showing the most substantial development during the perinatal period. Given the crucial role of sensory input in neurodevelopment . This maAs the preterm-born infants included in this study are predominantly moderate or late preterm, it is noteworthy that the differences in functional architecture are present even in this cohort. This evidences the importance of studying preterm-birth in a wide range of GAs at birth and not solely in infants born extremely preterm, particularly as 80% of preterm infants are born moderate or late preterm . It is, The fact that we did not observe any robust relationships between functional centrality and outcome at 18 months that survived multiple comparison correction is in line with the majority of neonatal fMRI studies, where clear relationships between functional connectivity patterns in the perinatal period and outcome at 1–2 years of age are seldom reported, particularly in term-born cohorts. Detecting links between brain imaging and general measures of behavior is difficult in the developing brain, as the changes associated with normal maturation are much larger than those associated with individual differences . The funOur study advances on previous work by providing a description of functional centrality in a large, representative sample. Our results offer complementary information to the findings by Our study has several strengths. We use a large cohort and employ a metric that does not rely on a particular parcellation template or selection of a priori ROIs. The imaging system for the dHCP is optimized for neonatal imaging. The close-fitting custom head coil provides exceptional SNR on the cortical surface . HoweverOur study is also limited by the fact that our outcome assessments are only available at 18 months of age, meaning we are unable to establish whether the patterns of atypical functional connectivity we observe to be associated with preterm birth translate into behavioral or socio-emotional difficulties commonly observed in preterm-born infants in later life, or whether they reflect a transient pattern of altered functional architecture. Further work is needed to understand whether DC at birth holds any predictive value for behavioral outcomes at later timepoints. Lastly, although DC is a widely used method to study functional network centrality, various other centrality measures exist and may capture other aspects of functional connectivity .In summary, this work contributes to our understanding of typical maturational trajectories of functional centrality at the voxel and RSN levels and explores how functional centrality is affected by preterm birth. Our findings suggest that the changes in centrality associated with typical maturation and preterm birth are both predominantly located within the primary motor and visual cortices. This suggests that preterm birth largely affects the pattern of functional centrality of the cortical regions that undergo the most substantial development in the perinatal period, highlighting both the sensitive and significant natures of these regions during early life. As functional centrality was not related to performance on outcome measures at 18 months, further work is needed to ascertain whether the alterations in functional centrality associated with preterm birth are adaptive and serve a compensatory role, or whether they are disruptive and potentially predictive of neurodevelopmental features that are more subtle than those measured here at 18 months, or that emerge later in life.DC_Manuscript_SupplementaryMaterial_Final_bhac444Click here for additional data file.


MT-SHLP6. The substitution occurring in several mammalian orders causes truncation of SHLP6 peptide size from twenty to nine amino acids. Public mass spectrometric (MS) data of human SHLP6 indicated a canonical size of 20 amino acids, but not the use of alternative translation initiation codons that would expand the peptide. The shorter isoform of SHLP6 was found in heterothermic rodents at higher frequency compared to homeothermic rodents (p < 0.001). In heterothermic mammals it was associated with lower minimal body temperature . In the thirteen-lined ground squirrel, brown adipose tissue—a key organ required for hibernation, showed dynamic changes of the steady-state transcript level of mt-Shlp6. The level was significantly higher before hibernation and during interbout arousal and lower during torpor and after hibernation. Our finding argues to further explore the mode of action of SHLP6 size isoforms with respect to mammalian thermoregulation and possibly mitochondrial retrograde signalling.Heterothermic thermoregulation requires intricate regulation of metabolic rate and activation of pro-survival factors. Eliciting these responses and coordinating the necessary energy shifts likely involves retrograde signalling by mitochondrial-derived peptides (MDPs). Members of the group were suggested before to play a role in heterothermic physiology, a key component of hibernation and daily torpor. Here we studied the mitochondrial single-nucleotide polymorphism (SNP) m.3017C>T that resides in the evolutionarily conserved gene  Tb of around 37°C during activity, heterothermic species are capable to enter controlled, hypometabolic phases of less or more than 24 h termed daily torpor or hibernation (multiday torpor), respectively   , electroate (MR)  and otheate (MR) , modificate (MR) . Hiberna(MR) (MR . The pha(MR) (MR . Althoug(MR) (MR  and asso(MR) (MR .https://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi) expand the coding capacity of the mitochondrial genetic code, with consequences for MDP size  server .Rodent orthologues of SHLP6 were identified using the sORF of human SHLP6 as query (GenBank accession number KX067784.1) for a Basic Local Alignment Search Tool (BLAST) search . Results were viewed in the Multiple Sequence Alignment Viewer of NCBI .To screen publicly available MS data for fragments of SHLP6, the commonly accepted amino acid sequences of human, rat and mouse SHLP6 were used as query. We also considered the option of an earlier translational start facilitated by the use of alternative codons for translation initiation used by mitochondria of Vertebrata . In detaatasets  and UNAfold [(http://www.unafold.org/].Secondary structure of the human 16S rRNA sequence (accession number NC_012920.1) was predicted without considering modulation by multiple proteins, in particular RNA-binding proteins , using TUNAfold . PTM was predicted using web servers with lowest threshold settings  addition (SUMOylation), and SUMO-interacting motif [ value  value  that wasT) value . Structuhttps://alphafold.ebi.ac.uk].Alternative prediction of human SHLP6 folding was derived from AlphaFold Protein Structure Database of UniProt . The transcript coding for the short, canonical 9mer of SHLP6 was not detected at all, likely also due to subcritical sequence length for the library. An RNA-seq library composed of shorter sequences that match in length would be a fitting alternative  is located within a loop of 16S rRNA according to prediction with the algorithms RNAfold3 and UNAfold, hence would likely not impair the folding of the transcript . None of the structural positions had a very low confidence (pLDDT score <50). The lower structural support obtained for the ends of the 20mers (pLDDT score <70) was attributed to intrinsic structural disorder . Similarly, non-helical predicted variants of SHLP6 structure were also found to be related to the heterothermic phenotype of the rodents. The concomitant occurrence of length and structural variants hampered clarifying whether alteration of length or structure alone or in combination associates with endothermic thermoregulation. The dichotomy of SHLP6 length was also observed in other orders of the taxon Mammalia (p > 0.1). This was attributed to the relatively low number of heterothermic species per order or an alternative adaptive strategy. Summarised, shorter SHLP6 size may mediate tolerance for low minimum Tb and/or better protect during rewarming.Here we focussed on the distribution of the Mammalia . Howeverhttps://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi], but not indicated in this study for human SHLP6 based on MS/MS data that would shift the N-terminus of the peptide. Resolution of MS analysis could be enhanced by combining immunoprecipitation with MS, hence help to overcome the failure of MS in detecting mouse and rat SHLP6  of action.It should also be addressed whether SHLP6 upregulation before torpor and during interbout arousal supports clearance of metabolites that accumulated due to torpor-induced low blood flow considering retrograde signalling by one or several MDPs as well as Tb of a heterothermic mammal. Reduction of SHLP6 size could modify the potential for oligomerisation (18F-fluorodeoxyglucose positron emission tomography/computed tomography (Rattus) that differ in the length of their SHLP6 isomers could be compared. In addition, species such as the gray squirrel (Sciurus carolinensis) having size variants encoded by mitochondrial and nuclear DNA, are promising models. In vivo experimentation could focus on the metabolic outcome of the 9mer of SHLP6 administered exogenously (This study highlighted the conservation and variation of the apoptotic regulator peptide SHLP6 across Rodentia and established the connection between heterothermy and a member of the class of MDPs. It also provided the gold-standard level of proof based on MS for the canonical variant of human SHLP6, and added evidence to the involvement of the peptide in hibernation physiology by demonstrating differential transcript abundance for a hibernating model rodent. SHLP6, putatively an extracellular communicator , has likrisation , extracerisation , and/or risation . Furthermography . For exagenously .A better understanding of SHLP size isomerisation and regulation can be of importance for therapeutic hypothermia, also known as targeted temperature management . It is i


Later-borns tend to be shorter than first-borns in childhood and adulthood. However, large-scale prospective studies examining growth during infancy according to birth order are limited. We aimed to investigate the relationship between birth order and growth during the first 4 years of life in a Japanese prospective birth cohort study. A total of 26,249 full-term singleton births were targeted. General linear and multivariable logistic regression models were performed and adjusted for birth weight, parents’ heights, maternal age at delivery, gestational weight gain, maternal smoking and alcohol drinking status during pregnancy, household income, breastfeeding status, and Study Areas. The multivariate adjusted mean length Z-scores in “first-borns having no sibling”, “first-borns having siblings”, “second-borns”, and “third-borns or more” were −0.026, −0.013, 0.136, and 0.120 at birth and −0.324, −0.330, −0.466, and −0.569 at 10 months, respectively. Results similar to those at 10 months were observed at 1.5, 3, and 4 years. The adjusted odds ratios  of short stature at 4 years in “first-borns having siblings”, “second-borns”, and “third-borns or more” were 1.08 (0.84–1.39), 1.36 (1.13–1.62), and 1.50 (1.20–1.88), respectively, versus “first-borns having no sibling”. Birth order was significantly associated with postnatal growth and may be a factor predisposing to short stature in early childhood. In Japan, the mean height of adults has been gradually decreasing for three decades ,2. As thSeveral studies have indicated that birth order is negatively associated with postnatal growth. Previous cross-sectional studies have reported that the height of first-borns is greater than that of later-borns in both early childhood ,17 and aOther studies reported that later-borns were shorter than first-borns at 6 months  and 12 mSeveral researchers have considered that the nutritional disadvantages in infancy, caused by unfairly divided family resources, in later-borns can result in a negative association between birth order and postnatal growth ,16,24. ITherefore, this study aimed to investigate the hypothesis that, even in Japan, later-borns are born larger than first-borns, but growth reverses during early childhood, which may be a risk factor for short stature. To this end, we examined the relationship between birth order (considering the existence of a sibling for first-borns) and postnatal growth using a Z-score of height at birth and at 10 months, 1.5 years, 3 years, and 4 years of age and the association between birth order and short stature among 4-year-old children, using large datasets from ongoing multicenter prospective birth cohort studies across Japan.The Japan Environment and Children’s Study (JECS) is an ongoing nationwide, multicenter, prospective birth cohort study in Japan, which examines the effects of environmental factors and medical, psychosocial, and lifestyle conditions during pregnancy and childhood on the infant’s health and development at birth and later in life. The details of the study design have been described elsewhere . The JECThe JECS protocol was reviewed and approved by the Ministry of the Environment’s Institutional Review Board on Epidemiological Studies  and the Ethics Committees of all participating institutions. Written informed consent was obtained from all participants. For this study, we used the dataset released in February 2022, which includes child growth information up to 4 years of age (dataset jecs-qa-20210401).n = 1891), delivery before 37 weeks (n = 4617), delivery after 42 weeks (n = 226), missing data for gestational age at delivery (n = 289), and two or more enrolled pregnancies to the same mother (n = 5195) were excluded, and 88,082 mother and infant pairs were extracted. Subsequently, the following cases were excluded: (1) 56,855 cases with missing and conflicting data for children’s length/height, such as ±5.0 standard deviation (SD), including birth length (n = 268), length at 10 months , length at 1.5 years , height at 3 years (n = 7803), and height at 4 years (n = 4835); (2) 4655 cases with missing and conflicting data for explanatory variables and covariates, including birth order (n = 289), parity (n = 897), the height of the mother (n = 2), the height of the father (n = 829), weight gain during pregnancy (n = 608), smoking status during pregnancy (n = 181), drinking status during pregnancy (n = 171), household income (n = 1467), and breastfeeding status at 2 years (n = 221); and (3) children with congenital cerebral abnormalities (n = 24), congenital cardiac abnormalities (n = 275), and chromosomal abnormalities (n = 14). Finally, a total of 26,249 mother and child pairs of singleton births at term were eligible for the study .We performed a sensitivity analysis of the exclusion procedure and found no significant differences in the sex or birth weight/height z-score of children between all JECS participants  and partThe birth order of the participants was reported in a self-administered questionnaire by mothers or caregivers 2.5 years after childbirth. The questionnaire asked questions regarding the presence and number of younger/older siblings living with the study participant. We classified participants into four groups: “first-borns who had no sibling”, “first-borns who had siblings”, “second-borns”, and “third-borns or more”. For some analyses, we renamed these four groups as “Group IA”, “Group IB”, “Group II”, and “Group III”, respectively, to simplify the detailed inter-group comparisons.Birth length was transcribed from the medical records of physicians, mid-wives/nurses, and/or research coordinators. Mothers or caregivers reported their children’s most recent length/height and the date of the measurement in questionnaires distributed regularly twice per year. Although the ages at the time of the reported measurements varied, only those within plus or minus two months of the aforementioned baseline ages were taken, converted to Z-scores, and then tabulated separately for each baseline age. The mean ages at measurements of length/height were 10.4 ± 0.8, 17.8 ± 0.8, 35.7 ± 0.8, and 47.8 ± 0.8 months. The Z-scores of length/height were calculated according to the growth standard charts for Japanese children using the lambda-mu-sigma (LMS) method, with the closest LMS values for each measurement age used ,30. ShorThe Japanese Maternal and Child Health Act obligates municipalities to issue all pregnant women a Maternal and Child Health Handbook for them to keep their medical information in. The handbook has pages for recording children’s weight and length/height measured at medical institutions or local government offices, as well as the date of the measurement. The act also obligates municipalities to implement free checkups for children at 1.5 and 3 years of age, and 72% of municipalities voluntarily provide additional checkups for 9- to 10-month-old children. The infant/child health checkup coverage at 9 to 10 months, 1.5 years, and 3 years is high at 85.7%, 95.7%, and 94.6%, respectively. Furthermore, the “Manual for the evaluation of infant and child physical development” reports that children’s height at 2 years or more must be measured by health care professionals in the standing position to the closest 0.1 cm . TherefoSocio-economic information and lifestyle-related data were self-reported during early and/or mid-pregnancies . The sexMaternal age at delivery , parentsThe sex of the child  was dichotomized. Maternal smoking status during pregnancy was classified as “Never”, “Previously did, but quit before recognizing current pregnancy”, “Previously did, but quit after finding out current pregnancy”, and “Yes, I still smoke”. Alcohol drinking status during pregnancy was classified as “never drinker”, “former drinker”, and “current drinker”. Household income levels (million JPY) were divided into less than 2, 2 to 6, 6 to 10, and 10 or greater. Breastfeeding status at 2 years was classified as “currently breastfeeding”, “stopped breastfeeding before 2 years”, and “never breastfed”. The Study Areas were divided into the locations of the 15 Regional Centers, which included “Hokkaido”, “Miyagi”, “Fukushima”, “Chiba”, “Kanagawa”, “Koshin”, “Toyama”, “Aichi”, “Kyoto”, “Osaka”, “Hyogo”, “Tottori”, “Kochi”, “Fukuoka”, and “South Kyushu/Okinawa”.p < 0.1 or p < 0.05) in the univariate analyses. Therefore, we concluded that a fully adjusted model reasonably well discriminated short stature at 4 years, with a rate of correct classifications of 90.2% and a p-value of 0.55 for goodness-of-fit using the Hosmer–Lemeshow test.These covariates were chosen based on recent biological and epidemiological insights regarding child growth or a strategy of selecting covariates according to a certain level of statistical significance  and 95% confidence intervals (CIs) of short stature at 4 years of age according to birth order were estimated using multivariable-adjusted logistic regression models. Model 1 was adjusted for the age of the mother at delivery. Model 2 was adjusted for the variable in model 1 and the Z-score of birth weight. Model 3 was the same as model 2 but with the parent’s height added as an additional variable. Model 4 was the same as model 3 but with weight gain during pregnancy, maternal smoking and drinking status during pregnancy, household income, breastfeeding at 2 years of age, and Study Areas  included as variables. Since postnatal growth patterns, such as catch-up and catch-down growth, may be influenced by the birth weight for gestational age, we further performed a stratified analysis by SGA, AGA, and LGA.To compare the adjusted means of the Z-score of length/height at birth and at 10 months, 1.5 years, 3 years, and 4 years of age according to the four birth order groups, multivariable general linear models were performed with post hoc Bonferroni tests. The p-value of less than 0.05 was considered significant.All statistical analyses were performed using IBM SPSS Statistics for Windows software version 28 . A A total of 26,249 children born at term were eligible for the present study: 9657 (36.8%) first-borns who had no siblings, 3463 (13.2%) first-borns who had siblings, 9240 (35.2%) second-borns, and 3889 (14.8%) third-borns or more. The study participants had a mean (SD) gestational age at birth of 39.07 (±1.14) weeks, and 50.7% were males. The Z-score of birth weight and length and the age of the mother at delivery were the highest in the third-borns or more. Maternal weight gain during pregnancy was higher in the group of first-borns than in the other groups. The proportion of current smoking and drinking status during pregnancy in first-borns was lower than that in the other groups. Household income tended to be lower in the groups of second-borns and third-borns or more. Regarding the breastfeeding status at 2 years, the proportion of those who were currently breastfeeding was higher in the group of third-borns or more than in the other groups. Significant differences were not observed in maternal height according to the birth order groups. A significantly higher paternal height was observed in the group of second-borns .p < 0.05; p for linear trend < 0.01), except at birth. A post hoc test using the Bonferroni test indicated no significant differences between second-borns and third-borns at birth and 1.5 years of age  children with a short stature were observed at 4 years of age. Although the proportion of those with a short stature increased with a later birth order, significant differences in ORs were not observed between first-borns with and without siblings. In contrast, even after the adjustment of all covariates, second-borns and third-borns or more had 1.36- and 1.50-fold higher ORs of short stature at 4 years than first-borns with no siblings. In addition, we observed that sequentially adding covariates into Models 1 to 4 resulted in marginally increased ORs .p for interaction >0.2)  . This large-scale prospective birth cohort study in Japan found that the Z-score of birth length in later-borns was greater than that in first-borns, but later-borns were shorter than first-borns at 10 months, 1.5 years, 3 years, and 4 years. At the ages of 10 months, 1.5 years, 3 years, and 4 years, birth order was negatively associated with the Z-score of length/height. Second-borns and third-borns or more had significantly higher ORs of short stature at 4 years than first-borns with no siblings, while no differences were observed between the presence or absence of siblings for first-borns. The relationship was independent of birth weight, such as SGA, AGA, and LGA. To the best of our knowledge, this is the first study to show a relationship between birth order and the Z-score of length/height, including short stature, at 4 years of age.The implications of the present findings are threefold. First, although the Z-score of birth length in later-borns was greater than that in first-borns, the Z-score of length in first-borns overtook that in later-borns by 10 months of age, and this relationship was maintained until 4 years of age. This finding is partly consistent with that in previous studies in Brazil  and the p for linear trend <0.05). The degree of differences was narrow until 1.5 years of age, but it slightly widened at 3 and 4 years. These findings are consistent with those of previous reports. A Swedish large-scale cross-sectional study reported that second-, third-, and fourth-borns were 0.4, 0.7, and 0.8 cm significantly shorter than first-borns in adulthood [Second, the adjusted mean Z-score of length/height decreased gradually with a later birth order from 10 months to 4 years of age  for low birth weight when the inter-pregnancy interval was less than 18 months compared with when it was not [A report in Nigeria found an OR of 7.331 ( was not . Moreove was not . These cThe strength of the present study is that it was a large-scale, nationwide prospective birth cohort research, which included important covariates, such as parents’ heights and birth size. Using this expansive dataset, we were able to report that second- and third-borns or more had higher ORs of short stature at 4 years than first-borns with no siblings. However, this study has some limitations. First, the participants had a significantly older maternal age at delivery and a higher proportion of first-borns than the original cohort. This may reflect the characteristics of the mothers or caregivers who reported the value of children’s length/height at 10 months, 1.5 years, 3 years, and 4 years of age. Second, the heights of the children and parents were self-reported; this may have created a response bias. Finally, birth spacing and underlying maternal diseases, such as gestational diabetes and pre-eclampsia, could not be adjusted for as potential confounders. Further large-scale studies are warranted to confirm our novel findings.The Z-score of birth length in later-borns was greater than that in first-borns. However, later-borns were shorter than first-borns at 10 months, 1.5 years, 3 years, and 4 years; furthermore, birth order was negatively associated with length/height. This is the first study to report that second-borns and third-borns or more had higher ORs of short stature at 4 years than first-borns with no siblings, regardless of birth size. Birth order may be considered one of the factors that predispose to short stature in early childhood.


We investigated the duration of invasive ventilation among very low birth weight (VLBW) infants to evaluate the current minimum time required for lung maturation to breathe without ventilator assistance after preterm birth.+6 weeks between 2013 and 2020 were enrolled. Clinical data were collected from the Korean Neonatal Network, a national prospective cohort registry of VLBW infants from 70 neonatal intensive care units. Differences in the duration of invasive ventilation according to gestational age and birth weight were investigated. Recent trends and changes in assisted ventilation duration and associated perinatal factors between 2017–20 and 2013–16 were compared. Risk factors related to the duration of assisted ventilation were also identified.A total of 14,658 VLBW infants born at ≤32+4 weeks of gestation. The median duration of invasive ventilation was 28.0, 13.0, 3.0, and 1.0 days at <26, 26–27, 28–29, and 30–32 weeks of gestation, respectively. In each gestational age group, the estimated minimum weaning points from the assisted ventilator were 29+5, 30+2, 30+2, and 31+5 weeks of gestation. The duration of non-invasive ventilation (17.9 vs. 22.5 days) and the incidence of bronchopulmonary dysplasia (28.1% vs. 31.9%) increased in 2017–20  than in 2013–16 . In contrast, the duration of invasive ventilation and overall survival rate did not change during the periods 2017–20 and 2013–16. Surfactant treatment and air leaks were associated with increased duration of invasive ventilation . We expressed the incidence proportion of ventilator weaning according to the invasive ventilation duration using Kaplan–Meier survival curves. The slope of the curve slowly decreased as gestational age and birth weight were low and risk factors were present.The overall duration of invasive ventilation was 16.3 days and the estimated minimum time required corresponded to 30This population-based data on invasive ventilation duration among VLBW infants suggest the present limitation of postnatal lung maturation under specific perinatal conditions after preterm birth. Furthermore, this study provides detailed references for designing and/or assessing earlier ventilator weaning protocols and lung protection strategies by comparing populations or neonatal networks. Bronchopulmonary dysplasia (BPD) is a serious lung disease in premature babies that affects not only the lungs but also growth and development –3. With The minimum time required for lung maturation sufficient enough to breathe without ventilator assistance after preterm birth is unclear. The duration of ventilator care appears to be mainly affected by the lung’s immaturity. Besides postmenstrual age (PMA), various perinatal conditions, ethnic differences, and epigenetic factors are thought to be related to weaning points. Practically and preferentially, improved pulmonary function results in weaning from assisted ventilation. Spontaneous respiration without a breathing apparatus is a common treatment goal in every neonatal intensive care unit (NICU). Earlier weaning from a mechanical ventilator has many advantages: reduced hospitalization days and costs, lower mortality, and decreased risk of long-term pulmonary sequela, cardiovascular impairment and growth/neurodevelopmental delay , 9, 10.Herein, we aimed to investigate the different duration of assisted ventilation and determine the current PMA limit for ventilator weaning for each gestational age and birth weight group. We analyzed the incidence proportion of invasive ventilator weaning over time for each gestational age and birth weight group of VLBW infants based on the Korean Neonatal Network (KNN) database, a national multicenter prospective registry of VLBW infants in Korea . We alsoClinical data from 70 NICUs of KNN-participating hospitals were prospectively recorded in the KNN database and analyzed retrospectively in this study. The KNN registry was approved by the institutional review board of each participating hospital, and informed consent was obtained from the parents upon enrollment in the NICUs of KNN-participating hospitals . This st+6 weeks and registered in the KNN database from January 1, 2013, to December 31, 2020, were included in this study. Infants with major congenital or chromosomal abnormalities were excluded. A total of 7,437 VLBW infants were enrolled in 2013–16 and 7,221 in 2017–20. We identified recent demographical and clinical trends by comparing the rate or distribution of perinatal characteristics among infants born in 2013–16 vs. 2017–20.A total of 14,658 VLBW infants born at ≤32Each study variable was defined according to the KNN manual of operation. “Surfactant treatment” was limited to the case of prophylaxis or rescue for respiratory distress syndrome, not for pulmonary hemorrhage or meconium aspiration syndrome. “Air leaks” was limited to a case that required chest tube insertion or needle aspiration. “Massive pulmonary hemorrhage” was severe bleeding enough to cause a cardiovascular collapse or acute respiratory failure. BPD was defined as the need for positive pressure ventilation or oxygen supplementation at 36 weeks PMA. This definition corresponded to the moderate to severe BPD definition from the National Institute of Child Health and Human Development , 13.The primary study outcome was the duration of mechanical ventilator support and the estimated weaning time from invasive ventilation according to different gestational age and birth weight groups. Assisted ventilation was defined as any type of invasive or non-invasive positive- pressure ventilation (PPV). Invasive ventilation includes conventional, high-frequency oscillatory ventilation or intermittent PPV through an endotracheal tube. Non-invasive ventilation includes nasal continuous positive airway pressure (CPAP), nasal NIPPV, and high-flow oxygen therapy delivered by a nasal cannula (HFNC) > 2 L/m. When more than one type of ventilator support was used on the same day, the longest-used ventilator was regarded as the main respiratory support mode. We investigated the difference in the duration of invasive and non-invasive ventilation between 2013–16 and 2017–20. We also identified the initial perinatal factors associated with the duration of invasive and non-invasive ventilation.The secondary outcome was the incidence proportion of ventilator weaning by invasive ventilation duration according to the gestational age (by 2 weeks) and birth weight (by 250 g) among the infants who received invasive ventilator care. The incidence proportion was expressed using the Kaplan–Meier survival curve. We observed that the change in the curve slope or curve shift depends on the presence of specific risk factors.P < 0.05 was considered statistically significant. All statistical analyses were performed using the SPSS version 26.0 software .Data were analyzed using the chi-square test for categorical variables and student’s t-test for continuous variables. The cumulative duration of assisted ventilation was described as the median and interquartile range (IQR). Kaplan–Meier survival curves were used to determine the weaning time of invasive ventilator treatment according to gestational age and birth weight groups. Cox regression analysis was used to identify maternal and initial neonatal factors affecting the required duration of PPV support (invasive and non-invasive). n = 14,658), maternal age, the rates of maternal diabetes and hypertension development during pregnancy, and cesarean section increased in the 2017–20 group than in the 2013–16 group . Also, the rate of death before 36 weeks of gestation and severe BPD increased in 2017–20 compared to 2013–16 . The mean duration of invasive ventilation according to the gestational age groups was as follows: 35.8 days at <26 weeks of gestation, 23.0 days at 26–27 weeks, 9.2 days at 28–29 weeks, and 4.3 days at 30–32 weeks  and birth weight (B) groups  weeks of gestation. The comparison of the duration of invasive ventilation among the gestational age subgroups showed differences except in the 28–29 and 30–32 weeks of gestation groups  was 4.0 (1.0–21.0) days. The estimated median (25–75 IQR) minimum time required for weaning from invasive ventilation was 30n groups . In contin Korea . This inWeisz et al.  reportedIn addition to gestational age, the presence of surfactant treatment and air leakage increased the risk of prolonged duration of invasive ventilation . The KapA limitation of this study is that the start and removal dates of each assisted respiratory ventilator and the exact intubation/extubation dates were not included in the data. Preterm infants born at <26 weeks of gestation or birth weight <750 g required longer invasive ventilation duration in 2017–20 compared to those in 2013–2016. Survival rates according to each gestational age group among 2013–14, 2015–16, 2017–18, and 2019–20 showed no difference including 22, 23, 24, and 25 weeks of gestation subgroups (In conclusion, the present data, a population-based, multicenter cohort study based on the KNN, provided current detailed references on postnatal lung maturation under specific perinatal conditions after preterm birth. VLBW infants require additional biological time for lung maturation and further tailored treatment to overcome immature lung function, especially those with RDS or air leaks. Our results are essential for assessing the therapeutic effects or quality of care for valid comparisons among populations or neonatal networks.