Caring for a child born preterm places significant emotional and financial burdens on family relationships. This paper examines (a) the extent to which children born very and extremely preterm are more likely to experience parental change/caregiver instability than children born full term, (b) predictors of parental change/s for preterm infants, and (c) whether exposure to parental change/caregiver instability increases child neurodevelopmental risk. Data were collected as part of a prospective longitudinal study of 110 very preterm and 113 full-term born infants and their parents studied from birth to corrected age 12 years. At ages 2, 4, 6, 9 and 12 years, detailed information was collected about the frequency and nature of all parent/caregiver changes for 3–6 monthly intervals of each child’s life. At age 12, all children completed a comprehensive neurodevelopmental evaluation of their emotional and behavioural adjustment, cognition, and educational achievement. Results showed that children born very preterm were at increased risk of experiencing parental/caregiver changes, with this risk being greatest for those born extremely preterm. Neonatal medical complexity, family socioeconomic disadvantage, maternal psychological wellbeing, and child neurodevelopmental impairment were associated with a higher risk of parental change. Preterm birth and exposure to parental change/instability contributed additively to poorer child outcomes. Findings support the need for family-focused neonatal and postnatal care strategies for high-risk infants, to support parents as well as their infants to optimize child health and developmental outcomes. Becoming the parent of a premature infant is a highly stressful life event for families ,2. Not oFurthermore, these challenges and stressors do not end at the hospital door. Children born VPT are at high risk for a range of health and neurodevelopmental impairments including feeding, respiratory and other health issues, more frequent hospitalisations, cerebral palsy, cognitive delay, executive dysfunction, and emotional and behavioural adjustment problems ,9,10. CoThere is some research to support the adverse impacts of preterm birth on family functioning, including disrupted social relationships, parental coping difficulties and increased financial costs particularly during early childhood (see Treyvaud  for a ren = 6016) found that by age 2, parents of very low birth weight  infants were two times more likely than parents of higher birth weight (>1500 g) infants to have separated or divorced . Odds ratios and significance levels for the predictors in each model are presented in The overall binary logistic models were significant at every age  and parental instability  but no interaction effect . Examination of the effects for each of the dependent variables indicated significant effects of preterm birth and parental change on both inattention symptoms and hyperactivity symptoms . In addition to SDQ scores, we examined the impact of preterm birth and exposure to parental change on symptoms of attention-deficit/hyperactivity disorder (ADHD). We used a multivariate analysis of variance to predict the two dependent variables of inattention and hyperactivity from preterm birth and parental instability. The overall multivariate tests (Pillai’s Trace) indicated significant main effects for preterm birth  and parental instability  on full-scale estimated IQ scores, but no interaction between these predictors . Preterm birth and parental instability were both predictive of lower cognitive ability at age 12.To understand the impact of preterm birth on educational outcomes we ran an analysis of variance predicting WISC-IV full-scale estimated IQ from preterm birth and parental instability. There was a significant main effect of both preterm birth  and parental instability , but no interaction between these predictors . A similar pattern was seen for broad reading skills, with significant main effects for preterm birth  and parental instability  but no interaction effect . The results indicate an additive effect whereby children are doubly disadvantaged if they are born preterm and have a history of parental instability.To understand the impact of preterm birth on educational outcomes we ran analyses of variance predicting the outcomes of WJ-III broad math standard scores and WJ-III broad reading standard scores from preterm birth and parental instability. When predicting broad math skills, there was a significant main effect of both preterm birth [This paper examined the longer-term impacts of very preterm birth on the partner relationships of parents of children born very preterm. Specifically, we examined whether very preterm birth may increase the likelihood that children will experience parental change/instability over their first 12 years, and in particular whether risks varied by infant gestational age at birth. Further, we examined potential risk factors for parental change and assessed the impact of this parental change/instability on children’s outcomes at age 12, above and beyond risks associated with preterm birth itself.Results indicate that very preterm birth is associated with an increased risk of parental change/instability from birth to age 12 years. These changes included parental divorce and separation, formation of new parental partner relationships, and adoption or foster care. There was a general tendency for the risk of any and multiple parental changes to be greatest for those born EPT, or those at the lower limits of viability. While children born extremely preterm had higher rates of both any and multiple parental changes than children born full term, children born very preterm were more likely than FT-born children to experience multiple or high levels of instability . By corrected age 12 years, nearly half of our sample of children born extremely preterm had experienced at least one parental change, and nearly a third had experienced two or more parental changes.Consideration of the predictors of parental change within the group of children born either very or extremely preterm showed that, in addition to gestational age at birth, early neonatal medical complexity was associated with increased risk of later parental instability at ages 9 and 12, suggesting longer-term impacts. Research on the impact of medical conditions such as autism spectrum disorder and ADHD has shown an extended period of the risk of divorce, persisting beyond early childhood and into early adulthood . Our resWe found that low SES was a significant predictor of parental instability at all ages. This is consistent with previous research showing that instability is more common within low SES families . FamilieOur findings also confirmed previous research suggesting higher rates of parenting stress in parents of children born very preterm, as compared to those born at full term ,4. FurthCollectively findings suggest that children born at lower gestations, subject to a more complex medical course during their neonatal hospital stay, living in lower socioeconomic conditions, and with mothers experiencing symptoms of depression, anxiety, and/or parenting stress are most at risk of exposure to instability of parents/caregivers.Finally, we examined the combined effects of preterm birth and parental change/instability on children’s neurodevelopmental outcomes at age 12. For most outcomes assessed, our results indicate an additive effect whereby children were doubly disadvantaged if they were born very preterm and then experienced subsequent parental change/s.An additive effect  was found for symptoms of inattention and hyperactivity (as assessed by both the SDQ and the SWAN), cognitive ability, educational outcomes, prosocial behaviour, and overall behavioural difficulties on the SDQ. There was an effect of preterm birth but not parental instability on peer relationship problems, and there was an effect of parental instability but not preterm birth on conduct problems. This latter finding is consistent with previous research that has found an inconsistent or lack of association between preterm birth and later conduct problems .These findings show that the family psychosocial context, in terms of maternal mental health, caregiver stability, and socioeconomic status, has long-term implications for the developmental outcomes of children born preterm. Strains on inter-partner relations and family stability may be greatest in families caring for a medically and neurodevelopmentally complex child, and in a context of limited financial and mental health/parenting resources and parenting/mental health stress. Taken together, our findings suggest a negative feedback loop in many families of children born very preterm, whereby the medical burden of preterm birth contributes to family breakdown and instability, which in turn negatively impacts children’s neurodevelopmental outcomes.Our results thus highlight the need for family-focussed neonatal and postnatal care strategies to support parents as well as their infants to ensure optimal child health outcomes. Monitoring of family and environmental factors ought to be a key component of neonatal and long-term follow-up. This is important as strategies to prevent parental and familial instability following preterm birth, such as support and resources directed at parental mental health needs and supporting their interparental relationships, will help to protect children from experiencing compounded risk to their cognitive, educational, and behavioural development.Our research focused on parental change and therefore does not take into account other types of family instability or disruption that children may experience. The comings and goings of other household members such as grandparents, other adult relatives and non-relatives, or siblings may also impact family functioning and child outcomes, particularly when household members are frequently changing. We chose to focus on parental change as it is likely of primary importance to child developmental outcomes and also most likely to be impacted by preterm birth. We also did not differentiate between the types of parental instability when examining outcomes. There may be differential effects based on the type of change experienced  but we did not have a large enough sample to explore the specific patterns associated with each type of parental change.Finally, in our consideration of psychosocial family factors, we focused on mothers  and have not examined the psychological well-being of fathers. Further work is required to better understand the contribution of paternal factors to parental instability in the families of children born very preterm.Our results indicate that preterm birth is associated with an increased risk of parental change/instability over the first 12 years of life. The risk of parental change was greatest for those born extremely preterm, with EPT children being 1.7 times more likely than FT-born children to experience any parental change and twice as likely to experience multiple (2 or more) parental changes. Neonatal medical complexity, family socioeconomic disadvantage, maternal psychological wellbeing, and concurrent child neurodevelopmental impairment were also associated with an increased likelihood of parental change. Furthermore, preterm birth and exposure to parental change/instability both contributed negatively to outcomes  in an additive, rather than an interactive, manner. These findings support the importance of family-focussed neonatal and postnatal care strategies to support parents as well as their infants for optimal child health outcomes.


We aimed to validate the predictive performance of the DIGIROP-Birth model for identifying treatment-requiring retinopathy of prematurity (TR-ROP) in Chinese preterm infants to evaluate its generalizability across countries and races.We retrospectively reviewed the medical records of preterm infants who were screened for retinopathy of prematurity (ROP) in a single Chinese hospital between June 2015 and August 2020. The predictive performance of the model for TR-ROP was assessed through the construction of a receiver-operating characteristic (ROC) curve and calculating the areas under the ROC curve (AUC), sensitivity, specificity, and positive and negative predictive values.Four hundred and forty-two infants (mean (SD) gestational age = 28.8 (1.3) weeks; mean (SD) birth weight = 1237.0 (236.9) g; 64.7% males) were included in the study. Analyses showed that the DIGIROP-Birth model demonstrated less satisfactory performance than previously reported in identifying infants with TR-ROP, with an area under the receiver-operating characteristic curve of 0.634 . With a cutoff value of 0.0084, the DIGIROP-Birth model showed a sensitivity of 48/93 (51.6%), which increased to 89/93 (95.7%) after modification with the addition of postnatal risk factors. In infants with a gestational age < 28 weeks or birth weight < 1000 g, the DIGIROP-Birth model exhibited sensitivities of 36/39 (92.3%) and 20/23 (87.0%), respectively.Although the predictive performance was less satisfactory in China than in developed countries, modification of the DIGIROP-Birth model with postnatal risk factors shows promise in improving its efficacy for TR-ROP. The model may also be effective in infants with a younger gestational age or with an extremely low birth weight.The online version contains supplementary material available at 10.1186/s12886-021-01952-0. Retinopathy of prematurity (ROP) is a retinal vasoproliferative disease that affects preterm infants. Although it is avoidable through early diagnosis and timely treatment, it is a leading cause of childhood blindness . The risLow gestational age and birth weight are the major risk factors for ROP, and form the basis of most established screening guidelines . PredictRecently, Pivodic et al. proposed an individualized risk prediction model for treatment-requiring retinopathy of prematurity (TR-ROP), called DIGIROP-Birth. The model was based on gestational age, birth weight, and sex and applicable for infants with a gestational age of 24 to 30 weeks . It has Therefore, the present study aimed to validate the DIGIROP-Birth model in Chinese preterm infants to evaluate its generalizability across countries and races.Preterm infants who received ROP screening examination between June 2015 and August 2020 in the neonatal intensive care unit of Zhujiang Hospital of Southern Medical University, Guangzhou, China, were retrospectively recruited. The study was approved by the hospital’s institutional ethics committee and adhered to the tenets of the Declaration of Helsinki. All parents or guardians of the recruited infants provided written informed consent prior to participation. Data were anonymized and de-identified before analysis. According to the Chinese guideline for ROP screening, eligible participants were preterm infants with gestational ages less than 32 weeks, birth weight less than 2000 g, or with risk factors for ROP as determined by a neonatologist . InfantsClinical data collected for our study related to maternal factors, neonatal factors, and neonatal interventions. These data were extracted from medical records by SZ and R. Specifically, maternal factors of interest included maternal age, mode of delivery , multiple gestations, in vitro fertilization, gestational hypertension, gestational diabetes mellitus, reproductive tract infection during pregnancy, and use of antenatal steroids. Neonatal factors included gestational age, birth weight, sex, apnea, respiratory distress syndrome, bronchopulmonary dysplasia, sepsis, necrotizing enterocolitis, intraventricular hemorrhage, patent ductus arteriosus, anaemia, and hyperbilirubinemia. Finally, neonatal interventions included invasive mechanical ventilation and blood transfusions.P < 0.05 (two-tailed) was considered statistically significant. To identify the independent risk factors for TR-ROP, univariate logistic regression analyses were performed for each variable. Variables with a P < 0.1 in univariate logistic analyses were included in multivariate logistic regression analyses. To assess the predictive performance of the model for TR-ROP, a receiver-operating characteristic (ROC) curve was constructed. In the ROC analysis, the areas under the ROC curve (AUC), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. The cutoff value that resulted in the maximal sum of sensitivity and specificity was chosen and assessed. In addition, the cutoff values previously tested by Pivodic et al. were also selected for validation [Categorical variables were expressed as numbers and percentages and analyzed using chi-squared tests. Continuous variables were expressed as means and standard deviations and compared using Wilcoxon’s rank-sum tests. lidation . The perP < 0.001) among TR-ROP cases. A significant difference in sex was also observed between the groups (P = 0.046), where the proportion of males was significantly higher in infants with TR-ROP. Finally, there was a higher occurrence of apnea (P = 0.041) and intraventricular hemorrhage (P = 0.034) in infants with TR-ROP.Of the 732 infants who underwent ROP screening, 11 had incomplete data and five had ocular diseases besides ROP. Among the remaining 716 infants, 274 were further excluded due to a gestational age less than 24 weeks or beyond 30 weeks, resulting in 442 infants being included in the study. Two hundred thirty-seven of 442 participants (53.6%) developed ROP of any stage, of whom 93 of 442 (21.0%) required treatment. The mean (SD) gestational age at birth was 28.8 (1.3) weeks, the mean (SD) birth weight was 1237.0 (236.9) g, and the sex distribution was 286 (64.7%) male and 156 (35.3%) female. Clinical characteristics of the study population are summarized in Table P = 0.082). Among the neonatal factors, infants with TR-ROP had a younger gestational age and lower birth weight (both P < 0.001) than infants without TR-ROP. In addition, male sex (P = 0.047), apnea (P = 0.043), bronchopulmonary dysplasia (P = 0.077) and intraventricular hemorrhage (P = 0.035) were associated with TR-ROP. As for the neonatal interventions, only blood transfusions were associated with TR-ROP (P = 0.082). Multivariate logistic regression analysis found that gestational age , birth weight , male sex , apnea , and intraventricular hemorrhage  were independently associated with TR-ROP.Univariate and multivariate logistic regression analyses were conducted to identify risk factors for TR-ROP  Fig. . The preThe performance of the DIGIROP-Birth model for identifying TR-ROP infants was also evaluated by gestational age and birth weight Tables , 6 and 7Visual loss from ROP may be prevented by early diagnosis and timely treatment, which emphasizes the importance of ROP screening in routine clinical practice . RecentlSeveral reasons could account for the discrepancy. First, there were only 83 (5.4%) Asians among the 1535 infants that comprised the US validation group in Pivodic et al.’s study , while oSeveral studies have shown that complications of prematurity, such as bronchopulmonary dysplasia, apnea, intraventricular hemorrhage, and sepsis, are associated with the development of ROP –35. GestThe sensitivity of the DIGROP-Birth model in infants with a gestational age < 28 weeks or a birth weight < 1000 g was satisfactory. This suggests that the DIGROP-Birth model may also be valuable as an auxiliary tool for ROP screening in extremely preterm infants and infants with extremely low birth weight. In infants with a gestational age ≥ 28 weeks or birth weight ≥ 1000 g, the DIGIROP-Birth model was less effective, but could be modified with postnatal risk factors. Thus, the DIGIROP-Birth model still has the potential to decrease the frequency of ROP examinations in less-developed countries.There was another factor impeding the generalization of the DIGROP-Birth model to Chinese preterm infants. The DIGROP-Birth model was developed in Swedish infants with gestational ages less than 31 weeks, thus this model could not be applied to infants with gestational ages of 31 weeks or more , resultiThis study has several limitations. First, this validation study was conducted retrospectively. Despite the retrospective nature, however, the clinical data included in our analyses are routinely recorded in the neonatal intensive care units and could be collected reliably. Second, the single-center study had a relatively small sample size compared with other validation studies of ROP prediction models. Future multicenter prospective studies with large cohorts will enhance our findings.This study validated the DIGIROP-Birth model in a cohort of Chinese preterm infants. Although the predictive performance of the model was lower than in developed countries, modification of the model with postnatal risk factors showed promise for improving its efficacy for TR-ROP. The DIGIROP-Birth model was also effective in infants with a younger gestational age or with extremely low birth weight. We believe that more studies in less-developed countries and other races will improve the efficacy and generalizability of the DIGIROP-Birth model, and subsequent adjustment of the easy-to-use model will help reduce the frequency of ROP screening examinations.Additional file 1: Supplementary Table 1. Univariate and multivariate analysis of risk factors for TR-ROP.


Cytokines facilitate the peripheral immune and cerebral response, through their ability to modulate the expression of brain derived neurotrophic factor (BDNF). Cytokines and BDNF are implicated in cancer-related cognitive impairment (CRCI), but their relationship has not been clearly defined for this condition. The aim of this study was to evaluate the associations of cytokines and BDNF among early stage breast cancer (ESBC) patients with different CRCI trajectories. This was a multicenter longitudinal study involving 136 ESBC patients. CRCI was assessed using the FACT-Cog (V3) questionnaire. Plasma cytokines and BDNF levels were quantified at three time points throughout chemotherapy. The associations between cytokines and BDNF were analyzed using linear mixed models, with interaction terms for CRCI status. All cytokines analyzed showed inverse associations with BDNF levels. There was a significant interaction between IL-6 and the persistent impairment trajectory, which would impact on BDNF levels (p = 0.026). The inverse associations with BDNF were more pronounced for IFN-γ, IL-1β, IL-4, IL-8, and GM-CSF in patients with persistent CRCI. The coefficient values for IL-2, IL-4, and TNF-α also indicate that there was a greater magnitude of decrease in BDNF level for every unit of cytokine increase in patients with acute and persistent CRCI, compared to patients without CRCI. The differential associations between cytokines and BDNF may be indicative of probable susceptibility to the elevation of cytokines. Further research is required to elucidate the specific associations of cytokines and BDNF, along with their contributions to acute and persistent CRCI. CRCI affects up to 65% of breast cancer patients during treatment and one third of survivors may suffer from lasting cognitive deficits 1 year following the completion of treatment ,54. DistThe etiology of CRCI is multifaceted with the involvement of psychological, biological and genetic determinants. One biological factor of interest is brain-derived neurotrophic factor (BDNF), a neurotrophin which is required for neurogenesis and the maintenance of neuronal plasticity . DysreguChemotherapy evokes a systemic immune response, leading to an increase in peripheral inflammatory cytokines which are able to cross the blood-brain barrier (BBB), whereas most chemotherapy drugs are impeded due to the larger molecular size . CytokinPreviously, we have shown that elevated levels of pro-inflammatory cytokines IL-1β and IL-6 corresponded with poorer cognitive response and higher BDNF levels were protective against persistent CRCI in a cohort of early stage breast cancer (ESBC) patients ,56. The 2.2.1.This was a prospective, longitudinal, cohort study and study participants were recruited from the National Cancer Center Singapore, Changi General Hospital and KK Women’s and Children’s Hospital. This study was granted ethical approval from the SingHealth Institutional Review Board (CIRB 2014/754/B) with recruitment and follow-ups from November 2014 to January 2020. All participants were consented prior to data collection. Patients who fulfilled the following criteria were recruited for the study: (i) at least 21 years of age, (ii) diagnosis of ESBC (stages I – IIIA), (iii) Have not undergone chemo/radiotherapy and scheduled to receive anthracycline- or taxane-based chemotherapy and (iv) able to read and understand English or Mandarin. Patients were excluded from this study if they were: (i) physically or mentally incapable of providing informed consent or (ii) diagnosed with neuropsychiatric or neurologic medical conditions that would interfere with cognitive functioning.2.2.Participants’ demographic and clinical information was acquired from electronic medical records and participant interviews. Participants were assessed at four time points: before chemotherapy initiation (baseline) (T1), 6 weeks after chemotherapy initiation (T2), 3 months after chemotherapy initiation (at the end of chemotherapy) (T3), and 12–24 months post-chemotherapy completion (T4). At each time point, subjective cognitive function, fatigue, anxiety, and depression were evaluated using patient-reported outcome questionnaires. Blood samples were taken for BDNF genotyping, plasma BDNF and cytokines quantification at T1, T2, and T3.2.3.Subjective cognitive function was assessed using the Functional Assessment of Cancer Therapy-Cognitive Function (FACT-Cog) version 3 questionnaire. The English and Mandarin versions used in this study have been validated in a population of breast cancer patients in Singapore . The queAnxiety, depressive symptoms and fatigue levels were assessed using the Beck Anxiety Inventory (BAI), Beck Depression Inventory (BDI), and Brief Fatigue Inventory (BFI), and respectively ,28,33.2.4.Whole blood was collected in an ethylene diamine tetraacetic acid (EDTA) tube and centrifuged at 2500 rpm for 10 min. The extracted plasma and buffy coat were stored at −80 °C until analysis.2.5.Plasma cytokine levels were determined using the Luminex multiplex panel  which included granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-γ), tumor necrosis factor (TNF-α), interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8 and IL-10. Plasma cytokine levels were determined from standard curves generated by standards of known cytokine concentrations . The con2.6.Genomic DNA was extracted from the buffy coat using the QIAmp DNA Blood Mini Kit . Genotyping of the Val66Met (rs6265) polymorphism was achieved by polymerase chain reaction (PCR) followed by Sanger sequencing using a 3730 xl DNA Analyzer . The primers for PCR were 5′-GGACTCTGGAGAGCGTGAA-3′ (forward) and 5′-CGTGTACAAGTCTGCGTCCT-3′ (reverse).2.7.Statistical analyses were conducted with the Stata version 16 software . Demographics, clinical data and the incidences of cognitive impairment were summarized using descriptive statistics. The Friedman test was used to analyze the changes in plasma cytokines and BDNF levels across time points, followed by the Wilcoxon signed-rank test with Bonferroni correction for post hoc pairwise comparisons.Linear mixed model analyses, with time point adjustments, were carried out to analyze the associations between plasma cytokines and BDNF levels, with cytokines as the independent variables and BDNF as the dependent variable. Analysis was first performed with interaction terms between cytokines and participants grouped by those without reported CRCI for all assessment time points and those with reported CRCI. Analysis was then performed with interaction terms between cytokines and the different cognitive impairment trajectories , with the group without reported CRCI acting as reference. Random intercept was included to account for individual-specific effects. Clinically important factors that could affect BDNF levels such as the rs6265 genotype status, age, BMI and depression status were included in the adjusted models ,44. The 3.3.1.A total of 217 participants were recruited for the study. After excluding participants with incomplete follow-up data or biomarker samples, 136 participants were included in the final analysis. The participants’ baseline characteristics are presented in 3.2.Among the cytokines analyzed, there was an overall significant increase in plasma concentrations of cytokines IFN-γ, IL-4, IL-6, and TNF-α, from the inception to the end of chemotherapy (T1 to T3) . IL-10 w3.3.Cytokines were evaluated for their associations with BDNF levels among ESBC patients with and without CRCI . All cytDue to the heterogenous nature of CRCI, the associations between cytokines and BDNF in patients with different CRCI trajectories  were then compared with patients without CRCI, as shown in 4.Building upon our previous analyses which demonstrated the protective effects of BDNF against CRCI and the associations of cytokines with cognitive function in ESBC patients, the present study is focused on the modulatory potential of cytokines on BDNF levels ,53,56. SThere was an overall trend of circulating cytokine increase, indicative of gross peripheral immune activation by chemotherapy, and a significant decline in BDNF levels throughout chemotherapy. While the mechanism of chemotherapy or cancer-induced inflammation is better defined, the exact cause of chemotherapy related BDNF decline is still unclear. BDNF is mostly secreted by neurons and is found abundantly in various regions of the brain, but is able to cross the BBB contributing to detectable levels in the blood . AlthougIn this study, we did not observe significant differential associations observed between cytokines and BDNF in patients without CRCI compared to patients who reported CRCI during the follow-up period. As the trajectory of impairment can be heterogenous among patients reporting CRCI, the associations between cytokines and BDNF were further evaluated in patients who experienced acute, persistent or delayed CRCI. Significant interaction was observed between persistent CRCI and IL-6, suggesting that there may be a divergence in the association between IL-6 and BDNF in this group of patients during chemotherapy. Previously, we have reported that a higher proportion of ESBC patients with persistent impairment had marked IL-6 increment compared to patients who had no impairment . AlthougThere is a paucity of clinical studies investigating the association or interaction between circulating cytokines and BDNF, and available literature has reported contrasting outcomes in different clinical conditions. Similar to the findings from our cohort of ESBC patients, BDNF was predictive of cognitive performance, and a negative correlation was seen between IL-6 and BDNF in depressive cancer patients undergoing chemotherapy . ConversSome cytokines may exhibit dual roles in the regulation of neurogenesis depending on the physiological condition, cell type involved, and signaling pathway activated ,34,35,46In the event of a pro-inflammatory condition, which can be a consequence of chemotherapy, the microglia switches pathways to release IL-6, TNF-α and IL-1β, exacerbating inflammation, oxidative stress and neurodegeneration in the brain microenvironment . In addiClinical evidence on the associations of cytokines and cognitive function in cancer patients, including the types of cytokines involved, has been disparate . This isAs this was an explorative analysis, the associations of the peripheral biomarker levels were only indicative of a potential relationship or regulatory role between cytokines and BDNF, and did not necessarily equate to a direct causative effect. Additionally, the sample size after classification into the various CRCI trajectories  may not be sufficiently powered to detect interaction effects of impairment status and cytokines. Subjective or self-reported measurement of CRCI was utilized in this analysis as our previous study has demonstrated a lack of significant association between BDNF and objective measures of CRCI . NumerouFuture work should include in vitro or in vivo functional studies to delineate the underlying mechanisms and draw conclusive inferences on the abilities of cytokines to modulate BDNF levels and the susceptibility to acute or persistent CRCI. Measurement of cytokines and BDNF levels after the completion of chemotherapy would also provide a clearer understanding of the relationship between these biomarkers especially in patients who develop delayed cognitive impairment. Nevertheless, the strength of this study lies in the longitudinal design as well as the wide array of cytokines investigated, which enabled the evaluation of cytokine levels in relation to BDNF and their trends during chemotherapy. Although the peripheral biomarker levels were only available throughout chemotherapy, the 12–24 months follow up period to assess cognitive function post-chemotherapy allowed for a comparison of the associations between cytokines and BDNF levels in different CRCI trajectories. Knowledge of the contributions of cytokines and BDNF dysregulation to CRCI could be leveraged for better management of this condition. Cytokines could represent valuable targets and biomarkers for the prevention of CRCI, as they can exert a direct or indirect impact, via BDNF, on cognitive function. For instance, physical exercise presents an attractive management strategy because it has been shown to improve cognitive function by restoring cytokine homeostasis and increasing BDNF levels ,37,41.In conclusion, cytokines were inversely associated with BDNF levels in ESBC patients with and without CRCI. The diverging trends for the associations between cytokines and BDNF were more distinctive when patients with CRCI were grouped by the different impairment trajectories. The differential associations between cytokines and BDNF throughout chemotherapy may be indicative of susceptibility for BDNF reduction to the increase of cytokines in patients experiencing acute or persistent CRCI. In particular, a significant interaction effect between IL-6 and persistent impairment was observed, which would impact on plasma BDNF levels. Findings from this study serve as a basis for future research to elucidate the specific interactions of cytokines and BDNF, along with their contributions to CRCI.Table S1 and S2


Evidence referring to the trade-offs between the benefits and risks of single embryo transfer (SET) versus double embryo transfer (DET) following assisted reproduction technology are insufficient, especially for those women with a defined embryo quality or advanced age.ClinicalTrials.gov were searched based on established search strategy from inception through February 2021. Pre-specified primary outcomes were live birth rate (LBR) and multiple pregnancy rate (MPR). Odds ratio (OR) with 95% confidence interval (CI) were pooled by a random-effects model using R version 4.1.0.A systematic review and meta-analysis was conducted according to PRISMA guidelines. PubMed, EMBASE, Cochrane Library and P < 0.001, n = 62), and lowered the rate of multiple pregnancy . In the sub-analyses of age stratification, both the differences of LBR  and MPR  between SET and DET groups became insignificant in patients aged ≥40 years. No significant difference in LBR for single GQE versus two embryos of mixed quality [GQE + PQE ] , nor any difference of MPR in single PQE versus two PQEs . Moreover, women who conceived through SET were associated with lower risks of poor outcomes, including cesarean section , antepartum haemorrhage , preterm birth , low birth weight , Apgar1 < 7 rate  or neonatal intensive care unit admission  than those following DET.Eighty-five studies  were eligible. Compared with DET, SET decreased the probability of a live birth (OR = 0.78, 95% CI: 0.71–0.85, In women aged < 40 years or if any GQE is available, SET should be incorporated into clinical practice. While in the absence of GQEs, DET may be preferable. However, for elderly women aged ≥40 years, current evidence is not enough to recommend an appropriate number of embryo transfer. The findings need to be further confirmed.The online version contains supplementary material available at 10.1186/s12958-022-00899-1. Increasing success following assisted reproduction technology (ART) has been accompanied by concerns about high rates of multiple pregnancies . MultiplIt is well known that many patients idealize that twins would be their optimum outcome following ART , therefoLarge observational studies focusing on the comparisons of different policies regarding the number of embryos transferred are emerging and do provide valid information –28. The This systematic review adhered to the PRISMA guidelines , and wasRCTs and observational studies comparing benefits and risks of SET versus DET in a single cycle, in infertile women using their own oocytes and embryos were deemed eligible for inclusion. To prevent any confusion between per person and per cycle, studies were excluded if it was not possible to clarify that each woman was included only once.A systematic electronic literature search was performed in PubMed, Embase, Cochrane Library, and RCT registries including ClinicalTrials.gov and WHO International Clinical Trials Registry Platform, through to February 9, 2021. The bibliographies of relevant studies and reviews were scrutinized for any additional eligible studies not covered by the literature search. The literature search combined the terms and descriptors related to human embryo transplantation concerning literature published in English , population , cycle , comparison categories and clinical outcomes  were extracted onto a piloted structured form by two reviewers, independently. The most comprehensive report would be given precedence if there were multiple publications from the same study or data source, while the others might be used as supplementary information. When studies had multiple comparisons, only the information and data of interest were extracted. Any uncertainty or disagreements were resolved through discussion and, if necessary, a consensus could be reached with the help of senior authors.< 35, 35-40, ≥40 years). Moreover, sensitivity analyses pooled of adjusted ORs, limited to first cycle or elective SET (eSET) cycles were performed to assess the robustness of the findings. Pooled effect size was deemed statistically significant at P < 0.05. Most data were dichotomous, we used the numbers of events in the groups of each study to calculate the ORs with 95% CIs. For continuous parameters, all the units had been harmonized by data conversion prior to analysis, the weighted mean differences (WMD) with 95% CIs were pooled to determine the effect size [I2 statistic [The quality of included RCTs was assessed using the Cochrane risk of bias tool . The Newtatistic . Publicatatistic . The leaThe literature search retrieved 14,938 citations. After removing duplicates, 11,847 abstracts were reviewed and 1071 full-text articles were further assessed for eligibility. Finally, 85 articles , 38–101 I2 = 91%, P < 0.001) .Sixty-two studies , 94, 99 I2 = 85%, n = 12) and 35–40 years , but no difference was observed for patients ≥40 years old , as well as a single PQE compared with two PQEs , 0.81 , 0.80  and 0.74 , respectively. Sub-analysis concerning study design indicated that the combination of RCTs resulted in a lower OR , compared with the result obtained from observational studies .Among subgroup analyses, when considering different age groups of included patients, an increase in LBR favoring the DET group was noted for patients aged < 35 years  .Forty-five studies –96, 98 wI2 = 0%, P < 0.001, n = 11) and 35–40 years , whereas the aggregate result from three studies found that the difference became insignificant in patients aged ≥40 years . Similarly, 10 observational studies investigated the differences in the association between MPR and the number of transferred embryos by embryo quality grade  and 0.12 , respectively. However, the difference in MPR between a single PQE and two PQEs was reduced and was no longer be statistically significant . Moreover, the pooled results did not materially change in the subgroup analyses regarding embryo stage , cycle type  and study design , with pooled ORs being 0.05 (P < 0.001) or 0.06 (P < 0.001).In the subgroup analyses of age stratification Fig. , the difTable I2 = 82%, P < 0.001, n = 60) was noted in the SET group compared with the DET group [I2 = 70%, n = 5), GQE versus GQE + PQE , and frozen cycles , neither in all the conducted subgroups . Compared with DET, mothers with SET had a lower risk of cesarean section . Only one included study [P = 0.623) and PE , meanwhile, APH rate was significantly lower in the SET group .A significant decrease in CPR . Moreover, 13 studies [I2 = 0%, P < 0.001). The overall findings did not materially change in all the conducted subgroup analyses . The pooled analysis of low birth weight rate generated a total of 3962 live births from nine studies [2 = 0%, P < 0.001), the finding was further confirmed by all the conducted subgroup analyses (Supplementary Table P = 0.414), while two other studies [I2 = 0%, P = 0.042, n = 2) and NICU admission rate .Eight studies , 65, 81, studies , 81 eval studies , 77, 81,P < 0.001, n = 12), limited to first cycle  and eSET cycles  confirmed the stability of the overall result , while the sensitivity analyses limited to first cycle  and eSET cycles  confirmed the stability of the overall result , no differences were found in all the other sensitivity analyses  of SET versus DET were consistent with current evidence. SET yielded less probability of a live birth  than DET in a single cycle, while simultaneously reducing the rate of multiple gestation . By contrast, the present study provided more comprehensive subgroup/sensitivity analyses and tracked more adverse fertility outcomes, further promoted the generation of individual program. Interestingly, changes emerged during several subgroup comparisons.An important factor that needs to be considered during embryo transfer is maternal age, particularly given the age-dependent decrements in ovarian function . RecruitRegarding embryo quality, compared with two GQEs, transferring a single GQE gained a significantly lower possibility of multiple pregnancy , as well as a lower rate of live birth . Since the chance of a live birth after a single PQE was only about 57% of that achieved by two PQEs, and there was no statistical difference in the MPR between groups, choosing to the DET might be beneficial in the absence of GQEs. The comparison between a single GQE and two embryos of GQE + PQE indicated that the PQE along with GQE did not improve the effectiveness of transfer, but significantly increased the risks of perinatal complications, including MPR, preterm birth rate and low birth weight rate. The interaction was proposed to explain that pre-implantation embryos could affect the development of surrounding embryos through the release of specific growth factors , 106. MoWith the apparent shift in the goal of ART, favorable perinatal outcomes have been regarded as crucial as a successful clinical pregnancy . CompareThe strength of this study is the big data from observational researches enabled us to address concerns about the generalizability of data from RCTs to routine clinical care . AdditioIn conclusion, based on the current evidence, SET yielded less probability of a live birth than DET in a single cycle, while simultaneously reducing the possibility of multiple gestation, as well as its related perinatal and neonatal complications. Subgroup analyses suggested that in women < 40 years old or any GQE is available, SET should be incorporated into clinical practice owing to the significant reduction in MPR and the acceptable LBR. Meanwhile, in the absence of GQEs, DET may be a preferable option because of the significant benefit of a live birth and the insignificant chance of multiple pregnancy. However, for elderly women ≥40 years old, current evidence is insufficient to recommend an appropriate number of embryos to transfer. Further high-quality RCTs or national registry-based cohort studies are still required to confirm these findings that will allow individualized transplantation strategies for different cohorts of infertile women.Additional file 1.Additional file 2: Supplementary Table S1. The characteristics of the included studies. Supplementary Table S2. Methodological quality of included studies. Supplementary Table S3. Sensitivity and subgroup analyses comparing clinical pregnancy rate after SET and DET in a single cycle. Supplementary Table S4. Sensitivity and subgroup analyses comparing miscarriage rate after SET and DET in a single cycle. Supplementary Table S5. Sensitivity and subgroup analyses comparing preterm birth rate after SET and DET in a single cycle. Supplementary Table S6. Sensitivity and subgroup analyses comparing low birth weight rate after SET and DET in a single cycle.Additional file 3: Figure S1. Forest-plot comparing live birth rate after single embryo transfer (SET) and double embryo transfer (DET) in a single cycle.Additional file 4: Figure S2. Forest-plot comparing multiple pregnancy rate after single embryo transfer (SET) and double embryo transfer (DET) in a single cycle.Additional file 5: Figure S3. Forest-plot comparing the clinical pregnancy rate between single embryo transfer (SET) and double embryo transfer (DET) based on maternal age and embryo quality stratification. G/GG, a single good quality embryo (GQE) versus two GQEs; P/PP, a single non-good quality embryo (PQE) versus two PQEs; G/GP, a single GQE versus two embryos of mixed quality (GQE + PQE).


Colloid cyst of third ventricle is a rare, benign, congenital lesion that usually presents with headache, and associated with altered cognition, nausea, vomiting, gait ataxia, and blurred vision. A large cyst/growing cyst can cause obstructive hydrocephalus leading to acute rapid neurological deterioration and sudden death.Here we report a classic clinical presentation and histopathological features of colloid cyst of third ventricle with specific emphasis on the importance of rapid diagnosis and management to avoid potentially fatal complications of this otherwise benign lesion. Newer modalities like neuroendoscopy or stereotactic aspiration of cyst are now the preferred choices of management.Awareness of this entity for early diagnosis and management with minimally invasive procedures such as neuroendoscopy or stereotactic aspiration of cyst is crucial for better prognosis and patient care. Colloid cysts of third ventricle are rare, benign, congenital lesions that comprise 0.5-2.0% of all intracranial lesions/tumors and 10-20% of all intraventricular lesions/tumors with an incidence of 3.2 cases per 100,000 people . Of all  A 24-year-old man presented with a short history of progressive headache, which was holocranial in nature with a feeling of heaviness, particularly aggravated in early morning since 15 days. This was associated with on and off type of vomiting, which did not subside with medication. He also complained of dizziness since five days. Patient was a known case of migraine, on treatment. There were no complaints of blurring of vision, seizures, or focal neurological deficits. Routine hematology and biochemical investigations were done and were normal.Patient was immediately taken for further radiological investigation. Non-contrast CT brain was performed and it showed a well-defined rounded hyperdense cystic lesion measuring 1 x 1 cm in the roof of third ventricle associated with mild to moderately dilated bilateral lateral ventricle. There was no evidence of periventricular cerebrospinal fluid ooze or intraventricular hemorrhage Figure , 1B. ResIntraoperatively cyst fluid was aspirated and was sent for fluid cytology. We received 0.5 mL slightly hazy fluid. The centrifuged smears examined show thick amphophilic proteinaceous fluidic material in the background. There were scattered inflammatory cells consisting of histiocytes, lymphomononuclear cells along with cholesterol crystals. Features were consistent with benign cystic lesion Figure .Histopathological examination showed a thin fibro-collagenous cyst wall lined by pseudostratified-to-simple columnar epithelium, focally ciliated Figure . The cysPostoperative period was uneventful. On close follow-up, patient was getting symptomatically better and no repeat imaging was required in this case.Colloid cysts of third ventricle are rare lesions arising from ectopic endodermal migration in the velum interpositum or from the primitive neuroepithelium of the tela choroidea. These cysts are usually located in the rostral aspect of roof of third ventricle. They also project inferiorly to occur in the antero-superior quadrant adjacent to the foramina of Monro . ColloidSudden death has happened in about 10% of patients with colloid cyst of the third ventricle. Symptomatic colloid cyst of the third ventricle has the highest risk of acute deterioration in 34% cases with a mortality rate of 12% .Both CT and MR imaging can be used in the diagnosis of colloid cysts. On CT scans, colloid cysts are slightly hyperdense compared to brain, but may occasionally be hypodense or isodense. Most colloid cysts are rounded to oval. Contrast-enhanced CT shows thin rim of enhancement and it represents the cyst capsule. Using MR imaging, colloid cysts usually have a variable appearance. MR imaging shows intracystic fluid levels or central and peripheral components in the lesion. Radiological differential diagnoses include small dermoid, craniopharyngioma, ependymoma, or an intraventricular glioma .On histopathological examination, colloid cysts are lined by pseudostratiﬁed epithelium along with scattered ciliated cells, interspersed with mucous goblet cells. The cyst content is amorphous periodic acid-Schiff positive. Ultra-structurally ciliated cells and non-ciliated cells having microvilli, basal cells, goblet cells with secretory granules, and undifferentiated cells with scanty organelles may be seen. These ciliated cells are endodermal in origin but the topographical distribution of the ciliated epithelial cells suggests respiratory origin and distally tapering cilia suggests olfactory origin. Histopathological differential diagnosis includes ruptured epidermoid cyst, choroid plexus papilloma, or degenerated choroid plexus .Some authors have suggested that cyst more than 1 cm should be surgically removed and neither the size, duration of symptoms, or ventricular dilatation seem to be reliable predictors. Microsurgical approaches like trans-callosal, trans-ventricular, and transcortical were traditionally the mainstay of treatment for colloid cysts. Newer modalities like neuro-endoscopy and stereotactic aspiration of cyst are minimally invasive procedures. Prognosis is excellent, if surgically managed on time .Thus, awareness of this entity for early diagnosis and management with minimally invasive procedures such as neuroendoscopy and stereotactic aspiration of cyst is crucial for better prognosis and patient care. Location, size, and direction of growth determine the prognosis of neglected cases. Preoperative and intraoperative diagnoses play a pivotal role. The entity of colloid cyst of third ventricle should always be considered in cases of acute headache, if other possibilities are ruled out. This also warrants further study to figure out the optimum timing, for surgical intervention in asymptomatic patients.


Previously we observed that maternal multiple micronutrient supplementation in pregnancy was associated with increased offspring size at birth and adiposity, as well as with maternal gestational diabetes risk, in the Cambridge Baby Growth Study. In this study we therefore investigated whether folic acid supplementation specifically is associated with similar changes, to test the hypothesis that folic acid supplementation mediates such changes.The majority of mothers who reported supplementing with folic acid in pregnancy  did so either from pre- (n = 139) or post-conception (n = 637) largely for all or just the first half of pregnancy. A minority of mothers (n = 198) reported not supplementing with folic acid. Folic acid supplementation in pregnancy was not associated with birth weight , height , head circumference  or adiposity . Neither was it associated with the development of maternal gestational diabetes . These results suggest that folic acid supplementation in pregnancy did not mediate the previously observed increases in offspring size at birth and adiposity, or the raised gestational diabetes risk, in response to supplementation with multiple micronutrients. Multiple micronutrient supplementation in pregnancy leads to a reduced risk of the baby being born with a low birth weight, and possibly one for being born small for its gestational age (SGA) . ConsistIn iron replete women in the U.K. the only micronutrients that are specifically recommended to supplement dietary intake with during pregnancy (and whilst attempting to get pregnant) are folic acid (at a dose of 400 µg per day) and, in certain months of the year, vitamin D , 6 and dThe initial phase of the CBGS involved recruiting 2229 participants from 2001–9 and has been described in detail elsewhere . Around From all the women recruited to the CBGS during this time the following were excluded from the present analysis: those that had already withdrawn from the study before the birth of their baby, twin pregnancies , and those that did not fill in  and return their pregnancy questionnaire.Offspring birth weights were recorded from hospital notes. Other newborn measurements  were made by trained paediatric nurses as soon as possible after birth [at a median (inter-quartile range) age of 2 (1–16) days], as described . Low birThis analysis, using results from the CBGS cohort, was run in an unmatched exposed v. non-exposed format as per that of our multiple micronutrient supplementation study . The exp2 or Fisher’s exact tests (as appropriate). Continuous variables were analysed using linear regression, adjusted for confounders where appropriate. Where the dependent variable residuals were skewed, the models were analysed with prior transformation of the data so that they were normally distributed. Missing data were treated by case or listwise deletion. P < 0.05 was considered statistically significant throughout. Stata  was used to perform the statistical analyses.Risk ratios of adverse pregnancy outcomes by folic acid supplementation were analysed by log-binomial regression. Other categorical variables were analysed using χThis analysis included 974 pregnancies: 776 with mothers that supplemented with folic acid and 198 that did not. Of those that supplemented with folic acid, most (n = 526) did so via the form of multiple micronutrient supplementation  could have introduced biases to the study.The lack of significant statistical associations observed could theoretically be caused by insufficient statistical power if the true effect sizes were very small . However, they still clearly do not explain the significant associations detectable with multiple micronutrient supplementation in pregnancy .


Litopenaeus vannamei (LvRab11). The interaction of 14-3-3ε and LvRab11 was confirmed by a GST pull-down assay. The LvRab11 open reading frame was 645 bp long, encoding a protein of 214 amino acids. Possible complexes of 14-3-3ε isoforms and LvRab11 were elucidated by in silico analysis, in which LvRab11 showed a better binding energy score with 14-3-3EL than with 14-3-3ES. In shrimp challenged with the white spot syndrome virus (WSSV), the mRNA expression levels of LvRab11 and 14-3-3ε were significantly upregulated at 48 h after challenge. To determine whether LvRab11 and binding between 14-3-3ε and LvRab11 are active against WSSV infection, an in vivo neutralization assay and RNA interference were performed. The results of in vivo neutralization showed that LvRab11 and complexes of 14-3-3ε/LvRab11 delayed mortality in shrimp challenged with WSSV. Interestingly, in the RNAi experiments, the silencing effect of LvRab11 in WSSV-infected shrimp resulted in decreased ie-1 mRNA expression and WSSV copy number. Whereas suppression of complex 14-3-3ε/LvRab11 increased WSSV replication. This study has suggested two functions of LvRab11 in shrimp innate immunity; (1) at the early stage of WSSV infection, LvRab11 might play an important role in WSSV infection processes and (2) at the late stage of infection, the 14-3-3ε/LvRab11 interaction acquires functions that are involved in immune response against WSSV invasion.The 14-3-3 proteins interact with a wide variety of cellular proteins for many diverse functions in biological processes. In this study, a yeast two-hybrid assay revealed that two 14-3-3ε isoforms (14-3-3ES and 14-3-3EL) interacted with Rab11 in the white shrimp  More than one hundred 14-3-3 binding partners are involved in cell cycle regulation, apoptosis, signal transduction, protein trafficking and stress responses3. A number of 14-3-3 isoforms are found in various organisms. In shrimp, two 14-3-3 epsilon (14-3-3ε) isoforms, 14-3-3ES and 14-3-3EL have been reported in Litopenaeus vannamei4. The expression of 14-3-3ε mRNA increases significantly after white spot syndrome virus (WSSV) infection. However, the function of 14-3-3 in shrimp is still unclear. In Drosophila melanogaster, 14-3-3ε is required for the Rab11-positive vesicle function, which in turn enables antimicrobial peptide secretion during an innate immune response5. 14-3-3ε mutants were present in accumulations of small Rab11-positive vesicles near the plasma membrane, but RNAi silencing of Rab11 significantly blocked the anterograde delivery of an antimicrobial peptide (drosomycin) from the perinuclear region to the plasma membrane.The 14-3-3 protein family is highly conserved and expressed in all eukaryotic organisms. 14-3-3 proteins are known for their ability to bind multiple cellular proteins6. Of the large number of Rab proteins, 11 isoforms are known to be involved in the immune response endocytic pathway, namely, Rab3a, Rab4a, Rab4b, Rab5a, Rab5b, Rab5c, Rab7a, Rab9a, Rab9b, Rab11a and Rab11b7. Rab11 mediates several cellular processes that involve intracellular vesicle trafficking, including the delivery of plasma membrane proteins to specialized sites and the secretion of various factors. Rab11-mediated trafficking events were reported to have a role in several aspects of innate immunity8, in Macrobrachium rosenbergii, MrRab11A was involved in antibacterial and anti-WSSV innate immunity in prawns9. Moreover, in Penaeus monodon, PmRab11 was required for yellow head virus (YHV) infection10.Rab proteins constitute the largest group of the Ras superfamily of small G-proteins found to be present in all eukaryotesL. vannamei using in vivo, in vitro and in silico analyses. In addition, molecular biological experiments were used to detail the characteristics and innate immune response function of LvRab11 in L. vannamei during viral infection.In the preliminary investigation of the function of 14-3-3ε in shrimp, a yeast two-hybrid assay was used to identify the binding ligands of the isoform, and Rab11 was found to be one of the binding partners. Therefore, this research focused on determining the relationship between 14-3-3ε and LvRab11 in Rab11. To determine the interaction, BD-14-3-3ES, BD-14-3-3EL and AD-LvRab11 were cotransformed into yeast cells. BD-14-3-3ES, BD-14-3-3EL and AD-LvRab11 protein interactions manifested as blue colonies on a high-stringency medium containing X-α-Gal, while a lack of interaction led to no growth  to detect functional sites of LvRab11. The search revealed a 14-3-3-binding phosphopeptide motif (129-RHLRSVP-135) in the LvRab11 protein  protein as the receptor molecule. The one-on-one simulation results found that the LvRab11/14-3-3EL complex shows a higher binding energy score of interaction than LvRab11/14-3-3ES, with the centre binding energy and the lowest binding energy being − 1000.60 kcal/mol and − 1021.10 kcal/mol, respectively, whereas the values for LvRab11/14-3-3ES were − 751.00 kcal/mol and − 867.70 kcal/mol, respectively  14-3-3ES/ES:LvRab11, 2) 14-3-3EL/EL:LvRab11, and (3) 14-3-3EL/ES:LvRab11  was identified and characterized (GenBank accession no. MW429225). The sequence of LvRab11 cDNA was 645 bp long, encoding 214 amino acids. The amino acid sequence of LvRab11 shared the highest similarity  with Rab11 from P. monodon (GenBank accession no. ASW35116.1), M. rosenbergii (GenBank accession no. NP_173136) and D. melanogaster (GenBank accession no. BAA21708), respectively. Alignment of the LvRab11 protein and other Rab11 proteins indicated that LvRab11 possesses many structural features of the Rab11 subfamily. LvRab11 contained five highly conserved GTP-binding sites, five Rab family motifs, four Rab subfamily motifs (RabSF1-RabSF4), and a Cys–Cys motif at the carboxyl-terminus   Figs. b, 6b. WhAn in vivo neutralization assay was set up to determine whether LvRab11 and 14-3-3ε are involved in WSSV infection in shrimp. There was no shrimp mortality in the PBS-infected group, but shrimp infected with WSSV alone showed an increasing mortality as the test progressed and 100% mortality was reached at day 6. However, low mortality was observed in the three remaining groups at 6 days post injection. For LvRab11 group, mortality was 22% at day 6 and reached 53% at day10. For the complex of LvRab11/14-3-3ES, mortality was 36% at day 6 and 89% at day 10. The complex of LvRab11/14-3-3EL, 29% mortality was observed at day 6 and 78% mortality at day10  LacZ dsRNA + WSSV, (2)14-3-3ε dsRNA + WSSV, (3) LvRab11 dsRNA + WSSV, and (4) LvRab11 and 14-3-3ε dsRNA + WSSV is shown in Fig. LvRab11 in LvRab11-silenced group and in both knockdown groups were significantly lower than that in shrimp from the control LacZ dsRNA + WSSV group, and the 14-3-3ε dsRNA + WSSV group. Using 14-3-3ε dsRNA, the results demonstrated that the expression of 14-3-3ES and 14-3-3EL transcripts were significantly decreased in the 14-3-3ε dsRNA + WSSV group and in both LvRab11 and 14-3-3ε silenced groups. However, the expression of the LvRab11 transcript was not affected by 14-3-3ε knockdown.To investigate whether ie1 mRNA expression level and WSSV copy number in LvRab11 knockdown group were significantly decreased after 48 h post WSSV infection compared to the other groups. This contrasts with the result of both the knockdown groups (LvRab11 + 14-3-3ε) where ie1 transcript and WSSV level were significantly increased after WSSV infection when compared to the control group and the LvRab11 knockdown group. However, the 14-3-3ε knockdown groups revealed that ie1 mRNA and WSSV level were significantly increased when compared to the LvRab11 knockdown group but not significant when compared to the control group  and, consequently, have an effect on viral infection 27.The proteins of the 14-3-3 family play critical roles in a wide range of cellular functions through interactions with client proteins. Their crucial role in health and disease is suggested by the association of 14-3-3 proteins with the regulation of a wide range of general and specific signalling pathwaysL. vannamei4. However, the function of 14-3-3ε that is required depends on the type of cell in which 14-3-3 is present. In this study, to understand the role of the 14-3-3ε isoforms, the binding partners during virus infection were investigated. The results obtained from the yeast two-hybrid (in vivo) and GST pull-down (in vitro) assay revealed that LvRab11 interacted with both 14-3-3ε isoforms. Two high-affinity phosphorylation-dependent 14-3-3 binding motifs, RSXpSXP (mode 1) and RXXXpSXP (mode 2), have been reported29. Analysis of the LvRab11 amino acid sequence revealed a 14-3-3-binding phosphopeptide motif (129-RHLRSVP-135) belonging to mode 2. These results imply that the 14-3-3ε isoform can bind both phosphorylated and nonphosphorylated LvRab11. Although most 14-3-3 proteins have been shown to interact with phosphoserine-containing peptides30, there are also several reports showing an interaction between 14-3-3 proteins and nonphosphorylated ligands32. There are structural similarities between the phosphorylated and nonphosphorylated ligands.In our previous study, 14-3-3ε formed both heterodimers and homodimers. The expression profiles of the two 14-3-3ε isoforms were altered after viral infection, indicating that the shrimp 14-3-3ε isoform is probably related to cellular processes that are often modulated during viral infection in 33.In the possible complexes of 14-3-3ε isoforms and LvRab11 obtained from our in silico analysis, LvRab11 molecules occupied most of the central space in the channel of the 14-3-3ε dimers. However the binding energy score of LvRab11 was better with 14-3-3EL than with 14-3-3ES. The simulated complexes suggest that amino acid Met1–Val30 at the N-terminus of the 14-3-3 structure is the recognition region for self-interaction at amino acids Ala55–Asp95. Moreover, the amino acid residues 54–GARRASWRIISSIEQKE–70 of 14-3-3ES and 14-3-3EL also present a pseudosubstrate domain. The serine residue at the C-terminus (Ser257 of 14-3-3ES and Ser273 of 14-3-3EL) may facilitate the formation of an active and inactive structure of 14-3-3ε for binding with its binding partner. Both the N-terminus and C-terminus of LvRab11 displayed regions that may play key roles in interacting with the 14-3-3 protein. Near the C-terminus of the 14-3-3ε isoform, the EF-hand motif domain with the pattern “LSEESYKDSTLI” (at Leu209-Ile220 of 14-3-3ES and Leu225-Ile236 of 14-3-3EL) forms a helix-loop-helix topology and may support the interaction of the 14-3-3 protein with ligand molecules within the HF-loop. The helix-loop-helix topology strongly supports the crystal structure of the 14-3-3ε protein, which exhibits a highly helical and cup-shaped dimerLvRab11 was identified from the hemocytes of L. vannamei. Sequence analysis revealed that the LvRab11 protein contains five GTP-binding sites (G1-G5). The Ras superfamily has the ability to bind GTP34, and Rabs are distinguished from other members of the Ras GTPase family by five Rab family motifs (RabF1-5) and four Rab subfamily motifs (RabSF1-4)35. Moreover, the LvRab11 protein exhibited a prenylation site (CC) at its C-terminus; prenylation is a crucial post-translational modification that enables Rab proteins to associate with and target cell membranes35. Based on homology searches in the GenBank database, the LvRab11 protein was highly homologous with Rab11 of other species and had the closest phylogenetic relationship with PmRab1110 and MrRab11A9. Thus, the LvRab11 protein might be closely associated with biological functions in cellular processes in shrimp. LvRab11 expression was observed in all the tissues tested in the shrimp but at different levels. Hepatopancreas tissue is important for the production of immune response molecules, it does not only initiate humoral immune, but also play an important role in cellular immune response37. In hepatopancreas, LvRab11 and 14-3-3ε expression profile were significantly increased in the late stage of WSSV stimulation (48 hpi), therefore, we imply that LvRab11 and 14-3-3ε represents a supplemental immune molecule that functions in the late stage of WSSV infection. Several studies have shown that the expression level of 14-3-3 isoform was upregulated after virus infection, and overexpression of 14-3-3 protein could reduce the production of virus particles39. Santos et al.36 reported that genes upregulated in hepatopancreas and in healthy shrimp (shrimps exposed to the virus but not infected) are likely to be involved in response to WSSV and are central ones in keeping shrimps WSSV free. Whereas downregulation of LvRab11 in early stage in this tissue may imply that the virus may inhibit this gene to favor its replication in the initial stage40.44. In addition, many Rab proteins play critical roles in the assembly and passage of enveloped viruses from cells45. In humans, Rab11 proteins have been implicated in the replication of many viral pathogens. For example, respiratory syncytial virus, cytomegalovirus and human immunodeficiency virus require Rab11 and its adapter to assemble in host cells45. Several viruses also use Rab11-dependent pathways for transport50.We also found that LvRab11 alone and the complex of LvRab11 and 14-3-3ε reduced shrimp mortality through neutralizing the WSSV infection. In agreement with in silico data, this results of an in vivo neutralization showed that complex of LvRab11 and 14-3-3EL delayed shrimp mortality more than complex of LvRab11 and 14-3-3ES. Several studies have confirmed that Rab proteins play a significant role in the immune systemLvRab11 by RNAi resulted in a decreased ie1 mRNA expression and WSSV replication. The ie1 is an early-immediate transcription gene that is highly expressed throughout the WSSV infection cycle. It plays an important role in promoting viral infection and replication51. Down-regulation of ie1 gene and WSSV level after LvRab11 silencing may indicate the possible role of LvRab11 proteins in intracellular trafficking of WSSV in the early stage of infection. Similar results were reported in PmRab11 and PmRab7. Silencing PmRab1110, PmRab753 or LvRab754 resulted in inhibition of viral replication in shrimp. For instance, the co-localization between PmRab11 and YHV was observed in hemocytes at 24 to 72 h post viral injection, whereas in PmRab11 knockdown hemocytes, a low level of co-localization of PmRab11 and YHV was detected at the perinuclear region at 24–72 h. These results revealed that PmRab11 is required for YHV infection in shrimp cells10. In addition, silencing of PmRab7 resulted in significant inhibition of WSSV-VP28 mRNA and YHV replication, suggesting that PmRab7 required for WSSV and YHV infection in shrimp cell52.In our RNAi experiment, silencing 14-3-3ε alone resulted in increased ie1 mRNA expression and WSSV level in shrimp compared to LvRab11 knockdown group but not significant when compared to the control group. However, when knockdown of both LvRab11 and 14-3-3ε were performed, the result showed that ie1 transcript and WSSV replication were significantly increased when compared to LvRab11 knockdown and control groups. In the same way, with in vivo neutralization, Rab11 and the complex of Rab11 and 14-3-3ε can delay mortality of shrimp after WSSV infection. The result of which may imply that LvRab11 might also have a role in the shrimp defense mechanism when binding to 14-3-3ε in the late stage of infection. After a virus is infected into the host cell cytoplasm, viral molecules may trigger binding of 14-3-3ε especially 14-3-3EL to LvRab11. Then, the LvRab11/14-3-3ε interaction may activate LvRab11 to turn on the specific function to protect the cell against WSSV infection. In most cases, 14-3-3 proteins bind onto target proteins at the original site, and upon certain stimulations, the 14-3-3 proteins will then bring their target proteins to specific locations where the proteins can properly functions57. Some Rabs such as Rab6 have been shown to play important roles in WSSV infection. It plays a critical role in the regulation of phagocytosis against WSSV by the rearranging the actin fibers59. In addition, suppression of Rab6 by specific dsRNA has significantly increased WSSV copy number in shrimp61.The silencing Hence, we can hypothesize that LvRab11 may have two functions during WSSV infection; (1) In the early stage, LvRab11 plays a role in the endocytic process and it is required for WSSV infection and (2) In the late stage, the interaction between 14-3-3ε and LvRab11, 14-3-3ε may be an activating binding partner LvRab11 protein and contribute an immune response against WSSV invasion.5. There are some reports about Rab proteins interacting with 14-3-3, such as 14-3-3 being an interaction partner for AS160 (containing a Rab GTPase-activating domain), indicating an important role for this protein in insulin-regulated GLUT4 trafficking63.However, this is the first report of the interaction between 14-3-3ε and LvRab11 in shrimp. In Drosophila, 14-3-3ε is required for Rab11-positive vesicle function, which in turn enables antimicrobial peptide secretion during the innate immune responseIn conclusion, knowledge of the viral infection mechanism and the associated cellular response is very important and provides insight into host–pathogen interactions that can be used to develop an improved approach for viral infection prevention in shrimp. Although we found an interaction between 14-3-3ε and LvRab11 in this study that may be related to an immune response after viral infection, further elucidation of the cellular mechanisms is needed to better understand the role of these interactions during viral infection and pathogenesis in host cells.P. monodon, that was constructed in pGADT7 and used to screen for interaction, was introduced into Saccharomyces cerevisiae strain AH109 using the Matchmaker GAL4 Two-Hybrid System 3 . Transformed yeast cells were spread on synthetic dropout selection medium such as low-stringency (SD/-leucine/-tryptophan), medium-stringency (SD/-leucine/-tryptophan/-adenine) or high-stringency (SD/-adenine/-histidine/-leucine/-tryptophan) medium at 30 °C. The interaction produced blue colonies in a high-stringency medium containing X-α-Gal. The cDNA library plasmids from interaction-positive colonies were sent for DNA sequencing. The full-length LvRab11 was cloned into pGADT7 (pGADT7-LvRab11) to confirm its interaction with pGBKT7-14-3-3ES and pGBKT7-14-3-3EL. The cotransformation containing pGBKT7-53 and pGADT7-T was used as the positive control, and that of pGBKT7 with pGADT7- LvRab11 or pGADT7 was employed as the negative control.The recombinant plasmids pGBKT7-14-3-3ES and pGBKT7-14-3-3EL were obtained from previous studies. Here, a cDNA library from hemocytes of WSSV-infected LvRab11 from L. vannamei was amplified using degenerate primers designed according to the conserved sequences of Rab11 genes such as the Homo sapiens (GenBank accession no. X56740), Mus musculus (GenBank accession no. AB232605), D. melanogaster (GenBank accession no. D84315) and Danio rerio (GenBank accession no. NM_001007359) in the GenBank database (http://www.ncbi.nlm.nih.gov). LvRab11 was amplified by PCR using the Rab11-F and Rab11-R primers. The PCR cycles were conducted as follows: 95 °C for 5 min, followed by 35 cycles of 95 °C for 1 min, 52 °C for 1 min, and 72 °C for 1 min and 72 °C for 10 min. The PCR product was purified and ligated into the pGEM-T easy vector (Promega) and sequenced.A partial fragment of LvRab11 gene, the open reading frame (ORF) of LvRab11 was amplified with LvRab11-specific primers (fullLvRab11-F and fullLvRab11-R) using the PCR conditions were as described above. Sequences were analysed with the BLASTX program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The LvRab11 protein motifs were analysed by InterPro (https://www.ebi.ac.uk/interpro/search/sequence-search). All primer sequences are presented in the supporting information  were obtained from Songkhla, Thailand. Various tissues, including the hemocytes, hepatopancreas, gill, intestine, lymphoid, muscle and stomach, were collected for RNA extraction with TRIzol reagent (Invitrogen). First-strand cDNA was synthesized from 1 µg of total RNA using the SuperScript III First-Strand Synthesis System kit (Invitrogen) according to the manufacturer’s protocols. The tissue distribution of LvRab11 in normal shrimp and in WSSV-infected shrimp (48 hpi) were analysed by semi-quantitative PCR using the primers Rab11-RT-F and Rab11-RT-R. The PCR conditions were as described above. The β-actin gene was amplified as an internal standard. The band intensity of LvRab11 and β-actin in gel was measured by densitometry through ImageJ software 1.37v . The mRNA expression levels of LvRab11 were normalized against those of β-actin.Healthy 3 copies). In the control group, 100 µl of PBS were injected into each shrimp. The expression pattern of the LvRab11 and 14-3-3ε genes in the hepatopancreas of WSSV-infected shrimp was investigated at 6, 12, 24, and 48 h to study the role of the gene in the innate immunity of L. vannamei. Reverse transcription quantitative real-time PCR (RT-qPCR) was performed using the CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories) with SensiFAST SYBR No-ROX Mix (Bioline). The thermal cycling profile used was 95 °C for 3 min, followed by 30 cycles of 95 °C for 30 s, 52 °C for 30 s (for LvRab11) or 60 °C for 30 s (for 14-3-3ES and 14-3-3EL) and 72 °C for 30 s. An additional temperature ramping step from 55 °C to 95 °C was utilized to produce melting curves for the reaction. EF-1α gene was amplified as an internal standard, and relative fold changes were calculated using the the formula 2−ΔΔCT method.The shrimp were divided into the control and WSSV-challenged groups. The shrimp in the WSSV-challenged group were injected with a 100 µl suspension of WSSV  vector (Novagen) that already contained an N-terminal 6xHis tag. The protein was expressed in Escherichia coli strain BL21 (DE3) and purified using HisPur Ni–NTA Superflow Agarose (Thermo Scientific) following the manufacturer’s instructions. GST-fusion 14-3-3ε proteins were subcloned into the pGEX-4T-1 vector (Amersham Biosciences). The recombinant GST-14-3-3EL and GST-14-3-3ES proteins were produced in E. coli strain BL21 and purified using Glutathione Sepharose 4 Fast Flow  following the manufacturer’s instructions. Purified recombinant proteins were analysed by SDS-PAGE. The protein concentration was measured with a dye-binding assay by the Bradford method and stored at − 20 °C.In the pull-down experiments, 30 µg of GST and the GST-14-3-3ES and GST-14-3-3EL fusion proteins were bound to glutathione Sepharose beads and washed five times with 1x PBS. Thirty micrograms of 6xHis-LvRab11 were incubated with GST or one of the GST-fusion proteins for 1 h. The complexed beads were washed ten times using 1x PBS. Finally, 5 × SDS sample loading buffer was added to the complexed beads, and the samples were boiled for 5 min.The proteins were separated by SDS-PAGE, and proteins were transferred to nitrocellulose membranes and blocked with 5% skim milk. The proteins were detected using the GST tag as follows. The membrane was incubated with primary antibody  for 1 h, washed three times with 1x PBS, incubated with alkaline phosphatase-conjugated secondary antibody , 1:10,000) for 1 h, and washed three times with 1x PBS. The substrate (NBT/BCIP) was added to the membrane and the proteins were visualized by color detection.Western blotting was performed according to standard procedures. The proteins were detected by using the His tag as follows. The membrane was incubated with mouse anti-His primary antibody  and goat anti-mouse horseradish peroxidase (HRP) secondary antibody . The ECL substrate was added to the membrane and the protein bands were visualized by exposing X-ray film to the membrane.https://swissmodel.expasy.org)13, the threading-based LOMETS resource on the I-TASSER server (https://zhanglab.ccmb.med.umich.edu/I-TASSER)16, and the pDomTHREADER search method on the PSIPRED server (http://bioinf.cs.ucl.ac.uk/psipred)18, were used for predicting the three-dimensional (3D) structures of the 14-3-3ε isoforms and the Rab11 protein. The online docking servers ClusPro 2.0 (https://cluspro.bu.edu)20, AutoDock suite (available for free download at http://autodock.scripps.edu)21 and AutoDock Vina (available for free download at http://vina.scripps.edu)22 were used for the lock-and-key model analysis of the interaction sites of the 14-3-3 isoforms and the Rab11 protein. The PyMol molecular viewer was used to visualize and analyse the details of the 3D structure models64.Freely available online tools, namely, the SWISS-Model server (L. vannamei specimens (approximately 3 g) were acclimated for at least 3–5 days. They were then divided into 5 groups (15 shrimps per group) for hemocoel injection with (1) 1x PBS as negative control, (2) WSSV (105 copies/shrimp) as positive control, (3) LvRab11 (5 µg/shrimp) plus WSSV, (4) LvRab11 (3 µg/shrimp) and 14-3-3EL (3 µg/shrimp) plus WSSV, and (5) LvRab11 (3 µg/shrimp) and 14-3-3ES (3 µg/shrimp) plus WSSV. Each group was done in triplicates. The experiments were carried twice. The shrimps were kept under standard conditions and mortality was recorded daily for 10 days.His-14-3-3ES, His-14-3-3EL and His-LvRab11 were expressed and purified in order to use in the in vivo neutralization assay. Before starting the experiments, the 3 copies of WSSV particles, and again 48 h later gill and pleopod tissues of shrimps were dissected and used to examine ie1 mRNA expression and the virus copies by RT- qPCR with specific primers  with a volume of 20 μl comprised of 200 ng cDNA, 10 μl of 2x SensiFAST SYBR No-ROX Mix (Bioline), and 400 nM of each primer , and cDNA was synthesized as mention above. RT-qPCR was conducted to detect the mRNA levels of 65. Briefly, VP28 gene was cloned into pGEM-T Easy vector (Promega). The quantification of purified recombinant plasmids were measured by NanoDrop 2000 spectrophotometer (Thermo Scientific) and its copy number was calculated. A ten-fold serially diluted solution of the plasmid DNA was used as standard sample to generate a standard curve in RT-qPCR, performed with VP28-F and VP28-R primers  following recommended protocols. Then, this DNA was used to evaluate the WSSV copy number using method as previously describedt-test. A measurement of p < 0.05 was accepted as statistically significant.The data are presented as the means ± standard deviation (SD) and were analysed using one-way ANOVA followed by an unpaired, two-tailed Supplementary Information.


Preterm labor is defined as a birth before 37 weeks of gestation and occurs in 5–20% of pregnancies. Preterm labor, as multifactorial entity associated with a high risk of neonatal morbidity and mortality, is influenced by maternal, fetal and environmental factors. Microbiological studies suggest that infectious pathogens may account for 25–40% of preterm birth. Infections of different sites, like genital, urinary tract infections, and pneumonia, are linked to the preterm labor. The most recent epidemiological studies consistently report that maternal periodontal disease is associated with preterm delivery, as well as the association between the presence of pathogenic oral bacteria in the placenta and adverse pregnancy outcomes. On the other hand, some previously published papers found periodontal bacteria in placentas of term pregnancies. In spite of a huge research done on the topic, both experimental and clinical, there are many controversial opinions about the role of periodontal infections in preterm birth. Thus, this comprehensive review addresses this very important topic and evaluates novel strategies of preventive and therapeutic approaches. Preterm labor is defined as a birth before 37 weeks of gestation and occurs in 5–20% of pregnancies ,2. It isPreterm births account for almost 75 percent of perinatal mortality and more than 50 percent of the long-term morbidity ,3,5,6,7.Preterm labor is a heterogeneous condition of multifactorial origin influenced by maternal, fetal and environmental factors . Due to The goal of this review is to evaluate the existing literature sources to identify the role of periodontal infection in the onset of preterm labor. Early identification of pregnancies at highest risk for preterm labor may help in the improvement of the existing and development of new therapeutic management options. It also could serve to improve strategies to prevent neonatal morbidity associated with preterm birth.www.cliniclatrials.gov, accessed on 29 August 2020). We used the medical subject heading (MeSH) term Premature Birth (MeSH Unique ID: D047928) in combination with Periodontal Abscess (MeSH Unique ID: D010508) and selected papers published in English, with no time restrictions about the year of publication. Titles and abstracts of studies retrieved using the search strategy, and those from additional sources, were screened independently by two review authors (M.T. and G.A.) to identify studies that potentially meet the objectives of this non-systematic review. The full text of the eligible articles was retrieved and independently assessed by other two review team members (S.T. and G.B.). Any disagreement between them over the eligibility of particular articles was discussed and solved with a third collaborator (A.S.L.). Two authors (G.A. and M.R.) independently extracted data from articles about study characteristics, populations, type of intervention and outcomes. Due to the nature of the findings, we opted for a narrative synthesis of the results from selected articles.A non-systematic review was performed through a search on the following databases: PubMed, EMBASE, The Cochrane Library , and Web of Science, research registers /Streptococcus agalacteae, Staphylococcus epidermidis, Chlamydia trachomatis, Micoplasma hominis and Ureaplasma urealiticum, Neisseria gonorrheae, Treponema pallidums, Trichomonas vaginalis, HIV, Hepatitis B and C, as well as bacterial vaginosis (BV). Some of them can be easily ruled out while others might remain undetected and a silent cause of infection.Multiple infectious agents could cause the vaginal infection and later via the above-mentioned routes the intrauterine/intra-amniotic infection: Mycoplasma spp., and, specifically, U. urealyticum [U. urealyticum often have spontaneous preterm labor or preterm premature rupture of membranes (PPROM) [The pathogens most commonly reported in the amniotic fluid are genital alyticum ,5,7. Wom (PPROM) . Importa (PPROM) ,5,22. Ma (PPROM) ,23,24. A (PPROM) ,23. BV h (PPROM) . Trichomonas vaginalis, Treponema pallidum, Neisseria gonorrheae, etc. are responsible for preterm birth [Trichomonas vaginalis seems to be responsible for some preterm labor cases with a relative risk (RR) of about 3 [Treponema pallidum, Neisseria gonorrheae and Chlamidia trachomatis are reported to be associated with preterm birth only in the presence of a maternal immune response with RR of 2 [It is not always clear whether other genital infections like rm birth ,5,25. Tr RR of 2 . MoreoveVaginal infections have been found to be linked with an increased risk of PPROM and preterm birth . Being iInfections of other sites, non-genital, like urinary tract infections, pneumonia, and periodontal infection, are also linked to the preterm labor ,10. SomeThere are two major pathways in biological mechanisms of APO related to oral pathogens defined by the consensus report from the joint European Federation of Periodontology/American Academy of Periodontology workshop on periodontitis and systematic diseases ,27: (1) Vaginal infections lead to the increased concentration of the inflammatory markers that can be identified in cervical and vaginal secretions and have been shown to be significantly associated with the preterm labor rates ,13,29. TWomen’s bodies undergo important adaptations in many organ systems and hormonal changes during pregnancy. As well as the other systems, gastrointestinal tract and the oral cavity as a part of it are also under this influence. In this period increased sensitivity to stimuli occurs in the gingiva . VomitinGingivitis is inflammation of the superficial gum tissue . GingiviThe decline and exacerbation in oral health during pregnancy depends on multiple factors . During As mentioned before, vomiting, especially during the first trimester of pregnancy, increases the acidity in the mouth. Because episodes of vomiting in cases of hyperemesis gravidarum are usually very frequent, the pregnant woman may not pay enough attention to oral care after each event ,31. SubsFor the past 30 years, it has been proposed that oral pathogens could act as an infectious reservoir and interfere with pregnancy outcomes. The first association between periodontal disease and preterm labor was investigated and reported in 1996 by Offenbacher . In latePeriodontal diseases represent one of the most common chronic infections in humans with a prevalence of 10 to 60% among adult population . PeriodoPeriodontitis is a chronic multifactorial inflammatory disease associated with dysbiotic plaque biofilms and characterized by progressive destruction of the tooth-supporting tissues . It is aPeriodontitis has been classified by different ways: (1) based on stages defined by severity, complexity and extent and distribution ; (2) based on grades that reflect biologic features of the disease including evidence of, or risk for, rapid progression, expected treatment response, and effects on general health  . There aPeriodontal pathology usually begins with a localized inflammation of the gingiva, called gingivitis, caused by dental plaques, microbial biofilms that form on the teeth and gingiva. If left untreated, the inflammation in gingivitis can lead to periodontitis . The infCampylobacter rectus, Fusobacterium nucleatum, Peptostreptococcus micros, Prevotella intermedia and Prevotella nigrescens. The biofilm is further colonized by the bacteria from the “red” cluster that are more aggressive, including Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola. Although many virulence factors of the pathogenic bacteria are already known, the exact contribution of each species in the development of periodontitis is still unknown [Periodontal disease initiates as infection caused by an overgrowth of certain bacterial species in the subgingival sites. However, most of the periodontal tissue destruction is caused by the persistent inflammation that develops as a host response ,10,46. THowever, microbial colonization and biofilm formation is just the initial step in the periodontal disease development. The disease develops when the host’s immune system overreacts to the presence of bacteria, a process called dysbiosis . This imApproximately, in 50% of cases of premature birth the etiological factors are known and infectious agents responsible for almost 75% of them. However, the other half of cases remain idiopathic ,52. Fusobacterium nucleatum, Treponema denticola, Tannerella forsythia, Campylobacter rectus, Eikenella corrodens, and Selenomonas sputigena [During pregnancy, due to hormonal changes, there may be a tendency towards development of periodontal disease. In particular, there is an increase of anaerobic gram-negative bacteria such as putigena ,51 that Fusobacterium nucleatum, Campylobacter rectus, Porphyromonas gingivalis, and Bergeyella spp. [Oral infections might be considered as one of the factors contributing to the preterm labor incidence, since commensal bacterial species of the oral cavity were found to be disseminating to the fetoplacental unit of women with term gestation and APO ,53. The lla spp. ,50.Many investigations have shown an association of periodontal disease, prematurity, and low birth weight ,36,38. OThe conclusions of many studies/systematic analyses could be contradictory due to the relative heterogeneity of studied populations according to ethnicity, different risk factors discussed, diversity in socio-economic and education levels, and periodontal status definition.During pregnancy, periodontal status changes. Due to the physiologic changes in the immune system, pregnant women are more prone to have inflammatory conditions and increased rate of gingival bleeding. It appears that pregnancy might lead to an increase of periodontal disease severity in women suffering from periodontitis before pregnancy ,10,22,55Pregnancy related nausea and vomiting are relatively frequent in early pregnancy. As a consequence, gastric acid would damage periodontal tissue barriers to various pathogens, which could be responsible for placental infection and systemic inflammation leading to preterm birth .Furthermore, hormonal fluctuations during pregnancy have been proposed to initiate changes in the composition of oral biofilm leading to exacerbation of gingival inflammation. Researchers discuss that hematogenous dissemination of microbes and pro-inflammatory mediators from sites of periodontal infection into the placenta, fetal membranes, and amniotic cavity induces pathological processes that lead to APO . HoweverThe biological plausibility of association between periodontal disease and APO is based in a hypothetical model of two major pathways (direct and indirect) responsible for the pathologic process ,60,61,62P. gingivalis induced preterm birth and low birth weight in pregnant mice and significantly elevated maternal levels of circulating TNF-α, IL-17, IL-6 and IL-1β [P. gingivalis in the placenta of P. gingivalis-infected pregnant mice [Relationship between pro-inflammatory cytokines, periodontal infection, and preterm birth is recognized in both experimental conditions and animal models. Most animal models show evidence of the causal link between APO and periodontal infection. In a study by Ao et al. 2015), nd IL-1β . Moreoveant mice . Some stant mice . On the ant mice . Another, nd IL-1ant mice . Some authors identified periodontal disease as the cause of more than 18% of all preterm birth cases. Therefore, there is a significant evidence that periodontal pathogens, its enzymes and toxins can induce inflammation in placental tissues and cells ,67. ThusThere is no evidence-based effective guideline developed for periodontal infection treatment in pregnancy. In general population the aim of periodontal treatment is to reduce periodontal tissues infection through a careful and continuous oral hygiene education, medical and a mechanic  treatment. In the severe chronic or aggressive forms of periodontitis the treatment includes the administration of systemic antibiotics . Pregnancy related guidelines on oral care varies from country to country. In some countries, recommendations on oral care and treatment are included in the pregnancy follow-up guidelines . In the A meta-analysis by Cochrane Oral Health Group has assessed the effects of treating periodontal disease in pregnant women in order to prevent or reduce perinatal and maternal morbidity and mortality . There wIn the Obstetrics and Periodontal Therapy (OPT) randomized controlled trial performed almost 15 years ago, periodontal treatment was not found to be associated with the increased risk of preterm labor . ContrarPublished guidelines on oral care during pregnancy highlight that it is an underdeveloped area and the relevance of oral hygiene during pregnancy is insufficiently acknowledged by dentistry and obstetric specialists. Recently published overview of systematic reviews demonstrates strong evidence for a link between periodontal disease and APO ,36,37,55The most important steps of fetal organogenesis take place in the first 12 weeks of gestation and is very sensitive to the influence of external factors, especially drugs. During the first trimester, only emergency dental treatment is indicated . Oral hyRegarding the systemic treatment with medications/antibiotics during pregnancy, the main concern is the possible teratogenic effect that might appear. Before prescribing a drug to a pregnant patient, the dentistry specialists follow the Food and Drug Administration (FDA) classification for the prescription of drugs to pregnant women based on the risk of adverse fetal outcomes ,78.As recommended by the recent guidelines, it is appropriate to undergo dental examination as a part of preconception counselling in order to prevent dental and periodontal diseases . The maiHaving the global burden of periodontal disease and the range of APO that have been associated with it, there is still a need to describe the role of local periodontal bacteria in the placenta and oral cavity in relationship to local and systemic inflammatory cytokines for the pathogenesis of preterm labor. Additionally, from the public health point of view, identification of the underlying mechanisms will enable development of preventive strategies aimed at reducing of preterm birth, as the most important APO. Thus, there is a need to establish a new preventive and therapeutic approaches at earlier stages of pregnancy, perhaps even for the period of preconceptional counselling.According to some authors, appropriate, timely and adequate periodontal treatment performed during pregnancy appears to be safe for the mother and the fetus ,52. It mFuture studies that will aim to confirm the positive association between periodontal disease and preterm birth should utilize a more thorough methodology, including the appropriate definition of the condition, adequate control of confounders for preterm labor, and application of effective interventions to eliminate periodontal infection . Multiple studies provide strong evidence role of the periodontal infection in preterm labor. Furthermore, conclusions of many studies reported that periodontal pathogens can be transferred from periodontal tissues to the feto-placental site and cause an infection. However, the exact mechanisms by which inflammation disseminated from the oral cavity to the placenta contribute to adverse pregnancy outcomes remain unclear and require further investigation. Moreover, there are many pieces of evidence supporting the importance of the establishment a prevention program to minimize problems during pregnancy. Prevention means scheduling the preconception counselling visits, timely examinations and reducing the presence of bacterial plaque, through professional hygiene sessions, education, motivation to proper oral hygiene and nutrition. For these reasons, it is essential to develop a more effective interdisciplinary collaboration between obstetricians and dentistry specialist in order to achieve an optimal health for pregnant women.