Objective: To evaluate the effect of dyslipidemia on the cumulative live-birth rate (cLBR) in patients without polycystic ovary syndrome (PCOS) undergoing in vitro fertilization/intracytoplasmic sperm injection–embryo transfer (IVF/ICSI–ET) cycles.Methods: A total of 1,132 patients from the Yantai Yuhuangding Hospital Affiliated to Qingdao University from January 2016 to December 2017 were retrospectively included. The subjects were distributed into two groups based on their lipid profiles, namely, dyslipidemia group (n = 195) and control group (n = 937). The clinical and laboratory parameters of the two groups were analyzed, and a multivariate logistic regression analysis of the cLBR was conducted. In addition, subgroup analysis was carried out to avoid deviation according to the body mass index (BMI).Results: Patients with dyslipidemia had significantly greater BMI and longer duration of infertility, as well as lower antral follicle count and basal follicle-stimulating hormone level compared with patients without dyslipidemia. Stratified analysis showed that dyslipidemia was associated with a significantly higher total gonadotrophin dosage required for ovarian stimulation as well as lower number of oocytes retrieved, independent of obesity. The live-birth rate in fresh cycle and cLBR were higher in the control group, although the difference between the groups was not significant . However, multivariate logistic regression analysis adjusting for potential confounders showed that dyslipidemia was negatively associated with cLBR .Conclusion: Our findings demonstrate for the first time that dyslipidemia has a deleterious impact on cLBR, independent of obesity, in non-PCOS population considered to have good prognosis. Assessment of serum lipid profiles as well as the provision of nutritional counseling is essential for increasing successful outcomes in assisted reproductive techniques. Recently, there has been a significant increase in the incidences of infertility and metabolic disorders, such as dyslipidemia and obesity, which has been associated with unhealthy lifestyles and poor living conditions . To dateDyslipidemia is clinically defined as abnormalities in one or more types of plasma lipids, such as elevated total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) concentrations, as well as reduced high-density lipoprotein cholesterol (HDL-C) levels . In ChinSeveral studies using mouse models have shown that chronic consumption of high-fat diet (HFD) results in metabolic and reproductive disorders independent of obesity. Specifically, mice on HFD ovulated less often , had dimIn the last few decades, most studies have generally focused on the effect of metabolic disorders, such as obesity  and polyThis was a retrospective cohort study. All women attending the Department of Reproductive Medicine at the Yantai Yuhuangding Hospital Affiliated to Qingdao University in the People’s Republic of China from January 2016 to December 2017 were screened for eligibility. The study received ethical approval from the Ethics Committee of Yantai Yuhuangding Hospital, and all the protocols were conducted in accordance with relevant guidelines and regulations. The need for informed consent for this study was waived due to the retrospective nature of the study.All patients aged 20–38 years, who had a normal ovarian reserve which is generally defined as the basal antral follicular count of more than seven . Our criThe exclusion criteria included the following: (1) history of chemotherapy, radiotherapy, or ovarian surgery; (2) endometriosis; (3) uterine abnormality; (4) indication for preimplantation genetic diagnosis or preimplantation genetic screening; (5) history of recurrent pregnancy loss; and (6) other medical conditions that are contraindicated in assisted reproductive technology and/or pregnancy.Blood samples were collected between day 2 and day 4 of the menstrual cycle before the IVF/ICSI protocol after an overnight fast of at least 8 h. The serum concentrations of lipoproteins, including total cholesterol (TC), LDL-C, HDL-C, and triglyceride (TG), were then evaluated by an automatic biochemical analyzer (Cardiocheck PA). All eligible individuals who had a known fresh cycle outcome (live-birth or not) were included in our study. Individuals who did not deliver a live baby in the fresh cycle were followed for at least 2 years (irrespective of the date of study entry), to estimate the outcomes of the frozen-thawed cycles. The study participants were divided into two groups based on their lipid profiles. According to the 2016 Chinese guidelines for the management of dyslipidemia in adults , particiIn our center, the standard GnRH agonist long protocol or GnRH antagonist protocol is used in patients with normal ovarian function. Briefly, for the GnRH agonist protocol, the women received a daily subcutaneous injection of 0.03–0.05 mg GnRH agonist, starting in the luteal phase of the previous cycle until the day of human chorionic gonadotropin (hCG) administration. After stimulation with GnRH for 14–21 days and satisfactory pituitary desensitization had been achieved , recombinant follicle-stimulating hormone (rFSH) was administered in concentrations ranging from 150 to 225 IU/day. This was adjusted according to follicular development monitored using transvaginal ultrasonography and assessment of the serum concentrations of sex hormones. For the flexible GnRH antagonist protocol, rFSH was started on days 2–3 of the menstrual cycle, and the doses were modulated based on the ovarian response of the patients. Human menopausal gonadotropin was co-administered at the discretion of the physicians. Ganirelix (0.25 mg) was administered daily when the lead follicle >12 mm, up to and including the day of hCG administration. Urinary hCG  was administered when at least two dominant follicles measured 18 mm or more in diameter. Oocyte retrieval was performed 34–36 h later.Based on sperm quality, oocytes were fertilized through conventional IVF or ICSI or via IVF/ICSI. The embryos were cultured up to day 3 or day 5/6. Embryo culture was carried out using sequential culture medium . Embryo assessment, including cleavage-stage embryo assessment and blastocyst assessment, was performed based on the recommendation of the Istanbul consensus workshop . RedundaOur primary outcome was the cLBR, which was defined as the delivery of a live-born infant (>24 weeks of gestation) in the fresh or in the subsequent frozen-thawed cycles in relation to the number of oocytes retrieved within two years . s was ct-test  or Kruskal-Wallis test . Categorical variables were analyzed using the Pearson χ2 test or Fisher’s exact test as appropriate and were expressed as frequency (percentages). Patients were divided into subgroups of BMI < 24 kg/m2 group and BMI ≥ 24 kg/m2 group for stratified analysis. Multivariate logistic regression was used to assess the association between different types of lipid metabolism and cLBR by adjusting the effect of baseline characteristics and groups to calculate the odds ratios (ORs) and 95% confidence intervals (CIs). All values of p < 0.05 for two-sided tests were considered to be statistically significant.All analyses were carried out using IBM SPSS Statistics . Normal distribution of quantity variables was tested using the Shapiro-Wilk test. Continuous variables were presented as means ± standard deviations (SDs), which were compared using the Student’s 2 vs. 22.08 ± 3.61 kg/m2, p < 0.001) and longer duration of infertility  compared with patents without dyslipidemia. In addition, patients with dyslipidemia had significantly lower bilateral antral follicle count  and basal FSH  than the control group. No differences were observed in terms of age, AMH, basal LH, basal FSH/LH ratio, basal E2, infertility type, causes of infertility, or ovarian stimulation protocol (p > 0.05).A total of 2,963 patients underwent the ovarian stimulation and oocyte retrieval during the study period. Of these, 1831 (61.8%) patients were excluded due to any of the following reasons: older than 38 years old , not the first stimulation cycle , no embryo transferred by the time of data collection , impaired ovarian reserve , and incomplete records . Therefore, 1,132 patients met the eligibility criteria, of whom 195 cases had dyslipidemia (17.2%) and 937 had normal lipid metabolism. The baseline characteristics are shown in p = 0.004) and the total stimulation dose  used were significantly higher in the dyslipidemia group, although peak E2 , the number of lead follicles on hCG day , and number of oocytes retrieved  were marked lower compared with patients in the non-dyslipidemia group. However, the fertilization rate, number of available embryos and embryos cryopreserved, the embryo utilization rate, proportion of fresh or frozen embryo transfer, and methods of endometrium preparation in FET cycles between two groups were comparable (p > 0.05).p = 0.043). Similarly, women with dyslipidemia had a higher chance of miscarriage compared with the women in the normal group .Notably, there was a significantly higher incidence of moderate-severe OHSS in the dyslipidemia group compared with the normal group, as shown in p = 0.116; 62.1% vs. 67.6%, p = 0.138). Other outcomes, including the rates of clinical pregnancy and twin birth, and pregnancy-related complications, including gestational diabetes, preeclampsia, and birth weights, showed no differences between the groups (p > 0.05).The live-birth rate in fresh cycle and cLBR were higher in non-dyslipidemia group although the difference between the groups was not significant  as normal (BMI < 24 kg/m2) and overweight/obese (BMI ≥ 24 kg/m2), as shown in 2 women with dyslipidemia was higher than other groups, although the difference was not significant, which may be related to the more dosage and days of Gn.To further determine whether the effect of dyslipidemia on reproductive outcomes was independent of obesity, patients were stratified based on their BMI . There was a positive correlation between the number of oocytes retrieved, available embryos, and embryos cryopreserved on the cLBR .Univariate logistic regression was first performed to evaluate the effect of each variable on cLBR . Thereafp = 0.044; p = 0.017; OR, 2.787, 95% CI, 1.691–4.283, p < 0.001; OR, 3.279, 95% CI, 2.353–5.432, p < 0.001; After adjusting for age, BMI, AMH, infertility type, total Gn dosage, number of oocytes retrieved, fertilization rate, number of available embryos, and number of embryos cryopreserved by multivariate logistic regression analysis, dyslipidemia was still significantly associated with decreased cLBR  was lower than the average incidence rate (40.7%) in the general population of China aged 18 and older , due to In our study, we found that patients with dyslipidemia had longer duration of infertility, and lower AFC and basal FSH levels. Notably, subgroup analysis demonstrated that dyslipidemia was associated with significantly higher total Gn dosage required for ovarian stimulation and lower number of oocytes retrieved, independent of obesity. In addition, there was a higher incidence of OHSS and miscarriage in patients with dyslipidemia compared to those with normal lipid metabolism. The live-birth rate in the fresh cycle and the cLBR were higher in the non-dyslipidemia group, although the difference was not significant. Multivariate logistic regression model showed that dyslipidemia alone is negatively associated with cLBR in non-PCOS patients.Previous studies have shown that dyslipidemia is positively correlated with body weight, and patients with dyslipidemia are more prone to obesity. These findings are consistent with results from our study in which patients with dyslipidemia were associated with significantly greater BMI, suggesting that obesity and dyslipidemia have a close relationship. We also found that patients with dyslipidemia required higher initial and total gonadotropin doses compared to patients with normal lipid metabolism during COH protocol. This is because the dose of gonadotropin required is usually adjusted by physicians based on the BMI of the patients. In other words, patients with higher BMI require a higher dose of gonadotropin to promote follicle development. Our findings on OHSS are corroborated by a recent study in which patients with dyslipidemia were reported to be more likely to develop severe OHSS in the freeze-all embryos group . There ahigh-fat diet (HFD). Physiologically, lipids are important for energy production during oocyte maturation, fertilization, and preimplantation development. Studies using mice models have shown that HFD causes lipotoxicity in granulosa and cumulus cells, resulting in accelerated ovarian follicle development and follicle loss, which eventually affects ovarian reserve (It is well known that disordered lipid metabolism usually occurs due to changes in lifestyle, including physical inactivity and excessive intake of  reserve . Likewis reserve . These f reserve . SimilarMoreover, a recent study found that the patients with accumulation of lipid droplets in cumulus and granulosa cells had lower IVF/ICSI pregnancy rates, regardless of their BMI . Excess The cumulative delivery rate was mainly affected by the age of patients and the number of oocytes retrieved during the COS process . RecentlThere are several strengths in this study, including the large sample size from a relatively homogenized cohort of people. In addition, we carried out subgroup analysis to eliminate the effect of obesity on the IVF/ICSI outcomes. Lastly, our study excluded the effect of PCOS, a common endocrine disorder contributing to subfertility, on reproductive outcomes. On the other hand, there are several limitations in our study. First, we could not investigate unknown confounding factors, such as dietary habits or medical interventions before or during IVF treatment due to the retrospective nature of the study. Secondly, the association between individual lipoproteins and IVF/ICSI outcomes could not be determined due to the sample size. Thirdly, while our results are convincing, they should be taken with caution since only the results of multivariate logistic analysis were used to conclude that dyslipidemia has detrimental effects on cLBR. Further longitudinal investigations and larger multicenter clinical investigations are required to validate these results and to explore the mechanism behind these effects.In conclusion, our findings demonstrate for the first time that dyslipidemia has a deleterious impact on cLBR, independent of obesity, in non-PCOS population considered to have good prognosis. Assessment of serum lipid profiles as well as the provision of nutritional counseling is essential for increasing successful outcomes in assisted reproductive techniques.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Yantai Yuhuangding Hospital. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.ZL and JC conceived and designed the study. ZL and XL took part in the clinical data collection and analyzed the data. ZL, HZ, SL, and SH wrote the manuscript. JC and HB supervised the entire work. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Compared with non-HT patients, HT patients had higher baseline NIHSS score  and serum occludin level . Moreover, receiver operating characteristic curve based on leave-one-out cross-validation showed that the combination of serum occludin level and NIHSS score significantly improved the accuracy of predicting HT . These findings suggest that the combination of two methods may provide a better tool for HT prediction in acute ischemic stroke patients with reperfusion therapy.Hemorrhagic transformation (HT) is a severe complication following acute ischemic stroke, particularly with reperfusion interventions, leading to poor prognosis. Serum occludin level is related with blood brain barrier disruption, and the National Institute of Health stroke scale (NIHSS) score reflects stroke severity. We investigated whether the two covariates are independently associated with HT and their combination can improve the accuracy of HT prediction in ischemic stroke patients with reperfusion therapy. Seventy-six patients were screened from the established database of acute ischemic stroke in our previous study, which contains all clinical information, including serum occludin levels, baseline NIHSS score, and hemorrhagic events. Multivariate logistic regression analysis showed that serum occludin level (OR = 4.969, 95% CI: 2.069–11.935,  Ischemic stroke accounts for 70–80% of all stroke patients, and reperfusion therapy has proved to be an effective treatment in patients with ischemic stroke . HemorrhBreakdown of the blood–brain barrier (BBB) is associated with the risk of HT in acute ischemic stroke . As a meAs it is well recognized, besides different treatment measures, there are many other risk factors associated with HT in clinic, such as age, ischemic severity, infarct size, hyperglycemia, and hypertension . Among tTherefore, the present study aims to determine whether serum occludin level combined with NIHSS score can improve the predictive value of HT in stroke patients with reperfusion therapy, utilizing our previous study database. The outcome of this study will be helpful for selecting the maximum number of patients who can benefit from reperfusion therapy while minimizing the risk of HT at the same time.The data in this study were obtained from our established database from a single-institute retrospective study , in whicn = 25), no reperfusion therapy (n = 91), and incomplete brain CT scans within 1 week  acute ischemic stroke patients with intravenous thrombolysis, mechanical thrombectomy, or bridging therapy (intravenous therapy followed arteries therapy); (2) the time from symptom onset to arriving at the emergency department < 24 h; (3) completed at least three times of brain CT scans within 1 week after reperfusion treatment. Exclusion criteria: (1) no baseline blood samples; (2) inflammatory or infectious diseases, cancer, hemorrhagic disease, and severe renal and liver failure. Informed consents were obtained for data analysis. According to inclusion and exclusion criteria of the study, among the 196 acute ischemic stroke patients in the database, 76 cases were included in this study after excluding patients with transient ischemic attack  was obtained according to our previous study , which wIn this study, the enrolled patients had undergone different reperfusion treatments for acute cerebral infarction. Depending on the specific condition of each patient, a variety of reperfusion procedures were used, including intravenous thrombolysis, mechanical thrombectomy, and bridging therapy .Diagnostic criteria for HT in this study followed the guideline of the American Heart Association/American Stroke Association. Briefly, HT was diagnosed in one of the following cases: (1) no intracranial hemorrhage on the first brain CT/MRI scan after cerebral infarction, but occurred on the second cranial CT/MRI examination ; (2) exiAccording to the European Cooperative Acute Stroke Study (ECASS) II, most HTs occur within 7 days post stroke onset or recanalization and are the major cause of death . Therefot-tests and Mann–Whitney U tests were used for comparing continuous variables. One-way analysis of variance, followed by LSD-t test, was used to examine the difference in each variable among the groups. The significance level of all the statistical tests was set at a type I error rate of 0.05.Statistical analysis was performed using SPSS 26.0, R software (version 3.5.1) and MedCalc software. Baseline characteristics were compared between patients with and without HT. Categorical variables were presented as frequencies (%), and continuous variables are shown as means ± SD for normal distribution or medians  for non-normal distribution. Chi-Squared test or Fisher exact tests were used to assess the association between categorical variables, while p < 0.1 from the univariate analyses were included in the multivariate logistic regression model. The variance inflation factor was estimated to check the presence of multicollinearity among variables included in regression model. Receiver operating characteristic (ROC) curve analysis was used to evaluate the prediction performance of individual variables and the model with multiple variables. The unbiased estimate for model prediction accuracy was obtained using leave-one-out cross validation. Area under the curve (AUC) of ROC curves were compared using De-long’s test. The goodness-of-fit of the model was examined using the Hosmer–Lemeshow C statistic.We utilized the univariate and multivariate logistic regression analyses to examine the associations of HT with the clinical risk factors, including baseline occludin level and NIHSS score. The variables with p = 0.003], and higher mean serum occludin level . There was no significant difference in other vital characteristics between HT and non-HT groups.In total, 76 patients were included in the study according to the inclusion and exclusion criteria. p < 0.1 from the univariate regression were included in the multivariate analysis. They are the time from onset to blood sampling, baseline diastolic blood pressure, hypertension, baseline NIHSS score, and baseline serum occludin level. Among the five risk factors, baseline serum occludin level and baseline NIHSS score were significantly associated with HT, as the confidence intervals of their odds ratio (OR) do not include one (p < 0.001) and baseline NIHSS score  were significantly associated with HT even after adjusting the effects of the other three factors , HI2 (n = 7), PH1 (n = 6), and PH2 (n = 2). The composition of different types of HT was shown in pie graph , HI2 , PH1  and PH2 , and Non-HT , respectively. As there were few cases in HI1 and PH2 groups, HI1 and HI2 was combined as HI, while PH1 and PH2, as PH group for statistical analysis.To further explore the relationship between baseline level of serum occludin and different types of HT, 17 patients with HT were further divided into four subgroups, according to the ECASS II criteria : HI1 . But there was no significant difference between HI and PH subgroups . These rAs for baseline NIHSS score, there was significant difference in PH subgroups, compared with the Non-HT group . There was no difference between the Non-HT and HI groups . These rlogit(π) = −10.052 + 1.32x1 + 0.256x2, where π is the probability of HT, and x1 and x2 are the baseline serum occludin level and NIHSS score. The Ngelkerke R squared is 0.565, and the p-value of the Hosmer-Lemeshow test is 0.755, suggesting the model is a good fit for the data. To obtain an unbiased estimate for the prediction accuracy of the combined model, we performed a leave-one-out cross-validation (LOOCV). The ROC curve based on the LOOCV prediction is presented in p-values of De-long’s test: 0.016 and 0.007)  . These rThis is the first clinical study to explore the value of serum occludin level combined with baseline NIHSS score for predicting the risk of secondary HT in patients with acute cerebral infarction after perfusion therapy. In this study, we found that serum occludin level and baseline NIHSS score were independent risk factors for HT, respectively, and the combination of the two factors could significantly improve the predictive effect for HT. The present study may provide a better method to evaluate the risk of HT before recanalization therapy, helping selecting patients with lower risk of HT.Reperfusion therapy is widely accepted as an effective treatment method for acute ischemic stroke patients . HoweverMany factors have been suggested to be associated with HT in clinic, such as age, stroke severity, infarct size, hyperglycemia, and hypertension . In thisBBB disruption is a critical step in the development of HT, and therefore, it is useful to evaluate the permeability of BBB as early as possible for predicting the risk of HT . Our preAs one of clinic related factors, NIHSS score is considered as a good and stable predictor of bleeding transformation . At presIn this study, ROC curve analysis showed that the combination of serum occludin and NIHSS score significantly improved predictive value of HT. In this analysis, we obtained an unbiased estimate for ROC and AUC of the combined model through the LOOCV procedure. De-long’s test results showed that, at 0.919, the AUC of the combined model is significantly larger than that of an individual predictor, suggesting that a combined diagnosis method is superior to a single variable (occludin level or NIHSS score) alone. Therefore, the combination of serum occludin levels (representing the extent of BBB disruption) and NIHSS score (reflecting stroke severity) markedly increased the predictive value for HT, providing a new method for predicting HT in stroke patients with reperfusion therapy.Although we have demonstrated that the combined diagnosis method for HT was better than each individual index in this study, the small sample size could bring about some limitations. These results, including risk factors for HT and the AUC of ROC curves obtained from the database may have false positives or biases. Therefore, these results need to be further confirmed in a prospective study with a larger number of patients. Moreover, a larger number of cases may include more HT cases, which would be more helpful to analyze the subgroup of symptomatic HT and provide a more useful guideline for clinical practice.Baseline serum occludin level and baseline NIHSS score were independent risk factors for secondary HT in patients with acute cerebral infarction following perfusion therapy. The combination of the two risk factors may provide an early prediction for HT in patients with acute cerebral infarction with reperfusion treatment.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by Ethics Committee of Xuanwu Hospital, Capital Medical University. The ethics committee waived the requirement of written informed consent for participation.SY: analysis of the data, design of the study, and drafting the manuscript for intellectual content. WL, CH, and HK: analysis of the data and revising the manuscript. QM and XJ: analysis of the data and interpretion of the data. ZQ and KL: design of the study, analysis of the data, and revising the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mother and newborn skin-to-skin contact (SSC) is an immediate postpartum intervention known to improve the health of newborn and mothers alike. Albeit, there is paucity of data that explored the coverage or factors associated with SSC in Nigeria. Therefore, we aimed to explore the coverage and hierarchical nature of the factors associated with SSC among women of reproductive age in Nigeria.p< 0.05.The 2018 Nigeria Demographic and Health Survey (NDHS) data was used for this study. Data on 29,992 women who had ever given birth were extracted for analysis. SSC was the outcome variable as determined by women’s report. A multivariable multilevel logistic regression model was used to estimate the fixed and random effects of the factors associated with SSC. Statistical significance was determined at The coverage of SSC was approximately 12.0%. Educated women had higher odds of SSC, when compared with women with no formal education. Those who delivered through caesarean section (CS) had 88% reduction in SSC, when compared with women who had vaginal delivery . Women who delivered at health facility were 15.58 times as likely to practice SSC, when compared with those who delivered at home . Adequate ANC visits and low birth weight significantly increased the odds of SSC. Women from richest household were 1.70 times as likely to practice SSC, when compared with women from poorest household . There was 65% reduction in SSC among women with high rate of community non-use of media, when compared with women from low rate of community non-use of media .SSC coverage was low in Nigeria. Moreover, individual, household and community level factors were associated with SSC. More enlightenment should be created among women to bring to limelight the importance of SSC specifically to newborn’s health. Globally, about 2.5 million newborn die in their first month of life, accounting for 47% of under-five death . AlthougSimilarly, SSC has been known as a safe substitute to reduce neonatal mortality in resource-poor settings . HoweverThe health care system of any setting has a role to play on the implementation of practices recommended for positive health outcomes. Unfortunately, Nigeria has a weak health care delivery system that contributes to adverse maternal and child health outcomes . PromotiWith efforts to meet the targets of the Sustainable Development Goals (SDGs) by 2030, to reduce neonatal mortality to as low as 12 per 1000 live births , it is chttp://dhsprogram.com/data/available-datasets.cfm. Details of DHS sampling procedure has been reported previously [In this analysis, we used cross-sectional, nationally representative data. For study, NDHS 2018 data was collected from 29,992 women who had ever given birth. A three-stage sampling stratification was involved in the sampling design, in which respondents were first stratified by urban versus rural dwellings, then randomly selected enumeration areas (EAs) within each stratum. Finally, using equal likelihood sampling, households within each EA were then chosen for the survey. In the calculation of survey weights, this three-stage sampling procedure was taken into account and applied to ensure the representativeness of the sample with respect to the general population. Data for this analysis is obtained from the questionnaire of individual women. The data is available in the public domain and accessed at; eviously .Was child put on mother's chest and bare skin after birth”. This was measured dichotomously and coded as “1” if a woman answered “yes” and “0” if otherwise.Mother and newborn skin-to-skin contact was derived from the question; “Maternal age: 15-19, 20-24, 25-29, 30-34, 35-39, 40-44, 45-49.Residential mobility: internal immigrant (if a respondent has lived less than 5 years in the current location) vs. indigenous (if a respondent has lived up to 5 years and above in the current location).Educational achievement: no formal schooling, primary and secondary or higher education.Religion: Christianity, Islam and the Traditions of Africa.Media exposure: was measured dichotomously (yes vs. no) whether a respondent used any newspaper/magazine, radio, TV or internet; Number of children ever born: 1-2, 3-4 and over 4 children.Child wanted while pregnant: then, later and no longer wanted.Health insurance: protected vs. not covered.Job status: working vs. not working.Childbirth mode: caesarean vs. vaginal.Location of delivery: health facility vs. home.ANC visits: no visit, 1-3, 4 and above.Type of birth: singleton vs. multiple.Sex of child: male vs. female.Preceding birth interval: <2years, 2-3years, >3-4years, >4years, firstborn.Birth weight: low birth weight (<2.5kg) vs. average (≥2.5kg). These factors were included based on previous studies which examined the factors associated with SSC –19.The size of the household was based on the total number of individuals residing together; 1-4, 5-8 and over 8 individuals. Household wealth quintile: To assign the wealth indicator weights, Principal Components Analysis (PCA) was used. This protocol assigned scores and standardized variables for the wealth predictor, such as; Main wall material, main roof material, sanitation facilities, water supply, radio, television, electricity, refrigerator, cooking fuel, furniture, number of people per room, bicycle, motorcycle/scooter, car/truck, main floor material, main wall material. Scores of the factor coefficient (factor loadings) and z-scores were determined. The indicator values have been multiplied by the loadings for each household and summed to produce the household’s wealth index value. The standardized z-score was used to disentangle the overall assigned scores to poorest, poorer, middle, richer and richest class , 21.Residential status: urban vs. rural. Geographical area: North Central, North East, South East, South South and South West, North East, North West. Non-exposure media concentration in the group . Uneducated women's concentration in the community . Poverty concentration in the society . This approach is similar to the methods of a previous study .http://goo.gl/ny8T6X.In this analysis, with all identifier information removed, we used population-based secondary datasets available in the public/online domain. Access was given to the authors by MEASURE DHS/ICF International to use the data. The DHS complies with requirements to ensure the security of the privacy of respondents. ICF International makes sure the survey is consistent with the U.S. Department of Health and Human Services regulations for the respect of human subjects. No further approval was required for this study. More details about data and ethical standards are available at p< 0.05. Data analysis was conducted using Stata Version 14 .For data strata, cluster and sample weights, we used the 'svy' module to modify. To estimate the fixed and random effects of the factors associated with SSC, a multivariable multilevel logistic regression model was used. For binary answer, we defined a 3-level model. A single woman (at level 1), a family (at level 2) living in a group (at level 3). We had five models built. To break down the amount of variance between group and household levels, the first model, an empty or unconditional model without any explanatory variables, was defined. In order to understand the variations of the community and households, the null or empty model is important and we used it as a guide to estimate how many household and community variables were able to explain the observed variation. Furthermore, we used it to explain the use of the multilevel statistical method, since it recommended the use of single-level logistic regression if the population variance was not important in the empty model. Statistical significance was determined at The results of fixed effects (association measures) with a 95 percent confidence interval were reported as adjusted odds ratios (AORs) (CI). The Intra-class Correlation (ICC) and Median Odds Ratio (MOR)  evaluateTable Results from Table Results from the full model (Model V) showed women with secondary  and tertiary education  were more likely to have SSC, when compared with women with no formal education. Women who delivered through caesarean section had 88% reduction in SSC, when compared with women who had vaginal delivery . Furthermore, women who delivered at health facility were 15.58 times as likely to practice SSC, when compared to those who delivered at home . ANC visits and low birth weight significantly increased SSC. Women from richest household were 1.70 times as likely to have SSC, when compared with women from poorest household . Geographical region of women was significantly associated with SSC. There was 65% significant reduction in SSC among women from high level community non-use of media, when compared with with women from low level community non-use of media . See Table 2 = 2.68; SE= 0.21) and households . The variance partition coefficient of 28.0% and 32.9% estimates for the null model implied that variance in the odds of SSC could be attributed to community and household level factors respectively. Results from the Median Odds Ratio also confirmed evidence of community and household-level factors influencing SSC. Based on estimates from the full model (Model V), it was estimated that if a women moved to another community or household with a higher probability of SSC, the median increase in their odds of SSC would be 10.88 and 8.79 fold respectively. The proportional change in variance (PCV), intra-class correlation (ICC) and model fit statistics were also estimated.We presented variance and standard error (SE) for the random effects in Table In this study, we explored the coverage, enablers and barriers of SSC in Nigeria, at individual, household and community levels. Based on the findings of the study, the coverage of SSC was low. Furthermore, the mode of delivery, educational attainment, place of delivery, attendance of ANC visits and LBW were significantly associated with SSC. At the household level, women belonging to the richest households were more likely to have SSC. While high rate of community non-use of media and geographical region were significantly associated with SSC at the community level.The prevalence of SSC was found to be 12%. This is slightly higher than the report from a previous study which showed that only 10% of newborns received SSC . This coAn individual-level factor in this study that was strongly associated with the practice of SSC was delivery at a health facility. Women who delivered in a health facility had 15 times higher odds of having SSC immediately after delivery with their newborns. This is consistent with the findings from previous studies which showed that the practice of SSC between the mother and the newborn was significantly more common in health facility based delivery compared with home births , 33. SkiFurthermore, higher odds of SSC was observed among women with higher levels of education compared to mothers with no education. Educated mothers may be more knowledgeable about SSC and its benefits, they may be more likely to discuss it with their health providers and request for it even when the information is not offered by the provider. Also, mothers who had attended ANC visits had increased odds of having SSC with their newborns immediately after delivery, compared with those who had no ANC visits. Similar findings have been reported in other studies , 35. ANCAt the household level, mothers from the richest household wealth quintile had higher odds of practicing SSC with their newborn immediately after birth. Women from rich households may be more educated, more likely to utilize maternal health services such as ANC, delivery in health facility , 43, 44 This study utilized the most recent NDHS 2018, a nationally representative population-based survey. The findings contributes to knowledge, given the paucity of studies on the subject area in Nigeria. Also the application of multilevel analysis addressed the hierarchical nature of NDHS data. However, the study does have some limitations. First, the cross-sectional nature of the data precludes conclusions about causality of the factors on the SSC cannot be made. Secondly, the construction of community variables from aggregation of individual level data means that inferences at a higher level are made based on individual level data . ThirdlyThe coverage of SSC in Nigeria is poor, despite evidence of its numerous benefits to the mother and the newborn. Moreover, this study revealed that the practice of SSC immediately after delivery between mothers and their newborn is associated with individual level factors such as place of delivery, mode of delivery, maternal educational level, birth weight of new born, ANC attendance during pregnancy; household wealth and community exposure to media.


This study was designed to explore the clinical efficacy of methotrexate combined with iguratimod on patients with rheumatoid arthritis (RA) and its influence on the expression levels of HOTAIR in serum.  A total of 268 RA patients were selected as research objects, 145 patients received methotrexate alone were used as a control group (CG), 123 patients received methotrexate combined with iguratimod were taken as a research group (RG), and serum of 60 healthy people undergoing physical examination was selected as a healthy control group (HCG). The therapeutic value of two therapeutic methods for RA was compared, and the HOTAIR expression in serum was detected by qRT-PCR.  Compared with methotrexate used alone, the joint use of methotrexate and iguratimod could provide better clinical efficacy for RA patients and would not increase the incidence of adverse events. HOTAIR was highly expressed in the serum of RA patients, and its expression decreased after treatment.  Combination therapy of methotrexate and iguratimod is a safe and effective way to treat RA patients, which can be popularized clinically. Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by inflammatory synovitis and progressive joint destruction . RA is tMethotrexate has been used to treat RA since the 1980s, and it is still the first-line drug for RA treatment to this day . Therapyα is a protein mainly produced by activated macrophages and monocytes. It participates in human inflammatory response and immune response and is essential to maintain homeostasis [Increased inflammatory mediator levels were linked to autoimmune diseases accompanied by chronic or repeated inflammation. Many cytokines, such as interleukin- (IL-) 6 and tumor necrosis factor- (TNF-) a, play a significant role in RA pathogenesis. TNF-eostasis .α, which is aimed at providing a safe and effective treatment method for RA patients.At present, there are few and incomplete researches on the efficacy and safety of methotrexate combined with iguratimod in treating RA. Therefore, this study mainly explored the efficacy and safety of the combination therapy of methotrexate and iguratimod and analyzed its influence on the expression levels of HOTAIR and TNF-A total of 268 RA patients admitted to the People's Hospital of Wenjiang from July 2017 to January 2019 were selected as the research objects. Among them, 145 patients received methotrexate alone were selected as the control group (CG), and the remaining 123 patients received methotrexate combined with iguratimod were selected as the research group (RG). Inclusion criteria were as follows: patients all conformed to RA-like diagnostic criteria [Patients in the CG were treated by methotrexate . The method of use is as follows: 10 mg/time, once a week; 12.5 mg/time after two weeks, twice a week; and 15 mg/time after four weeks, once a week until the end of the experiment. And it was taken orally. On the basis of the CG, those in the RG were treated by iguratimod , twice a day, 25 mg/dose. Every 12 weeks is one course of treatment, and patients from both groups continue to treat for two courses of treatment.After the treatment, the efficacy was evaluated: markedly effective: symptoms disappeared completely, physical signs improved by more than 75%, and C-reactive protein (CRP) and erythrocyte sedimentation rate decreased significantly and fell to the normal range; effective: symptoms partially improved, physical signs improved by more than 30%, and CRP and ESR decreased to some extent, but did not fall within the normal range; ineffective: symptoms and signs did not improve, and CRP and ESR did not significantly decrease. Total effective = markedly effective + effective.The clinical symptoms of the two groups were observed and recorded, including the number of joint swelling, joint tenderness, and morning stiffness time before and after treatment.DAS28 evaluation standard  was usedVisual analogue scale (VAS)  was usedBarthel index  was usedAdverse events. The incidence of adverse events after treatment between the two groups was compared, mainly including transaminase elevation, leukopenia, renal function and liver function damage, dizziness, diarrhea, nausea and vomiting, hypertension, and infection.QLQ-C30  was usedα in patients' serum before and after treatment. The experimental process was strictly carried out in accordance with the kit instructions of TRIzol extraction kit , reverse transcription kit  Co., Ltd., China, FP209), human TNF-α ELISA kit , etc.The serum of patients in the CG and the RG was collected, respectively, before and after treatment. qRT-PCR and ELISA were used to detect the expression levels of HOTAIR and TNF-t test, and paired t test was used for comparison before and after treatment in the group. Comparison of the measurement data from more than two groups was conducted by one-way analysis of variance, correctness of statistical value was verified by post hoc test (Tukey HSD method), and Pearson method was used for correlation analysis. P < 0.05 means differences were statistically significant.SPSS 21.0  was used for statistical analysis, and GraphPad Prism 7 was used to draw the data picture. The counting data were under chi-square test, comparison of the measurement data between the two groups was under independent-samples P > 0.05)  .P > 0.05) , with statistically significant difference ( > 0.05) .P > 0.05). After treatment, the three abovementioned symptoms in the two groups significantly improved compared with those before treatment, and the improvement in the RG was more significant than that in the CG (P < 0.05)  .P > 0.05). After treatment, VAS and DAS28 scores of those in both groups decreased significantly, and the decrease in the RG was more significant than that in the CG, while Barthel score was opposite (P < 0.05)  .P > 0.05)  .P < 0.05), as shown in The QLQ-C30 scale in the RG was significantly higher than that in the CG (α in serum of patients in the CG and the RG were significantly higher than those in the HCG (P < 0.05), while there was no significant difference between the CG and the RG (P > 0.05). After treatment, the expression levels of HOTAIR and TNF-α in serum of patients in the two groups decreased significantly, and their expression in the RG was lower than that in the CG (P < 0.05)  .α in 268 RA patients before treatment was analyzed by Pearson analysis. The results showed that HOTAIR was positively correlated with TNF-α  (The correlation between serum HOTAIR and TNF-< 0.001) .α level in serum of both groups and found that its level of the RG was significantly lower than that of the CG after treatment, which indicated that methotrexate combined with iguratimod could jointly inhibit the production of inflammatory factors.Methotrexate mainly inhibits the synthesis of thymine and purine by inhibiting dihydrofolate reductase, thus inhibiting the activity of immune cells and achieving the therapeutic purpose . IguratilncRNA is considered to be a universal regulator of gene expression, and thousands of lncRNAb have been discovered at present . HOTAIR α in patients in the CG before treatment, suggesting that HOTAIR might participate in the pathogenesis of RA, and methotrexate, iguratimod, and HOTAIR might have certain regulatory relationships. Inflammatory cytokines, especially TNF-α, and two interleukins, IL-1β and IL-6, are key in driving inflammation and joint damage. Cytokines such as IL-23, IL-17A, as discussed above, and interferon gamma (IFN-γ) also play crucial roles in the pathogenesis of RA. IL-4 and IL-10, on the other hand, have been suggested to improve arthritis. While there is overall consensus on the inflammatory role of IL-6 in the pathogenesis of RA, there are some studies pointing out that its role is not clear-cut. It has emerged that IL-1 signaling reduces IL-6 signaling in RA, overall worsening patients' conditions [According to our research results, the HOTAIR expression in serum of patients in the CG and the RG was significantly higher than that in the HCG. After treatment, its expression decreased markedly, and there was a remarkable positive correlation between serum HOTAIR and TNF-nditions .There are some shortcomings in this study. For instance, the optimal dosage of the joint use of methotrexate and iguratimod and the relationship between HOTAIR and RA, as well as methotrexate and iguratimod, have not been deeply explored. Future studies are needed to explain how the immune cell functions are regulated by methotrexate and iguratimod. The participation of methotrexate and iguratimod and its dysregulation in RA pathogenesis are needed to be investigated on a larger scale. Exploring the role of them in RA pathogenesis is essential for the deep understanding and, therefore, effective treatment of this disease.In conclusion, compared with methotrexate used alone, the joint use of methotrexate and iguratimod can provide better clinical efficacy for RA patients, improve their symptoms and life treatment, and will not increase the occurrence of adverse events. Besides, HOTAIR may participate in the pathogenesis of RA.


Background: Major depressive disorder (MDD) and general anxiety disorder (GAD) share many common features, leading to numerous challenges in their differential diagnosis. Given the importance of the microbiota–gut–brain axis, we investigated the differences in gut microbiota between representative cases of these two diseases and sought to develop a microbiome-based approach for their differential diagnosis.Methods: We enrolled 23 patients with MDD, 21 with GAD, and 10 healthy subjects  in the present study. We used 16S rRNA gene-sequencing analysis to determine the microbial compositions of the gut microbiome based on Illumina Miseq and according to the standard protocol.Results: GAD showed a significant difference in microbiota richness and diversity as compared with HC. Additionally, Otu24167, Otu19140, and Otu19751 were significantly decreased in MDD relative to HC, and Otu2581 and Otu10585 were significantly increased in GAD relative to MDD. At the genus level, the abundances of Sutterella and Fusicatenibacter were significantly lower in MDD relative to HC, and the abundances of Fusicatenibacter and Christensenellaceae_R7_group were significantly lower in GAD than in HC. The abundance of Sutterella was significantly higher whereas that of Faecalibacterium was significantly lower in GAD relative to MDD. Moreover, we observed that Christensenellaceae_R7_group negatively correlated with the factor score (Limited to Hopelessness) and total score of HAMD-24 (p < 0.05), whereas Fusicatenibacter negatively correlated with FT4 (p < 0.05). Furthermore, the GAD group showed significant differences at the genus level for Faecalibacterium, which negatively correlated with PTC (p < 0.05).Conclusions: This study elucidated a unique gut-microbiome signature associated with MDD and GAD that could facilitate differential diagnosis and targeted therapy. Major depressive disorder (MDD) is characterized by deep sadness, reduced energy, vegetative nervous system dysregulation, cognitive dysfunction, and even a high suicidal tendency . GeneralFaecalibacterium negatively correlating with symptom severity   at the fAll procedures contributing to this work comply with the ethical standards of national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. All procedures involving human subjects and patients were approved by the Ethics Committee of West China Hospital of Sichuan University . Written informed consent was obtained from all study subjects.via electrochemiluminescence. All reagents and calibrations were performed according to manufacturer instructions.The HPT axis test indicators include thyroid-stimulating hormone , triiodothyronine , thyroxine , free triiodothyronine , and free thyroxine . The HPA axis test indicators include ACTH  and 8:00 A.M. cortisol . Fasting venous blood was taken by drawing 4 mL of cubital venous blood at 8 A.M. after overnight fasting. All analyses were performed using a Roche Cobas e601 module  https://www.hmpdacc.org).Fecal samples were immediately frozen upon collection in a sterile plastic cup and stored at −80 °C before analysis. Microbial genomic DNA was extracted using the QIAamp DNA stool mini kit  according to manufacturer instructions. The 16S rRNA V3-V4 amplicons were generated using the National Institutes of Health (NIH) Human Microbiome Project protocols  software program. Chimeric sequences were identified and removed using UCHIME . The taxonomy of each 16S rRNA gene sequence was analyzed with RDP Classifier (http://rdp.cme.msu.edu/) using the SILVA (SSU 138) 16S rRNA database at a confidence threshold of 70% , emphasizing both statistical significance and biological relevance. ANOVA was used to compare the relative abundance of microbes identified with 16S rRNA sequencing.p < 0.05 was considered significant. The false recovery rate representing the threshold correction was generated using the Benjamini–Hochberg method . One-way ANOVA was used to compare the continuous variables, including age, BMI, and clinical scales. Fisher's exact test was used to analyze contingency tables, and the chi-squared method was used to compare the variables of all three groups. A g method .U tests were used to identify differences between the two groups. The β-diversity was calculated using the Bray–Curtis index as the distance method and reported according to principal component analysis. A hierarchical clustering tree was used to describe similarities among different data-point categories using the Bray–Curtis distance method and visualized using iTOL (https://itol.embl.de/).The α-diversity was calculated by the ACE, Chao, Simpson, and Shannon indices. Mann–Whitney PICRUSt, a bioinformatics software package that predicts metagenome functional content from marker gene  surveys and full genomes , was usehttp://circos.ca/) visualizes data in a circular layout , and 21 (19 newly diagnosed and two relapsed) subjects in the healthy crowd (HC), MDD, and GAD groups, respectively. The mean age at assessment was 30.04 ± 5.90, 30.43 ± 7.95, and 30.22 ± 6.50 years for the MDD, GAD and HC groups, respectively, with no significant difference found between groups according to ANOVA. Moreover, the BMI, sex ratio, marital status, family history, and levels of neuroendocrine hormones did not differ significantly among the three groups .Accounting for 70% of the valid sequences, we obtained 1,620,000 high-quality sequences from the 54 fecal samples of all participants: the HC, MDD, and GAD groups contained 630,000, 690,000, and 300,000 sequences, respectively. The richness of gut bacterial communities in all three groups was estimated by ACE and Chao indices, and the diversity was estimated using the Shannon and Simpson diversity indices. ACE and Chao analysis showed that most of the gut microbial diversity in each sample had been captured with the current sequencing depth. After rarefying the sequencing depth among all samples using a bootstrap method , the Shannon and Simpson diversity index estimates were calculated, revealing no significant difference in richness and diversity between HC and MDD. However, GAD showed a significant difference in microbiota richness and diversity as compared with HC . To explBacteroides and Prevotella were abundant in the three groups. The abundance of Faecalibacterium decreased and Sutterella, Fusicatenibacter, and Christensenellaceae_R7_group increased in MDD relative to HC. The abundance of Fusicatenibacter and Christensenellaceae_R7_group decreased in GAD relative to HC. Compared with that in the MDD group, we found an increase in the relative abundance of Fusobacteria, Tenericutes, Verrucomicrobia, and Bacteroidetes but a decrease in that of Proteobacteria, Actinobacteria, and Firmicutes in the GAD group  significantly correlated with Fusicatenibacter, and its levels were higher in MDD and lower in GAD relative to HC. We used a network diagram to explain the relationship between the significantly different KOs and OTUs/species  with differences in MDD and GAD relative to HC or between them. These four bacterial genera can be considered as important genera that affect the disease phenotype. To further explore the functions of these different bacterial genera, we evaluated correlations among the relative abundance of bacteria, hormones , and the total and factor scores of HAMD  in the MDD group and those of HAMA (Psychic anxiety and Somatic anxiety) in the GAD group. We found significant differences in correlations at the genus level for Fusicatenibacter and Christensenellaceae_R7_group in MDD patients (Christensenellaceae_R7_group negatively correlated with the HAMD factor score (Limited to Hopelessness) and total score (p < 0.05), Fusicatenibacter negatively correlated with FT4 (p < 0.05), and other key phylotypes showed no strong correlation. Further, the GAD group showed significant differences at the genus level for Faecalibacterium, which negatively correlated with PTC (p < 0.05), whereas other key phylotypes showed no strong correlation . Various studies also reported that MDD is associated with aberrant branched-chain amino acid and energy metabolism  and K02067 . Niacin deficiency is reportedly a contributing factor in mental-illness development and symptom alleviation  and total scores of HAMD, suggesting that although Christensenellaceae_R7_group has not been observed as enriched in patients with MDD, it might affect the clinical manifestations and severity of MDD. Similar conclusions have been confirmed in other studies  can be found in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of West China Hospital of Sichuan University. The patients/participants provided their written informed consent to participate in this study.ZD and WK: conception of the work, final approval of the version to be published, and agreement to be accountable for all aspects of the work. ZD, XS, YH, JL, HL, HX, and LY: acquisition of data. ZD and LY: analysis and interpretation of data for the work. ZD: writing. WK: revising the work. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China  and the Key R&D Projects of Science and Technology Department of Sichuan Province (Grant No. 2019YFS0217).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


However, DF samples presented the best predictive performance values for the main sugarcane quality parameters, and required only minimal sample preparation. The results contribute to advancing the development of on-board quality monitoring in sugarcane, indicating better sampling strategies.Proximal sensing for assessing sugarcane quality information during harvest can be affected by various factors, including the type of sample preparation. The objective of this study was to determine the best sugarcane sample type and analyze the spectral response for the prediction of quality parameters of sugarcane from visible and near-infrared (vis-NIR) spectroscopy. The sampling and spectral data acquisition were performed during the analysis of samples by conventional methods in a sugar mill laboratory. Samples of billets were collected and four modes of scanning and sample preparation were evaluated: outer-surface (‘skin’) (SS), cross-sectional scanning (CSS), defibrated cane (DF), and raw juice (RJ) to analyze the parameters soluble solids content (Brix), saccharose (Pol), fibre, pol of cane and total recoverable sugars (TRS). Predictive models based on Partial Least Square Regression (PLSR) were built with the vis-NIR spectral measurements. There was no significant difference ( Near-Infrared (NIR) spectroscopy is a well-established technique to monitor the quality of raw sugarcane received by sugar mills , and conDifferent wavelength regions of the electromagnetic spectrum can be used in spectroscopy, such as visible 400 to 750 nm), near-infrared , shortwave near-infrared , and visible and near-infrared  0 nm, nea,7,8. HowDespite the advances in industrial sugarcane quality monitoring, spectral methods are still a distant reality for in-field measurements in line with precision agriculture practices. Current proximal sensing technologies applied at the canopy level allow only monitoring crop yield ,17. HoweNawi et al. 2014) indicated that the ideal place for implementing an on-board sugarcane quality monitor would be in the elevator of the harvester, where the sugarcane material is partially cleaned and processed in the form of billets. In this context, some studies have reported promising results on the prediction of sugarcane Brix from sensor readings made on the outer-surface (‘skin’) 14 indica of sugarVariability of sugarcane quality parameters throughout a harvest occurs due to diverse environmental conditions, mainly temperature and precipitation, during the harvest . Based oThe data collection was carried out in the quality laboratory of a sugar mill. Three hundred and two samples were collected, and different levels of processing were applied. Also, all data acquisition was performed in a controlled temperature environment (20 ± 5 °C), minimizing the effects of sugar degradation by microorganisms. In addition, we collected the daily results of conventional analysis performed by sugar mill quality laboratory over the same months in which the samples were collected for spectral analysis.The sampling procedure for vis-NIR analysis was carried out sequentially and simultaneously to the sample preparation for conventional analysis, as described:An oblique probe collected a sample of sugarcane billets in each truckload to proceed with the conventional analyzes of the sugarcane transported to the sugar mill a. BeforeThe remaining sample of billets was milled in a mechanical knife crusher and homogenized in a mixer c. Then, −2) for 60 s to obtain the raw juice for conventional analysis  was determined by pouring raw juice into a refractometer probe  with a maximum resolution of 0.1 Brix. Then, 14 g of a mixture composed of a 4:2:1 proportion of Celite , aluminum chloride, and calcium hydroxide, respectively, was added to 200 mL of raw juice homogenized by a magnetic stirrer until the solution was well-mixed. The solution was filtered through filter paper to obtaining clarified juice. A volume of 70 mL was added in a digital polarimeter  to the saccharimetric reading. The result was obtained as percentage of apparent sucrose in the juice (Pol).After the juice extraction, the remaining fibrous cane residue j was traThe Pol of cane and TRS were calculated for each sample from the previous parameters as described by CONSECANA (2015). The triplicate values obtained from each sample were averaged.It is important to mention that reducing sugars (fructose and glucose) and purity (apparent sucrose in the soluble solids content) were also determined in the laboratory. However, while these parameters are not the subject of the present study, they were used to calculate TRS by the reference method.The spectral measurements were performed with a Veris vis-NIR spectrometer . This equipment was developed for on-the-go soil measurements mounted on a platform, connected to a three-point hitch, and pulled by a tractor . HoweverThe three billets of each sugarcane sample were cut transversely at both extremities, and their skin was lightly cleaned with paper to remove residues from harvest b. An ad The spectral scan method on billets was adapted from Nawi ,21 and PThe spectral measurements of defibrated cane and raw juice were performed in the same manner. A recipient available from the equipment itself with a volume of around 3 mL was used. The recipient was filled with sample   to elimiFirstly, the spectral data of the four sampling conditions were concatenated. Then, the data was divided into calibration  and external validation  data sets, based on the Kennard-Stone method . This prn is the number of samples, iy is the reference measurement of sample i, and i.The models were calibrated using the venetian blinds cross-validation method with 10 splits. The optimal PLS models were determined based on the lowest number of latent variables (LV), in which the root mean square error of cross-validation (RMSECV) was not significantly higher than the minimum RMSECV . The roo2 and Q statistics, respectively [The outliers were evaluated during the calibration step for the reference lab values and spectral data. The presence of outliers in the spectral data was evaluated by the “influence plots” based on high leverage and unmodeled residuals by Hotelling Tectively . Samplesectively . The extectively .2) for calibration and prediction , and the ratio of performance to the interquartile range (RPIQ), which is calculated by the ratio between the interquartile difference and the RMSEP. Also, a randomization test [The model accuracy was evaluated based on the RMSE for calibration, cross-validation, and prediction . Prediction performance was evaluated based on the determination coefficient (Rion test  with 0.00): RMSEPsample type 1 = RMSEPsample type 2 (accuracy is similar);Null hypothesis (H1): RMSEPsample type 1 ≠ RMSEPsample type 2 (accuracy is not similar).Alternative hypothesis  . More dep is the number of wavelengths (dimensionless), h is the number of latent variables (dimensionless), Z is the fraction of variance in the prediction explained by the latent variable (dimensionless), and w is the loading weight (dimensionless).Moreover, the variable importance for the projection (VIP) was calculated to verify the wavelengths with a more significant impact on the external validate models  for eachAll models, routines, and data processing were performed in Matlab R2015a  and PLS Toolbox 8.9 .From the daily results of analyses performed by conventional methods at the mill, it was possible to characterize the variation of the main parameters determined analytically , as well as for TRS, throughout the months in which the experiment was performed .It is possible to observe an increasing trend in all parameters from June to October. Afterward, there is a tendency to decrease, except for fibre. Weather is highly influential on sucrose storage . In the On a first view, the Kennard-Stone method provides a representative calibration data set, with external validation data set between its range . The samp < 0.05). On the other hand, the other parameters showed a positive correlation higher than 0.94 (p < 0.05). The highest correlation was observed between Pol of cane and TRS, close to 1.00 (p < 0.05). Higher correlation values are observed between the TRS with parameters analytically determined such as Brix and Pol . The correlation values for these attributes are firstly explained by the composition of the soluble solids content of sugarcane, measured by Brix, in which the largest proportion corresponds to sucrose (about 15–18%), measured by Pol [The distribution of all quality parameter values had wide distribution . Fibre cd by Pol ,39, and d by Pol ; note thThe vis-NIR raw spectral data obtained for 302 samples of each sugarcane sample type are shown in A PCA analysis performed an exploratory overview of the data structure. The spectral data were only mean-centered, and the classes were identified by sample type. Two principal components, PC1, and PC2, explained 98.6% of the data variance . The firVis-NIR spectroscopy may be used in a number of applications, including the classification of sugarcane varieties, with promising results . The samThe vis-NIR technique principle is based on the detection of compounds and molecules through their molecular vibration states . Differep-value < 0.05) for Fibre, Pol of cane, and TRS .More LVs were necessary to explain the variance of the data for models constructed from SS samples (between 7 and 10) than those obtained to predict the same parameters from other sample types . Also, i and TRS . Moreove and TRS . The RPIp-value > 0.05) and very close values of R2p and RPIQ. Moreover, from DF samples, it was possible to obtain a satisfactory performance to predict Fibre content; this was not possible for RJ samples. On the other hand, the models performed for SS samples presented a higher number of LV than for a prepared sample. The model performance for predicting parameters related to sucrose  was not satisfactory, with R2p and RPIQ below 0.5 and 2.0, respectively, except for Fibre prediction. The prediction results from SS samples for Fibre were close to those obtained for DF samples, as shown by the values of R2p and RPIQ. However, the results were less promising than those obtained by Phuphaphud et al. (2019) [2p and 0.63 for RMSEP. Although Fibre content is an important attribute for sugarcane quality determination, it is not essential for sucrose estimation. Fibre content has no relation with some important attributes, such as Brix and Pol, and only minimally impacts TRS calculation. The prediction of this parameter is important for producing energy cane and breeding programs, as in work developed by Phuphaphud et al. (2019) [The model performance for DF and RJ samples was equivalent for practically all parameters evaluated. There was no significant difference between the model’s accuracy (. (2019) , which o. (2019) .2p and RPIQ. In a first investigation, Nawi et al. (2013a) obtained values of 0.87 for R2p. The excellent performance of this index can be explained by the method of data acquisition adopted by the authors, with individualized samples according to the stem portion  and only three varieties of cane. Sucrose accumulation occurs in an ascending manner, with more accumulation in the lower portion and less in the internodes of the upper portion, close to the leaves [Some models developed for CSS samples were similar to those developed for SS, as for Brix and Pol prediction. However, its predictive performance was lower than those obtained for Fibre, Pol of cane, and TRS predicted by SS samples, with worse results for Re leaves ,44,45. TThe RPIQ values for the SS method were higher than those obtained by the CSS method. The SS method on billet samples on the harvester conveyor would be the most practical method, due to the better operability of sample acquisition in that portion of the harvester . HoweverThere are common waxy materials on the cane surface that affect vis-NIR measurements by the SS method. Maraphum et al. (2018) evaluated the effect of the waxy material on the cane surface to eliminate or avoid getting low accuracy of the models for Pol measurements. They obtained RMSEP values were around 1.20 to 1.50%, i.e., close to those found by the present study. The authors concluded that spectra acquisition by removed-wax samples was convenient for the measurement of Pol. However, other compounds could affect vis-NIR spectroscopy measurements, such as cellulose and lignin .2p and RPIQ) indicate that DF and RJ samples presented similar performance and provide the best results. However, models built from DF samples require less preparation, i.e., by avoiding juice extraction, making them more attractive for an on-board system. Moreover, CSS samples presented worse performance than all other samples. SS samples presented higher values of R2p and RPIQ than CSS samples for all quality parameters. Furthermore, SS samples showed a nonsignificant difference  of accuracy (RMSEP) with models built from DF samples, but worse results for performance (R2p and RPIQ), except for Fibre. Possibly DF results were satisfactory due to the exposure of the internal constituents, which overlapped concerning the waxy material that becomes visually negligible. On the other hand, the organic compounds in the sugarcane outer-surface may have interfered in the prediction models. Future studies using nonlinear processing methods [Overall, the predictive performance results of the models , especially related to the prediction of Brix, Pol, Pol of cane, and TRS by CSS and RJ samples, corresponding to the second and third overtone of O-H and C-H stretching, respectively . The regAt 1139 nm, there is a small band with VIP values higher than 1.0 for all parameters predicted for four sample types, except for Fibre content. On the interval between 1100 and 1230 nm occurs the second vibrational frequency overtones associated with C-H stretching . Also, aAt 1360 nm, there is another expressive region with high VIP values, possibly related to C-H combinations and the O-H first overtone, respectively . Then, aThe scores of the models for all measured quality parameters are displayed by their first PLS loadings , accountThe gap between two spectrometers, starting at 1011 nm until 1070 nm, is not related to significative known vibrational frequency overtones associated with some bands related to sugars or fibres [Processed samples allowed a more significant interaction of electromagnetic radiation corresponding to vis-NIR bands with matter constituents. This physical effect resulted in more prominent signals from specific vibrational frequency bands related to the chemical constitution of sugarcane quality parameters. Overall, defibrated samples (DF) showed performance prediction results that were close to raw juice samples (RJ). Also, the DF sample allowed us to predict Fibre content as well as other parameters, which is not possible with RJ samples. The prediction of sugarcane quality parameters from less processed samples is a desirable characteristic for mechanization of on-the-go measurements of crops, thus promoting spatial information of crops based on quality. DF samples may partially satisfy this requirement; however, this is a destructive sampling technique.Improving the predicting performance of sugarcane quality parameters from billets for on-the-go systems may be possible . Some efp-value > 0.05) in the accuracy (RMSEP) of prediction of whole cane samples when compared to processed samples, such as defibrated cane (DF) and extracted raw juice (RJ), for all evaluated quality parameters. Also, outer-surface measurements of sugarcane billets presented a better accuracy  and performance (R2p and RPIQ) than measurements on the cross-section.This study demonstrates that vis-NIR spectroscopy could be used as a quick method to assess the abundance of chemical compounds of sugarcane related to its quality. There was no significant difference , DF and RJ sampling presented better performance than outer-surface measurements of sugarcane billets. Moreover, the performance of the models from DF and RJ samples were similar, but DF samples involve less preparation, as they do not require juice extraction of the sample.Despite the similar accuracy , saccharose (Pol), Fibre, Pol of cane, and total recoverable sugars (TRS), all of which are used for pricing and trading between mills and sugarcane producers. The DF models presented RMSEP varying between 0.72% and 0.87% for Brix, Pol, Fibre, and Pol of cane, and 6.71 kg MgThe results in this study contribute to advancing the development of on-board quality monitoring in sugarcane. This information shows the spatial variability of crop quality and helps guide site-specific management of sugarcane fields.


Zea mays and Fagopyrum esculentum were selected as model plants, and after harvest, the element uptake was compared between plants grown on inoculated versus reference soil. The results indicate an enrichment of B. amyloliquefaciens in inoculated soils as well as no significant impact on the inherent bacterial community composition. For F. esculentum, inoculation increased the accumulation of most nutrients and As, Cu, Pb, Co, and REEs , while it slightly decreased the uptake of Ge, Cr, and Fe. For Z. mays, soil inoculation decreased the accumulation of Cr, Pb, Co, Ge, and REEs (significant for Co with 57%) but showed an insignificant increased uptake of Cu, As, and nutrient elements. Summarily, the results suggest that bioaugmentation with B. amyloliquefaciens is safe and has the potential to enhance/reduce the phytoaccumulation of some elements and the effects of inoculation are plant specific.Bioaugmentation promises benefits for agricultural production as well as for remediation and phytomining approaches. Thus, this study investigated the effect of soil inoculation with the commercially available product RhizoVital Soil pollution majorly arises from the dumping of waste from natural or anthropogenic sources in soil, thereby causing undesirable impacts on the chemical, biological, and physical properties of air, soil, and water . In addiThe environmental presence of these elements of interest has implications that are either negative or positive, depending on their concentration and the sensitivity of the living organisms in the environment. Potentially toxic elements and some CRMs have negative consequences on living organisms when they exist in concentrations that are beyond permissible limits, as has been revealed by some studies ,9. TheirPhytoextraction is among the several techniques that can be used to remediate the high presence of PTEs in soil and biologically extract CRMs (phytoremediation for PTEs and phytomining for CRMs). It is cost effective and has less environmental impact . It invoThe improvement of soil health and the bioavailability of elements can be done via bioaugmentation using soil microbes . The bioIn addition, many studies have reported these PGPR strains as being capable of solubilizing phosphate in soil, including a recent one by Schwabe et al. . HoweverPseudomonas maltophilia was reported to have reduced the toxicity of chromium (Cr) in soils by reducing the toxic Cr6+ to nontoxic and immobile Cr3+ and to have restricted the mobility of toxic ions like cadmium (Cd2+), lead (Pb2+), and mercury (Hg2+) [Ricinus communis with Pseudomonas sp. PsM6 or P. jessenii PjM15 increased plant biomass production and enhanced the phytoextraction efficacy for nickel (Ni), copper (Cu), and zinc (Zn) by the production of indole-3-acetic acid (IAA) and solubilizing phosphate. Bacillus amyloliquefaciens BSL16 was reported to increase Cu accumulation and the growth of rice seeds and tomato plants under Cu stress [Bacillus lichenformis was reported to have enhanced the accumulation of Cu, Cd, Pb, and Cr [Several studies have demonstrated the involvement of beneficial micro-organisms, such as rhizobacteria or endophytes associated with plant roots, for the extraction or accumulation of elements of interest or for reducing toxicity and the immobilization of elements in soil . Pseudomy (Hg2+) ,22,23. Ry (Hg2+)  also obsu stress . Furtheru stress  reported, and Cr . In addi, and Cr  reported, and Cr  reportedB. amyloliquefaciens FZB42 inoculated via the commercially available formulation RhizoVital®42 on the simultaneous uptake of PTEs, CRMs such as Ge and REEs, nutrients, shoot yield, and bacterial community composition using Fagopyrum esculentum cv Moench and Zea mays cv Badischer Gelber as test plants and for the purpose of phytomining and phytoremediation have not been studied. Therefore, the main aim of this study was to evaluate the effects of bioaugmentation using inoculum from a commercially produced microbial formulation of B. amyloliquefaciens FZB42 on the phytoextraction of PTEs (arsenic (As), lead (Pb), cobalt (Co), copper (Cu)) and CRMs (germanium (Ge), and the sum total of REEs (REET)), as well as iron (Fe), silicon (Si), calcium (Ca), and phosphorus (P)—regarded as the nutrient elements in this study—from soil. We hypothesized that the inoculation of soil with Rhizovital 42 (bioformulated B. amyloliquefaciens FZB42) inoculum will enrich the strain in soil, and improve plant shoot yield and the aboveground phytoaccumulation of elements, given the reports of the effects of PGPR on element accumulation from previous studies.These studies have highlighted the roles that PGPR plays in plant element accumulation. However, to the best of our knowledge, the effects of bioaugmentation by The analyses of the bacterial community at the end of the experiment revealed no significant differences between the studied treatments. Neither the crop nor the application of Rhizovital showed significant effects on the relative abundance of main bacterial phyla  or on thBacillus amyloliquefaciens FZB42, the results of Illumina sequencing show that compared to reference soils for both plants, soils inoculated with B. amyloliquefaciens generated a lower number of sequences  than uninoculated soils  and had a lower number of operational taxonomic units (OTUs)  than inoculated soils . In addition, the results show that soils in which F. esculentum was grown generated a higher number of sequences and had higher OTU numbers compared to the soils planted with Z. mays. For F. esculentum, inoculated soils generated 764 and 77 fewer sequences and OTUs, respectively, than uninoculated soils, while for Z. mays, soils inoculated with PGPR generated 4250 and 3 fewer sequences and OTUs, respectively, than uninoculated soils. In reference soils in which F. esculentum was grown, no sequences related to the inoculated strain were found, whereas in soils inoculated with the PGPR, approximately 510 sequences were generated. Similar observations were found for the reference soils (four sequences generated from just a single replicate) versus inoculated soils (383 sequences generated) in which Z. mays was grown. Therefore, the results demonstrate that the strain B. amyloliquefaciens was present in the inoculated soils with average relative abundances of 0.85% and 0.75% for the bacterial soil communities of F. esculentum and Z. mays, respectively.Regarding the investigated target strain Z. mays and F. esculentum, there were no significant differences between the biomass produced by plants grown on reference soils and soils inoculated with B. amyloliquefaciens. Inoculation with PGPR only slightly affected the shoot yield of F. esculentum and Z. mays. Inoculated plants showed an 8% higher shoot yield for F. esculentum and an 18% higher yield for Z. mays compared to the reference plants  and 1-aminocyclopropane-1-carboxylic acid deaminase (ACC deaminase) activity, some of which promote increased photosynthetic rates [Z. mays upon inoculation with endophytic PGPR, which colonized the plants. In addition, an increased acquisition of nutrients may have contributed to the slight increase in the biomass observed, but this would be mostly true for F. esculentum, where inoculation increased the accumulation of most nutrients  between 22% and 25% compared to Z. mays, where the slight percentage increase upon inoculation was not more than 8%. The increased accumulation of nutrients might be a result of a B. amyloliquefaciens-induced increase in the nutrient element solubilization and the mobility of these nutrients in the rhizosphere, thus making these elements bioavailable for plant uptake. A Bacillus species was reported by Jamil et al. [B. amyloliquefaciens being a siderophore-producing bacterium, may be because the siderophore produced under the conditions in the substrate favored the solubility and binding of metals other than Fe, hence the decrease in the accumulation of Fe [In this study, we used fertile PTE-polluted soil from the post-mining area of Freiberg. Thus, it was not surprising that the biomass production (shoot yield) was only slightly affected by inoculation under the conditions of adequate nutrient supply, as evident in the slight increase in the biomass of the inoculated plants compared to the non-inoculated reference plants. This slight increase, although insignificant, could be due to the plant growth-promoting properties of ic rates ,38,39,40ic rates  reportedic rates  reportedl et al.  to have on of Fe .B. amyloliquefaciens on plant growth is of interest for plant growth promotion in agriculture and biomass production for bioenergy purposes, especially on marginal soils characterized by high concentrations of PTEs. However, beyond these reasons, there is interest in the effects of B. amyloliquefaciens on the phytoextraction of elements from soil, for example, PTEs [The effect of le, PTEs  and CRMsF. esculentum (a forb and strategy 1 plant with respect to Fe acquisition) and Z. mays (a grass and strategy 2 plant with respect to Fe acquisition) differed for some elements and were similar for others. These differences in the observed effects may be related to the plant species’ characteristics, such as growth habits, element acquisition strategy, and colonization of the plant roots by bacteria [F. esculentum and Co phytoextracted by both test plants were significant. The increased accumulation of Cu and As in Z. mays, as well as Cu, As, Co, and REET in F. esculentum upon inoculation with B. amyloliquefaciens may be connected with the solubilization of these elements by substances produced by the bacteria, such as carboxylic acids, indole acetic acids, and siderophores, as well as root exudates produced by plants, which solubilize these metals and facilitate their uptake by the plant roots [Bacillus amyloliquefaciens BSL16 increased the accumulation of Cu in rice and stated the production of organic acids, biosurfactants, and siderophores as possible reasons for the increased Cu accumulation, as suggested by Sheng et al. [In this study, the observed effects of inoculation on element accumulation by bacteria . In addint roots . The fornt roots ,46. Thesnt roots , who repg et al. . Additiog et al. , who repZ. mays, as well as of Cr and Ge in F. esculentum may be due to a possible immobilization of these elements in the soil upon inoculation with bacteria, thus limiting uptake by Z. mays. It is possible that B. amyloliquefaciens used polymeric substances, exopolysaccharides that are capable of forming biofilms around plant roots, and other chemical substances, such as some carboxylates it produces to immobilize these elements by forming stable complexes with their ions in the soil solution, thus limiting their uptake by plants [Bacillus spp. Fan et al. [Z. mays with some PGPR strains reduced the accumulation of Cr in Z. mays, and this reduction in the accumulation of Cr may be due to the reduction of the mobile Cr6+ to the immobile toxic Cr3+ ions, as reported by Jing et al. [Bacillus [The contrasting results of the decreased accumulations of Cr, Pb, Co, Ge, and REET in y plants ,49,50,51y plants  reportedn et al.  reportedn et al.  reportedg et al. . This agBacillus .Z. mays upon inoculation may be connected to the chemical relationship between As and P [Furthermore, element accumulation patterns upon inoculation may have been due to chemical relationships or similarities in origin that resulted in simultaneous accumulation by plants, as the plant may not have easily taken them up differentially or, in some cases, because of competition for the same transport channels or sites. For example, the observed higher accumulation of As and P in As and P . In addiAs and P ,57,58 anAs and P , P and CAs and P , Ca and As and P .Z. mays and F. esculentum as test plants and commercially available B. amyloliquefaciens FZB42 bioformulation as the inoculant. We demonstrated that it is possible that upon inoculation of soil with bacteria, biomass production by Z. mays and F. esculentum can be enhanced, while phytoextraction can be enhanced or impeded depending on several interacting factors related to plant species characteristics, such as growth habits, element acquisition strategy, and the colonization of plants by bacteria, which could differ between the two plant species [B. amyloliquefaciens FZB42 as the PGPR, as well as for phytoremediation purposes, is safe, as the B. amyloliquefaciens FZB42 establishes itself well in soil and does not majorly affect the structure of the indigenous soil microbial composition. Although the above-mentioned effects of inoculation might not all be significant, we think that they are meaningful, as they indicate what possibilities of element accumulation there could be upon the inoculation of soils in which F. esculentum and Z. mays are grown, using B. amyloliquefaciens as the microbial inoculant. Thus, the findings of this study may provide useful information when planning agricultural projects that intend to use microbes to boost plant growth and nutrient content, for environmental remediation projects that intend to use plants and microbes to enhance the extraction of economically valuable elements and contaminants from soil, and for biomass for bioenergy projects that intend to use microbes to enhance plant biomass production.Conclusively, our study has highlighted the possibilities of enhanced biomass production and phytoextraction of elements, including nutrients, PTEs, and elements of economic value, using  species . In addiZea mays cv Badischer Gelber and Fagopyrum esculentum cv Moench, which were grown under constant laboratory conditions of a temperature of 25 °C and light exposure time of 12 h per day. The plants were grown in 3 replicates, each in 2 kg of potted soils obtained from the vicinity of Technische Universität Bergakademie Freiberg, which represent typical soils of the Freiberg area of Germany [Bacillus amyloliquefaciens. An inoculation rate of approximately 0.4% (0.4 mL of inoculum in 100 mL) per pot was used, and the soil was inoculated twice (100 mL of 0.4% inoculum mixture each time) within the 53-day growing period of the experiment, with a time interval of 2 weeks between inoculations. Rhizovital 42 (bioformulated Bacillus amyloliquefaciens), supplied by ABiTEP GmBH Berlin and containing 2.5 × 1010 CFU/mL (colony-forming units per milliliter) of Bacillus amyloliquefaciens, was the source of inoculum.The plant species used as test plants in this study were  Germany . Five se2CO3 and K2CO3 were placed in a nickel crucible and thoroughly mixed for melting digestion, according to the methods by Alfassi and Wai [According to Du Laing , readily and Wai . The mix and Wai . The resThe physico-chemical properties of the uninoculated soil, the concentrations of the readily available soil element fractions, and the total element concentrations are reported in During harvest, the plants were cut off at heights between 2–3 cm above ground level, weighed, and dried at 60 °C in an oven  for 48 h to obtain a constant weight. Subsequently, the dry mass of the samples was determined and pulverized to a fine powder using an ultra-centrifugal mill . Then, 100 mg of the dried pulverized plant samples were weighed out for digestion in a microwave  according to the methods by Krachler et al. . Before Microbial DNA was extracted from approximately 250 mg soil, which had been collected immediately after plant harvest and preserved at −80 °C. The extraction procedure was done using a QIAGEN DNeasy Power Soil kit and based on the specifications of the manufacturer. Before storing the DNA extracts at −20 °C, the DNA concentrations in the extracts were examined with a NanoDrop ND-8000 spectrophotometer . For the PCR, the DNA concentrations of the extracts were adjusted to 10–15 ng/µL. Amplification of the bacterial 16S rRNA gene V4 region was performed in triplicate for each sample with the universal primers 515f and 806r , which wDemultiplexed sequences were processed using the “dadasnake” pipeline , which ip < 0.1 using IBM SPSS Statistics 26 software. Significant differences (p ≤ 0.1) between the means indicated are indicated by an asterisk * in the figures. The bar plots and PCoA were created with R, version 4.0.5, using the “vegan” and “ggplot2” packages.The statistical differences between the treatments for each plant species for shoot contents (amount accumulated), element concentrations, and shoot yield were evaluated using Welch’s analysis of variance (ANOVA) at a significance level of