Nonalcoholic fatty liver disease (NAFLD) is a complex metabolic disorder that increases the risk for cardiovascular disease in patients with type 2 diabetes mellitus (T2DM). Global longitudinal strain (GLS) is an indicator of left ventricular (LV) mechanics and can detect subclinical myocardial dysfunction. We compared the effects of pioglitazone and empagliflozin on GLS in patients with T2DM and NAFLD without established atherosclerotic cardiovascular disease.This study was a 24-week randomized, single-blind, and parallel-group (1: 1 ratio) clinical trial. Seventy-three participants with T2DM (being treated with metformin) and NAFLD but without established atherosclerotic cardiovascular disease (ASCVD) were randomized to empagliflozin or pioglitazone. Liver steatosis and fibrosis were measured using transient elastography, and GLS was measured by echocardiography. The primary endpoint was the change in GLS from baseline to week 24. Secondary end points include changes in controlled attenuation parameter (CAP) and Liver stiffness measure (LSM).± 2.34% (P < 0.01) in the pioglitazone group and 1.06 ± 1.83% (P < 0.01) in the empagliflozin group without a significant difference between the two groups (P = 0.31). At baseline, GLS was inversely associated with the severity of liver fibrosis: r = − 0.311, P = 0.007. LSM in the pioglitazone and empagliflozin group [(-0.73 ± 1.59) and (-1.11 ± 1.33)] kpa (P < 0.01) decreased significantly. It was without substantial difference between the two groups (P = 0.26). Empagliflozin and pioglitazone both improved controlled attenuation parameter. The improvement was more critical in the empagliflozin group: -48.22 + 35.02 dB/m vs. -25.67 + 41.50 dB/m, P = 0.01.In this study, GLS improved by 1.56 Subclinical cardiac dysfunction is highly important in patients with T2DM and with NAFLD. Empagliflozin and Pioglitazone improve LV mechanics and fibrosis in patients without established ASCVD. This has a prognostic importance on cardiovascular outcomes in high-risk patients with T2DM. Moreover, empagliflozin ameliorates liver steatosis more effectively them pioglitazone. This study can serve as a start point hypothesis for the future. Further studies are needed to explore the concept in larger populations.https://www.irct.ir/search/result?query=IRCT20190122042450N5.: This trial was registered in the Iranian Registry of Clinical Trials (IRCT): “A Comparison between the Effect of Empagliflozin and Pioglitazone on Echocardiographic Indices in Patients with Type 2 Diabetes Mellitus and Nonalcoholic Fatty Liver Disease” IRCT20190122042450N5, 29 November 2020. The online version contains supplementary material available at 10.1186/s12876-023-02948-4. The prevalence of Nonalcoholic fatty liver disease (NAFLD) is increasing in parallel with the worldwide increase in obesity and type 2 diabetes mellitus (T2DM) . NAFLD iThe relationship between NAFLD and CVD is complex , and NAFGlobal longitudinal strain (GLS) is one of the most sensitive and accurate echocardiographic measures for assessing LV mechanics and determining subclinical left ventricular systolic dysfunction . In addiTo the best of our knowledge, no previous study has compared the effects of empagliflozin and pioglitazone on subclinical cardiac dysfunction that is associated with NAFLD. The present study was designed to compare the impact of these two agents on echocardiographic indices in individuals with T2DM and NAFLD who did not have established atherosclerotic cardiovascular disease (ASCVD).This study was a 24-week prospective, 1:1 ratio randomized, single-blind clinical trial. It was registered in the Iranian Registry of Clinical Trials (IRCT): IRCT20190122042450N3. The study protocol was approved by the Ethics Committee (EC) of the Institutional Review Board (IRB) of the Iran University of Medical Science; ethical code: IR.IUMS.REC.1398.1408. Iran University of Medical Science (IUMS) funded the study. All of the participants signed a written informed consent before enrollment and after a face-to-face discussion about the study procedures and outcomes. Abidi pharmaceutical company supplied the medication. However, they had no role in data management, analyses, and writing final report.We invited patients with T2DM aged 20 to 80 to participate in this study. In a 3-week run-in period, volunteers were screened using standard medical history, biochemical tests, liver elastography, and Echocardiography  Standards of Medical Care in Diabetes  .Liver elastography was performed at baseline and the end of the study. Liver fibrosis was defined according to the liver stiffness measurement (LSM).Hepatic steatosis was measured based on the controlled attenuation parameter (CAP) using Fibroscan® 502 Touch equipped with both M and XL probes. The procedure was performed by an expert physician (blinded to the study protocol) trained and certified by the Iranian Association of Gastroenterology and Hepatology (IAGH). Participants with CAP greater than 302 dB/m were enrolled in the study.Transthoracic Echocardiography (TTE) was performed using echocardiography equipment, Acuson SC2000TM ultrasound system , with a 4 MHz probe (Acuson 4V1c). The images were obtained while subjects were at rest and in the left lateral decubitus position. All images acquisition and measurement were performed according to the recommendations of the American Society of Echocardiography (ASE) guidelines.Standard 2D-TTE and strain imaging were performed at baseline and week 24 by an expert cardiologist who was blinded to the study protocol. Two-dimensional images were optimized to achieve the highest frame rate possible. Tissue Doppler Imaging (TDI) of the interventricular septum and lateral wall was recorded using the apical 4-chamber view with the narrowest sector angle feasible and a high frame rate. The images were digitally captured for subsequent analysis. LV systolic and diastolic function parameters including the global LV ejection fraction (LV EF) were assessed. LV EF was calculated by modified Simpson’s rule after measuring the end-diastolic volume (EDV) and the end-systolic volume (ESV), from both four-chamber and two-chamber views. LV diastolic function was assessed via pulsed-wave (PW) Doppler of the mitral inflow. Indices were measured by placing the pulsed-wave Doppler at the tip of the mitral valve leaflets during diastole, and mitral inflow velocities were recorded from the apical 4-chamber view (A4C). These included measurements of peak E-wave velocity (E), peak A-wave velocity (A), mitral E/A ratio, mitral E-wave deceleration time (DT), and isovolumic relaxation time (IVRT), which was recorded as the interval between aortic valve closure and mitral valve opening at a sweep speed of 100 mm/s. The Tissue Doppler Imaging (TDI)-driven mitral annular e’ velocity was measured as the average of septal and lateral e’ velocities. The E/e’ ratio was calculated as an index of LV filling pressure. We also measured LA volume at end of systole using Simpson rule. It was indexed for body surface area to drive LAVi. In addition, estimated pulmonary systolic pressure was determined.Strain imaging was performed semi-automatically. Three standard apical views were recorded  at the end of expiration in three consecutive cardiac cycles, with a frame rate ranging between 40 and 60 frames per second. Offline analysis was conducted using approved software available on the Acuson ultrasound equipment. Endocardial borders were meticulously traced manually in each image. Aortic valve closure was automatically identified by the software and edited if necessary. Longitudinal strain values were calculated by the software and presented in the bull’s-eye tomogram, numeric values and as the average global longitudinal strain (GLS) among the 16 segments. GLS was measured in two cardiac cycles, and the average value was used for the final analysis. The normal value for GLS was considered as the absolute value greater than 18% .Follow-up visits include in-person visits at baseline and weeks 12 and 24. Remote visits via phone were done by week 4, 8, 16, and 20. Anti-hyperglycemic treatments were changed, as necessary, according to the recommendations of the American Diabetes Association Guideline (ADA) 2019. Before enrollment, moderate-intensity statin was initiated for patients who were not receiving a statin, according to the American Diabetes Association’s (ADA’s) Standards of Medical Care in Diabetes [The International Physical Activity Questionnaire (IPAQ) assessed the participants’ physical activity . PatientThe primary outcome was the change in GLS compared to the baseline. Secondary outcomes included changes in diastolic parameters as well as changes in CAP, LSM, fibrosis-4 (FIB4) index, and NAFLD fibrosis score from the baseline.Sample size was calculated based on the study of Akelbrom A et al., which explored the effect of dapagliflozin on GLS in patients with T2DM . To indiBaseline characteristics are shown as means ± standard deviation (SD) for continuous variables and percentages for categorical variables. Within-group changes from baseline were examined by paired t-test. We used analysis of variance (ANOVA) to assess the between-group comparisons. Correlations were evaluated by Pearson’s correlation test for parametric variables and Spearman’s correlation test for nonparametric variables. One participant in the pioglitazone group and two in the empagliflozin group withdrew during the treatment period. Analysis of the data was by intention to treat (ITT) method. P-values less than 0.05 were considered statistically significant. The analyses were done using SPSS software version 25.0.Seventy three participants (37 patients in the empagliflozin group and 36 patients in the pioglitazone group) were enrolled to the two study arms. Data from these patients were used for an intention to treatment analysis. Three patients (one in the pioglitazone group and two in the empagliflozin group) did not complete the study due to non-adherence to the study medications. The flowchart of patient enrollment is shown in Supplement ± 2.40% and − 19.65 ± 2.15% in the pioglitazone and empagliflozin group, respectively with no difference between the two (P = 0.28). There was an inverse linear relationship between the GLS and liver fibrosis (LSM) at baseline . We found statistically significant improvement in GLS in both treatment groups. GLS improved by (1.57 + 2.34%) and (1.07 + 1.83%) in the pioglitazone and empagliflozin group respectively, (P < 0.01). It was no statistically significant difference between the two groups (P = 0.31). In addition, there was no linear correlation between the improvement of GLS and the changes in the LSM.At baseline, GLS was − 19.07 ± 41.5 dB/m (P < 0.01) in the pioglitazone group and by 48.2 ± 35.0 dB/m (P < 0.01) in the empagliflozin group. Empagliflozin was more effective than pioglitazone in reducing hepatic steatosis (P = 0.01). Furthermore, both pioglitazone and empagliflozin improved liver fibrosis by 0.7 ± 1.5 kpa, and 1.1 ± 1.3 kpa, respectively . However, the reduction was insignificant in the pioglitazone group . Diastolic blood pressure was reduced in both groups with no statistically significant difference between the two groups. EF at baseline was 58.7% ± 8.1% and 57.9% ± 6.9% in the pioglitazone and empagliflozin respectively.Systolic blood pressure was reduced by 3.57 ± 2.08 at baseline and 9.45 ± 1.68 by the end of the study (P = 0.39). On the other hand, it decreased significantly from 9.93 ± 2.49 to 9.09 ± 1.89 (P = 0.04) in the empagliflozin group. Pioglitazone and empagliflozin did not affect cardiac dimensions and pulmonary artery pressure after 24 weeks . Also, only in the pioglitazone group did the TG level decrease significantly (P < 0.01). Both pioglitazone and empagliflozin significantly reduced the insulin resistance measured by the TyG index at study end, although the difference between the two groups was not significant, (P = 0.16).Changes in BMI, FBS, HbA1c, Total cholesterol, LDL, HDL, AST, ALT, and TyG index from baseline are shown in Table Nine non-severe adverse events (AEs) occurred during the study. These included cystitis, gross hematuria, minor hypoglycemia, and lower extremity edema. In addition, two severe AEs happened in two patients, namely, cholangitis and transient ischemic attack that required hospitalization. The severe AEs resolved entirely (Supplement Left ventricular ejection fraction (LVEF) is a central measure of global systolic function . HoweverIt is also an independent predictor of CVD, cardiac death, and ventricular arrhythmias . MoreoveIn patients with diabetes mellitus, abnormal GLS may be a marker of subclinical LV systolic dysfunction, even in asymptomatic patients . On the In the present study, we showed that in patients with T2DM with NAFLD and without established ASCVD, liver fibrosis is associated with reduced GLS at baseline. Our results are in parallel with the CARDIA study . They reWe also demonstrated that both empagliflozin and pioglitazone significantly improve liver fibrosis and ameliorate LV remodeling and subtle myocardial dysfunction. However, there was no statistically significant relationship between GLS, LSM, and CAP changes. The relatively small sample size and short duration of the study might act as interfering factors. Moreover, the changes in LSM could be partly related to changes in CAP. Nevertheless, improvement of cardiac structure and function in this study appear to be independent of the improvement of liver steatosis and fibrosis. The greater numerical improvement of GLS in the empagliflozin group compared to pioglitazone has a clinical importance. However, it did not reach the limit of statistical significance. This might be explained by low power of the study to detect statistical significance between the two groups.de-novo synthesis by the liver is increased [Using an animal model, Santos-Gallego et al. showed that empagliflozin in the non-diabetic model reduces LV remodeling and improves myocardial strain by decreasing neuro-hormonal activation. They suggested that the effects of empagliflozin are independent of its impact on glucose homeostasis . In patincreased . Contribncreased .Empagliflozin improves cardiac energetics and metabolism and reduces LV remodeling by increasing the oxidation of free fatty acids, ketone bodies, and branched-chain amino acids . Also, aThe present study demonstrates that pioglitazone improves liver fibrosis and ameliorates subclinical myocardial dysfunction. In addition, empagliflozin and pioglitazone improved diastolic function in our patients with T2DM and NALFD. We showed in our previous studies that empagliflozin effectively improves liver steatosis in patients with and without T2DM , 42. ThiIn addition, Yoneda et al. evaluated the changes in hepatic steatosis after 24 weeks of pioglitazone and tofogliflozin treatment using MRI-PDFF. No significant differences were observed between the groups concerning relative changes from the baseline .We do have some limitations in our study. It was a single-center study with small number of patients. All patients were asked to follow lifestyle modification recommendations to control HbA1c as an interfering factor. However, although we managed lipid and glycemic control according to the standard guideline, the impact of other unmeasured confounders can never be overemphasized. In addition, we did not perform a liver biopsy as the gold standard method to evaluate liver steatosis and fibrosis.Subclinical cardiac dysfunction is highly important in patients with T2DM and with NAFLD. Empagliflozin and Pioglitazone improve LV mechanics and fibrosis in patients without established ASCVD. This has a prognostic importance on cardiovascular outcomes in high-risk patients with T2DM. Moreover, empagliflozin ameliorates liver steatosis more effectively them pioglitazone. This study can serve as a start point hypothesis for the future. Further studies are needed to explore the concept in larger populations.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4


The impact of small-for-gestational-age (SGA) on very-low-birth-weight (VLBW) premature infants remains inconclusive. This study aimed to assess the effects of being born SGA status on the short-term and long-term outcomes in VLBW preterm infants.We conducted a population-based, prospective cohort study on VLBW preterm infants born in Taiwan between 2012 and 2017. Sociodemographic, neonatal, growth and neurological data at 2 years of corrected age were collected. A total of 4243 VLBW infants born at 24 through 32 completed weeks' gestation participated in this study, of whom 1,005 had SGA status defined as a birth weight <10th percentile of gestation, and 3,238 did not (the non-SGA group).We compared the risks of short-term outcomes , long-term outcomes . Subgroup analysis was performed by stratification of gestation age (GA): GA 24–26, 27–29 and 30–32 weeks.p < 0.05). The SGA group had a significantly increased risk of bronchopulmonary dysplasia in GA 27–29 and 30–32 weeks . We also found that there was an increased risk of severe retinopathy of prematurity in GA 24–26 and 27–29 weeks in the SGA group compared with the non-SGA group .In the analysis of short-term outcomes, the SGA group had an increased risk of neonatal mortality  in all GA subgroups in comparison with the non-SGA group (p < 0.05).In the analysis of long-term outcomes, the SGA group had a significantly increased risk of NDI throughout all GA subgroups  in comparison with the non-SGA group. The SGA groups also had an increased risk of growth status <10th percentile at 2 years of age (SGA VLBW premature infants had higher risks of neonatal death, growth status <10th percentile, and NDI at 2 years of corrected age compared with the non- SGA premature infants. Prenatal surveillance, postnatal attention, and long- term follow-up are warranted to improve the outcomes of VLBW SGA premature infants. The prevalence of preterm birth and VLBW preterm birth were 9.31% and 0.88% in Taiwan between 2012 and 2017, respectively . In neonPreviously, the two terms “intrauterine growth restriction (IUGR)” and “SGA” were used interchangeably –8. HowevThe etiologies of IUGR and SGA are diverse , 15. ToxHeterogeneity exists in growth and neurodevelopmental outcomes in SGA preterm infants compared to those in the non-SGA group. This is due to different definitions of SGA as well as inclusion criteria of the studies. Children born SGA were unable to have catch-up-growth ranging from 13 to 40% in different reports . Hokken-Because of countless prenatal, perinatal and postnatal variables, VLBW premature neonates born SGA are especially at risk of neurodevelopmental impairment (NDI) until later life , 23. ManThis study aimed to assess the effects of being born SGA status on short-term outcomes  and long-term outcomes (growth status <10th percentile and NDI at 2 years of corrected age) among VLBW preterm infants compared with the non-SGA VLBW premature infants.2.2.1.VLBW premature neonates born between 2012 and 2017 at 22 neonatal care centers were enrolled in the Taiwan Premature Infant Follow-up Network (TPFN) database. This database covers more than 70% of all tertiary medical centers which are capable of caring for VLBW preterm neonates in Taiwan. This prospective, longitudinal, multicenter cohort study evaluated VLBW premature neonates admitted to neonatal wards including intensive care units (NICUs) or newborn general wards within 1 week after birth until they survived to discharge. Sociodemographic, perinatal, and neonatal data during hospitalization were collected. The exclusion criteria were infants with major congenital anomalies or chromosomal disorders. SGA infants were defined as having a birth weight less than the 10th percentile for gestational age (GA) according to Taiwanese infants' sex-specific standards for singleton  or twinsThis study was approved by the Institutional Review Board of Mackey Memorial Hospital (IRB number 20MMHIS277e). All parents signed an informed consent form designed by the Premature Baby Foundation of Taiwan.Infants born with a GA of 24–32 weeks in the TPFN database who survived to discharge and completed follow-up to a corrected age of 2 years were enrolled into the final analysis. Among these participants, those who were SGA were compared with those who were non-SGA in neonatal and 2 years' neurological outcomes. Subgroup analysis was performed by stratifying the SGA and non-SGA groups in to three groups by GA: 24–26, 27–29 and 30–32 weeks. 2.2.Neonatal data, including the details of neonatal morbidities and management during NICU hospitalization, were recorded. Bronchopulmonary dysplasia (BPD) was defined as oxygen requirement at the postmenstrual age of 36 weeks. Severe intraventricular hemorrhage  was diagnosed using brain sonography. Cystic periventricular leukomalacia (cystic PVL) was detected by brain sonography or cranial magnetic resonance imaging (MRI) scan. Necrotizing enterocolitis (NEC) (≥Stage 2A) was defined according to modified Bell's criteria. Advanced retinopathy of prematurity (ROP) (≥Stage III or requiring laser or injection treatment) was classified according to international guidelines.2.3.All study participants were followed until a corrected age of 2 years in the TPFN. Growth status was assessed, including body weight (BW), body length (BL), and head circumference (HC); these parameters <10th percentile at 2 years of corrected age were defined as <10.5 kg, <83.9 cm, and <46.5 cm, respectively, in boys, and <9.9 kg, <82.3 cm, and <45.4 cm, respectively, in girls . Neurode2.4.All statistical analyses were conducted using R version 4.0.2 . Continuous data were presented as mean and SD, and categorical data were presented as numbers and proportions. Unadjusted comparisons between categorical variables were performed using the chi-square test, and continuous data were compared using the independent t-test. Multivariable logistic regression was used to estimate the relative risk of poor cognitive development in VLBW SGA infants compared with non-SGA preterm infants. Potential confounders including sex, use of prenatal steroids, preterm premature rupture of membranes, being the first-child, and paternal and maternal educational status less than 12 years were included in the model –39. Adju3.n = 5,679) were enrolled, of whom 29 died after discharge and 1,407 were lost to follow-up by a corrected age of 2 years. The remaining 4,243 VLBW infants completed the follow-up course (follow-up rate = 74.71%), of whom 1,005 (23.69%) had SGA status and 3,238 (76.31%) did not   .3.1.p < 0.001).Sociodemographic, perinatal, and neonatal data of the SGA and non-SGA groups were shown in 3.2.Subgroup analyses by GA  were performed to investigate the effects of SGA on the incidence of various neonatal outcomes . Multivap < 0.01). Although an increased risk of BPD was observed in all SGA subgroups, the risk was significantly higher only in the GA 27–29 and 30–32 subgroups . There were also increased risks in cystic PVL and NEC in all SGA subgroups, however none of them were statistically significant. An increased risk of severe ROP requiring treatment was observed in the SGA subgroups with GA at 24–26 and 27–29 weeks ; however, a non-adverse effect was observed in GA 30–32 weeks .The results showed that the SGA group was associated with an increased risk of death in all GA subgroups  compared with the non-SGA group (3.3.The incidence rates of binocular blindness were 0% (=0/78), 0% (=0/278), and 0.15% (=1/649) in the SGA subgroups , compared to 0.12% (=1/840), 0.06% , and 0% (=0/805) in the non-SGA subgroups.The incidences of hearing impairment requiring hearing aids or deafness were 5.13% (=4/78), 2.52% (=7/278), and 0.77% (=5/649) in the SGA subgroups , compared to 2.74% (=23/840), 1.95% , and 0.99% (=8/805) in the non-SGA subgroups.p < 0.001) (p < 0.05).Multivariable analyses were performed to explore the association between SGA and neurodevelopmental outcomes at 2 years of corrected age after adjusting for sex, use of prenatal steroids, preterm premature rupture of membranes, being the first-child, maternal education ≤12 years and paternal education ≤12 years according to the literature review. < 0.001) . SGA stap < 0.01).p < 0.05). Of note, that among SGA VLBW preterm infants, those at GA 24–26 weeks tended to have motor composite score (MCS) <85  and NDI . In addition, those at GA 30–32 weeks tended to have delays in development in multiple domains: cognitive composite score (CCS) <85 , MCS <85  and NDI .In the analysis of neurodevelopmental outcomes at 2 years of corrected age, the SGA group was associated with an increased risk of NDI in all GA subgroups . Although the SGA group had an increased risk of BPD in all GA subgroups, the risk was significantly higher only in the GA 27–29 and 30–32 subgroups , but not at GA 24–26 weeks (p = 0.57). Preterm neonates born at GA 24–26 weeks were relatively small and fragile and tended to have a higher risk of developing BPD. Our observation was that the BPD rates were both high in the SGA and non-SGA groups at GA 24–26 weeks (75.64% vs. 72.63%), so no statistical significance existed between the two groups (p = 0.54). Additionally, the nutritional status and degree of ventilator support could not be obtained in the TPFN database, and further large-scale surveys are warranted to evaluate the possible mechanisms.Bronchopulmonary dysplasia (BPD) is a neonatal lung problem with multifactorial etiologies, including possible pulmonary ontogenic changes at higher gestational ages related to nutrition or growth in animal model , 46, durSimilar to previous studies , 50, we p < 0.05), however there were non-adverse effects in GA 30–32 weeks   compared with the non-SGA subgroup  . This fi = 0.66) . The mec = 0.66) . Further4.2.Consistent with Itabashi et al.’s report , we demoOur results showed a significantly increased risk of NDI in the SGA subgroup after adjusting for confounding factors in all GA subgroups  –6. This Children born very preterm, especially before a GA of 32 weeks, are at particularly high risk of long-term disabilities because of various prenatal, perinatal, and neonatal factors . Male inp < 0.05). Higher rates of cystic PVL and NEC ≧ stage IIA were observed in all SGA subgroups, although the differences were not significant. We also found that all SGA subgroups had a significantly higher incidence of NDI at 2 years of corrected age compared with non-SGA subgroups. Moreover, the SGA group with a GA of 24–26 weeks had a higher rate of MCS <85 in BSID-III than the non-SGA group at 2 years of corrected age (p < 0.05). Furthermore, the SGA group with a GA of 30–32 weeks had CCS < 85, MCS <85 in BSID-III, and NDI at 2 years of corrected age (p < 0.05).To identify potential associations between neonatal morbidities and neurodevelopmental outcomes at 2 years of corrected age between the SGA and non-SGA groups, we performed a subgroup analysis according to GA at 24–26, 27–29 and 30–32 weeks. SGA VLBW preterm groups had a significantly higher rate of severe ROP ≧ stage3 or requiring treatment in both GA 24–26 and 27–29 weeks. SGA infants also had a higher incidence of BPD at GA 27–29 and 30–32 weeks , which may be associated with the higher risk of NDI in the SGA group. Further large-scale study in maternal pathology and neurodevelopmental outcomes in SGA neonates are warranted.Second, the increased neonatal morbidities resulting from SGA could have adverse effects on cognitive, motor, vision, hearing or overall NDI toward at least 2 years of age . Regev ep < 0.05). Severe ROP is strongly associated with blindness  in SGA preterm infants, especially the persistence of microcephaly reflecting cerebral volume at 2 years of corrected age, has been reported to determine poor intelligence and neurosensory or motor developmental outcomes , 60, 61.It has been proposed that brain maturation was related to retarded motor development in preterm neonates at GA < 28 weeks with severe SGA (Three possible explanations for these results are as follows:vere SGA . We founvere SGA .Our results differ from those of other reports , 26, 63,Our findings also have important public health implications. The higher risk of NDI in VLBW preterm neonates born SGA indicates that they may need more resources for rehabilitation including occupational, physical, and speech therapies. Additional attention may also be required when this vulnerable group enters school. This study suggests that government health agencies, including the Ministry of Health and Welfare, and the Bureau of National Health Insurance in Taiwan, should provide more resources to SGA VLBW preterm infants to improve their survival rate and neurodevelopmental outcomes. We also suggest that our government health agencies provide more supports for high-risk pregnant women with fetal growth restriction in their prenatal examinations, including Doppler sonographic examinations for umbilical blood flow to detect ADEV or REDV, biophysics, and fetal growth velocity, which provide very important information for the timing of delivery, either iatrogenic delivery or expectant management , 64.The strength of this study is the prospective, multicenter cohort design with the inclusion of 22 neonatal care centers, which covers approximately 70%–80% VLBW preterm neonates born in Taiwan. This is the first population-based cohort study on growth and neurodevelopmental outcomes at 2 years of corrected age in SGA VLBW preterm infants compared with non-SGA VLBW preterm infants in Taiwan. However, this study had some limitations. Although the TPFN database includes very detailed and complete perinatal and neonatal data, the incidence of SGA preterm birth in Taiwan could not be obtained by exploration in this study, and some obstetric sonographic parameters, such as symmetric or asymmetric IUGR status, placental blood flow and fetal cerebral blood flow patterns, were not available. Detailed obstetric sonogram data can help assess fetal growth restriction, which may predispose individuals to SGA.Another limitation of this study was the case distribution of the cohort. By definition, SGA is the 10th percentile by birthweight, which means it should make up about 10% of the general VLBW population. However, the TPFN databank was not a nationwide population, so the overall case number was not large enough to achieve normal distribution in each GA subgroup. Further large-scale population-based cohort study is warranted.5.This study provides important information for prenatal counseling as well as evidence of the public health impacts of SGA births. Our results showed a strong association between SGA birth status and neonatal mortality in VLBW premature infants born at GA of 24–32 weeks. Being born with SGA status was also associated with increased risks of BW, BL, and HC remaining less than the 10th percentile as well as NDI at 2 years of corrected age. Antenatal close surveillance, postnatal comprehensive care, and long-term follow-up are warranted to improve the outcomes of premature VLBW SGA infants.


To determine the characteristics of longitudinal choroidal thickness (CT) and axial length (AL) changes in a group of Chinese young adults with various refractive errors.In this 2 year prospective cohort study, a total of 291 (314 enrolled at baseline) Chinese medical freshmen aged 18 to 22 years  underwent eye examinations at baseline and follow-up visits, including cycloplegic refraction, ocular biometry measurements, and swept-source optical coherence tomography. Choroidal thickness measurements were performed at nine locations in the macular area.p < 0.001) and parafoveal CT of 7 locations between emmetropia and mild to moderate myopia, and the changes of the subfoveal CT (p = 0.002) and parafoveal CT of 6 locations between emmetropia and high myopia. But there were no differences for AL and SE (p > 0.05). The multivariable linear regression analysis showed that baseline subfoveal CT (per 1 μm) was a significant factor affecting the changes of subfoveal CT (p < 0.001), whereas age, gender, and baseline AL were not significantly associated.At baseline, the CT and AL was significant differences among groups of emmetropia, mild to moderate myopia and high myopia. During a two-year period, there were significant differences found in the changes of the subfoveal CT (The longitudinal change in CT varies with refractive errors in Chinese young adults aged 18 to 22 years over a two-year period. The changes of subfoveal CT were significantly associated with the baseline subfoveal CT, but not associated with baseline AL. The study participants were first-year university students from the School of Medicine, Sun Yat-sen University, Guangzhou, China. Those students with systemic diseases, intraocular lens or aphakic eyes, astigmatism ≥1.50 diopters (D) in both eyes, anisometropia ≥1.00 D, severe ocular abnormalities, dysfunction in binocular vision, history of ocular surgery, or those who underwent medical therapy (such as atropine treatment), were excluded from the study. A total of 367 first-year medical students in 2017 were screened, of which 307 (83.7%) were eligible and participated in the study at baseline. Of 307 participants, 291 (94.8%) attended the 2-year follow-up visit. Ethics approval was obtained from the human ethics committee of the Zhongshan Ophthalmic Center. The study was conducted in accordance with the Declaration of Helsinki. For each participant, written consent was obtained prior to baseline examination.Trained ophthalmologists and optometrists conducted the following procedures at the Zhongshan Ophthalmic Center Clinic for all visits. All the subjects underwent cycloplegic autorefraction using  on both eyes, after three instillations of 1% tropicamide eyedrops, the mean of 5 consecutive measurements was recorded as the final value for each eye. Refractive error was analyzed using spherical equivalent (SE), defined as the spherical power plus half of the cylindrical power. Emmetropia was defined as SE > −0.5 D, low to moderate myopia was defined as SE between −0.5 D to > − 6.0 D, and high myopia was defined as SE worse than or equal to −6.0 D.Monocular visual acuity was measured at 4 m with the Logarithmic Visual Acuity Chart. Best-corrected visual acuity (BCVA) was also measured. The mean of 5 ocular biometry measurements was collected, including axial length (AL) and anterior chamber depth (ACD), using the Lenstar LS-900 . The mean of 3 consecutive intraocular pressure (IOP) was measured with a non-contact tonometer (TOPCON CT-80A/DKT-18).’s membrane and the choroidal-scleral interface. A three-dimensional macular scanning was performed, the ETDRS grid was applied in the final analysis of CT, and the mean CT in each grid sector was calculated by built-in software. A built-in software completed segmentation of different layers on the OCT images and construction of topographic maps. When the process was inaccurate, leading to measurement artifacts, manual segmentation was carried out. The scan protocol was repeated three times consecutively on the same visit. The participant and device were repositioned after each scan. The diameters for the inner circle , middle circle and outer circle of the ETDRS grid were 0.5, 1.5, and 3 mm, respectively. Nine locations were measured .According to a previously established method, Swept-source OCT  was used to evaluate CT in both eyes after cycloplegia . In detaThe right eye was used in the analysis .p values less than 0.05 were considered statistically significant. All the statistical analyses were conducted using SAS 9.4 software .Baseline characteristics were compared using one-way ANOVA or Chi-squared test among three refractive error groups. Linear mixed models were performed to calculate and compared the rate of ocular biometry change and choroid thickness change over 2 years, with follow-up duration, group and a group and duration interaction as fixed effects, and follow-up duration at participants level as random intercept. The follow-up duration was adjusted as categorical variable in the mixed-effect model. Multiple group comparisons adjustment was performed to preserve the family-wise error rate of 0.05 using Bonferroni correction. A multivariable linear regression was fitted to identify factors associated with the 2-year cumulative change in subfoveal choroidal thickness. Two-tailed Two hundred and twelve (72.9%) of 291 participants had mild to moderate myopia, whereas only 20 (6.9%) were emmetropic subjects and 59 (20.2%) were high myopic subjects. Of the 291 participants at baseline, 132 (45.4%) were males and 159 (54.6%) were females, with a mean age of 18.8 ± 2.4 years . The meap < 0.001) and highly myopic subjects (p = 0.005). Similarly, significant differences were also found in the changes of parafoveal CT, the emmetropic subjects showed the significant choroidal thinning in five locations compared with mild to moderate myopic subjects and three locations compared with highly myopic subjects. However, there were no significant differences between moderate myopic subjects and highly myopic subjects.No statistically significant differences were found in the changes of SE, AL, keratometric power, lens thickness, IOP among emmetropic, mild to moderate myopic and high myopic groups . Howeverp < 0.001), whereas age, gender, and baseline AL were not significantly associated (p = 0.010), −0.37 (p < 0.001), and − 0.44 (p < 0.001) in emmetropia, mild to moderate myopia and high myopia, respectively. And the positive correlations between the change in the subfoveal choroidal thickness and change in the refractive error were found for both refractive error groups (p = 0.193), 0.20 (p = 0.005), and 0.27 (p = 0.042) in emmetropia, mild to moderate myopia and high myopia, respectively.After multivariable linear regression analysis, the baseline subfoveal CT (per 1 μm) was a significant factor in determining the changes of subfoveal CT, with a regression coefficient of −0.09 , allowing greater insight into the changes in choroidal thickness with age and eye growth in an age range of 18 to 22 years. The main limitations of our study include the relatively small sample size with unequal numbers of emmetropic, mild to moderate myopic and highly myopic subjects, and short follow-up time, as emmetropia is in the limited number in this group of population. The recruited participants were Chinese medical students. It may limit the generalizability of our findings to Chinese young adults in general from various regions in China and other countries.In conclusion, it is the first time that the longitudinal change in CT varies with diverse refractive errors in Chinese young adults aged 18 to 22 years over a two-year period were reported. The CT of emmetropic eyes at both subfoveal and parafoveal locations thinned at a relatively faster rate than that of myopic eyes, with no significant differences in changes in SE and AL between refractive error groups in young adulthood. The baseline subfoveal CT was a significant factor for evaluating the longitudinal change in subfoveal CT among young adults. Choroid seems continually play a role in this age group of adults’ refractive error development.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 human ethics committee of Zhongshan Ophthalmic Center. The patients/participants provided their written informed consent to participate in this study.FD, XC: conception and design. JD, ACY, AY, and YW: data collection. ZW: analysis and interpretation. ZC, FD, and XC: manuscript draft, review, and revision. All authors contributed to the article and approved the submitted version.This work was supported by funds from Natural Science Foundation of Guangdong province (2020A1515011364) and Essilor International.ACY is an employee of Essilor AMERA, and AY, BD, and YW are employees of Essilor International.The remaining 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.


Rhizosphere bacterial communities play a crucial role in promoting plant and soil ecosystem health and productivity. They also have great potential as key indicators of soil health in agroecosystems. Various environmental factors affect soil parameters, which have been demonstrated to influence soil microbial growth and activities. Thus, this study investigated how rhizosphere bacterial community structure and functions are affected by agronomic practices such as organic and conventional fertiliser application and plant species types.Rhizosphere soil of vegetable crops cultivated under organic and conventional fertilisers in different farms was analysed using high-throughput sequencing of the 16S rRNA gene and co-occurrence network pattern among bacterial species. The functional structure was analysed with PICRUSt2 pipeline.4, PO4, pH and moisture content largely driving the variations across the farms. Organic farms had a higher diversity richness and more unique amplicon sequence variants than conventional farms. Bacterial community structure in multivariate space was highly differentiated across the farms and between organic and conventional farms. Co-occurrence network patterns showed community segmentation for both farms, with keystone taxa more prevalent in organic than conventional farms.Overall, rhizosphere bacterial communities varied in response to fertiliser type, with soil physicochemical parameters, including NHAgromyces, Bacillus and Nocardioides. The results revealed that organic fertilisers support high functional diversity and stronger interactions within the rhizosphere bacterial community. This study provided useful information about the overall changes in soil microbial dynamics and how the changes influence ecosystem functioning under different soil nutrient management and agronomic practices.Module hub composition and identity varied, signifying differences in keystone taxa across the farms and positive correlations between changes in microbial composition and ecosystem functions. The organic farms comprised functionally versatile communities characterised by plant growth-promoting keystone genera, such as  Soil microbes participate in nutrient cycling, organic matter decomposition and energy flow, which are key in sustaining soil ecosystem functions . HoweverCurrently, organic and conventional fertilizers are the main agro-inputs for improving soil nutrient content . ImproveFurthermore, plant species or genotypes influence the synthesis of unique exudates and extracellular enzymes, which impact microscale spatial patterns in soil bacterial communities and differentially stimulate the growth of specific microbial groups under different ecosystems across geographic locations . UsuallySoil parameters vary by geographic location and primarily provide similar conditions that affect the activities of plants and their associated microbiome to soil microbes . StudiesConsequently, this study assessed the effects of fertilizer type  and plant species type on the microbial dynamics (including keystone taxa) and ecosystem functions of vegetable rhizosphere soil across different farms. It is hypothesized that fertilizer type in each of the farming systems predisposes the selection of certain rhizobacterial communities, which possess diverse but unique functional gene repertoire of agronomic importance. Results from this study could provide baseline information useful in predictive modeling for manipulating soil bacterial communities to improve agroecosystems. Similarly, the approach used in this study could unravel keystone taxa with unique biomarker potential for monitoring soil health and productivity in a given nutrient management system.www.sa-venues.com/weather/). Average annual temperature and rainfall are 22.36°C and 36.6 mm for North West province and 20.64°C and 56.11 mm for Gauteng province. The average annual relative humidity for North West and Gauteng provinces are 37.8 and 50.22%, respectively (Rhizosphere soils were collected from four vegetable farms located in the North West (farms B and S) and Gauteng (farms J and T) provinces of South Africa. Farm B (26°42′55.0″S 27°04′59.6″E) and farm S (26°47′43.5″S 27°02′18.3″E) are situated in JB marks municipality, while farm J (26°11′40.1″S 28°03′58.3″E) and farm T (26°03′16.0″S 27°40′13.3″E) are in Johannesburg and West Rand District municipalities, respectively. The North West province is characterized as a semi-arid climate while the Gauteng province is classified as a mild climate that is neither humid nor too hot (ectively . Vegetabectively . The farAllium cepa (onion), Brassica oleracea (cabbage), Lactuca sativa (lettuce), and Spinacia oleracea (spinach), were sampled from each farm as described by Sampling was conducted in the summer (October–February) of 2021. Based on visual inspection, rhizosphere soils of healthy vegetables of the same age and size including, 2+, Mg2+, K+, and Na+) in the soil were determined using the inductively coupled plasma mass spectrometry (ICP-MS), following standard procedure , while the electrical conductivity (EC) was determined with an EC meter. The amounts of macronutrients , micronutrients , heavy metals  and exchangeable cations  following the manufacturer’s instructions. The DNA concentration was measured with a Qubit fluorometer  and the integrity was checked using gel electrophoresis with a 1% agarose gel. Extracted DNA was normalized to equimolar concentrations (5 ng/μl) using 0.1 M Tris-HCl (pH 8.5) and the partial 16S rRNA gene (V3-V4 region), a universal barcode for the characterization of bacteria, was amplified in a polymerase chain reaction (PCR) using Illumina-barcoded 341F and 805R primers . The 16SThe 16S rRNA gene sequence reads were demultiplexed and trimmed of barcodes using MiSeq Reporter software  and then quality checked with FastQC (v. 0.11.5) . Low-quality reads were trimmed with trimmomatic software (v. 0.38)  and readrunRefBlast function. Thereafter, the functional prediction was evaluated using the makeFunctionalPrediction, where the ASVs were summarized based on the results of the next neighbor search -based method in the molecular ecological network analysis (MENA) pipelinep < 0.05 and the data was tested across fixed factors: farms, fertilizer types  and vegetable species. Data normality was tested using the Shapiro–Wilk test and non-normal data was transformed with log10, square root or sine to fit a normal distribution. Normal or non-normal data were analyzed using parametric or non-parametric tests, respectively. Correlation between soil physicochemical and enzyme activity data was tested using Pearson or Spearman rank correlation for normalized or non-normal datasets. The community structure across the farms, and between organic and conventional farm soil was visualized with a nonmetric multidimensional scaling (NMDS) plot and a cluster dendrogram using vegan and dendextend (v. 1.12.0)  between the farms, organic and conventional farms and plant species using microeco package in R software  was performed in R software to show the physicochemical parameters that best explain the variations in the microbial community composition.All statistical analyzes were performed in R software (v.4.1.1) unless otherwise stated. The significance for all tests was set at  1.12.0)  packages 1.12.0)  was perfsoftware . Subsequhttps://www.ncbi.nlm.nih.gov/bioproject/904574).The 16S rRNA gene sequence data for this study are available in the sequence read archives (SRA) of the National Centre for Biotechnological Information under a BioProject with SRA accession no PRJNA904574 (p < 0.05) differed across the farms  across the fertilizer types.The soil physicochemical parameters varied across the fixed factors with no particular trend. A near-neutral pH range was observed across the farms, with the conventional farms having a higher pH than the organic farms . The moihe farms , while op < 0.05) different across the farms and fertilizer type, except for alkaline phosphatase, which was not influenced by the fertilizer type. Mostly, the enzyme activities were higher for organic farms compared to conventional farms (p > 0.05).The soil enzyme activities were significantly  across the farms, with the pairwise comparison  showing differences between farms T and S. In addition, the combined effects of the fixed factors (fertilizer type and plant species) and their interactions had no significant effect  on the alpha diversity indices measured.The alpha diversity indices measured were not significantly influenced by the main effect of fertilizer type and plant species, except for the inverse Simpson index, which was significant  and across organic and conventional farms , contributing about 39.01 and 11.74% of the variations in the models and a PERMDISP of 0.073 and 0.269, respectively. Significant variation was observed between farms B and T, J and T, and S and T. However, the plant species had no significant  effect on the bacterial community structure, which was not clearly differentiated  are somewhat differentiated, they are jointly less similar to conventional farms (farms J and T) ; the comentiated . The nonentiated  suggestsentiated . The effentiated .Bacillus, Rubrobacter, Gemmatimonas, Solirubrobacter, RB41, Nocardioides and Bryobacter  phyla were Actinobacteria (32.3%), Proteobacteria (25.4%), Acidobacteria (8.4%), Firmicutes (7.6%), Chloroflexi (7.6%), and Planctomyces (5.7%) . The relyobacter . Bacilluin onion  comparedrobacter .p < 0.05; LDA score > 2.0; p > 0.05). The genera Rubrobacter and Microlunatus were highly discriminant in organic farms  at the genus taxa across the farms are shown in Rubrobacter and uncultured Conexibacteraceae were more discriminant in farm B, while Pira4lineage and uncultured bacterial Clone C112 were discriminant in farm T. In farm S, Uncultured Actinomycetales, Thermoactinomyces, and Tumebacillus were found to be discriminant. The discriminant features in conventional farms, Marmoricola and Lysobacter, were mainly from farm J. Before multiplicity adjustment of the LEFSe results for plant species type, Pseudomonas, Paracoccus and Candidatus Udaeobacter were discriminant  and abundant in cabbage, onion, and spinach, respectively  effect size (LEFSe) showed that a total of 205 features were discriminant across the farms  after multiplicity adjustment . Organic farms had more differentially abundant pathways compared to conventional farms. Amino acid, arginine, proline metabolism, C-fixation and citrate cycle pathways were discriminant in the organic farm  and PO4  but negative correlation with OM , NH4+  and K . Both alkaline and acid phosphatase significantly (p < 0.05) correlated with TOC, positively and Cl, negatively. Moreover, alkaline phosphatase correlated positively with NO3 while acid phosphatase correlated positively with pH and EC. In addition, dehydrogenase had significant correlations that were negative with pH, PO4, NH4 and K but positive with OM and TOC. Although there exists either a positive or negative correlation between the soil enzymes and moisture content, Ca and sand, the correlations were not significant. Similarly, though not significant, there were correlations between the species function percentage of bacterial community with the soil parameters  (p < 0.05) .The RDA plot showed that the soil physicochemical properties contributed 34.5%  of the variation in the bacterial community structure. Farms and fertilizer type-specific clustering were observed, especially for the organic farms . Among t < 0.05) . Similar < 0.05) . Overall < 0.05) . PO4, Nar = 0.70, p < 0.05) positive correlations with the environmental traits, which produced a significant  effect on the network connectivity. The significant ASVs and node connectivity were found to be significant for two major phyla: Actinobacteria  and Acidobacteria . From the topological metrics  and the higher values of some of the structural features in the empirical compared to the random networks  associations occurred among the bacterial communities in the organic farms compared to conventional farms, which had a higher negative (~33%) association. Though community segmentation was apparent in both networks, the organic farms had a more profound fragmentation. Moreover, a higher network complexity was observed for organic farms compared to conventional farms, which had more numbers of smaller modules and edges than organic farms. The module hubs were not of similar parameters, with organic farms  having mhagaceae .Soil microbes significantly contribute to ecosystem functions through organic matter decomposition, nutrient cycling, and mineralization. These functions and plant ecosystems are influenced by agronomic practices such as soil nutrient management and cropping systems. Thus, this study provided insights into the rhizosphere soil bacterial community structure and functions of vegetable crops cultivated under organically and conventionally managed soil in different farms. Functional profiling and differentially abundant taxa of rhizosphere bacteria allowed for the identification of rare microbial communities with unique potentials in agroecosystems, emphasizing the benefits of understanding soil microbiome dynamics to reveal specific patterns, functions and strategies used by microbes under different agronomic conditions.4+ and NO3− were not significant across the farms, However, except for pH and P, the measured values were relatively not within the optimal range required for the productivity of most crops, including vegetables  can be found below: RA: conceptualization, methodology, validation, formal analysis, investigation, resources, writing: review and editing, project administration. AR: validation, resources, writing—original draft, review and editing, methodology, validation, formal data analysis, and investigation. OE: validation, data analysis, writing—review and editing. All authors contributed to the article and approved the submitted version.This research was funded by the National Research Foundation (NRF) South Africa, under grant number 116251 and the Technology Innovation Agency with grant number NWU SF2210012/TIA1396/01.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.


Breast Tanner stage , ovarian volume , and Σ4DEHP  were independent risk factors for IPT progression. For each 10 µg/g/Cr increase in the urine level of Σ4DEHP, the risk of progression from IPT to CPP/EP within one year increased by 20%. This study demonstrated that the breast Tanner stage, ovarian volume, and Σ4DEHP in urine were independent risk factors for IPT progression, and Σ4DEHP may be associated with the progression of IPT to CPP or EP.Phthalate esters (PAEs) may act as estrogen receptor agonists, and their relationship with precocious puberty is a global health concern. However, their role in isolated premature thelarche (IPT) progression remains unclear. We conducted a cohort study investigating the relationship between IPT progression and urinary PAE metabolites. Girls with IPT aged 6–8 years were regularly followed up every three months for one year. Clinical data and urine PAE metabolite levels were collected. Participants who progressed to central precocious puberty (CPP) or early puberty (EP) had significantly higher ovarian volume, breast Tanner stage, and levels of the creatinine-adjusted urinary secondary oxidized di-2-ethylhexyl phthalate (DEHP) metabolites (Σ Isolated premature thelarche (IPT) is defined as unilateral or bilateral breast development in girls before age eight1. IPT appearing before the age of 2–4 may be attributable to mini-puberty2. However, IPT presented at an older age (mostly after age six) may progress to central precocious puberty (CPP) or early puberty (EP)3. These conditions may have a detrimental effect on the final adult stature and psychology4. At present, relevant studies have mainly focused on indicators of clinical transformation, such as serum levels of estradiol, luteinizing hormone (LH)5, insulin-like growth factor-1 (IGF-1) standard deviation score (SDS)6, age at onset7, absolute growth velocity5, weight8 and estrogen effects on target organs, such as uterus length and breast Tanner stage3. However, the results varied, and few studies have explored the underlying mechanism.9, and PAEs are omnipresent: in food, water, breast milk, soil, breathing air, dust, dress materials, dwellings, hospitals, jails, etc.10. Di-2-ethylhexyl phthalate (DEHP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP), and di-isobutyl phthalate (DiBP) are the four PAEs with relatively high detection levels in indoor environments, river sediments, and aquaculture fishponds in the Pearl River Delta region, where Shenzhen is located13. Furthermore, the levels of PAEs in water and sediments in the Pearl River Delta Basin recently showed an increasing trend15. More importantly, the metabolites of these four PAEs are also higher than those of other PAE types in the urine of school students in Shenzhen, with a detection rate of 80–100%16, indicating that children in Shenzhen have relatively high exposure to PAEs.Exposure to phthalate esters (PAEs) is of great concern19, while no relationship with puberty20 or an association with the later onset of puberty22 has also been reported. A meta-analysis showed that DEHP and DEP may lead to precocious puberty23. Our published study presented that low-molecular-weight PAEs metabolites were associated with CPP by comparing the age-matched and BMI-matched groups of IPT, CPP, and girls with no breast development24. Although the mechanism remains to be further elucidated, endocrine-disrupting chemicals (including PAEs) might act through a number of mechanisms at some level in the hypothalamic-pituitary–gonadal (HPG) axis and peripheral endocrine organs25. For example, PAEs might act as estrogen receptor agonists or androgen receptor antagonists and might also interfere with androgen synthesis27. Therefore, we speculate that PAEs may be associated with progressing from IPT to CPP or EP. However, the role of PAEs in the progression of IPT has not been previously reported. Therefore, we conducted this cohort study to investigate the relationship between urinary metabolites of the four relatively high-exposure PAEs  and the progression of IPT to CPP or EP in girls within one year.The relationship between PAEs and precocious puberty is a widespread concern. Different studies have shown significant associations with precocious puberty29. All of the IPT girls had no pubic hair and armpit hair growth. Initially, 84 girls diagnosed with IPT were included in the study.Girls aged 6–8 years with isolated breast development underwent their first evaluation in the outpatient clinic of Shenzhen Children's Hospital from December 2018 to June 2019. They were hospitalized for a GnRH stimulation test when they presented with no breast tissue regression for at least three months and/or rapid progression in the breast Tanner stage, significantly advanced bone age, and/or linear growth acceleration. IPT was based on the following diagnostic criteria: peak LH level lower than 5 IU/L and ratio between peak LH and peak FSH in the GnRH stimulation test lower than 0.6. Meanwhile, uterine length was less than 3.5–4 cm, and ovarian volume was less than 2 mlThe exclusion criteria were as follows: other endocrine diseases , neurological disorders , local mammary gland hyperplasia, history of hormone treatment, history of nutritional tonic  and traditional Chinese medicine intake, family history of CPP, premature birth, and small size for gestational age. Ultimately, 74 participants were enrolled in this study. Clinical data and urine specimens examined for PAEs were collected from all the children at baseline.http://www.chictr.org/cn/). The study was approved by the Ethics Committee of Shenzhen Children's Hospital (No. 2018031). All girls participated voluntarily, and written consent was obtained from their parents.This cohort study is registered as ChiCTR1900020492 in the Chinese Clinical Trial Registry  patients who missed the follow-up or failed to cooperate during the follow-up period, (2) patients who received any sex hormone treatment, and (3) patients suffering from endocrine diseases, as mentioned above.The outcome of this study was whether IPT progressed to CPP/EP within the one-year ± two months follow-up period. Following the Society of Pediatrics, Chinese Medical Association (2015) guidelinesPatients were divided into two groups according to the follow-up results Fig. . The tra2). The values were expressed as age- and sex-specific SDS based on national survey data from 200531. Two trained pediatric endocrinologists evaluated the staging of pubertal development according to the Tanner criteria32. A larger breast was the determinant of breast stage in the participants.Height and weight were measured, and the body mass index was calculated as body weight (kg)/height (m33. The lower limit of detection (LOD) for estradiol was 20 pmol/L, and data below 20 pmol/L were expressed as half of the lower LOD (10 pmol/L). The GnRH stimulation test  was performed between 8 and 10 am. Plasma gonadotropin concentrations were determined at time zero and 30, 60, and 90 min after gonadorelin administration .Levels of serum hormones, including LH, FSH, estradiol, dehydroepiandrosterone sulfate, and IGF-1, were measured at 8 am and 9 am using an electrochemiluminescence immunoassay . IGF-1 levels were standardized according to reference data for different age groups in China34, the standard method for determining bone maturity in Chinese children based on the TW3 RUS method. Pelvic ultrasound evaluations were performed by an experienced pediatric radiologist using an ultrasound machine (GE logic E9) with a 3.5–12 MHz probe and a 9–12 MHz linear probe. Ovarian volume was calculated using the formula for a prolate ellipsoid and the mean of the right and left ovaries.Bone age was evaluated using the China 05 (CH05) method on a radiograph of the left handThe participants' first-morning urine sample was collected in a fasted state. Urine samples were collected in polypropylene tubes (50 mL) and transported to the laboratory at the Shenzhen Center of Disease Control and Prevention on ice within 4 h. Urine samples were stored at − 40 °C and tested within one year.4DEHP) include MEHHP, MEOHP, MCMHP, and MCEPP. Σ4DEHP means the summation of the four secondary oxidized DEHP metabolites.Eight PAE metabolites were assessed in this study: monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), monoisobutyl phthalate (MiBP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono-[(2-carboxy methyl)hexyl] phthalate (MCMHP), mono(5-carboxy-2ethylpentyl) phthalate (MCEPP), and mono-2-ethylhexyl (MEHP). Among these, MEHP is the primary metabolite of DEHP. Secondary oxidized DEHP metabolites  coupled with a Shimadzu 20AD UHPLC system was used in instrumental analyses of PAE metabolites. Quality control samples were analyzed for each batch of 40 samples, including two program blanks and one matrix-spiked sample. We subtracted the mean concentration of the program blanks from the final concentration of PAE metabolites for each specific batch. The concentrations of the PAE metabolites below the LOD were replaced with 1/2 LOD. The LOD of PAE metabolites ranged from 0.1 to 0.3 μg/L. Urinary creatinine was analyzed using UHPLC-MS/MS to correct urine dilution.PAEs with side chain lengths of 1–4 carbons are classified as low molecular weight (LMW)-PAEs, and those with side chain lengths of more than four carbons are classified as high molecular weight (HMW)-PAEs. LMW is the sum of three LMW-PAE metabolites , and HMW is the sum of the concentrations of five DEHP metabolites. All urinary PAE metabolites were analyzed using ultra-performance liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS), according to a previously reported method8, and that of p2 was 13% (the lowest one of IPT progression)35. With 90% power and 5% significance, the total sample size was 61, and the actual power was 90.8%. Based on a 20% loss rate, 76 cases should be included. However, a total of 74 participants were included in the study. After various measures to improve compliance, 61 patients completed the follow-up, which met the sample size requirements.Sample size calculations were performed using G*Power . According to previous clinical observational studies and literature reports, the proportion of p1 was 30% (the highest ratio of IPT progression that we had searched)p-value of < 0.05 was considered statistically significant for all analyses. Numerical variables are presented as means ± standard deviations or medians and quartiles. Categorical variables are presented as numbers and percentages. The independent samples t-test assessed the differences in numerical variables between the two groups when parametric test assumptions were met. In contrast, the Mann–Whitney U test was used when they were unmet. The association between significant bone age advancement and the status of obesity or being overweight was calculated using the chi-square test.Statistical analyses were performed using IBM SPSS version 26.0 (IBM Cor. A p < 0.05) in the comparison between the two groups  and other factors that may be clinically correlated .To identify independent risk factors that significantly affected IPT progression, we performed a binary logistic regression analysis (forward likelihood ratio stepwise method) with progression from IPT to CPP/EP (yes/no) as the dependent variable. The covariates were those shown to be significant (http://www.chictr.org/cn/) on January 2, 2019. The study was performed in line with the Declaration of Helsinki and approved by the Ethics Committee of Shenzhen Children's Hospital (No. 2018031).This cohort study was registered as ChiCTR1900020492 in the Chinese Clinical Trial Registry  completed the one-year follow-up period. As shown in Table p = 0.032) and breast Tanner stage (p = 0.043). There were no differences in chronological age; the levels of basal LH, follicle-stimulating hormone (FSH), and IGF-1 SDS; or uterine length between the two groups (Table There were 22 (36.1%) participants in the transformed group and 39 (63.9%) in the untransformed group. Compared with the untransformed group, the transformed group had a higher ovarian volume (p < 0.05). However, the other creatinine-adjusted urinary PAE levels were not significantly different between the two groups. The details are listed in Table The detection rates of PAE metabolites in the urine of the 61 participants were as follows: MBP (100%), MECPP (100%), MiBP (100%), MEHHP (100%), MEP 93.4%), MEHP (91.8%), MEOHP (88.5%), and MCMHP (86.8%). The creatinine-adjusted urinary MCEPP, MEHHP, MEOHP, and MCMHP levels were significantly higher in the transformed group than those in the untransformed group , ovarian volume , and Σ4DEHP  were independent risk factors for IPT progression. Compared to IPT with breast Tanner stage 2, IPT with breast Tanner stage 3 had a 7.041-fold higher risk of progressing to CPP/EP within one year. For each 1 mL increase in the ovarian volume and 10 µg/g/Cr increase in the urine level of Σ4DEHP, the risk of progression from IPT to CPP/EP within one year increased by 260% and 20%, respectively.The results of binary logistic regression analysis (forward LR stepwise method) are presented in Table 4DEHP in urine were independent risk factors for the progression of IPT in the present cohort.This is the first cohort study investigating the relationship between IPT progression and urinary PAE metabolites. Urine PAEs were detected using UHPLC-MS/MS and corrected by creatinine to reduce the error caused by the urine concentration in the test. Our results showed that among the 61 participants with IPT, 22 (36.1%) progressed to CPP/EP during the one-year follow-up period. Breast Tanner stage, ovarian volume, and Σ3 found that 21.5% of participants with IPT converted to CPP at a chronological age of 7.1 + / − 0.7 years. In our study, the ratio of IPT progression was 36.1%, which is also higher than that in the studies of Lee et al. 36, Volta et al. 7, de Vries L et al. 35, Uçar et al. 5, and Dilek Ç et al. 8. These differences may be related to the inclusion criterion of age (onset at 6–8 years), which excluded any interference by mini-puberty and might have increased the probability of progression. In addition, the criteria requiring further investigation indicate that the breast might progress easily.The reported rates of IPT progression to CPP vary. In a Chinese retrospective cohort study, Zhu et al.37, this study presented the independent risk of breast Tanner stage and ovarian volume in IPT progression. Estrogen target organs are also associated with estrogen exposure. Long-term exposure to estrogen levels higher than prepubertal levels may be important in converting IPT to CPP. Similar to congenital adrenal hyperplasia and McCune–Albright syndrome, higher exposure to estrogen is highly susceptible to the transition from peripheral precocious puberty to CPP3. However, de Vries et al.35 showed that the levels of estradiol and adrenal hormones had no relationship with IPT progression to CPP, which was similar to the results of the present study. This may be associated with the volatility of estradiol itself, and different batches and testing methods could affect the test results, resulting in poor comparability. Thus, more attention should be paid to girls with IPT with higher breast Tanner stage and ovarian volume during the first visit and follow-up period.In contrast to previous studies4DEHP levels in the urine are independent risk factors for IPT progression. For each 10 µg/g/Cr increase in the Σ4DEHP level, the risk of progression from IPT to CPP/EP within one year increased by 20%. DEHP, an estrogenic environmental endocrine disruptor, is widely used as a plasticizer to manufacture polyvinyl chloride38. Because it is not covalently bound to plastic, it is easily emitted into the environment. The human body can absorb DEHP through the skin, gastrointestinal tract, respiratory tract, and other ways39. In prepubertal children, the clearance rate of estradiol metabolism is considered low. Therefore, exposure to estrogen-like chemicals may have significant biological effects on breast development prior to puberty.In addition, the most important finding of the present study is that Σ40. Moreover, DEHP may act as an obesogen and alter metabolic programming during fetal and early childhood development, resulting in alterations in the metabolic and peripheral hormones associated with the onset of puberty27. Intermittent fasting can ameliorate precocious puberty induced by DEHP exposure via the HPG axis41. As IPT is caused by the partial activation of the HPG axis, DEHP may promote the progression of IPT to CPP via the HPG axis. Studies have shown that PAEs can exert estrogen-like functions by binding to estrogen receptors42 and increasing estrogen synthesis by inducing aromatase gene expression43. Breasts and gonads are estrogen-target organs; therefore, DEHP may act directly on these organs to promote breast and ovarian development. More in-depth research is needed to reveal the possible mechanism of DEHP in the progression of IPT to CPP/EP.DEHP may be associated with the progression of IPT to CPP through various central HPG axis and peripheral pathways. By gavage of DEHP in rats, DEHP was found to promote the release of gonadotropin-releasing hormone (GnRH) by interfering with the IGF-1/PI3K/Akt/mTOR signaling pathway in the hypothalamus, thereby affecting the function of the HPG axis and finally resulting in precocious puberty4DEHP exhibited considerably longer elimination half-lives (10–24 h vs. 4 h) and later maxima of urinary excretion than the simple monoester MEHP44. Thus, the Σ4DEHP levels in urine were several times higher than those of MEHP after DEHP intake for 24 h. These represent the major share of DEHP metabolites excreted in urine (70% for Σ4DEHP vs. 6% for MEHP)44. They are immune to external contamination and possibly the ultimate developmental toxicants45. Therefore, Σ4DEHP can reflect the exposure level of DEHP in the human body.DEHP is extensively metabolized after all routes of uptake. In the first and fastest step, DEHP is metabolized into primary MEHP and further metabolized by different oxidation reactions. Σ48 have shown that single urinary PAEs may reflect exposure levels for months or even years. Teitelbaum et al.46 collected 2–6 urine samples from each 6–10-year-old child within six months for environmental endocrine disruptor analysis, indicating that a single urine sample could moderately predict the average concentration of six months. Hauser et al.47 showed a moderate predictive effect on exposure to PAEs over three months in adult males. Similarly, a study48 revealed that DEHP levels in women's urine PAEs remained steady over 1–3 years (nurses). Also, the amount of PAEs in the environment that kids spend a lot of time in is rather steady. Examples include the furnishings and dust in homes and schools, water cups, skin care products, and other items that could be exposure sources11. If multiple urine samples from a single participant can be measured, the exposure level of PAEs may be more accurate49. Thus, our study has some limitations. First, the number of participants was small. Hence, expanding the sample size may yield more informative results. Second, collecting multiple urine samples from the same child during follow-up may have further increased the accuracy of PAE exposure.Some studies4DEHP in urine are independent risk factors of IPT progression. In addition, Σ4DEHP may be associated with the progression of IPT to CPP or EP, which suggests that children with IPT should minimize exposure to DEHP.This study reveals that the breast Tanner stage, ovarian volume, and Σ


Oreochromis niloticus) culture, Streptococcus agalactiae, causes economic harm. Infected fish’s immune systems worked to eliminate of the infection. This study demonstrated the effect of different bacterial concentrations on tilapia immunity and optimal vaccine concentration to induce immunity in Nile tilapia. The experiment was performed at 102, 104, 106, 108, and 1010 CFU/fish of S. agalactiae compared with the control (PBS) through intraperitoneal injection for 72 h. Fish that survived employed to gather blood, and immune responses were assessed through measures of the survival rate include blood smears, antibody titers, and immunoglobulin gene expression. The vaccine experiment investigated formalin-inactivated S. agalactiae vaccination and administered S. agalactiae injections for 14 days. The statistic revealed a significant difference (p < 0.05) in the 108 and 1010 CFU/fish injections with high survival rates . Immunoglobulin gene expression was highly represented in the 1010 CFU/fish injection; antibody titers were significantly improved from the control group, and antibody levels were high in the 1010 CFU/fish injection. The analysis of blood cell types using the blood smear method revealed a progressive increase in leucocytes, particularly lymphocytes, neutrophils, and monocytes, in the treatment group compared to the control group. Moreover, the erythrocyte/leucocyte ratio decreased significantly in response to the high bacterial injection, indicating an increase in leucocytes. Conversely, the erythrocyte level stayed ed within at the 7.03–9.70 × 102 cell/ml and shown no significant difference (p > 0.05). The lymphocytes were almost two-fold in 1010 CFU/fish compared to 108 CFU/fish. As depicted in the lowest concentration of 106 CFU/fish, the vaccine performance had a high relative percent survival (RPS) at 86.67%. This research suggested that the tilapia infected with high S. agalactiae concentrations did not affect the mortality of the tilapia, and vaccine concentration was effective in 106 CFU/fish.The main pathogen in the Nile tilapia ( In Thailand, Chitralada 3 tilapia is a strain of tilapia developed from GIFT (Genetic Improvement of Farmed Tilapia), a Philippine ICLARM unit’s fifth generation . Since breeding via the group selection approach was said to have been successful most recently in 2007, “Chitralada 3” has been consistently bred. Noteworthy for its impressive yield, excellent survival rate, and robust growth, “Chitralada 3” is characterized by its small, thick, dense, and highly meaty heads .Streptococcus agalactiae, stands out as one of the most detrimental bacterial infections affecting Chitralada 3 strain Nile tilapia. This infection has the potential to result in a mortality rate as high as 90%  × 100.Tilapias were divided into seven groups for testing with p < 0.05.The software SPSS Statistics for Windows (Version 20.0) was utilized to analyze cumulative mortality, survival rate, antibody titers, blood cell and immunoglobulin gene expression data through one-way analysis of variance (ANOVA) and Duncan’s multiple range test. The results were presented in triplicate, depicting mean and standard deviation values. Significant differences among treatments were determined at S. agalactiae, the death of tilapia occurred only around 3–12 days  difference compared to the control group. Figure 10 CFU/fish of the S. agalactiae injection, but no significant (p > 0.05) differences were found in 104, 106, and 108 CFU/fish. The 102 CFU/fish injection revealed a higher antibody level than the control group (PBS).We observed that the various antibody levels in the tilapia having a significant (p < 0.05) difference between the control and injection treatments. The high bacterial inoculum concentration impacted the increase in lymphocyte, neutrophil, and monocyte counts  of each type Fig. . Increasnts Fig. . The con4-108 CFU/fish did not reveal a significant difference (p > 0.05) among the groups. The higher significance levels (p < 0.05) were distinguished in the 1010 CFU/fish injection. The expression of 102 CFU/fish also did not significantly (p > 0.05) differ from the control groups.Figure S. agalactiae vaccine was demonstrated in 106 CFU/fish; the survival rate was 86.67%. The vaccination with the 102 and 104 CFU/fish illustrated that no fish survived after the S. agalactiae injection. The RPSs were highly demonstrated in 106 CFU/fish vaccination, but statistically significant (p > 0.05) differences did not occur with 108 and 1010 CFU/fish. The corresponding RPSs were 86.67, 83.33, and 76.67%, respectively.As depicted in Table p < 0.05) among the treatments, determined through one-way ANOVA, followed by Duncan’s multiple range test.*The data is presented as mean ± standard deviation (n = 3 fish). Distinct superscript letters in the same row signify statistically significant difference  existed among 104–1010 CFU/fish. The lymphocyte and immunoglobulin produced antibodies with diverse functions in serum and mucus, and they participated in the B cell surface complex and antigen signaling function [Antibody titers demonstrated that varying bacterial concentrations increased the augmentation of antibody levels; however, no statistically significant differences , and every thrombocyte was sharp on blood smear slides . A smallS. agalactiae resistance 14 days following the induction of immunity. The intraperitoneally vaccinated injection with the whole-cell inactivated S. agalactiae vaccine resulted in high survival at 106 CFU/fish concentration and no significant difference (p > 0.05) at 108 and 1010 CFU/fish. In contrast, no fish survived in 102 and 104 CFU/fish after the S. agalactiae injection for 14 days of observation. Through intraperitoneal injection, formalin-killed vaccines exhibited excellent protection and a high relative percent survival (75–100%). Although no finding regarding the immune system existed, the fish experiment can be seen as a sign of success [Moreover, vaccination demonstrated the effectiveness of  success .S. agalactiae injections on immunological induction and survival, including an increase in the immunoglobulin gene expression and antibodies and an increase in leucocytes. The research indicated that injections of highly concentrated S. agalactiae reduce virulence and reverse immunological activation. Therefore, different vaccination concentrations had varying effects. We advocate vaccinating juvenile tilapia with 106 CFU/fish of S. agalactiae vaccination to reduce S. agalactiae outbreaks in the tilapia farm industry and for convenience. Furthermore, we propose vaccination as an alternative method to safeguard tilapia population against S. agalactiae infection.Our experiment has demonstrated the effect of highly concentrated


To investigate correlates in hippocampal subfield volume and verbal and visual memory function in patients with temporal lobe epilepsy (TLE), mild amnestic cognitive impairment (MCI) and heathy participants (HP).50 right-handed participants were included in this study; 11 patients with temporal lobe epilepsy (TLE), 18 patients with mild amnestic cognitive impairment (MCI) and 21 healthy participants (HP). Verbal memory performance was evaluated via the verbal memory test (VLMT) and visual memory performance via the diagnosticum for cerebral damage (DCM). Hippocampal subfield volumes of T1-weighted Magnetic Resonance Imaging (MRI) scans were computed with FreeSurfer version 7.1. Stepwise correlation analyses were performed between the left hippocampal subfield volumes and learning, free recall, consolidation and recognition performance scores of the VLMT as well as between right hippocampal subfield volumes and visual memory performance.β = 0.284; p = 0.042) and free recall performance in the VLMT . The volume of the left CA3 subfield showed a significant correlation to the consolidation performance in the VLMT  and recognition performance in the VLMT . There was no significant correlation identified between the right hippocampal subfields and the visual memory performance.The volume of the left subicular complex was highly correlated to learning performance (The results of this study show verbal memory correlates with hippocampal subfields and support the role of left subiculum and left CA2/CA3 in verbal memory performance. The domin vivo correlations of memory performance with hippocampal atrophy patterns , 18 patients with mild cognitive impairment (MCI) and 21 healthy participants (HP) were included in the study. All participants were recruited in the Department of Neurology, Christian Doppler University Hospital Salzburg, Austria and received a multimodal neuropsychological evaluation and a three-dimensional (3D), T1-weighted MRI scan. The TLE diagnosis was based on neurological assessment by experienced epileptologists, including video-EEG examination for up to five days. There was no evidence of hippocampal sclerosis in the included TLE population. The MCI diagnosis was based on Petersen’s criteria . All MCI2.2.All participants underwent a multimodal neuropsychological evaluation including verbal and non-verbal memory performance assessment using the verbal memory test (VLMT)  and the 2.3.All three-dimensional (3D), T1-weighted MRI scans of the participants were performed with a 3T Siemens  Magnetom TrioTim syngo MR B17 scanner, a 12-channel head coil and the following parameters: sagittal orientation, 192 slices per slab, 256 mm FoV read at 93.8%phase, voxel dimension 1 × 1 × 1 mm, repetition time (TR) 2,300 ms, echo time (TE) 2.91 ms, inversion time (TI) 900 ms, flip angle (FA) 9°.2.4.https://surfer.nmr.mgh.harvard.edu/fswiki/HippocampalSubfields. The detailed output volumes are presented at https://surfer.nmr.mgh.harvard.edu/fswiki/HippocampalSubfieldsAndNucleiOfAmygdala. The pipeline shows a good test–retest reliability and reliability across vendor platforms and field-strengths and has been applied in several neurocognitive studies, which examine hippocampal pathologies including patients with TLE and MCI  and MCI . All raw2.5.All subfield volumes were corrected for total brain volume before the analysis. For the correction, we used a covariance approach , 22, 23 2.5.1.In the next step, the relationship between corrected subfield volumes and neuropsychological variables was examined. The analyses were performed with the IBM SPSS Statistics program version 26.0. To determine whether the volumes of certain hippocampal subfields were related to neuropsychological outcomes, regression analyses were computed separately for the left and right hippocampal subfield volumes. To avoid problems of multicollinearity, stepwise regression methods were used in entering the predictors. For verbal memory, T-values of learning performance, free recall, consolidation performance and recognition of the VLMT were used as outcome variables and all subfields of the left hippocampi were entered into the regression analyses as predictors. For figural memory, the percentile ranks of the DCS served as outcome measure and all subfield volumes of the right hippocampi were entered into the regression analysis as predictors. The following subfields were entered into regression analyses: subiculum, presubiculum, and parasubiculum added to subicular complex (SUB) , 26, cor2.6.Anonymized data can be made available to any qualified investigator upon request.3.3.1.n = 29) was included in this study. The age of the participants was significantly different among groups , primarily due to the younger age of TLE patients. The median disease duration of epileptic seizures was 16 years (range 3–51 years) in the TLE group, and the median duration of amnestic symptoms was 12 months (range 6–24 months) in the MCI group. The patients’ characteristics are presented in A total of 50 participants , F = 4.37; p = 0.042]. The volume of the left subicular complex was a significant predictor of learning performance in the VLMT  while no other subfield volumes were significant predictors (ps > 0.369).A stepwise linear regression was computed using T-values of the learning performance as outcome variables and subfields of the left hippocampi as predictor variables. The regression model was statistically significant . The volume of the left subicular complex was a significant predictor of free recall performance in the VLMT  while no other subfield volumes were significant predictors (ps > 0.112).The regression model using T-values of the free recall performance as outcome variable and subfields of the left hippocampi as predictor variables was statistically significant . The volume of the left CA2/CA3 subfield was a significant predictor of consolidation performance in the VLMT  while no other subfield volumes were significant predictors (ps > 0.177).A stepwise linear regression using T-values of the consolidation performance as outcome variable and subfields of the left hippocampi as predictor variables was statistically significant . The volume of the left CA2/CA3 subfield was a significant predictor of recognition performance in the VLMT  while no other subfield volumes were significant predictors (ps > 0.284)  , 2.3.2.2.A stepwise linear regression was computed using percentage rank of the non-verbal memory test DCS as outcome variable and subfields of the right hippocampi as predictors. The regression model was not significant.4.in vivo identification of neuroanatomical correlates of specific memory functions. In this study, participants with reduced left subicular complex volumes showed significantly reduced learning and 30-min free recall performance. Left subiculum volumes have already been associated with verbal memory functions (The development of more accurate measurement methods of hippocampal subfield volumes enables the unctions , 27–29. unctions . Similarunctions , 28, 29.The subiculum is considered the main output formation of the hippocampus with projections to several cortical and subcortical regions such as prefrontal and entorhinal cortex, amygdala, nucleus accumbens and hypothalamus, receiving input mainly from CA1 and the entorhinal cortex . Due to Reduced CA2/CA3 volumes were significantly correlated with lower consolidation and recognition performance, while there were no associations identified between DG and CA1 volumes and the four scales of VLMT. The results of previous studies concerning the above mentioned subfields vary; Mueller et al.  have preConcerning the figural memory, there were no significant correlates with the right hippocampal subfields and the non-verbal memory test DCS. Previous studies have demonstrated correlations of figural memory with different hippocampal subfield volumes and visual memory recall such as presubiculum , CA4 4141, subicThis study has some limitations. First, although high resolution MRI and advanced segmentation methods have contributed to greater accuracy in measuring the hippocampal subfield volumes, these might differ from the real subject’s subfield volumes. Moreover, the results should be interpreted with caution due to the small sample size, different age range and disease duration of the included patients. Finally, we aimed to study a mixed population in order to identify non-disease specific but rather neuropsychological specific correlates; however, the different disease pathomechanisms might influence the hippocampal subfield volumes differently, possibly affecting the correlations identified.5.The findings of this study support previous evidence that hippocampal subfields play an important role in specific memory functions. Identifying neuroanatomical correlates of verbal memory can be useful for applying more targeted therapies. In epilepsy patients, for instance, techniques like stereo-encephalography and highly selective laser surgery techniques might contribute to preserving unaffected mesiotemporal substructures crucial for verbal performance resulting into both seizure freedom and good postsurgical neuropsychological outcome in some cases. Moreover, the estimation of specific hippocampal atrophy patterns might enable the clinical monitoring of verbal memory performance in patients with MCI or Alzheimer’s disease as well as the prediction of postoperative verbal memory outcome in patients with temporal lobe epilepsy.The anonymized data supporting the conclusions of this article will be made available by the authors, upon reasonable request.The studies involving human participants were reviewed and approved by Ethics Commission Salzburg/Ethikkommission Land Salzburg; approval number 415-E/1429. The patients/participants provided their written informed consent to participate in this study.P-ET: study concept and design, data analysis, interpretation, statistical analysis, manuscript writing, and revision. C-JM: data acquisition, interpretation, statistical analysis, and manuscript revision. MB, FZ, and KM: data analysis and manuscript revision. ET: data acquisition, analysis, interpretation, and manuscript revision. SK and AT: study concept and design, data acquisition, analysis, interpretation, and manuscript revision. All authors contributed to the article and approved the submitted version.