Objective: Examine connections between mothers’ adult attachment and subjective birth experience in the context of parity and mode of delivery.Background: Research has established a clear connection between adult attachment and birth experience. This study extended previous research with an in-depth self-report attachment measure examining different dimensions of mothers’ attachment representations and their relation to subjective birth experience.  Interactions between mode of delivery and parity were also considered. Method: Participants were 257 mothers who gave birth 4 days to 12 months prior to the study. Mothers’ mean age was 30.5 years, 61% primiparas, and 26% delivered by caesarean.  Participants completed an online survey with the Birth Experience Questionnaire, the Reciprocal Attachment Questionnaire, and demographic information.Results: Hierarchical moderated regression analyses showed direct effects from adult attachment dimensions to mothers’ subjective birth experiences, specifically perceived availability, feared loss, separation protest, angry withdrawal, and compulsive careseeking. Interactions emerged for parity and/or mode of delivery for overall subjective birth experience, perceived control, perceived social support, and satisfaction.Conclusion: Adult attachment representations related to subjective birth experience, indicating that attachment figures serve as secure bases and safe havens for mothers during childbirth. These results have implications for practitioners and provide direction for future research. Based on repeated experiences with attachment figures, individuals develop and carry forward expectations about the self and others in regard to whether others can be counted on to provide support when they are distressed  that mothers’ adult attachment would relate to their subjective birth experience, and that relations between attachment and birth experience would differ based on (b) parity and (c) delivery mode.M = 6.8, SD = 3.53) with approximately equal numbers of girls and boys . Mothers’ ages ranged from 16 to 45 years . All participants had healthy, singleton births; no infants were admitted to the neonatal intensive care unit. Participant descriptive information can be seen in Participants were 257 mothers living in the USA who gave birth within 12 months of the study. Infant age ranged from 4 days to 12 months (Birth Experience Questionnaire (BEQ). The BEQ was adapted from the Green et al. . There are four subscales associated with factors important to the subjective birth experience: accuracy of expectations , perceived control , social support (e.g. ‘I feel that my companion(s) was aware of my needs during childbirth’, α = .75) and satisfaction with the birth . Subscale summary scores are the mean of the subscale items and the BEQ overall summary score was calculated as the mean of the subscales (α = .76). This study examined both mothers’ overall birth experience as well as the specific subscales.n et al.  postpartReciprocal Attachment Questionnaire . The questionnaire has 43 items that are rated using a five-point rating scale (1 = strongly disagree to 5 = strongly agree). The measure assesses five attachment goal-related dimensions: proximity-seeking , separation protest , feared loss , perceived availability  and use of attachment figure . Four additional dimensions assess insecure attachment-based personality clusters: compulsive care-giving , compulsive care-seeking , compulsive self-reliance  and angry withdrawal . Traditionally, alpha levels are considered acceptable above .60 for this number of items . The RAQ dimensions reduce the tendency to view individuals in the context of broad categories and permits evaluating fine-grained aspects of mothers’ attachment representations, following Bowlby . RAQ valParity was measured by asking mothers to indicate whether they had previous children and the ages of their other children.Income was assessed by asking mothers to indicate their household income range in USD: Under $10,000, $10,000–$25,000, $25,000–$50,000, $50,000–$75,000, $75,000–$100,000, or over $100,000.Mothers were recruited using postcard advertisements with the study web link and web announcements posted on parent-oriented websites, such as parenting blogs. Postcards were distributed in toy stores and community centres in major US urban areas. The first page of the survey described the research purpose, terms of participation and required participant consent to continue. Mothers could end the survey at any time. Participants did not receive any compensation for their participation in this study. Data were collected as part of a master’s thesis project, so as many people as possible were recruited in the time frame allotted. The final sample comprised the 257 mothers who completed all of the relevant measures, which is large enough for us to detect at least medium effect sizes for the relationships examined Cohen, .A series of hierarchical moderated regression analyses were conducted to examine relations between attachment and subjective birth experience and interactions with parity and delivery mode. Parity and delivery mode were dummy coded such that primiparous = 0 and multiparous = 1, and vaginal = 0 and caesarean = 1. Infant age was included as a control to account for time since delivery. Controls were entered in the first step of each regression. Parity and mode of delivery were entered in step 2. RAQ dimensions were entered in step 3. Interaction terms were created by multiplying the grand centred mean for each attachment dimension by those of parity and mode of delivery and were entered in step 4 of each regression.n = 157). Delivery modes were 73.9% vaginal (n = 190) and 26.1% caesarean (n = 67). Of the caesareans, 35 were planned (primipara = 17) and 32 were unplanned (primipara = 26). Correlations among variables of interest are reported in t-tests showed no significant demographic differences between mothers who did and did not complete the survey. There were no significant demographic differences between mothers who delivered vaginally and by caesarean.Primiparas comprised 61.1% of the sample , explaining 18% of the variance in subjective birth experience. Perceived availability and feared loss of attachment figure both related to subjective birth experience and angry withdrawal was also related once interactions were included. The only significant interaction was feared loss × multiparous, indicating that feared loss was a more relevant attachment dimension for primiparas than multiparas.The first moderated regression used overall subjective birth experience as the outcome variable see . HousehoThe second moderated regression examined accuracy of expectations see . Parity The third moderated regression examined perceived control during labour and delivery see . HousehoR2 from .01 to .23 , indicating that 23% of the variance in social support could be attributed to attachment. Perceived availability, feared loss and compulsive care-seeking all significantly related to social support. Mode of delivery interacted with perceived availability and was more prominent for women who delivered vaginally. Mode of delivery also interacted with separation protest, which was more salient for mothers who delivered by caesarean.The fourth moderated regression examined social support see . The RAQThe last moderated regression examined overall birth satisfaction see . Women wThis study explored the relations between adult attachment and subjective birth experience, examining interactions between attachment, parity and mode of delivery. Mothers’ adult attachment was significantly related to subjective birth experience, and there were interactions between attachment, parity and mode of delivery. Further, different interactions emerged related to different aspects of birth experience, based on parity and mode of delivery.Perceived availability, separation protest and feared loss of attachment figure were the attachment dimensions that stood out as being consistently related to aspects of subjective birth experience. These dimensions are especially meaningful because they tap into the secure base and safe haven aspects of attachment – protesting separations and confidence that your attachment figure will be present and accessible. These results suggest that birthing women need both a secure base from which to engage in this stressful experience and a safe haven for retreat when the pain and momentousness of the task becomes overwhelming.Feared loss of attachment figure emerged as more salient for primiparas than multiparas. Primiparas have never given birth before and their lack of experience may result in fears about what is happening to them and the amount of control they have during childbirth. They are also becoming parents for the first time and may be anxious or frightened about changes in their adult attachment relationship in light of the family transition. Having a child for the first time is meaningfully different from having a second child . Subjective birth experiences following planned versus emergency caesareans differ  since delivery or changes in mothers’ memories of their birth experiences cannot be accounted for in our analyses, although we did control for time since delivery. The self-report nature of the data also limits the generalisability of these results to actual maternal behaviour. Additionally, it would be ideal for data to be collected from fathers as well. Birth is an event that usually includes the entire family and examinations of fathers’ experiences of birth to date indicate that their experiences are meaningfully different from mothers (Hasman, Kjaergaard, & Esbensen, Adult attachment and birth experience is a small but promising area within the broader empirical exploration of reproductive psychology and this study adds to the growing literature on attachment and birth experience. Findings from the present study demonstrated that the safe haven and secure base functions of adult attachment are meaningful for the subjective experience of women giving birth. For practitioners working with birthing and new mothers, attachment should be considered an important factor and support from her attachment figure should be facilitated wherever possible. Future research will hopefully continue to expand our understanding of the complex connections between mothers’ attachment and their subjective birth experiences.


The impact of the consistent implementation of “aggressive” nutrition by means of intensive early neonatal nutritional support up to 40–44 weeks postmenstrual age and the use of nasal continuous positive airway pressure (nCPAP) within the first hour of life on the respiratory function of very premature neonates (VPN) at school age is unclear.Respiratory function was evaluated in 108 VPN and 70 term controls. Growth, frequency of lower respiratory tract infections, re-hospitalization, and spirometry were recorded up to 8–10 years of age. Comparison was carried out between the two study groups.There was no significant difference in forced expiratory volume in 1 s and forced vital capacity at 8 years of age, and also in lower respiratory tract infections and re-hospitalization due to them, up to 8 years of age between preterm and term neonates. No significant difference was found in spirometry measurements neither between premature neonates with and without BPD nor between the two subgroups of preterms and term neonates.“Aggressive” nutrition, persistent nCPAP use, and their impact on early postnatal growth probably positively affect the respiratory function of our study population. These very encouraging results need to be confirmed by larger studies. Lemons, et al.6 reported that 97% of VLBW neonates born in 1995–1996 were below 10th percentile at 36 weeks postmenstrual age (PMA). From the early 2000s, two policy changes have determined a new era in neonatology. In 2002, Ziegler et al.7 reported that “aggressive” nutrition, with early increased quantity of protein and calories, resulted in better growth. At the same time, early use of water-seal nasal continuous positive airway pressure (nCPAP)8 even in extremely low birth weight (ELBW) neonates resulted in decreased frequency and duration of invasive ventilatory support, reducing incidence of BPD and increasing weight gain.9 In the mid-2000s, Ehrenkranz et al.10 established that growth velocity, as expressed by weight and head circumference (HC), significantly affects psychomotor development. These are observations that support the hypothesis that nutritional interventions early in life probably decrease the risk of pulmonary complications (BPD) of very preterm neonates.11Respiratory system immaturity is the main cause of morbidity and mortality in very preterm infants, while bronchopulmonary dysplasia (BPD) represents a significant cause of long-term morbidity. In the “pre-surfactant era,” very preterm neonates with BPD would demonstrate increased airway obstruction compared to healthy term controls at school age.Taking into account all the above data, we hypothesized that “aggressive” nutrition by means of intensive early neonatal nutritional support during neonatal period as well as early and persistent nCPAP in very preterm neonates would probably affect their short- and long-term respiratory outcome. The primary aim of the study was to evaluate respiratory function of very preterm neonates at school age by performing spirometry and to compare these measurements with term controls. As a secondary aim, it was decided to assess the growth and long-term pulmonary outcome as expressed with the frequency of lower respiratory tract infections and hospital readmissions up to 8–10 years of age. Furthermore, the impact of the presence of BPD on lung function in school age was investigated.A short not validated questionnaire was developed, and in 2012 after approval of the hospital Ethical Committee and obtaining parental consent, the data for the study were collected. Data from the admission file and the outpatient long-term follow-up files of all study neonates admitted to our Neonatal Intensive Care Unit (NICU) between 2007 and 2009 were retrospectively collected. After 2012, data regarding the respiratory course of study neonates in the first 8–10 years of life were collected prospectively with the use of the questionnaire, clinical examination, and spirometry. The initial study population consisted of very preterm neonates with birth weight (BW) <1500 g and gestational age (GA) <32 weeks, without congenital abnormalities, admitted to our NICU during the study period (group A) and term neonates hospitalized in level 1 NICU for maximum 4 days in the same period (group B). Effort was made to avoid selection bias by including a sample of term neonates with minor medical problems, such as feeding difficulties during the 2 first days of life or neonatal jaundice, and excluding participants with major clinical entities, considered as confounding factors. Term neonates with congenital anomalies, respiratory problems, suspected or confirmed sepsis, and also small for gestational age were excluded. Data from 108 very preterm neonates and 70 term neonates were collected. Very premature neonates were divided into two subgroups: preterms with BPD (P-BPD) and preterms without BPD (P-non-BPD). As shown in the flow chart  with initial protein content of 2.5 g/kg/day and final protein content of 3.5–4.5 g/kg/day according to the GA in the first 5 days of life. Fat was added from the second day of life with final amount of 3 g/kg/day. For the uninterrupted administration of TPN, a central line catheter was inserted on day 1 of life, either umbilical venous catheter or peripherally inserted central catheter. In very premature neonates, milk feeds were introduced from the first or second day of life, mainly maternal milk or, if not available, preterm formula (Nestle Stage 1 with protein content 2.9 g/100 ml) as trophic feeds for 1–3 days, then increasing the amount with a rate of 24–36 ml/kg/day until achieving full enteral feeding at 200 ml/kg/day. Breast milk fortifier  was added at 100 ml/kg/day of enteral feeds. If the growth rate achieved was unsatisfactory, especially of the HC, milk feeds were gradually increased up to 220–240 ml/kg/day. Very preterm neonates continued to receive fortified breast milk or post-discharge preterm formula after their discharge from NICU up until 44 weeks PMA or until HC reaches the 25th percentile.9 Very preterm neonates were started on water-seal nCPAP on admission to NICU within the first hour of life or the ones requiring intubation right after extubation. Very preterm neonates transferred from peripheral hospitals were started on nCPAP on admission to NICU. In 19 cases, there was a delay of up to 7 h as these neonates were transferred from islands of the Aegean Sea. Neonates remained on bubble nCPAP for the whole duration of their respiratory symptoms, while applying permissive hypercapnia . Weaning attempts started after 2 weeks. Neonates developing tachypnea (respiratory rate >50/min), apneas, or FiO2 requirement >0.25 after discontinuation were placed back on nCPAP and further weaning was attempted after 48–72 h. Neonates remained on bubble nCPAP up until 33–34 weeks PMA (weight 1300–1400 g) or until they were in air or FiO2 <0.25. The prolonged use of nCPAP facilitated stable nutrition. BPD was defined as the need for O2 after 28 days of life and its severity was defined as mild, moderate, or severe according to the needs for O2 or respiratory support at 36 weeks PMA.12 Neonates with BPD and very preterm neonates <29 weeks GA received full course of palivizumab (Synagis).Our respiratory policy was based on the introduction of early nCPAP.2 administration, diagnosis, and severity of BPD were collected. The BW and HC of every preterm neonate of the study were retrospectively plotted on the Fenton Preterm Growth Charts13 and the mean growth of HC and body weight from birth to discharge (HC in cm/week and weight in g/day) were recorded. The very preterm neonates with BW and HC <10th percentile at birth and at discharge were noted. For all preterm neonates, weight, height, and HC at corrected age of 2 years and at the age of 8 years were recorded. Data regarding lower respiratory tract infections  and hospital admissions due to respiratory causes at 3 intervals were obtained. Initially and up to 2 years of age, data from their routine 3 monthly outpatient clinic follow-up appointments were collected. The second interval was between 2 and 5 years of age and the third between 6 and 8–10 years of age when spirometry was performed. During these periods, a dedicated member of the team was in regular contact with the parents and collected data via a questionnaire and from the mandatory pediatrician entries in the child’s health care record. For term controls, the same data for growth and respiratory infections and readmissions at the same intervals were documented.Data regarding the duration of nCPAP after seventh day of life, duration of O14 by the same technician who is an official technician of the Hellenic Respiratory Society and performs >2000 measurements/year. Respiratory function was expressed as the percentage of predicted values for height, weight, and sex. Flow–volume curves were obtained. Only forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1) in each neonate of the two groups were recorded. Children with spirometry values <70% predicted repeated the measurements in 3–6 months, assuming an issue with the child’s cooperation with the procedure.The Spirolab spirometer of Mir technology was used for the spirometry. Ninety percent of all the measurements were performed at the neonatal outpatient clinic of the “Agios Panteleimon” Hospital according to the American Thoracic Society and the European Respiratory Society guidelinesz-score points, with statistical power 80%, using a significance level a = 0.05. All the continuous variables are presented as mean and standard deviation (SD), if normally distributed, or as median and range, otherwise. Categorical variables are presented as absolute and relative frequencies. Associations between continuous and categorical variables were assessed with t test  and analysis of variance (for three group mean comparison), if the assumptions were met, or with Wilcoxon signed-rank test, if not. Chi-Square test, or Fisher’s exact test, where applicable, was used in order to assess possible relations between preterm vs term infants or in preterm infants between infants with BPD or not (non-BPD) with several anthropometric  or medical history characteristics  from birth up to 2 years, from 2 to 5 years, and from 6 to 8 years. All reported probability values  were based on two-sided tests and compared to a statistically significant level of 5%. STATA 15 software was used for all the calculations .Prior to the implementation of the study design, sample size calculation was performed. An estimated sample size of at least 64 participants in each of the two study groups (preterm vs term neonates) was necessary in order to detect standardized differences between the means of the two groups of 0.5 P < 0.001, P < 0.001) and developed more severe complications such as patent ductus arteriosus, sepsis, necrotizing enterocolitis stage IIB—surgical, retinopathy of prematurity requiring laser therapy, and intraventricular hemorrhage grade >II during their NICU hospitalization compared to the rest of the premature neonates.Table P = 0.000] and their hospital stay was longer . The PMA of preterm neonates with BPD at discharge from the NICU was 38.6 (3.98) weeks, while preterm neonates without BPD were discharged at 36.2 (2.34) weeks (P = 0.001).Preterm neonates with BPD received mechanical ventilation for more days than those without BPD [days 9.5 (13.9) vs 0.91 (1.46), P = 0.515) and the HC by 0.81 (0.18)cm/week and by 0.82 (0.29)cm/week, respectively (P = 0.727).At birth, 7.4% of very premature neonates of our study had BW <10th percentile and 8.3% had HC <10th percentile. At discharge from NICU, 25% of very preterms had body weight <10th percentile and only 4.6% had HC <10th percentile. There were no significant differences at discharge regarding the percentage of very preterm neonates <10th percentile for body weight and HC between the two subgroups of very premature neonates. Neonates with BPD increased their body weight  from birth up to discharge by 21.8 (3.8)g/day, whereas neonates without BPD increased their weight by 21.1 (5.7)g/day  6.9 (10.1) days,  and 3 newborns without BPD for 1, 10, and 18 days. Five very preterm neonates received corticosteroid therapy to aid extubation. None of the neonates with BPD received any corticosteroids for the prevention or treatment of BPD.P = 0.09). The growth of very premature neonates with BPD compared to term infants was also significantly delayed at the age of 2 and 8 years (Table Growth of very premature neonates compared to term infants was significantly delayed at the age of 2 and 8 years as shown in Table P = 0.03).Table Statistical analysis of respiratory function at the mean age of 8.4 years (SD 0.8 years) for preterm and 8.3 years SD 1.2 years) for term showed no significant difference in FEV1 and FVC between preterm and term neonates, preterm neonates with and without BPD, and the two subgroups of premature neonates and term neonates ,6 and to 44.05% at 36 weeks in a retrospective study of VLBW infants born during 2010–2014.21 The apparent close correlation of “aggressive” nutrition with neurodevelopment did not allow us to feed our very preterm neonates in a different way, so a group of preterms not subjected to “aggressive” nutrition could not be included in the study. Post-discharge growth was not significantly different between very premature neonates with or without BPD at 2 and 8 years, whereas it was significantly delayed compared to term infants, with the exception of HC at school age. Our results differ from those of Schmalisch et al.,22 who noted that the extent of somatic growth of very preterm infants with previous BPD lags behind those of preterm infants without BPD for the first 15 months of life.In our study, 25% of very premature neonates were <10th percentile for weight at discharge vs 7.6% at birth and 4.6% were <10th percentile for HC at discharge vs 8.3% at birth, with figures for weight at discharge improved compared to 97% in the NICH neonatal research network 1995–1996 data23 but are well in line with Pramana et al.,24 who showed no significant difference between the pulmonary outcomes  of VLBW infants with and without BPD.Despite later delayed growth, the effect on the respiratory system, as determined by lower respiratory tract infections and hospital admissions up to the age of 8 years, was not significantly different both between the two subgroups of premature neonates and between them and the group of term neonates. These results differ significantly from those reported by Lamarche-Vadel et al.Our results were confirmed by the respiratory evaluation at 8 years of age and showed no significant difference in FEV1 and FVC between very premature and term neonates, very premature neonates with or without BPD, and between the two subgroups of premature and term neonates. These results can be interpreted as a combined effect of the “aggressive” nutrition and early nCPAP policies.25 had noted significant improvement in the intelligence quotient (IQ) and neurodevelopment at the age of 7 years of very premature neonates receiving milk feeds with increased protein in up to 4 weeks after NICU discharge compared to neonates receiving the standard formula of that era. These findings led them to the theory that “early nutrition during critical windows in early life may have “programming” effects on long-term outcomes.”Since the nineties, Lucas et al.10Feeding policies up to 2000 resulted in significant postnatal growth retardation, as expressed by body weight, and HC during hospitalization was significantly correlated with adverse neurodevelopment outcomes in ELBW neonates.26 and “aggressive” nutrition7 for the prevention of postnatal growth retardation. According to many studies, the positive impact of “aggressive” nutrition on growth up to discharge from NICU but especially between 26 and 34 weeks PMA results in significant improvement in IQ and neurodevelopment.28 Early aggressive parenteral and enteral nutritional support was associated with lower rates of death and short-term morbidities and improved growth and neurodevelopmental outcomes.29 That is likely to reflect improved alveolarization and respiratory function.The situation changed in early 2000s after the introduction of increased protein and calorific intake through TPNNutrition influences prenatal lung development, directly affecting mechanisms of lung growth, but this also continues in postnatal life, especially in early infancy.30 The postnatal undernutrition negatively affects lung maturation as this has been reported in rodent models, disrupting alveolarization and development of the bronchiolar epithelium.31 To what extent these experimental findings can be translated to humans is not known, but there is some evidence indicating that nutritional management of prematurely born infants may be involved in the pathogenesis of BPD and in greater long-term consequences on lung development as alveolarization continues until at least 2 years of age.32 Filbrun et al.33 concluded that “infants with BPD above average somatic growth showed significant greater improvement in respiratory tests (P < 0.05) than their peers.”It has been long known through animal studies even after 2000 that intrauterine growth retardation showed to have long-term effect on the pulmonary structure. In neonates, intrauterine growth retardation was the sole factor implicated in the increased incidence of BPD in a study correlating intrauterine and perinatal factors with long-term respiratory prognosis.8 and its duration in some preterms with unstable respiratory system until the 33–34 weeks PMA34 helped the uneventful continuation of enteral nutrition and possibly affect lung function through mechanisms already proven in animal models. The use of nCPAP for 2 weeks in an animal study has shown to increase the volume, weight, protein, and DNA content of the lungs comparing with controls (P < 0.01).35 More recently, daily CPAP use in newborn mice exposed to hyperoxia resulted in better respiratory mechanics , increased alveolarization, and reduced macrophage infiltration compared to the ones not on daily CPAP.36The early nCPAP implementation in our babies using the “Columbia method”2 levels during nCPAP resulted in decreased incidence of BPD, and in addition, these neonates had improved growth compared to controls.9 These results confirmed a recent meta-analysis as well as some previous ones that show a small but significant effect of nCPAP in the reduction of the incidence of BPD.37 nCPAP is known to improve gastric emptying in VLBW neonates and this mechanism potentially promotes feeding stability and growth of very premature neonates who remain on nCPAP.38In VLBW neonates, the early use of nCPAP for extended periods of time during respiratory instability and tolerance of higher PaCO39 Also permissive hypercapnia does not increase the incidence of early complications and does not influence neurodevelopment at 18–22 months of corrected age of intubated neonates GA <29 weeks even in levels of PaCO2 up to 75 mm Hg.40Permissive hypercapnia, the policy used during the nCPAP support in our study, is well documented as safe. It is considered appropriate for the weaning of intubated very preterm neonates from the ventilator.Limitations of the present study are its observational and retrospective design, the limited number of participants, and data from one single NICU. Strength of the study: single-center data ensure the consistent use of the policies and data recorded regarding growth, respiratory infections, hospital admissions, and spirometry up to 8 years.In conclusion, although it is difficult to precisely determine the factors that affect the long-term outcome of BPD, due to its multifactorial nature, it appears likely that the “aggressive nutrition” of very premature neonates, the persistent use of nCPAP, and their impact on the achievement of early postnatal growth up to 40–44 weeks corrected age played an important role in the results of this single-center study. To the best of our knowledge, this is the first time that such encouraging results are shown regarding the long-term respiratory prognosis of very premature neonates (with or without BPD), although further large-scale comparative studies are necessary to verify these findings.


Congenital birth defects may result in a critical condition affecting the baby, including severe fetal/neonatal handicap and mortality. Several studies have shown that genetic, nutritional, and environmental factors may have an impact on fetal development and neonatal health. The relevance of essential and toxic elements on fetal development has not yet been fully investigated, and the results of recent research indicate that these elements may be crucial in the assessment of the risk of malformations in neonates. We determined the association between essential and toxic elements and the level of folate in maternal serum (MS) and amniotic fluid (AF), along with neonatal abnormalities. A total of 258 pregnant Polish women in the age group of 17–42 years participated in this study. AF and MS were collected during vaginal delivery or during cesarean section. An inductively coupled plasma mass spectrometry technique was used to determine the levels of various elements in AF and MS. The results of this exploratory study indicate that the levels of essential and toxic elements are associated with fetal and newborn anatomical abnormalities and growth disorders. The frequency of congenital abnormalities in Poland is approximately 2–4% of live births . They arSeveral studies have shown that micronutrient status plays an important role in the health and well-being of pregnant women and in the development and long-term health of their offspring ,4. In a Environmental pollutants including heavy metals may also play a contributory role in reproductive and developmental abnormalities . EpidemiThe impact of essential and toxic elements on fetal development has not yet been fully investigated and the mechanisms are poorly understood. The results of recent research indicate that the role of some micronutrients may be crucial in assessing the risk of malformations in infants. Therefore, the aim of this exploratory analysis was to determine the association between essential, toxic elements, and folate; and fetal congenital anatomical defects, fetal growth abnormalities, as well as neonatal disorders.This was an exploratory study to determine the levels of elements and folate in AF and MS between 27 and 42 weeks of gestation. The AF and maternal blood were collected during routine diagnostic and treatment procedures in pregnant women hospitalized in the Polish Mother’s Memorial Hospital Research Institute. The samples were stored at −80 °C.The study was conducted on pregnant Polish women (white/Caucasian) who were under routine obstetric care. Women voluntarily participated in the study after signing an informed consent form. The participants consented to the secondary use of their samples. The study was conducted on patients qualified to participate based on the inclusion and exclusion criteria.The inclusion criteria were normal single intrauterine pregnancy and signing an informed consent form. The exclusion criteria were vaginal bleeding, pelvic pain, chorionic hematoma, premature rupture of membranes, placental insufficiency, multiple pregnancies, and genetic conditions in the current pregnancy or present in the women.ClinicalTrials.gov, ID: NCT03598361.The study protocol was approved by the Bioethics Commission of the Research Ethical Committee, Polish Mother’s Memorial Hospital Research Institute in Poland  and The University of British Columbia Clinical Research Ethics Board . This study was conducted in accordance with the Declaration of Helsinki. The study has been registered in A routine medical interview was conducted to collect data on maternity, pregnancy, family history of illnesses and operations, drugs and medications used, and the occurrence of allergies. Maternal weight, to the nearest 0.1 kg, was measured prior to the delivery using a calibrated digital weight scale while women were wearing lightweight clothing. Maternal height was measured in centimeters and pre-pregnancy weight was self-reported. Pre-pregnancy body mass index was calculated. Birth weight of newborns was assessed using a digital weight scale at the third stage of delivery. The week of gestation was determined by first trimester obstetric ultrasonography.Congenital birth defects were diagnosed prenatally by performing an ultrasound scan to detect anomalies at 20–24 weeks of gestation according to the guidelines of the Polish Society of Gynecologists and Obstetricians, which are based on the protocols of the International Society of Ultrasound in Obstetrics and Gynecology and Fetal Medicine Foundation. Abnormal prenatal findings and suspected congenital defects were re-evaluated in the neonatal period by pediatricians performing clinical examinations and using ultrasound  and X-ray (chest and lungs) techniques.Fetal growth disorders, such as macrosomia and fetal growth restriction (FGR)/hypothropy, were established when ultrasound-estimated fetal weight according to the Hadlock formula exceeded the 90th or 10th percentile for gestation, respectively . GestatiNeonatal disorders found during the pediatric postpartum medical examination were classified as neurologic, respiratory, or cardiovascular according to standard newborn assessment protocols .Blood samples were collected from the antecubital vein of the pregnant women in the first stage of labor (latent phase) during admission to the delivery ward. Monovette test tubes  were used without any anticoagulants to obtain blood serum and the tubes were spun for 30 min (3000 rpm/min at 4 °C) and then frozen. All serum samples were stored at −80 °C.Samples of AF were obtained (5 mL) via transabdominal puncture at the surgical wound during cesarean section or transvaginal puncture using a speculum when cervical dilatation was above 4 cm during vaginal birth. The membranes were intact in both situations; sterile needles and syringes were used just before delivery. The samples were centrifuged (3000 rpm/min for 10 min at 4 °C), frozen, and stored at −80 °C.Lactobacillus casei as the test microorganism. The inter-assay variations for serum folate concentrations was 5.9%. Whole blood hemolysate (NIBSC code: 95/528) served as an external quality control; the measured mean folate concentration was 12.8 ng/mL  with an inter-assay variation of 7.8%. Measurement of essential and toxic elements in AF and MS has been described by Markiewicz et al. [R2—was acceptable for all analyzed elements (R2 > 0.999). The trueness of the analytical method was assessed by analyzing the certified reference material (CRM) Seronorm™Trace Elements Serum L-2. The values of recovery are within an acceptable range for all analytes, which demonstrates that the described analytical procedure is fit for the intended purpose.Folate analysis in AF and MS was performed according to the method described in our previous article . We usedz et al.  and Kocyz et al. . Samplesp < 0.05.All statistical analyses were performed with Statistica 10 for Windows and RStudio software . Data were tested for normal distribution using the Shapiro–Wilk test. The Mann–Whitney test was used to compare the differences between groups with defects and a healthy group for all the studied parameters. Logistic regression analysis was used to assess the relationships between variables . The proIn this study, we observed a neonatal abnormality rate of nearly 30% in the study population, including congenital birth defects (5.4%), macrosomia (8.1%), hypotrophy (10.9%), and newborn disorders (8.9%). In five cases, macrosomia or birth defects co-occurred with newborn disorders. Nearly 50% of the birth defects were cardiac-related, 30% skeletal defects, and 20% were other disorders . Newborn disorders included cardiovascular, neurological, and respiratory dysfunctions.p = 0.08). Comparison of levels of different trace elements in AF showed the greatest differences in the newborn disorders group (IV), where Al and Ba levels were higher than normal.The levels of study elements and folate in AF and MS in groups with abnormalities were compared with healthy deliveries as previously published ,23. The −1 was related with a 20% probability of the occurrence of birth defects. In maternal serum the probability of macrosomia increased with a decrease in cobalt concentration. A cobalt level below 0.5 µgL−1 was related with a nearly 15% probability of the occurrence of birth defects. Moreover, the probability of newborn disorders increased with an increase in cadmium concentration in MS. A cadmium level over 0.5 µgL−1 was related with a nearly 20% probability of the occurrence of newborn disorders  depending on the values of the elements and folate in the AF and MS was analyzed . It was isorders .−1 was related with a nearly 10% probability of the occurrence of birth defects. It was found that the probability of birth defects increased with an increase in aluminum concentration in AF. In this study, an aluminum level over 300 µgL−1 was related with an over 10% probability of the occurrence of birth defects. The probability of macrosomia increased with a decrease in vanadium concentration in AF. In the study group a vanadium level below 0.2 µgL−1 was related with an over 10% probability of the occurrence of birth defects. Moreover, it was shown that the probability of newborn disorders increased with an increase in lead concentration in AF. A lead level over 50 µgL−1 was related with an over 10% probability of the occurrence of newborn disorders (In AF the probability of birth defects decreased with an increase in folate concentration. A folate level below 10 µgLisorders .p = 0.02) and low level of folate in AF  were the best predictors of birth defects (I). In MS, a low concentration of Mg was related to an increased risk of birth defects (I) . Low levels of V in AF  and low levels of Co in MS  were associated with macrosomia (II). Moreover, high levels of Pb in AF  and Cd in MS  were predictors of newborn disorders (IV). The results of the logistic regression analysis showed that, among the selected factors, a high level of Al  increased with low levels of Mg in MS. Mg is known to play a crucial role in maternal health and fetal development, which is confirmed by the results of this study. This essential mineral is necessary for the growth of the body and plays an important role in the metabolism of the fetus. Its deficiency in pregnancy may increase the risk of health disorders for both the mother and the newborn, such as mortality and morbidity, gestational diabetes, preterm labor, preeclampsia, and small for gestational age or intrauterine growth restriction ,25. It ind heart . Anothernd heart . It seemnd heart . AnotherWe found that a high level of Al in AF is related to newborn disorders and that the probability of birth defects increases with increasing levels of Al in AF. This suggests that maternal exposure to Al increases the risk of birth defects and other abnormalities. Al is a toxic metal which easily accumulates in the organism due to its fast intestinal absorption, organic distribution, and rather low excretion. It is transported across the placenta and then to the fetus, and the heart is a potential target of Al toxicity . ResultsThe results of this study indicate that high levels of Pb and Cd in AF and MS are associated with newborn disorders . Results from human studies have shown an association between As, Cd, Mn, and Pb pollution and their high levels in MS and congenital heart defects in the fetus ,30,31. EIn this study, we found that V and Co are the important elements needed for fetal growth, and a low level of these elements is related to a high probability of macrosomia. V is an essential element and is necessary for the normal growth and development of bone and reproductive systems . To our The results of our study have confirmed that low maternal folate status during pregnancy is associated with a high risk of birth defects. Several studies have indicated that folate deficiency during embryogenesis in pregnancy is related to neural tube defects ,43,44. IThe limitations of the study are: firstly, analysis of elements and folate only in serum and AF, not umbilical cord blood, which may better clarify the impact of essential and toxic elements and folate on the fetuses. Secondly, in this study, we included only women at delivery and a rather heterogeneous group of birth defects and fetal growth abnormalities. Furthermore, in the present study, we did not determine nutritional factors and environmental factors that may affect the concentrations of elements in the analyzed samples. Another limitation involves the maternal and obstetrical backgrounds which differ between the present and previous studies. The former study was designed to create reference ranges based only on healthy non-smoking women with normal growth pregnancies. The latter research also included smoking patients, women with chronic diseases, and pregnancy-related conditions. These are potential confounding factors which may affect essential and toxic elements and folate, and thus should be considered in the interpretation of the results and in future study designs.In conclusion, low concentrations of Mg and folate in the MS and AF increase the risk of birth defects. Macrosomia is related to low levels of V in AF and Co in MS. Maternal exposure to Al is associated with birth defects, especially cardiac dysfunction. Furthermore, high levels of Pb and Cd in AF and MS increase the risk for neurological, cardiovascular, respiratory, and other disorders in newborns after birth. The results of this study indicate that essential and toxic elements are associated with fetal congenital anatomical defects and growth disorders. Our results provide directions for further research.


Previous studies have reported an intergenerational association between maternal and offspring preterm birth (PTB) but the nature of the association remains unclear. We assessed the association between maternal and offspring preterm birth using a quasi-experimental sibling design and distinguishing between preterm birth types.We conducted a retrospective intergenerational cohort study of 39,573 women born singleton in Manitoba, Canada (1980–2002) who gave birth to 79,198 singleton infants (1995–2016). To account for familial confounding we defined a subcohort of 1033 sisters with discordant PTB status who subsequently gave birth and compared offspring PTB rates between 2499 differentially exposed cousins using log-binomial fixed-effects generalized estimating equation models. PTB was defined as a delivery < 37 gestation weeks, divided into spontaneous and provider-initiated.In the population cohort, mothers born preterm were more likely to give birth preterm  and very preterm birth . However, in the siblings cohort, the intergenerational association was not apparent among births to sisters with discordant PTB status . Mothers born at term with a sister born preterm had a similarly elevated risk of delivering a preterm infant (10%) than their preterm sisters. Intergenerational patterns were observed for spontaneous PTB but not for provider-initiated PTB.Our findings suggest that it is not the fact of having been born preterm that puts women at higher risk of delivering preterm, but the fact of having been born to a mother who ever delivered preterm. Consideration of a female family history of PTB may better identify women at higher risk of preterm delivery than relying on maternal preterm birth status alone. Further research may benefit from distinguishing preterm birth types. Preterm birth (PTB) is one main factor associated with infant mortality and morbidity in industrialized countries , 2. PretThe etiology of preterm birth is multifactorial, complex and not well understood . One impOur goal was to assess whether maternal preterm birth status is an individual risk factor per se for delivering preterm. To distinguish the risk of delivering preterm among women born preterm themselves from that of women born at term but with a family history of preterm birth, we used fixed-effects methods to compare preterm birth rates among the offspring (cousins) of sister mothers of discordant preterm birth status. Furthermore, we distinguished between spontaneous and provider-initiated PTB, which may be differentially involved in the recurrence of PTB across generations –19.This retrospective cohort study used linked population-based health and social registers, accessed through the Manitoba Population Research Data Repository. Manitoba is Canada’s central province with an area slightly higher than France and 1.28 million inhabitants by the 2016 Census, 72% of whom live in urban areas. All Manitoba residents are covered by the publicly funded provincial health insurance plan. The study population included all women born singleton in a Manitoba hospital from 1980 to 2002 who subsequently gave birth to a singleton live birth in the province at or after 15 years of age, from 1995 to 2016. Mother-child clusters including multiple births, missing or extreme (< 24 or > 42 weeks) gestational age were excluded.We used two cohorts to make comparisons. First, to allow comparability with previous studies, we assessed the association among all women giving birth in the general population. Like in most previous studies, PTB rates of children born to mothers who were born preterm were compared to the PTB rates of children born to mothers born at term. Most mothers in this population cohort (71%) did not have a sister giving birth in the study period, which makes it impossible to separate individual from familial influences due to the absence of a within-family comparator, that is, a sister who shares the genetic and family environment but differs in preterm birth status. Secondly, to overcome this limitation, we used a subset of the population cohort composed of sisters of discordant preterm birth status . This sibling subcohort allows for comparison of risk of PTB between the offspring (cousins) of sisters of discordant PTB status while controlling for stable family characteristics shared by sister mothers using fixed-effects analysis –22.We used deterministically linked province-wide health and social databases. Maternal obstetrical and newborn hospital discharge abstracts are cross-referenced  and were used to identify sisters born to the same biological mother, PTB status, date of birth, birth order, maternal age and infant sex. A validation study found the hospital abstracts to be adequately accurate to assess perinatal morbidity . PTB wasThe other linkable databases in the Manitoba repository are also of good quality and have evolved over time , 27. InfPhysician claims for visits in offices, hospitals and outpatient departments are compiled by the provincial Ministry of health in the Permanent Medical Statistical file and were used to assess maternal substance abuse in the year preceding the birth of the child, in combination with hospital records. Substance abuse was defined as one or more physician visits with a diagnosis for alcohol or drug psychoses, alcohol or drug dependence, or nondependent abuse of drugs . The same codes were used for hospitalizations occurred before April 1, 2004. ICD-10-CA codes F10-F19, F55 were used for hospitalizations occurred on or after April 1, 2004.Descriptive frequencies were provided according to maternal sibling patterns. A few variables were re-categorized to prevent small cell counts, which cannot be disclosed according to provincial privacy legislation. Remaining cells with small counts were suppressed in our tables.To assess the association between maternal and offspring PTB in the population cohort Tables  and 3 weIn the sibling subcohort we conducted a matched cohort analysis Tables  and 4. EIn sensitivity analyses restricted to a subset of women giving birth at or after 2000 with complete information on additional potential confounders, we further controlled for individual-level characteristics that may differ between sister-mothers, such as adequacy of prenatal care based on the Revised-Graduated Prenatal Care Utilization Index, smoking during pregnancy, maternal education, single marital status and receipt of employment income assistance. A second sensitivity analysis was restricted to one randomly selected child per mother to account for the recurrence of PTB among different pregnancies of a given woman, which could inflate the numerator and therefore the effect estimates.N = 17,687), extreme maternal gestational age (N = 242), mother born from multiple pregnancy (N = 356), missing child’s gestational age (N = 4613), child is a multiple (N = 2735) and extreme child’s gestational age (N = 4824). After exclusion of 19,055 mothers (32.5%) and 37,592 children (32.2%), the population cohort for analyses was composed of 39,573 mothers and 79,198 children. Of these mothers, 28,133 (71.1%) did not have a sister giving birth during the study period.There were 58,628 mothers born on or after April 1st, 1980 who gave birth to 116,790 children from April 1st, 1995 to March 31, 2016. We excluded mother-infant pair records that met one or more of the following exclusion criteria. Missing maternal gestational age  were born at term. Overall, offspring preterm birth rates were 6.2 per 100 live births in the population. Mothers without sisters giving birth in the cohort were more likely to deliver preterm if they themselves were born preterm. Among sister mothers with discordant PTB status, offspring preterm birth rates did not differ (10.2% among sisters born preterm and 10.0% among sisters born at term). The highest PTB rate occurred in a small number of families where all sister mothers were born preterm (13.1%) and the lowest rate where all sister mothers were born at term (6.1%).Among the 11,440 sisters, 1033 (9.0%) had discordant preterm birth status (455 born preterm and 578 at term) , history of substance abuse, smoking during pregnancy, inadequate prenatal care, receiving income assistance and low maternal education Table . InfantsIn the population cohort analysis, infants exposed to maternal preterm birth had higher risk of preterm birth before, and after adjustment  , similar to that of a meta-analysis involving seven studies with low heterogeneity   [9]. LiWe also observed that sisters of concordant PTB status exhibited extreme outcomes. PTB rates were lowest among sisters all born at term (6.1%) and highest among sisters all born preterm 13.1%). These findings are consistent with polygenic theory according to which higher recurrence rates occur among close relatives if more than one family member is affected . Althoug.1%. ThesIn our analyses stratified by preterm birth types, there was no intergenerational association for infant provider-initiated PTB in the population cohort or in the sibling cohort. Infant spontaneous PTB exhibited clear associations with maternal spontaneous PTB in the population cohort across all models. In the sibling analysis, these associations were positive but not statistically significant presumably due to reduced statistical power to detect associations. Given this limitation, the possibility of a true maternal transmission of spontaneous PTB cannot be ruled out. Further studies may clarify this issue.Our study has limitations. The use of administrative data collected for other purposes restricted the information available for analyses. At the same time, routine data collection on a relatively stable and large population over decades made this study possible. Our sibling design is particularly suited to draw causal inferences regarding an individual exposure that differs between siblings, provided that there is no exposure measurement error or confounding due to factors not shared by siblings . GestatiOur findings provide new insights into the study of intergenerational effects of PTB. First, our study provides additional evidence that PTB clusters within families and that maternal preterm birth status may not be a predictor of preterm delivery as sensitive as a family history of PTB. Interestingly, the risk of offspring PTB within families in which at least one mother-sister was born preterm was higher (≥10%) than among women born preterm without sisters (7.7%). Part of this excess risk may be due to poorer sociodemographic profiles aggravated by larger family size, so that the environmental factors contributing to PTB among women born preterm seem to affect equally their sisters not born preterm themselves. The association between socioeconomic disadvantage and adverse birth outcomes is well established not only cross-sectionally  but alsoSecond, both the population and the sibling analyses indicated that provider-initiated PTB was not influenced by maternal or familial PTB. The possibility that the event of being born preterm, irrespective of the cause, may physiologically predispose women to deliver preterm later in life  is not supported by our findings. A focus on spontaneous PTB may better serve to identify mechanisms involved in familial patterns of PTB.Our findings do not support the notion of seeing maternal PTB status per se as an individual risk factor for preterm delivery. Rather, consideration of a female family history of PTB may better identify women at higher risk of preterm delivery and perhaps other pregnancy complications. The clustering of PTB within families suggests that family-based approaches to understanding the occurrence of preterm birth and its sequelae may be more fruitful than solely focusing on individual risk factors.


WRKY genes in wintersweet (Chimonanthus praecox) are still unknown. In this report, a group IIc WRKY gene, CpWRKY71, was isolated from wintersweet. CpWRKY71 was localized to the nucleus and possessed transcriptional activation activity. qRT-PCR  analysis showed that CpWRKY71 was expressed in all tissues tested, with higher expression in flowers and senescing leaves. During the flower development, the highest expression was detected in the early-withering stage, an obvious expression of CpWRKY71 was also observed in the flower primordia differentiation and the bloom stage. Meanwhile, the expression of CpWRKY71 was influenced by various abiotic stress and hormone treatments. The expression patterns of the CpWRKY71 gene were further confirmed in CpWRKY71pro:GUS (β-glucuronidase) plants. Heterologous overexpression of CpWRKY71 in Arabidopsis caused early flowering. Consistent with the early flowering phenotype, the expression of floral pathway integrators and floral meristem identity (FMI) genes were significantly up-regulated in transgenic plants. In addition, we also observed that the transgenic plants of CpWRKY71 exhibited precocious leaf senescence. In conclusion, our results suggested that CpWRKY71 may be involved in the regulation of flowering and leaf senescence in Arabidopsis. Our study provides a foundation for further characterization of CpWRKY genes function in wintersweet, and also enrich our knowledge of molecular mechanism about flowering and senescence in wintersweet.The WRKY transcription factors are one of the most important plant-specific transcription factors and play vital roles in various biological processes. However, the functions of  Chimonanthus praecox), a perennial deciduous shrub of the Calycanthaceae, is native to China and widely distributing in south-central and southwest China [APETALA3 (CpAP3) gene was found to partially rescue stamen development in the Arabidopsis ap3 mutant, and caused numerous morphological changes and homeotic conversions to flowers [AGAMOUS-like6 (CpAGL6) gene was reported to promote flowering through inhibition of FLOWERING LOCUS C (FLC) and activation of APETALA1 (AP1) and FLOWERING LOCUS T (FT) and cause abnormal stamen and carpel development in later developing flowers [CpCZF1 and CpCZF2, two C3H type zinc finger protein genes of wintersweet, were revealed to affect stamen development and caused the formation of abnormal flowers in transgenic Arabidopsis plants [Wintersweet  was isolated from sweet potato [WRKY genes have been found subsequently in various plants [Arabidopsis WRKY41 was reported to control Arabidopsis seed dormancy by directly regulating the expression of ABI3 [GhWRKY17 accelerated leaf senescence in A. thaliana [Dlf1, a WRKY transcription factor gene which was isolated from rice, was reported to participate in the regulation of flowering time and plant height in rice [CmWRKY48 enhanced aphid resistance of transgenic chrysanthemum [TaWRKY93 in A. thaliana enhanced multiple abiotic stress tolerances [In the past decades, a growing number of transcription factor genes have been isolated and proved to widely participate in various developmental and physiological processes. Among them, the WRKY transcription factor family is one of the largest plant-specific transcription factor families. They are named after the conserved motif WRKYGQK and contain one or two conserved WRKY domains which are comprised of 60 amino acids at the N-terminal end and a zinc finger-like motif either C2H2 or C2HC at the C-terminal. Based on the number of WRKY domains and the type of zinc-finger motif, the family can be classified into three subfamilies, namely, group І (two WRKY domains and C2H2 zinc-finger), group ІІ (one WRKY domain and C2H2 zinc-finger) and group Ш (one WRKY domain and C2HC zinc-finger). Additionally, based on additional structural motifs outside the WRKY domain, group ІІ can be further divided into five subgroups . WRKY proteins regulate the expression of downstream genes by recognizing and binding to the W-box (TTGACC/T) present in their promoters . Since St potato , a larges plants ,16,17,18 of ABI3 . Overexpthaliana . Dlf1, a in rice . Overexpanthemum . Overexplerances .WRKY genes in wintersweet. We noted a novel WRKY gene, CpWRKY71, appeared to be differentially expressed during flower development, based on the information from the wintersweet transcriptome database [WRKY gene CpWRKY71 from wintersweet. The expression pattern of CpWRKY71 was investigated. Ectopic expression of CpWRKY71 in Arabidopsis resulted in an early flowering and precocious leaf senescence phenotype.At present, the function of WRKY transcription factor has been well elucidated in model plants. However, to some extent, this family has not been well studied in some non-model plants or woody plants. To our knowledge, no information is available about the isolation and functional analysis of database . Here, wCpWRKY71 was obtained. Sequence analysis showed that CpWRKY71 contained a complete open reading frame of 951 bp, which encodes a protein of 316 amino acids. Its predicted molecular weight and theoretical isoelectric point were 34.96 kDa and 6.72, respectively. Subsequently, we isolated 2231 bp genomic DNA fragment of CpWRKY71. Compared with cDNA sequence of CpWRKY71, the genomic DNA of CpWRKY71 contained two introns (417 bp and 677 bp) and three exons   A. Sequen 340 bp) B, indica 340 bp) , so we dCpWRKY71 was fused to the N-terminus of GFP (green fluorescent protein) gene. The Agrobacterium tumefaciens cells harboring 35S:CpWRKY71-GFP or 35S:GFP constructs were infiltrated to young leaves of tobacco (Nicotiana benthamiana) plants, respectively. The GFP fluorescence of CpWRKY71-GFP was observed in the nucleus, whereas that of GFP control was expressed in the cytoplasm and nucleus. These results indicate that CpWRKY71 is a nuclear-localized protein , and show α-galactosidase activity on the plates containing X-α-gal. On the contrary, the yeast cells transfected with pGBKT7 could survive on the SD/-Trp medium only. This result indicates that CpWRKY71 possesses transcriptional activation activity in yeast , followed by flower primordia differentiation stages  and the bloom stage (S4), and the lowest level was present in the flower-bud (S1), the petal-display (S2) and the initiating bloom stage (S3)  B.CpWRKY71 gene, a 3777 bp promoter fragment of CpWRKY71 was fused to the GUS reporter gene to generate transgenic Arabidopsis plants (named CpWRKY71pro:GUS). Histochemical GUS staining showed that faint GUS activity was observed in the roots, stems and young leaves  and hormone treatments  were detected by qRT-PCR. These treatments were chosen according to the cis-acting elements present in the promoter region of CpWRKY71  were selected for further phenotypic analysis , SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1), AP1, FRUITFULL (FUL) and CAULIFLOWER (CAL) in OE, WT, and EV plants. The result shows that all these genes except SOC1 were upregulated in OE1 and OE2 plants (CpWRKY71 in Arabidopsis promoted flowering.Under long day (LD) conditions, compared with EV and WT plants, OE1 and OE2 showed an early flowering phenotype A. OE1 an2 plants E. These CpWRKY71 accelerated leaf senescence. OE1 and OE2 exhibited an early leaf senescence phenotype compared with the EV and WT plants  were increased in transgenic lines  under greenhouse conditions .CpWRKY71, the roots, stems, and young leaves were collected from six-leaf stage plants, senescence leaves and flowers at stage 4 (bloom period) were collected from the adult wintersweet plant [For the tissue-specific expression pattern of CpWRKY71 gene in different flower development stages, samples at different flower developmental stages were collected as previously described [For the expression pattern of escribed . Three bA. thaliana ecotype Columbia were sown on solid Murashige and Skoog (MS) medium before vernalization at 4 °C for three days and then grown in a growth chamber at 22 °C with the16 h light/8 h dark photoperiod (120 µmol photons·m−2·s−1).Seeds of the CpWRKY71-F/R , then cloned into the pMD19-T vector  and sequenced by TsingKe Company . The multiple alignments were performed using BioEdit software. MEGA 5.0 software with the NJ method (1000 BootStrap replicates) was used to construct the phylogenetic tree of CpWRKY71 and WRKY proteins from other species.Total RNA isolation and the first-strand cDNA synthesis were performed as described by Liu et al. . Total gKY71-F/R  were desCpWRKY71 was isolated according to the protocol of the Universal Genome Walker Kit . Specific primers GSP1 and GSP2 (CpWRKY71 gene sequence to clone the promoter. The purification and sequencing of the PCR products were performed as described above. The putative cis-acting regulatory elements were searched with the PLACE database (http://www.dna.affrc.go.jp/PLACE) and PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [The 5’-upstream sequence of and GSP2  were dese/html/) .CpWRKY71 without termination codon was cloned into the modified pCAMBIA 1300 vector [35S:CpWRKY71-GFP. For transcriptional activity analysis, the ORF of CpWRKY71 was inserted into pGBKT7 via XmaI and Sal I sites, to create the plasmid pGBKT7-CpWRKY71. For GUS histological assay, CpWRKY71 promoter region was cloned into the vector pBI121 to replace the CaMV35S promoter via HindIII and BamHI sites, to construct the plasmid CpWRKY71pro:GUS. The plasmid 35S:CpWRKY71-GFP was also used to generate CpWRKY71 overexpression plants. The primers are listed in For the subcellular localization assay, the open reading frame (ORF) of 0 vector  via SacI35S:CpWRKY71-GFP and the empty vector 35S:GFP were separately introduced into A. tumefaciens strain GV3101. The young leaves of tobacco (N. benthamiana) plants were infiltrated with A. tumefaciens cells harboring 35S:GFP and 35S:CpWRKY71-GFP, respectively. Confocal laser microscopy  was used to observe the GFP fluorescence.The recombinant plasmid CpWRKY71 and pGBKT7 were transformed into the AH109 yeast strain, respectively. SD/-Trp medium was used to select the positive transformants. SD/-His (containing 10 mM 3-AT) and SD/-His/X-α-gal medium (containing 10 mM 3-AT) were used for transactivation analysis. The pGBKT7 empty vector was used as a negative control.The recombinant plasmid pGBKT7-In order to understand gene expression patterns under different abiotic and hormone treatments, wintersweet plants at the six-leaf stage were used . For theGUS gene driven by the CpWRKY71 promoter in Arabidopsis, ten-day-old homozygous Arabidopsis T3 seedlings were used [Arabidopsis seedlings were sprayed with 50 µM ABA, 100 µM SA, or 100 µM MeJA for 6 h [To understand the effects of different treatments on ere used . For droere used . For theere used . For exo for 6 h ,52, respCpActin, CpTubulin, and AtActin were used as the internal reference for the gene expression analysis in wintersweet and Arabidopsis, respectively [qRT-PCR was performed on a Bio-Rad CFX96 Real-time system machine using the Ssofast EvaGreen Supermix . The PCR procedure was as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 5 s and 72 °C for 5 s, and a melt cycle from 65 to 95 °C. CpWRKY71pro:GUS was introduced into A. tumefaciens strain GV3101 and then transformed into Arabidopsis via the floral dipping transformation method [CpWRKY71 overexpression plants were generated as described above. The positive plants were selected on MS media containing 25 µg/mL hygromycin. The seedlings with the resistance grown in a growth chamber under long-day conditions (22 °C with the 16 h light/8 h dark photoperiod and 120 µmol photons·m−2·s−1 light intensity).For GUS histological assays, the recombinant plasmid n method . T0 seedHistochemical staining was performed according to the method described by Khan et al. . Samplesd-glucuronic acid  was used as the substrate. The GUS activity was measured using a Varioskan Flash Spectral Scanning Multimode Reader  with 365 nm excitation and 455 nm emission. GUS enzyme activity was expressed as pmol 4-MU produced per mg protein per minute.The procedure of the GUS fluorometric assay was performed as described by Niu et al. . The proThe flowering time was measured as described by Zhang et al. . The dayt-test. It was considered significant with a p-value of 0.05, or very significant with a p-value of 0.01.Statistical analysis was conducted using SPSS 17.0 software . The significance of differences was analyzed by Student’s


Growing body of evidence shows their higher risk of adverse outcomes at any period of life, including increased rates of neurologic delay, noncommunicable chronic diseases , and mortality. Although there is no consensus regarding its definition, birthweight centile threshold, or follow-up, we believe birthweight <10th centile is the most suitable cutoff for clinical and epidemiological purposes. Maternal clinical factors have modest predictive accuracy; being born SGA appears to be of transgenerational heredity. Addition of ultrasound parameters improves prediction models, especially using estimated fetal weight and abdominal circumference in the 3rd trimester of pregnancy. Placental growth factor levels are decreased in SGA pregnancies, and it is the most promising biomarker in differentiating angiogenesis-related SGA from other causes. Unfortunately, however, only few societies recommend universal screening. SGA evaluation is the first step of a multidimensional approach, which includes adequate management and long-term follow-up of these newborns. Apart from only meliorating perinatal outcomes, we hypothesize SGA screening is a key for socioeconomic progress.The actual burden and future burden of the small-for-gestational-age (SGA) babies turn their screening in pregnancy a question of major concern for clinicians and policymakers. Half of stillbirths are due to growth restriction  The intrauterine environment influence on fetus development is a well-known determinant of individual's long-term health and quality of life. From the initial description of 23 infants being born at term weighing less than 2000 g, Warkany et al.  introducIn fact, human development goes far beyond genetic inheritance. Lessons learned from pregnancies subjected to smoking , 6 or inTherefore, considering the long latency of some events, such as cognitive delays and cardiovascular diseases, SGA has impacts of public health magnitude, especially in low- and middle-income countries (LMICs) , 25. In th century, gestational age at birth and birthweight concepts overlapped; the World Health Organization recommended a birth weight of 2500 g or less to characterize prematurity [The identification of FGR as a distinct pathophysiological entity is merged with preterm birth history. In the first half of the 20maturity . Howevermaturity . Only inmaturity . Apart fmaturity  and thismaturity –13, due maturity –30.th centile, either by population-based or customized charts, is the most accepted definition for SGA infants [th and the 90th centiles and (ii) the agreement on the 10th centile among studies up to the 1960s [th [rd or even ≤2, and 3rd centile [th centile seems the most suitable cutoff for epidemiological and clinical purposes, and it is the adopted threshold in this review.Currently, the birthweight <10 infants . This mahe 1960s . There a960s [th , ≤3rd or centile , are conThe SGA prevalence varies according to the reference standards applied; it tends to be higher with customized curves , 12, 14.The “great obstetrical syndromes” are related to defective deep placentation , and stuAdditionally, some clinical risk factors are similar between the “great obstetrical syndromes.” Multiple pregnancies and maternal chronic conditions, such as previous hypertension, systemic lupus erythematosus, and diabetes mellitus, are all associated with them –47. Nullth percentile (compared to the 10–90th centiles) [Regarding the outcomes of SGA newborns, extensive investigation has been performed on immediate , 32, 50 entiles) . Besidesentiles) –18, 55, entiles) , 11, 14,entiles) . Interesentiles) , which tentiles) , 58 and rd centile [rd centile also have higher levels of leptin [Unfortunately, the higher risk of mortality goes beyond the neonatal period. Data from Sweden show a hazard ratio (HR) of 1.37 (95% CI 1.28–1.47) of death up to 18 years old, which increased to 2.61 95% CI 2.19–3.10) for those neonates born <3 centile . Additio–3.10 for centile , and hyp centile . If SGA  centile . Regardif leptin .in utero shows increased odds for metabolic syndrome [Evidence from adults exposed to famine syndrome  and obessyndrome  in SGA nsyndrome . Althougsyndrome , higher syndrome , and incsyndrome  in adultsyndrome .Therefore, timely recognition of SGA—still in pregnancy—is a real concern for obstetricians, perinatologists, health workers, and policymakers. Unfortunately, only a small proportion of SGA babies are suspected before birth , 57, leaClinical risk assessment is the first approach to antenatal care. A detailed maternal history at booking can identify several risk factors and guide referencing to tertiary care facilities.st century, still demonstrates effects on fetal growth [Single maternal clinical factors demonstrate poor prediction accuracy , and, asl growth , 6, 48 al growth , 14, 48 l growth . Body mal growth . The perl growth , but it l growth . Howevernd trimester, proteinuria, daily vigorous exercise, and diastolic blood pressure ≥80 mmHg, combined with the protective factors rising random glucose, recreational walking (≥4x/week), and Rhesus negative blood group, provided an AUC of 0.63 [Other maternal factors have been combined differently, evidencing how SGA syndrome can be heterogeneous in distinct settings. In a multicenter international nulliparous cohort, a family history of coronary heart disease, maternal birthweight <3000 g, infertility, college student, smoking at the 2 of 0.63 . In the  of 0.63 . In thisrd) [Adding ultrasound scan (US) parameters to maternal clinical factors improves the performance of prediction models, although not consistently , 71. Crord) , 76. Unfrd) . In Tablst trimester, decreased values of NT were associated to lesser odds for SGA , but the CRL has shown no relationship  [nd trimester, McCowan et al. [z-score <10th centile, AC z-score <10th centile, and UtA-RI ≥ 0.05. The higher the UtA-RI, the higher the OR for SGA, reaching 4.56 (95% CI 2.45 to 8.48) when 0.8–1.0. At 35–37 w, Fadigas et al. [z-score, which improved AUC from 0.81 (95% CI 0.802–0.824) to 0.98 (95% CI 0.98–0.98) for delivering an SGA infant <3rd centile in less than two weeks. In this cohort, adding mean arterial pressure and UtA-PI has not improved the prediction performance .In the 199–1.00) . In the n et al.  demonstrs et al.  have comth centile is associated with increased odds for IUGR or SGA  [th centile at 35–37 w in predicting delivery within two weeks is better for SGA <3rd  than for SGA <10th  [th centile is related to a higher risk of any adverse perinatal outcomes [rd delivering in 2 w) [rd , when compared to the 4–10th centiles  but reached a disappointing AUC of 0.54 (95% CI 0.48–0.61) for predicting adverse perinatal outcomes [nd trimester) [rd centile or suspected before birth by abnormal Doppler results. This finding partially contradicts the Pregnancy Outcome Prediction (POP) Study, which found a relative risk of 17.6 (95% CI 9.2–34.0) for delivering an SGA infant when both EFW and ACGV (between 28 and 36 w) were <10th centile [th centile was higher with universal screening for SGA <10th (57%) or <3rd centile (77%) than with clinically oriented US evaluation  [Interestingly, a single measurement is better than longitudinal follow-up , 73, 75.34–4.48) . The perI 67–71) . Althougoutcomes , it demo in 2 w) . In anotoutcomes . This isimester) . The detrd or ≤5th centile by US or MRI before delivery (48 h). However, DR of SGA ≤10th centile was superior with MRI  than the US .More recently, magnetic resonance imaging (MRI) has been explored in maternal-fetal surveillance. Carlin et al.  have demBiomarker measurements of placental functioning-related substances have had significant development in the last three decades. Many of these compounds are also involved with antenatal detection of chromosomal anomalies, or preeclampsia, such as PAPP-A, AFP, PlGF, or sFLt-1  Table 2Table 2. rd centile [nd trimester (15 w), serum levels of PAPP-A, PlGF, and insulin are significantly lower in SGA pregnancies [The AFP: PAPP-A ratio >10 at 12 w of pregnancy provided a risk ratio of 3.74 (95% CI 2.3–6.09) for SGA <3 centile . In earlgnancies , while ignancies . Conversgnancies .nd and 3rd trimesters [th or <10th centiles. For higher sFlt-1/PlGF ratios, there is better AUC for preeclampsia-associated SGA [PlGF has consistently lower levels in SGA pregnancies, in 2imesters –84, espeated SGA , 41. Theated SGA . Unfortuated SGA . Perhapsnd trimester (19–24 w), PlGF and AFP, combined with maternal factors and fetal biometry, made up an AUC of 0.96 for birth below 32 weeks in SGA newborns [After all, better accuracy was achieved by combining multiple maternal, ultrasonographic, and biochemical clinical factors. In an international cohort of nulliparous women , PlGF hanewborns .Fetal growth restriction is related to adverse outcomes in the perinatal period, childhood, and adulthood; the estimated actual burden of SGA , 25 mighrd trimester US seems the best approach for SGA prediction [Although the cost-effectiveness of short-term pregnancy-related adverse outcomes is still a matter of debate , little ediction . In nearediction , aiming ediction  effect o


To predict gait impairment in toddlers born preterm with very-low-birth-weight (VLBW), from near-term white-matter microstructure assessed with diffusion tensor imaging (DTI), using exhaustive feature selection, and cross-validation.Near-term MRI and DTI of 48 bilateral and corpus callosum regions were assessed in 66 VLBW preterm infants; at 18–22 months adjusted-age, 52/66 participants completed follow-up gait assessment of velocity, step length, step width, single-limb support and the Toddle Temporal-spatial Deviation Index (TDI). Multiple linear models with exhaustive feature selection and leave-one-out cross-validation were employed in this prospective cohort study: linear and logistic regression identified three brain regions most correlated with gait outcome.Logistic regression of near-term DTI correctly classified infants high-risk for impaired gait velocity , right and left step length , single-limb support , step width , and Toddle TDI . Linear regression of near-term brain DTI and toddler gait explained 32%–49% variance in gait temporal-spatial parameters. Traditional MRI methods did not predict gait in toddlers.Near-term brain microstructure assessed with DTI and statistical learning methods predicted gait impairment, explaining substantial variance in toddler gait. Results indicate that at near term age, analysis of a set of brain regions using statistical learning methods may offer more accurate prediction of outcome at toddler age. Infants high risk for single-limb support impairment were most accurately predicted. As a fundamental element of biped gait, single-limb support may be a sensitive marker of gait impairment, influenced by early neural correlates that are evolutionarily and developmentally conserved. For infants born preterm, early prediction of gait impairment can help guide early, more effective intervention to improve quality of life.• Accurate prediction of toddler gait from near-term brain microstructure on DTI.• Use of machine learning analysis of neonatal neuroimaging to predict gait.• Early prediction of gait impairment to guide early treatment for children born preterm. At near-term age, the infant brain is rapidly developing . Brain mDiffusion tensor imaging (DTI) is a promising neuroimaging technique that reflects white matter (WM) microstructural injury and can be used to assess early brain development. DTI reveals the amount and direction of water diffusion. In the brain, water diffusion is restricted by neural development, in particular, the presence and isotropy of cellular membranes and myelination. Thus, brain DTI can be used as a metric of brain neurodevelopment and organization . DTI quaFor preterm infants, prognosis based structural brain MRI findings have demonstrated partial success . ChildreWe previously reported on neonatal correlates of toddler gait, analyzing near-term DTI in six subcortical WM regions. We analyzed four bilateral regions and two regions of the corpus callosum (CC) which were selected based on previously reported relevance, using standard statistical techniques in the same cohort of VLBW preterm children . The curThe current study applies exhaustive feature selection and leave-one-out cross-validation of WM in 99 brain regions, including 48 bilateral regions and three regions of the CC, in order to investigate the use of linear statistical models on DTI metrics for early prognosis of toddler gait impairment. A prior study of this cohort employed similar statistical learning methods to investigate prediction of cognitive and motor neurodevelopment, as measured by the Bayley Scales of Infant Development-Third Edition . In thisParticipants born with VLBW (birth weight ≤1500 g), gestational age at birth ≤32 weeks, and no evidence of genetic disorder or congenital brain abnormalities were recruited. 102 infants treated at Lucile Packard Children’s Hospital (LPCH) NICU from 1/1/10-12/31/11 participated, representing 76% of eligible infants admitted over the 2-year period. All parents of eligible infants were approached prior to scheduled routine MRI and written informed consent was obtained for this IRB-approved prospective cohort study. 66 of 102 neonates had successful DTI scans at near-term age, collected at end of routine MRI scan, prior to discharge from the NICU.n = 42) at 18–22 months adjusted age, previously published  at LPCH. A 3-plane localizer was used and an asset calibration was prescribed to utilize parallel imaging. Sagittal T1 FLAIR image parameters were: TE = 9l ms, TR = 2200 ms, FOV = 20 cm, matrix size = 320 × 224, slice thickness 3.0 × 0.5 mm spacing, NEX = 1. T2, DWI, and DTI axial scans were prescribed using a single acquisition full-phase field of view (FOV). The axial fast spin echo T2 imaging parameters were: TE = 85 ms, TR = 2500 ms, FOV = 20 cm, matrix = 384 × 224; slice = 4.0 × 0.0 mm spacing. Axial T2 FLAIR parameters were: TE = 140 ms, TR = 9500 ms, FOV = 20 cm, slice = 4.0 × 0.0 mm, inversion time 2300 ms, fluid-attenuated inversion recovery matrix = 384 × 224. Axial DWI parameters were: TE = 88.8 ms, TR = 10000 ms, FOV = 20 cm, slice = 4.0 × 0.0 mm spacing, matrix = 128 × 128. Coronal T1 SPGR parameters were: TE = 8 ms, TR = 3 s, slice = 1.0 × 0.0 mm spacing, FOV = 24 cm, matrix = 256 × 256.Structural MRI was assessed for degree of White Matter Abnormality (WMA) and significant cerebellar abnormality. Radiological evaluation was performed by an experienced pediatric neuroradiologist (XS) and confirmed by a second (KY), both were masked to all other participant data. A form validated for near-term neuroradiological assessment  was usedWoodward et al., 2006Woodward et al., 2006Diffusion tensor imaging was calculated based on diffusion-weighted images (DWI) obtained along 25 orientations with slice thickness of 3 mm, matrix size of 128 × 128, and 90-degree flip angle on a 3T MRI  at LPCH at the end of routine MRI acquisition. A repetition of DTI sequence was successfully collected in 64 of 66 cases. Thus, in 64 subjects, a full scan was motion free. For 2 out of 66 cases, a composite image was generated by selecting the best slices out of two repetitions manually and combining them to a composite image. Infants were swaddled and fed and typically remained asleep during the scan. Sedation typically was not utilized for routine near-term MRI and was not utilized as part of the research protocol.A trained inspector selected the best DTI repetition to eliminate MRI scans with artifacts or evidence of motion. As noted above, for 2 out of the 66 cases, due to the lack of usable full repetition, a composite repetition was generated from the best image slices. Eddy current distortions were corrected by applying affine transformation. Skull stripping was performed based on B0 and trace  maps using a ROI editor, and manually rotated to align with the JHU neonatal template, which is a template based on a neonatal brain atlas integrating DTI data with co-registered anatomical MRI . Scans w2/s and FA < 0.15 were considered cerebrospinal fluid (CSF) and gray matter, respectively, and were used to obtain the mask of WM regions. WM regions were then segmented into 126 regions based on the JHU parcellation atlas, and the average FA, MD, AD, and RD values were calculated for each region. The number of regions was then narrowed to apical regions ensuring quality of registration, resulting in 48 regions of both sides in addition to the splenium and genu of the CC, and the overall CC  for linear regression, and leave-one-out cross-validated area under the curve (AUC) of the receiver operator characteristic (ROC) for the binary classification with logistic regression. Logistic regression models were also evaluated on the structural MRI for presence of WMA, cerebellar signal abnormality, cerebellar asymmetry, and intraventricular hemorrhage  on structural MRI.For each temporal-spatial gait metrics, including velocity, step length, step width, SLS, and Toddle TDI, distinct linear models were generated to examine their correlations with DTI measures. Using an exhaustive search in the feature space, multiple linear regression models were evaluated with leave-one-out cross-validation, L2 regularization, and regularization strength 1.0 to find a set of 3 regions (features) most correlated with gait metrics. Logistic regression models were evaluated with leave-one-out cross-validation, L2 regularization and regularization strength 1.0 on DTI to find a set of 3 regions that best classified high-risk infants scoring worse than one standard deviation from typically developing mean values previously reported . Best moR2 and AUC, were evaluated with leave-one-out cross-validation (LOOCV), such that for both regression and classification tasks, N distinct models (N = number of subjects) using the same set of features were evaluated leaving out the n-th subject during the determination of the model parameters. Ultimately, each model was evaluated on the left-out subject, and the N distinct results in pair with their ground truth values were used to calculate the cross-validated performance metrics. For the classification tasks (high-risk vs. low-risk), balanced sensitivity and specificity were determined by maximizing the sum of the squares of sensitivity and specificity (sensitivity2 + specificity2). Coefficients of logistic regression reported in Diffusion tensor imaging scalars were adjusted for PMA at scan and normalized to have zero mean and unit variance. To ensure model generalization and robustness, and avoid overfitting, performance measures, i.e., adjusted Near-term MRI and DTI were collected at 36.6 ± 1.8 weeks postmenstrual age in 66 children born preterm  with very-low-birth-weight (1090 ± 266 g). Follow-up gait temporal-spatial parameters were collected in 52 children at 20.2 ± 1.0 months adjusted age; all participants had complete neuroimaging and gait data sets. Gait impairment was correctly classified for: velocity with 93% sensitivity and 79% specificity ; right sClinical findings on structural MRI were also evaluated for their predictive value  using loGait temporal-spatial values were predicted using cross-validated linear regression on near-term DTI with exhaustive feature selection of three brain regions . The thrStatistical learning is an area in statistics, referring to a set of tools for modeling complex datasets. It blends parallel developments in computer science, in particular to machine learning, and has been successfully applied to numerous fields. It is also a promising method to improve prognostic accuracy and guide early treatment of preterm infants. We built supervised statistical models using exhaustive feature search applied on near-term brain microstructure assessed on DTI to predict temporal-spatial gait in preterm toddlers at 18–22 months adjusted age. Due to the multiple comparisons inherent to exhaustive search, leave-one-out cross-validation was applied to reduce over-fitting and optimize robustness of generalization. Application of exhaustive feature search with cross-validation on DTI generated relatively high predictive values, compared to standard techniques using structural MRI at near-term age.p = 0.003).Infants were classified with high sensitivity and specificity as high-risk for gait impairment based on near-term WM microstructure . Most coBoth the hippocampus and the inferior frontal gyrus contributed to several best performing logistic and linear models. The hippocampus contributed to logistic regression of 4/6 gait parameters  and is iToddler’s with the gait impairment of SLS time were most accurately predicted from near-term brain microstructure. This may be explained because as a toddler learns to walk, achieving sufficient SLS requires single limb strength and balance as well as bilateral stability and symmetry. As a fundamental element of human biped gait, SLS may be a sensitive marker of toddler gait impairment influenced by early neural correlates that are evolutionarily and developmentally conserved. Classification with the logistic function fitted on the right fusiform gyrus FA, splenium AD, and genu FA predicted right SLS with 100% sensitivity and 100% specificity; logistic function fitted on the right middle frontal gyrus RD, right superior occipital RD, and left lateral fronto-orbital gyrus FA predicted left SLS with 100% sensitivity and 100% specificity . In addiStep width was also well predicted in the present study, the linear regression with exhaustive feature search and cross-validation found that the left and right globus pallidus, along with the right tapetum, were predictive of step width, a gait metric that typically reflects development of postural balance.Coefficients of logistic regression models  reinforcWe found that prediction by the models using DTI outperformed models using structural MRI , consistWe previously reported data from the same cohort, and evaluated velocity and SLS with respect to WM and cerebellar abnormality as assessed on structural MRI, and in 6 different subcortical WM regions assessed on DTI, using standard partial correlation analyses . The MRIIn the current study, the genu and splenium of CC, as well as fusiform gyrus, superior-occipital gyrus, lateral fronto-orbital gyrus, and right middle frontal gyrus contributed to 100% accurate prediction of SLS impairment. Further, the anterior limb of the internal capsule and retrolenticular part of the internal capsule and inferior frontal gyrus also contributed to explaining approximately 15% of the variation of toddler SLS, a sensitive gait metric that reflects gait stability, weight bearing, and symmetry. These findings suggest that a set of brain regions taken together may be more sensitive to outcome than a single region in isolation.To ensure ease of interpretability we used linear models, which limit accuracy due to the highly non-linear nature of the solution space. Study limitations also include that we analyzed a relatively small cohort which requires the use of statistical tools that are less robust compared to state-of-the-art machine learning approaches , and that classification on imbalanced dataset can be biased, even when using ROC-AUC or precision and recall as performance metrics. Furthermore, evaluation of cortical WM can be confounded by imaging resolution and signal-to-noise ratio. Methods outlined in this study need to be validated on larger preterm populations.Applying machine learning algorithms on near-term regional WM microstructure may help identify risk of neurodevelopmental delay, guide early intervention and ultimately, may inform neuroprotective treatment to improve quality of life for preterm children. In this study, we employed an exhaustive feature selection algorithm to identify a set of 3 brain regions that best predicted outcomes. Results indicate a relatively high prognostic value for temporal-spatial gait parameters, in particular SLS, and warrant further investigation in larger preterm populations.The datasets generated for this study are available on request to the corresponding author.This study was approved by the Stanford University Institutional Review Board and consent was obtained from parents or guardians.KC-R, RV, JR, and KS contributed to the concept, data collection, data analysis and interpretation, and writing the manuscript. KS, KY, and DS contributed to the concept, data analysis and interpretation, and writing 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.