Previous studies indicate a role of immune disturbances during early development in the etiology of autism spectrum disorders (ASD). Any potential disturbances during fetal development are best addressed by prospective evaluation of maternal markers of inflammation. Previous studies have investigated maternal cytokines, a group of powerful effectors of the immune system, with inconsistent results. In this study, we aimed to clarify the relationship between maternal cytokines and ASD by evaluating levels of 17 cytokines in first trimester maternal serum samples, from 318 mothers to ASD-cases and 429 mothers to ASD-unaffected controls, nested within the register-based Stockholm Youth Cohort. Overall, we observed no consistent associations between levels of maternal cytokines and ASD. While we observed a number of individual associations, the patterns varied across the diagnostic sub-groups. Levels above the 90th percentile of IL-1β , IL-7 , IL-13 , and MCP-1  were associated with increased odds of ASD with co-occurring intellectual disability (ID), whereas GMCSF  and TNF-α  were associated with increased odds of ASD with ADHD but none survived correction for multiple comparisons. Also, none of the measured maternal cytokines were associated with ASD without co-occurring ID or ADHD. Implementing a data-driven approach using machine learning (Random Forest's Variable Importance measurement), we found no evidence to suggest that adding these cytokines and other markers of maternal immunity, to register-based maternal factors  improves prediction of ASD. In summary, we found no robust evidence of an association between maternal immune markers during early pregnancy and ASD. Autism Spectrum Disorder (ASD) is a heterogeneous group of neurodevelopmental disorders characterized by social impairments, restricted and repetitive patterns of behavior, and atypical sensory responses . The diaSeveral factors related to the prenatal environment have been linked to ASD in observational studies . A numbeCytokines are a group of powerful regulators of the immune system, secreted mainly by immune cells such as tissue resident macrophages and lymphocytes, but also by other cell types, including endothelial cells and adipocytes. Cytokines play important regulatory roles at different stages of pregnancy, e.g., embryo implantation and placental development , 16, andSeveral previous studies have investigated maternal cytokines during pregnancy related to offspring risk of ASD, all use samples collected in mid-pregnancy –26. The Cytokines and other immune markers measured in early life may be useful as biomarkers for ASD, with potential implications for early detection and development of novel preventive strategies –29. In tThis study is nested within the Stockholm Youth Cohort (SYC), a population-based cohort including all individuals born in Sweden and resident in Stockholm County for ≥4 years  and coven = 98,597). We collected archived Neonatal Dried Blood Spots (NDBS) from a national biobank at Karolinska University Hospital, Solna, from nearly all children with a diagnosis of ASD  and a random sample of ASD-unaffected control individuals  (n = 430) and mothers to controls (n = 549). Ethics approval was obtained by the Stockholm regional review board (DNR 2010/1185-31/5). Individual consent was not required for this anonymized register-based study.The study design and collection of biological samples have been described in detail previously . Briefly= 1,847) . For a sNational and regional registers were used for the ascertainment of ASD, ID and ADHD, covering all pathways to care and diagnosis in Stockholm County. The case-finding procedure has been described in detail previously , 32. ASDArchived maternal serum samples initially drawn as part of an antenatal screening program for maternal infections, were collected from regional biobanks at Karolinska University Hospital, Solna and Huddinge. Samples are usually, but not always, drawn at the first antenatal visit, near the end of the first trimester . In order to compare samples from the same stage of development, the samples were restricted to those drawn within the first trimester, resulting in a final study population of 318 ASD and 429 control mothers for the primary statistical analyses . After tBased on previous associations with ASD, and a plausible relationship with cytokines, we considered the following covariates as potential confounders: maternal age , psychiaData management and analyses were performed using Stata (v14.1) with external package xbrcspline , and R SDue to skewed distributions, the concentrations of cytokines were log2-transformed . Plate sIn our previous study of maternal APP in these same samples, the exposure variables were categorized into tertiles . In the p < 0.2) with any of the outcomes and at least one of the cytokines among controls. For the cytokines with a low proportion of samples falling within the limits of detection and thus a high proportion (>70%) of imputed values  is divided by the number of independent tests conducted. We calculated a Bonferroni-corrected p-value considering either 17 tests  or 68 tests . Bonferroni correction is considered to be highly conservative (p < 0.05) which are false positives (Type I errors in which the null hypothesis was incorrectly rejected), to levels of 5, 10, and 25%, respectively, and considered 68 tests.In the categorical analyses, we used logistic regression models to estimate the odds of ASD associated with dichotomized levels of each cytokine (based on the 90th percentile), with the lowest category (≥90th percentile) as the referent level. The analyses were repeated after stratifying on the co-occurrence of ID and ADHD. After dichotomizing the sample at the 90th percentile based on the distribution of the measured cytokines, we calculated that we would be able to detect an odds ratio of 1.92 at 80% power given the size of our study at an α level of 0.05. Because numerous statistical comparisons were made, we considered two approaches to correct for multiple comparisons. For the Bonferroni correction, the specified α  to re-construct the covariation between all the biomarkers into independent components. To explore the full range of available immune markers, we combined the cytokines measurements with the previously reported first trimester maternal APP , measuren = 747). We used the Mean Decrease Accuracy measurement for the variable importance, which employs an algorithm to replace a variable with a randomly generated number and examine the decrease in the new prediction accuracy. To assess the predictive performance of the respective models, we randomly divided the data into a training set (2/3 of the sample) and a test set (1/3 of the sample) and ascertained the overall accuracy. We explored the performance of different sub-sets of our data by using as predictors: (a) only the non-biomarker  covariates used in the fully adjusted regression models , (b) only the six largest PCA-components, (c) the six largest PCA-components together with the non-biomarker covariates. Because random forest models can in principle deal with a very large number of correlated covariates and therefore does not require dimension reduction techniques , we alternatively fit random forest models including as predictors: (d) the original cytokine and APP z-score variables only, and (e) the original cytokine and APP z-score variables together with the register-based covariates. We repeated these steps 1,000 times with randomized training and test sets and estimated the mean accuracy together with the 2.5th and 97.5th percentile value .Finally, we used the Random Forest Classifier to assess the predictive performance of the biomarker measurements in comparison to other covariates and to compute the variable importance on the full sample size (n = 979) and evaluated any associations with ASD.In the main analyses, the samples were restricted to those drawn in the first trimester of pregnancy, in order to reduce variation in cytokines related to gestational age and improve interpretability. However, this resulted in the exclusion of 232 individuals for whom maternal sera were available. As a sensitivity analysis, we therefore repeated the categorical analysis (using the 90th percentile cutoff for the cytokines) including all available serum samples and additionally adjusted for gestational week at sampling . We also repeated the PCA using information from all available serum samples  varied from 0% (MIP-1β) to 93.3% (IL-5) . Seven cp < 0.05) linear relationship between IL-8, IL-17, GCSF, MCP-1 and MIP-1β, and gestational age, using all available samples . Among mothers to ASD-cases, some of the associations overlapped with the control group , whereas others were unique for the ASD-case group   .p < 0.2) with at least one of the cytokines (testing the proportion of samples in the highest decile), among mothers to controls  larger for IL-10 in cases compared to controls (p < 0.05) were observed between IL-7, IL-10 and IL-13, and odds of ASD with ID, and between TNF-α and ASD with ADHD (p = 0.030), IL-6 , and TNF-α , and the inhibitory cytokine IL-10  at or above the 90th percentile were associated with increased odds of any ASD diagnosis. Further, IL-1β , IL-7 , IL-10 , IL-13 , and MCP-1 , were associated with increased odds of ASD with ID (p = 0.041) and TNF-α  were associated with ASD with ADHD  was observed between IL-7 and ASD with ID  would only reach statistical significance  at an FDR of 41.7%.Although several of the associations between cytokines and the outcomes were significant at p < 0.05 , none suThe first four principal components, each explaining >5% of the variation, together explained over 50% of the variation in cytokines and APPs . PC1 expp = 0.041) association between low levels of PC2 and any diagnosis of ASD (We retained the first four components (PC1-PC4), which were approximately normally distributed  and exams of ASD , and sims of ASD . BecauseThe random forest prediction models (number of trees = 100) including only the register-based covariates  had an overall prediction accuracy of ASD of 60.7% , the observed associations were generally consistent with the main categorical analyses (restricted to the first trimester) regarding direction and magnitude, although some differences were observed (p = 0.05), whereas the associations between IL-12 and any ASD diagnosis, and GMCSF and ASD with ID, fell below the threshold for statistical significance , though no convincing separation of cases and controls. Only one extracted principal component, whose loading was dominated by APP, was associated with ASD. Using the immune markers collectively did not improve the prediction of ASD in our sample, beyond that observed for models including only maternal and child characteristics obtained from register data.n = 84), ID without ASD (n = 49), and population controls (n = 159) (n = 415), developmental delay (n = 188) and controls (n = 428)  compared to the present study . One of n = 159) . IFN-γ, n = 428) . No signn = 428) , but theIn the “Seychelles Child Development Study Nutrition Cohort 2”, Irwin et al. measured 13 inflammatory markers including 10 cytokines in a cohort of 788 mother-child pairs, at a mean of 28 weeks of gestation, and related these to the trait scales Social Communication Questionnaire (SCQ) and Social Responsiveness Scale (SRS), at age 7 . Both thIn a recent case-control study, Carter et al.  investigThis is the first study to date to investigate maternal serum cytokines during the first trimester of pregnancy and risk of ASD in the offspring. In the present study, we analyzed cytokines in a study sample nested within a large population-based cohort. None of the significant associations in the present study overlapped with those reported previously. There are methodological differences between the studies that may explain these discrepancies. As in previous case-control studies, our archived serum samples were collected as part of an antenatal screening program, and handling of samples before final storage at −80°C cannot be accounted for. This might have influenced the reliability of the measurements . FinallyIn this cohort of pregnant women, we measured a wide range of cytokines, including mediators of both innate and adaptive immune pathways. We observe several associations between individual immune markers and ASD. However, the associations were weak to moderate in general, and none survived correction for multiple comparisons. This is in line with previous studies on maternal cytokines and ASD, although none of the previous studies corrected for multiple comparisons. Overall, we found no strong evidence for maternal immune activation among mothers to individuals with ASD in this cohort, although there was a tendency for odds ratios estimates to be above one, similar to other studies , 24.Although we observe increased levels of several of the pro-inflammatory cytokines and ASD, the specific results differ considerably according to diagnostic sub-group. Indeed, recent data suggests that ASD without ID is more familial and may have a different genetic architecture than ASD with ID , 49, 50.If any of the cytokines are causally related to ASD, the mechanism by which they can potentially affect children's neurodevelopment remains to be established. In experimental animals, IL-6 can cross the placental barrier and influence fetal brain development potentially through transmission of an inflammatory signal to the fetal compartment , 51. MatThe overall picture indicates that instead of relying on individual markers, some general aspect of the maternal immune function and its interaction with the developing fetus may be relevant for ASD. To integrate the biological information from all our measured immune markers in one model, we employed an approach using PCA, also including measurements of maternal APP measured in the same cohort of women. A similar approach has been used in a few previous studies of cytokines in archived maternal or neonatal samples. Jones et al. used separate PCA for each diagnostic sub-group  to investigate the presence of specific aggregated patterns of maternal cytokines. The authors concluded that there was no obvious specific cytokine-profile for any of the stratified groups. Krakowiak et al. conducted a PCA of 14 cytokines, though these were measured in neonatal samples from ASD-affected and unaffected children. Overall, there were no specific clustering patterns across diagnostic sub-groups . Finally, Heuer et al. used partial least squares discriminant analysis (PLS-DA) to investigate if neonatal cytokine levels separated diagnostic groups . The authors concluded that there was no outcome-specific profile based on a plot of cases and controls in component space, but a variable importance analysis suggested that the cytokines IL-6 and IL-8 were the most important cytokines, which corresponded to their logistic regression analyses. In our study, maternal levels both IL-6 and IL-8 were among the most important predictors in our random forest models, although the confidence intervals overlapped with zero.We observed a non-linear relationship between one of the extracted principal components, PC2, and odds of ASD, with increased odds of ASD associated with low PC2 scores. Low PC2 scores correspond to high levels of several of the APP, given the direction of the loading of the APPs on the second principal component, indicating that children's odds of ASD generally increase with increasing levels of maternal APP. The remaining components, including the cytokine-dominated PC1, showed no significant associations with ASD-case status, and cases and controls were not otherwise separated based on the first four components. This indicates that maternal APP levels may contain more information relevant for the children's risk of ASD than maternal cytokine levels. This may relate to their longer half-lives, higher base-line concentrations, and effector functions within the maternal innate immune system .Finally, adding the immune markers or their derived principal components to other registered covariates regarding maternal and child characteristics did not improve the prediction of ASD-case status in Random Forest prediction models. This indicates that knowledge regarding maternal immune biomarkers (including cytokines and APP) are unlikely to be informative in terms of early detection of ASD. However, the weak predictive capability does not rule out a role for maternal immune status in the etiology of ASD. The potential dysregulation of immune processes may be more subtle than what can be detected in our current sample, and the maternal serum samples from early pregnancy may not reflect the conditions in the developing fetal brain. There is a possibility that each of the markers has a very small effect size that we are unable to capture given our restricted sample size, and that each of the small effects add up to a cumulative effect that is only detectable in a substantially larger population, analogous to single nucleotide polymorphisms in genome wide association studies.We use a validated case-finding procedure within a healthcare system with universal coverage and regular developmental screening. This increases the likelihood of identifying ASD cases in the population. Using our large, well-characterized population-based cohort, we were able to assess confounding by a range of different environmental factors and investigate how they influence the serum levels of cytokines. By analyzing multiple immune markers, we increased the likelihood of detecting a signal of activation of the maternal immune system. On the other hand, by doing multiple statistical comparisons, we also increased the probability of chance findings. None of the associations survived correction for multiple comparisons, either using a traditional (Bonferroni) or a less conservative (FDR) approach. A strength of our study is the PCA and Random Forest analyses, which move beyond the conventional one-by-one analyses and allows for an integrated interpretation of the immune markers.We could adjust for a wide range of potential confounders, such as maternal BMI and fetal sex, with documented associations with both the maternal immune status and autism , 30, 53.By restricting the analysis to the first trimester, we reduced heterogeneity and variation in cytokines related to the progression of pregnancy. On the other hand, the strategy also reduced the sample size and statistical power and prevented detection of differences during later stages of pregnancy. The majority of serum samples (76.3%) in this study were drawn in the first trimester. The observed associations between several of the maternal cytokines and gestational age, as well as other covariates, stress the importance of taking key covariates into account in any assessment. Including samples from all trimesters in a sensitivity analysis yielded results similar to the main analysis in terms of the direction and magnitude of the estimated odds ratios, though some relationships were less apparent when considering the full cohort and a few became more apparent.Cytokines are powerful regulators of the immune system and generally have short half-lives and low baseline levels at homeostatic conditions. Since cytokines are often involved in local (paracrine) cell-signaling pathways, systemic levels may not necessarily reflect concentrations at peripheral sites of inflammation, or in the embryo and the fetal/placental unit. Thus, measuring concentrations in blood/serum samples can be both technically challenging and difficult to interpret. Overall, there was a large proportion of non-detectable values for many of the cytokines. The inter-assay coefficient of variation was markedly higher for IFN-γ compared to the other cytokines, and the results for IFN-γ must therefore be interpreted with some caution.Moreover, because these samples were collected as part of a clinical screening program, storage procedures may have varied. Samples could be stored at room temperature or in a refrigerator up to several days before freezing at −80°C, though this procedure was not within our control, and we do not have information on the handling of individual samples prior to their arrival in our laboratory. Since cytokines are vulnerable to degradation at ambient temperatures, there is a possibility that the levels measured in the samples do not reflect the actual levels at the time of sampling/venipuncture. The degradation of cytokines in the samples due to these issues would attenuate any real associations toward the null, and further reduce our ability to detect case/control differences.While we observed a number of individual associations between maternal cytokines measured in early pregnancy and children's risk of ASD, none survived corrections for multiple comparisons. Considering the individual associations, our results do not provide strong support for the maternal immune activation hypothesis in ASD, especially when compared to the often divergent results of previous studies. The relationships we observed varied with the presence or absence of co-occurring neurodevelopmental diagnoses, including both ADHD and ID. We also observed variation in the maternal cytokine levels with key covariates, such as maternal characteristics and the gestational week at serum sampling. This emphasizes the importance of considering both potential genetic and environmental influences on the maternal immune system when attempting to interpret associations between maternal immune biomarkers and children's risk of ASD in future studies.When taking a more integrated view of biomarkers reflecting the maternal immune response in the first trimester, we observed that maternal cytokines as a class were not strongly associated with children's risk of ASD, though higher levels of another class of immune biomarkers, the acute phase proteins, were associated with children's risk for ASD. Using all of these markers together did not markedly improve prediction models for ASD, indicating a limited utility for maternal immune biomarkers in early detection strategies for ASD given the limitations of studies to date in terms of sample size and the relatively small effect sizes for the individual markers.The Swedish health and population register data used in this study are available from Statistics Sweden and the Swedish National Board of Health and Welfare. The authors are not allowed to distribute the data according to the ethical approval for this study and the agreements with Statistics Sweden and the Swedish National Board of Health and Welfare.The studies involving human participants were reviewed and approved by Stockholm Regional Review Board. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.MB contributed to data acquisition, data analysis, and drafted the first manuscript. HK and RG contributed to study design, data acquisition, data analysis, and manuscript preparation. CD contributed to data acquisition, study design, and manuscript preparation. HS contributed to data acquisition, data analysis, and manuscript preparation. BL contributed to study design and manuscript preparation. All authors contributed to the article and approved the submitted version.This work was supported by grants from the Swedish Research Council , Autism Speaks [Basic and Clinical Grant No. 7618 (to BL)], and the Stanley Medical Research Institute (to HK). The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, and approval of the manuscript; or decision to submit the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To explore the effect of mother's own milk (MOM) feeding time on the risk of moderate and severe bronchopulmonary dysplasia (BPD) in infants with very low birth weight (VLBW).n = 397) and the late mother's own milk (LMOM) feeding group . Differences in the incidence of moderate and severe BPD among the two groups were analyzed using the chi-square test. Effects of MOM feeding time on the incidence of moderate and severe BPD were evaluated using univariate and multivariate logistic regression analysis.Clinical data from 630 infants with VLBW were retrospectively analyzed. Participants were divided into the early mother's own milk (EMOM) feeding group . Variate logistic regression analysis showed that the LMOM feeding group had an increased risk of moderate and severe BPD compared with the EMOM feeding group . The results of multivariate logistic regression analysis showed that the LMOM feeding group had an increased risk of moderate and severe BPD compared with the EMOM feeding group .The incidences of moderate and severe BPD in the EMOM feeding group and the LMOM feeding group were 13.9% (55/397) and 21.0% (49/233), respectively (The first time of MOM feeding within 72 h after birth and the persistence of mother's own milk feeding during hospitalization can reduce the incidence of moderate and severe BPD in infants with VLBW. Bronchopulmonary dysplasia (BPD) is a common respiratory disease in preterm infants. Due to its many complications, high fatality rate, and lack of effective treatment, it has become an important issue affecting the survival rate and quality of survival of preterm infants, especially in infants with very low birth weight (VLBW). Existing research shows that MOM feeding can reduce the risk of BPD in infants with VLBW , 2. In nThis is a retrospective analysis of the infants with VLBW admitted to the Department of Pediatrics, Women's Hospital of Nanjing Medical University/ Nanjing Maternity and Child Health Care Hospital from January 2015 to December 2019.Inclusion criteria: (1) birth weight <1,500 g and gestational age at birth <34 weeks; (2) admission within 24 h after birth; and (3) intestinal nutrition within 72 h after birth, and mother's own milk feeding accounted for >90% of participants.Exclusion criteria: (1) hospital day ≤ 14 days; (2) non-cured infants who did not reach 28 days, lost after discharge; (3) congenital malformation or genetic metabolic diseases; and (4) incomplete data.The aim of this study was to provide a retrospective analysis of the effect of MOM feeding time on the risk of moderate and severe bronchopulmonary dysplasia (BPD) in infants with VLBW.http://www.ningbx.com) was an online database where we got the infants' information from. The hospital established a human milk bank in August 2013. All nutrition types and feeding volumes of infants included in the study were recorded in the nursing list. The variables in this study included sex, gestational age, birth weight, delivery, polycyesis, 1-min Apgar score, antenatal steroid, surfactant use, mechanical ventilation (MV), time on MV ≥7days, and total MOM feeding. Neonatal outcomes examined included the incidences of BPD, necrotizing enterocolitis (NEC), retinopathy of prematurity (ROP), grade III or IV intraventricular hemorrhage (IVH III/IV), sepsis, late-onset sepsis (LOS), neonatal respiratory distress syndrome (NRDS), and patent ductus arteriosus (PDA).Paper or electronic scanned versions of the medical records of infants with VLBW admitted to the NICU of Nanjing Maternity and Child Health Hospital, affiliated to Nanjing Medical University, from January 2015 to December 2019 were used. The NingBX neonatal homogeneity platform . The median and interquartile range represent skewed variables; and frequencies and percentages represent categorical variables. The chi-square test and the Kruskal–Wallis test were used to compare the risk of BPD with neonatal data and clinical information. Univariate logistic regression was used to analyze the relationship between related variables and moderate and severe BPD occurrence; multivariate logistic regression analyzed the relationship between first mother's own milk feeding time and BPD occurrence, adjusting for risk factors closely associated with outcome. The risk was reported as an odds ratio (OR) with 95% CI. All tests were two-sided tests with P < 0.05). There were no significant differences between gestational age, birth weight, cesarean section, 1-min Apgar score <7, prenatal hormone use, surfactant use, MV, and time on MV ≥7 days, (P > 0.05)  .P < 0.05), and no significant differences were found in NEC (≥ stage 2), ROP, IVH III/IV, sepsis, LOS, NRDS, and PDA (p > 0.05) (The incidence of moderate and severe BPD was lower in EMOM feeding ( > 0.05) .There was a higher risk in the LMOM feeding group, with an OR value of 1.656 (95%CI: 1.083–2.532) .Screening factors were statistically significant as independent variables together with the LMOM feeding group, which showed that the morbidity rate in moderate and severe BPD was higher than that in the EMOM feeding group with an OR value of 1.894 (95% CI: 1.127–3.185) .Bronchopulmonary dysplasia is a common respiratory complication affecting the survival rate and quality of survival of infants with VLBW. Because infants with moderate and severe BPD still need to use oxygen at specific times, it often affects the severity of the disease and prognosis, which not only is the focus of clinicians but also affects the treatment plan.An observational cohort study of 16,407 infants with VLBW in five countries showed that the incidence of moderate and severe BPD in infants with VLBW was 15.7% 2,580/16,407) 0/16,407 –11. The Research primarily focused on the occurrence of BPD; however, the relationship between first MOM feeding time and the occurrence of moderate and severe BPD has not been reported. This study found that first MOM feeding time in infants with VLBW >72 h after birth was a high-risk factor for moderate and severe BPD, suggesting that MOM feeding within 72 h of the birth of infants with VLBW reduces the risk of moderate and severe BPD. Breast milk reduces oxidative stress and provides antioxidant protection, vitamin A, and scavenger enzymes, which play an important role in reducing oxidative damage, protecting, and promoting immune system maturation in premature infants , 14. HowThe preventative role of breast milk for BPD has been confirmed by systematic review and meta-analysis , but no The particularity of maternal lactation with premature infants affects the time when some premature infants receive maternal breast milk. Preterm birth is an independent risk factor for delayed lactation initiation . The resThe results provide a practical basis for promoting active breast sucking and instant milk feeding in infants with VLBW, while highlighting the importance of the time for first MOM feeding of infants with VLBW. MOM feeding of infants with VLBW within 72 h can reduce the occurrence of moderate and severe BPD. Early exposure to various beneficial components in maternal breast milk can promote the maturation of the immune system and improve the body's antioxidant capacity. However, when the time was further reduced, the difference was not statistically significant between the two groups, which may relate to insufficient sample size and uneven distribution of age at the time of the first maternal breastfeeding. The results showed a higher maternal milk intake during hospitalization than the LMOM feeding group, although the occurrence of moderate and severe BPD was independent of maternal breast milk aggregates during hospitalization. Earlier MOM seems to indicate that infants with VLBW can obtain more adequate maternal milk supply during hospitalization, partly because normal lactation starts to reduce the risk of insufficient lactation and partly because early delivery shows that the mother can actively pump milk and chooses to participate in the breastfeeding of the infant with VLBW; these factors complement each other and jointly improve MOM feeding time of infants with VLBW.There are some limitations to this study. First, it is a single-center study with limited data that needs confirmation through multi-center studies; second, there are many factors affecting BPD, and we only selected some related factors, despite the limited number, it can still illustrate the protective effect of early mother's own milk feeding on BPD, which can also be easily understood clinically; finally, the 72-h criterion was subjective, and more data is needed to verify this time period.In conclusion, the first MOM feeding time within 72 h after birth and the persistence of MOM feeding during hospitalization can reduce the incidence of moderate and severe BPD in infants with VLBW. Hospitals and medical staff should follow guidelines, which can improve the enthusiasm of maternal milk absorption by maternal separation, and give infants with VLBW MOM feeding in good time. More multi-center large-sample clinical trials are needed to validate the outcomes of the relationship between first MOM feeding time and infants with VLBW.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of the Maternity Hospital Affiliated to Nanjing Medical University. Written informed consent to participate in this study was provided by the participants' legal guardian or next of kin.YZ and AS conceptualized and designed the study, drafted the initial manuscript, reviewed, revised the manuscript, supervised data collection, and critically reviewed the manuscript for important intellectual content. XC, JZ, and CJ collected data and carried out the initial analyses. All authors approved the final manuscript as submitted and agreed to be accountable for all aspects of the work.This work was supported by the Six Top-Notch Talent-funded Projects (LGY 2019008), the Nanjing Medical Science and Technology Development Fund (ZKX 19045), and the Nanjing Medical University's Specialized Disease Cohort -funded Project (NMUC 2020037).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To investigate the association of crown-rump length (CRL) during the first trimester of pregnancy with neonatal outcomes.+0 to 13+6 weeks during pregnancy and transformed to a standard deviation score (SDS) adjusted for gestational age. Linear regression was used to explore risk factors for CRL. A generalised linear model was used to evaluate the association between CRL and neonatal outcomes.A total of 15,524 women with a reliable first day of the last menstrual period and a regular menstrual cycle (28 ± 4 days) were included from January 2015 to November 2016. CRL was measured by ultrasound from 7P < 0.001; 0.04 SDS, 95% CI = [0.03–0.04], P < 0.001), multipara  and folic acid supplement use  were positively associated with CRL, while pre-pregnancy BMI  was negatively related to CRL. For neonatal outcomes, CRL was negatively associated with small for gestational age (SGA) ([risk ratio] (RR) = 0.733, 95% [CI] = 0.673–0.8004, Padjusted < 0.001) and neonatal intensive care unit (NICU) admission , and preterm birth , but positively related to large for gestational age (LGA) . When stratified by pre-pregnancy BMI, the risk of SGA and LGA remained significant in all groups, while the increased risk of preterm birth was only observed in the lean group (BMI < 18.5 kg/m2) and decreased risk of NICU admission rate in the normal group (BMI 18.5–24 kg/m2).In the multivariate analysis, maternal age (0.25 mm, 95% CI = [0.22–0.28], Maternal characteristics were independently associated with CRL in the first trimester, which was negatively related to foetal size, SGA, preterm birth, and admission rate to the NICU, but positively related to LGA.The online version contains supplementary material available at 10.1186/s12887-022-03426-8. Crown-rump length (CRL), measured using ultrasound as early as the first prenatal visit, is generally used to assess gestational age in the first trimester . It is aMost studies investigated the role of CRL in the assessment of foetal size such as birth weight, small for gestational age (SGA) and large for gestational age (LGA), which has a close relationship with foetal, neonatal, and adult health , 10. ForIn this study, we investigated the correlation between CRL in the first trimester and neonatal outcomes in natural singleton pregnancies to offer insights for the early recognition of adverse outcomes .+6 weeks were included  in Shanghai between January 2015 and November 2016 were enrolled in the study. A total of 29,448 women with natural singleton pregnancies were included. After excluding 6,052 cases without complete medical records, 67 with pre-pregnancy diabetes, 1,853 with a history of thyroid disease, and 762 with in vitro fertilization, 20,714 mother-infant pairs remained. The gestational age was calculated based on the last menstrual period. Subsequently, 5,063 women with neither a known first day of the last menstrual period nor a regular menstrual cycle of 28 ± 4 days were excluded. To investigate CRL in the first trimester, 127 cases of CRL measured beyond 14 weeks of gestation were excluded. Ultimately, 15,524 mother-infant pairs with confirmed gestational ages of 7–13All foetal biometry measurements were recorded by qualified technicians according to the clinical guidelines recommended by the International Society of Ultrasound in Obstetrics and Gynecology (ISUOG) . Standarth Revision, Clinical Modification.Maternal characteristics: gestational diabetes ; intrahepatic cholestasis , and pre-eclampsia (high blood pressure and excess protein in the urine after 20 weeks of pregnancy). The serum vitamin D concentration was described in our previous study , P < 0.001) per year in age, pre-pregnancy BMI  per kg/m2 increase in BMI, multipara , and folic acid supplement use  with CRL remained significant  and the birth weight by 32.78 ± 2.51 (g) (P < 0.001). As expected, each SDS increase in CRL was associated with a 25.2% decrease in the odds of SGA  and a 19.6% increase in LGA . Furthermore, newborns with higher CRL in early pregnancy were more likely to have preterm birth, as a 1-SDS increase in CRL resulted in 9.9% higher odds of preterm birth . We also found that CRL was negatively associated with NICU admission rate .For anatomical parameters, the average birth length was 49.9 ± 1.3 (cm) and the average birth weight was 3342 ± 438 (g)  per SDS, Padjusted < 0.001], SGA , and LGA  with CRL were more obvious. Preterm birth  remained significant. A similar result was observed for NICU admission . Since SGA newborns were apt to be transferred to the NICU, we included SGA as a covariate in Model 2. The results showed that an increase in SDS was associated with a 6.6% decrease in the NICU admission rate . The incidence rates of the other neonatal outcomes are shown in Table SIn the multivariate analysis, the associations of birth length [0.15 ± 0.01 (cm) per SDS, 2  per kg/m2 Table .Table 4A2) had the same trend as previous results for SGA , LGA , and admission to the NICU . However, an association between preterm birth and CRL was not observed in women with normal pre-pregnancy BMI. In the lean group (< 18.5 kg/m2), CRL was strongly related to the incidence rate of SGA, with 31.1% decrease per SDS , and LGA with 28.1% increase . Moreover, 1-SDS increase was associated with a 47.7% increase in the odds of preterm birth . In Figure SP for non-linearity = 0.067). Interestingly, for the obesity group (BMI > 24 kg/m2), correlations of CRL with neonatal outcomes were only observed in anatomical factors such as birth weight, birth length, SGA, and LGA, where the effect size or RR was relatively smaller than the other two groups. The risk ratios of all outcomes are shown in Fig. Women with normal BMI 18.5–24 kg/m4 kg/m2 hIn this study, we investigated the risk factors contributing to CRL, as well as the correlation between CRL in the first trimester and neonatal outcomes. First, maternal age and multipara and folic acid supplement use were positively associated with CRL, whereas pre-pregnancy BMI was negatively related with CRL. Second, CRL was positively associated with birth length, birth weight, incidence rate of LGA, and preterm birth, while it was negatively related to the incidence rate of SGA and NICU admission. Furthermore, when stratified by pre-pregnancy BMI, the correlation between CRL and neonatal outcomes was distinct in the different groups.Previous studies have indicated that male sex , maternaCRL, which serves as an early predictor of foetal growth, is related to neonatal outcomes. Several studies have explored the correlation between first trimester CRL and foetal growth. Leung et al. found that the CRL of the foetus in the first trimester was related to birth weight but was not an independent predictor of SGA in a study of 2,896 cases . A case–th percentile of CRL, was associated with an increased risk of preterm birth (adjusted OR = 2.12) [P = 0.019) [th percentile of CRL) was not associated with preterm birth in our study . Given the inconsistency in the association between CRL and preterm birth, we performed a subgroup analysis based on pre-pregnancy BMI. Interestingly, a correlation between CRL and preterm birth was observed  only in the lean group, but not in the normal or obese group. Preterm birth is a complicated process that may be affected by social stress and race [In addition to foetal size, we found that CRL was slightly yet positively related to preterm birth . Inconsistent with our findings, a prospective cohort study indicated that first-trimester growth restriction, defined as less than the 20 = 2.12) . Kwak et= 0.019) . Neverth= 0.019) . In the and race , infectiand race , and genand race . In certand race . TherefoInterestingly, a larger CRL in the first trimester was a protective factor against admission to the NICU. The trend remained significant only in the normal BMI group, which accounted for most of the population. The SGA was conventionally transferred to the NICU; thus, we constructed another model including the SGA. The trend was retained . Admission to the NICU is a comprehensive and robust indicator of newborns’ health.One of the strengths of our study was its large sample size. The SDS based on ‘GAMLSS’ method was also applied to enable adjustment for gestational age GA to avoid the inclusion of non-linear functions of GA in models. However, owing to the retrospective study design, this study had some limitations. We have not differentiated the aetiology and severity of preterm birth to perform a subgroup analysis, which will be our future interest.In conclusion, several maternal characteristics contribute to first trimester CRL. CRL might be considered a potential early predictor of foetal size at delivery, preterm birth, and admission to the NICU, since differences in growth trajectories may be expressed in early pregnancy.Additional file 1: Figure S1. Centile curves of CRL measured in 7–13+6 gestational weeks fitted with BCGo model . Figure S2. Restricted cubic spline plot of risk ration of preterm birth. Table S1. Selection of models of crown-rump length. Table S2. Neonatal outcomes of the population.


Participants with live births (n = 1130) were propensity score matched to participants who experienced miscarriage (n = 132), stillbirth (n = 72), preterm birth (n = 243), and neonatal mortality (n = 20). Logistic regression was used to examine drinking water As recommendations of 50, 10, 5, 2.5, and 1 µg/L on the odds of adverse birth outcomes. (3) Results: The odds of miscarriage were higher for pregnant women exposed to drinking water ≥2.5 versus <2.5 µg As/L . (4) Conclusions: These preliminary findings suggest a potential threshold where the odds of miscarriage increases when drinking water As is above 2.5 µg/L. This concentration is below the World Health Organizations and Bangladesh’s drinking water recommendations and supports the re-evaluation of drinking water regulations. (1) Background: Arsenic (As) is a common drinking water contaminant that is regulated as a carcinogen. Yet, As is a systemic toxicant and there is considerable epidemiological data showing As adversely impacts reproductive health. This study used data from a birth cohort in Bangladesh (2008–2011) to examine associations between drinking water As levels and reproductive outcomes. (2) Methods: Pregnant individuals ( Groundwater is an important source of potable water yet arsenic, a geogenic compound, contaminates groundwater in 107 countries ,3,4,5. TThe World Health Organization (WHO) also based its provincial drinking water guideline at 10 µg/L based on arsenic’s carcinogenicity . As addiThe human health effects of arsenic exposure have continued to be studied in populations around the world that rely on groundwater. Subsequently, there are considerable epidemiological data indicating that arsenic is a reproductive toxicant. However, it is unclear if drinking water regulatory standards established using cancer data are also protective against miscarriages, stillbirths, premature births, and neonatal mortality. These adverse reproductive outcomes are widespread and affect approximately 48 million pregnancies every year ,14. BangThe improvements Bangladesh made to these maternal child outcomes were largely based on efforts to address the major risks of adverse reproductive outcomes such as low nutritional status, limited birth spacing, and limited access to obstetric care ,19. HoweUsing an established prospectively enrolled cohort, we examined the associations between arsenic exposure at different regulatory cutoff levels and adverse pregnancy outcomes in Bangladesh. The regulatory level cut offs were used to compare risks of neonatal mortality, stillbirth, miscarriage, and preterm birth between different arsenic exposure groups. Propensity score methodology was used to control for confounding by matching pregnant women on predictors of arsenic exposure status. We hypothesized that the odds of neonatal mortality, stillbirth, miscarriage, and preterm birth would be higher for pregnant women who were exposed to arsenic above 10 µg/L compared to those below this WHO and US EPA regulatory cut off level. Thresholds below these guidelines were also examined to understand if there was a potential threshold below which the odds of adverse pregnancy outcomes remain stable.From 2008–2011, mothers were recruited in two study areas (Sirajdikhan and Pabna Sadar Upazilas) of Bangladesh in partnership with Dhaka Community Hospital Trust (DCHT) . The stuParticipants were included in this analysis if they had an ultrasound at the time of enrollment (<16 weeks gestation), had a singleton pregnancy, and provided a drinking water sample that was measured for arsenic. Participants with missing data at the time of enrollment were excluded because propensity score matching requires complete data. Of the 1613 pregnant women enrolled in the original cohort, 4 women were excluded for not having an ultrasound at enrollment, 2 women were excluded for lacking exposure data, 8 women were excluded for having twins, and 2 women were excluded for missing enrollment data. n = 132) and stillbirth was the loss of a fetus during a viable birth period . Neonatal mortality was determined by mortality of newborn within the first 30 days after live birth from womb (n = 20). Preterm birth was defined as a viable birth < 37 weeks gestation and lived past one-month post-partum (n = 243). Gestational age was determined at enrollment via ultrasonography at the first visit and calculated by mean gestational sac diameter at 4-6 weeks or by crown-rump length at 7–16 weeks. Ultrasounds were conducted with two technicians for each participant and verified by an obstetrician at DCH. Trained field staff confirmed all neonatal mortalities with the parents within the first 30 days of life for n = 1150 live births. Fetal loss was defined as a fetus that was not alive outside of the womb. Miscarriage was the loss of a fetus before a viable birth  (continuous), monthly family income reported by the financial provider of the mother , clinic location (Pabna or Sirajdikhan), previous miscarriage or stillbirth if not first child (yes/no), and parity (1st child or 2nd and greater). Models for neonatal mortality and preterm birth were controlled for the above covariates as well as: birth gestational age , birth type , sex of child , and birthweight of child (kg).Covariates were chosen tal loss ,30,31. TDescriptive statistics  were calculated for continuous variables. Study participants were matched using propensity scores. Rosenbaum and Rubin  developeSensitivity analyses evaluated the robustness of our findings for the adverse outcome of miscarriage. Specifically, results from unadjusted and adjusted logistic regression analyses of the unmatched data were compared to results from full and nearest-neighbor propensity score matched analyses. n = 1354). The characteristics of mothers with and without adverse birth outcomes are presented in Of the 1597 women who met the inclusion criteria of this analysis; 1130 women had live births, 132 women experienced a miscarriage, 72 women experienced a stillbirth, and 20 infants were lost due to neonatal mortalities one month after birth. Within the live births and neonatal mortalities there were 243 preterm births. Additionally, 215 women withdrew or were lost to follow up prior to birth, and 28 women withdrew from the study after birth. We used optimal full propensity score matched samples to compare different drinking water arsenic levels against adverse pregnancy outcomes . These aSensitivity analyses were conducted to determine the robustness of the drinking water arsenic threshold on miscarriage between different modeling parameters . Comparisons between model types, matching diagnostics, and descriptive statistics between matched and unmatched cohorts is tabled in the This preliminary analysis of the effect of arsenic exposure in drinking water and adverse birth outcomes observed an increased risk of miscarriage in this Bangladesh cohort when drinking water arsenic concentrations were above 2.5 µg/L. This risk dissipated at higher concentrations which could indicate a non-linear or perhaps U-shaped exposure-response relationship between drinking water arsenic concentrations and miscarriage. Alternatively, the attenuation of the risk of miscarriage at higher concentrations could also be a function of the underlying sample distribution. However, this finding is important because it indicates that the Bangladesh drinking water regulatory levels of 50 µg/L (and the WHO 10 µg/L guideline) was not protective against an elevated risk of miscarriage. Our analysis did not find a protective drinking water level for neonatal mortality, stillbirth, or preterm birth. These adverse outcomes were rare in this population-based prospective cohort of healthy women which resulted in a relatively limited sample size. Thus, our findings should be considered preliminary with larger studies needed to confirm our findings.Our analysis focused on reproductive health outcomes which have been shown to be influenced by arsenic and suggests that pregnant women are a particularly vulnerable sub-group of the population that may not be protected by current drinking water arsenic regulations. Currently, the human health risk assessment process that yields regulatory drinking water recommendations in Bangladesh is based on the older US EPA cost–benefit analysis that did not include epidemiological data from Taiwan. Additionally, the WHO and US drinking water regulations are based on a cost–benefit that only included the risk for lung and bladder cancer . While tn = 150), a sample size of miscarriages similar to our study [Our findings are consistent with other research groups in Bangladesh who have shown that drinking water arsenic exposures are associated with adverse birth outcomes ,45. Mostur study . Within ur study ,48,49, wur study . Outsideur study ,52. Specur study ,57,58,59Propensity score matching was used within this cohort to address residual confounding. As mentioned previously, our non-matched logistic regression models did not show significant odds of miscarriage compared to those regressions using propensity score matching. The one significant predictor in our models with arsenic exposure was location of clinic for either Pabna or Sirajdikhan. While Bangladesh tends to have a fairly homogenous culture and practices, especially among these more rural areas, they do differ. Pabna is a more rural community with more farmers while Sirajdikhan is more peri-urban and closer to the capital city of Dhaka. Exposure profiles also differ between these two locations beyond arsenic. The use of propensity score matching by full matching allowed for more specific comparisons of location and exposure to measure odds of miscarriage in our models. Additionally, full matching allowed for more flexibility of matching where all data was used and matching at least one control to each case. Propensity score matching is not without its downsides. Researchers pointed out that “greedy” propensity score matching such as nearest-neighbor can exclude many data points but also reduce the bias of having lessor matches of controls in cohort studies . Our appIt is also worth noting that our population-based sample included a largely healthy birth cohort of pregnant women. There was no tobacco use and very little preexisting conditions such as underweight or overweight, diabetes, or hypertension. Very few women were hypertensive during pregnancy, developed gestational diabetes, or developed pre-eclampsia. Our exposure assessment utilized individual level drinking water arsenic measurements. This does introduce some exposure misclassification because it does not include dietary intake. However, previous studies in the cohort demonstrated the high correlation between drinking water and internal toenail arsenic exposures, identifying drinking water as one of the main sources of exposure . There in = 20), we were only able to compare proportions of matched cases and controls in association models. Future studies with larger sample sizes of adverse birth outcomes such as neonatal mortality and stillbirth could possibly see a threshold value in the examples we used. We were unable to use toenail arsenic exposures in our study as individuals that experienced miscarriage or stillbirth did not have measurements taken after birth. The use of propensity scores allowed our results to be significant with an alpha of 0.05, where traditional adjusted logistic regression models were null. The direction of the effect size was similar between the two methods, the use of propensity scores creates dependent study samples which are not comparable to the entire population. There is a chance that k:1 propensity score matching could increase bias of the estimation with additional matches beyond 1:1, yet full matching relaxes that assumption [There are several limitations in this study. Due to low number of neonatal mortality cases (sumption . AdditioWomen during pregnancy are vulnerable to environmental contamination through ingestion of drinking water and this vulnerability is not currently taken into consideration when establishing drinking water guidelines. We observed that the odds of miscarriage increased when drinking water arsenic was above 2.5 µg/L, a concentration that is well below the current WHO’s provisional drinking water guideline and Bangladesh’s drinking water recommendations. Our findings are preliminary because of the relatively small number of adverse birth outcomes observed in this cohort but it helps support the understanding of low-level arsenic exposures and associations with adverse pregnancy outcomes, not just at higher levels of over 50 µg/L. While pregnant women are still exposed to unsafe levels of arsenic around the world, clinicians and providers need to be aware of all levels of arsenic exposure to reduce adverse effects and support healthy pregnancies.