Multivariate logistic regression analyses were performed to determine the relationship between RDS and TSH. A total of 146 infants were enrolled. Of these, 60 had RDS, 72 had no RDS, and 14 were excluded. T3 and TSH were lower in the RDS groups (p < 0.05) on the day of birth. Multivariate logistic regression analysis indicated that lower serum TSH levels immediately after birth were associated with a higher incidence of RDS . The TSH level was associated with the incidence of RDS. This suggests that suppression of the hypothalamus–pituitary axis function contributes to RDS, which is the result of surfactant deficiency.Thyroid hormones are known to influence the production and secretion of pulmonary surfactant. The objective of this study was to explore the relationship between respiratory distress syndrome (RDS) and thyroid hormones. This was a retrospective study of preterm infants at 24–33 weeks gestational age from April 2017 to February 2019. T Respiratory distress syndrome (RDS) is a disease caused by inadequate production of pulmonary surfactant and structural immaturity of the lung . Althoug4 (fT4) with RDS in premature infants [4, triidothyronine (T3), and TSH levels were analyzed based on gestational age to assess the relationship between hormone levels and RDS.In animal studies, Warburton et al. noted that thyroid hormone promotes fetal lung maturation and has a synergistic effect with glucocorticoids . An ante infants , and Ryc infants . In 2007 infants . In thisThis study was a retrospective analysis of premature infants between 24 weeks 0 days and 33 weeks 6 days of gestational age who were born and admitted to the neonatal intensive care unit (NICU) of Wonju Severance Christian Hospital of South Korea between April 2017 and February 2019. Infants with congenital anomalies and congenital hypothyroidism, outborn infants, or those lost to follow-up were excluded. Detailed patient information was extracted from the electronic medical records for analysis. 3, fT4, and TSH are measured on cord blood on the day of birth and on venous blood 1, 3, and 6 weeks after birth. The NICU has a protocol for screening preterm infants for thyroid dysfunction. TRDS was defined as those infants with air-bronchograms on chest X-ray and respiratory distress symptoms including chest retractions, nasal flaring, and at least a 40% FiO2 requirement. The case group was those infants with RDS, whereas the control group was those without RDS. Bronchopulmonary dysplasia (BPD) was defined as the need for supplementary oxygen at 36 weeks postmenstrual age (PMA) or discharge, whichever came first. Severe BPD was defined as the need for ≥30% oxygen and/or positive pressure at 36 weeks PMA or discharge, whichever came first . Necroti3, fT4, TSH, birth weight, and gestational age were presented as the mean ± SD. Categorical variables were analyzed using the chi-squared test or Fisher’s exact test. Continuous variables were analyzed using the independent t-test, the Mann–Whitney U-test, and repeated measures of ANOVA. The p-value for statistical significance was <0.05.The Statistical Package for the Social Science  was used for data analysis. Categorical variables were presented as numbers and percentages. Continuous variables such as T3 and TSH. The 5 min APGAR score, birth weight, and gestational age were adjusted as confounding variables. The results were expressed as odds ratios and 95% confidence intervals. A p-value < 0.05 was considered to indicate statistical significance.Multivariate logistic regression was performed using stepwise selection to analyze the association between the incidence of RDS and TOf the 146 premature infants enrolled in the study, 132 were included for analysis. A total of three infants with congenital anomalies, six infants with congenital hypothyroidism, three out-born infants, and two infants who were lost to follow-up were excluded. There were 60 infants with RDS and 72 infants without RDS .p < 0.05; 1328.3 ± 503.1 vs. 1692.9 ± 320.8, p < 0.05, respectively). The 5 min APGAR score of the RDS groups was lower than that of the control groups , and there were no significant differences in the delivery mode, labor pain, gender, small for gestational age (SGA), maternal age, gestational diabetes mellitus, preeclampsia, chorioamnionitis, or exposure to antenatal steroids. When intergroup comparisons were made based on gestational age, there were no differences in delivery method, gender, multiple births, and SGA, but the 5 min APGAR score tended to be lower for the groups with lower gestational age (p < 0.0). The baseline characteristics of the two groups according to RDS are presented in 3 levels from birth to week 6. However, the T3 levels of RDS groups at every time point were significantly lower . There were no differences between the groups in fT4 levels from birth to 6 weeks. TSH levels tended to decrease in both groups following birth. The TSH levels of the RDS groups were significantly lower than that of the control groups at birth (p = 0.048), and the TSH level of the RDS groups was increased gradationally. At PNA 3 weeks, levels in the RDS groups were significantly higher than that in the control group (p = 0.002). When serum thyroid hormone levels were compared , both grp < 0.05). However, no differences were observed for pulmonary hypertension, periventricular leukomalacia, intraventricular hemorrhage, and retinopathy of prematurity requiring panretinal photocoagulation. There was no difference in mortality between the RDS and control groups (p = 0.196).3 level of the RDS groups was significantly lower than that of the control groups after multivariate logistic regression analysis controlling for gestational age and birth weight. On the day of birth, a lower TSH was significantly associated with an increased risk of RDS after adjustment for significant baseline characteristics, including gestational age, birth weight, and 5 min APGAR . At 3 we3 and fT4 levels. As a result, serum fT4 levels increase to the highest levels in life and then slowly decrease after 1 week [3 and fT4 secretion does not begin until 12 weeks of gestational age [4 is the primary hormone secreted, and the concentration of T3 remains relatively low [3 and fT4 levels were lower among infants of lower gestational ages. However, as gestational age increased, the significant difference in fT4 disappeared over time [This study confirms that serum TSH levels immediately after birth were significantly lower in infants with RDS. Immediately following birth, significant changes occur in the thyroid hormone axis. TSH secretion by the pituitary gland increases up to 70 mIU/L in response to environmental temperature changes within 30 min after birth. This phenomenon is called the TSH surge and stimulates the thyroid to increase serum Tr 1 week ,14. The onal age . Howevervely low . Shortlyver time .4 and T3, which is qualitatively similar to, but quantitatively smaller than, that of term infants [Preterm infants have a postnatal TSH surge and rise in serum T infants ,18,19,20 infants ,18,19. P4 is thought to promote the translocation of lipids into the lamellar body and increase the influx of phosphatidylcholine precursors to facilitate fetal lung maturation [3 is much higher than that of fT4 [3 level at birth in the RDS groups was lower in our study. Additionally, this study also confirmed a significant difference in the TSH level even after adjustment for other confounders, which was different from those in studies of Tanaka et al. [Previous studies have demonstrated that various hormones such as corticosteroids, thyroid hormones, TRH, prolactin, catecholamines, cAMP, r-interferon, and estrogen affect the surfactant metabolism and lung development in the fetus and newborn ,5,6. Thet of fT4 ,22,23. Ta et al.  or Tawfia et al. . These fa et al. . Tawfik a et al. . Tawfik’The hypothesis of this study is supported by the ACTOBAT study, which reported in 1995 that prenatal TRH administration could increase RDS . In 19983 and fT4 increased after birth. At 3 weeks of age, TSH appeared to be higher in the RDS groups, but there was no difference after adjustment with gestational age and birth weight. Additionally, this inexistence of difference may be related to the normalization of pituitary gland function by 3 weeks of age or the timing of discontinuation of temperature control of the incubator.In the present study, TSH tended to decrease after the initial surge following birth, whereas TThe strength of this study is that it is the first report of an association between TSH and RDS after confounding factors have been eliminated and consistency of care by a single neonatologist in a single center. However, because this is a single-center study, the small sample size is a limitation, and additional studies with a larger number of infants are warranted. 3 and fT4 were not associated with RDS. Surfactant deficiency, the cause of RDS, was not affected by thyroid hormone but was associated with suppression of pituitary gland function at birth. Based on these findings, additional research is required to identify the specific endocrinological function that is related to surfactant deficiency.In this study, lower TSH levels immediately after birth were associated with an increased risk of RDS, but T


Higher birth order is associated with increased risks of adverse health outcomes attributable to alcohol or narcotics in adolescence, but it remains unclear whether these observed birth order effects are also present in midlife. Drawing on a national Swedish cohort born in 1953 and their siblings, we estimate associations between birth order and alcohol- or narcotics-attributable hospitalization or death with a 25-year follow-up to assess whether birth order differences are observed during this life course period. Health events attributable to alcohol or narcotics use were identified using the Swedish National Patient and Cause of Death registers, respectively. We apply Cox proportional hazards models to estimate average birth order differences in hazards for alcohol- or narcotics-attributable hospitalization or death between ages 30 and 55. We estimate birth order differences between families, and use two fixed-effects approaches to estimate birth order differences within families and within families of the same type. Bivariate results indicate increased hazards for both outcomes with higher birth order; however, these results are no longer observed after adjustment for familial background characteristics in all models. Our results thereby show limited evidence for birth order differences in midlife. This study highlights that shared factors within the family of origin may be stronger predictors of adverse health outcomes attributable to substance use among siblings during this life course period. Future research should disentangle the contributions of the social environment within the family of origin for adverse health outcomes attributable to alcohol or narcotics among siblings. •We estimate birth order differences for alcohol or narcotics use outcomes between ages 30-55.•Birth order differences are not observed for alcohol- or narcotics-attributable events in midlife.•Family background characteristics may be stronger predictors of these outcomes. In particular, prior research indicates that higher birth order is associated with higher likelihood of suicide attempts , psychiaThe strong birth order effects observed during the teenage years aligns with previous scholarship on alcohol and narcotics use among siblings during this period in the life course. Evidence suggests that use of alcohol or narcotics among earlier-born siblings is predictive of use among later-borns . The assModeling of earlier-born siblings’ substance use is associated with a developmentally premature initiation of use among their later-born counterparts . In turnThe unilateral influence of earlier-born siblings’ substance use behavior on their later-born counterparts may decrease as individuals transition into adult social roles. The observed transmission of alcohol or narcotics use between earlier- and later-born siblings occurs when offspring are ostensibly living in the parental home. The transition to adulthood is typically characterized by such demographic transitions as entry into marriage or childbearing; these transitions are also associated with a decline in substance use . SourcesWe use a unique, high-quality Swedish data register to ascertain birth order differences in hospitalization or death attributable to alcohol or narcotics within a cohort of individuals born in 1953 and their siblings. All children in each family were followed from ages 30–55, from the onset of adulthood through middle age, allowing us to harmonize the follow-up data on alcohol- and narcotics-attributable hospitalization or death for 25 years. Building on previous research that found strong birth order effects before reaching young adulthood, we estimate the extent to which birth order is associated with alcohol- or narcotics-attributable events in midlife to discern whether birth order differences for these outcomes are observed during this discrete life course period.22.1The data were derived from the RELINK53 data material. Created in 2017/2018, RELINK53 is comprised of a birth cohort of individuals born in 1953 and living in Sweden in 1960, 1965, and/or 1968. The register also includes the index individuals’ antecedent, contemporaneous, and descendant family members, the linkages for which were obtained from the Swedish Multigenerational Register . The Sto2.2The study population consisted of the index individuals  from the 1953 birth cohort and their linked biological siblings , comprising 107,462 unique families. Linkages to the egos' siblings were ascertained via the biological mother's assigned identification number. Each mother's full fertility history was included to determine the size of each respective sibling group of which the ego is a part; however, siblings' inclusion in RELINK53 was conditional upon their survival until 1960 .The analytical sample was first restricted to families with two or more children, all of whom were born between 1943 and 1960 . We subsequently excluded families of more than five children  as such large families are relatively uncommon in Sweden; these families comprised only 5.5% of the total sample. We further excluded families with monozygotic or dizygotic twins  due to the lack of clarity with which birth order could be assigned. Finally, we excluded individuals who died or emigrated from Sweden prior to the start of the observation period (n = 6020). These criteria yielded a final analytical sample of 57,575 families .2.3Hospitalization and death attributable to use of alcohol or narcotics were identified using records from the National Patient and Cause of Death registers, respectively. The 25-year measurement period started in 1973 for the oldest cohort and in 1990 for the youngest. The hospitalization variables were each operationalized as an inpatient event with an underlying or secondary diagnosis attributable to use of alcohol or narcotics, respectively, and included the following codes from the tenth revision of the International Classification of Diseases (ICD-10): E244; F10–16; F18–19; G312; G621; G721; I426; K292; K70; K852; K860; O354; O355; T40; T423–424; T426; T436; T51; Y90–91; Z502–3; Z714–15; Z721–22 . An exteThe cause-specific mortality variables were each operationalized as a primary or underlying cause of death attributable to use of alcohol or narcotics, respectively, in accordance with the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) guidelines. The measures included the above ICD-10 codes, as well as the external cause codes for alcohol  and narcotics  .Conversion tables available from the Swedish National Board of Health and Welfare were used to translate codes between the ICD-10 and the eighth , which is expected as narcotics use is subject to period effects  is used for the entire analytic population, the 17-year difference between the oldest (1943) and youngest (1960) birth cohorts could introduce unmeasured cohort effects into the analysis. We therefore estimated birth order differences between families using only the index cohort sample  to minimize cohort effects; the results are presented in Finally, some misclassification of sibling group size, and therefore birth order, could have resulted from the reduced coverage of deceased individuals in the Multigenerational Register between 1968 and 1991 . We cond4Prior research on birth order indicates that later-born children have a higher risk for adverse mental and behavioral health outcomes, relative to their earlier-born siblings, before reaching young adulthood. This study adds a unique contribution to the literature by using high-quality Swedish register data over a 25-year period to estimate the effects of birth order between and within families on health outcomes attributable to use of alcohol or narcotics during midlife, a discrete age period for which birth order effects have not been estimated.Here, and in other birth order studies, the aim was to estimate the direct effects of birth order, or the variability in alcohol- or narcotics-attributable events by birth order. In comparisons between families, our unadjusted results suggest a positive relationship between birth order and alcohol- and narcotics-attributable events, wherein we generally observe increasing hazards with increasing birth order. However, the direct effects of birth order were reduced when holding familial background factors constant. The results from both fixed effects approaches show similarly higher hazards among higher birth order individuals, relative to first-borns, but the 95% CIs largely overlap between higher-order births when adjusting for time-invariant family and family type characteristics, respectively. Our findings therefore suggest that, for these outcomes, the previously observed birth order differences prior to young adulthood may be less evident in later life course stages, and differences between siblings in midlife can, to a large degree, be explained by other variables within the family of origin.Aligning with a prior study that uses contemporary Swedish register data and family fixed effects to estimate birth order effects on these outcomes , we explOur results highlight the importance of considering the social environment in a given period in the life course. Social influence is one theoretical mechanism attributed to birth order differences in the uptake and continuity of alcohol or narcotics use within sibling groups . Our folFinally, these results underscore the potentially dynamic influence of familial characteristics on alcohol and narcotics use across the life course. Though direct effects of birth order may be more evident at younger ages, our results suggest that familial background factors beyond that of birth order may contribute to adverse health outcomes among siblings, which may include both environmental and genetic factors. Substantial evidence shows that there are significant environmental and genetic influences on the development and transmission of substance use disorders , 2021. F4.1This study uses unique data material to estimate associations between birth order and hospitalization or death attributable to use of alcohol or narcotics for a national Swedish birth cohort and their siblings for 25 years during the same stage of the life course (ages 30 to 55). This study is based on high-quality data and we use appropriate methods to address our research aim; however, it is not without limitations.The limited evidence of direct birth order effects on the outcomes could partly be a result of the observation period under study. It is possible that direct birth order effects could have been observed for this population had we been able to measure hospitalizations occurring before age 30 for all siblings and subsequently compare observed birth order differences between this age period and midlife. However, rather than the acute intoxication often reflected in adolescent diagnoses, our measure of first hospitalization or death likely captures substance use-related disorders and diseases that mainly affect adults. As morbidity and mortality attributable to substance use are increasing among middle and older aged individuals , disentaThe higher hazards generally observed among individuals of higher birth order may partly reflect the sample. As birth order was based on the mother's full fertility history, sibling groups may include children of differing paternity . The commonality of familial complexity in Sweden has been increasing since the 1950s , and priDespite the high validity of diagnoses in the National Patient Register , it is pThe generalizability of our findings to contemporaneous Swedish cohorts, or cohorts in other contexts, may be limited by the data material. There are 17 birth years represented among the sibling cohorts, which could result in unmeasured cohort effects. However, restricting these samples to include families where all children were born between 1943 and 1960, and thereby allowing for the same age at entry and exit, aims to mitigate this issue. Further, given our sensitivity analysis using the single birth cohort yielded similar patterns by birth order, the variation in risk for these outcomes is unlikely to be purely the result of cohort effects. Finally, distinctive age, period, and cohort trends have been observed regarding hospitalization or death due to use of alcohol and narcotics in Sweden during our observation period , which m4.2In summary, our study finds limited evidence for the direct effects of birth order for both outcomes in midlife. These results further support the idea that the risks for adverse mental and behavioral health events are dynamic, and familial characteristics may differentially influence these outcomes during different life course stages. Future research should further disentangle the time-varying contributions of background variables in the family of origin, including family instability and complexity, for birth order differences in alcohol- and narcotics-attributable events among siblings during discrete periods in the life course.Contributors: LB and KB planned and designed the study. KB advised on methodology. LB analyzed the data, and LB and KB interpreted the results. LB drafted the original manuscript; LB and KB reviewed and revised the manuscript, and approved the final manuscript for submission. LB attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted.None.


This study investigates the impact of antenatal and postnatal infection or inflammation on the onset and progression of Retinopathy of Prematurity (ROP). We retrospectively collected clinical and demographic data of preterm infants with birth weight ≤ 1500 g or gestational age < 30 weeks admitted to the neonatal intensive care unit of Verona from 2015 to 2019. Uni- and multivariable analysis was performed to evaluate the potential effect of selected variables on the occurrence of any stage ROP and its progression to severe ROP, defined as ROP requiring treatment. Two hundred and eighty neonates were enrolled and 60 of them developed ROP (21.4%). Oxygen need for 28 days and late-onset sepsis (LOS) increased the risk of any grade ROP after adjusting for birth weight and gestational age . Days of mechanical ventilation and of non-invasive ventilation increased the risk of progression to severe ROP after adjusting for birth weight and gestational age . Exposure to infection with production of inflammatory mediators may contribute to increase the risk of ROP occurrence in very preterm neonates. Following the increased survival of more preterm neonates, due to advances in neonatal care, ROP continues to be one of the primary causes of potentially treatable lifelong visual impairment and blindness in childhood, in both high and middle-income countries2.Routine screening program based on birth weight and gestational age allows timely diagnosis of neonates and prompt treatment in those with sight-threatening ROP, thus limiting ROP-related morbidity4.Recently, predictive algorithms that take into account postnatal risk factors have been proposed to individualize risk prediction and allow better risk stratification. These algorithms early identify infants that will eventually develop a severe form of ROP, far in advance compared to appearance of retinal lesions. Although further validation is needed before incorporation in clinical practice, predictive algorithms have the potential to reduce both the number of neonates undergoing screening and the number of missed or late diagnosis5.ROP pathogenesis is divided into two sequential phases. In the first phase, the immature retina is exposed to higher levels of oxygen (relative hyperoxia). The latter causes an arrest of retinal vascularization. In the second phase, the undeveloped retinal blood vessels are unable to provide an adequate oxygen supply for the increased metabolic demand of the more mature retina, leading to an upregulation of retinal growth factors (including VEGF) and an aberrant neo-vascularization. This may ultimately lead to retinal detachment and permanent vision loss2. Recent studies suggest a potential role of both prenatal and postnatal infection, however both the occurrence of ROP and its progression from the first to the second phase has not been completely clarified6. In fact, infection could trigger the production of inflammatory markers  that contribute to both arrest of vascularization in the first phase of ROP and aberrant neo-vascularization in the second phase7.Although risk factors for ROP, such as low gestational age, low birth weight and oxygen requirements, are well known, the precise mechanisms leading to occurrence of ROP have not been fully understoodThough these initial findings are promising, a better understanding of the role of infection in the ROP pathogenesis is needed. The latter would eventually allow to better individualize risk prediction on the basis of prenatal and postnatal infections and to identify potentially preventable risk factors. Finally, identification of the specific molecular pathways involved in ROP pathogenesis may help the future development of new treatments.Aim of this study was to investigate the impact of antenatal and postnatal infections on the onset and progression of ROP in a cohort of very preterm neonates.This was an observational retrospective study carried out at the level three Neonatal Intensive Care Unit (NICU) of Verona , from January 2015 to December 2019. Ethics approval was obtained from our local research ethics committee (“Comitato etico per la Sperimentazione Clinica delle Province di Verona e Rovigo”). The parents or guardians of babies enrolled gave their written informed consent. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All infants admitted within the study period who met the eligibility criteria were enrolled.8. Exclusion criteria were: death or transfer before ROP screening, major congenital malformations, chromosomal disorders, inherited metabolic diseases.No formal sample size calculation was done. Inclusion criteria were the ROP screening criteria according to the American Academy of Pediatrics recommendation: birth weight ≤ 1500 g or gestational age ≤ 30 weeks8. Retinal screening examinations were performed after pupillary dilation with 2.5% phenylephrine galenic eye drops by using binocular indirect ophthalmoscopy. The international classification of retinopathy of prematurity revisited (ICROP3) was used for disease staging9. Timing of first eye examination was based on gestational age at birth and follow-up examinations were based on retinal findings, following the AAP recommendation. All patients were followed up until completion of vascularization, even after discharge from the NICU. Infants with stage 1 ROP or incomplete vascularization were evaluated every 2 weeks. Patients with stage 2 or higher were evaluated weekly. In the presence of plus sign, the newborns were evaluated several times a week. The RetCam Imaging System III  was used in any grade ROP to confirm diagnosis, monitor disease and treatment, evaluate retinal neovascularization and to take a fundus photo.ROP screening was performed by two board certified consultant ophthalmologists , according to the recommendation of the American Academy of Pediatrics (AAP)10.All diagnosed ROPs were included as the disease progresses in stages, except for aggressive ROP, which does not follow the classic pattern of progression. It was further distinguished between severe ROP (or Type 1 ROP) and Type 2 ROP. The former includes ROP in zone I of any stage with plus sign, stage 3 with and without plus sign and zone II stage 2 or 3 with plus sign. The second includes ROP zone I stage 1 or 2 without plus and zone II stage 3 without plus. The above-mentioned distinction is pivotal, as it allows to distinguish between Type 1 ROP or severe ROP, that requires treatment, and Type 2 ROP, that requires only monitoring, until completion of the vascularization. This distinction is also one of gravity, as Type 2 ROP typically has minor consequences11, necrotizing enterocolitis (NEC), according to Bell’s diagnostic criteria and need of surgical intervention12, intraventricular hemorrhage (IVH), according to Papile’s classification13, death before discharge. For each infant, we reported the presence of ROP, stage and treatment, if any.Clinical and demographic data were collected from electronic medical records. The following data were collected: antenatal steroid use, antenatal antibiotics administration, intrauterine growth restriction (IUGR), preterm premature rupture of membranes (PPROM), clinical and histological chorioamnionitis, maternal C-reactive protein (CRP), maternal rectal and vaginal swab, gestational age at birth, birth weight, length and head circumference, sex, Apgar score at 5 min, mode of delivery, single or multiple birth, admission blood culture, surface  swabs at admission, number of surfactant doses, duration of non-invasive respiratory support and mechanical ventilation (days); duration of parenteral nutrition (days), number of red blood cell (RBC), fresh frozen plasma (FFP) and platelet (PLT) transfusions, occurrence of early-onset sepsis (EOS), late-onset sepsis (LOS), patent ductus arteriosus (PDA) requiring medical and/or surgical treatment, bronchopulmonary dysplasia (BPD), defined by oxygen need for 28 days and an assessment of respiratory support at 36 weeks’ postmenstrual age3), maternal tachycardia (heart rate > 100 beats/min), fetal tachycardia (heart rate > 160 beats/min), uterine tenderness and foul-smelling vaginal discharge14. EOS and LOS were defined when a bacterial pathogen was isolated from a blood and/or cerebrospinal fluid culture before and after the first three days of life, respectively15. If a Coagulase negative staphylococcus (CONS) was detected, sepsis diagnosis was confirmed if there was CONS growth in two consecutive blood cultures taken within two days or with CONS growth in a single blood culture along with CRP elevation16.Clinical chorioamnionitis was defined according to the conventional Gibbs criteria: the presence of fever (≥ 37.8 °C) and more than two of the following: maternal leucocytosis , median (interquartile range (IQR)) or count (%) for parametric, non-parametric and categorical variables respectively. To compare the two groups of neonates independent-sample t-test, Mann–Whitney U test and χPotential independent effect variables on the occurrence of any grade of ROP and of ROP progression to severe ROP or Type 1 ROP were selected by univariable analyses. Variables that were highly correlated with others (variance inflation factors or VIF above 5) and those with no predictive value  were removed from the final model. A multivariable stepwise logistic regression analysis was performed to evaluate the potential independent effect of the selected variables on the occurrence of any stage ROP and of severe ROP. Effect estimates were expressed as odds ratio (OR) and 95% confidence interval (95% CI). Data were analysed with IBM SPSS 23 . P value < 0.05 was considered significant.During the study period 321 neonates with birth weight ≤ 1500 g and/or gestational age ≤ 30 weeks were admitted to our NICU. Forty-one neonates were excluded due to major congenital malformations/chromosomal abnormalities (n = 6) and death or transfer before ROP screening (n = 35). A total of 280 neonates were studied . Stage of ROP at diagnosis and at discharge according to ICROP-3 is reported in Table .4%. StagAmong neonates with stage 3 ROP (n = 15), 6 regressed to lower stages until healing and completion of vascularization, 9 were treated with laser photocoagulation after the onset of plus sign. Infants with stage 4 ROP (n = 3) were treated with pars plana vitrectomy with endo-laser photocoagulation.Clinical characteristics of the two cohorts of neonates with and without ROP are presented in Table 2(4) = 112.948, p < 0.05. The area under the ROC curve (AUC) showed an excellent level of discrimination . Predictor variables are reported in Table Univariable analysis identified the following potential predictors of any grade ROP: gestational age, birth weight, oxygen need for 28 days, PDA, late onset sepsis, clinical chorioamnionitis, PLT, FFP and RBC transfusions. Four variables were included in the final model: birth weight, gestational age, LOS and oxygen need for 28 days. The other variables were excluded due to collinearity (VIF > 5). The logistic regression model was statistically significant, χ2 = 112.94When evaluating the predictor variables of severe ROP among neonates with any grade ROP, we found that days of mechanical ventilation and of non-invasive ventilation increased the risk of severe ROP , after adjusting for birth weight and gestational age. Clinical chorioamnionitis was not associated with increased risk of severe ROP .In the current study, we evaluated the association between ROP and maternal and neonatal infection or inflammation in a cohort of very preterm neonates.17.In our cohort of very preterm neonates ROP prevalence was 21.4%, similar to what has previously been reported19. On the contrary, Cantey et al. in a retrospective cohort of 2242 neonates found that LOS caused only by CoNS and not by all pathogens was not associated with increased risk of any stage ROP 20.In our population we found that LOS increased more than twice the risk of developing any stage ROP. The latter is in agreement with previous studies. Reyes et al. in a retrospective cohort of 171 neonates and Chen et al. in a case–control study of 622 neonates found an OR between late-onset sepsis and any stage ROP of 7.47 (95% CI 1.21–46.04) and of 2.1 (1.4–3.2), respectively21. In conclusion, our finding that LOS increased the risk of ROP development supports prior evidence, although the underlying physiological mechanisms are yet to be clarified. We speculate that inflammatory factors triggered by postnatal sepsis, such as growth factors and cytokines , may modulate retinal angiogenesis and alter vessels development22.In a recent meta-analysis by Huang et al. aiming to evaluate the association between sepsis and ROP, the pooled OR of the association between any stage ROP and LOS was 1.81 (95% CI 0.74–4.43); however, among 34 included studies, only three discriminated between EOS and LOS24.However, we did not find any association between any grade ROP and EOS or other risk factors of intrauterine infection , as previously reported25.It has been proposed that prenatal inflammatory exposure has a limited impact on ROP development, because it occurs in an early phase of retinal development, prior to the first phase of ROP pathogenesis26. However, there may be an association between prenatal infection or inflammation and ROP that was not captured by the risk factors we examined.Our data seem to support this theory. A recent meta-analysis by Villamor-Martinez et al. found that chorioamnionitis did not increase the risk of severe ROP, after controlling for gestational age. Part of the effects of chorioamnionitis on the development of ROP may be mediated by its role as an etiological factor for very preterm birth27.In conclusion, multiple exposures to infection or inflammation in postnatal life may contribute to gradually increase the risk of ROP development (multi-hit hypothesis)The important impact of LOS on ROP development underlines the importance of developing strategies to prevent nosocomial infections in very preterm babies during their NICU stay. Moreover, we feel that, if an episode of LOS occurs, neonates should be screened more frequently, due to the increased risk of ROP development, although more prospective data are needed before this may be universally recommended in clinical practice.In the near future, individualizing risk prediction may allow better risk stratification and more sustainable screening program. According to our findings, LOS, that so far has not been included in any algorithm to predict ROP risk, should be further investigated. Larger datasets and further validation are needed before incorporating LOS in ROP score predictive algorithms.The main limitation of the current study is related to its observational design: although we confirmed an association between LOS and ROP, causality should be still considered uncertain. Moreover, sample size was relatively small. On the other hand, our cohort was homogenous. In fact, during the study period, institutional protocols of stabilization and management of VLBW infants, ventilation and oxygen target saturations did not change.Another limitation was the lack of severe ROP cases, that reduces the reliability of ROP progression prediction. Further data are needed to confirm the role of infection or inflammation in ROP progression and the multi-hit hypothesis.Finally, in the current study ROP assessment was based only on disease stage without considering presence and location of the plus disease and zone, to indicate the degree of retinal vessel immaturity. In a future study, we would like to explore if infection or inflammation leads to different subtypes of severe ROP in a larger cohort of patients.Supplementary Information.


The neonatal mortality rate in South Africa is lower than the global average, but still approximately five times higher than some European and Scandinavian countries. Prematurity, and its complications, is the main cause (35%) of neonatal deaths.To review the maternal, delivery period and infant characteristics in relation to mortality in very low birth weight (VLBW) infants at Charlotte Maxeke Johannesburg Academic Hospital (CMJAH).This was a retrospective descriptive study of VLBW infants admitted to CMJAH between 1 January 2017 and 31 December 2018. All infants with a birth weight between 500 to ≤ 1,500 grams were included. The characteristics and survival of these infants were described using univariate analysis.Overall survival was 66.5%. Provision of antenatal steroids, antenatal care, Cesarean section, female sex, resuscitation at birth, and 5-min Apgar score more than five was related with better survival to discharge. Among respiratory diagnoses, 82.8% were diagnosed with RDS, 70.8% received surfactant therapy and 90.7% received non-invasive respiratory support after resuscitation. At discharge, 59.5% of the mothers were breastfeeding and 30.8% spent time in kangaroo mother care.The two-thirds survival rate of VLBW infants is similar to those in other developing countries but still remains lower than developed countries. This may be improved with better antenatal care attendance, coverage of antenatal steroids, temperature control after birth, improving infection prevention and control practices, breastfeeding rates and kangaroo mother care. The survival rate was lowest amongst extremely low birth weight (ELBW) infants. In South Africa, the under-5 mortality rate in 2017 was 37.1 per 1,000 live births  of neonatal deaths according to estimates developed by the United Nations Inter-agency Group for Child Mortality Estimation in their report from 2019. The second and third largest reasons contributing to neonatal mortality are intrapartum-related complications (24%) and sepsis (15%) % of neon.The complications of preterm birth are many, due to immaturity of multiple organ systems . Some ofThis study aimed to review the maternal, delivery period and infant characteristics in relation to mortality in VLBW infants at the CMJAH. The acquired information will be useful to improve the neonatal care and neonatal survival at CMJAH.This was a retrospective observational study of VLBW infants admitted to CMJAH. All infants with birth weight between 500 to ≤ 1,500 grams, born between 1 January 2017 and 31 December 2018 and admitted to the neonatal unit at CMJAH were included. Infants with incomplete data for birth weight, place of birth and date of outcome were excluded.Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) is a large tertiary referral hospital within the public hospital framework in South Africa. South Africa is considered a developing country and has limited health resources and high patient numbers. In this setting it is not possible to provide full support to every very low birth weight (VLBW) infant due to necessity of rationing care. Therefore, intermittent positive pressure ventilation (IPPV) after the resuscitation period is not routinely provided to infants who weigh <800 grams in this institution based on anticipated poor outcome, prolonged ventilation, and high use of resources.At CMJAH there is a quality improvement project called PRINCE—the Project to Improve Neonatal Care—with the purpose to develop targeted interventions to improve the neonatal care and clinical outcome of infants at CMJAH. PRINCE has been accredited as a research programme within the Faculty of Health Science at the University of Witwatersrand.Data was managed using the Research Electronic Data Capture (REDCap) computer database, hosted by the University of Witwatersrand , 9. The Variables were selected from amongst those available from the database and considered as of possible importance for preterm delivery and the short-term outcome. The studied maternal characteristics were: maternal age, multiple gestation, parity, maternal HIV status, maternal syphilis, antenatal care, antenatal steroids. The studied characteristics for the delivery period were: place of birth (inborn or outborn), mode of delivery , 5-min Apgar score. The studied infant characteristics were: gestational age, birth weight, head circumference at birth, sex, birth HIV PCR, initial resuscitation in the delivery room, and hypothermia.Factors related to hospital stay and neonatal complications that were studied included age on admission, age at outcome, respiratory diagnosis , surfactant therapy at any time, respiratory support after initial resuscitation (non-invasive or invasive), any respiratory support after 36 weeks, steroids for BPD, screening for ROP, ROP stage 3 or 4, IVH grade 3 or 4, PDA, NEC grade 2 or 3, early-onset sepsis, late-onset sepsis, surgery for NEC, other surgery, blood transfusion, neonatal jaundice requiring phototherapy, congenital anomalies, Kangaroo Mother Care (KMC), and breastfed at discharge.VLBW infants were defined as birth weight ≤ 1,500 grams. Extremely low birth weight (ELBW) infants were defined as birth weight ≤ 1,000. Short-term outcome was defined as death or survival to discharge. For diseases, standard definitions as per the Vermont Oxford Network were used . Sepsis Attendance at antenatal care constituted at least one antenatal visit during pregnancy. Place of birth was divided into inborn (infants born within CMJAH) and outborn, including infants born before arrival, at midwife obstetric units or born at other hospitals and referred to CMJAH. Mode of delivery was divided into vaginal delivery (including vertex and breech presentation) and Cesarean section (including elective and emergency cases). Gestational age at birth was decided from the best obstetric estimation available . If none of the above was available, gestational age was estimated using the Ballard Score .Hypothermia was defined as body temperature <36.5 degrees Celsius, measured within 1 h of admission to the neonatal unit. Respiratory support after initial resuscitation was divided into non-invasive and invasive ventilation. Non-invasive respiratory support included nasal-prong oxygen, nasal continuous positive airway pressure (NCPAP) and high flow nasal cannula oxygen, whereas invasive respiratory support included conventional mechanical ventilation and high frequency ventilation. Similarly, respiratory support at 36 weeks was used to determine severity of BPD, based on the level of support of the earlier mentioned alternatives. During the study period, there was a weight cut off for invasive ventilation at 800 grams and for NCPAP at 750 grams. ELBW who did not qualify for invasive or non-invasive ventilation due to their birth weight, were offered surfactant then placed on nasal prong oxygen.Mothers were screened for HIV at antenatal care visits and again at delivery. Babies born to HIV-positive mothers had an HIV PCR test performed at birth and were put on to HIV prophylaxis dependent on their level of exposure risk. The requirements for surfactant therapy were preterm infants who were hemodynamically stable, with changes suggestive of RDS on chest X-ray, who had respiratory distress and who required fraction of inspired oxygen ≥40% oxygen to keep oxygen saturations >89%. Cranial ultrasound was performed within the first seven days of life and repeated at 10–14 days of life and then again prior to discharge.All babies with a birth weight ≤ 1,500 grams or gestational age at birth below 32 weeks, were screened for ROP, at 4 to 6 weeks chronological age. Breastfed on discharge included breastmilk only, fortified breastmilk or breastmilk and formula together. KMC included both intermittent and continuous. KMC was introduced once a baby weighed over 1,200 grams, tolerated full enteral feeds, had an adequate weight gain (>15 g/kg/day), was off supplemental oxygen and could maintain temperature and glucose levels. Survival as outcome included discharged to home or transferred to another hospital. Babies were discharged home once they had achieved a weight of 1,600 grams, established enteral feds, were off supplemental oxygen, maintained temperature and glucose levels.t-tests were used comparing normally distributed continuous variables and Mann-Whitney U tests for skewed distribution. Chi-Square tests were used to compare categorical variables. A p-value of <0.05 was considered significant. Only valid cases were analyzed for each variable . Thereafter a multiple logistic regression model with mortality as the binary outcome variable was performed. Variables that were (1) significantly associated with mortality in univariate analysis, (2) had a sufficient number of valid cases, (3) passed the assumption of linearity using Box-Tidwell procedure, (4) were not transformed as part of the initial univariate analysis, and (5) were appropriate were included in the model.Statistical analysis was performed using IBM SPSS Statistics version 25. Frequencies and percentages were used to describe categorical variables. Continuous variables were described using mean and standard deviation if they were normally distributed, and median and interquartile ranges (IQR) if they were skewed. Univariate analysis was performed to determine significant associations of various factors with survival at discharge. Unpaired Ethical approval for the study was obtained from the Human Research Ethics Committee of the University of the Witwatersrand (clearance certificate number M190874). As this was a retrospective audit of an existing database, informed consent was waived. All methods were carried out in accordance with relevant guidelines and regulations.There were 946 VLBW infants admitted during the study period. Eight infants were excluded , 938 VLBW infants were included in the study. The overall survival rate was 66.5% (624/938). The mean birth weight was 1,093.6 grams (SD: ± 249.9), mean gestational age at birth was 28.9 weeks (SD: ± 2.8) and median age at outcome was 27.0 days (IQR: 38).The mean maternal age was 28.8 years (SD: ± 6.4) and 28.5% (222/780) were primiparous. There were 16.6% (152/913) multiple gestation. Risk factors with significant result for mortality related to antenatal care, labor and delivery are presented in Among the mothers 29.6% (275/929) were HIV positive and 1.7% (16/868) positive for syphilis. A total of 78.6% (737/938) infants were inborn. Temperature measured within 1 h of admission showed 60.8% (463/762) of the infants had hypothermia. These results were not significant in relation to mortality.The birthweight, gestational age and head circumference of survivors were significantly greater compared with the infants who died see . The 5-mp < 0.001) compared to the group of extremely low birth weight infants (ELBW) at 35.1% (110/313).Survival by birth weight category is shown in Treatments and complications during the neonatal period and hospital stay with statistically significant result for mortality are shown in There were 90.7% (851/938) of infants who received non-invasive respiratory support after initial resuscitation. Under respiratory diagnosis, 82.8% (777/895) of the infants were diagnosed with RDS and 0.5% (5/935) babies with congenital pneumonia. These results were not significant in relation to mortality. Other respiratory diagnoses which were significant in relation to mortality are included in PDA was reported in 9.2% (85/927) of cases. Among the infants with sepsis there were 4.5% (42/902) with early-onset sepsis and 32% (298/930) with late-onset sepsis. 43.2% (399/924) of the infants received blood transfusion. These results were not significant in relation to mortality.Screening for ROP occurred in 30.1% (273/906) of the infants and among these 2.9% (8/273) of infants had ROP stage 3 or 4. Breastfeeding at discharge occurred in 59.5% (322/541) of the infants and 30.8% (283/920) spent time in KMC.There were 22 significant variables on univariate analysis. Six variables  were excluded as there were not enough valid cases for both outcomes to include in the logistic regression model. The three continuous variables  all failed the Box-Tidwell procedure for linearity and were thus excluded from the model. 5-min Apgar score was presented as both an ordinal variable and a transformed categorical variable and was excluded as neither were appropriate for this model. NEC surgery was excluded as it is dependent on NEC 2 or 3, to avoid circular reasoning.Of the 10 predictor variables included , seven were statistically significant as shown in This retrospective review showed an overall two-thirds survival of VLBW infants. This is decreased when compared with the results from previous studies at the same unit where the overall survival of VLBW infants in 2006/2007 was 70.5% and in 2013 was 73.4% , 13. TheThe survival of ELBW in this study is comparable to that found in the 2006/2007 study , but lowA possible reason for the lower survival rate could be the increase in late-onset sepsis in this study. In 2013, 19% of the VLBW infants at CMJAH had late-onset sepsis, whereas in this study there was an incidence of 32% . This maIn this study 78.6% of the infants were inborn which could be compared with 81.5% in 2006/2007 and 84.3% in 2013 , 13. TheWhen it comes to antenatal steroids, the coverage in this study had improved slightly (from 39.1 to 48%) compared to 2013 . HoweverDespite the modest improvement in antenatal steroid coverage, the rates of NEC (12.3% vs. 7.3%) and IVH (11.5% vs. 7.9%) in this study has increased compared to 2013 . Earlier studies done at the same department had not stated their definition of hypothermia, so no comparison could be made. However, this is interpreted as a large number and needs to be improved. Hypothermia increases morbidity (of which RDS is one) and mortality . This maSeveral diseases showed significant association with mortality, such as pulmonary hemorrhage, congenital anomalies, IVH and NEC. This indicates the severity of these conditions. In addition, surgery for NEC and other surgery were also significantly associated with mortality, although that may be related to the severity of the condition rather than the surgery itself. Predictably, infants with congenital anomalies had higher mortality. It is unit policy not to offer invasive ventilation to infants with congenital anomalies with poor prognosis .Taking logistic regression into consideration, receiving any respiratory support during initial resuscitation, surfactant at any time, as well as invasive ventilation after initial resuscitation was associated with higher odds of mortality. The high use of surfactant replacement therapy found in this study is concerning. During the study period, delays were frequently encountered when instituting NCPAP due to equipment shortages. This may have caused infants to deteriorate sufficiently to necessitate surfactant replacement therapy. Efforts have been made to address the delays and improve the use of NCPAP, thereby decreasing the use of surfactant therapy.Several improvements can be made in the neonatal care prior to discharge. For instance, screening for ROP is supposed to be done on all VLBW infants and was only performed in one third of them prior to discharge in this study. This is still an improvement compared to 2006/2007 when 18.3% of the infants were screened for ROP prior discharge . Since tIn recent years, efforts have been made to increase the number of breastfed infants at discharge, considering the well-documented benefits of breastfeeding and its affordability. To this end, several lactation consultants have been employed. As a result, the number of breastfed infants at discharge has nearly doubled from 2013 (30.5%) to the current rate in this study, which is commendable .On the other hand, KMC has decreased in this study compared with 2006/2007 (30.8% vs. 44.5%) . The limWe acknowledge several limitations to this study. This was a retrospective analysis of an existing database, so some information was missing. Additionally, certain data such as usage of parental nutrition and antibiotics usage, is not routinely collected in the database and were therefore not analyzed. Our study favored maternal dates and Ballard score to determine gestational age as access to early ultrasound is poor. The short-term outcome of survival to discharge included infants discharged home and to other hospitals. We did not look at categorizing the timing of when deaths took place which would be useful in identifying trends. Importantly, this is not a population-based study. This study was conducted in a referral hospital with access to the appropriate level of care, as per national guidelines.This study showed a two thirds survival rate in VLBW infants, which is similar to those in other developing countries (like India and Iran) but still remain lower than developed countries. A large number of patients are placing a burden on a hospital with limited resources. The survival rate can be improved by increasing antenatal care attendance, coverage of antenatal steroids, improving temperature control to decrease the high rates of hypothermia, improving infection prevention and control practices, breastfeeding rates and the use of KMC. Improved screening for morbidities such as ROP and IVH should also be emphasized.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by University of the Witwatersrand Human Research Ethics Committee. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.KI conceptualized and designed the study, collected data, carried out data analysis, drafted the initial manuscript, revised the manuscript, and approved the final manuscript. AE, EH, and RS participated in protocol development, supervised the study, reviewed and revised the manuscript, and approved the final manuscript. DB supervised the study, revised the manuscript, and approved the final manuscript. All authors contributed to the article and approved the submitted version.This study was financially supported by Stena A. Olssons Stiftelse för Forskning och Kultur and Adlerbertska Scholarship Foundation with their scholarships. Further, this work was supported by grants from the Swedish state under the agreement between the Swedish government and the county councils, the ALF agreement (ALFGBG- 117661).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Maternal childhood trauma (MCT) is an important factor affecting offspring size at birth. Whether the effect of MCT persists during the subsequent development remains unclear. We present the results of a semi-longitudinal investigation examining the physical growth of infants born to mothers with high (HCT) and low (LCT) childhood trauma during the first year of life. One hundred healthy mother-infant dyads were included based on following criteria: exclusive breastfeeding, birth on term with appropriate weight for gestational age. MCT was assessed using the Early Life Stress Questionnaire. The weight, length, and head circumference of the infant were taken at birth, 5 and 12 months postpartum. Separate MANCOVA models were run for infant size at each age. We found an association between MCT and infant size at 5 and 12 months. The children of mothers with HCT had higher weight and greater head circumference than the children of mothers with LCT. These results suggest that MCT might contribute to developmental programming of offspring growth during the first year of life. From an evolutionary perspective, the larger size of HCT mother's offspring might represent an adaptation to potentially harsh environmental conditions. This effect might be mediated by epigenetic changes to DNA and altered breast milk composition. This period is characterized by increased vulnerability and sensitivity to environmental disruptors that, if persistent, can disrupt prenatal development and result in fetal growth retardation4. One of such factors that influence pre- and postnatal development is maternal stress5.The prenatal period is a crucial stage of individual development with long-term consequences for subsequent well-being and health6. For example, the placental enzyme 11β-hydroxysteroid dehydrogenase, which converts cortisol to its less physiologically active form cortisone8, only does so to a certain extent; therefore, maternal and fetal cortisol levels during high stress periods correlate with each other8. The observed effects of maternal stress on the development of offspring include increased reactivity of the hypothalamus–pituitary–adrenal (HPA) axis 10, changes in body size and composition at birth and during postnatal life12. More specifically, a high level of maternal stress during pregnancy has been found to be associated with a lower weight, length, and head circumference at birth in several studies14, however, some authors have shown a positive or nonsignificant effect17.Studies have shown that the placental mechanisms can protect the fetus against high maternal stress to some extent; however, these mechanisms are insufficient to completely diminish the effect of increased stress18. In particular, extreme traumatic stress during childhood can affect the sensitivity of the HPA axis and, consequently, lead to its long-term dysregulation20. For example, pregnant women who experience traumatic stress during childhood have a higher awaking cortisol response22. Recent studies investigating the effects of maternal childhood trauma (MCT) suggest that these developmental effects may be transgenerational (transmitted to the offspring). Although most studies have focused on the behavioral and emotional consequences of MCT, some of them suggest that the biological development of the offspring might be also affected25. For example, MCT, including violence, is associated with preterm birth26, lower birth weight27 , lower infant intracranial volume25, and a higher cephalization index23.Interestingly, a new body of evidence suggests that some maternal stressors experienced even before pregnancy might influence the growth and well-being of an offspring29. Simultaneously, the study also suggested that neither catch-up growth nor breast milk energy density contributed to the observed growth effects29. While MCT-associated changes in infant body size have been detected in the very early stages of development–at birth and early postpartum, it remains unclear whether these changes persist in later stages of development. To address this, we studied body size in relation to the MCT level in the same breastfed infants during the first year of life. We hypothesized that the differences in the growth pattern between infants of mothers with high childhood trauma (HCT) and low childhood trauma (LCT) observed during the initial months would persist further in their development.Our preliminary results showed that body weight and head circumference are positively associated with increased maternal childhood trauma among exclusively breastfed infants32, especially during the first year of life. Most of them experience catch-up growth, but some remain consequently smaller33 during this period. Thus, including prematurely born infants in the study group could potentially confound the results.The study group consisted of 100 mother-infant dyads from Poland. The recruitment of dyads took place when the babies were about 5 months old and was based on the following inclusion criteria: (a) for mothers: age older than 18 years old; not taking steroid medication, smoking, or drinking alcohol during pregnancy or lactation; without metabolic or congenital diseases (b) for infants: being born from a single and uncomplicated pregnancy; at least 37 weeks of gestation with birth weight at least 2600 g and exclusively breastfed for at least 5 months. The above criteria where established based on the literature indicating that children born prematurely or small for gestational age have a different developmental patternThe study protocol included two meetings with mothers and infants. The first meeting occurred when the babies were approximately 5 months old. During this meeting, we collected maternal and infant measurements, information on maternal socioeconomic status and life satisfaction, birth outcomes, and postpartum depression. At the second meeting that took place when the children were about 12 months, maternal and infant measurements were collected again. At this point, mothers were also asked about their traumatic experience during childhood. This research protocol was approved by the Bioethical Committee of Lower Silesian Medical Chamber in Wroclaw (protocol code 1/NT/2016 from 10.02.2016).Each participant in the study received information about the course and purpose of the study, giving informed, written consent to participate in the study in accordance with the tenets of the Declaration of Helsinki. Respondents were allowed to opt out of the study at any stage of the study, and there were no legal or financial consequences for opting out.34, which is an international psychological tool constructed based on the Child Abuse and Trauma Scale35. Women were asked to indicate their childhood traumatic events up to the age of 12 years. The questionnaire included 19 events such as peer bullying, domestic violence, sexual harassment, long-term illness, or natural disasters. Each event scored 1 point.The MCT was evaluated using the Polish version of the Early Life Stress Questionnaire (ELSQ)36. Following other studies, including those conducted on Polish samples, the cut-off point for higher risk of postpartum depression was defined by a score value of at least 1436. Additionally, the participants were asked to assess their financial satisfaction on a 7-point Likert scale  and declare their educational status (higher education with at least Bachelor's or nonhigher education).Experiencing symptoms of maternal postnatal depression  was evaluated using the Polish version of the Edinburgh Postpartum Depression Scale (EPDS). This 4-point and 10-item questionnaire is a widely used tool in clinical and nonclinical settingsAnthropometric measurements of the dyads were taken twice. First, when the infants were approximately five months old and second, at twelve months. Maternal body weight was measured with a Tanita SC-240 MA scale (accuracy of 0.1 kg) and height with a stadiometer (accuracy of 0.1 cm). The mothers also reported their pre-pregnancy body weight. The measurements were used to calculate body mass index . For infants, measurements included body length using a Seca measuring board , weight using an analog hospital scale (with an accuracy of 0.1 kg), and head circumference using a measuring tape (with the accuracy of 0.1 cm). Birth outcomes  were taken from the child's health record.2 test. The association between infant weight, length, head circumference, and maternal childhood trauma was tested using General Linear Models. The separate multivariate analyses of covariance (MANCOVA) models were built for growth parameters at each infant age  with all size parameters as dependent variables, the level of MCT (low–high), infant sex (boy-girl) and risk of maternal postpartum depression (low–high) as categorical predictors, and maternal BMI and infant age as covariates. Following the MANCOVA models, we also ran separate univariate analyses of covariance (ANCOVA) to test which of the dependent variables were statistically significant. In addition, Cohens’ d values (group comparisons with different sample size)37 was calculated to quantify the effect of MCT on infant body size parameters.Mothers were divided into LCT or HCT groups according to the median value (Me = 2) of the ELSQ score. Differences in mean values of main study variables between LCT and HCT mothers were tested using t-test. Differences in the number of mothers according to increased depression risk and infant sex between LCT and HCT group was tested using chiwww.statsoft.com), and the R statistical environment (version 3.6.0). The statistical significance level was established at p < 0.05, however, we also reported a marginally significant effect at 0.06 > p > 0.05.The Henze-Zirkler test indicated multivariate normality of the dependent variables, and the Box M test confirmed the homogeneity of the variance–covariance matrices. Since the deviations from normality were relatively minor and our sample size was sufficient to obtain robust results (n > 30 in both groups), we followed the standard parametric procedure for the univariate analysis. Statistical analysis was performed using StatSoft STATISTICA , version 12 . Furthermore, neither maternal economic satisfaction nor education was associated with MCT (Table The ELSQ score in the study group ranged from 0 to 11 traumatic events during childhood. 46% (n = 46) of the participants suffered from more than 2 traumatic events up to 12 years of age, while only 14% (n = 14) of the women in the study group did not experience any traumatic events. Therefore, mothers who experienced more than 2 traumas were included in the HCT group. Out of all participating mothers, 14 (14%) had an increased risk of postpartum depression according to the defined cut-off point when their babies were five months old. The results of the chip = 0.008; t = −2.91, p = 0.005 at 5 and 12 months respectively) and had larger head circumference than infants of LCT mothers . No significant differences were found in infant body size at birth and the gestational age. Maternal characteristics also did not differ between the HCT and LCT groups, excluding maternal age when the children were 5 months old  were found at the age of 5 and 12 months  = 3.57, p = 0.0 17) remained significant at the age of 5 months and marginally significant at the age of 12 months  = 2.61, p = 0.056) after controlling for maternal BMI and risk of postpartum depression as well as infant sex, and age  = 0.64, p = 0.592).The MANCOVA models show, that the differences between LCT and HCT in infant growth characteristics  = 8.06, p = 0.006, Cohen’s d = 0.53) as well as head circumference  = 6.17, p = 0.015, Cohen’s d = 0.42) was found at 5 months  = 7.17, p = 0.009, d = 0.60; F  = 5.06, p = 0.027, d = 0.45 for weight and head circumference, respectively)  via maternal postpartum depression. Almost 30% of the participants in this study suffered from postpartum depression, which in turn predicted a negative infant development outcome at the age of 12 months28. The low prevalence of postpartum depression observed in our study (about 14% of the participants) could be related to a common practice of universal and exclusive breastfeeding among the study participants, which has been previously shown to have beneficial effects on maternal well-being39 and infant development40. Contrasting findings also came from the study by Smith et al.24, who reported a lower birth weight in infants born to mothers who experienced HCT than those with LCT. The discrepancies in the findings between this and our study could be due to the fact that the former did not control for preterm birth and/or low gestational age, both associated with decreased weight at birth24.The presented results corroborate the results of an experimental study in hens showing that traumatic stress experienced during early life and puberty is related to earlier hatching of offspring and their increased body weight at the age of one month42 which in the future might result in an increased risk of glucose intolerance and metabolic syndrome. Such effects usually co-occur with overweight and obesity43 and have been previously reported in adult descendants of parents with higher trauma44. In addition, higher body weight during infancy is related to the risk of being overweight and obesity during adulthood45. Thus, the results of our study suggest that the tendency towards an increased weight among children born to HCT mothers might be long-lasting, and probably result in a higher risk of metabolic conditions in adulthood.The higher weight in babies born to HCT mothers observed in our study might be associated with impaired glucose metabolism23, who found that infants born to women with HCT had a higher cephalization index. An increase in the value of this index is usually associated with an increase in head circumference.Our analysis also showed a larger head circumference among infants of HCT mothers compared to the infant of LCT mothers. This result is in line with a study by Appleton et al.46. These effects are hypothesized to be mediated by epigenetic effects on germline and somatic cells, including DNA methylation and histone and RNA modifications46. Breast milk, which contains noncoding RNAs, such as microRNAs, serves as epigenetic vectors in molecular communication between mother and offspring and constitutes the first vital gate allowing these developmental effects46. Furthermore, HCT may program HPA axis reactivity to produce an increased level of stress hormones, including cortisol, many years after exposition47. Thus, cortisol transmitted from serum to breast milk might serve as a second gate48. Glucocorticoid levels during the perinatal period demonstrate a long-term programming effect on growth and health during later life49. Recent literature has shown, that higher values of head circumference and body weight are related to altered levels of fatty acids51 and glucocorticoids54 in breast milk. Additionally, our previous research showed that maternal stress reactivity is positively associated with the level of polyunsaturated fatty acids in milk55, which are crucial for brain growth and development56. It is important to note that the infants in our study group were breastfed exclusively for at least 5 months. Thus, it is possible that maternal HCT was reflected in a modified level of polyunsaturated fatty acids and higher cortisol in milk and, as a result, a faster increase in body mass and head circumference.Overall, these results suggest that MCT experience may induce intergenerational changes in physical development even without trauma present in the next generation57. Individuals that exist under harsh environmental conditions, higher levels of stress and increased risk of mortality must adapt, so such conditions would result in accelerated sexual maturation  and earlier successful reproduction58. On the other hand, it would be associated with significant costs to health and longevity59. For example, early life adiposity predisposes to obesity and other metabolic disorders in adulthood, while earlier and faster reproduction generates considerable metabolic cost to the individual60. According to this theory we postulate that the MCT can be, to some extent, treated as an indicator of a stressful environment and harsh condition within the meaning of the Life History Theory. It is also possible that the observed effect may represent a mismatch between maternal and offspring developmental environments, where the maternal adaptive response to a harsh environment might induce long-term changes in offspring development, even in the absence of ecological obstacles.From the evolutionary perspective, a larger size of offspring born to HCT mothers might result from a faster life pace, as posited by the Life History Theory. The term Life History was introduced by Stearns in 1992 and emphasizes that environmental conditions can push individuals into two types of life strategies: fast and slow63. Furthermore, we did not control for post-traumatic stress disorder (PTSD) and resilience as the additional factors in the analysis. Several studies have underlined the effect of childhood trauma on the prevalence of PTSD64. The latter has been shown to have a long-lasting effect on maternal metabolism and as a consequence, on the development and health of the offspring65. Whereas resilience is postulated as a protective factor with the potential to decrease the negative effect of childhood trauma on the development of offspring and future health66. Finally, the information about birth outcomes was collected from child health records instead of being measured, so the measurement protocol could not have been entirely consistent between different hospitals.One of the limitations of our study might be the fact that participants childhood traumatic events retrospectively. However, this limitation is difficult to avoid due to the extended period between maternal childhood and pregnancy. We also did not know how often and for how long women had been exposed to the reported experiences. To minimize the recall bias, we assessed MCT using a standardized psychological questionnaire, which was successfully applied in several other studiesOur study demonstrates that MCT is significantly associated with the size of the offspring during the first year of life even after adjusting for other significant factors that influenced body size such as maternal BMI and postnatal depression, infant age, sex, and age. Children of mothers with HCT had higher weight and larger head circumference than peers born to mothers with LCT, and this effect was independent of body size at birth. These results suggest that MCT might contribute to alterations in the maternal physiology of the HPA axis, which in turn program the development of offspring in the long-term perspective. We propose that this effect may arise from the faster pace of life syndrome mediated by epigenetic changes to DNA and the altered composition of breast milk.Supplementary Information.


Here we present a theoretical-computational study dealing with the evaluation of the pKa of the Cysteine residues in Thioredoxin (TRX) and in its complex with the Thioredoxin-interacting protein (TXNIP). The free energy differences between the anionic and neutral form of the Cysteine 32 and 35 have been evaluated by means of the Perturbed Matrix Method with classical perturbations due to both the environment and an exogenous electric field as provided by Molecular Dynamics (MD) simulations. The evaluation of the free energies allowed us to show that the effect of the perturbing terms is to lower the pKa of Cysteine 32 and Cysteine 35 with respect to the free amino-acid. On the other hand, in the complex TRX-TXNIP, our data show an enhanced stabilization of the neutral reduced form of Cys 35. These results suggest that external electric stimuli higher than 0.02 V/nm can modulate the Cysteine pKa, which can be connected to the tight regulation of the TRX acting as an antioxidant agent. The evaluation of the protein residue pKa due to the environment is an intriguing challenge from both an experimental and theoretical viewpoint. In this context, the tendency to lose or gain a proton by specific Cysteine residues in Thioredoxin is known to be related to its biological function. Thioredoxin (TRX) takes part in the Thioredoxin system together with its reductase (TrxR) and a molecule of NADPH ,2; such  helices ,6. The a helices ,5. The r helices ,7, whereadiation ,8,9, redadiation ,11,12, aadiation ,14,15 anadiation ,17,18,19The Perturbed Matrix Method ,17,18,19 systems ,23,24,25In this context, the electronic Hamiltonian operator, Equation .(1)H^=Hvia a multipolar expansion centred in the QC centre of mass, The jth particle, and Nth atomic region around the corresponding atomic centre Nth atom of the QC), as defined in the following equation:j running over all QC nuclei and electrons, N running over all QC atoms, and Nth atomic region. The expansion of the perturbing term is used in this work only for the Hamiltonian matrix diagonal elements, whereas the other Hamiltonian matrix elements are obtained by using the QC-based perturbation operator expansion within the dipolar approximation  , is usedEquation . For eacNote that, in cases where an exogenous electric field is present, it is classically imposed to each atom of the MD simulation box and its associated perturbation is included in the environmental electric term The acid/base reaction of deprotonation, considered for the QC, can be expressed as follows:The Helmholtz Free Energy change for the reported reaction is obtained by considering the average of both the protonated and the deprotonated ensemble, each with its own ionic environment. The expression of Equation , is baseFurthermore, The ensemble average is evaluated by means of the PMM-MD procedure, within the two ensembles depicted by MD trajectories, containing the proper number of counterions to make the overall environment neutral.Finally, for the pKa estimates , the folIn Equation , the eleThe standard chemical potential of the solvated proton is determined by experimental estimates of the solvation free energy of the proton, Here, the terature ,26,27, win Refs. ,28.The Essential Dynamics (ED) of proteins principal collective motions is a multivariate statistical method, based on the diagonalization of a correlation matrix of the atomic positions . The metThe unperturbed gas-phase properties  were calculated for the ground state by means of Density Functional Theory (DFT), with B3LYP ,31 as fuFurthermore, B3LYP was chosen as functional in virtue of its feasibility and widespread use in describing biological systems ,35,36,37The Cysteine pKa may vary according to the different protein environments. That is, charged groups, positive electron density at the N-terminal part of Ka value ,44,45,46As reference value, the computed pKa of ≈8.2 for the Cysteine (in its zwitterionic form) in water was taken into account and the results are reported in As noted by the analysis of the TRX-TXNIP  complex, the presence of the inhibitor involves large conformational changes in TRX, which can affect the pKa of Cys 35 [Therefore, we applied a Principal Component Analysis, i.e., the Essential Dynamics analysis, on the C-To evaluate the effect of an external electric field on the acidity of TRX Cysteine 35, we applied static electric fields at increasing amplitude values. The lowest electric field intensity was set to 0.02 V/nm, while the highest intensity was set to 0.12 V/nm, with increasing steps of 0.02 V/nm. Note that in this work we implicitly consider the electric field intensity to fulfil the so-called ‘weak field conditions’, i.e., to produce a linear response in the system. The application of a static electric field at increasing field intensities decreases the pKa values, as reported in To further investigate the effects of external electric fields on our system, we performed the PCA analysis with three different exposure conditions: (i) in absence of any applied electric field; (ii) in presence of an applied field of 0.06 V/nm; (iii) in presence of an applied field of 0.12 V/nm. Results indicated that, by increasing the field intensity, the protein gradually changes its accessible conformational space, as shown by the trajectory projection on the C-pace see .On the contrary, the temperature increase does not provide a clear trend on the pKa, suggesting that the effect of the temperature is quite limited see . What emTherefore, the pKa decrease due to the applied electric field is not a thermal effect but is due to its direct effect on the dynamical behaviour of the system.The methodology proposed in this paper allowed for an initial estimation of the value for the pKa of Cysteine in water, found to be ≈8.2 units. This value matches the experimental data well ,12,47, cAfter proving the validity of the PMM methodology for this kind of studies, we estimated the pKa values of Cysteines in the complex environment represented by a solvated protein. Thus, for Cys 32 and Cys 35 in TRX we obtained a in water ,49,50,51proteins , while dproteins ,11,12 inGiven the importance of TRX functioning in cellular antioxidant defence, we investigated the effect of the TXNIP inhibitor on the Cys 35 pKa variation. The conformational structural change in the enzymatic loop closure, where the disulphide bridge exchange reaction occurs, is able to facilitate the covalent bond break between TRX and TXNIP. In terms of If we assume that such variation is mainly localized on the active site  we can circumscribe the analysis on the C-The analysis of hydrogen bonds between the sulphur atom of Cys 35 and the surrounding solvent molecules, conducted with Vmd  HydrogenOne of the goals of the present manuscript was to study the response of TRX protein  in reFurthermore, the presence of the helix residues ,44,46 juIn conclusion, what has emerged from this study is a defined characterization of TRX Cys 35 pKa variation, with the explanation of the role of the natural inhibitor TXNIP in closing the active site to solvent access, reducing thiolate stabilization. By considering the effect of an applied electric field, as an additional source of perturbation, TRX has revealed itself as a molecule susceptible to an exogenous applied field, able to lower its pKa value with it, thus enhancing its ability to use Cys 35 as resolving residue in the reaction of disulphide bridges exchange. On the other hand, temperature variation does not provide a clear effect on the pKa, thus suggesting that the electric field effect is not related to the thermal effect, but is due to a perturbation of the dynamical behaviour of the system in the presence of the electric field.Therefore, we can conclude that TRX activity, under electric field exposure, is correlated to the field intensity, defining a cellular response pathway, via TRX enzymatic role, that acts in defence of stress conditions. To fully understand this intracellular mechanism, our future aim will be a deeper investigation on the effect of an applied electric field, by focusing on the thiol/disulphide interchange reaction, at the basis of TRX active site catalytic functioning.


The temporal growth of the infant microbiome in the early years of life influences short- and long-term infant health. The aim of this longitudinal study was to investigate bacterial dynamics in the microbiome of preterm infants during tooth eruption.n = 24) and preterm infants (n = 31) were collected 30 days after birth and after the eruption of two primary mandibular incisors. Based on Illumina MiSeq Sequencing of the 16S rRNA gene, the dynamic microbial changes of newborns at two-time points were investigated. Meanwhile, the Human Oral Microbiome Database was adopted for assigning taxonomy.Saliva samples from normally delivered (Gemella spp., Rothia mucilaginosa, Veillonella atypica, etc. Several microorganisms colonize later in the oral microbiome development of premature babies, such as Gemella spp. In addition to teeth eruption, the growth of the saliva microbiome in preterm infants could be influenced by breastfeeding durations and birth weight.Using alpha and beta diversity analyses, different shift patterns of microbiome structures in preterm and healthy participants and bacterial diversity over time were observed. The relative abundance and shifts trend, along with the two lower primary central incisors eruption, of core oral flora varies in full-term and preterm groups, including This study provided insights into how the oral microbiota changes during tooth eruption in preterm infants and how the colonization of the oral cavity with bacteria in preterm infants differs significantly from that in full-term infants. The human oral cavity refers to a complex microbial community consisting of bacteria, fungi, viruses, archaea, and protozoa, and the microorganisms constantly interact with each other . EubiosiThe mother plays a crucial role in shaping the oral environment in early life , which sPorphyromonas gingivalis, Treponema denticola, Fusobacterium nucleatum, Prevotella intermedia, and Tannerella forsythia, in saliva or dental plaque samples have recently been shown to be strongly associated with preterm low birth weight . Before recruitment and routine examination, informed consent was acquired from all parents of the enrolled infants. Complete clinical examinations of all infants included were performed, and their caregivers were asked to complete the questionnaire at recruitment and follow-up. We also followed specific regulations and institutional guidelines.This longitudinal study was conducted at Children’s Hospital of Shanghai and Huinan Community Health Service Center, Department of Child Healthcare, Shanghai, China, from June 2018 to November 2020. The inclusion criteria for the healthy newborn group (CT group) included the following: (1) birth at full term ; (2) standard birth weight (2.5–4.5 kg); (3) age between 30 and 40 days at the time of enrollment; (4) no antibiotic use within 30 days; and (5) no oral or systemic disease during the entire period. The inclusion criteria for the preterm group (PT group) included: (1) gestational age between 28 and 35 weeks at birth; (2) low birth weight (less than 2.5 kg); (3) the age range of 30–40 days at the enrollment time; and (4) no present malformations or syndromes. All participating infants were followed up at 3, 6, 12, and 18 months of age until the eruption of deciduous teeth. All preterm infants included in the present work received the standard care offered by the hospital medical staff according to stability and degree of preterm birth.Non-stimulated saliva specimens were collected during baseline and follow-up in this study. All infants were not allowed to eat or drink for 2 h before sampling. Under the assistance of one assistant and their parents, the same pediatric dentist was responsible for collecting oral samples. Saliva samples were first collected using two sterile cotton swabs that were carefully dipped into the saliva pool at the base of the mouth. Later, the tongue and buccal mucosa were gently wiped so that the saliva soaked the swab for approximately 60 s and transferred to an empty sterile Eppendorf tube.Among the 168 newborns meeting the inclusion criteria, salivary samples of 135 children were collected. During follow-up, 20 infants were excluded because they had a new-onset systemic disease or had taken antibiotics in the previous 30 days. A total of 15 children were not willing to be involved because of individual reasons, and 45 were absent during follow-up after the deciduous teeth eruption. Finally, a pool of saliva samples was obtained from these remaining infants in two specific dentition states : first, We collected the specimens in the morning (9–11 a.m.). In addition, all samples were immediately positioned in the bubble chamber, including dry ice for 4 h, which were subsequently kept at a − 80°C freezer. Using the Soil DNA Kit protocol , microbial DNA was extracted from saliva samples. We also used the NanoDrop 2000 UV–Vis spectrophotometer  to evaluate the purity and final content of the DNA. Subsequently, the quality of DNA was assessed with 1% concentration of agarose gel electrophoresis. Moreover, V3-V4 primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′), 806R (5′-GGACTACHVGGGTWTCTAAT-3′), and a thermocycler polymerase chain reaction (PCR) system  were adopted for amplifying bacterial 16S-rRNA gene (V3-V4 hypervariable segments). We also performed all experiments in triplicate with 20 μL aliquots containing 4 μL 5× FastPfu buffer, 2 μL 2.5 mM deoxynucleoside triphosphate, 0.8 μL of each primer (5 μM), 0.4 μL FastPfu polymerase, and 10 ng DNA template. In addition, the whole PCR process included the following steps: denaturation , annealing , elongation , and final extension . This work employed 2% agarose gels for extracting PCR products as well as AxyPrep DNA Gel Extraction Kit , and QuantiFluor™-ST  to perform further purification and calculation, respectively. Subsequently, we used Illumina MiSeq  for pooling the amplicons in equimolar paired-end sequencing (2 × 300) according to the instructions from Majorbio Bio-Pharm Technology Co Ltd. . The data presented in the study are deposited in the SRA repository, accession number PRJNA876796.Each 16S-rRNA gene sequence was categorized with the RDP Classifier algorithmOn the basis of the alpha (α) index, the bacterial richness diversity of the saliva microbiota was evaluated. In this study, R-forge was used to assess beta diversity. Following the normalized weighted UniFrac distance matrices, a principal coordinate analysis (PCoA) was performed based on QIIME . Apart ft-test and paired t-test to examine the significance of the difference in continuous variables along with alpha diversity between the two groups. In addition, the chi-square test was used to examine the differences in the discontinuous numerical variables between the two groups involved. To evaluate the correlation of the associated elements with the relative abundance of each species, Spearman’s correlation analysis was used.We carried out the statistical analysis with the application of SPSS v20 software . In addition, as all the tests were two-sided, the significance level was determined at 5%. Meanwhile, descriptive statistics was adopted for children’s sociodemographic features and clinical measurements. We used Student’s n = 31) and full-term control (n = 24) groups. We also compared all samples in the following four subgroups: preterm birth group before teeth eruption , preterm group after teeth eruption , control group before teeth eruption , and control group after teeth eruption .16S rRNA gene sequencing of saliva samples was performed in a total of 55 infants, including the preterm (p = 0.439) and enrollment month age . Moreover, 11 out of 24 and 25 out of 31 babies were born by cesarean section (p < 0.001). The babies in the PT1 group were notably older (age in months) than the CT1 group , indicating a later teeth eruption age in preterm infants. Mean birth weight was significantly lower in the preterm group (1.67 ± 0.31) than in the control group . In addition, the mean weeks of gestation were significantly lower in the preterm group than in the control group . The babies of the two groups presented no obvious differences regarding gender . The results of the questionnaires are shown in In addition, there were no apparent differences in either group with respect to maternal demographic and socioeconomic characteristics, including age and education level, and oral hygiene and infant feeding habits at follow-up  identified a total of 13 phyla, 31 classes, 58 orders, 107 families, 196 genera, 372 species, and 849 OTUs. Moreover, the rarefaction curve suggested the near-complete sampling of the saliva community . AccordiThe distribution of relative abundances of bacteria from the phylum to species level is shown in p = 0.491). After tooth eruption, the preterm group had significantly higher OTU richness than the full-term group (p < 0.001). Based on a comparative assessment of the Shannon index, the preterm group initially had significantly higher microbial diversity than the control group (p = 0.031), and the difference was not significant after tooth eruption (p = 0.559).To evaluate the alpha diversity of the saliva bacterial microbiome, the richness index (Chao) and diversity index (Shannon) were used. Apart from that, the Chao index presented a higher OTU richness in the preterm infants at baseline, although no significant difference existed .The overall microbial composition and structure between preterm and control groups were compared with the use of PCoA and ANOSIM following the weighted normalized UniFrac distance (observed at the OTU level). Significant differences in salivary microbiota structure were found between the two groups at two stages of dentition . Of note, some individual samples had a lower Chao index and a higher Shannon index while growing up and after tooth eruption, suggesting a more balanced environment.The comparative assessment of the Shannon and Chao indexes using paired t-tests revealed a generally statistically increased tendency in the alpha diversity in both full-term and preterm infants along with the first two lower primary central incisors eruption . These preterm infants showed irregular variations in microbiome structures and shifted in various directions while growing up. Most of the samples in the control group could shift in the same direction. In addition, PC1 levels increased in 20 of 24 samples after tooth eruption, whereas PC2 levels decreased in 16 of 24 samples during follow-up.PCoA and ANOSIM confirmed intraindividual variations in the structure of the tongue-coating microbiota in all samples during tooth eruption and normal growth. According to the findings, the microbiome in the two groups revealed obvious shifting patterns  and V. atypica decreased significantly (p < 0.05) in preterm infants at the rate of tooth eruption, whereas it remained relatively stable in healthy control subjects during tooth eruption. Longitudinal changes also showed that R. mucilaginosa decreased in preterm infants and increased in full-term infants. Veillonella spp. oral taxon 780 (sp._HMT_780) and Veillonella dispar were both abundantly enhanced among preterm and full-term babies over time  of mothers at delivery, gestational weeks, birth weight, breastfeeding habits, and incubator living days. Factors with a VIF >10 were excluded from the study. In addition, the VIF for maternal BMI, birth weight, duration of breastfeeding, length of hospital stay, and weeks of gestation for preterm infants was 1.32, 1.31, 1.00, 1.41, and 2.06, respectively. The VIF for days in the incubator was greater than 10 (VIF = 20.00) and was removed from the study.r2 = 0.34, p = 0.005; r2 = 0.39, p = 0.003, respectively). After teeth eruption, the db-RDA showed significant correlations between birth weight and gestational weeks at birth and the saliva bacterial community composition .The db-RDA on the basis of the weighted normalized UniFrac distance matrix was used for analyzing whether these environmental elements exerted any extra impact on the saliva microbiota communities and composition in preterm infants. At the predentation stage, birth weight and breastfeeding duration showed significant associations with salivary flora composition  as well as a negative relationship to birth weight . The relative abundances of S. salivarius  presented the reducing trend with the increasing length of stay in the hospital at birth in PT1 samples.In addition, Spearman’s correlation analyses were performed between the top 30 species  in the salivary microbiota and these environmental factors in preterm infants ,D. At thp < 0.05) in the network.A microbial correlation analysis was performed among the 30 most abundant species to investigate relationships in the salivary microbial community, finding apparent associations  and C. gilardii. In addition, S. salivarius showed a strong positive correlation with R. mucilaginosa, V. atypica, and V. dispar  but also in richness (Chao) in the upbringing of both groups. The teeth eruption into the infant oral cavity starts at around 6 months of age in healthy infants and significantly later in preterm babies; thus, explaining a higher Chao diversity in PT1 than CT1 infants in this study. In the PT1 group, community development was more advanced. Streptococcus spp. salivarius, Pseudomonas spp., R. mucilaginosa, V. atypica, V. dispar, Gemella spp. and Neisseria spp.  can be identified in the preterm group. While in the healthy birth group, Streptococcus, especially S. salivarius, V. atypica, Gemella spp. and Staphylococcus spp., dominated the infant oral microbiota. Streptococcus accounted for more than 70.0% of the total microbiota at the predental stage and then decreased to 44.9% at the CT1 stage. The obtained result is consistent with other studies , Shanghai Commission of Science and Technology (No. 22Y11903200), and Shanghai Municipal Health Commission (Nos. 2019SY027 and 2020YJZX0114).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.