Various methods have been developed for the isolation although each method differs in the number and purity of exosomes they yield. In melanoma, tumor-derived exosomes drive immunosuppression within the tumor microenvironment. The co-elution of exosomes and soluble factors such as cytokines during isolation, however, make it difficult to ascertain the contribution of exosome cargo, as soluble cytokines are equally capable of immune suppression. In this review we will expound upon the biological relevance that exosome-associated cytokines possess. Furthermore, we discuss the technical challenges that arise during exosome isolation and what this means for further studies into the TME and  The tumor microenvironment (TME) has been the mainstay of tumor biology research for over 20 years. Cross talk between the immune system and the TME promotes immunosuppression, proangiogenic tendencies, and the inhibition of tumor cell death. Cytokines facilitate intercellular communication between immune cells as well as to other cells within the tumor microenvironment. Therefore, most efforts have focused on the exploitation of the immune system to eliminate cancer using cytokines. Multiple approaches including immunotherapy and cytokine therapy have been used as potential strategies to treat cancer.Cytokines are categorized as immune-modulating, soluble factors that are less than 30kDa in size and include chemokines, interferons, interleukins, lymphokines and the tumor necrosis factor family of proteins . These mvia blebbing, a hallmark of cell death and the consequence of the disintegration of the cytoskeleton  carry a variety of cargo that includes lipids, proteins and nucleic acids. EVs can be divided into three categories: microvesicles, apoptotic bodies, and exosomes, which differ in the method of biogenesis, release, size and content . A singlskeleton . Distincskeleton .in vitro work. Nevertheless, the ability to package a variety of critically bioactive molecules within a stable, lipid membrane-bound vesicle makes exosomes appealing to study as vehicles for therapeutics  and various lipid species capable of translating immunomodulatory effects to cells . The secapeutics .in vivo studies.With the increasing potential for the use of exosomes clinically, it has become important to optimize exosome isolation methods to ensure maximum purity, yield and reproducibility. Due to the variety of exosome isolation methods utilized, the question arises as to whether current techniques can isolate exosomes devoid of cytokines and other soluble factors, which can confound the causative associations reported in literature. In this review, we will assess the biological role of EACs. Furthermore, we will discuss the technical challenges that preclude us from excluding soluble cytokines from exosome isolates and how this can complicate studies of the TME as well as other Multiple factors including cell origin, health status and environmental factors prior to exosome isolation can impact the variety and quantity of secreted cytokines present in exosome samples –15. EstrTo decrease a cytokines’ effective range and thereby maintain specificity, many cytokines also function as a membrane-bound protein. Several cytokines and their receptors including IL-1, M-CSF, TGFβ, and TNFα have been reported in other studies to exist in both soluble and membrane-bound forms, both which are biologically active –23. MostRapid Exosome Isolation using Ultrafiltration and Size-exclusion chromatography (REIUS) method, led to an 836-fold reduction in 13 cytokines including IL-10 compared to the more commonly used ultracentrifugation (UC) isolation method, suggesting that the cytokines detected may not be physically associated with exosomes , are released by melanoma, driving immunosuppression . Murine EACs cannot be excluded from eliciting toxic effects. The stability and high biocompatibility of exosomes have made them an attractive vehicle for chemotherapeutics and other drugs. As such, research efforts have focused on determining which cell types are the most ideal source of exosomes. As exosomes embody a similar molecular profile to the originating cell, retention of the lipid and surface protein profile may prove important for proper exosome function , 53. ExoCurrent technical limitations related to the isolation method prevent us from completely removing soluble factors from exosome isolates, making it difficult to distinguish whether downstream effects are due to exosome function or that of the co-isolated cytokines. Moreover, they also prevent us from fully distinguishing between exosomes and other EVs. For instance, microvesicles bud off from the plasma membrane as opposed to being secreted into the extracellular space. As they overlap in size with exosomes and share common surface markers and cargo, they are commonly co-eluted with exosomes. These limitations prove detrimental to the understanding of the intricacies of the TME, particularly the contribution of exosomes in the maintenance of the TME.The TME consists of tumor cells, vascular cells, fibroblasts and immune cells and the subsequent interaction of all these cell types contributes to the progression of cancer. HMEX are immunosuppressive and contain functional EACs , 57. As Exosomes contain specific protein markers corresponding to the originating cell type. Therefore, it would be hard to decipher a mixture of exosomes derived from various cell types from patient blood, which theoretically can serve as a representation of the TME . Sharma The presence of EACs, specifically those within the payload of exosomes, is strongly supported by the literature. Numerous cytokines that include IL-2, IL-4, IL-10, IL-18, and IL-33, TNF-α, TGF-β, M-CSF were found to be preferentially enriched within exosomes , 61–63. Many factors including cell type and stimulus can affect the level of cytokine presence in an exosome sample as well as the form in which the cytokines are secreted, whether for local or more distant signaling. Understanding these interactions within the milieu that is the ‘tumor exosome microenvironment’ and further developing technologies to be able to tease them apart is critical to more targeted therapies.SB-D: contributed to the general idea, wrote and edited the manuscript, and created figures. MK: contributed ideas to, compiled information for manuscript, and edited figures and manuscript. CA: contributed ideas to and edited manuscript. ME: provided expert opinion and input and edited manuscript. SL: conceived the general idea, created figures, and edited the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by institutional funds provided by Roswell Park Comprehensive Cancer Center.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.


Lipids (fatty substances) and cytokines are molecules that affect how the immune response works. The measurement of the amounts of lipids and cytokines in blood might give clues about how prostate cancers grow or respond to treatment. This study looked at the blood levels of lipids and cytokines in men with advanced prostate cancer that was growing despite standard treatment . We found that certain lipids were consistently associated with poorer clinical outcome, while cytokines were not. The levels of a type of lipid (ceramide) were associated with some cytokines. This lipid is known to activate the immune system and is associated with poor outcomes in mCRPC. A change in lipid profiles was associated with better response to treatment. Overall, our findings suggest that blood lipids might be more informative than cytokines, might influence the immune response, and might help predict treatment response.n = 146, 139) having life-prolonging treatments were subjected to lipidomic and cytokine profiling . Higher baseline levels of sphingolipids, including ceramides, were consistently associated with shorter overall survival in both cohorts, whereas the associations of cytokines with overall survival were inconsistent. Increasing levels of IL6, IL8, CXCL16, MPIF1, and YKL40 correlated with increasing levels of ceramide in both cohorts. Men with a poor prognostic 3-lipid signature at baseline had a shorter time to radiographic progression (poorer treatment response) if their lipid profile at progression was similar to that at baseline, or their cytokine profile at progression differed to that at baseline. In conclusion, baseline levels of circulating lipids were more consistent as prognostic biomarkers than cytokines. The correlation between circulating ceramides and cytokines suggests the regulation of immune responses by ceramides. The association of treatment response with the change in lipid profiles warrants further research into metabolic interventions.Circulating lipids or cytokines are associated with prognosis in metastatic castration-resistant prostate cancer (mCRPC). This study aimed to understand the interactions between lipid metabolism and immune response in mCRPC by investigating the relationship between the plasma lipidome and cytokines. Plasma samples from two independent cohorts of men with mCRPC ( Androgen-deprivation therapy (ADT) is the standard of care for the initial diagnosis of metastatic prostate cancer, as prostate cancer growth relies on testosterone and ADT lowers circulating testosterone levels to castrate levels . When prIn the past decade, the therapeutic landscape of mCRPC has shifted from docetaxel chemotherapy as the only effective standard of care to multiple life-prolonging options that are clinically approved. These include targeted agents such as the novel androgen receptor signalling inhibitors (ARSI) , lutetium-177-prostate-specific membrane antigen, and PARP inhibitors, as well as another taxane, cabazitaxel . HoweverEvidence for the role of dysregulated lipid metabolism in the clinical outcomes of metastatic prostate cancer is increasing . ObesityCirculating lipids may be contributing to prostate cancer progression through the modulation of the immune response ,10. For Several lines of evidence indicate an interplay between the immune response and lipid metabolism in prostate cancer progression. For example, a high fat diet was sufficient to drive metastasis in the non-metastatic Pten-null mouse model of prostate cancer . AdditioThe role of IL6 and other cytokines in driving prostate cancer progression have been demonstrated by studies with cell lines and mouse models ,21,22,23Studies on the interaction between the immune response and lipid metabolism rely on in vitro and animal models, whereas the tumour microenvironment and host metabolism in humans are more heterogenous and complex. It is essential to understand how these different biological systems interact within human cohorts. Therefore, our study aims to examine the relationship between the plasma lipidome and multiple cytokines in men with mCRPC.Plasma samples used in this study were from two cohorts of patients with mCRPC in Australia, referred to as Cohort 1 and Cohort 2. Cohort 1 consisted of 146 patients from the study described by Lin et al. (2017)  and was g for 15 min, and the separated plasma was transferred to a fresh tube and centrifuged again at 5000× g for 10 min. Aliquots of the plasma after the second spin were then stored at −70 °C.Blood was collected in EDTA-containing tubes from the participants prior to commencing a new line of treatment, according to a standardised blood collection protocol that differed in the plasma separation technique for both cohorts. For Cohort 1, the blood was centrifuged at 3000 g for 5 min, and the separated plasma transferred to a fresh tube and stored as aliquots at −70 °C. For Cohort 2, the blood was centrifuged at 1600× Clinical data for Cohort 1 from the first study  were updLipidomic analysis of plasma samples from Cohort 1 was described in Lin et al. 2017). In that study, the plasma samples were analysed as two separate cohorts and included samples from 13 other patients. The normalised lipidomic datasets from that study were aligned using the ComBat algorithm , resulti. In thatMultiplex analysis of cytokines in plasma samples from Cohort 1 and Cohort 2 were analysed separately, 2 years apart, using the following Milliplex Map assays  according to the manufacturer’s instructions with the Bio-Plex MAGPIX system  and Bio-Plex Pro-Wash Station (Biorad)—Human High Sensitivity T Cell Magnetic Bead Panel , Human Th17 Magnetic Bead Panel , and Human Cytokine/Chemokine Panel IV Magnetic Bead Panel . Cohort 1 samples were analysed in two batches, whereas Cohort 2 samples were analysed in three batches. The same sample layout on the multi-well plate was used for each type of assay. Data was generated using the Bio-Plex Manager MP and Bio-Plex Manager 6.1 software. Cytokine levels were exported as the observed concentration and converted to ng/mL.The datasets from both cohorts were pre-processed separately as follows. Cytokines with more than 50% missing values (zero or below range) were removed. Missing values of remaining cytokines were replaced with half of the lowest value for that cytokine. Concentrations for values above range (three samples in Cohort 2) were estimated from interpolation of their standard curve using Prism . Next, each batch of samples was normalised by the Probabilistic Quotient Normalisation method, using the average of each cytokine to create the reference, and the final values transformed into log2 scale. Finally, the different batches were combined with batch correction performed with the ComBat algorithm  .Statistical analyses were performed with R (version 3.6.0) using the R packages listed accordingly. Plasma samples of Cohort 2 were classified by the poor prognosis three lipid signature (3LS) that was originally derived in the study involving Cohort 1 by Lin et al. (2017) . First, Enrichment of lipid types was determined by one-sided Fisher’s exact tests using bc3net (v1.04). Kaplan–Meier curves were drawn with survminer (v0.4.6). Pearson correlation analysis between lipid and cytokine levels was performed with Hmisc (v4.2-0). Heatmaps and hierarchical clustering (complete linkage) were produced with pheatmap (v1.0.12).The cohorts and study schema are summarised in n = 128 men) was studied as baseline plasma samples from first line therapy were available from these men. The clinical characteristics of Cohorts 1 and 2a are summarised in For all comparisons to Cohort 1, a subset of Cohort 2  plasma samples. The EOT samples were collected at disease progression or at baseline of the subsequent treatment . There wp < 0.0001, p = 0.002, p = 0.009, p = 0.004, In our previous study involving Cohort 1 , we had p < 0.05, p < 0.05, p ≤ 0.04, Cox regression analyses of baseline levels of individual lipids for first line therapy identified 80 lipids for Cohort 1 and 128 lipids for Cohort 2a that were significantly associated with OS .Higher levels of free cholesterol were associated with shorter OS in both cohorts .In contrast, the baseline levels of only a few cytokines were associated with OS or rPFS. In Cohort 1, higher levels of IL8 were associated with shorter OS  compared to men who had the 3LS together with low levels (<median) of any of these cytokines  of either IL6, CXCL16, IL27, or CCL28 compared to men who had the 3LS together with lower levels (<median) of any of these cytokines , IL17a, IL1B, and IL2 correlated with increasing levels of ceramides . The correlation analyses of 121 prognostic lipids or cytokines with NLR showed that only 21 lipids and 2 cytokines were correlated with NLR, of which only one of them is a sphingolipid—a ganglioside  is a known marker of poor prognosis in cancer, and comprehensive data of this was only available for Cohort 2. A high NLR was associated with shorter OS in Cohort 2a  of the EOT samples by the profiles of 121 prognostic lipids or 40 cytokines showed that the EOT lipid or cytokine profiles do not group together by treatment . The hiep = 0.02, p = 0.03, To determine if the similarity of lipid or cytokines profiles at baseline and EOT were related to treatment response, the rPFS of treatment lines with the poor prognostic 3LS at baseline were examined. There were 16 pairs of matched baseline EOT samples with the 3LS at baseline, of which matched samples with similar lipid profiles tend to have different cytokine profiles, and vice versa a. Treatmt-test p ≥ 0.05, t-test p = 0.002), and the levels of three T-helper-17 (Th17) cytokines  were lower at EOT   species tend to be lower at EOT for lipid profiles that differed at EOT from baseline ( ≤ 0.04) e. The leThis study identified a significant relationship between circulating lipids and cytokines in mCRPC. We confirmed that increased levels of circulating sphingolipids, particularly ceramides, were associated with poor clinical outcomes in another independent cohort of mCRPC (Cohort 2a). The association between the baseline levels of cytokines and OS were inconsistent in both cohorts, indicating that circulating lipids perform better as prognostic biomarkers than the cytokines. However, although the cytokines do not perform as well as lipids, the cytokines are still important in prostate cancer progression as other studies provide evidence on their pathological role in prostate cancer, and the levels of some cytokines were correlated with that of the ceramides  in the two independent cohorts.The correlation between certain cytokines and prognostic ceramides suggests that there may be an interplay between the cytokines and lipids. Possible scenarios would be induction of cytokine expression by ceramides directly or indirectly in immune cells, cancer cells or other cell types, which could be occurring in the tumour stroma, circulation, or organs. Alternatively, perhaps the relationship between cytokines and ceramides are independent, where the weak correlation exists simply from a weak prognostic relationship between cytokines and clinical outcome. In the following paragraphs, we present the existing knowledge on the connection between ceramides and cytokines.In both cohorts of our study, elevated levels of IL6, IL8, CXCL16, and YKL40 were consistently significantly correlated with elevated levels of ceramides. Other studies have shown that elevated circulating levels or tumour expression of these four cytokines were associated with poorer clinical outcomes in prostate cancer ,24,33,34p ≤ 0.003, Increasing plasma levels of IL6 was correlated to increasing plasma levels of ceramide in patients with cardiovascular disease . IL6 expOther cytokines that may be regulated by ceramide are IFNγ and IL2, which are typically associated with Th1 cell responses . Th1 celThe independent relationship of circulating lipids and cytokines in our study was demonstrated by the association of treatment response with the overall profiles of prognostic lipids and cytokines at radiographic progression, for patients with the poor prognostic 3LS at the start of treatment. The prognostic lipid profiles with the 3LS at baseline have shorter rPFS if their lipid profile at progression (EOT) was similar to that at baseline or if their cytokine profile at progression was different to that at baseline. Specific lipid types with significant changes that relate to a better treatment response could not be identified and are likely to involve the combined minute effects of multiple lipids. A poorer treatment response was associated with an increase in the levels of IL8 and the decrease in the levels of several Th17 cytokines at EOT. In this situation, the relationship between IL8 and Th17 response appears to be independent, given that IL17 can stimulate cells to produce IL8 ,51,52. OWe also found that higher levels of free cholesterol were associated with shorter OS in both cohorts, although total cholesterol was not. Other studies have reported that total cholesterol levels in patients undergoing radical prostatectomy were not associated with recurrence, except for men with dyslipidemia ,56. HoweIn summary, higher levels of circulating sphingolipids including ceramides were associated with shorter OS in mCRPC and were correlated with elevated pro-inflammatory cytokine levels across two independent patient cohorts. Circulating lipids appeared to be better prognostic biomarkers than cytokines, as the baseline levels of the lipids were more consistently associated with OS. These data support the concept of the circulating lipid profile as a potentially druggable target, which may also affect the immune environment downstream.


Low birth weight  and macrosomia (> 4000 g) are both adverse birth outcomes with high health risk in short- or long-term period. However, national prevalence estimates of LBW and macrosomia varied partially due to methodology limits in China. The aim of this study is to estimate the prevalence of LBW and macrosomia after taking potential birth weight heaping into consideration in Chinese children under 6 years in 2013. The data were from a nationally representative cross-sectional survey in mainland China in 2013, which consists of 32,276 eligible records. Birth weight data and socio-demographic information was collected using standard questionnaires. Birth weight distributions were examined and LBW and macrosomia estimates were adjusted for potential heaping. The overall prevalence of LBW of Chinese children younger than 6 years was 5.15% in 2013, with 4.57% in boys and 5.68% in girls. LBW rate was higher for children who were minority ethnicity, had less educated mothers, mothers aged over 35 years or under 20 years, or were in lower income household than their counterparts. The overall prevalence of macrosomia of Chinese children younger than 6 years was 7.35% in 2013, with 8.85% in boys and 5.71% in girls. The prevalence of macrosomia increased with increasing maternal age, educational level and household income level. Both LBW and macrosomia varied among different regions and socio-economic groups around China. It is found that estimates based on distribution adjustment might be more accurate and could be used as the foundation for policy-decision and health resource allocation. It would be needed to take potential misclassification of birth weight data arising from heaping into account in future studies. Globally, the prevalence of LBW was 14.6% and about 20.5 million babies were born with LBW in 2015, of which 91.0% were in developing countries2. Macrosomia was associated with increased risks of infant morbidity, and obesity and metabolic complications in childhood and adulthood5. The prevalence of macrosomia ranged from 5 to 20% in developed countries and from 0.5 to 14.9% in developing countries, while an overall 15–25% increase around the world was also found in the past two to three decades7.Birth weight is a strong predictor for mortality and morbidity during infancy and later life. Both low birth weight and macrosomia are great concerns to public health. LBW is thought to place a high risk on children survival, growth, development and has long-term impact on health outcomes such as diabetes and cardiovascular diseases in later life8. Another study reported a LBW rate of 2.87% among 16,954 infants in 5 provinces around China between 2006 and 20089. A 39-hospital based study showed a LBW rate of 6.1% in 14 provinces of China  in 201110. China’s National Maternal Near Miss Surveillance System (NMNMSS) of 441 health facilities indicated a LBW rate of 5.36% in China between 2012 and 201411. The prevalence of macrosomia varied across different studies in China too. Perinatal Health Care Surveillance System in 12 cities and counties in southeast China showed the macrosomia rate of 7.83% in 200512 and a cross-sectional study of 14 provinces in China reported a macrosomia rate of 6.5% in 200613. Another studies reported a macrosomia rate of 10.44% in 2006–20089, and 7.3% in 201114. Halves of these studies were health facility-based studies and the other were community-based studies.The prevalence of LBW and macrosomia in China varied across different studies in the past two decades. For example, a study of 18,554 livebirths from 14 provinces found a LBW rate of 4.6% in 200615. As a result, a certain proportion of infants whose birth weight are exactly 2500 g (the cut-off point for LBW) actually < 2500 g or > 2500 g and those are exactly 4000 g (the cut-off point for macrosomia) actually > 4000 g or < 4000 g. This could dramatically misestimates the prevalence of LBW and macrosomia16. However, most LBW and macrosomia estimates did not take potential misclassification of birth weight data arising from heaping into account. A modified and reliable estimate of LBW and macrosomia is needed to ensure accuracy at the national and international levels to guide future interventions and policies.It is found that when information on birth weight is collected from either health facility records or self-reported by mothers or other caregivers, the data tend to heap around multiples of 500 gThe purpose of this study was to estimate the prevalence of LBW and macrosomia by geographic areas, age and socio-demographic accounting for potential birth weight heaping, using a representative survey conducted in China in 2013.Overall, 32,276 records were included in the analysis Fig. , Table 1The overall prevalence of low birth weight of Chinese children younger than 6 years was 5.15% (95% CI 5.01–5.33%) in 2013, with 4.57% (95% CI 4.39–4.80%) in boys and 5.68% (95% CI 5.41–5.98%) in girls  in 2013, with 8.85% (95% CI 8.53–9.01%) in boys and 5.71% (95% CI 5.59–5.94%) in girls  or younger  mother, were in lower income household, or lived in west China or in rural areas. The macrosomia rate was particularly high for children who were Han ethnicity, were from higher income household, or lived in central or east China and urban areas, or whose mother had higher education degree or was older .15. The birth weight data from maternal or other caregivers’ recall showed heaping to multiples of 500 g, which were influenced by their age, wealth index quintile, marital status and education18. As a result, some of the infants with birth weight exactly 2500 g will actually have lower or higher birth weight than 2500 g and those are exactly 4000 g will actually have lower or higher birth weight than 4000 g. This further underestimates or overestimates the prevalence of LBW and macrosomia16. In order to address the challenge of heaping in survey-based estimates of the prevalence of LBW, Boerma et al.19 and Blanc and Wardlaw17 developed a method to combine reported birth weights with additional variables, such as mothers’ perception of the child’s size at birth, which could increase the proportion classified as having low birth weight by 25%17. Another adjustment method of heaping issue is birth weight distribution method for health-facility records or caregivers’ reports, which was used to estimate national, regional, and worldwide estimates of low birthweight in 20152. This method is also applicable to estimate the prevalence of macrosomia, which might provide more reliable estimates of the prevalence of LBW and macrosomia. Therefore, we fitted normal distributions to the birth weight data to produce more reliable estimates for the proportion of infants born with LBW and macrosomia.In most cross-sectional studies, birth weight data were recalled by caregivers. Clustering of birth weight data in multiples of 500 g affects estimates of the incidence of low birth weight and macrosomia20. Compared with the prevalence of LBW by the use of traditional calculation method (LBW (%) = total numbers of LBW/total numbers of surveyed children, 3.60%), the estimate using this heaping adjustment method (5.15%) was closer to that based on birth weight records in health facilities (5.36%)11. So does macrosomia in China. Compared with the prevalence of macrosomia with using traditional calculation method (5.30%), the adjusted estimate of 7.35% was closer to the estimate based on birth weight records in health facility (7.30%)14. Birth weight distribution adjustment method was more reliable than traditional calculation method for estimating prevalence of LBW and macrosomia based on recalled birth weight data or combined data of recalls and records.Birth weight records were less clustering on certain digits than recalled birth weights11. The macrosomia rate varied between 6.5% and 10.44% in China14. Different types of data and estimate methods may contribute to the variation. For example, Hu9 estimated the prevalence of LBW (2.77% in 2006–2008) and macrosomia (10.44% in 2006–2008) by the use of traditional method based on recalled birth weight data. Yu et al.13 reported a prevalence of LBW and macrosomia of 4.60% and 6.50% respectively in 2006, Chen et al.10 reported a LBW rate of 6.10% and Li et al. reported a macrosomia rate of 7.30% in 2011 based on recorded data. In addition, different study areas and study population may partially explain the variation. One study was from 14 provinces in China13. Another study enrolled subjects from 5 provinces9. The third study included 12 cities and counties in southeast China12. The remaining studies were from 39 hospitals of 14 provinces14 or China’s National Maternal Near Miss Surveillance System (NMNMSS) in 441 health facilities11. Given that the lower hospital delivery rate in developing countries, especially in poor rural areas, and bias in the selection of delivery hospitals, estimates of the LBW and macrosomia rate from health-facility-based data are subject to bias and selection problems17. In 2014, more than 99.5% of newborns were born in hospital in China21. Although data on macrosomia was scarce, it was found that the prevalence of LBW varied in different class hospitals and the prevalence was higher in tertiary care hospital than that in secondary care hospital. Women with high-risk pregnancies or complications were transferred to high level hospital due to its advanced equipment and technology10. One of the advantages of survey data on birth weight is that they are from probability samples, which might reduce bias due to hospital selection and non-hospital delivery in developing countries. Therefore, nationally representative household surveys could improve the estimates of LBW and macrosomia rate with heaping adjustment.Recent studies reported that LBW ranged from 2.77 to 6.1% in China17. The LBW rate of 5.15% in China was lower than the one in developed countries such as the United States (8.00%), the United Kingdom (7.00%) and Japan (9.60%)2. The macrosomia rate of 7.35% in China was lower than the one in the United States (8.23%)22 and England and Wales (8.80%)23, but much higher than Japan (0.90%)24 and Spain (0.65%)25.Prevalence of LBW and macrosomia in developed countries were mainly based on health-facility recorded birth weight. Demographic and Health Survey (DHS) and Multiple Indicator Cluster Survey (MICS) collected birth weight recall data in developing countries and traditional estimates without adjustment of heaping issue may cause the prevalence of LBW and macrosomia substantially downwards9 and 2012–201411 corroborated with our results. In addition, poor rural areas have higher macrosomia rate than medium and small cities and non-poor rural areas in our study, which may be related to higher percentage of missing birth weight records. Nearly 40% of 2059 ineligible or missing birth weight records was from poor rural areas.Disparities existed across different regions of China in both LBW and macrosomia rate. The prevalence of LBW in economically underdeveloped western China was significantly higher than in central and eastern China and was also higher in rural areas than in urban areas. The prevalence of macrosomia was higher in relatively developed central and east China than in west China, and was also higher in urban areas than in rural areas. Previous studies in 200626 or over 35 years28, less educated mother30, low household income32, or minority) tend to have higher LBW rate. By contrast, subpopulation  tend to have higher macrosomia rate. This observation was consistent with previous studies’ findings in China14 and other countries39. Dietary, nutrition and educational interventions could significantly reduce LBW rate43, and diet and exercise interventions could reduce macrosomia rate45.Vulnerable subpopulation  to control LBW or macrosomia. It would be needed to take potential misclassification of birth weight data arising from heaping into account for LBW or macrosomia estimation.46. Written informed consent was obtained from each child’s caregivers before the survey.Children under 6 years of ages who were free of birth defects and other health problems  were included, and they were categorized into 7 age groups . The study was approved by the Ethics Review Board of the National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention (Number: 2013-018) and all methods were performed in accordance with the relevant guidelines and regulations47. Briefly, this was a cross-sectional study on the nutrition and health status of children under 6 years and lactating women. A multi-stage stratified cluster random sampling was used to select participants under 6 years of ages from 30 provinces, autonomous regions and municipalities in mainland China (Tibet autonomous region was not included in the survey). A total of 55 national representative survey sites were selected from the 2865 districts/counties/county-level cities using proportion to population size sampling method. Then three sub-district/townships were selected in each survey sites with using population size sampling method and three neighborhood/village committees were randomly selected in each sub-districts/townships. Overall, 495 neighborhood/village committees were selected in this study. Finally, 70 children  with equal number of girls and boys were selected in each neighborhood/village committees using a cluster sampling method. Totally, 34,650 records were collected in the survey.The data of this study was from Chinese National Nutrition and Health Survey in 2013 (CNNHS 2013). Methods  have been previously described in detailA face-to-face interview was conducted to caregivers of children under 6 years. Birth weight data was collected by checking the birth certificate records or by caregivers’ recalls using a standard format and was recorded in grams in a questionnaire for children under 6 years. LBW is defined as a weight at birth < 2500 g and macrosomia is defined as a weight at birth > 4000 g. The household and individual’s demographic and socioeconomic information was collected using a household questionnaire, including infants’ age, gender, ethnicity, residential area, and maternal age, educational level, and household income level.17. It is found that birth weight is approximately normally distributed under ideal conditions such as low-risk full-term singleton livebirths included in the WHO child growth standards48. In order to address the challenge of heaping, we adopted an adjustment method to fits birth weight data to a normal distribution to calculate the prevalence of LBW and macrosomia.One of the methodological issues when analyzing birth weight data is heaping of reported birth weights. Previous studies have found that respondents in national health surveys show a tendency to round birth weight to multiples of 100 and 500 g, for example 2500 g instead of 2485 g2. First, we fitted two normal distributions of the data (full-term babies and preterm babies). Second, we calculated the LBW Z-score for each of the two normal distribution curves (full-term babies and preterm babies): Z2500 = (2500 g − Mean Birthweight)/SDBirthweight. Third, we calculated the percentage LBW for each of the two distribution curves: LBW (%) = probability of being below Z2500 (% area under the curve below Z2500). Finally, we calculated the overall LBW prevalence: Overall LBW (%) = (LBW (%)term baby population × total number of term babies + LBW (%)preterm baby population × total number of preterm babies)/total subjects. The proportion of macrosomia was calculated by the similar steps except a single normal distribution was fitted to the data. First, we fitted one normal distribution to the data. Second, we calculated the macrosomia Z-score for the normal distribution curve: Z4000 = (4000 g − Mean Birthweight)/SDBirthweight. Finally, we calculated the percentage macrosomia: macrosomia (%) = probability of being beyond Z4000 (% area beyond the curve of Z4000). The 95% confidence interval of LBW and macrosomia were also calculated by the same way.In summary, the proportion of low birth weight could be estimated in four stepsData were entered via a standardized data management platform and were cleaned for all variables. The prevalence is shown as percentage . We examined prevalence of LBW and macrosomia in the total population of and in sociodemographic subpopulations for children under 6 years. Student’s t-test and ANOVA was used to compare the means of each group and chi-square test to compare prevalence or proportion. All the data were analyzed using SAS 9.4 release . Statistical tests were two-sided and a P value below 0.05 was considered statistically significant.Supplementary Information.


This cohort study compares the risk of preterm birth among mothers with singleton live births who conceived naturally and those who used assisted reproductive technology or other fertility treatment. Are singleton pregnancies with fertility treatment associated with an increased risk for preterm birth?In this cohort study of 14 370 920 mother-newborn pairs, fertility treatment, including assisted reproductive technology and other procedures, was associated with preterm singleton births. Neonates who were conceived by fertility treatment had higher rates of very preterm and extremely preterm birth.The findings suggest an association between fertility treatment and preterm birth in singleton-birth neonates, warranting additional investigation. Preterm birth is a global health issue. The association between fertility treatment and preterm singleton births has not been clarified.To examine the association between fertility treatment and preterm birth.This population-based cohort study used birth data that were submitted by 50 states and the District of Columbia to the National Vital Statistics System database. All mothers in the database who had a singleton live birth from January 1, 2016, to December 31, 2019, were included. Those who had preexisting hypertension or diabetes were excluded.Fertility treatment categorized as assisted reproductive technology (ART) or non-ART treatment.The main outcome was a diagnosis of preterm birth, which was defined as birth before 37 complete weeks (<259 days) of gestation. Gestational age was calculated by obstetric estimation at delivery and was collected from the database.P < .001) and non-ART treatment  had significantly higher risk for preterm birth after full adjustment. These associations were similar in subgroups of participants as defined by baseline characteristics.The final sample consisted of 14 370 920 mothers  with singleton live births. Of these women, 122 944 (0.9%) conceived by ART and 71 176 (0.5%) received non-ART treatment. The prevalence of preterm birth was 7.6% (n = 1 071 994) in natural conception, 10.7% (n = 13 205) in ART, and 9.3% (n = 6629) in non-ART groups. Compared with neonates who were naturally conceived, newborns who were conceived with ART (adjusted risk difference [aRD], 3.10% ; adjusted odds ratio [aOR], 1.49 ; This study found that singleton neonates who were conceived by fertility treatment had higher rates of preterm birth. Further investigations are warranted into the association between ART or non-ART treatment and the risk of preterm birth in singleton neonates. Prematurity and its associated complications are the leading cause of death in neonates and children younger than 5 years worldwide.3 Furthermore, preterm neonates are at a higher risk for lifelong health and developmental challenges compared with term newborns.5 It is imperative to identify the essential risk factors for preterm birth and to enhance perinatal and neonatal management.Preterm birth is a crucial global health issue. The worldwide incidence of preterm birth was 10.6%, equating to 14.84 million live-born preterm babies in 2014,6 and 8% to 12% of the childbearing population worldwide.8 The World Health Organization recognizes infertility as a global public health issue.9 With the rapid development of fertility treatment, which includes assisted reproductive technology (ART)10 and non-ART medically assisted treatment, conceiving a child has been possible for infertile couples. Assisted reproductive technology is defined as treatment in which eggs or embryos are handled for the purpose of achieving pregnancy. In the US, the number of fertility treatments and fertility treatment–conceived births has increased steadily since the early 1980s.12 At present, approximately 2% of pregnancies are conceived through ART, and its use has been increasing in recent years.14 Non-ART treatment, which includes fertility-enhancing drugs, artificial insemination, and intrauterine insemination, accounted for even a higher proportion of pregnancies.15 Although greater availability of fertility treatments can help reduce the burden of infertility worldwide, some studies found that fertility treatments were associated with an increased risk of adverse health outcomes for both mothers and neonates, including preterm birth.16Infertility, defined as the failure to achieve pregnancy after 12 months of regular unprotected sexual intercourse, affects 12.1% of US women aged 15 to 49 years14 Compared with singleton pregnancies, multiple pregnancies may be at greater risk for preterm birth. The association between fertility treatment and preterm birth in singleton neonates has not been clarified. Most studies have suggested that even singleton neonates who were conceived with ART may also be at higher risk for preterm delivery compared with neonates in the general population.18 However, a prospective cohort study published in 2020 found no substantial difference in adverse outcomes associated with ART in singleton pregnancies.19 Furthermore, the association between non-ART treatment and preterm delivery has not been comprehensively investigated.A consequence of the increased use of fertility treatment, especially ART, is a substantially higher rate of multiple pregnancies.The association between fertility interventions and preterm birth needs to be explored in a large population with substantial available information for potential confounding factors to enable physicians to provide tailored prepregnancy and postpregnancy advice and care to women undergoing fertility treatment. In this cohort study, we used US birth certificate data to examine the association between fertility treatment and preterm birth.STROBE) reporting guideline.This prospective, comparative, population-based cohort study used the National Vital Statistics System, a database that contains birth and death records that are electronically submitted by the 50 states and District of Columbia to the Centers for Disease Control and Prevention and National Center for Health Statistics. All US states had implemented the revised birth certificate as of January 1, 2016. Specifically, for this study, we obtained maternal and neonatal demographic and health characteristics for all mothers in the database with singleton live births from January 1, 2016, to December 31, 2019, excluding those with prepregnancy hypertension or diabetes. Records with no information on gestational age or fertility treatment were also excluded. The Children’s Hospital of Fudan University Institutional Review Board deemed the study exempt from review and waived the informed consent requirement because the data used were publicly available and the study did not involve human participants. We followed the Strengthening the Reporting of Observational Studies in Epidemiology , very (delivery at 28-31 weeks and 6 days), and moderately and late (delivery at 32-36 weeks and 6 days). Birth weight was collected directly from the database and was converted from pounds and ounces to grams by the National Center for Health Statistics.The main outcome was diagnosis of preterm birth, which was defined as birth before 37 complete weeks (<259 days) of gestation according to the Exposure measurement in this study was pregnancy that resulted from fertility treatment, including (1) ART, such as in vitro fertilization, gamete intrafallopian transfer, and zygote intrafallopian transfer, and (2) non-ART treatment, such as fertility-enhancing drugs, artificial insemination, and intrauterine insemination. Mothers who received both ART and non-ART treatment were classified under the ART group. The exposure information was collected directly from the medical and health information section of the US Standard Certificate of Live Birth.Variables that were considered as potential confounding factors included maternal age, race and ethnicity, educational level, marital status, parity, smoking status before pregnancy and during pregnancy, history of preterm delivery, history of cesarean delivery, prepregnancy body mass index (BMI), timing of initiation of prenatal care, gestational hypertension, eclampsia, gestational diabetes, and neonate sex.Maternal age was defined as age at the time of birth and classified as younger than 30, 30 to 34, 35 to 39, or 40 years or older. Maternal race and ethnicity were self-reported in the database and included Hispanic, non-Hispanic Black, non-Hispanic White, and other individuals . Maternal educational levels were recorded as lower than high school diploma, high school diploma, or higher than high school diploma. Marital status was categorized as married or unmarried. Parity, defined as the total number of live births excluding the current delivery, was classified as 0, 1, 2, 3, or 4 or more. Smoking status before pregnancy and during pregnancy was classified as yes or no. Time of initiation of prenatal care was categorized by the trimester of the first prenatal visit as no prenatal care, first to third month, fourth to sixth month, and seventh to ninth month. Maternal prepregnancy BMI was calculated as prepregnancy weight in kilograms divided by height in meters squared and classified as less than 18.5, 18.5 to 24.9, 25.0 to 29.9, 30.0 to 34.9, 35.0 to 39.9, or 40 or higher. Gestational diabetes, gestational hypertension, and eclampsia were each categorized as yes or no. History of preterm delivery was classified as yes or no. These maternal variables were identified from the facility worksheet of the US Standard Certificate of Live Birth.Neonate sex was categorized as male or female. Birth weight was converted from pounds and ounces to grams by the National Center for Health Statistics. We subdivided birth weight into low birth weight (<2500 g), very low birth weight (<1500 g), or extremely low birth weight (<1000 g). Variables including birth weight and neonate sex were collected from the database. A missing category for covariates was added when necessary.P values were 2-sided, and P < .05 was considered to be statistically significant. Means and SDs were reported for continuous variables, and frequencies and percentages were reported for categorical variables. Differences were tested using the χ2 test for categorical variables and an unpaired, 2-tailed t test or analysis of variance for numerical variables.Statistical analyses were performed using Stata, version 15.0 (StataCorp LLC). All 24 we covered multiple potential confounding factors with 3 models. Model 1 was not adjusted. Model 2 was adjusted for sociodemographic characteristics, including maternal age, race and ethnicity, educational level, and marital status. Model 3 was model 2 but further adjusted for potential pregnancy-related confounders, including parity, smoking status before and during pregnancy, history of preterm delivery, history of cesarean delivery, prepregnancy BMI, timing of initiation of prenatal care, gestational hypertension, gestational diabetes, eclampsia, and neonate sex. Furthermore, all results in the main analyses were provided with an adjusted risk difference (aRD) and 95% CI using the postestimation margins command in Stata.25 The analyses were repeated for the finer categorization of preterm birth. We corrected for multiple testing using the Bonferroni method in the main analyses; therefore, only unadjusted P > .004 was considered to be significant at the 95% CI level.We performed a series of logistic regression models to estimate the odds ratios (ORs) with 95% CIs of preterm birth vs fertility treatment. In the main analyses, we estimated the association between fertility treatment (ART and non-ART treatment group separately) and preterm birth, with neonates who were conceived naturally as the reference group. Informed by previous research,In the stratified analysis, we subdivided women according to baseline characteristics . Among these subgroups, we analyzed the association between fertility treatment and preterm birth after adjusting for other potential risk factors to explore potential disparities in the association between fertility treatment and preterm birth.In addition, to examine the robustness of the results, we performed a series of sensitivity analyses. First, we reran the model after excluding potential risk factors whose effect sizes were significant in the main analyses  to assess the implications of these complicated pregnancy conditions for the association between infertility treatment and preterm birth. Second, to examine the implication of missing covariate data for the analysis, we conducted a sensitivity analysis in which mothers with incomplete covariate data were omitted.The final sample consisted of 14 370 920 mothers  with singleton live births . A compaP < .001) and extremely preterm births  were significantly higher in the ART and non-ART treatment groups compared with the natural conception group (P < .001). Baseline characteristics are shown in Of the 1 091 828 preterm births (7.6%), 948 714 (6.6%) were classified as moderately and late, 90 873 (0.6%) as very, and 52 241 (0.4%) as extremely preterm. The prevalence of preterm birth was 7.6% (n = 1 071 994) in those with natural conception, 10.7% (n = 13 205) in those who received ART, and 9.3% (n = 6629) in those who used non-ART treatment. Incidence rates of very preterm  or non-ART treatment  had a higher risk of preterm birth. After (model 3) adjustment for maternal age, race and ethnicity, educational level, marital status, parity, smoking status before and during pregnancy, history of preterm delivery, history of cesarean delivery, prepregnancy BMI, timing of initiation of prenatal care, gestational hypertension, eclampsia, gestational diabetes, and neonate sex, the odds of preterm birth were 49% higher in the ART group than in the natural conception group  and 35% higher in the non-ART treatment group compared with the natural conception group . The full logistic regression model output for all covariates is provided in eTable 2 in the P < .001) and 46% higher in the non-ART treatment group  after full adjustment (model 3). Risk of extremely preterm birth increased by 88% in the ART group  and by 83% in the non-ART treatment group  compared with the natural conception group after full adjustment (model 3)  . These rStratified analyses by maternal age, race and ethnicity, prepregnancy BMI, gestational hypertension, gestational diabetes, eclampsia, history of preterm delivery, history of cesarean delivery, and neonate sex were conducted. The neonates who were conceived with ART or non-ART treatment had a higher risk of preterm birth in almost all subgroups .Sensitivity analyses, from which we excluded women with potential risk confounders such as cesarean delivery history, preterm delivery history, gestational hypertension or eclampsia, and gestational diabetes, yielded similar results as the main analyses . After e26 Despite a large number of people worldwide who have conceived through fertility treatments, the potential adverse effects of these procedures, including preterm birth, have not been fully explored. We believe this study has added clinical and public health implications to the knowledge of fertility treatment outcomes.In this nationwide population-based cohort study of more than 14 million multiracial and multiethnic pairs of mothers and singleton newborns, we found that conception using fertility treatment, either ART or non-ART, was significantly associated with preterm birth compared with natural conception after adjusting for all covariates. However, this result does not rule out the association of infertility with preterm delivery, and the risk outcome might be overestimated. Infertility is becoming a common health care problem and has drawn widespread attention in recent decades.28 A meta-analysis that included 8044 singleton births assisted by either in vitro fertilization or intracytoplasmic sperm injection and 53 633 singleton births by spontaneous conception found that the risk of preterm birth was significantly increased in ART-conceived singleton births.18 A cohort study in Finland reported that medically assisted reproduction, including ART and non-ART fertility drugs, was associated with an elevated risk of preterm birth outcomes.29 However, Martin et al30 found no association between increased risk of preterm birth and single-embryo transfer for singleton pregnancies, but double-embryo transfer was associated with a significant increase in the risk of preterm birth. This finding is not contrary to the results of the present study in that we did not conduct a subgroup analysis of detailed ART because of lack of proper data. A prospective cohort study that recruited 4252 women who conceived using ART observed no association between ART and preterm birth in singleton births.19 This result differed from the findings of this study, partly because of the limited sample size of the previous investigation.The results of this study are consistent with those of existing studies that found an increased risk of preterm birth associated with fertility treatment–assisted pregnancies. Several cohort studies have compared the perinatal outcomes between singleton births using ART and singleton births from spontaneous conception, with adjustment for relevant confounders.31 reported that low reproduction technology, such as intrauterine insemination and donor insemination, was not associated with an increased risk of very preterm and extremely preterm birth, which contradicts the results of the present study. A potential reason for the difference might be the confounder of multiple pregnancies, and singleton pregnancies were the focus of this study.We also analyzed the associations between non-ART treatment in singleton pregnancy and preterm birth. We found that non-ART treatment might be associated with an increased risk of preterm delivery after adjusting for all confounders. This result is partly consistent with findings from previous studies except that the subgroup analysis in the current study revealed a significant increase in the risk of extremely preterm, very preterm, and moderately preterm events. Wang et al24 However, in the stratified analyses, we observed no fertility treatment–associated increase in the risk of preterm birth among women with a history of preterm delivery. One explanation might be that women undergoing fertility treatment who have a history of preterm delivery might pay more attention to perinatal care, which could reduce the risk of preterm birth. Another possible explanation is that women with a history of preterm delivery are less likely to be infertile, and thus it might be reasonable to assume that the group with no history of preterm delivery comprises more women with infertility. Furthermore, in the sensitivity analyses, the results remained robust after excluding women with a history of preterm delivery.Studies have found that women with previous preterm deliveries had a higher risk of preterm birth in their next pregnancy.32 A meta-analysis of 7 studies reported an increased risk of preterm births associated with singleton births by spontaneous conception in women with subfertility compared with singleton births in fertile women.33 In the present study, we lacked the data to distinguish the association of infertility with preterm birth from the association of fertility treatment with preterm birth, and the risk associated with fertility treatment might be overestimated.Preterm birth in neonates who were conceived with fertility treatment is likely multifactorial. A major challenge for research that reports on this association is separating the contribution of infertility from that of ART. The fertility treatment may alter the endocrine profiles in pregnancy, which may affect placental and fetus development. Meanwhile, increasing evidence indicates that subfertility itself, including parental characteristics, may also have adverse implications for preterm birth.34 Dysregulation of DNA methylation patterns associated with different ART methods may affect the development of placenta and fetus in the early embryonic stages and may introduce changes in growth patterns, and DNA methylation has already been found to contribute to the formation of congenital malformation.35 Although no manipulation of embryos was involved in non-ART treatment, the use of pharmacological agents to stimulate the production of oocytes was associated with poor-quality embryos.36 In addition, processing of sperm for artificial insemination might be associated with oxidative stress, which could potentially lead to chromosomal damage. Further research into the potential biological mechanisms of fertility treatment–induced preterm birth is warranted.The potential mechanisms of the association between ART or non-ART treatment and preterm birth need to be elucidated. DNA methylation is a crucial element in the epigenetic regulation of mammalian embryonic development.This study has some strengths. It analyzed a nationwide population of mother-newborn pairs, included a large sample size, and had low levels of missing data, which provided sufficient statistical power to enable the examination of the association between maternal infertility treatment and preterm birth in subpopulations. Furthermore, the study excluded multiple pregnancies, pregestational hypertension, and pregestational diabetes. This approach limited confounding from these known preterm risk factors.38 In this study, mothers with fertility treatment tended to be older and were more likely to have gestational hypertension and gestational diabetes. Although we adjusted many maternal characteristics and complicated pregnancy conditions to mitigate confounding, we used administrative data and thus cannot guarantee complete and accurate data collection. We conducted a series of sensitivity analyses to verify the robustness of the main results, although residual confounding may still exist, and we could not ascertain that the adjustment was adequate. Furthermore, there were missing values for the variables included in the logistic regression analysis. However, a sensitivity analysis that deleted records with missing variables yielded similar results, suggesting the robustness of the data used in this study.This study also has several limitations. First, a challenge in studying pregnancy outcomes in women who conceived by fertility treatment is selecting the appropriate comparison group. In this study, we used mothers with spontaneous conception as a reference. We assumed those who met the criteria of infertility while conceiving without fertility treatment would be a better comparison group. The 3 groups in this study  differed by age, socioeconomic status, educational level, and reproductive history, and we performed a series of logistic regression analyses and subgroup analyses to verify the reliability of the results. Second, with such an observational study, a central concern was unmeasured or uncontrolled confounding.This cohort study found that singleton births after fertility treatment had a higher risk of preterm birth. Understanding this risk is essential for individuals who are considering using fertility treatment to conceive and for physicians who provide prepregnancy and postpregnancy advice and care to these individuals. Further investigations are warranted into the mechanisms of the association between ART or non-ART treatment and the risk of preterm birth.


Objective: This study investigated changes of plasma cytokines and aimed to build a dynamic nomogram for diabetic macular edema (DME) in type 2 diabetes mellitus (T2DM).Methods: In a pilot cohort, plasma samples were selected from 9 T2DM patients and 9 DME patients to screen for cytokine differences. The screening cytokines were then validated by enzyme-linked immunoassay in a cohort, which contained 100 DME (DME group) and 100 T2DM patients without DME (T2DM group). A dynamic nomogram for predicting DME was developed, based on the plasma cytokines.Results: In the pilot cohort, 11 plasma cytokines were significantly increased in the DME group. In the validation cohort, platelet-derived growth factor (PDGF)-BB, tissue inhibitors of metalloproteinase (TIMP)-1, angiopoietin (ANG-1), and vascular endothelial cell growth factor receptor (VEGFR)-2 were confirmed to be significantly elevated in the DME group. The dynamic nomogram demonstrated good calibration and discrimination, with an area under the receiver operating characteristic curve (AUC) of 0.88. In the test set, sensitivity, specificity, and AUC were 73.3%, 80.0%, and 0.84, respectively.Conclusion: Plasma cytokines were closely associated with DME. A novel dynamic monogram including ANG-1, PDGF-BB, TIMP-1, and VEGFR2 was a novel tool for predicting DME. Diabetic macular edema (DME) is one of the most common microvascular complications in patients with type 2 diabetes mellitus (T2DM) and is a major cause of vision loss worldwide. The prevalence of DME depends on the type and duration of diabetes, and the incidence of DME is higher in individuals with type 1 diabetes than in those with T2DM. Approximately 27% of patients develop DME within 9 years of diabetes onset . AlthougAngiogenesis and inflammation response are key mechanisms of DME development . ANG-1 eTo explore the potential role of plasma angiogenesis and inflammation cytokines in DME, we investigated the levels of vascular and inflammatory cytokines by protein-chip screening and enzyme-linked immunosorbent assays (ELISAs). A dynamic nomogram for predicting the risk of DME in patients with T2DM was developed and was based on the DME-associated plasma cytokines.The general characteristics of the study subjects are shown in p < 0.05, To profile plasma cytokines, blinded screening by protein microarray analysis was performed, and semi-quantitative results were obtained for 60 plasma proteins. The relative changes of plasma cytokines are shown in the heatmap in p < 0.001). Between-group differences in CXCL16 (3776.43 pg/ml vs. 3794.39 pg/ml) were not significant (p = 0.791).To further determine whether ANG-1, CXCL16, PDGF-BB, TIMP-1, and VEGFR2 levels were significantly increased in DME, 100 DME patients and 100 T2DM patients were recruited to validate the array data using ELISA. As shown in r < 0.3). Focusing on the inner relationship of cytokines, there was also no obvious correlation among these 5 cytokines  accounted for 41.7%, and 20.3% of the variation, respectively, in the dataset. The projection of samples in the PCA revealed relatively little overlapping of areas. CXCL16 contributed more to the second than to the first principal component. ANG-1, PDGF-BB, TIMP-1, and VEGFR2 contributed more to the first principal component . As showp = 0.026], PDGF-BB , and TIMP-1  were identified as significant risk factors. VEGFR (p =0.061) did not reach statistical significance. However, as Akaike information criterion (AIC) increased from 129.65 to 131.32 when removing VEGFR2 from the model, it was retained in the analysis. Multivariate logistic regression was used to develop a model to predict DME. As shown in Based on the results of multivariate logistic regression, a nomogram including ANG-1, PDGF-BB, TIMP-1, and VEGFR2 was constructed for DME risk prediction . The poihttps://doctorcao.shinyapps.io/DynNomapp/).The decision curve demonstrated that in both the training and test datasets, the net benefit was greater than with an all-or-none patient intervention scheme if the threshold probability was less than 60%, which supports use of the nomogram in clinical practice . A cliniDME is a type of retinal vascular disease. Angiogenic and inflammatory processes mediate the genesis of DME, and it is important to define which processes are enhanced or decreased in clinical practice. In the pilot cohort, we analyzed the expression changes of 60 angiogenic and inflammatory factors in DME by protein-chip screening. Eleven cytokines, including PDGF-BB, TIMP-1, ANG-1, and VEGFR2 were significantly increased in DME compared with those T2DM group. We expanded the sample size in the validation cohort and obtained consistent results that were used to develop a dynamic online nomogram for DME prediction. The user-friendly digital interface makes it easier to predict risk and to make better clinical decisions.In this study, we highlighted the significance of increased expression of inflammatory and angiogenic factors in DME. Studies found that cytokines were elevated or decreased in the DME group and were closely related to the pathogenesis of vascular permeability and inflammation during the development of DME –13. Our In this study, the characteristics of elevated vascular inflammatory factors in DME were used to establish a prediction model for DME. Previous clinical studies have reported elevated VEGF, MCP1, IL-6, and ICAM-1 were detected in vitreous fluids of DME patients, which is consistent with our results –22. TherThe use of OCT-imaging biomarkers for evaluating therapeutic response has been described. Ceravolo et al.  demonstrWe did not observe positive correlations between cytokines and indexes of glucose metabolism such as HbA1c or C-peptide in the DME group. The possible cause was that the study subjects were all hospital inpatients in the department of endocrinology for improved glucose management and control, which may have eliminated the effects of glucose level on the results. The association of cytokines with the course of disease or diabetic complications needs further investigation.There were some limitations. First, we included about 200 subjects for model development and verification. Although the results were clear, there was no external cohort to validate the performance of the model. A large prospective study is needed for further validation. Second, coronary heart disease (CHD) may also result in abnormal expression of plasma proteins. However, the diagnosis of CHD was based on a history of disease, and as CHD in diabetes patients may be asymptomatic, a few CHD patients may have been included in this study.In conclusion, plasma ANG-1, TIMP-1, PDGF-BB, and VEGFR2 were increased and were comprehensive predicators of DME when included in a dynamic nomogram. The nomogram needs further confirmation in large populations.The study received ethical approval from the competent Institutional Review Boards of Beijing Luhe Hospital. All procedures complied with the ethical principles of Helsinki Declaration for studies of human subjects. The study was registered on clinicaltrials.gov (NCT03811470).Patients were recruited at the Center for Endocrine Metabolism and Immune Diseases of Beijing Luhe Hospital, Capital Medical University, Beijing, China from January 2017 to December 2018. All patients participated in the program of the National Metabolic Management Center (MMC) . 9 T2DM For the antibody arrays and ELISA, blood samples were collected at our center with ethylenediaminetetraacetate as the anticoagulant. The plasma was isolated by standard blood processing and the aliquots were frozen and stored at −80° C, avoiding freeze-thaw cycles.Cytokines were assayed in duplicate with RayBio R Human Cytokine Antibody Array . Spiking and recovery tests were performed to ensure a linear concentration response. As determined by densitometry, the interarray coefficient of variation of spot signal intensities was 20%. Cytokine results were presented as a heatmap using the “pheatmap” package.Plasma PDGF-BB, TIMP-1, ANG-1, CXCL16, VEGFR2 levels were determined with human ELISA kits (Mlbio) following the manufacturer’s instructions. The intra-assay coefficient of variation was 20%, and the intra-assay coefficient of variation was 12%. No significant cross reactivity or interference was observed.The patients in the validation cohort were randomly divided into training and test sets, which included 70% and 30% of the data, respectively. The cytokines most predictive of DME were selected by PCA using the data included in the training cohort. Multivariate logistic regression identified the cytokines significantly associated with DME development and they were used to construct a nomogram presenting a specific system for calculating the risk of DME. The performance of nomogram was evaluated by sensitivity, specificity, discrimination, and calibration. Discrimination, which is the predictive accuracy to distinguish patients with DME from those without DME, was measured by the receiver operating characteristic (AUC). A calibration curve, which reflected the consistency between the predicted probability and the actual probability, was plotted using 1000 bootstrap resamples. Decision curve analysis (DCA) was used to assess clinical usefulness of the nomogram. For access to risk estimation, a dynamic nomogram having a user-friendly digital interface was created online using the DynNom package .t-tests for normally distributed data; otherwise, the nonparametric Mann-Whitney test was used to analyze the data. Chi-square tests were performed for categorical variables. Pearson’s correlation analysis was performed to assess the relationships of the plasma cytokines. In all cases, p-values < 0.05 were considered statistically significant.All data analysis and visualization were performed with in R software, version 3.6.3 . Significance was evaluated by The study received ethical approval from the competent Institutional Review Boards of Beijing Luhe Hospital.Supplementary Figures


Low birth weight and prematurity remain leading causes of infant mortality and morbidity globally. Although extensive literature has highlighted the importance of socioenvironmental characteristics for birth outcomes, the role of indirect violence on health remains fairly understudied.Região Oeste Coorte – ROC-Cohort) of infants born between 2012 and 2014 and geocoded crime reports, we assessed the associations between exposure to violent crimes during pregnancy within a 1-km radius of the mother’s residence and low birth weight, preterm delivery, and being born small-for-gestational-age. Violent crime exposure was categorized into quintiles. Multivariate logistic regressions were used to examine the associations between violence exposure and birth outcomes. Models were adjusted for sex, maternal age and education, socioeconomic status, and risk factors such as hypertension, diabetes, smoking, and drinking during pregnancy.Using geocoded birth records from the ongoing Western Region Birth Cohort , preterm (1.35[1.01–1.80]), and low birth weight (1.42[1.03–1.98]). While socioeconomic status and maternal education were positively associated with lower violence exposure, no associations were found between these characteristics and birth outcomes.Higher exposure to violent crimes in the close vicinity of pregnant women’s residence is associated with substantial increases in the odds of adverse birth outcomes. Policies to improve neighborhood safety can potentially contribute not only to the short-term wellbeing of populations but may also have large social, economic, and health benefits in the long term.The online version contains supplementary material available at 10.1186/s12889-021-10900-y. Low birth weight accounts for more than 80% of all neonatal mortality globally . In 2015Violence is a growing public health concern with immediate and long-term influences on population health –10. ContExposure to violence has been consistently associated with mental health problems and high-risk behaviors such as alcohol, smoking, and drug abuse, which threaten the wellbeing of both adult and child populations , 12, andChronic exposure to such environmental stress can trigger neural or neuroendocrine responses, change the functioning of the hypothalamic-pituitary-adrenal (HPA) axis and lead to increased allostatic load . Stress A growing body of literature has tried to link external violence’s to adverse birth outcomes at the area level –33. The In this paper, we aim to explore the relationship between exposure to violent crime in close vicinity and three of the most common adverse birth outcomes: preterm birth, small-for-gestational-age, and low birth weight in one of the largest metropolitan areas of the world . Using aThis study was designed as a retrospective cross-sectional study linking data collected as part of a prospective cohort study to geocoded administrative data on violent crime.This study was conducted in the municipality of São Paulo, Brazil. In São Paulo State, the proportion of preterm births increased from 7.7% in 2004 (7.7%) to 12.4% in 2013 . At the Like other LMICs, Brazil has experienced a rapid and mostly unmanaged urbanization process in the past decades, accompanied by large increases in social and economic inequality , 42. TheRegião Oeste Coorte – ROC-Cohort) enrolled locally resident babies born at the University Hospital of the School of Medicine of the University of São Paulo between April 1, 2012, and March 31, 2014. The study’s main objective was to evaluate the impact of environmental and social determinants of health on child development. A total of 7066 births occurred in the period, and 6207 mother-child pairs who resided in the studied setting were enrolled in the cohort. Children were followed-up at 6 months, 12 months, and 36 months. The cohort is still active, and the 72 months (6 years) follow-up was paused due the ongoing COVID-19 pandemic. The follow-up rounds targeted all enrolled children who were alive at the time of the follow up. Assessments involved child development, mental health, health habits and status, parenting practices and general living conditions. A full description of the cohort can be found elsewhere [The São Paulo Western Region Birth Cohort , preterm delivery (PT), and small-for-gestational-age (SGA). Birth weight was measured by the Hospital’s neonatology team immediately after birth using standard hospital equipment. Gestational length in weeks was estimated using the New Ballard Score . LBW waswww.ssp.sp.gov.br. The system collects detailed information on all willful murders, femicides (gender-based murder), robberies followed by death, bodily injuries followed by death, deaths resulting from police intervention, suspicious deaths, fatal vehicle accidents, and cellphone and car theft occurring in São Paulo State. Data on other crimes such as intimate partner abuse , rape, and child sexual assault are not collected in this system.Data on violence is routinely collected and made publicly available by the Secretariat of Public Safety of the State of São Paulo and can be accessed on Each reported incident record contains the date, time, and address of the crime. Following most of the external violence literature, we focused on violent crime in our analysis, which includes willful murder, robbery followed by death, bodily injury followed by death and death resulting from police intervention but excludes other crimes such as robberies without injuries. Feminicide was not included in the analysis because this data was only available after 2015.ggmap package [We extracted data on violent crimes between 2011 and 2014 to cover all ROC-Cohort children’s pregnancy period. The address of each reported crime was geocoded with latitude and longitude coordinates using the  package , and a p package . The matWe first computed the average number of crimes for each of the exposure quintiles. Next, we tested crude associations of exposure to violence quintiles with each outcome . In the fully adjusted multivariable models, we included all covariates highlighted in the existent literature as risk factors for adverse birth outcomes, including maternal age , educational level , alcohol consumption (yes/no), smoke (yes/no), diabetes (yes/no), hypertension (yes/no) and experience of physical violence (yes/no) during pregnancy – reported by the mother in the postpartum questionnaire, and socioeconomic (SES) index. The construction of a socioeconomic (SES) index was based on the approach proposed by Vyas and Kumaranayake , which rGiven that the postpartum questionnaire was not available for the whole sample, we initially screened the dataset for missing data and patterns of non-responsiveness in the postpartum questionnaire using t-test and chi-square to test if the assumptions of missing at random (MAR) were met. The reasons for not completing the postpartum questionnaire vary from being too young to consent to being discharged before the interview could be completed. The details can be found elsewhere . As the mi package [We performed the MICE using Stata’s  package . The var package . All ana package . FurtherAmong the 6207 children in the ROC-Cohort, 44 were stillbirths and excluded from the analysis. The likelihood of being stillborn was not associated with violence exposure during pregnancy in our sample . Additionally, 895 mother-child dyads were excluded due to an address outside of the studied setting. Overall, 5268 children and their mothers met the inclusion criteria for this study.n = 2008) did not differ from those who completed (n = 3260) the survey with respect to the type of birth, gestational length category, birth weight and length, and child sex.Mothers who did not respond to the postpartum questionnaire (398 (7.8%) births were classified as SGA, 369 (7.0%) as PT, and 355 (6.7%) as LBW. Table Figure p = 0.014), while PT and SGA displayed positive but not statistically significant associations with violent crimes. Upper middle  and high  SES status was negatively associated with violent crimes. Physical violence during pregnancy reported in the postpartum questionnaire was also negatively associated with violent crimes .The prevalence of SGA, PT and LBW were 6.7, 7.9, and 9.5% (SGA), 6.5, 6.4, and 6.3% (PT), and 5.4, 7 and 7.5% (LBW) in the first, third, and fifth exposure quintiles, respectively. Table Figure 2 - experiences, on average, 0.94 violent crimes per month within 1-km walking distance from home. This implies 5.6 crimes in close vicinity within the first six months of pregnancy for the average pregnant mother. Even mothers will, with all likelihood, personally witness only a small fraction of these events, the threat of external violence is, without any doubt, real and constantly present.This study uses a novel data set from Brazil to explore the empirical associations between neighborhood-level exposure to violent crime and birth outcomes. Our study yields three main findings: first, exposure to violent crimes is very high among pregnant women in the urban areas studied. Based on the São Paulo estimated population by the Brazilian Institute of Geography and Statistics and violent crimes reports extracted by us from the Secretariat of Public Safety of the State of São Paulo, from 2011 to 2014, there were on average 2.87 violent crimes per 10,000 inhabitants in the city of São Paulo and an average of 3.62 violent crimes per 10,000 inhabitants in the study area. While these numbers may be somewhat abstract, they imply that each person living in a densely populated area like the one studied - with an estimated density of 7770 inhabitants per kmSecond, our results suggest that external violence should not be interpreted as homogeneous characteristics of entire cities or larger census tracts, but something highly heterogeneous within relatively small spatial areas. As shown in Fig. Third, and maybe most importantly, we found that exposure to crime is associated with substantial SGA, PT, and LBW increases even after controlling for SES status and maternal education, and other risk factors. Our findings that violence increases adverse birth outcomes are consistent with a previous municipality-level comparison from Brazil  and highThe general literature on violence exposure and birth outcomes has been more mixed with some studies such as Messer et al. , findingOur results corroborate the previous literature and add to the understanding of violence and its influence on health in a densely populated and heterogenous setting in an LMIC. Although our study did not explore the direct violence against pregnant women, previous findings suggest that domestic violence against pregnant women and mental disorders during pregnancy can have detrimental effects on birth outcomes in the Western Region of São Paulo . The patRapid rates of urbanization combined with increasing economic and gender inequalities are likely to trigger increased rates of violent crime and lead to a higher prevalence of risk factors in many settings, especially in areas where access to basic education and health care services remains limited , 64. HigThe analysis presented in this paper has some limitations. The ROC-cohort enrolled mother-child dyads at the delivery, and important risk factors such as diabetes, hypertension, and physical violence were assessed only through the postpartum questionnaire. Those variables were in the analysis as “yes/no” did not assess the extent or in which period of the pregnancy it occurred. Moreover, smoking and drinking were assessed in five categories ranging from “never” to “every day”, and in both variables, almost 90% fell in the first category (never). Therefore, we decided to recode to “yes/no”, since we also could not know which period of the pregnancy occurred. Another limitation is that in our post-partum questionnaire we did not collect information related to intimate partner abuse , rape, and child sexual assault which could influence women’s sense of safety that could impact pregnancy outcomes.Although violence exposure during the third trimester of pregnancy is likely to trigger adverse birth outcomes, our study only considered violence exposure during the first two trimesters of pregnancy. The primary reason for this was the presence of early preterm children  in our sample, which did not allow us to compute third-trimester exposure variables. Excluding these cases or computing only partial exposures would likely have resulted in substantial estimation bias. It is also not clear how complete the violence data is. The National System of Statistics on Public Security and Criminal Justice  classifiThe results presented in this paper suggest that living in a neighborhood with high rates of violent crime is associated with substantial increases in small-for-gestational-age, preterm and low birth weight births. Actions to improve neighborhood safety and to prevent violence could reduce inequalities among disadvantaged populations with likely substantial long-term benefits on population health and wellbeing.Additional file 1 Supplementary file 1: Postpartum questionnaire.Additional file 2 Supplementary file 2: Multiple imputations. Table S1. Missingness among variables used in the multiple imputation models.


Objectives: This study aimed to explore the changes in infant vitamin A (VA) status and the effect of early VA supplementation on VA level throughout the first 6 months of life.Methods: A prospective cohort study was conducted in Chongqing, China. A total of 1,016 healthy infants were enrolled at birth. Then, 930, 882, 854 and 822 healthy infants were followed up at postnatal day 7 and postnatal months 1, 3, and 6, respectively. Blood samples and dietary survey and physical development data were collected. Serum VA was measured by chromatography tandem-mass spectrometry and was classified according to the VA deficiency (VAD) criteria for older children aged 6–70 months . Normally distributed continuous variables are presented as the mean ± standard deviation. The categorical variables are described by the frequency and percentage (%). The reference interval for the VA level was the 2.5th−97.5th percentile. Changes in VA status with age and the relationship of VA supplementation with VA level were investigated by generalized estimating equations followed by Bonferroni post hoc test, controlling for the effects of feeding pattern and sex.Results: Infant VA levels increased significantly from 0.499 ± 0.146 to 1.061 ± 0.414 μmol/L with age at 6 months, even without VA supplementation (P < 0.05). From birth to 6 months, the percentage of infants with a VA level <0.70 μmol/L decreased from 88.6 to 19.5%. During follow-up, no infant demonstrated clinical VAD conditions, such as night blindness, conjunctival xerosis or Bitot's spots. Less than 7.0% of infants were underdeveloped in terms of weight, length and head circumference. The VA status of infants with VA≥0.588 μmol/L at birth gradually increased to adequate VA (VA ≥ 1.05 μmol/L) at 6 months. For these infants, there was no significant difference in VA level between the VA supplementation and non-supplementation groups (P > 0.05). Infants with VA <0.430 μmol/L at birth still had VA <0.70 μmol/L at 6 months; in this group, VA levels increased by 0.08 μmol/L more among supplemented infants than among non-supplemented infants (P < 0.05).Conclusions: A low VA level among neonates at birth may be a normal physiological state and may increase with age; thus, not all neonates may need early VA supplementation. More multicenter studies are needed to determine a new cutoff point for the diagnosis of neonatal VAD and the administration of nutritional intervention. Vitamin A (VA) is an essential fat-soluble micronutrient for normal functioning of the visual system as well as for immune response, gene expression, reproduction, embryogenesis, hematopoiesis, normal growth and neurocognitive development –4. VA nuAccording to the World Health Organization (WHO) , a serumIn addition, there are no clear clinical guidelines on whether neonates should be administered VA supplements in the early postpartum period , 19. On Therefore, we first followed up the VA nutritional status among healthy infants throughout the first 6 months of life by a large prospective longitudinal study to prove that the low VA level of neonates at birth may be a normal physiological state and that the percentage of neonates with VAD may be overestimated. Moreover, we explored the influence of postnatal VA supplementation on VA levels throughout the first 6 months of life to provide solid evidence regarding whether early VA intervention is necessary for infants.A population-based, prospective longitudinal study following infants throughout the first 6 months of life was conducted between May 2018 and May 2019 in Chongqing, China. This study was carried out in two representative large tertiary grade A hospitals in Chongqing, the Second Affiliated Hospital of Chongqing Medical University, located in an urban area, and Qianjiang Central Hospital, located in a suburban area. Chongqing is located in southwest China and is one of the most important central cities in China, with a total area of 82,400 square kilometers, a permanent resident population of 31,243,200 and a birth population of 326,200 per year.All infants born in these two hospitals from May 2018 to May 2019 who met the following inclusion criteria were enrolled in this study from birth: (1) parents who agreed to participate in this study; (2) gestational age ≥37 weeks and <42 weeks; (3) infant birth weight ≥2,500 g and ≤ 4,000 g; (4) singleton pregnancy; (5) prenatal examination showing no abnormalities; and (6) no metabolic or infectious diseases at birth. The exclusion criteria for the infants were as follows: (1) family disapproval of participation and (2) hospitalization for infectious diseases after birth.n = p, the estimated prevalence, was 18%  μmol/L, and the maternal VAD rate was 23.3%. The intake of maternal dietary VA was  μg RAE/d, and the insufficiency rate of dietary intake was 27.6% see .All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Medical Ethics Committee of Children's Hospital Affiliated with Chongqing Medical University (022/2014).After obtaining informed consent, the history of pregnancy and delivery, gestational age, and sex of the participants enrolled at birth were collected from the electronic medical records of the participants. Infant physical development was monitored by measuring weight, length and head circumference at birth and at postnatal day 7 and postnatal months 1, 3, and 6. Weight was measured to the nearest 10 g with an electronic baby scale . Length was measured using a measuring bed , and head circumference was measured using a flexible ruler , both with a precision of 1 mm. The measured value was the average value after three repeated measurements.Infant dietary information, including feeding patterns and VA supplementation, was collected using a questionnaire. At the 7-day, 1-month, 3-month, and 6-month scheduled visits, the diets of the infants since their last visits were reviewed and recorded. Feeding patterns were categorized into three groups . According to the infant nutrition and health guidelines , infantsSamples of cord blood at birth and capillary blood at 7 days, 1 month, 3 months, and 6 months postnatally were collected. Blood samples (0.1 ml) were collected, centrifuged, and stored at −80° within 12 h. The retinol concentration was detected within 2 months.The measurement of retinol level in serum is a common way to assess the level of VA in the body. The samples were analyzed at the Pediatric Research Institute, Children's Hospital Affiliated with Chongqing Medical University. The retinol concentration in serum was measured by high-performance liquid chromatography tandem-mass spectrometry (HPLC-MS/MS) using an API3200 . Briefly, serum samples (20 μl) were deproteinized with methanol containing an internal standard (0.5 μg/ml d6-retinyl acetate), extracted with hexane, evaporated to dryness under nitrogen, and reconstituted in methanol. The retinol in serum was separated by HPLC on a Shimadzu C18 75 ×2.0 mm column and quantitated by MS. All procedures were performed in a dark room to protect the samples from light. The lowest sensitivity of the measurements was 0.014 μmol/L for retinol.Infants with weight, length or head circumference below the third percentile were considered underweight, stunted or underdeveloped for head circumference, respectively. According to the Growth Standards and Growth Charts for Chinese Children , the thiP < 0.05.Statistical analysis was performed using the SPSS software system, version 20.0. Figures were drawn using the GraphPad Prism software system, version 5.0. Normally distributed continuous variables are presented as the mean ± standard deviation (SD) (2.5th−97.5th percentile). Categorical variables are described by the frequency and percentage (%). The reference interval for the VA level was the 2.5th−97.5th percentile. Changes in VA level and distribution with age were determined with generalized estimating equations. These equations were also used to investigate the relationship between VA supplementation and serum VA levels with age, controlling for the effects of feeding pattern  and sex . The Bonferroni method was used to correct for multiple comparisons following the use of the generalized estimating equations. Statistical significance was set at The mean gestational age at birth was 39.5 ± 1.1 weeks . The ratP < 0.05) (P < 0.05). Similar trends were also observed when analyzed after stratification by VA supplementation (P < 0.05)  . Since t < 0.05) , it was  < 0.05) . The VA  < 0.05) .P < 0.05)  . In addiP < 0.05)  at 6 months and which cannot (VA <1.05 μmol/L) to find cutoff points for grouping neonatal VA levels. First, the infants were divided into three groups according to the VA level at 6 months  . Then, w < 0.05) . TherefoP > 0.05)  . The cur > 0.05) .P < 0.05) (P > 0.05)  . VA leve > 0.05) . These rAccording to the WHO , a serumHowever, it may be inappropriate to use the VAD diagnostic criteria for adults and older children aged 6–70 months . It is nVA supplementation in children aged 6–59 months has been demonstrably associated with a significant reduction in mortality by 23–30% –30. The One major limitation of the present study is that this population was sampled from only one representative corner of China. In the future, a large number of multicenter studies are needed. Due to safety and ethical concerns, this study is not a randomized controlled trial, which may have caused some bias.In conclusion, the low VA levels of neonates at birth may be a normal physiological state that increases with age, suggesting that the VAD criteria for adults and older children (VA <0.70 μmol/L) may overestimate the percentage of neonates with VAD. The VA status of infants with VA ≥0.588 μmol/L at birth gradually increased to adequate VA (VA ≥1.05 μmol/L) at 6 months; these infants thus may not need special nutritional intervention. Infants with VA <0.430 μmol/L at birth still had VAD at 6 months, however, and their VA levels can be significantly increased by early VA supplementation. More multicenter studies are needed to determine a new cutoff point for the diagnosis of neonatal VAD and the administration of nutritional intervention.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 Medical Ethic Committee of Children's Hospital affiliated with Chongqing Medical University (022/2014). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.TL, JM, and JC designed research. JM provided technical guidance and financial support for the study. HL, QC, LY, and TY conducted research. HL analyzed data and wrote the paper. TL and JM had primary responsibility for final content. All authors read and approved the final manuscript. All authors agreed on the order in which their names were listed in the manuscript.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.