The highest specificity was achieved by the prothrombin time (69%), and the HALP score was also 69%. The FAR score had an AUC of 0.646, with a sensitivity of 68%, and specificity of 64% . All other variables were significant estimates for the risk of preterm birth, although with lower accuracy. Pregnant women with a hemoglobin level below 12.0 g/dL had a 3.28 higher likelihood of giving birth prematurely. A prothrombin time below 12.5 s determined a 2.11 times higher risk of preterm birth. Similarly, the aPTT below 25 s was linked with 3.24 higher odds of giving birth prematurely. However, the strongest predictors were the D-dimers above 250 ng/mL (OR = 4.26), the FAR score below 0.1, with an odds ratio of 5.30, and the HALP score with a 6.09 OR for a cut-off value above 24. It is important to determine these parameters in pregnant women at risk for giving birth prematurely, but further external validation is required to confirm these findings.Premature birth is a worldwide health issue, posing a high mortality risk for newborns, as well as causing emotional and financial difficulties, and long-term health issues for patients. Identifying effective predictors for preterm birth is essential for prolonging gestation or improving obstetric care. As invasive methods are costly, risky, and not universally available, we aim to assess the predictive capacity of various serum parameters in pregnant women during the third trimester, as a non-invasive alternative. Based on previous studies, it was hypothesized that hemoglobin, the association of hemoglobin, albumin, lymphocyte, and platelets’ (HALP) score, and coagulation parameters such as the prothrombin time (PT), activated partial thromboplastin clotting time (aPTT), D-dimers, and fibrinogen to albumin ratio (FAR) have significant prediction capabilities. With a retrospective design, a total of 161 patients with a history of preterm birth were included in the analysis, being matched 1:1 with a control group of women who gave birth at term. All laboratory samples were collected during the third trimester of pregnancy. The computed area under the curve (AUC) ranged between 0.600 and 0.700 in all six studied parameters, suggesting a fair discrimination. The highest predictive value for preterm birth was observed to be represented by the HALP score with AUC = 0.680 and the highest sensitivity (75%,  Premature birth, which is considered as giving birth before 37 weeks of pregnancy, is a global issue of the maximum importance . It impaIncreasing data reveal that preterm birth is a condition caused by the interaction of various variables, including heredity, inflammation, disturbance of maternal hormones, placental vascular abnormalities, and numerous more hypotheses ,8,9. DueSo far, the use of non-invasive methods to determine the risk of premature birth was established on the accuracy of serum parameters responsible for anemia and different scoring methods based on the inflammatory status of the pregnant woman, such as the Neutrophil–Lymphocyte Ratio, Monocyte-to-Lymphocyte Ratio, Platelets-to-Lymphocyte Ratio, Systemic immune–inflammation index, or the systemic inflammatory response index ,15,16. TThe current study was designed as a retrospective cohort at the Clinic of Obstetrics and Gynecology “Odobescu” from Timisoara, Romania, in affiliation with the Victor Babes University of Medicine and Pharmacy and the Emergency Municipal Clinical Hospital from Timisoara, Romania. The research design and methodology were approved by the two affiliated institutions to collect, analyze, and publish patient data from 1 January 2020 until 31 December 2021, in accordance with the guidelines of the Declaration of Helsinki, with the approval number E-2814 from 19 May 2022.Patients considered for inclusion were pregnant women identified in the hospital database during the aforementioned study period. The inclusion criteria comprised the following: (1)—history of preterm birth; (2)—age over 18 years; (3)—consent for personal data utilization; (4)—complete medical records; (5)—all laboratory samples were measured during the third trimester of pregnancy. Among the third-trimester measurements in the prematurity group, one-third were in the early third trimester (between 28 and 31 weeks of gestation), and the other two-thirds were measured in the late third trimester, between 32 and 36 weeks of gestation. Patients were excluded from this study based on the following criteria: (1)—being underage; (2)—missing consent for private data utilization; (3)—incomplete medical records regarding the analysis of a complete blood count, albumin, and the coagulation parameters ; (4)—laboratory data from the first- or second-trimester of pregnancy. To avoid confounding factors, patients were also excluded if they had a diagnosis of coagulation disorders, or infections and inflammatory illnesses. Other exclusion criteria were the use of anti-inflammatory and corticosteroid medications, which might have affected the serum parameters during pregnancy. A control group of pregnant women without a history of preterm birth was collected and was matched with the patients from the preterm birth group based on age, number of gestations, and number of comorbidities at a 1-to-1 ratio, as described in According to existing guidelines, preterm delivery was considered when the birth happened before 37 weeks of gestation . The thiThe variables proposed for analysis in the current study included background characteristics of the pregnant women  and serum parameters .t-test, the mean and standard deviation of Gaussian variables were compared. The proportions of categorical variables were compared with the Chi2 test. The chance of preterm was analyzed and described by a Kaplan–Meier curve. To determine the risk of preterm delivery, a multiple linear regression analysis was conducted, that was adjusted for confounding factors. The risk of preterm birth prediction performance was examined using the receiver operating characteristic (ROC) curve method by calculating the area under the curve (AUC) and its significant related value. The ideal cut-off values for inflammatory indices were determined using Youden’s index.The sample size was determined using a convenience sampling method. It was calculated that 128 cases represent the ideal sample size, considering a rate of premature births in the general population of up to 10%, a margin of error of 5%, and a confidence level of 99%. The threshold for statistical significance was 0.05. The statistical power (1-β) calculation was 80%, for a type I error rate of 5%. Data were sorted by variables based on the type of their distribution and evaluated with the Kolmogorov–Smirnov test. Using the Student’s 2 vs. 24.3 kg/m2, p-value < 0.001). Another significant difference was the proportion of smokers, since 11.2% of women in the prematurity group were smoking during pregnancy, compared to 5.0% in the control group .At the end of the patient selection process, a total of 322 eligible pregnant women were included in the analysis with a complete background profile and laboratory profile. p-value = 0.003). The onset of preterm labor was at 35.3 weeks in the prematurity group, while the term labor occurred at around 37.9 weeks of gestation in the control group . On the other side, there were significantly more c-sections performed in the control group . It was also observed that pregnant women in the prematurity group suffered from urinary tract infections significantly more often than patients in the other group .Regarding the obstetrical characteristics, it was observed that premature rupture of membranes occurred significantly more often in the prematurity group . Additionally, hemoglobin levels were significantly decreased among women from the prematurity group . The coagulation parameters PT and aPTT were also decreased in the prematurity group  and 26.7 s vs. 27.9 s , respectively. The average albumin levels were 31.4 g/L among women who gave birth prematurely, compared with 36.6 g/L among those who gave birth at term . Fibrinogen and d-dimers, on the other hand, were significantly more elevated in the prematurity group  and 5.13 g/L vs. 3.10 g/L in the “no prematurity” group , respectively. The mean HALP score value among women who gave birth prematurely was 12.82, compared to 23.96 in the control group . The FAR score was above the threshold in the prematurity group .The serum parameters analyzed to assess the risk of preterm birth are presented in p-value = 0.001). The highest specificity was achieved by the prothrombin time (69%), and the HALP score was also 69%. The FAR score had an AUC of 0.646, with a sensitivity of 68% and specificity of 64% . All other variables were significant estimates for the risk of preterm birth, although with lower accuracy.p-value < 0.001). A prothrombin time below 12.5 s determined a 2.11 times higher risk of preterm birth . Similarly, the aPTT below 25 s was linked with 3.24 higher odds of giving birth prematurely. However, the strongest predictors were the D-dimers above 250 ng/mL (OR = 4.26), the FAR score below 0.1, with an odds ratio of 5.30, and the HALP score with a 6.09 OR for a cut-off value above 24 .The risk assessment for preterm birth based on maternal serum parameters is presented in The presented diagnosis scores might have the potential to be applied in clinical practice to prevent premature birth. Invasive methods for predicting preterm birth are costly, risky, and not universally available, and thus, identifying effective non-invasive predictors is crucial. The parameters assessed in this study, including hemoglobin, HALP score, and coagulation parameters, could be measured during routine prenatal care visits and may offer a non-invasive method for identifying women at high risk of preterm birth. Another possible application is to monitor women who are already at high risk of preterm birth due to underlying medical conditions or previous history. Regular measurement of these parameters during prenatal care visits may allow for the earlier detection of changes in these scores, indicating an increased risk of preterm birth. This may prompt healthcare providers to offer closer monitoring or earlier intervention to prevent preterm birth. However, it is important to note that further external validation is required to confirm the findings of this study and determine the optimal cutoff values for each parameter. Additionally, cost-effectiveness and accessibility of these non-invasive methods in different healthcare settings need to be considered before implementing these diagnosis scores in clinical practice.The present research found that hemoglobin levels below 12.0 g/dL, prothrombin time below 12.5 s, aPTT below 25 s, D-dimers over 250 ng/mL, a FAR score below 0.1, and a HALP score above 24 were significant predictors of preterm delivery in the third trimester. In this context, values that fall above the cut-off values are statistically insignificant predictors for preterm birth. The HALP score also had the highest sensitivity for preterm birth, with 75%, and a likelihood of 6.09 times higher than the reference range. However, the HALP score has so far mostly been investigated in correlation with malignancies. One study found that the association of hemoglobin, albumin, lymphocyte levels, and platelets represents a good predictor for negative outcomes in patients with cancer . A HALP Multiple processes may describe the effects of inflammation and dietary status on the HALP score and preterm birth. A greater proinflammatory condition reduces erythrocyte survival, inhibits bone marrow activity, and induces hypoferremia, leading to decreased hemoglobin concentration. Moreover, low albumin levels, a component of the HALP score, have been described during pregnancy as accounting for adverse events. In pregnancy-related hypertension, serum albumin level is a strong predictor of illness severity and may be regarded as a valuable measure for predicting time to delivery, proteinuria severity, and pregnancy outcomes . In addiOne of the strongest predictors identified during the third trimester for preterm birth was hemoglobin. We identified that anemia with a hemoglobin level below 120 g/L was associated with a 3.28 higher likelihood of premature delivery. One meta-analysis observed that there was no significant correlation between maternal anemia during pregnancy and preterm delivery . The resAlthough our study described a high proportion of pregnant women with anemia, the type of anemia was not investigated, even though different types of anemia might be stronger determinants for preterm labor. In another research study, the anemia was primarily of the normocytic normochromic variation with a prevalence rate of more than 50% . SimilarMaternal anemia is a risk factor for adverse pregnancy outcomes and poses a danger to the fetus’s survival. The preponderance of fetal and maternal problems, including abortions, obstructed labor, postpartum hemorrhage, and pre-eclampsia, were found in anemic women . AnotherEarlier research indicated that hypercoagulation might be linked to premature delivery. In agreement with our results, additional research studies found substantially shorter PT and aPTT in women with PUC who had preterm labor compared to those with normal term birth, indicating that both the intrinsic and extrinsic processes are active in preterm birth . ShorterOther studies propose the evaluation of coagulation parameters such as PT and aPTT for the risk of preterm birth, but also for the risk of intraventricular hemorrhage . The sigCompared to term newborns, moderately preterm infants have lower plasma concentrations of both procoagulant and anticoagulant vitamin K-dependent coagulation factors . HoweverThe last phase of the coagulation cascade is the stimulation of fibrinogen by thrombin to produce a fibrin plug. Another research evaluated fibrinogen levels between women of the same gestational age who went into preterm labor and those who delivered at term . Due to Beginning with the study’s retroactive approach, this article was limited by several drawbacks. Because the data were obtained from a local hospital, the examined sample may be more homogenous and less applicable to the greater group of pregnant women. Blood specimens were not collected at particular times to avoid certain confounding factors, as one third of all samples in the prematurity group were measured during the early third trimester (weeks 28 to 31), while the other two thirds were measured between 32 and 36 weeks of gestation, based on the moment of birth of each infant. Smoking and being overweight are both known risk factors for preterm birth. While the frequent smoking status and overweight status was significantly more frequent in the preterm birth group, the statistical analysis was adjusted for these confounding factors. Furthermore, we were unable to completely exclude the impact of a particular medication on the observed serum parameters. However, the study concluded with 161 premature newborns and a total size of 322 participants, which provides a sufficient statistical power. Nevertheless, the findings of this study need to be validated in external populations to ensure that the diagnosis methods have a broader applicability. Studies with larger sample sizes and diverse populations are necessary to confirm the accuracy and reliability of these methods.Regarding the future perspectives and issues of attempting to use these methods in current clinical practice, the cost-effectiveness of using these diagnosis methods in clinical practice should be evaluated. The cost of laboratory testing for these parameters may vary in different healthcare settings, and it is essential to consider the cost-effectiveness of these methods relative to the benefit gained in preventing preterm birth. In addition, the accessibility of these diagnosis methods in low-resource settings may be a challenge. These diagnosis methods require laboratory testing, which may not be easily available in all healthcare settings. Ensuring the availability and accessibility of these methods in all settings is crucial to prevent disparities in preterm birth prevention. The optimal timing of measurement for these diagnosis methods needs to be determined, as in the current study the parameters were measured during the third trimester of pregnancy, but it is unclear whether earlier measurement would provide a better prediction accuracy. Further research is needed to determine the optimal timing of measurement for each parameter. Lastly, it is important to consider the ethical implications of using these diagnosis methods. The diagnosis methods may lead to increased anxiety and stress for women identified as being at high risk of preterm birth. Healthcare providers should ensure that appropriate counseling and support are available to women identified as being at high risk of preterm birth.Since prematurity puts the newborn at significantly greater risks, it is important to identify risk factors that are easy to assess. In this study, hemoglobin levels, the association of hemoglobin, albumin, lymphocyte, and platelets’ (HALP) score, and coagulation parameters such as the prothrombin time (PT), activated partial thromboplastin clotting time (aPTT), D-dimers, and fibrinogen to albumin ratio (FAR) during the third trimester of pregnancy, proved to be significant determinants for the risk of preterm birth. However, due to financial constraints, it would be more appropriate to assess these predictors only in pregnancies that are at risk. Nevertheless, further external validation is required to confirm the accuracy of these predictors for premature delivery, since they all showed a poor level of discrimination. It is recommended that these parameters not be applied until more data are presented to support them.


This randomized clinical trial investigates the effect of extrauterine placental perfusion compared with delayed cord clamping on mean hematocrit levels in infants with very low birth weight. Does a physiological-based cord-clamping approach by extrauterine placental perfusion (EPP) increase mean hematocrit levels or improve oxygenation or neonatal outcomes in resuscitation of infants with very low birth weight (VLBW) compared with time-based delayed cord clamping (DCC)?In this randomized clinical trial among 60 infants with VLBW, mean hematocrit levels did not differ between groups, but infants who received EPP had significantly improved peripheral and cerebral oxygenation during transition.In this study, neonatal resuscitation of infants with VLBW using EPP resulted in comparable hematocrit levels as DCC but improved oxygenation during transition. An extrauterine placental perfusion (EPP) approach for physiological-based cord clamping (PBCC) may support infants with very low birth weight (VLBW) during transition without delaying measures of support.To test whether EPP in resuscitation of infants with VLBW results in higher hematocrit levels, better oxygenation, or improved infant outcomes compared with delayed cord clamping (DCC).This nonblinded, single-center randomized clinical trial was conducted at a tertiary care neonatal intensive care unit. Infants with a gestational age greater than 23 weeks and birth weight less than 1500 g born by cesarean delivery between May 2019 and June 2021 were included. Data were analyzed from October through December 2021.Prior to cesarean delivery, participants were allocated to receive EPP or DCC. In the EPP group, infant and placenta, connected by an intact umbilical cord, were detached from the uterus and transferred to the resuscitation unit. Respiratory support was initiated while holding the placenta over the infant. The umbilical cord was clamped when infants showed regular spontaneous breathing, stable heart rates greater than 100 beats/min, and adequate oxygen saturations. In the DCC group, cords were clamped 30 to 60 seconds after birth before infants were transferred to the resuscitation unit, where respiratory support was started.The primary outcome was the mean hematocrit level in the first 24 hours after birth. Secondary prespecified outcome parameters comprised oxygenation during transition and short-term neonatal outcome.Among 60 infants randomized and included, 1 infant was excluded after randomization; there were 29 infants in the EPP group  and 30 infants in the DCC group . The mean (SD) birth weight was 982.8 (276.6) g and 970.2 (323.0) g in the EPP and DCC group, respectively. Intention-to-treat analysis revealed no significant difference in mean hematocrit level . During transition, infants in the EPP group had significantly higher peripheral oxygen saturation as measured by pulse oximetry  and regional cerebral oxygen saturation . Neonatal outcome parameters were similar in the 2 groups.This study found that EPP resulted in similar hematocrit levels as DCC, with improved cerebral and peripheral oxygenation during transition. These findings suggest that EPP may be an alternative procedure for PBCC in infants with VLBW.NCT03916159ClinicalTrials.gov Identifier:  Delayed cord clamping (DCC) of at least 30 seconds compared with immediate cord clamping in infants born preterm has been shown to improve survival and reduce intraventricular hemorrhage (IVH) of any grade, rate of red blood cell transfusion, and risk of death or major disability at 2 years.5 However, the recommendation for DCC in infants born preterm is based mainly on studies that excluded infants who did not breathe spontaneously. Animal studies suggest that adequate aeration of the lungs before umbilical cord clamping, also termed physiological-based cord clamping (PBCC), may be the key for benefits of DCC.7 Although most infants with VLBW start to breathe within 30 to 60 seconds of DCC,8 initiation of spontaneous breathing is not equivalent to adequate aeration of the lungs and thus is not an indicator of successful transition.9 Over the last decade, mobile resuscitation units for PBCC have been developed to provide lung aeration in resuscitation of infants born preterm using continuous positive airway pressure (CPAP) during DCC. A study10 evaluating these devices revealed challenges in temperature management, space issues, and incomplete equipment sterility in the setting of cesarean delivery. Therefore, the applicability and effectiveness of such devices remain to be determined and alternative approaches for PBCC should be investigated.Transition from intrauterine to extrauterine life is a critical process in which clamping of the umbilical cord represents one of the most significant events, particularly for infants born preterm.11 studied the potential benefit of DCC after removal of the placenta from the mother in cesarean delivery of infants born preterm decades ago. This approach, in which infants are born by cesarean delivery with the placenta still connected via an intact umbilical cord and transferred to the resuscitation unit, may work as an alternative approach for PBCC. It was also shown that such an approach for PBCC is safe for mother and infant in babies born at term and provides placental perfusion independently of the mother.12 Our center has been using this approach in neonatal resuscitation of infants with VLBW born by cesarean delivery for more than a decade and has extended the method by simultaneously applying mask CPAP for respiratory support. We termed this modified approach extrauterine placental perfusion (EPP). A first retrospective analysis revealed that EPP was not associated with adverse outcomes for infants with VLBW.13It is assumed that by performing PBCC, blood from the placenta stabilizes hemodynamic transition from prenatal to postnatal circulation when the cord is not clamped prior to lung aeration. However, it is unknown if a placenta detached from the uterus has the same hemodynamic effects given that there is still ongoing placental circulation from the extrauterine placenta to the infant. DunnThis is the first randomized clinical trial of EPP to our knowledge. We hypothesized that providing respiratory support with an intact umbilical cord by performing EPP for PBCC would increase mean hematocrit levels on the first day of life compared with DCC. In addition, we hypothesized that the EPP procedure would stabilize the circulation during neonatal transition and would not adversely affect short-term neonatal outcomes.NCT03916159) was a nonblinded, single-center randomized clinical trial conducted at the University Hospital of Cologne, Germany  reporting guideline.The Extrauterine Placental Transfusion in Resuscitation of VLBW Infants (EXPLAIN) trial , placental abruption or placenta previa with hemorrhage, placenta accreta or increta, monochorionic multiples, congenital anomalies, and lack of consent to the study from parents.Participants were randomly assigned using opaque, sealed envelopes, stratified by gestational age (24 0/7 to 27 6/7 weeks or ≥28 0/7 weeks) and type of pregnancy (singleton or twin pregnancy). The allocation sequence was based on computer-generated pseudorandom numbers (permuted blocks of varying length). The research team opened envelopes in the theater immediately before the cesarean delivery. The randomization result was then announced to the entire team during the team time-out prior to the start of the surgery.15 Briefly, all infants received mask CPAP starting with a positive airway pressure application of 8 to 10 cm H2O with stepwise increase up to a maximum of 30 cm H2O applied using the Benveniste valve as CPAP generator. Initially, fraction of inspired oxygen (Fio2) was set in the range of 0.21 to 0.30 in infants at less than 28 weeks’ gestational age.16Infants randomized to the control group (DCC) were born by cesarean delivery, placed in a sterile plastic suit , and gently stimulated by rubbing the back or feet if apneic until breathing commenced. Infants were held at placental level during the DCC procedure .Infants randomized to the intervention group (EPP) were born by cesarean delivery with gentle detaching of the placenta from the uterus along with the infant and intact umbilical cord. All involved obstetricians and surgical staff (including B.G.) were experienced in this technique. After delivery, infants with the placenta still attached were placed in a sterile plastic suit by the obstetrician. The time to detach the placenta in the EPP cohort was not recorded but comprised only a few seconds. While mothers received oxytocin directly after delivery of the placenta, infants were immediately transferred to the resuscitation unit, where the neonatal staff  resuscitated the infant according to the same protocol as described in control group. The placenta was placed in a bowl, covered with a sterile plastic bag, and held 40 to 50 cm above the babies’ heart level while lung recruitment by respiratory support was performed  and heart rate were measured with a Masimo 7 SET pulse-oximeter . Cerebral oxygenation was measured by near-infrared spectroscopy , with sensors placed on infants’ foreheads after transfer to a resuscitation bed. Regional cerebral oxygen saturation (rcSo2) was recorded synchronously with cardiorespiratory parameters.Oxygen saturation as measured by pulse oximetry  additionally recorded respiratory rate, airway pressure, and tidal volume in both groups during neonatal resuscitation. In all twin births, only the second newborn was randomized to the control or intervention group, while the firstborn was always intended to have a DCC of at least 30 seconds.In our center, hematocrit level is routinely determined in every infant with VLBW at 15 to 60 minutes after birth and at 12 and 24 hours after birth. The primary outcome was the mean value of these measurements during the first 24 hours after birth.o2 and rcSo2 levels during transition, neonatal and maternal complications, and parameters related to the study procedure, were collected from infant medical records. All study data were collected and managed using REDCap electronic data-capture tools.19 Bronchopulmonary dysplasia was categorized according to the definition of Walsh et al.20 A summary of secondary outcome parameters is provided in the study protocol  to 57.3% (7.3%) in the EPP group. The 2-sample t test requires a sample size of 2 × 29 evaluable patients  to reach a power of 80% at a 2-sided significance level of 5%. We used the power twomeans command in Stata statistical software version 15.1 (StataCorp).We hypothesized that EPP would increase the mean (SD) hematocrit level in the first 24 hours after birth by a relative 10%, from 52.1% (6.6%) in the DCC group  was performed by analysis of covariance (ANCOVA) with fixed effects for group, gestational age (24 weeks 0 days to 27 weeks 6 days vs >27 weeks 6 days) and gravidity (singleton vs multiples). Quantitative variables were described with the number of valid and missing values, mean, SD, and percentiles ; group comparison was done analogously to the primary outcome. Qualitative variables are reported with absolute (number) and relative (percentage) frequencies; group comparison was done based on relative risk (with 95% CIs). A 2-tailed Among a total of 156 eligible pregnant individuals, 60 individuals were randomized and enrolled in this study between May 2019 and June 2021. A total of 30 individuals were assigned to the control group (DCC) and 30 to the intervention group (EPP), respectively . One newIn the intervention group EPP was feasible in 24 of 24 singletons and in 3 of 5 second twins. In 1 twin pair, EPP was not feasible because the second twin’s placenta was attached to first twin’s placenta; the procedure was switched to DCC according to the study protocol. In the other case of twin birth, the attending obstetrician opted for immediate cord clamping in both infants due to severe chorioamnionitis and maternal instability. In the control group, DCC was performed as randomized in 24 of 25 singletons and 5 of 5 twin births. In 1 case, the placenta was accidentally detached during cesarean delivery before birth, and thus the infant received EPP instead of DCC.o2 levels  ; age 8 minutes: 76.3% [14.5%] vs 65.1 [15.9%]; AMD, 10.9 percentage points ) (io2 levels were lower in the EPP group (P = .05) but was not confirmed by ANCOVA   hematocrit level was 56.0% (1.8%) compared with 53.9% (1.8%) in the DCC group . Subgroup analyses by gestational age, sex, and mode of pregnancy also showed no significant differences . With repoints])  and rcSopoints])  during tPP group  during nPP group E. There PP group . A signi3.5 mL]) . A secon3.5 mL]) .23 The absence of uterine contractions during elective cesarean delivery and the cutting of the uterus during this type of delivery itself may contribute to a generally reduced placental transfusion in infants delivered by this method.24 Of note, most infants in both groups studied in our trial were delivered by elective cesarean delivery. However, it was interesting to us that infants receiving EPP, in whom the cord was clamped after successful lung aeration, did not show higher hematocrit levels in the first 24 hours after birth compared with infants receiving DCC. While lung aeration prior to cord clamping is thought to be responsible for a significantly greater amount of transfused blood,6 most studies have failed to find higher postnatal hematocrit levels in infants after PBCC, which is consistent with our findings.25To our knowledge, this is the first randomized clinical trial of EPP as an alternative approach for PBCC instead of DCC in infants with VLBW. Our study found that the EPP approach resulted in similar mean hematocrit levels 24 hours after birth and was safe given similar neonatal outcomes in EPP and DCC groups. While numerous studies have shown a significantly higher hematocrit level after DCC in babies born spontaneously, studies in infants born by cesarean delivery are controversial.o2 and rcSo2 levels during transition than infants receiving DCC. A faster increase in arterial oxygenation in the setting of PBCC has been observed in animal studies26 and confirmed in a clinical study of PBCC in infants born preterm.27 In addition to these observations, we found significantly higher cerebral oxygenation during the first minutes after birth. Prolonged placental perfusion in PBCC optimizes tissue perfusion and may influence cerebral perfusion, with the potential to reduce the risk of IVH.28 Our study demonstrates that this effect is also observed in PBCC with an extrauterine placental circulation. This supports the hypothesis that the main benefit of PBCC with sustained placental perfusion may not be in the higher number of red blood cells but in the provision of preload volume stabilizing cardiac output during neonatal transition. This may be the key to avoiding disturbances in cerebral blood flow and preventing neonatal complications. In a 2020 study, Padilla-Sánchez et al29 found significantly higher Spo2 values in infants who were mature and late preterm with sufficient respiration receiving DCC compared with reference values in infants who underwent immediate cord clamping. This underscores the importance of successful lung aeration and adequate placental perfusion during transition. Our finding that infants in the EPP group showed better oxygenation parameters raises another interesting question: whether oxygen transfer from mother to infant across the placenta, which does not occur in EPP because the placenta is detached, may be less important than an effective maintenance of venous return to the heart, which is supported by our EPP approach.An intriguing observation of our study was that infants receiving EPP had significantly higher Sp12 and supports the finding that EPP may be considered as an alternative to the currently recommended practice of DCC.In this trial, the EPP approach was feasible in all singleton and most multiple births. This finding is consistent with published data in neonates born at term12 and our own experience with the EPP procedure showed that times were much shorter than in DCC when staff were experienced with the technique. Second, the procedure does not conflict with the sterility of the operating room.30 Third, most current resuscitation platforms are not certified for transport, requiring transfer of the infant after initial stabilization.10 Fourth, the EPP approach is a cost-effective and technically simple approach for PBCC. Therefore, it may be particularly attractive in resource-limited settings where mobile resuscitation units are not available. Moreover, a 2021 survey31 confirmed the need for alternative procedures to achieve placental perfusion given that delayed cord clamping was performed in 60% of infants born preterm who were stable and 6% of infants born preterm who were unstable in low- and middle-income countries.Compared with PBCC approaches using mobile beside resuscitation platforms, EPP has several advantages: first, the procedure does not delay the course of cesarean delivery compared with DCC, in which surgical procedures are stopped until the cord is clamped. Although we did not record time from delivery of the infant to placental detachment, data from Welsh et alAlthough there was a statistically significant difference in maternal blood loss during cesarean delivery, the higher blood loss in mothers in the EPP compared with DCC group is not considered clinically significant by obstetricians. In addition, significance was not confirmed in ANCOVA. The higher rate of blood loss in mothers in the EPP group may be explained by differences in the surgical procedure when performing EPP. Whereas in EPP oxytocin is administered after the placenta has been detached, in DCC it is usually administered immediately after umbilical cord clamping but before the placenta has been detached. Further studies on the EPP technique should address the issue of maternal blood loss in terms of safety.Our EXPLAIN study has several limitations. First, restricting a study to participants giving prenatal consent may limit external validity because mothers who were not approached were in advanced labor. Thus, a large proportion of infants at a higher risk of neonatal complications may not have been enrolled, also resulting in a relatively old patient population. Second, this was a monocentric trial comprising 60 infants in a center with long experience in EPP and a different strategy of respiratory support during neonatal resuscitation based on titration of high continuous airway pressures rather than the use of positive pressure ventilation. Therefore, our findings regarding feasibility may not be generalizable to other settings.Another important limitation of our trial is that we did not calculate sample size with regard to safety aspects of the EPP procedure. Therefore, our study did not have sufficient power to conclude with certainty that these 2 interventions were interchangeable with respect to adverse maternal and neonatal outcomes in the short and long term. Given that the incidence of major neonatal complications, such as bronchopulmonary dysplasia, IVH, or necrotizing enterocolitis, is low in our center, a higher number of participants may be needed to detect significant differences in these outcome variables. In this context, further multicenter trials are mandatory to determine feasibility and reproducibility in other centers less experienced with EPP.Findings from this first monocentric randomized clinical trial of EPP suggest that it may be an alternative procedure for PBCC in infants with VLBW. EPP resulted in similar postnatal hematocrit levels compared with DCC but significantly higher peripheral and cerebral oxygenation levels during transition. Further, larger trials are needed to evaluate the impact of EPP on neonatal outcomes.


To define the relationship between sex, residence, maternal age, and a broad range of birth defects by conducting a comprehensive cross-analysis based on up-to-date data. Data were obtained from the Birth Defects Surveillance System in Hunan Province, China, 2010–2020. Prevalences of birth defects ) with 95% confidence intervals (CI) were calculated by sex, residence, maternal age, year, and 23 specific defects. Cross-analysis of sex, residence, and maternal age was conducted, and crude odds ratios (ORs) were calculated to examine the association of each maternal characteristic with birth defects. A total of 1,619,376 fetuses and 30,596 birth defects were identified. The prevalence of birth defects was 188.94/10,000 (95% CI 186.82–191.05). Birth defects were more frequent in males than females , in urban areas than in rural areas , and in mothers ≥ 35 than mothers 25–29 . Cross-analysis showed that the prevalence of birth defects was higher in urban females than in rural males , the difference in prevalence between males and females was more significant for maternal age < 20 compared to other age groups , and the prevalence difference between urban and rural areas is more significant for maternal age 25–34 compared to other age groups . Cleft palates were more frequent in males, and nine specific defects were more frequent in females. Five specific defects were more frequent in rural areas, and eight were more frequent in urban areas. Compared to mothers 25–29, five specific defects were more frequent in mothers < 20, seven specific defects were more frequent in mothers 20–24, two specific defects were more frequent in mothers 30–34, and ten specific defects were more frequent in mothers ≥ 35. Our data indicate that sex, residence, and maternal age differences in the prevalences of birth defects and most specific defects are common. We have found some new epidemiological characteristics of birth defects using cross-analysis, such as residence is the determining factor for the prevalence of birth defects, the difference in prevalence between males and females was more significant for maternal age < 20 compared to other age groups, the prevalence difference between urban and rural areas is more significant for maternal age 25–34 compared to other age groups. And differences in the epidemiological characteristics of some specific defects from previous studies. Future studies should examine mechanisms. Our findings contributed to clinical counseling and advancing research on the risk factors for birth defects. The accepted prevalence of birth defects is about 2–3% worldwide2. Birth defects are associated with many adverse pregnancy outcomes, such as preterm birth, stillbirths, and child mortality5. WHO estimated that about 12.6% of neonatal deaths worldwide each year are related to birth defects6, and 240,000 newborns worldwide die each year from birth defects within the first 28 days of life, and birth defects cause 170,000 children deaths between the ages of 1 month and 5 years7. Birth defects have been a significant problem for health care in terms of the resources they require because of their longer life expectancy, especially in low- and middle-income countries7. Therefore, the study on birth defects is significant and deserves more attention.Birth defects are structural or functional anomalies at or before birth8; the epidemiology of chromosomal abnormalities (2016–2019)9; the characteristics of the prenatal diagnosis of birth defects and termination of pregnancy for fetal anomalies (2015–2018)10; the prevalence of birth defects between 2005 and 201411. However, no studies have analyzed the relationship between birth defects and sex, residence, and maternal age in depth. In addition, many of the previous studies had data limitations or needed to be updated. Therefore, an update from a cohort study for birth defects is needed.Instead, there have been fewer national studies on birth defects in China recently. There have been some studies on birth defects in Hunan Province, China. E.g., study on the association between ambient air pollution and birth defects (2014–2016)13. Risk factors for birth defects may change over time and vary between regions and populations. Epidemiological studies on birth defects help advance research on the risk factors for birth defects. Previous studies have shown that many risk factors were associated with birth defects, such as environmental factors , genetic causes , and socioeconomic factors14. Among them, sex, residence, and maternal age are the most common epidemiological characteristics, which can also be used as proxies for additional epidemiological characteristics, such as healthcare and economic conditions. There were some previous studies on the relationship between sex, residence, maternal age, and birth defects. In general, birth defects were more common in males, urban areas, or fetuses of advanced maternal age18. However, there are also limitations in these studies. First, some studies only analyzed the prevalence of all birth defects overall, not by disease type. Second, some studies were limited in data, such as relatively few cases included or surveys conducted in unrepresentative districts or hospitals. Third, some studies needed to be updated. Fourth, there is a lack of in-depth analysis methods, such as cross-analysis, in most studies. In addition, some studies have different findings that diverge. E.g., Ahn et al. found that very low-quality evidence suggests that women in the older maternal age group increased the risk of birth defects19, and Goetzinger et al. found that advanced maternal age was associated with an overall decreased risk for major anomalies20. Xiong et al. found a higher prevalence of birth defects in rural than urban areas21. It is essential to conduct systematic research using the latest representative data.Although in many birth defect cases, the cause is still unknown, many researchers believe that birth defects may result from hereditary polygenic defects or a gene-environment interactionTherefore, we conducted a comprehensive cross-analysis based on the data from the Birth Defects Surveillance System in Hunan Province, south-central China (from 2010 to 2020), to define the relationship between sex, residence, maternal age, and a broad range of birth defects (including 23 types of specific defects in detail). Our study is an essential update on birth defects research. It will provide additional information on birth defects, such as epidemiological characteristics of various specific defects, cross-risk factors for birth defects, etc. Our study may contribute to clinical counseling and advancing research on the risk factors for birth defects.This study used data from the Birth Defects Surveillance System in Hunan Province, China, 2010–2020, which is run by the Hunan Provincial Health Commission and involves 52 representative registered hospitals in Hunan Province. Surveillance data of fetuses (births and deaths at 28 weeks of gestation and beyond) and all birth defects (between 28 weeks of gestation and seven days after delivery) included demographic characteristics such as sex, residence , maternal age, and other key information. The 52 hospitals are required to report all cases of birth defects at birth and at 28 weeks of gestation and beyond in medical records to the Birth Defects Surveillance System.According to the WHO International Classification of Diseases , birth defects were classified into 23 subtypes: anencephaly (Q00), spina bifida (Q05), encephalocele (Q01), hydrocephalus (Q03), cleft palate (Q35), cleft lip (Q36), cleft lip-palate (Q37), anotia/microtia , other external ear defects (Q17), esophageal atresia (Q39), anal atresia (Q42), hypospadias (Q54), bladder exstrophy (Q64.1), talipes equinovarus (Q66.0), polydactyly (Q69), syndactyly (Q70), limb reduction , diaphragmatic hernia (Q79.0), omphalocele (Q79.2), gastroschisis (Q79.3), conjoined twins (Q89.4), Down syndrome (Q90), congenital heart defects (Q20-26) or ‘other’ .We confirmed that informed consent was obtained from all subjects and/or their legal guardian(s). Doctors obtain consent from pregnant women before collecting surveillance data, witnessed by their families and the heads of the obstetrics or neonatal departments. Doctors obtain consent from their parents or guardians for live births, witnessed by their families and the heads of the obstetrics or neonatal departments. Since the Health Commission of Hunan Province collects those data, and the government has emphasized the privacy policy in the “Maternal and Child Health Monitoring Manual in Hunan Province”, there is no additional written informed consent.The Medical Ethics Committee of Hunan Provincial Maternal and Child Health Care Hospital approved the study. (NO: 2022-S013). It is a retrospective study of medical records. All data generated or analyzed during this study were from the Birth Defects Surveillance System. All data were fully anonymized before we accessed them. Moreover, we de-identified the patient records before analysis. We confirmed that all experiments were performed following relevant guidelines and regulations.To carry out surveillance, the Hunan Provincial Health Commission formulated the "Maternal and Child Health Monitoring Manual in Hunan Province". Data were collected and reported by experienced doctors. To reduce integrity and information error rates, the Hunan Provincial Health Commission asked the technical guidance departments to conduct comprehensive quality control each year.2trend) were used to determine trends in prevalence by year. Crude odds ratios (ORs) were calculated to examine the association of each maternal characteristic with birth defects.The prevalence of birth defects is defined as the number of birth defects per 10,000 fetuses. Prevalences and 95% confidence intervals (CI) were calculated for any defect and 23 specific defects. If the number of birth defects was tiny (≤ 50), we calculated the 95%CI by the Robust Poisson method; if the number of birth defects was > 50, we calculated the 95%CI by the log-binomial method. Chi-square trend tests .2trend = 246.44, P < 0.01) . From 2010 and 2020, the prevalences of birth defects were 187.28, 227.81, 204.96, 189.61, 221.87, 218.39, 182.03, 179.96, 163.14, 160.34, 164.75 per 10,000 fetuses, respectively, and showed a decreasing trend , in urban areas than in rural areas , and in mothers ≥ 35 compared to mothers 25–29 . Male fetuses, urban residents, and mothers ≥ 35 were risk factors for birth defects , polydactyly , other external ear defects , syndactyly , hypospadias , cleft lip, palate , cleft lip , anal atresia  and anotia/microtia . The prevalence of cleft palate was lower in males than females . It is the only specific defect that is lower in males than females , hydrocephalus , cleft lip , spina bifida , and gastroschisis .The prevalence of the following specific defects was higher in urban than rural areas: congenital heart defect , polydactyly , other external ear defects , syndactyly , hypospadias , cleft palate , anal atresia , and Down syndrome  . In comparison to mothers 25–29, the following specific defects were more frequent in mothers < 20: polydactyly , talipes equinovarus , cleft lip-palate , hydrocephalus , gastroschisis  and anencephaly .Compared to mothers 25–29, prevalences of the following specific defects were lower in mothers 20–24: congenital heart defect  and hypospadias . And the following specific defects were more frequent in mothers 20–24: talipes equinovarus , cleft lip-palate , hydrocephalus , limb reduction , cleft lip , gastroschisis , and encephalocele .Compared to mothers 25–29, prevalences of the following specific defects were lower in mothers 30–34: talipes equinovarus , hydrocephalus , spina bifida , and gastroschisis . And prevalences of the following specific defects were higher in mothers 30–34: congenital heart defect  and anal atresia .Compared to mothers 25–29, mothers ≥ 35 had a higher prevalence of the following specific defects: congenital heart defect , polydactyly , cleft lip-palate , limb reduction , cleft lip , anal atresia , Down syndrome , diaphragmatic hernia , omphalocele , and anencephaly  , and some specific defects were shown to correlate with sex, residence, and maternal age. Based on a representative sample size of the most recent decade of long-term birth defect surveillance data, this study is an important update on the prevalence and epidemiology of birth defects. In this study, we found that the prevalence and epidemiology of some specific defects differed significantly from previous studies, and some other findings rarely mentioned in the previous literature by applying cross-analysis. Therefore, our discovery makes a significant original contribution to the field. In the following discussion, we will examine these differences and discuss the causes in detail.2. However, there were differences in prevalence between this study and others. e.g. 23.9 per 1000 births in Europe (2003–2007)22, 298.6 per 10,000 pregnancies in Japan (2011–2014)23, 446.3 per 10,000 births in Korea (2008–2014)24, 1.1% of malformed newborns in the Latin American network for congenital malformation surveillance (2017–2019)25, 66.2 per 10,000 births in Uganda (2015–2017)26, and 184.48 per 10,000 births in India (reported in 2018)27. Our prevalence is generally lower than in some high-income countries and higher than in some low- and middle-income countries. There has been no national prevalence of birth defects reported in China recently. Some regions in China reported lower prevalences of birth defects than in this study, such as 13.55 per 1000 births in Guilin, Guangxi Zhuang Autonomous Region (2018–2020)28, and 71.51 per 10,000 fetuses in Southern Jiangsu (2014–2018)29. We infer that the main reason for these results is differences in diagnosis and reporting rates30, as pregnant women had better access to diagnostic and therapeutic services in high-income countries than in low- and middle-income countries. Over the past few decades, there have been significant improvements in diagnostic and therapeutic services for birth defects in China. In addition, birth defects result from hereditary polygenic defects or a gene-environment interaction13. Differences in the prevalence of birth defects reported in different studies may also be related to differences in genetic and environmental factors34. From 2010 to 2020, the prevalence of birth defects decreased. It is inconsistent with previous studies36. It may result from many birth defects diagnosed and terminated before 28 weeks of gestation10. In our study, these prematurely terminated fetuses are not used to calculate the prevalence of birth defects.The overall prevalence of birth defects  is consistent with the reported or accepted global prevalence (about 2–3%)47. However, the prevalences of some specific defects seem to be lower than the reported or accepted global prevalences, such as hydrocephalus 48, Down syndrome 49, spina bifida 50, diaphragmatic hernia 51, esophageal atresia  52, gastroschisis 53, anencephaly 54, encephalocele 55, and conjoined twins 56. Moreover, the prevalences of some specific defects were more than tenfold lower than were generally accepted, such as Down syndrome, encephalocele, and conjoined twins. In contrast, the prevalence of several specific defects was higher than in other studies. E.g., Shin et al. reported lower prevalences of polydactyly and syndactyly in Korea (1.157‰ and 0.309‰)57.The prevalence of many specific defects appeared to be consistent with the reported or accepted global prevalence, including congenital heart defects, talipes equinovarus, hypospadias, cleft lip-palate, cleft lip, cleft palate, limb reduction, anal atresia, anotia/microtia, omphalocele, and bladder exstrophy58. Second, as is discussed, birth defects may result from hereditary polygenic defects or gene-environment interactions. e.g. polydactyly and syndactyly may be mainly related to chromosomal or genetic abnormalities and are more common in some ethnic groups59. Third, the attitudes of pregnant women and their families, affected by the treatment and financial conditions, significantly impact the survival of some fetuses with severe defects, which may also be important factors for these results60. Pregnant women in high-income countries are likelier to give birth to babies with defects because there are advanced therapeutic tools and better economic conditions to cure them61. Fourth, public health measures were adopted for some specific defects. e.g. the prevalence of neural tube defects  has decreased because of the use of folic acid62.We infer that several factors may be related to these differences. First, as discussed, differences in diagnosis and reporting rates may be one of the most critical factors for these results. e.g. with improvements in prenatal screening and diagnosis technologies, most Down syndrome fetuses (which one could argue is not a birth defect but rather a genetic syndrome constituted of numerous co-occurring birth defects) are diagnosed and terminated in the second trimester, resulting in a low prevalence64. The following are accepted explanations of this. First, sex differences in the prevalence of birth defects may be related to the male gonad differences during fetal development and the subsequent hormonal and physiologic differences in male and female fetuses65. Second, urban–rural differences in the prevalence of birth defects may be related to etiologies, diagnosis, or surveillance, similar to the analysis above63. Third, the risk of aneuploidy or non-chromosomal abnormalities increased with maternal age. It is the reason for a higher prevalence of birth defects in fetuses of advanced maternal age14. However, several studies have obtained different findings. e.g. very low-quality evidence suggests that advanced maternal age increases the risk of birth defects19, low prevalences of major anomalies in advanced maternal age20, and a higher prevalence of birth defects in rural areas66. It may be partly related to no adjustment of confounders or surveillance methods . In addition, some new features were found in the cross-analysis in this study. First, cross-analysis of sex and residence showed that residence is the main determinant. As discussed, diagnosis and reporting rates may be the main determinant. Second, cross-analysis of sex and maternal age showed that the difference in prevalence between males and females was more significant for maternal age < 20 compared to other age groups. Third, cross-analysis of residence and maternal age showed that the difference in prevalence between urban areas and rural areas is more significant for maternal age 25–34 compared to other age groups. These findings have been rarely covered in previous studies. And the mechanisms are unclear.Birth defects were more frequent in males, urban areas, or fetuses of advanced maternal age (≥ 35). It is consistent with many previous studies68. However, the epidemiological characteristics of some specific defects differed from other specific defects or previous studies or have rarely been reported. First, cleft palate was the only defect higher in females than males. The possible explanation for this fact may be sex differences in development69. Cleft lip-palate and cleft lip were higher in rural than urban areas. There were similar reports70. It may be related to material deprivation and parental agricultural work72, while the cleft palate is not. Second, some specific defects were more frequent in fetuses of low maternal age, including polydactyly, talipes equinovarus, cleft lip-palate, cleft lip, hydrocephalus, limb reduction, gastroschisis, anencephaly, and encephalocele. There were some similar reports80, but also there are considerable differences19. It may be related to early exposure to risk factors such as tobacco, alcohol, and illicit drugs19 and may be easier to influence by confounding factors. Third, some specific defects have rarely been reported. e.g. hydrocephalus was higher in rural than urban areas, which is rarely reported. Jeng et al. found that low socioeconomic status was associated with an increased risk of hydrocephalus, but the reason for this phenomenon is unknown81. Fourth, some studies have found different sex characteristics from this study. e.g. Siffel et al. found that bladder exstrophy was almost twice as high in males than in females47; Tovar et al. reported a higher prevalence of diaphragmatic hernia in males than females51. Fifth, some studies have found different residence characteristics from this study. e.g. several studies found gastroschisis, omphalocele, and anotia/microtia were more frequent in rural areas82; Luben et al. found no correlation between residence and spina bifida83. Sixth, some studies have found different maternal age characteristics from this study. e.g. several studies reported no correlation between talipes equinovarus, hydrocephalus, and low maternal age85; several studies reported higher prevalences of hypospadias and esophageal atresia in fetuses of advanced maternal age84. In general, the mechanism for most of the results is unknown. Differences from previous studies and new findings in this study may be related to the location site, sample size, year, or cross-risk factors and bioinformatics testing multi-factors significant level.Some specific defects were related to maternal age, sex, and residence. Some specific defects were reported in previous studiesThe above discussion on the relationship between birth defects and sex, residence, and maternal age suggests that birth defects may result from gene-environment interaction. Although the relationship mechanisms between birth defects and sex, residence, and maternal age were unclear, our study described the phenomenon in detail. It will be beneficial for conducting mechanistic studies in the future.Some things could be improved. First, data on fetuses under 28 weeks of gestation needed to be included. Second, we did not examine the prevalence of birth defects separately among the live-born or dead infants. Third, some specific defects may be diagnosed after the seventh day, and there is a possibility of underdiagnosing some specific defects, such as congenital heart defects.In summary, our data indicate that sex, residence, and maternal age differences in the prevalences of birth defects and most specific defects are common. We have found some new epidemiological characteristics of birth defects using cross-analysis, such as residence is the determining factor for the prevalence of birth defects, the difference in prevalence between males and females was more significant for maternal age < 20 compared to other age groups, the prevalence difference between urban and rural areas is more significant for maternal age 25–34 compared to other age groups. And differences in the epidemiological characteristics of some specific defects from previous studies. Future studies should examine mechanisms. Our findings contributed to clinical counseling and advancing research on the risk factors for birth defects.


Depression during pregnancy was found to be strongly associated with delivering an infant with a birth defect . Conclusion: Depression during pregnancy and its interaction with smoking and diabetes are vital in determining birth defects in infants. The results indicate that birth defects in the United States can be reduced by lowering depression in pregnant women.Objective: To assess both individual and interactive effects of prenatal medical conditions depression and diabetes, and health behaviors including smoking during pregnancy on infant birth defects. Methods: The data for this research study were collected by the Pregnancy Risk Assessment Monitoring System (PRAMS) in 2018. Birth certificate records were used in each participating jurisdiction to select a sample representative of all women who delivered a live-born infant. Complex sampling weights were used to analyze the data with a weighted sample size of 4,536,867. Descriptive statistics were performed to explore frequencies of the independent and dependent variables. Bivariate and multivariable analyses were conducted to examine associations among the independent and dependent variables. Results: The results indicate significant interaction between the variables smoking and depression and depression and diabetes (OR = 3.17;  Birth defects are a primary cause of fetal death, infant mortality and morbidity, and long-term disability. Birth defects affect the quality of life of these infants and pose a burden for their families and society. According to the World Health Organization (WHO), about 300,000 newborns diagnosed with birth defects die within the first 28 days of life . ApproxiDiabetes mellitus (DM) is a metabolic disease that results in hyperglycemia and is caused by either the low level of insulin in the body or resistance to insulin . The oveSeveral studies indicate that birth defects are associated with maternal smoking ,5. In a Likewise, prenatal depression among pregnant women poses a comprehensive public health problem and is a potential risk factor for adverse birth outcomes . A crossAlthough previous studies have addressed the impact of prenatal health conditions and health behaviors on birth defects, they did not assess the interactive effect of these variables on birth defects. This study aims to address the gaps in the literature by understanding both the individual and interactive effects of smoking during pregnancy, diabetes, and prenatal depression on birth defects. The study aims at finding the risk of delivering a child with birth defect in the women with depression, diabetes, and health behaviors such as smoking.The primary objective of this research was to assess both the individual and interactive effects of prenatal depression, diabetes, and smoking in pregnant women on infant birth defects. The study hypothesized that birth defects are associated with two-way or three-way interactions of prenatal depression, smoking, and diabetes during pregnancy.The research proposal was approved by Institutional Review Board at U.T. Health San Antonio on 2 March 2021. The IRB number is HSC20210029N. This secondary research planned to analyze the data collected through the Pregnancy Risk Assessment Monitoring System (PRAMS) survey datasets. PRAMS is a joint research project between the state, territorial, or local health departments and the Centers for Disease Control and Prevention, Division of Reproductive Health. The PRAMS survey dataset is a multistate analytic dataset created by the stratified sampling technique .The dataset contains demographic and clinical information collected through the state’s vital records system, birth certificate, and other variables such as operational, weighting, questionnaire, and analytic variables. Topics addressed in the PRAMS survey questionnaire included prenatal care, obstetric history, maternal habits, physical abuse, contraception, economic status, maternal stress, and early infant development. Each year, the data are collected through surveys and are available publicly after 14 months for a specific year. This study used the PRAMS data collected for the year 2018. The birth defect information is retrieved by the PRAMS through the birth certificate record and linked with the survey responder. The data received from PRAMS include information about birth defects which is classified as the binary variable of Yes or No. This birth defect binary variable includes all the birth defect-related anomalies .The Pregnancy Risk Assessment Monitoring System (PRAMS) combined two modes of data collection, which were a survey conducted by mailed questionnaire with multiple follow-ups and a telephone survey . OverallThe data obtained from CDC-PRAMS for 2018 were used to create a new data subset for analysis following initial data cleaning and merging. The independent or exposure variables included prenatal conditions and health behaviors such as depression, smoking, and diabetes. The birth defect variable, dependent or outcome variable, was dichotomous and classified as “Yes” for the presence of a birth defect and “No” for the absence of a birth defect. The “diabetes during pregnancy variable” classification is different. The PRAMS dataset combined all the types of diabetes together including Type 1, Type 2, and gestational diabetes. The diabetes during pregnancy variable in this dataset is binary showing “Yes” and “No”. The pregnant mother who responded yes reflects diabetes which includes Type 1, Type 2, and gestational diabetes. The data were analyzed using SPSS, Version 26 .Univariate analyses were used to explore the frequencies for the dependent, independent, and demographic variables. Chi-square tests were conducted to test the associations between the birth defect variable with independent variables such as smoking, depression, and diabetes. Subsequently, the logistic regression model was used for diabetes during pregnancy and testing the interactive effects among two or more covariates. The effect modification was determined in the logistic regression model. A multiplicative model was the model of choice to determine the interactive effect of smoking, depression, and diabetes variables, and to assess various risk factors of birth defects, including depression, smoking during pregnancy.The average survey response rate of the PRAMS survey for all states was 56.81%. The total sample of respondents is 89,839. This sample size is all the respondents of the study survey. After applying the complex sampling weight to the survey data, the total sample size is 4,536,867.The response rate varied from as high as 80.4% for Puerto Rico to as low as 39.4% for Nevada . After ap-value < 0.001), race , body mass index , maternal smoking habits during pregnancy , depression during pregnancy , diabetes during pregnancy , abuse , folic acid intake , vitamin intake , and hypertension   and having an income of USD 57,000 to 85,000 (38.6%). The data indicate that 7.2% of pregnant women smoked during pregnancy, 14.2% were diagnosed with depression, and 9.6% were diagnosed with diabetes. The bivariate analysis revealed statistically significant Chi-square values for demographic variables, including age (< 0.001) .The multivariate binary logistic regression model for the PRAMS data indicates that age, race, depression during pregnancy, maternal smoking, abuse during pregnancy, hypertension, and smoking e-cigarettes are significantly associated with birth defects . The stup-value <0.001, OR = 1.17). The women in the age group of 25–29 years show lower odds of delivering a child with birth defect compared to maternal age groups of 18–19, 20–24, and 40 years and above .After adjusting the model for covariates, Asian women were at a higher risk of birth defects compared to White women . Depression during pregnancy was significantly associated with infants born with birth defects with odds of 1.31 compared to women with no depression during the prenatal period . Diabetes during pregnancy was negatively associated with birth defects compared to non-diabetic women when the model was controlled for covariates. .The odds of delivering an infant with a birth defect in mothers who smoke during pregnancy are approximately two times the odds for mothers who do not smoke during pregnancy . The interaction of smoking and depression resulted in higher odds of delivering the child with a birth defect compared to mothers who did not report smoking or depression when a multivariable model was controlled for covariates  . The interaction of smoking and diabetes was negatively associated with birth defects when other variables were constant   . The int< 0.001) .This research to analyze both individual and interactive effects of smoking, diabetes, and depression during pregnancy on birth defects using recent data from all PRAMS active sites in the United States for 2018 . The comp-value = 0.966) in predicting birth defects in children. The findings are inconsistent with the study hypothesis that these variables show an interactive effect in determining birth defects, thus rejecting this hypothesis. Insignificant results could be due to different reasons, such as study design and selection bias. The sample of women who answered yes to smoking, diabetes, and depression during pregnancy was very small to identify the effect of exposure in the population  and depression (OR = 1.72) in pregnant mothers, on delivering children with birth defects . The resp-value < 0.001). This study used a cross-sectional study design and larger sample, which provided the necessary number of subjects to analyze the effect of smoking and depression on birth defects. Another prospective cohort study proposed that diabetes, including gestational diabetes and its interaction with obesity, was significantly related to birth defects and increased birth defects by 65% [Previous studies did not analyze the interaction of smoking and depression, but the present study provided significant evidence of the interactive effects of smoking and depression during pregnancy on children with birth defects  or HIPAA  possibility due to linkage of birth defect data individually with birth records. If given a chance to repeat this study, more emphasis should be placed on understanding the individual and interactive impact of different types of birth defects.This study provided strong evidence that depression during pregnancy is associated with birth defects and variables leading to depression, including abuse or other mental health issues that can be related to childbirth defects ,9. The cThe awareness of depression and its possible impact on birth defects in children should be prioritized. More efforts are needed to educate pregnant women about managing stress, reporting abuse, and maintaining mental health to avoid depression during pregnancy. To mitigate birth defects, federal programs for maternal and child health, including HRSA  grants “Maternal, Infant, and Early Childhood Home Visiting Program”, should focus more on providing resources for depression during pregnancy and conditions responsible for depression ,19.


Starting Parkinson’s disease (PD) patients on subcutaneous apomorphine (APO) infusion is generally undertaken on a hospital day-case basis. During the COVID-19 pandemic, day-case facilities were unavailable. To avoid delays in treatment, a new procedure was developed for initiation of APO therapy in the patient’s home. A home initiation protocol was developed and followed for each patient in this analysis. The hospital team worked in collaboration with APO nurses provided by the manufacturer of APO therapies to implement initiation and undertake follow-up. In this analysis, 27 PD patients were initiated onto APO infusion and 21 (77.8%) achieved a therapeutic response. Home initiation of APO infusion can be undertaken successfully and has benefits for both patients and healthcare teams. This protocol will now continue as a standard of care at our centre. Apomorphine (APO) infusion is a potent dopamine receptor agonist with an established role for the treatment of motor symptoms of Parkinson’s disease (PD), including tremor, bradykinesia, and rigidity Muller . APO is APO infusion has been used successfully in clinical practice use for the management of PD for over 30 years and is generally well tolerated by patients  initiation between June 2020 and June 2022. Duration of treatment and outcomes were assessed up to December 2022. No institutional review board or ethics committee approval was necessary for this work as this was a clinical service evaluation. Informed patient consent was not required as the data were analyzed by members of the treating clinical team .Patients were selected for APO infusion therapy based on the consensus suitability criteria published for pump therapy  was used by patients to support dose changes and to assess clinical benefits and any adverse effects. The PKG measures movement accelerations of the wrist, providing objective information on bradykinesia and dyskinesia which has been shown to closely correlate with Unified Parkinson's Disease Rating Scale (UPDRS) motor scores  achieved a therapeutic response. Mean (± SD) duration of treatment was 365 ± 264 days. PKG was used for motor response assessment in 10/27 patients (37%). There was one failed titration, and four patients withdrew from treatment , whilst one died of pneumonia shortly after starting treatment; this was not deemed related to the treatment. The median initial infusion rate was 1.0 mg/h (range 0.75–2.0) and the median maximum infusion rate was 4.0 mg/h (range 1.0–6.0). Seven of the 27 patients used APO infusion at night as well as during the waking day. A total of 22/27 (81.4%) of patients were able to reduce their oral/transdermal medication use once established on APO infusion therapy.The change in the APO initiation procedure used at Salford from a hospital-based to home-based setting arose from the need to radically change clinical practice to meet patients’ needs during the COVID-19 pandemic. The significant impact that the pandemic has had on patients with PD treated with device-aided therapies has been recognized around the world  financial and capacity pressures that were in place during the pandemic. Our results show that home initiation of APO infusion can be undertaken successfully and has benefits for both patients and healthcare teams, in accordance with the license for this product. Home initiation resulted in a significant reduction in the waiting list for APO infusion and waiting time for initiation, as well as allowing day-case beds to be prioritized for admissions for other device-aided therapies. A focus on initiation in the home rather than the hospital setting, and the collaborative working partnership with the APO nurses, also released the hospital’s Advanced Therapy Specialist Nurse capacity to undertake more movement disorder clinics and thereby reduced the clinic waiting list. Patient expectations of treatment and follow-up appointments can be managed and supported by a collaborative and shared team effort comprising APO nurses, NHS specialist nurses, and clinicians.Our findings are in line with a recent large, prospective, longitudinal study of home APO infusion initiation in 106 PD patients, in which a more rapid improvement in quality of life and autonomy was seen compared to 38 in-hospital admissions (Zagnoli et al. We acknowledge limitations to this work. First, this was a retrospective analysis and therefore no formal outcome measures were recorded. Second, limitations on nurse autonomy to make dose changes may limit the introduction of such protocols in some healthcare settings. However, as described in Table Due to the success of the home initiation process in place during the pandemic, this will now continue as a standard of care, with day-case admission being reserved for cases not felt suitable for home initiation. It is hoped that our experience and practices with home initiation of APO infusion will encourage other centres to implement similar procedures.


Priestia megaterium ZS-3(ZS-3) can effectively help plants tolerate salinity stress. However, how ZS-3 regulates its metabolic adaptations in saline environments remains unclear. In this study, we monitored the metabolic rearrangement of compatibilisers in ZS-3 and combined the findings with genomic data to reveal how ZS-3 survives in stressful environments, induces plant growth, and tolerates stress. The results showed that ZS-3 tolerated salinity levels up to 9%. In addition, glutamate and trehalose help ZS-3 adapt to osmotic stress under low NaCl stress, whereas proline, K+, and extracellular polysaccharides regulate the osmotic responses of ZS-3 exposed to high salt stress. Potting experiments showed that applying the ZS-3 strain in saline and neutral soils could effectively increase the activities of soil acid phosphatase, urease, and invertase in both soils, thus improving soil fertility and promoting plant growth. In addition, strain ZS-3-GFP colonised the rhizosphere and leaves of Cinnamomum camphora well, as confirmed by confocal microscopy and resistance plate count analysis. Genomic studies and in vitro experiments have shown that ZS-3 exhibits a variety of beneficial traits, including plant-promoting, antagonistic, and other related traits . The results support that ZS-3 can induce plant tolerance to abiotic stresses. These data provide important clues to further reveal the interactions between plants and microbiomes, as well as the mechanisms by which micro-organisms control plant health.Approximately one-third of agricultural land worldwide is affected by salinity, which limits the productivity and sustainability of crop ecosystems. Plant-growth-promoting rhizobacteria (PGPR) are a potential solution to this problem, as PGPR increases crop yield through improving soil fertility and stress resistance. Previous studies have shown that  Salinity has become a major contributor to land degradation and the decline in plant productivity, posing a serious threat to sustainable agricultural production and global food security . The rhiBacterial survival in challenging high-salt environments is possible . BacteriPriestia megaterium is a typical gram-positive strain with numerous patents and industrial applications in biotechnology [P. megaterium for plant growth promotion (PGP) and biotic/abiotic control has been characterised, as repeatedly demonstrated in different crops [P. megaterium ZS-3 (ZS-3) improves plant growth under salt stress by adjusting the root architecture, promoting photosynthesis, alleviating oxidative and osmotic stress, and inducing systemic resistance [chnology . In agrint crops ,15,16. Psistance . HoweverPriestia strains to determine genome-scale metabolic models for industrial micro-organisms [Bacillus [Priestia strain’s ability to biotransform soil contaminants [The ongoing exploration of whole-genome sequences of individual strains, as well as gene function prediction and characterisation, will unveil new frontiers for understanding how bacterial species thrive in different environments, as well as for understanding their physiology and evolution in general. Recently, researchers have elucidated the genomes of several rganisms ,19, elucBacillus , investiaminants ,22, or daminants . In thisTo study the salt tolerance of strain ZS-3, we evaluated the growth of the strain on solid and liquid media at different salt concentrations. Strain ZS-3 was a moderately salt-tolerant bacterium, as it showed colony formation on LB plates supplemented with 0–9% NaCl a. When tSince a devastating effect on ZS-3 growth was observed with 9% NaCl stress, trehalose and aa levels were measured at 0–7% NaCl only. The trehalose content increased by 56.24%, 87.43, and 466.67% in the 3%, 5%, and 7% treatment groups, respectively, compared to the 0% treatment group a. SimilaEPS production by plant-growth-promoting rhizobacteria (PGPR) is often reported as a growth strategy that forms a protective film around cells to protect the cells from external influences in the presence of biotic and abiotic stresses. Therefore, EPS production by ZS-3 under salt stress was assessed. As shown in + concentration was significantly higher in the 3%, 5%, and 7% treatment groups than in the 0% treatment group. This indicates that ZS-3 converts and absorbs Na+ from the environment, which is dependent on high accumulation of intracellular K+ (synchronisation). In vivo K+ concentration was significantly higher in the 5% and 7% treatment groups compared to the 0% treatment group , and membrane integrity and protective mechanisms   . Furtheraturase) . These iPolyamines such as putrescine (put), spermidine (spd), and spermine (spm) play important roles in plant growth promotion. Several polyamine metabolism and transporter genes were identified in the genome of strain ZS-3 ; these gThe production of indole-3-acetic acid (IAA) directly promotes the growth of plant roots. Whole-genome sequencing confirmed that strain ZS-3 possesses genes associated with IAA synthesis and tryptophan (trp) synthesis , such asBacillus strains and positively affect plant growth and defence responses. Genomic analysis revealed genes and pathways involved in the metabolism of acetyl urea and butanediol. In addition, DMS-, 1-3-propanediol-, and nitric oxide-related genes were identified in the genomic data  are produced by mic data .phoA and phoD  assay indicated the presence of siderophores in the ZS-3 fermentation product  Presl was examined by colony counting and fluorescence microscopy. The population dynamics of the GFP-tagged ZS-3 strain (ZS-3-GFP) were determined via resolution of the GFP-tagged bacterial population from the roots and leaves at different time points. ZS-3-GFP was detected in the leaves and roots of seedlings . ACP activity increased by 14.29% (30 d), 8.45% (60 d), and 10.49% (90 d) in neutral soil inoculated with ZS-3 (N-ZS-3) compared to that in soil inoculated with ddH2O (N-CK). In neutral soils, ACP activity increased by 14.29% (30 d), 8.45% (60 d), and 10.49% (90 d) in the N-ZS-3 treatment compared to the N-CK treatment. However, the effect of ZS-3 on soil urease activity was only monitored in neutral soils, and no significant effect on urease activity in saline soils was observed. As shown in Under salt stress, strain ZS-3 significantly increased the biomass of th soils . ZS-3 alBacillus species must undergo many molecular and metabolic changes to survive hyperosmotic stress [Pontibacillus species [Proteobacteria and many gram-positive species [Bacillus species and the related genera Salinicoccus and Halobacillus [+, and EPS are involved in regulating the stress response of this strain at high salt stress levels.The challenges faced by bacteria that inhabit soil include frequent fluctuations in osmotic stress . Bacilluc stress . This st species . The com species . Ectoine species . Pro, Nδbacillus . The abuB. subtilis is reported to physiologically prefer the uptake of betaine from the external environment to the ab initio synthesis of pro [gbsB and betB, which are involved in glycine betaine biosynthesis. Furthermore, ornithine-oxo-acid transaminase was localised in the pro synthesis pathway of ZS-3. This transaminase encodes a protein that catalyses the biosynthesis of pro- and polyamines via the ornithine pathway, potentially conferring resistance to drought and salt stress in plants [Glu functions as the central metabolite that links carbon and nitrogen metabolism, as well as the main precursor for the de novo synthesis of pro . This kns of pro . Genomicn plants .+ and/or K+ for H+ from outside the cell [+/H+ antiporters participate significantly in conserving intracellular pH, cellular sodium amount, homeostasis, and cell volume [+/H+ antiporters are known in prokaryotes, including NhaA, NhaB, NhaC, NhaD, and NapA [+/H+ antiporter , multicomponent Na+/H+ antiporter subunit (mnhA/B/C/D/E/F/G), Ktr system potassium uptake protein (ktrB/C/D), and Ktr system potassium uptake protein (ktrB/C/D), which are genes involved in ion transport and osmoregulation. Adaptation to ionic hyperosmotic stress in B. subtilis, B. cereus, and B. licheniformis is a two-step process in which cells introduce K+ rapidly and transiently as an initial emergency response to rapid osmotic stress; then, cells replace these ions with compatible substances that do not interfere with cell physiology and biochemistry [+ in ZS-3 accumulated substantially at salt concentrations greater than 5% rather than 3%. Therefore, the adverse physiological effects caused by 3% salt stress are tolerable for strain ZS-3. Strain ZS-3 only shows a stress response at salt concentrations above 5%; under this environment, strain ZS-3 integrates more cellular responses involved in the regulation of osmotic stress, such as the massive synthesis of EPS. Our results are similar to those from several studies in which salt stress was shown to enhance EPS production [+ uptake and increase the water-holding capacity through EPS production to protect plants from abiotic stress [The majority of bacterial genomes include five to nine different genes that encode monovalent cation/proton antiporters, which trade cytoplasmic Nathe cell . Transmel volume . Five Naand NapA . Strain hemistry . The conoduction ,33. The oduction ,35. In aBurkholderia pyrrocinia JK-SH007 contains all the genes needed for the IAM and TAM pathways but lacks key genes for the IPyA and IAN pathways [B. pyrrocinia JK-SH007 , tryptamine (TAM), indole-3-acetonitrile (IAN), and indole-3-acetamide (IAM) . Burkholpathways . AlthougJK-SH007 . Many baPR1b and PBZ1 and the phytoalexin genes CPS4 and NOMT [Penicillium expansum by promoting the accumulation of endogenous GABA in apple fruit, which is regulated by enzyme activity and gene expression in polyamine anabolism and catabolism [Endogenous and exogenous polyamines can positively affect plant growth, yield, and stress resistance . For exaand NOMT . Genomicand NOMT . Exogenotabolism . In additabolism , salt [4tabolism , PEG [49tabolism . There iA. thaliana [Bacillus amyloliquefaciens MBI600 has been reported to produce the catecholate siderophore bacillibactin, which directly inhibits the growth of Pseudomonas syringae under iron-deficient conditions [F. oxysporum and Rhizoctonia solani [The production of vocs by PGPR is an important mechanism for its contribution to plant growth; 2,3-Butanediol and acetoin produced by PGPR promote growth in thaliana . Previouthaliana ,53. Bacinditions . In addia solani . In thisPseudomonas, Rahnella, Burkholderia, and Bacillus, utilise direct  and indirect  mechanisms to improve plant growth [Klebsiella spp. showed significant differences in their strain activity despite containing the same PGP gene [B. pumilus and B. safensis show more adaptations to plant-associated lifestyles than P. megaterium 16 PB based on an analysis of the genomic biosynthesis gene cluster and the amount of annotation of antifungal metabolites [P. megaterium ZS-3, which has a significantly better potential for promoting plant growth than potential for biocontrol. The number of antimicrobial metabolite genes in the core genome is limited.Genomic and physiological studies have shown that various bacteria, such as t growth ,56,57,58PGP gene . B. pumiabolites ,61. ThisP. megaterium varies between 0 and 10 [B. megaterium NCT-2 [P. megaterium ZS-3 in the development of eco-friendly biofertilisers that can contribute to the improvement of soil fertility and crop yield. The data also lay a foundation to further unravel plant–microbiome interactions and the mechanisms of microbial control of plant health.Genomic data revealed that strain ZS-3 contains one circular chromosome and zero plasmids. This species has a highly variable number of plasmids. The NCBI genome database shows that the number of plasmids in 0 and 10 ,16,62. F0 and 10 , which ium NCT-2 ,64. GeneLB medium was prepared with 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl (pH 7.4). Different salt concentration media were prepared based on LB medium. Briefly, LB medium was supplemented with 0.51, 0.86, 1.20, and 1.54 mol/L NaCl to obtain media with salt concentrations of 3%, 5%, 7%, and 9%, respectively. LB medium was used as a control. Solid LB medium was supplemented with 15 to 20 g of agar.600 = 0.2) was inoculated into 10 mL of medium with different salt concentrations, mixed thoroughly, and added to a 96-well plate. The 96-well plates were placed in a Bioscreen C instrument  at 37 °C to determine the growth curve, and OD600 measurements were collected automatically every 2 h.To prepare bacterium-only samples, ZS-3 was grown overnight in LB until it reached the exponential growth phase. The bacterial suspensions were inoculated into solid and liquid LB media and incubated at 28 °C. The cells of ZS-3 were collected by centrifugation and dried to constant weight, and the cell dry weight was determined. Then, 100 μL of the bacterial suspension  at low temperature. Centrifugation was performed at 4 °C and 6000 rpm for 15 min. The supernatant was retained and used to determine the content of trehalose at different salt concentrations with a trehalose content test kit .The overnight cultured bacteria were re-inoculated into 50 mL of LB different salinities and incubated at 28 °C for 48 h at 200 rpm. Subsequently, 50 mL of bacterial suspension was collected and placed in an ice bath and centrifuged at 8000 rpm for 10 min, and the supernatant was discarded. The precipitated bacterial bodies were rewashed twice with isotonic NaCl solution and centrifuged at 8000 rpm for 10 min at 4 °C to collect the cells. The cells were lysed with 5 mL of precooled 0.25 mol/L perchloric acid, mixed well, and left for 10 min. After centrifugation at 8000 rpm for 10 min at 4 °C, the supernatant was filtered through a membrane . The aa content was determined by an automatic amino acid analyser . The volume flow rate of the buffer was 0.25 mL/min, the volume flow rate of ninhydrin (pH 5.2) was 0.45 mL/min, and the detection wavelengths were 570 nm and 440 nm.The overnight cultured bacteria were re-inoculated into 50 mL of LB different salinities and incubated at 28 °C for 72 h at 200 rpm for EPS quantification. Extraction procedures were performed according to a previously established method . The ext+ and K+ concentrations in vitro. Precipitated bacterial cells were rewashed twice with PBS buffer  and then centrifuged at 8000 rpm for 10 min. The collected cells were broken by an ultrasonic crusher  and centrifuged at 8000 rpm for 10 min. Then, the supernatant was used to determine the Na+ and K+ concentrations in vivo. LB medium without bacterial inoculum was used as a control. Na+ and K+ were extracted according to a previously established method [+ and K+ were determined by flame photometry .The overnight cultured bacteria were re-inoculated into 50 mL of LB and incubated at 28 °C for 48 h at 200 rpm. Subsequently, 50 mL of the bacterial suspension was collected. Centrifugation was performed at 8000 rpm for 10 min, and the supernatant was retained to measure Nad method . The cong for 3 min, and the supernatant was extracted and passed through an organic phase filter membrane . Characterisation of put, spd, and spm levels in the supernatant was performed using HPLC-MS  as described previously [Extraction of biogenic amines was carried out according to previously described methods . Brieflyeviously . The amiPurified ZS-3 was grown in TSB medium with or without 5 mM L-Try  and incubated in the dark at 37 °C. TSB medium with or without L-Trp addition was used as a negative control. IAA production and secretion were measured in culture supernatants using Salkowski reagent as described previously . BrieflyEscherichia coli DH5α. The gfp gene was ligated into the corresponding restriction site (BamHI) of the pHIS1525 Plasmid and then transformed into Escherichia coli DH5α. By screening positive transformants containing the gfp gene, the pHIS1525-gfp plasmid was obtained. The pHIS1525-gfp plasmid was transfected into ZS-3 cells according to a polyethylene glycol-mediated procedure [−1 tetracycline) and fluorescence microscopy  [The GFP gene sequence was amplified by PCR using pFB01 as a template, and the GFP primers were GFP-f (TCTCCGGAGCTCCCGGGATCCTTTGTAGGGCTCATCCATGCC) and GFP-v (CATGCGGGCCGGGTACCGGATCCGTTGTTGACTTTATCTACAAGGTGTGG). The pHIS1525-gfp plasmid was constructed by ligating the gfp gene into the corresponding restriction site (BamHI) of the pHIS1525 plasmid and then transformed into rocedure . The traGermany) . All con8 cfu mL−1. To observe the dynamics of colonisation, annual C. camphora were used as sample, and each pot was inoculated with 100 mL of ZS-3-GFP bacterial suspension (1 × 108 cfu mL−1). An equal volume of sterile water was used as the control. C. camphora were grown in nursery pots containing sterile soil (pH 6.8) with one seedling per pot. Each treatment had five biological replicates and three technical replicates. On the 5th, 10th, 20th, and 30th days post inoculation, fresh leaves and roots were collected. ZS-3-GFP strains were isolated from fresh leaves and roots on LB plates containing tetracycline. A fluorescence microscope  was used to determine the number of colonies of ZS-3-GFP. Images of the roots were captured under a confocal microscope  equipped with a 20× objective and an emission wavelength of 488 nm, and fluorescence was measured in the 500–550 nm range.ZS-3-GFP was inoculated into LB medium containing tetracycline (10 µg/mL) and incubated at 200 rpm for 24 h at 37 °C in a shaker. ZS-3-GFP cultures were centrifuged at 8000 rpm for 10 min, and then the cell pellet was suspended in sterile distilled water at a final concentration of 1 × 10C. camphora seedlings were transplanted into pots containing saline soil (pH 8.15) and neutral soil (pH 6.8), and one seedling was planted in each pot. Each pot was inoculated with 100 mL of a cell suspension of the ZS-3 strain (1 × 108 cfu mL−1), and the treatment inoculated with an equal amount of sterile water served as a negative control. The following treatment groups were used: S-CK, S-ZS-3, N-CK, and N-ZS. Fifteen replicates were set up for each treatment. All other management practices were the same for each group during potting, and soil ACP, urease, and invertase activities were measured at 30 d, 60 d, and 90 d. Soil ACP, urease, and invertase activities under different treatments were measured by ACP activity test kits, soil urease test kits, and soil sucrase activity test kits .Two-year-old The ZS-3 genome was sequenced by the third-generation (Pac-Bio SMRT) sequencing technology method. Hifiasm was used for assembly, Circlator v1.5.5 was used for circularisation and adjustment of start sites, and Pilon v1.22 was used for further error correction using second-generation sequencing data, resulting in a final high-precision genome for subsequent analysis.Prodigal v2.6.3 was used for gene prediction in the genome. RepeatMasker v4.0.5 was used to search the genome for repetitive sequences by comparing it with a database of known repetitive sequences. Infernal v1.1.3 was used to accurately predict the three classes of rRNA in the genome based on a covariance model. tRNAscan-SE v2.0 was used to search for tRNAs in the genome with high accuracy based on the sequence and secondary structure features of tRNAs and the corresponding promoter features. Infernal v1.1.3 predicts the presence of other ncRNAs in the genome based on covariance models. CRT v1.2 software was used to predict CRISPR.P. megaterium ZS-3 is stored as No: M20231406 in the China Centre for Type Culture Collection (CCTCC).Predicted gene sequences were compared with the nonredundant (Nr), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), direct homology cluster (eggNOG), protein family (Pfam), Swiss-Prot, TrEMBL, and other functional databases by BLAST and gene function annotation. The Draft Genome Shotgun sequence project of ZS-3 has been deposited under the CP129891 accession number at GenBank. t test (t test), and Duncan’s multiple range test were performed. The data represented in the graphs are means ± standard deviations (SDs) of at least three replicate biological samples (n ≥ 3).Statistical analysis of the data was performed using SPSS . One-way analysis of variance (ANOVA), Student’s