Birth defects are the main cause of fetal death, infant mortality and morbidity worldwide. However, the etiology of birth defects remains largely unknown. Maternal folate status during periconception plays an important role in organogenesis and folic acid supplement reduces the risk of neural tube defects, congenital heart diseases, and several other birth defects. This trial seeks to evaluate the effectiveness of folate-oriented tertiary interventions during periconception on the incidence of fetus and birth defects.This is a single-blind, two-arm cluster randomized controlled trial in Shanghai, China. Eligible women from 22 clusters are recruited at pre-pregnancy physical examinations clinical settings. Compared to the routine perinatal care group (control arm), folate-oriented tertiary interventions will be provided to the intervention arm. The core interventions consist of assessments of folate status and metabolism, folate intake guidance, and re-evaluation of folate status to ensure red blood cell folate level above 400 ng/ml (906 nmol/L) before pregnancy. Screening and consulting of fetus and birth defects, and treatments of birth defects during pregnancy and afterward will be provided to both arms. The primary outcome is a composite incidence of fetus defects, stillbirth, and neonatal birth defects identified from the confirmation of pregnancy to 28 days after birth. Secondary outcomes include maternal and offspring adverse complications and cost-effectiveness of folate-oriented tertiary interventions. This protocol adheres to the SPIRIT Checklist.To achieve the recommended folate status before or during pregnancy is still a challenge worldwide. This community-based cluster-randomized controlled intervention trial will evaluate the effectiveness of a package of interventions aiming at achieving recommended maternal folate status covering pre- and during pregnancy in reducing fetus and birth defects. Our study has the potential to improve the community-based practice of reducing modifiable risk factors of disease and improving primary prevention of the defects in China. The procedures would formulate the policy on folic acid supplementation during periconception against birth defects in primary care settings.NCT03725878. Prospectively registered on 31 October 2018.Clinical Trial Registry,  Birth defects, known as congenital anomalies or congenital malformations, are structural or functional anomalies that occur during intrauterine and can be identified before and after delivery . Birth dThe protective effect of folic acid supplementations on several birth defects is well-established –6. The eMTHFR) gene which encodes the pivotal metabolic enzyme can reduce the MTHFR enzyme activity and lower maternal folate status, and increase the susceptibility of NTDs and CHDs [Insufficient folic acid intake and abnormal folate metabolism are the main determinants to folate status. Multiple single nucleotide polymorphisms (SNPs) in folate metabolism pathway genes are common causes of unclear low folate status in women who take sufficient folic acid. The C677T polymorphism on 5, 10-methylenetetrahydrofolate reductase . A total of 22 community-based primary health care centers will be recruited as clusters in Minghang and Songjiang districts in Shanghai, China. Both of the two districts contain rural areas and urban areas, selected health care centers routinely provide service for over five million local residents . A SPIRIT flow diagram see Fig. The study population consists of women preparing for pregnancy. Participants meeting the following criteria are eligible to this study: (1) women planning for pregnant within a year; (2) women aging 18 to 45 years. (3) Females and their husbands attend premarital or pre-pregnancy physical examinations from Minhang and Songjiang districts in Shanghai. There are no exclusion criteria.Eligible women are recruited at pre-pregnancy physical examinations clinics or premarital health checkup centers in Minhang and Songjiang districts in Shanghai. A written informed consent would be obtained from each participant during recruitment. The recruitment will be stopped until 2420 participants (> 110 pregnant women for each cluster) getting pregnant.The participants would only receive routine perinatal care according to the national guidelines , includi(i)Preconception proceduresParticipants in the intervention arm would be offered a package of folate-oriented tertiary interventions, including preconception, pregnant and neonatal procedures. The details are following as:(ii)Prenatal proceduresThe intervention arm will receive folate status evaluation by serum and RBC folate concentrations measurement. Combined the self-administered questionnaire and the folate metabolism ability , an individualized folic acid supplementary guide by phone counseling and a follow-up of folate status would be provided to whom with RBC folate below 400 ng/ml. The core rationale of preconception intervention following advises: (1) to take folic acid if one did not; (2) to take sufficient dose of folic acid if one did not; (3) to take sufficient dose of folic acid at least 3 months if one did not; (4) to take larger dose of folic acid if one with risk genetic variants; (5) to reevaluate RBC folate levels until one’s RBC folate levels achieve recommended levels before getting pregnant.(iii)Postnatal proceduresParticipants will be suggested to take folic acid until the end of the first trimester. Participants who will be screened with a fetus with defects will be referred to an authorized tertiary obstetric hospital and pediatric hospital, and receive prenatal consultations and treatments provided by a multi-disciplinary clinical team.The postnatal with defects will be transferred to Children’s Hospital of Fudan University to receive treatments and rehabilitation.The referral procedures during prenatal and postnatal periods will also be provided to participants in the control arm considering ethics. This will attract more couples taking part in the study and stimulate participants’ adherence to the protocol.The primary outcome is defined as the occurrence of fetus defects, stillbirth, and neonatal birth defects identified from the confirmation of pregnancy to 28 days after birth. This composite outcome covered a total of 24 types of defects according to the national birth defects surveillance policy  the proportion (%) of total abortions that related to congenital defects. ; (2) the incidence (%) of infant death or severe organ dysfunctions (composite outcome) (Time Frame: From birth to 6 months after delivery (can be expanding to the end of the 7th month)); (3) extra medical cost that related to fetus and birth defects during pregnancy and after birth (Time Frame: from confirmation of pregnancy to one-year-old after birth).Sample size calculation is performed based on the primary outcome: the incidence of a composite rate of fetus defects, stillbirth, or birth defects. The proportion of primary endpoint was conservatively estimated to be 8% in the control arm according to two observational studies , 23, andPermuted block randomization method with a block size of 4 will be used. Stata 15 was used for randomization code generation. Twenty-two community health care centres (clusters) will be allocated to the intervention and control arms. The randomization list will be generated by an independent statistician.The study is single-blinded. The investigators who are involved in data collection, laboratory examination, and outcome assessments will be blinded. The statisticians will also be blinded when they perform analysis.Two questionnaires will be used for nutrient supplementary data collection. Questionnaire one will be administered at recruitment before pregnancy to collect information on folic acid and vitamins supplement, the brand and content of nutrient supplements, maternal education, socio-demographic status, occupation, smoking status, alcohol consumption, and BMI. Questionnaire two will be used at early pregnancy. Besides the information in Questionnaire one, medications, reproductive history and health status will be included. The details of the information collected at each time-point are shown in Table In this study, the remaining blood samples (serum and Ethylene Diamine Tetraacetic Acid (EDTA) anticoagulation) for routine clinical examinations will be collected and divided into light-proof tubes within 6 h and temporarily stored in a − 20 °C refrigerator at sites in both arms. Samples will be transported to the central laboratory for storage in microfuge by three trained investigators. Only samples in the intervention arm will be conducted the nutrient evaluation, and others will be stored for a future test.Only samples in the intervention arm will be conducted the nutrient evaluation, and others will be stored for a future test. EDTA anticoagulation blood will be used for measuring RBC folate and serum for folate, homocysteine, vitamin D, vitamin B12 and ferritin assays using an electrochemiluminescence assay . RBC folate concentrations will be adjusted using hematocrit if the RBC folate concentration is below 150.0 ng/ml and the serum folate level is over 3.5 ng/ml. Examinations will be performed in the central laboratory of Children’s Hospital of Fudan University. The serum concentration of vitamin A and vitamin E will be quantitatively detected by liquid chromatography-tandem mass spectrometry . Fasting serum cholesterol, high-density lipoprotein, low-density lipoprotein, triglyceride, and glucose levels will be measured using the Beckman Coulter AU chemistry analyzers  in the central laboratory of the Children’s Hospital of Fudan University. Serum levels of Mg, Fe, Zn, Se, Mn, As, Cu, and Ca be analyzed by inductively coupled plasma mass spectrometry  in the Instrumental Analysis Center of Shanghai Jiao Tong University.BHMT (rs3733890), CBS , FIGN (rs2119289), MTHFD1 (rs2236225), MTHFR , MTR , MTRR , RFC1 (rs1051266), and SHMT (rs1979277) [The SNPs involved in folate metabolism pathway will be genotyped: FIGN rs21289, MTHF1 rs22362, MTHFR (1979277) . GenotypAll data is managed centrally in the project management office. STATA 15.0 will be used for statistical analyses. An ID number will be used as the unique code to connect the various stages of the database. Data will be stored at the research team’s office. Daily data checking will be carried out by the research coordinator. A database will be built using Access 2013 ; double entry and checking will be performed by an assigned data entry team. Only the data managers can access to the database by passwords and back up the database daily.The findings of the trial will be reported according to the CONSORT guidelines for cRCT. An intention-to-treat (ITT) analysis will be used for the primary outcome. The primary analyses will be performed by generalized estimating equation (GEE) model with intervention as fixed effects, baseline measurements as covariates, and clusters and patients as random effects. GEE model will be performed to estimate risk ratio and 95% confidence intervals (CI), as well as a risk difference and 95%CI, by adjusting cluster with link of identity. The clinical and healthcare factors will be identified as the covariates since that would influence the probability of receiving intervention and outcomes according to previous studies and biological plausibility.All secondary outcomes will be analyzed based on the intention-to-treat (ITT) population. Binary secondary outcomes will be analyzed with the same strategy and method as that used for the primary outcome. For the analysis of continuous secondary outcomes, a general linear model (GLM) model will be employed with treatment as a fixed effect and with normal distribution and identity link function. The mean difference of outcomes with 95%CI will be derived from the GLM model. For repeated measurement variables, such as infant weight at a different visit, or maternal body weight during gestation, will be analyzed using a mixed-effect linear regression model.yuan (¥) and then converted to international dollars using purchasing power parities and gross domestic product (GDP) deflators. The estimates include five costs: folate evaluation, folic acid supplements, fetus and birth defects diagnosis, the treatment related to fetus and birth defects, and intervention procedures. Cost-effectiveness analyses will be conducted in agreement with the Consolidated Health Economic Evaluation Reporting Standards [The cost will be collected in Chinese tandards  and willA research management team will be set up for this study to monitor the process and verify protocol compliance. The team is also responsible for data, biological samples, quality control, and participant management. All investigators responsible for recruiting has been trained before the project started and strictly follows the standard recruiting procedures and criteria. Blood samples will be transported using cold chain by trained carriers to control the quality. A pilot study was conducted before starting the trial to assess the feasibility and reliability of the procedures. Data correctness will be assessed and serious adverse events will be reported to the ethics committee of the particular center and Children’s Hospital of Fudan University, who has the authority to terminate the trial according to local laws or institutional regulations. The investigators should keep each care center, ethics committee, and the journal informed of any major protocol modifications.It is a consensus that peri-conceptional folic acid supplementation can prevent fetus and birth defects, and many countries adopted different strategies and procedures to improve population folate nutrition levels during periconception period. However, few studies focused on achieving the optimal RBC folate status before pregnancy. The present study will evaluate the effectiveness of the package of interventions to a high risk group among the entire population based on the current individual folate examinations on the reduction of the incidence of fetus and birth defects. The used intervention procedures that were integrated into the community-based routine peri-conceptional primary care service will be generalizable and helpful for improving practice of primary prevention of the defects.Our primary outcome may be influenced by several factors. Firstly, since some pregnancy is un-planned, participants in the intervention arm may fail to achieve the optimal folate level before pregnancy and may reduce the intervention effectiveness. Secondly, some participants receiving individual folate guidance may poorly comply with our suggestion of reevaluating folate status to ensure RBC folate concentrations > 906 nmol/L before they plan to get pregnant. This will reduce the intensity and effectiveness of interventions because our critical interventions focused on peri-conception. Considering these situations, we collect participants’ blood samples when they attend the first antenatal care at early pregnancy  for examination of folate levels. This will help us to obtain a RBC folate levels that are close to the critical window of defects occurrence, since RBC folate is stable and can reflect the folate status of the latest two-three months. Besides, for humanitarian reasons and maternity and child health, we provide interventions during pregnancy and after delivery to both groups, doing this may also reduce the chance of finding group difference in effectiveness of interventions. In the pre-conception recruitment, only participants who get pregnant during the study period will be valid study subjects and included for the outcome analysis. Since the low birth rate in Shanghai, we will expand recruitment to ensure enough sample size for the primary outcome.The use of individual randomization would be impractical because participants tend to be contaminated and interfere with the interventions. Therefore, a cluster-randomized clinical trial study is performed. The trial is conducted based on community-based health care centers that provide routine primary maternity and child care service. This system will improve the compliance of participants because the general practitioner has close connections with the women from their community.The intervention has several advantages over routine health care: (i) Pre-pregnancy interventions are an individualized, comprehensive intervention program that references the questionnaire, genes, and nutrient levels in the blood; (ii) This intervention study builds a complete health-care network from pre-pregnancy to childbirth, from community to hospital, which could be widely applied in the future; (iii) This community-based cRCT study could provide a good representation of the study population. The results of this trial will provide evidence on the prevention of fetus and birth defects in primary care settings.ClinicalTrials.gov with identifier number NCT03725878 on Oct 31, 2018. Recruitment started on Nov 1, 2018, and the study is currently recruiting participants and collecting pregnancy associated information.The Protocol Version is 1.0. The trial is prospectively registered at Additional file 1 Table S1. A list of the participating hospitals and community care center in Songjiang and Minghang district of Shanghai.Additional file 2. SPIRIT Checklist.Additional file 3 Table S2. Types of fetus defects and birth defects. Notes: Defects were detected by Down’s syndrome screening, NT examination and Ultrasound examination during the second trimester; and the number and type of birth defects after childbirth are diagnosed by professional clinical team.Additional file 4. Statistical analysis plan (SAP).


This research studied the relationship between maternal exposure to polychlorinated biphenyls and neonatal birth weight through systematic review and meta-analysis of existing literature. We searched for all the studies published in MEDLINE / PUBMEDN / EMBASE  by June 2018, and seven studies had been selected. The results showed that there was significant correlation between birth weight reduction and PCBS exposure throughout pregnancy . There was a negative correlation between birth weight and PCBs exposure and umbilical cord serum (β=-0.833g) and maternal serum (β= -0.504g).Subgroup analyses showed significantly different effects of PCBs exposure on birth weight in different regions, stages of pregnancy and study designs. It was thought the heterogeneity was mainly caused by geographical regions, stages of pregnancy, and the assessment methods. The meta analysis revealed a negative correlation between PCBs exposure and birth weight but there was significant difference in the correlation between birth weight loss. Polychlorinated biphenyls (PCBs) are lipophilic and poorly metabolized compounds, which tend to be accumulated in adipose tissue of aquatic organisms in polluted water. These compounds may have many adverse effects on infant development through placenta and breast feeding  Birth weight is an important indicator of fetal growth, and Low birth weight (LBW) is associated with perinatal morbidity and mortality. Moreover it was also found that the adolescent and middle-aged health issues including asthma, low IQ, and hypertension could be caused by LBW or premature birth . Therefore, it was important to find the risk factors which caused LBW,and it was significant to reduce the LBW incidence for public health. In the past, more and more studies had estimated the association of PCBs exposure with birth weight and LBW. Many studies had reported inconsistent findings regarding the relationship between maternal PCBs exposure and birth weight. Some studies have suggested a negative correlation between PCBs and birth weight in newborns , while others had shown that the relationship between PCBs and birth weight was not clear . These inconsistent and controversial results suggested there was an urgent need to quantitatively synthesize and explain the evidence available in order to promote public health policy and ensure informed clinical decisions. PCBs were lipophilic compounds and contain different amounts of polychlorinated biphenyls in different tissues. So there was a lot of controversy when referred to the different sample extraction. And no previous research has shown the effect of PCBs in different tissues on birth weight. Therefore, we carried out meta- analysis to quantitatively synthesize the results of various studies. In this analysis, we systematically collected previously published studies to estimate the effects of PCBs exposure in different tissues on birth weight and/or LBW. Meta-analysis model was used to quantitatively evaluate the effect of PCBs exposure and the exposure evaluation method on birth weight and LBW. Finally we further discussed the effects of different regions, research settings and research designs in subgroup analysis. Study designWe searched all studies published in MEDLINE, PubMed, Embase, China Biomedical and Wanfang Electronic Databases before June 2018. Our search strategy combined the following keywords: Biphenyls, Polychlorinated, Polychlorinated biphenyls Compounds, Polychlorinated biphenyls PCBs, Birth Weight Synonyms, Low Birth Weights, Infants Weight, change in birth weight, LBW, adverse birth outcomes, and adverse pregnancy outcomes. We also manually searched for additional publications for each major research reference. Further publications are also determined through reviewing articles, but only in English or Chinese publications.Inclusion and exclusion criteriaFirstly we screened the abstract of all research articles to exclude studies which were not involve in the relationship between PCBs and birth weight. The remaining studies were labeled as potential and further evaluated by two independent authors. Studies which met the following criteria were included into the meta analysis: (a) Studies included PCBs exposures during pregnancy, and pregnancy outcomes measured birth weight and/or LBW; birth weight was measured as a continuous variable; LBW was defined as live births with a weight of less than 2500g, including terminology LBW (TLBW) and preterm LBW(PLBW); (b)The study provided LBW birth weight, sample size, partial regression coefficient (β) odds ratio (OR), and 95% confidence intervals (CI), which could also be used to infer the results of the study; (c) If more than one study was indentified for the same population, only student that included the latest population or the most information were selected. Accordingly, studies those didn’t conform to the above criteria were excluded. Data ExtractionThe following information was extracted from each study: year and source of publication, study period, study setting, study plan, PCBs exposure detection method, data source, sample size, PCBs exposure window, exposure mean and range,β/OR and 95% CI. If the study provided an association between PCBs exposure throughout pregnancy and birth weight during specific periods of pregnancy, all estimates were extracted. Some studies assessed PCBs exposures based on monitoring network data and remote sensing data, and the estimates based on the monitoring network were preferred, because it was more commonly used and potentially reduced the heterogeneity among studies in the meta-analysis. In addition, if other covariances were fully adjusted, estimates could only be extracted from a single contaminant model because there was a considerable collinear relationship in the model of the contaminants. Sometimes there was considerable collinearity in the pollutants from the same source, as a result, estimates were extracted from a single pollutant model only when covariance had been fully adjusted. If the pollutant didn’t exist alone, there would be combined effect. However, it wasn’t every studies adjusted those contaminants except PCBs. Qualification assessments and all data extraction in standard forms were performed by two authors, and differences were resolved after discussion.Meta-analysis and Statistical AnalysisBefore meta-analysis were carried on, we converted all risk estimates (β and OR) of PCBs volume and chemical composition to common exposure unit, which allowed us to pool the estimates from different studies. To compare exposure level in cohort results with different matrices, we showed the contaminants levels in wet-weight umbilical cord serum levels, which directly reflected fetal exposure during childbirth (some compounds do not effectively cross the placenta). If there were no umbilical cord blood results, we estimated the concentrations in maternal serum, maternal whole blood, and breast milk. And the following conversion factors were used .Level of umbilical cord serum (ng/L) = 0.20 × level of pregnancy serum (ng/L) = 1.20 ×level of breast milk (ng/g fat) = 0.36 x level of materials’ whole blood (ng/L).oC. The levels of PCBs in umbilical cord serum is a good indication of the infant exposures to PCBs, and the results were reproducible . PCBs levels in pregnancy serum were detected in blood samples taken from pregnant women during the first trimester, the second trimester, or the third trimester of pregnancy respectively . However, thePCBs levels in pregnant women appeared to be relatively stable throughout pregnancy. All studies included in the study were divided into two categories: retrospective and prospective. Meta-regression analyses were also used to explore the revision effect of the study period, exposure measurement methods, study design and study settings on the association between PBCs exposure and birth weight.Moreover, we conducted several meta-analysis studies to quantitatively estimate the relationship between different PCBs concentrations and birth weight. Minor analyses were also performed to estimate the summary effects of PCBs exposure on birth weight throughout pregnancy in the subgroups with different exposure measurement methods , study design and study settings. These subgroup analyses were designed to investigate the what important role these characteristics play in the effect of PCBs exposure on birth weight ,and to further detected whether the characteristics took effect on the heterogeneity in the reported association. The studies included four detection methods to evaluate PCBs levels ,including umbilical cord serum, maternal serum, maternal serum and breast milk. Cord blood serum was collected from cord blood samples at the time of birth, and the serum fraction was removed after centrifugation and stored at -20To investigate the heterogeneity of the results, we assumed that the effect magnitude may changed with the study method. The inclusion heterogeneity of the studies was assessed via the Q and I² statistics. Cochran’s Q statistic was calculated by the square of the summation, and weight were conducted with each study’s contribution , each study’s estimate and the deviation of overall meta-estimation P values were obtained by comparing Q statistic with a chi-square distributionwith k-1 degrees of freedom, where k was the number of studies included . If the P-value was less than 0.05, then random effects model was selected on the other side the fixed effects model was selected. The I² statistic [I² = (Q-df) / Q × 100] described the percentage of inter-study variation caused by heterogeneity rather than occasionality. If I²>50%, it indicated that there was significant statistical heterogeneity . We also used the asymmetry of funnel plot to detect potential publication bias. The symmetry of funnel diagramwas tested with Egger regression, where the reciprocal of the standard error was an independent variable and the standardized estimate of effect size was the dependent variable .Finally, a series of sensitivity analysis were conducted to test the robustness of our results. Because some subgroup analyses contained few studies, only sensitivity analysis were performed in those subgroup with more than three studies. For each sensitivity analysis, we removed the largest and the smallest estimates from a single study separately from the meta-analysis. All statistical tests were bilateral, and P <0.05 was considered statistically significant. STATA software and “mate for” package were used in the analysis.Search results and research featuresThe search strategy identified 1,516 articles, and 334 articles were duplicates among those. The titles and the abstracts of the remaining 1,182 studies were reviewed, and 1,119 studies  were excluded. The full text of 63 studies were reviewed and 56 studies were further excluded, because 22 articles are mainly analysis and summary previous studies or lack of specific data and he research direction of 19 studies was irrelevant, the research objects of 10 articles were irrelevant, single or multiple homologues of PCBs were measured in 2 articles, 2 articles was published a long time ago and 1 articles was animal experiments. Finally, 7 studies was included in the meta-analysis, including Matthew et al., 2005; Xijin et al., 2015; Marina et al, 2014; Lars et al., 2000; Kanae et al., 2009; Halldorsson et al., 2008; Sharon et al., 2007). The selection process was shown in Comprehensive estimation of the effect of PCBs exposure on birth weightIt is estimated that there is a significant negative correlation between birth weight loss and PCBs exposure, as shown in There was significant heterogeneity in these 7 studies (P < 0. 853). In order to explore the source of heterogeneity in the study, a series of subgroup analysis was carried out. The results show that race is the biggest cause of heterogeneity. In particular, the largest factor contributing to the study of heterogeneity in Europe (P=0.839) . Three sSensitivity Analysis and Publication Bias AnalysisFinally, we conducted a series of sensitivity analyses to determined the robustness of the results. An American study was excluded in our meta-analysis which detected the PBCs level in cord blood and had maximum impact, and we found no change . The robustness of a single study with the maximum and minimum impact on estimating the relevance of correlation. There was statistically significant  in estimated aggregate effects of birth weight (β= -0.319g), as shown in In this meta-analysis, we collected seven eligible studies and quantitatively assessed the relationship between PCBs mass concentration and birth weight and/or LBW risk, involving a total of 8,054 subjects. The results showed that the exposure of PCBs was negatively correlated with the risk of low birth weight but there was significant difference in the correlation between birth weight loss  and PCBs exposure during pregnancy (P < 0.001). It was consistent with previous meta analysis results . Karmauspointed out that the birth weight reduced about 500g if PCBs level in mother was more than 25μg/L . Eva’s meta analysis found, the birth weight loss was 150g (95% CI: -250 -50 g) as 1 μ g/L PCB-153 increasing . Nina’s meta analysis found that when PCB-153 concentration was beyond 183ng/g, newborn birth weight loss was about 140g . These results further revealed the toxicity of PCBs to fetal growth during pregnancy. Nowadays, PBCs was found in all of the world, and most pregnant woman faced exposed to PBCs more and less, as a result, these fetuses who were more susceptible to pollutants and were in a critical development stage, could be most risk situation. Therefore, it was critical to eliminate environmental PCB exposure to pregnant womanin in order to improve fetal health.It was not sure the relative fators which effect the toxic mechanism of PCBs until now. Previous studies found that male infants were more susceptible to PCBs than female infants. However, there was few strong experimental evidences to prove this phenomenon. During the literature screening process, we found that the effects of PCBs, dioxin, polychlorinated biphenyls (PCDF) on birth weight in most studies, but there was a few studies to explore the combined effects of these pollutant with PCBs. Therefore, it will pay more attention to joint exposure of PBCs, the genes relative to PBCs in humans, or environmental gene interaction between gene an PBCs.It was still controversial which was the most right and sensitive tissue to detect pops pollution. It is well known that human body were composed with all kinds of substances and the distribution of substances in human body were inconsistent. Therefore it is reasonable to conduct a meta-analysis to evaluat the analytical differences of PCBs from different tissue. In this study, we divided all of the studies into two groups . For the analysis of, we observed that the there was more significant correlation between birth weight and PCBs levels in cord blood than in the blood of pregnant women. Our study also found that the birth weight was different at different concentration of PCB in cord blood while the PCB concentration of maternal serum were almost similar in prenatal . This difference may has relationship with the oganizational distribution of PCBs in vivo. However, PCB were detected in placental tissue in some studies , and we believe that new method will appear in other tissue in the future. In addition, studies have shown that the serum concentrations of Polychlorinated biphenyls congeners at 6 weeks after delivery and in early pregnancy were significantly lower than those before conception, so that different exposure items were detected at the same time. The content of PCBs was also detected at different periods of time.Several subgroup analyses were performed to determine the heterogeneity sources in the studies. Heterogeneity test showed that the most likely source of heterogeneity between PCBs exposure and birth weight was different ethnic groups. The toxicity of PCBs to humans may vary with race. There could be some other factors, such as gene, heredity, individual differences and so on. Different food habits may also be the cause of this difference. The eating habits in different countries and regions was one of the reason that cause different PCBs level in human. For example, the femal subjects were divided into subgroups depended on fish consumption in some studies.It was found a correlation between fish intake and PCB concentration in body . And Xijin et al.,’s (2015) study also showed that the similar effects of dietary intake on PCBs level. In addition, different standards and methods for the PCBs detection in various countries were different , which may have an impact on the results of the study. In the subgroup analysis, our results showed that there was a stronger aggregate association between PCBs exposure and LBW in late pregnancy than in the evaluation of PCBs exposure in the second and early stages of pregnancy. In Marc-Andr é et al.,’s (2013) study, the link between PCBs levels and birth weight may be due to weight gain during pregnancy. However, studies have also shown that the increase in PCBs concentration is associated with decreased sympathetic activity up to the second trimester of pregnancy . Because of the lack of data, we can’t estimate the PCBs exposure by using two classification variables, which leads to the occurrence of low birth weight infants. In addition, the reasons for the difference in the assessment of PCBs exposure in early and second trimester may be that the stage of some studies is not clear, which leads to subjective consciousness in the process of collecting data. For pregnancy staging, our study was divided into three stages, 1-12 weeks, 13-27 weeks, and 38-42 weeks, respectively. Different countries and regions have different regulations on pregnancy staging, which is one of the effects of the differences.Our study also showed that PCBs exposure in Europe and America had a more statistically significant impact on birth weight than in Asia. The reason for this problem may be that the differences in analysis and measurement of exposure data are difficult to compare PCB levels from different regions, and different studies use different methods of detection. In the kanae study, the average total TEQ level  of the mother’s blood was 17.5 TEQ pg / g lipid, which was lower than that of the mother’s plasma TEQ (28.4 TEQ pg / g lipid) in another study in her area . T. I. et al. Determination of PCBs content by Gas Chromatography and Electron capture. In Marina dengr, the content of PCBs was measured by using an Agilent 7000B gas chromatograph triple quadrupole mass spectrometer (G) . Although the studies involved are few, can not fully confirm our conclusion, but can provide a direction for future research.In addition, this meta-analysis has certain limitations. We found high or moderate heterogeneity in most subgroup meta-analysis. These results suggested that heterogeneity in the study may also be affected by other factors, such as economic status, mother’s education, and fetal sex, and We didn’t take into account the limitation of the number of relevant studies in this study. Therefore, further meta-analysis is necessary to explore the source of heterogeneity and more original research in the future.In Conclusion, the meta analysis revealed a negative correlation between PCBs exposure and birth weight but there was significant difference in the correlation between birth weight loss . Race and exposure assessment items both were the main source of heterogeneity. These results extend our understanding of the adverse effects of PCBs on fetal health and it should be paied more attention for the importance of reducing environmental PCBs pollution and reducing maternal PCBs exposure during pregnancy to improve birth outcomes. More research is needed to further evaluate the health effects of a single PCBs homologue, as well as the effects of different detection methods such as gas chromatography or electron capture.


This study aimed to investigate the effect of the duration of embryo culture on clinical outcome in vitrified-warmed cycles.This retrospective cohort study enrolled 10,464 infertile patients, with a total of 18,843 vitrified-warmed day 3 embryos from 2012 to 2017 at a single center. The patients were divided into 2 groups: 9470 cycles in the short-term culture group (0.5–8 hours of post-thaw culture) and 994 cycles in the 48 to 72 hours culture group. The independent effect of the following variables on clinical outcomes was determined: duration of post-thaw culture, maternal age, transferred embryos, embryo quality, and endometrial thickness.We found that the pregnancy rate was positively associated with the post-thaw culture time. Ordinary least square regression analyses showed that the duration of post-thaw culture was positively associated with implantation and live birth rates overall. However, the implantation and live birth rates were not significantly associated with the post-thaw culture time in the short-term culture group. Additionally, maternal age and the number of transferred embryos were independent predictors of the implantation and live birth rates. Moreover, the duration of post-thaw culture did not affect live birth weight.These results indicated that the pregnancy rate is positively associated with the duration of post-thaw culture. Therefore, under the condition of not affecting work shifts, properly prolonging the duration of post-thaw culture to improve the outcome of frozen-thawed embryo transfer should be considered. With regard to an favorable outcome obtained with FET, an increasing amount of reproductive centers tend to adopt the freeze-all strategy. With the advent of a freeze-all policy, exploration of the risk factors associated with pregnancy and implantation rates generated from vitrified-warmed cycles is important.Refinement in cryopreservation techniques and improvement in the pregnancy rate after frozen embryo transfer have facilitated the increasing use of frozen-thawed embryo transfer (FET) globally. Various factors are known to affect the outcome of FET. Among the clinical factors, maternal age at oocyte retrieval, embryo quality, and the number of embryos transferred could independently affect the clinical outcomes. Furthermore, the duration of post-thaw culture could be involved.,8 Existing data concerning the effect of culture duration on outcomes are conflicting. Rato et al found higher implantation and live birth rates after a short post-thaw culture period compared with overnight culture. However, other studies showed no differences in these variables between the short culture and long culture groups.,9 Nonetheless, these studies mainly focused on comparison between short culture and long culture, without analyzing the effect of different periods of a short duration of culture on the outcomes of FET. In a short culture, embryos are evaluated immediately after thawing and are transferred on the same day of thawing if ≥ 50% intact blastomeres were observed. Whether immediate transfer after thawing (culture time < 2 hours) affects the success rate and whether transfer after a certain time of culture can improve the outcome remain inconclusive. Without considering the duration of culture, the relative effect of embryos transferred at any time after thawing on clinical outcomes has not been evaluated.FET is currently mainly performed at the cleavage stage (days 3) or at the blastocyst stage (days 5–6). Vitrified-warmed day 3 embryo transfer is still routinely applied in the clinical setting.The present study aimed to evaluate the importance of the duration of post-thaw culture in FET and identify which other characteristics in FET affect the clinical outcome after FET. Specifically, the association of short-term culture duration and clinical outcomes after FET were analyzed.22.1This study was approved by the institutional ethical review board of Sir Run Run Shaw Hospital, The Affiliated Hospital of the College of Medicine, Zhejiang University. In this retrospective cohort study, all women who underwent first vitrified-warmed day 3 embryo transfer treatment between January 2012 and November 2017 were included for analysis. Those cycles with missing data and without accurate medical record numbers were excluded. A total of 10,464 vitrification cycles were included in this retrospective cohort study.2.2Morphological survival was analyzed according to the cell stage before cryopreservation. Embryos were evaluated for morphological warm survival immediately after warming. Embryos with at least 50% of their cells intact were considered to have survived and able to be transferred. An embryo with 6to 12 equal blastomeres and ≤ 20% fragmentation was defined as good quality.2.3The vitrification procedure was performed using Cryotop strip (open vitrification procedure) or CBS High Security Vitrification straws (closed vitrification procedure) in combination with ethylene glycol-dimethylsulfoxide-sucrose  as the cryoprotectant.2.4 For patients with regular menstrual cycles, the natural cycle was proposed and embryo transfer was performed 3 days after ovulation, or 4.5 days after the peak of luteinizing hormone. In patients with irregular ovulation or an anovulatory cycle, hormone replacement treatment or an HMG-stimulation protocol was used.In our center, 3 main types of clinical protocols were used for endometrial preparation as follows: the natural cycle, hormone replacement cycle, and human menopausal gonadotrophin (HMG)-stimulated cycle.2.5Patients were divided into the overall group (0.5–72 hours of post-thaw culture) and the short-term culture group (0.5–8 hours of post-thaw culture). Moreover, to clarify whether the factors associated with clinical outcome were different in younger patients and older patients, we divided the patients into the 2 following groups: ≤ 35 and > 35 years, according to the patients’ ages. Additionally, to examine the results more specifically, patients were stratified into 7 groups according to the post-thaw culture time as follows: group 1, the culture time was < 2 hours; group 2, the culture time was ≥ 2 hours and < 3 hours; group 3, the culture time was ≥ 3 hours and < 4 hours; group 4, the culture time was ≥ 4 hours and < 5 hours; group 5, the culture time was ≥ 5 hours and < 6 hours; group 6, the culture time was ≥ 6 hours and < 8 hours; and group 7, the culture time was ≥ 48 hours and ≤ 72 hours.2.6Biochemical pregnancy was assessed by detecting serum β-hCG levels at 12 days after transfer and β-hCG levels ≥ 50 IU/L were considered positive. The pregnancy rate was defined as the number of β-hCG-positive patients divided by transferred cycles. Five weeks after transfer, the number of gestational sacs and the presence of fetal heart motion were evaluated by ultrasound. The implantation rate was calculated using the number of observed intrauterine gestational sacs divided by the number of embryo transferred. The live birth rate was defined as the number of newborns per number of embryos transferred.2.7P value of < .05 was considered to be statistically significant.Binary logistic regression analysis was used to identify independent correlates between the pregnancy rate and possible confounding factors. Ordinary least square regression models were conducted to evaluate the associations of variables of interest and the implantation rate, live birth rate, and live birth weight. All statistical analyses were performed using Statistical Package for the Social Sciences Version 22.0 . A 3Between January 2012 and November 2017, a total of 10,464, first vitrified-warmed transfer cycles were analyzed. There were 9470 cycles in the short-term culture group and 994 cycles in the 48 to 72-hours culture group , the number of transferred embryos , the number of transferred good quality embryos , and endometrial thickness  . In each age group (≤ 35 and > 35 years), the clinical pregnancy rate was significantly associated with the number of transferred embryos, the number of transferred good quality embryos, endometrial thickness, and maternal age. Interestingly, the association between the post-thaw culture time and pregnancy rate remained significant in the overall and short-term culture groups of ≤ 35 years and the overall group of > 35 years. However, in the short-term culture group of > 35 years, the pregnancy rate was not significantly associated with the post-thaw culture time with adjustment for the same factors (P > .05).Binary logistic regression analysis was used to analyze the associations between pregnancy rates and risk factors. In the overall and short-term culture groups, after adjusting for known confounding factors such as maternal age, duration of infertility, endometrial preparation protocol, number of transferred embryos, number of transferred good quality embryos, and endometrial thickness, the pregnancy rate was positively associated with the post-thaw culture time  and live birth rate (P < .001). However, the implantation and live birth rates were not significantly associated with the duration of post-thaw culture in the short-term culture group. Additionally, maternal age and the number of transferred embryos were independent predictors of implantation and live birth rates. In subanalysis of factors associated with implantation and live birth rates in the ≤ 35 and > 35 years groups, multiple linear regression with the same factors as in the main analysis showed that the duration of post-thaw culture was not associated with implantation and live birth rates in the > 35 years group. Moreover, the duration of post-thaw culture did not affect live birth weight. The number of live births was the only independent predictor of live birth weight . Only a small number of day 3 embryos (9.5%) were transferred after 48 to 72 hours of culture and no embryos were transferred after overnight culture. In the current study, the overall pregnancy rate of vitrified-warmed day 3 embryo transfer after short-term culture was 59.6% (5645/9470), which is in line with previous studies. Our results reinforce the accuracy and advantages of a frozen-thawed embryo selection approach strictly based on cryosurvival of blastomeres. In all 9470 cycles of day 3 embryos transferred on the day of thawing, the duration of post-thaw culture ranged from 0.5 to 8 hours. We found that the duration of post-thaw culture had a positive correlation with the pregnancy rate, but had no significant correlation with embryo implantation and live birth rates. However, in group analysis, except for group 1, we found an increasing trend of embryo implantation and live birth rates in the groups. The live birth rate in group 1 was higher than that in the other groups. Furthermore, the age of patients in group 1 was younger than that in the other groups. Additionally, the mean number of transferred good embryos per cycle was relatively high, which may have led to higher live birth rates in group 1. Moreover, the present study showed that, except for the duration of culture, maternal age was an independent predictor of pregnancy, implantation, and live birth rates. Therefore, we divided the patients into 2 groups of ≤ 35 and > 35 years, according to the patients’ age. We found that there was a positive correlation between the duration of culture and pregnancy rate in the younger age group, but no correlation in the advanced age group. This finding suggested that appropriately prolonging the duration of culture could improve the pregnancy rate when vitrified-warmed day 3 embryos were transferred on the day of warming.To the best of our knowledge, this is the first large retrospective study to evaluate the association of the duration of post-thaw culture with clinical outcome after vitrified-warmed day 3 embryo transfer. There are 3 options for vitrified-warmed day 3 embryo transfer. One option is blastocyst transfer after culture for 48 to 72 hours, another is overnight culture transfer after culture for 24 hours, and the third is transfer on the day of thawing. The first 2 transfer methods may have the possibility of elimination of an undeveloped embryo, which may reduce the number of transferred embryos or cancel embryo transfer. Therefore, compared with the third method, the first 2 methods can improve the pregnancy rate because of elimination of embryos without developmental potential. showed that the culture period had a significant and strong effect on birth weight of singleton newborns. Therefore, the live birth weight increases with a longer culture period, However, a recent study by de Vos et al reported a significantly lower birth weight in singletons born after vitrified-warmed blastocyst transfer compared with cleavage-stage transfer. In the present study, we did not find a correlation between the duration of culture and fetal weight, which may be because the culture durations are much the same. Although the difference in duration of post-thaw culture was nearly 8 hours, this was still not long enough to affect the live birth weight. Moreover, the present study showed that even though frozen-thawed day 3 embryos were cultured to the blastocyst stage, the live birth weight did not increase or decrease. These contradictory findings could be largely attributed to the limited population size of live birth obtained after blastocyst transfer. Furthermore, possible factors known to affect intrauterine fetal growth, such as pregnancy-induced hypertension, gestational diabetes, and preeclampsia, were not excluded from our study.We also wanted to examine whether the duration of post-thaw culture is associated with the live birth weight. Therefore, we analyzed the factors associated with live birth weight. We found that the duration of post-thaw culture did not affect live birth weight and this result could not be overcome by prolonging the in vitro culture time. In contrast to our findings, Zhang et alThe design of the current study may have resulted in some biases that were inherent because of its retrospective nature, which might have affected our results. Therefore, we meticulously screened the data with strict criteria and without a single case lost to follow-up. One of the main strengths of our study is the large sample size, including 10,464 cycles, with first vitrified-warmed transfer cycles within a single institution. This provided useful information on the rates of pregnancy, implantation, and live birth. Most importantly, in the present study, laboratory conditions, the embryo selection criteria for cryopreservation, and procedures, as well as culture medium, remained consistent during the study period.In conclusion, our study shows that the pregnancy rate is positively associated with the duration of post-thaw culture. The probability of pregnancy becomes greater with a longer duration of post-thaw culture. Therefore, under the condition of not affecting work shifts, properly prolonging the duration of post-thaw culture to improve the outcome of FET should be considered. Large prospective studies should be conducted to confirm this finding in the future.www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.The authors sincerely thank other investigators and physicians who made contributions to this study. The authors thank Ellen Knapp, PhD, from Liwen Bianji, Edanz Group China (Analysis: Yamei Xue.Data curation: Haiyan Zhu, Wen Xu, Xiaoying Jin.Formal analysis: Xiaoying Jin.Funding acquisition: Wen Xu.Investigation: Xiaomei Tong.Project administration: Songying Zhang.Software: Yamei Xue.Supervision: Songying Zhang.Writing – original draft: Haiyan Zhu.Writing – review & editing: Haiyan Zhu.


The prevalence of preterm birth and low birth weight has been increasing slightly in recent years. A few studies have suggested that psychosocial stress during pregnancy may increase risk for these adverse birth outcomes. To extend those observations, we analyzed various major life event stressors separately and cumulatively as potential risk factors for preterm birth and low birth weight using granular categories of each outcome in a large, population-based study. Additionally, we assessed if greater social support buffered any effects.Data were from a nested prevalence study of 4395 women in the National Birth Defects Prevention Study who delivered live-born non-malformed infants (controls) between 2006 and 2011. Participants completed a standardized, computer-assisted interview between 6 weeks and 24 months after delivery that included questions on stress and social support from 3 months before pregnancy to the 3rd month of pregnancy. Cumulative stress and support indices were also calculated. Preterm birth was divided into “early preterm” (< 32 weeks), “late preterm” (32–36 weeks) and “term.” Low birthweight was divided into “very low birth weight” (< 1500 g), “low birth weight” (1500–2499 g) and “normal birth weight” (≥2500 g). Relative risks and 95% confidence intervals (95% CI) were calculated using Poisson regression.For women reporting relationship difficulties, there was a suggestive risk of early preterm birth  and very low birthweight . For women reporting that they or someone close to them were victims of abuse, violence, or crime, there was an increased risk of low birthweight  and late preterm birth . There were no strong associations observed between social support questions and the various outcomes.Our results add some support to prior evidence that certain stressors may be associated with increase selected adverse birth outcomes risk. We did not find strong evidence that social support buffered the observed risks in our study. Roughly 10% of births in the United States are delivered preterm (before 37 weeks of gestation), and 8% of infants are born at low birthweight < 2500 g); the prevalence of both has been increasing slightly in recent years [500 g; thIn a recent US study of 27 states, 70% of women reported experiencing at least one stressful life event in the year before giving birth . MaternaThe impact of individual stressors may be exacerbated in the presence of other stressors or abated by effective coping mechanisms or more social support. A prospective pregnancy cohort study in Canada observed a measure of cumulative psychosocial stress to be associated with an increased risk of late preterm birth, independent of other preterm birth risk factors . When stThe current study explored stressful major life events and lack of social support (examples of data that can be efficiently collected) as potential risk factors for the outcomes of preterm birth and low birthweight employing data from controls in a large, population-based, U.S. case-control study of birth defects – The National Birth Defects Prevention Study. To explore whether different severities of preterm birth and low birthweight may have different etiologies, we used more specific sub phenotypes of these outcomes.Data for these analyses were from the National Birth Defects Prevention Study (NBDPS). The NBDPS is a population-based, multi-center, case-control study conducted at 10 sites located in Arkansas, California, Georgia, Iowa, Massachusetts, North Carolina, New Jersey, New York, Texas, and Utah. The NBDPS methods have been described in detail elsewhere . BrieflyIn this analysis, we included only women delivering control infants due to the association between birth defects and preterm birth  and pote2), smoking, alcohol use, and intake of folic acid-containing vitamin/mineral supplements from the month before pregnancy through the first trimester of pregnancy.Outcomes of interest were preterm birth and low birthweight. Although preterm birth often accounts for many low birthweight infants, they were examined separately. Preterm birth was divided into “early preterm” (< 32 weeks gestation), “late preterm” (32–36 weeks gestation) and “term,” and low birthweight was divided into “very low birthweight” (< 1500 g), “low birthweight” (1500–2499 g) and “normal birthweight” (≥2500 g) . Women wPoisson regression with robust standard errors was used to calculate relative risks and 95% confidence intervals. Each stress and support question was analyzed individually with “no” as the reference group. The question regarding feelings of nervousness and stress was analyzed categorically with “sometimes” as the reference group since it tended to be the most frequent response. The stress and support indices were analyzed continuously and categorically with the stress reference group being “low stress” and the support reference group being “low support.” The combined index was analyzed categorically using low stress/high support as the reference group. Relative risks were calculated after adjustment for the previously selected covariates using complete case analysis.Finally, a sensitivity analysis was performed excluding women who were taking benzodiazepines or selected antidepressants (based on those most frequently used in NBDPS , 25 to rMaternal characteristics by high or low cumulative stress scores are displayed in Table Adjusted relative risks of early and late preterm birth are presented in Table Adjusted relative risks of very low birthweight and low birthweight are presented in Table Overall, we did not observe individual stressful events to be moderate-to-strong risk factors for shortened gestation or having a low birthweight infant except for an increased risk of having a low birthweight infant if the woman or someone close to her was a victim of violence or crime. There were no significant associations observed between responses to social support questions and these adverse birth outcomes.Stress could affect fetal development through a variety of mechanisms, including impacts on thrombotic and inflammatory pathways, hypoxia, oxidative stress, placental development and uterine contractility –28. StreThe associations observed between women who reported they or someone close to them were victims of abuse, violence, or a crime and low birthweight and the suggestive associations between relationship difficulties and very low birthweight and preterm birth support results from the literature. Previous studies have reported associations between abuse and adverse pregnancy outcomes, with both physical and psychological mechanisms proposed , 33. ExpPrevious studies of maternal social support and low birthweight have had conflicting results , 35. We Medication for stress-related disorders such as anxiety and depression have been associated with preterm birth and low birthweight in some studies. In one review, antidepressant use later in gestation was associated with preterm birth, independent of depression . In anotOne limitation of this study is that the interview asks about the presence or absence of selected stressors without taking into account duration or chronic nature of the stressor and thus may be a better measure of acute versus chronic stress. It is possible that some of the women responding yes to certain questions did not experience a level of stress of sufficient intensity to impact pregnancy outcomes. The questions also ask if the woman or someone close to her experienced some of the stressful situations. It is possible that stress in response to the situation of another person may impact a pregnancy in different ways than direct stress to oneself. The questions also asked for a participant’s experience during a specific point in gestation from pre to early pregnancy. One study observed that only perceived stress during the second trimester was associated with preterm birth , which wStrengths of this study include the population-based sample and standardized interview examining multiple possible situations of stress and social support. The questionnaire examined these situations utilizing major life events that are relatively common and easy to recall among women who are representative of the general population. While we did not observe specific associations for many of the separate stress or support questions, we gained some insight regarding the potential association with cumulative stressors. The indices were created based on the assumption that effects of stressful and supportive life events are additive , and we Given that exposure to stress is relatively common, and that social support may not be adequate to attenuate potential harm due to stress, it is important to continue to understand the effects of various types of stress, both acute and chronic, in different populations and what methods of support or treatment may buffer its negative effects. It is important to keep developing ways to improve mental wellbeing, particularly during pregnancy.Our results add some support to prior evidence that certain stressors, like being or knowing a victim of violence or a crime, may be associated with an increased risk of selected adverse birth outcomes. We did not find strong evidence that social support buffered the observed risks in our study but given the high levels of perceived social support in our population, we may not have had an adequate distribution to fully investigate its potential effects.Additional file 1. Questions Related to Stressful Life Events and Social Support in the National Birth Defects Prevention Study from 3 months before pregnancy through the 3rd month of pregnancy.


The literature has recognized premature birth as a risk factor for infant development and maternal anxiety. This study investigated the impact of the severity of birth weight, as well as of maternal anxiety at 3 months of infants’ corrected age, on infants’ outcomes during the 1st year postpartum. Moreover, it described the longitudinal trajectories of developmental outcomes, additionally exploring the impact of anxiety.The study compared 147 mothers and their 147 newborns, differentiated in 25 Extremely Low Birth Weight (ELBW), 41 Very Low Birth Weight (VLBW), and 81 Full-Term (FT) infants. At 3, 9, and 12 months (corrected age in the case of preterm infants) the level of infants’ development was investigated according to the 5 quotients  of the Griffiths Mental Development Scales (GMDS-R). During the assessment of 3 months, mothers fulfilled Penn State Worry Questionnaire (PSWQ) to evaluate the presence of generalized anxiety.Among the 5 GMDS-R quotients, significant effect of severity of birth weight emerged only for Performance quotient: preterm infants  showed lower scores than FT ones. Moreover, this quotient decreased from 3 to 9 and to 12 months for VLBW and FT infants, while it was stable for ELBW ones. A significant interaction between severity of birth weight and maternal anxiety emerged for Hearing and Language and Locomotor quotients. In the first case, scores for ELBW infants, independently from maternal anxiety, decreased from 9 to 12 months. The same results emerged for VLBW infants, in the case of non-anxious mothers. Regarding Locomotor quotient, mean scores decreased from 3 to 9 and to 12 months for all groups in the case of non-anxious mothers. Conversely, when mothers were anxious, this decrease emerged only for VLBW infants. Lastly, ELBW, VLBW and FT showed difference in the growth and slope of the trajectories of different quotients.The severity of birth weight for preterm infants, also in interaction with maternal anxiety, had significant and specific impact on different dimensions of infants’ development. Clinical implications of these results underline the need for individualized interventions. Prematurity is an unexpected and traumatic event during childbirth  and it rA preterm birth also negatively influences the transition to parenthood , as pareAccording to an estimation by the World Health Organization, each year approximately 15 million babies are born prematurely, worldwide . In ItalLow Birth Weight-LBW) and “high-risk” preterm infants, with birth weight less than 1500 grams (Very Low Birth Weight-VLBW), and specifically less than 1000 grams (Extremely Low Birth Weight-ELBW)  .The risk of sequelae, including neurodevelopmental delays, is inversely proportional to infant birth weight  and is sRecent studies and reviews on ELBW or VLBW sequelae report evidence regarding different developmental domains, such as: neurosensory , motor , linguisFurthermore, most studies have often focused on one specific population (VLBW or ELBW), neglecting the comparison between the 2 groups. It is reasonable to speculate that ELBW and VLBW infants may show specific and somewhat different profiles concerning impairments, needs and resources, as we have already discovered in previous studies , 2017. SIn fact, due to the influence of multiple and heterogeneous variables, the trajectories of preterm infant development may show a wide range of variability from child to child.Other than considering the role played by the biological and neurodevelopmental factors, we have to include environmental variables, which may interfere with/or positively influence preterm baby growth. For example, we can acknowledge the quality of the care provided by both the hospital environment and the staff, as well as the way in which the preterm baby’s parents react to the unexpected birth of their infant.Indeed, many studies have, in the last years, focused on the investigation and description of emotional reactions and stress experienced by the mother after a premature childbirth. One of the most frequent consequences for maternal mental health is a heightened risk of experiencing different kinds of symptoms, such as traumatic stress symptomatology, depression, anxiety and acute stress disorder . In factConcerning the premature babies, the degree of severity of prematurity seems to be related to a high risk of maternal symptomatology. In fact, VLBW has been recognized as a relevant risk factor for preterm babies’ mothers, increasing the risk of being postnatally depressed from 4 to 18 times . Also, VPerinatal anxiety has been defined as anxiety experienced during the antenatal and/or postpartum period (first 12 months after birth) . DespitePostnatal anxiety, in association with the possible negative consequences of a preterm birth, may impact on the child development. Indeed, we may hypothesize that maternal anxiety is expected to be particularly intense in the first postpartum months, usually corresponding to the period of NICU stay. Some empirical evidence would support this; for example, It is notable that a debate on the specificity and characterization of perinatal anxiety, with obvious implications on the tools to use for the assessment, has received a growing interest from the scientific literature in the last few years . Based oThis is the case, for example, with the studies on “pregnancy-specific anxiety” , 2016, oTo sum up, despite the evidence of the influence of postnatal anxiety on parent-infant-relationships and child outcomes, the literature still shows a lack in investigating this issue. Moreover, the above-mentioned studies on preterm birth and maternal anxiety did not compare different subpopulations of preterm infants. In a previous study , we analThe literature has developed knowledge and findings on macro areas of child development in the case of preterm infants, such as language, attention and motor skills. However, there still is a lack of investigation regarding specific developmental dimensions, considering the severity of prematurity and specific maternal symptoms, especially in a longitudinal perspective.Based on this, we developed a longitudinal study considering 3 specific time points that represent significant steps  for the progress of infant development during the 1st year of life. Three months represent an important step for the detection of the early skills of the baby . At 9 months, new skills are supposed to emerge, like crawling and joint attention. At the end of the 1st year of life (12 months), the infant’s autonomy may be observed by the development of deambulation and/or the occurrence of the first words.The first aim of this study was to investigate the impact of severity of preterm birth on specific areas of infant development , at 3, 9, and 12 months of age, corrected for preterm infants. We hypothesized that, according to a higher degree of severity of premature birth (that is the case of ELBW), infant development would be worse compared to VLBW and FT. According to previous literature, we supposed that in ELBW infants, but not in VLBW and FT ones, scores in all dimensions would significantly decrease across the 1st year.Secondly, we investigated whether maternal anxiety, at 3 months of the infant’s corrected age, could influence infant development in the different dimensions considered. Specifically, we hypothesized lower quotient scores according to the presence of both low birth weight and maternal anxiety.Thirdly, we aimed at giving a description, through growth trajectories, of the different areas of development in ELBW, VLBW and FT infants from 3 to 12 months, considering also the effect of maternal anxiety.This study was part of a wider longitudinal research aimed to assess the course of infants’ development from 3 to 24 months postpartum.All mothers were recruited according to the following exclusion criteria: presence of previous or present psychiatric illness, lack of fluency in Italian, presence of infants’ chromosomal abnormalities, cerebral palsy, malformations, fetopathy, severe complications . In the case of twin birth, only the first-born one was included.At the end of the recruitment, our sample included 147 mothers and their 147 newborns.The Preterm (PT) group, recruited at Neonatal Intensive Care Unit (NICU) of Bufalini hospital , was composed by 66 mothers and their 66 preterm infants (44.9% of the infants’ sample), with a birth weight under 1500 g and gestational age < 32 weeks. This group was differentiated in two groups: 41 mothers and their 41 babies with weight between 1000 and 1500 g and gestational age < 32 weeks (27.9% of the infants’ sample) constituted the VLBW group; 25 mothers and their 25 babies with weight under 1000 g and gestational age < 28 weeks (17% of the infants’ sample) constituted the ELBW group.The Full Term (FT) group, recruited at the antenatal classes held in Cesena  during the third trimester of pregnancy, was composed by 81 mothers and their 81 full term healthy infants (55.1% of the infants’ sample), that had a birth weight > 2500 g and gestational age > 36 weeks.All the assessments took place at “Anna Martini” University Laboratory  at 3 months (T1), at 9 months (T2), and at 12 months postpartum (T3) (corrected age for preterm infants). During all the assessments, the level of infant development was evaluated by a trained psychologist according to the 5 quotients of the Griffiths Mental Development Scales GMDS-R; .ad hoc questionnaire regarding socio-demographic variables  and infant information . They were also asked to complete a self-report questionnaire aimed to assess the level of anxiety, while a trained psychologist assessed their infants’ development.At T1, all mothers, after providing their written informed consent, were asked to complete an The Ethical Committee of the Department of Psychology (University of Bologna) approved the design of the study.The Griffiths Mental Development Scales GMDS-R- is a welThe presence of maternal anxiety was investigated by the Penn State Worry Questionnaire PSWQ; . The PSWThe PSWQ is composed by 16 items, rated on a Likert scale between 1  to 5 . Eleven items are positively worded , while five items are negatively worded . The sum of all items provides a total score that ranges from 16 to 80, where the higher the value, the higher the levels of pathological worry. In the present study, we administered the Italian version of PSWQ that showed good internal consistency (0.85), suggesting a clinical cut-off score ≥ 57 to discriminate anxious from non-anxious subjects .Statistical analyses were carried out using the IBM SPSS statistical package version 25.0.To verify the homogeneity among ELBW, VLBW and FT dyads regarding of socio-demographic and clinical variables, we performed Pearson’s Chi Square Test and Univariate Anova.For the first and second purposes, Repeated Measures Manova were used to investigate the influence of specific factors , and of their interactions, on the 5 quotients of GMDS-R continuous scores at T1, T2, and T3.For the third purpose, growth curve analysis was used to describe trajectories of each GMDS-R quotients from T1 to T3 in ELBW, VLBW, and FT babies as a function of time and maternal anxiety at T1. With three repeated measures  of outcome variables, analyses were limited to linear and quadratic models . TherefoModeling took place in two steps. Model 1 was fit as an unconditional growth model, where only the intercept, linear slope, and curved slope were specified in order to determine the trajectories of each GMDS-R quotients in ELBW, VLBW and FT babies irrespective of maternal anxiety at T1. Model fit was evaluated using the −2 log likelihood difference test (−2LL).Model 2 was fit as a conditional growth model for exploring the effect of maternal anxiety at T1 on trajectories of each GMDS-R quotients in the three birth weight groups.p-values were lower than 0.05.Significant results were considered when 2(2) = 18.11; p < 0.0001), level of education (χ2(2) = 13.12; p < 0.0001), and anxiety (χ2(2) = 6.36; p = 0.042). In particular, FT mothers, compared to VLBW and ELBW ones, were primiparous, had graduated and were non-anxious in a higher percentage  = 36.19; p < 0.0001), twinning (χ2(2) = 28.60; p < 0.0001), gestational age  = 1066.80; p < 0.0001), days of hospitalization  = 325.30; p < 0.0001), and small gestational age (χ2(2) = 4.26; p. 039). Specifically, in FT mothers, cesarean section delivery and twinning were less frequent, compared to VLBW and ELBW mothers  = 6.413; p = 0.002): FT infants had significantly higher mean score than those observed in VLBW and ELBW infants   .F = 3.365; p = 0.038). Looking at the differences between groups, at T1, ELBW infants showed a significantly lower mean score than that reported by FT and VLBW infants ; while, at T3, VLBW infants showed a significantly lower mean score than that observed in FT infants (Bonferroni post hoc test p = 0.033). Looking at the differences within groups, mean scores of both FT and VLBW groups significantly decreased from T1 to T2  and from T1 to T3 . No differences emerged in the ELBW group  = 5.052; p = 0.008): ELBW infants showed a significantly higher mean score at T2 than that observed at T3 (Bonferroni post hoc test p < 0.0001)  .No significant differences emerged for the other GMDS-R quotients .The majority of VLBW and FT infants did not show delays at T1, while a small percentage was observable at T2 and T3. Conversely, ELBW showed delays in all three assessments.When specific quotients are observed, a low number of cases emerge in Hearing and Language scores, while a high rate emerges in Locomotor ones.In line with the second aim, we investigated the interaction between birth weight, maternal anxiety and time of assessment on infants’ GMDS-R quotients. All results are shown in Regarding the interaction between birth weight and maternal anxiety, no significant differences emerged.F = 3.274; p = 0.041) and Hearing and Language quotients  = 3.255; p = 0.042).When the interaction among birth weight, maternal anxiety and time of assessment was considered, significant results emerged on Locomotor (post hoc test p = 0.022)  (post hoc test p < 0.005)  . Lookingctively) . In the < 0.005) .post hoc test p = 0.017). Considering the differences within groups, ELBW infants showed a mean score at T2 significantly higher than that reported at T3 in both cases of non-anxious and anxious mothers  , though only in the case of non-anxious mothers  ; moreove mothers .In line with the third aim, we explored the trajectories of each GMDS-R quotient in ELBW, VLBW and FT infants as a function of time (model 1) and maternal anxiety (model 2).2(1) = 10.72; p < 0.01), VLBW (χ2(1) = 24.18; p < 0.01) and FT infants (χ2(1) = 34.85; p < 0.01) were characterized by a significant negative linear slope, followed by a positive quadratic (curved) slope, indicating a U-shaped pattern , the −2 log likelihood model comparison tests indicated that the average trajectories in ELBW , results showed that in the case of FT infants, maternal anxiety, even if it did not predict change of the intercept, showed a significant main effect on the linear slope: FT infants with non-anxious mothers had a significantly greater negative linear slope than those with anxious mothers. No significant effect emerged on the quadratic curve.No significant change of the intercept, as well as of the linear and the quadratic slope emerged in the case of VLBW and ELBW infants.2(1) = 19.63; p < 0.01), VLBW (χ2(1) = 19.47; p < 0.01) and FT (χ2(1) = 52.77; p < 0.01) infants were characterized by a significant negative linear slope and a positive quadratic (curved) slope, indicating a U-shaped pattern  = 9.86; p < 0.01) and VLBW infants (χ2(1) = 15.19; p < 0.01) were characterized by a significant positive linear slope followed by a negative quadratic (curved) slope, indicating an inverted U-shaped pattern; while the trajectory of FT infants (χ2(1) = 0.33; p < 0.05) was characterized by a non-significant negative linear slope, indicating a linear pattern  = 10.67; p > 0.05) was best described by a significant negative linear slope, indicating a linear pattern, while those of VLBW (χ2(1) = 4.08; p < 0.05) and FT infants (χ2(1) = 9.43; p < 0.01) were characterized by a significant negative linear slope and a positive quadratic (curved) slope, indicating a U-shaped pattern  = 0.01; p > 0.05) was best described by a non-significant negative linear slope, indicating a linear pattern, while those of VLBW (χ2(1) = 23.62; p < 0.01) and FT infants (χ2(1) = 58.03; p < 0.01) were characterized by a significant negative linear slope and a positive quadratic (curved) slope, indicating a U-shaped pattern . No significant change emerged in the case of VLBW and FT infants.This study aimed at assessing preterm infants’ outcomes in different developmental areas  during the 1st year of life, exploring the impact of severity of birth weight, also in relation to postnatal maternal anxiety. A further aim was to describe trajectories of these developmental dimensions in ELBW, VLBW and FT infants from 3 to 12 months postpartum. One of the main strengths of this study was to explore the impact of the severity of prematurity on each GMDS-R quotient in order to highlight possible areas of vulnerability in specific phases of development.Different results emerged in relation to specific developmental areas, as measured by GMDS-R quotients.Regarding the Performance quotient, which measures skills in manipulation, speed of working and precision, as well as the ability to apply them in novel situations , a firstThe presence of maternal anxiety did not seem to have a significant impact on infants’ mean scores of performance quotient, independently from the birth weight classification.Furthermore, growth trajectories analyses underline that ELBW infants showed a non-significant negative linear pattern of growth, while VLBW and FT infants demonstrated a U-shaped pattern of growth. These findings suggest that, during the 1st year postpartum, VLBW and FT infants had a similar trend of development, even if VLBW infants showed lower scores across time. The only significant result in the case of maternal anxiety emerged on the intercept of ELBW infants: ELBW with anxious mothers showed significantly lower scores than those without anxious mothers.The decrease of quotients observed in the study is somehow unexpected. However, a possible explanation could be given by the increase of the complexity of task demands required by the GMDS-R . Indeed,ad hoc interventions; in particular, in the case of ELBW babies, interventions aimed at promoting very simple and general abilities should start since the first postpartum months of life, while in the case of VLBW babies, the therapeutic interventions, aimed at building more complex abilities, could start later.These results may suggest that the development of ELBW and VLBW infants could benefit from To our knowledge, no previous studies have explored ELBW, VLBW and FT infants’ performance development across time, nor their trajectories of growth, by also considering the role of maternal anxiety. Therefore, further studies are recommended.Regarding the Hearing and Language quotient, it is relevant to note that most of the infants did not present an index of delay, defined as a score < 1 DS. This result is unexpected and is not in line with previous studies , in whicA first consideration is about the significant decrease in scores from 9 to 12 months shown by ELBW infants. This effect emerged both in the case of anxious and non-anxious groups. Conversely, we observed this decrease in VLBW only if their mothers were non-anxious and it never emerged for FT infants. These results could be explained considering the steps of the acquisition of hearing and language skills during the 1st year: the period between 9 and 12 months is a sensitive and pivotal time in which a baby should show more complex linguistic abilities, such as using gestures  and vocalizations . For this reason, during the first postpartum months of an infant’s age, items of GMDS-R scales are mainly focused on hearing skills, while at the end of the 1st year the quantity and quality of infant vocalizations are deeply assessed. So, the decrease in the scores from 9 to 12 months could show how the adaptation to new skills is highly demanding in the case of preterm infants, especially for high-risk babies like ELBW ones, as we found in a previous research . This trConversely, full-term infants might have already found an adjustment on these abilities, thus showing more stability in their scores throughout all the assessments.At 9 months postpartum, VLBW infants showed a higher quotient compared to the FT group and this result emerged only in the case of non-anxious mothers. This somehow unexpected result may possibly be explained considering the fact that the VLBW group represents a low risk sample and it could have taken advantage of the supportive interventions realized in NICU and during the follow-up programs , aimed aFurther studies including the role of maternal anxiety are recommended.To sum up, our results suggest that more evident difficulties for hearing and language development manifest at the end of the 1st year postpartum, when a baby should start to use gestures, vocalizations and single words to communicate. These findings are important because they highlight the importance of planning early language-focused interventions in order to limit these subsequent difficulties .Regarding the Locomotor quotient, ELBW, VLBW and FT babies had similar scores, independently from time of assessment. This result is not in line with previous studies, highlighting that preterm infants, compared to FT ones, had significantly more difficulties in acquiring gross motor skills in the 1st and 2nd year of life . RegardlWhen maternal symptomatology was also included, specific patterns relating to anxious and non-anxious groups emerged. When mothers were non-anxious, the mean scores observed in ELBW, VLBW and FT infants significantly decreased from 3 to 9 months, followed by relatively stable scores from 9 to 12 months. In this case, the Locomotor quotient tends to show a specific trend of development across time, independently from the severity of birth weight.Conversely, when mothers were anxious, ELBW and FT infants showed similar scores from 3 to 12 months postpartum, while only VLBW infants maintained the decrease observed from 3 to 9 and 12 months. It is unexpected for VLBW infants to reach good performance at T1 despite maternal anxiety. Previous literature has suggested that, in the case of preterm birth, anxiety could function as an adaptive response to traumatic condition , helpingThis result confirms the way in which ELBW and VLBW may show different profiles in the acquisition of Locomotor skills, with different time of improvement and different resources to environmental stimulations.To our knowledge, no previous studies have explored the differences between ELBW, VLBW and FT infants on locomotor development at 3, at 9 and at 12 months of life, considering the presence of maternal anxiety and this study represents a first step in this direction.Lastly, considering Eye-hand Co-Ordination and Personal and Social quotients, significant results did not emerge according to severity of birth weight and maternal anxiety.In the case of the Eye-hand Co-Ordination quotient, ELBW infants presented a small prevalence of delays in all the assessments and a negative linear pattern, different from the U-shaped pattern reported by VLBW and FT infants. This result is in line with previous studies , reportiIn the case of the Personal and Social quotient, no significant differences emerged when parametric analyses were run; nevertheless, relevant changes were observed in the rate of delay in the 3 groups. In particular, during the assessment at T2, 9 months, a very high rate of delay was present in the preterm group, especially in the case of ELBW infants (44%). Nine months represent a sensitive period for development, as babies more actively interact with the surrounding environment and new skills are learned, especially in the food area; e.g., the consolidation of weaning leads to increasing autonomous behaviors, like taking foods by hands, attempting to drink from the bottles alone, etc. The acquisition of these skills could be more difficult for severely preterm infants and for their parents; indeed, memories of previous experiences during hospitalizations  could interfere with the scaffolding role that parents could play. Therefore, the clinicians need to pay specific attention to the meaning that feeding has for these families. Furthermore, results of growth curve analysis showed that, despite not significant, preterm infants obtained lower mean scores than FT ones, with a U-shaped pattern.A relevant consideration is required about the use of developmental scales for the assessment of relational skills, rather than the evaluation by interactive scales, as in previous literature . As suggTo our knowledge, this is the first study that longitudinally explored the impact of the severity of birth weight along with maternal anxiety on each GMDS-R quotient, in order to understand whether specific areas of development are more exposed to impairments across the 1st year postpartum.Taken together, the results may suggest a discrete instability of the scores at 3-, 9-, and 12-months, as assessed by GMDS-R, strengthening the evidence from previous research that the assessment of infant development by developmental scales would show lower sensitivity in the case of high risk infants , compareThough this may suggest a limit regarding the methodology of the study, some clinical implications for intervention may arise. First, the assessment of high-risk infants and first signs of delay in the context of preterm birth should benefit from the inclusion of a series of diagnostic and observative instruments , have significant impact on infant development across the first postpartum year.ad hoc tailored interventions should be promoted to assess the risk of preterm infants’ delay and anxiety symptoms with adequate tools, to offer special support and treatment for symptomatology and to enhance parental functioning. This could help to implement more accurate interventions, as suggested by Developmental Care . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.EN prepared the study design, organized the sample recruitment, collected data, performed statistical analysis, and contributed to the writing of all the sections of the manuscript. FG performed statistical analysis and contributed to the writing of all the sections of the manuscript. FM, ET, AB, and MS contributed to the preparation of the study design and supervised data collection and the research team. FA prepared the study design, supervised all the phases of the research study and contributed to the writing of all the sections of the manuscript. All authors reviewed and approved the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.