Association between poor infant and young child feeding (IYCF) practices and malnutrition in infants and young children (IYC) is well established. Furthermore, appropriate IYCF practices are important during the first 1,000 days of life to ensure optimal health and development. Understanding IYCF practices and associated socioeconomic and demographic factors will inform interventions to achieve the UN 2030 Sustainable Development Goal (SDG) target to end malnutrition in all forms.This study estimates the prevalence of Minimum Dietary Diversity (MDD), Minimum Meal Frequency (MMF), and Minimum Acceptable Diet (MAD), and examines their association with socioeconomic and demographic characteristics among children aged 6–23 months in Ghana.We used data from the Ghana Multiple Indicator Cluster Survey 6 (GMICS6) conducted in 2017–18. Participants were recruited through multi-stage stratified cluster sampling. Information on caregiver’s self-reported breastfeeding status and 24-hour dietary recall of foods IYC were fed with were collected through face-to-face interviews. We estimated the prevalence of MDD, MMF and MAD with a 95% confidence interval (CI). We investigated the socioeconomic and demographic determinants of MDD, MMF and MAD using univariate and multivariable logistic regression analyses.Among 2,585 IYC aged 6–23 months, MDD, MMF and MAD were estimated as 25.46%, 32.82% and 11.72% respectively. Age of the IYC, educational status of the mothers/primary caregivers, and resident regions were found to have positive associations with MDD, MMF and MAD. In addition, the richest household wealth index and urban area of residence were found to have significant positive associations with MDD.We report a low prevalence of MDD, MMF and MAD. Efforts to improve IYCF practices among children aged 6–23 months in Ghana should focus on multi-sectorial approaches including increasing access to formal education, income-generating activities and addressing regional and rural-urban inequity. Appropriate feeding practices for infants and young children (IYC) reduce the risk of diarrhoea  and acutTo assess infant and young child feeding (IYCF) practices, WHO and UNICEF (2021) developed 16 core indicators. These indicators include ever breastfed, early initiation of breastfeeding, exclusively breastfed for the first two days after birth, exclusive breastfeeding for the first six months, feeding formula and/or animal milk in addition to breast milk under six months, continued breastfeeding till 12–23 months, the introduction of solid, semi-solid or soft foods (ISSSF) at six months, minimum dietary diversity (MDD) during 6–23 months, minimum meal frequency (MMF) during 6–23 months, minimum milk feeding frequency (MMFF) for non-breastfed children aged 6–23 months, minimum acceptable diet (MAD) during 6–23 months, egg and/or flesh food consumption during 6–23 months, sweet beverage consumption during 6–23 months, unhealthy food consumption during 6–23 months, zero vegetable or fruit consumption during 6–23 months and bottle feeding during 0–23 months. MDD, MMF and MAD are among the important indicators recommended by WHO for assessing complementary feeding practices among IYC aged 6–23 months .Despite the WHO/UNICEF recommendation and the critical benefits related to IYCF, most studies have recorded suboptimal rates of appropriate IYCF practices. Analysis of the UNICEF global database reports global rates of 52.2% and 29.4% for MMF and MDD respectively, among children aged 6–23 months . SynthesIn sub-Saharan Africa, data from demographic and health surveys between 2010 and 2016 revealed that 41.9% of children aged 6–23 months met MMF, 21% met MDD and 9.8% met MAD . A similIn Ghana, the prevalence of appropriate IYCF practices for IYC aged 6–23 months is far below the WHO recommendation. As of 2008 in Ghana, the prevalence of MAD for children aged 6–23 months was 29.9% , but thiGovernments and stakeholders need nationally representative accurate and timely evidence to understand the current situation of IYCF practices for IYC aged 6–23 months and its associated factors. Knowing the factors influencing IYCF practices will help shape the development and implementation of evidence-based and effective interventions. In this context, our study aimed to provide information about the prevalence of MDD, MMF and MAD among IYC aged 6–23 months and their association with socioeconomic and demographic characteristics of the IYC in Ghana using nationally representative data from the most recent Ghana Multiple Indicator Cluster Survey 6 (GMICS6), conducted in 2017–18. The findings of this study are expected to assist key policymakers and programme implementers to identify areas requiring further improvements and design need-based and evidence-driven interventions. This will help improve appropriate IYCF practices for IYC aged 6–23 months in Ghana and in the long run contribute towards the target of ending malnutrition in all forms under the UN 2030 second Sustainable Development Goal (SDG) of zero hunger.A secondary data analysis of the Ghana Multiple Indicator Cluster Survey 6 (GMICS6) was done to estimate the prevalence of MDD, MMF and MAD and investigate its associated socioeconomic and demographic factors. The GMICS6 was carried out from October 2017 to January 2018 by the Ghana Statistical Service (GSS) in partnership with the Ministry of Health, Ministry of Education, Ministry of Sanitation and Water Resources, Ministry of Gender, Children and Social Protection, Ghana Health Service and the Ghana Education Service  with tecThe global MICS programme is an international multi-purpose household survey initiated by UNICEF in the mid-1990s with the goal of assisting countries to collect internationally comparable data on varied characteristics of women and children such as breastfeeding status and dietary practices. This data is used for evidence-based national development planning and initiatives and for monitoring progress made towards national goals and global commitments such as the SDGs .Ghana is a middle-income West African country which currently has 16 administrative regions. However, at the time GMICS6 was conducted, there were 10 administrative regions namely: Western, Central, Greater Accra, Volta, Eastern, Ashanti, Brong Ahafo, Northern, Upper East and Upper West .This study utilised data from the mother/caregiver-child pair about breastfeeding and dietary practices of 2,585 IYC aged 6–23 months.The GMICS6 employed a multi-stage stratified cluster sampling approach using a sampling frame based on the 2010 Population and Housing Census (PHC) . GMICS6 The overall sample size of the GMICS6 was 13,202 households with a response rate of 99.4% . All chiThe sample flow details are outlined in Mother’s or caregiver’s self-reported breastfeeding status and all foods that children under five years of age were fed with and frequency of feeding within the last 24 hours before the survey alongside socioeconomic and demographic characteristics were collected using the under-5 questionnaire of the GMICS6.Data for the GMICS6 was collected via face-to-face interviews using Computer-Assisted Personal Interviewing (CAPI) technique.A training of 30 days for field investigators was conducted from 10th September to 10th October 2017. The content of this training included interviewing techniques, contents of the questionnaires, CAPI application, skills in asking questions and mock interviews, 5 days of field practice and a one-day full pilot survey in localities around Winneba in the Central Region. Mandatory re-interviewing was implemented in one household per cluster using both random and purposive sampling techniques. Daily observations of the interviewer’s skills and performance were done .We utilised secondary data from GMICS6 whose ethical considerations have been published elsewhere .Minimum Dietary Diversity (MDD): IYC aged 6–23 months who were fed foods and beverages from at least five of eight food groups during the previous day. The eight food groups are: breastmilk; dairy products ; grains, roots, tubers and plantain; legumes or pulses , nuts and seeds; flesh foods ; eggs; vitamin-A rich fruits and vegetables and other fruits and vegetables [Minimum Meal Frequency (MMF): IYC aged 6–23 months who were fed foods and beverages 2 times and 3 times for 6–8 months and 9–23 months, respectively, and for non-breastfed IYC aged 6–23 months, 4 times including milk feeds and at least one solid, semi-solid or soft food during the previous day [Minimum Milk Feeding Frequency (MMFF) for non-breastfed children: Non-breastfed IYC aged 6–23 months who were fed with at least two milk feeds  during the previous day [Minimum Acceptable Diet (MAD): Breastfed IYC aged 6–23 months who received MDD and MMF per age during the previous day and non-breastfed children aged 6–23 months who received MDD, MMF per age and MMFF during the previous day [The following socioeconomic and demographic characteristics of IYC aged 6–23 months were included in the analysis: sex of the child; place of residence; administrative region ; age in months ; mother’s or caretaker’s educational attainment ; household ethnicity  and household wealth index which captures the underlying long-term wealth of the household ranked according to the wealth score of the household based on urban and rural factor scores regressed on assets owned by that household and finally divided into 5 equal parts or quintiles .The GMICS6 dataset was accessed at mics.unicef.org. Each IYCF indicator or outcome variable  was dichotomised into a binary variable as “yes = 1” for a child who meets the indicator and “no = 0” for a child who does not meet the indicator. Frequencies, percentages/prevalences and means (± standard deviation) were used to describe the socioeconomic and demographic characteristics and outcome variables. Firstly, the Chi-square test was done to investigate the association between socioeconomic and demographic variables as well as the outcome variables. Socioeconomic and demographic variables that showed statistically significant (p ≤ 0.05) association with the outcome variables were also investigated using univariate (unadjusted) logistic regression. Statistically significant (p ≤ 0.05) socioeconomic and demographic variables in the univariate models were then included in the multivariable logistic regression model to get the adjusted association of selected socioeconomic and demographic factors after controlling for other potential confounders. The regression outputs were reported in the form of Odds Ratios (ORs) with 95% confidence intervals (CIs). The level of significance was determined at p-value ≤ 0.05. The data were analysed using Stata/SE version 17.0 . Stata syntax developed for calculating IYCF indicators  was custOver a quarter  of IYC in this study met MDD . Almost The percentage of IYC who achieved MMF in the 24 hours preceding the survey was found to be 32.8%  . ApproxiThe prevalence of MAD 24 hours prior to the survey was 11.7% (95% CI: 9.7–13.7) . Among mCompared to the Western region, IYC from the Volta , Brong Ahafo , Northern  and Upper West  regions respectively had 49%, 44%, 8% and 51% lower odds of achieving MDD. IYC from rural areas had 44% reduced odds of achieving MDD compared to IYC from urban areas.Our multivariable logistic regression analysis suggests that IYC with mothers or caregivers having secondary education  and above secondary education  were 1.92 and 3.23 times more likely to achieve MMF respectively compared to the IYC with mothers or caregivers with no formal education. The odds of achieving MMF among IYC from the Central region  was 1.74 times those from the Western region .On the other hand, IYC in the age groups of 9–11 months , 12–17 months  and 18–23 months  had 58%, 59% and 80% reduced odds of achieving MMF respectively compared to IYC aged 6–8 months. IYC from the Greater Accra region  had a 42% reduced odds of achieving MMF compared to IYC from the Western region.Our analyses suggest that IYC aged 9–11 months  and 12–17 months  were 1.86 and 2.13 times more likely to achieve MAD respectively compared to IYC in the age group of 6–8 months . Having On the other hand, IYC from the Greater Accra , Brong Ahafo  and Upper West  regions had 63%, 56% and 59% reduced odds of achieving MAD respectively compared to IYC from the Western region.We investigated the prevalence and socioeconomic and demographic determinants of MDD, MMF and MAD based on the GMICS 6 (2017/18). The prevalence of MDD, MMF and MAD was found to be 25.4%, 32.8% and 11.7% respectively. Socioeconomic and demographic characteristics having a positively significant association with MDD, MMF and MAD were the age of the IYC, educational status of the mothers/primary caregivers, and resident regions. In addition, the household wealth index was also found to have a positive significant association with MDD.The percentage of IYC who achieved MDD in this study was slightly lower than 29.4% which was reported through an analysis of UNICEF global database  but compAlso, this study used the WHO and UNICEF revised definition of MDD (≥ five out of eight food groups) considering breastmilk as a distinct food group. Other previous studies however used the old definition (≥ four out of seven food groups). The use of the revised definition has been shown to result in a decrease in the prevalence of MDD . This shHowever, considering the results of this study, about a quarter of Ghanaian IYC aged 6–23 months were fed foods from at least five food groups during the previous day. This means about 75% of the IYC in this study are at risk of not meeting nutrient requirements which are essential to their physical and mental development. Even though quantities of the foods IYC were fed with were not included in this study and dietary intake was assessed on a one-day basis which may not reflect usual intake, nonetheless MDD has been shown to be a good predictor of nutrient adequacy and nutritional status .This study also found a low intake of egg and flesh food among the IYC during the previous day (20% and 45% respectively). Many countries have also recorded similar low consumption of egg or flesh food . This isThe percentage of IYC who achieved MMF in the 24 hours preceding the survey in this study is lower than 41.9% found in sub-Saharan Africa using DHS data  but simiGhanaian IYC aged 6–23 months in this study who achieved MAD compares favorably with the average of 9.8% found in sub-Saharan Africa using DHS data  and 9.2%The association of socioeconomic and demographic characteristics with MDD, MMF and MAD found in this study may help inform specific interventions to improve IYCF practices among children aged 6–23 months in Ghana. IYC in the age groups of 9–11, 12–17 and 18–23 months were 2.39, 4.48 and 3.54 times more likely to achieve MDD than those aged 6–8 months. Similarly, IYC aged 9–11 and 12–17 months were 1.86 and 2.13 times more likely to achieve MAD than those aged 6–8 months respectively. This is similar to that found in Dabat District, Ethiopia , India , Ghana  and NortIYC of mothers/caregivers with secondary and/or higher education were more likely to achieve MDD, MMF and MAD than IYC of mothers/caregivers with no formal education. These results corroborate earlier studies such as that found in Ethiopia , 43–46, IYC from the richest households were 2.28 times more likely to achieve MDD than their counterparts from the poorest households. This finding is in agreement with that found in Pakistan , EthiopiIYC from the Central region were more likely to achieve MMF and MAD than those from the Western region. Also, IYC from Volta, Brong Ahafo, Northern and Upper West regions were less likely to achieve MDD compared to IYC from the Western region, IYC from Greater Accra region were less likely to achieve MMF compared to those from Western region and those from Greater Accra, Brong Ahafo and Upper West regions were less likely to achieve MAD compared to those from the Western region. Region of residence as a significant factor to meeting IYCF practices has also been found in previous studies in Ghana , 47 and Surprisingly, in this study, only MDD was significantly associated with the area of residence. The urban area of residence has however been shown to be associated with IYCF practices in previous studies in Ethiopia , 26, 39,This study utilized the most recent nationally representative GMICS6 data with a large sample size  and high response rate (99.4%). This study used the WHO and UNICEF definition of MMD which has been recommended for measuring intermediate outcome indicators for tracking progress towards the 2025 global nutrition targets . The datThe prevalence of MDD, MMF and MAD among Ghanaian IYC aged 6–23 months in this study was low . Socioeconomic and demographic characteristics of IYC having positively significant associations with MDD were the age of IYC, educational status of mothers/caretakers, household wealth index, administrative region and area of residence. Additionally, the age of IYC, educational status of mothers/caretakers and administrative region were socioeconomic and demographic characteristics of IYC that showed significant positive associations with MMF and MAD. Surprisingly, unlike other previous studies, only MDD had a significant association with the area of residence in this study.Based on the evidence generated in this study, we recommend that interventions towards MDD, MMF and MAD in Ghana should focus on improving access to formal education, income-generating activities and strengthening behaviour change communication using available channels in the communities to improve awareness and knowledge of IYCF.


This study aimed to elucidate the algorithm of various influential factors relating to the association between 25-hydroxyvitamin D (25(OH)D) concentration at delivery and mode of delivery. The investigation constituted a secondary analysis using data collected as part of the Khuzestan Vitamin D Deficiency Screening Program in Pregnancy, which is a stratified randomized vitamin D supplementation-controlled trial comprising 1649 eligible pregnant women. The Bayesian Network (BN) method was utilized to determine the association algorithm between diverse influential factors associated with maternal vitamin D and mode of delivery. The optimized intelligent BN algorithm revealed that women presenting with moderate  and severe vitamin D deficiency  at delivery were more likely to undergo cesarean section than those presenting with normal concentrations of this nutritional hormone . The occurrence probabilities of preeclampsia in mothers with normal, moderate, and severe vitamin D deficiency at delivery were , , and , respectively. Additionally, mothers with moderate  and severe  vitamin D deficiency exhibited a higher probability of preterm delivery in comparison to those presenting with normal concentrations . This study demonstrated that the vitamin D status of pregnant women at delivery could directly affect the mode of delivery and indirectly through maternal complications, such as preeclampsia and preterm delivery, leading to a higher occurrence probability of cesarean section. However, epidemiologic studies have demonstrated that pregnant women are at a higher risk of vitamin D deficiency6. A systematic review estimates that the prevalence of vitamin D deficiency, defined as serum 25(OH)D < 20 ng/ mL (50 nmol/L), ranges from 64 to 83% depending on geographic region7.Vitamin D is recognized as a crucial nutrient, that plays a pivotal role in various physiological processes such as cell function, bone mineralization, calcium homeostasis, and muscle performance11, and an increased risk of cesarean Sect. 17. The mechanisms behind the association between 25(OH)D and increased cesarean section risk are complex and not fully elucidated. Vitamin D is important for smooth muscle performance and myometrium contractility which plays a critical role in the initiation of labor18. The deficiency of vitamin D can also lead to endothelial dysfunction, impaired vascular health19, and glucose homeostasis abnormalities21, which can predispose to developing GDM22. The levels of vitamin D can also affect preterm delivery through inflammation and immune system modulation. Additionally, vitamin D deficiency can increase the risk of infection, which is known to be a major cause of preterm delivery10. Maternal complications such as preeclampsia, GDM, and preterm delivery can indirectly lead to cesarean section due to fetal distress25. Studies suggest that 25(OH)D levels during delivery may influence the rate of cesarean and operative deliveries by increasing birth weight26, independently of gestational age27. Birth weight is linked to GDM, which can increase the risk of cesarean section due to cephalopelvic disproportion (CPD) and prolonged labor (dystocia)28.In addition to the role of vitamin D in maintaining calcium homeostasis and bone metabolism, a deficiency in vitamin D during pregnancy can result in various complications such as preeclampsia, gestational diabetes mellitus (GDM), and preterm delivery32. Some studies have suggested that mothers with vitamin D deficiency are at an increased risk of cesarean Sect.17, while others have found no link between serum levels of 25(OH) D and type of delivery32. This discrepancy may be due to various confounding factors that were not accounted for in most of these studies.Several studies have examined the relationship between 25(OH)D concentrations and mode of delivery, but their findings have been inconsistent and inconclusiveTo better understand and simplify the complex association between 25(OH)D concentration at delivery and mode of delivery, we utilized data from a stratified randomized controlled field trial of vitamin D supplementation. We employed a Bayesian Network (BN), which is a popular data mining algorithm to predict the likelihood of cesarean section in mothers with vitamin D deficiency. This innovative approach allowed us to elucidate the relationships among the variables in our analysis.34.This study presents a secondary analysis of data collected from the Khuzestan Vitamin D Deficiency Screening Program in Pregnancy, which was a stratified randomized controlled field trial. The study protocol and procedures have been previously publishedThis study was conducted in two phases. The first phase involved a population-based cross-sectional study that recruited 1,600 and 900 first-trimester pregnant women from health centers in Masjed-Soleyman and Shushtar, respectively. The sampling method used was stratified multistage cluster sampling with probability proportional to size (PPS). Inclusion criteria include age range 18–40 years, gestational age < 14 weeks, singleton pregnancy, not consuming multivitamins containing 400 IU/d of D3, and no previous history of chronic diseases. Participants consuming multivitamins containing more than 400 international units (IU) per day of vitamin D3 and anticonvulsants and those with a history of chronic diseases like diabetes, hypertension, renal dysfunction, liver diseases, and complicated medical or obstetrical history were excluded from the study. The study data was collected from 1800 first-trimester pregnant women referred to health centers in Masjed-Soleyman (intervention) and Shushtar (control), from whom fasting blood samples were collected. Serum samples of participants from Shushtar were stored and kept frozen at -80° C until further assays at the end of the study, according to the original study protocol. The vitamin D status of participants from Masjed-Soleyman was measured immediately, and those found to have vitamin D deficiency were randomly assigned to a treatment regimen consisting of vitamin D3 supplementation until delivery. During the second phase of the study, based on 25(OH)D levels, mothers were divided into severe deficient (< 10 ng/ml), moderate deficient (10-20 ng/ml), and normal status (> 20 ng/ml) (6). A total of 800 pregnant women with vitamin D deficiency (defined as serum concentrations of 25(OH)D < 20 ng/ml) from Masjed-Soleyman were included in the intervention group and received vitamin D supplementation according to the assigned protocol. Participants were randomized into groups of four using a computer-generated list. The physicians involved in the study were not informed about which group each woman was placed in, but the midwife who was not involved in any phase of the study knew. It was not possible to hide the treatment allocation from healthcare workers, but those who assessed pregnancy outcomes were unaware of the treatment allocation. The remaining women with vitamin D deficiency were referred to specialists for further treatment. Participants from Shushtar served as the control group and did not receive any vitamin D supplementation. Women with severe and moderate vitamin D deficiency from Masjed-Soleyman were randomly allocated to one of four treatment modalities: A1 , A2 , A3 , and A4 . Women with moderate vitamin D deficiency were assigned to one of four study groups: B1 , B2 , B3 , and B4 . Serum levels of 25 (OH)D of all participants (from both cities) were measured at delivery. All participants (intervention and control groups) received routine prenatal care and followed till delivery.For this analysis, elective and repeat cesarean section cases, those without reliable data about the type of cesarean section n = 143), IUFD (n = 2), and incomplete data on predictors (n = 6) were excluded. As a result, a total of 1488 pregnant women were included in the analysis  by height (m2). Systolic and diastolic blood pressures (SBP and DBP) were measured twice in a sitting position with a standard mercury sphygmomanometer after a 15-min rest, and the mean of the two measurements was considered as SBP or DBP. Serum concentrations of 25(OH)D were assayed for all participants (both Masjed-Soleyman and Shushtar) at baseline and at delivery using the ELISA method and a kit of Immunodiagnostics Systems by AutoAnalyzer . The interassay and intraassay coefficients of variation were 3.891% and 3.37%, respectively (sensitivity of 5 nmol/L). The instruments were calibrated as per the manufacturer's instructions, and validation studies were performed before the test. Samples were analyzed by a single technician using the same equipment throughout the study in a reference laboratory and were measured according to standard operating procedures.Clinical and anthropometric parameters were measured for all participants D between 10 and 20 ng/mL diagnosed as moderate vitamin D deficiency and those with concentrations < 10 ng/mL considered as severe vitamin D deficiency.Preeclampsia was defined as having systolic blood pressure > 140 mmHg or diastolic blood pressure 90 mmHg and 24-h proteinuria 0.3 g, initiated at > 20 weeks of gestationThe study outcome was the mode of delivery which was classified as either vaginal or emergency cesarean section. According to the ICD-10 classification system, an emergency cesarean section included all cases where such delivery was undertaken after the onset of labor. Dystocia refers to prolonged or slowly progressing labor and is defined as abnormal or difficult labor and may be associated with factors of the passenger (fetus), the passage , the forces (powers), or a combination of these factors. CPD refers to the inability of the fetus to pass through the pelvis.The available data appears to be appropriate for achieving research goals; however, some methodological considerations are necessary to address the complexity of the design. To simplify the relationships between variables, we used the BN method, one of the most well-known algorithms in data mining.The statistical method employed to evaluate the generalizability of our findings was a tenfold cross-validation technique. The dataset was randomly divided into ten equal parts, with nine parts being utilized for model training in each iteration. The remaining part was used for evaluating the predicted values. To obtain a comprehensive performance evaluation, we calculated the sensitivity, specificity, and accuracy metrics by averaging these values across all ten iterations.38.We employed the Hill-Climbing (HC) algorithm to achieve the best Directed Acyclic Graph (DAG) corresponding to our data. HC is a score-based structure learning algorithm that progressively restricts the neighborhood. Compared with constraint-based algorithms, score-based algorithms result in entirely directed DAG, which is their primary advantage39. We predicted the type of delivery for individuals in the test set and compared it with the actual observations. To evaluate the validity of predictions, we used the Receiver Operating Characteristic (ROC) curve and the Area Under Curve (AUC). The conditional probability tables for the primary exposure and outcome variables were derived from the BN, and the chi-squared statistics were used to test the significance of relations.To fit the parameters of BN corresponding to DAG achieved in structure learning, we used the Bayesian estimationFinally, we fitted several logistic regressions adjusted for potential confounders as the alternative approach for modeling the data. We compared the prediction power of these models with the suggested BN using the AUC of these models in the test datasets.40. The type one error for statistical inferences was defined at 0.05.The statistical analysis, data preparation, and graphical representation were conducted using the R statistical software environment. We used the "bnlearn" package to fit the BN modelThis research project was approved by the Medical Ethics Committee of the Research Institute for Endocrine Sciences (IR.SBMU.ENDOCRINE.REC.1400.042). All methods were performed in accordance with the relevant guidelines and regulations. All study participants provided written informed consent.P-value = 0.03), preeclampsia , low birth weight , and preterm  were significantly higher in the control city than in the intervention city. The prevalence of moderate and severe vitamin D deficiency at baseline was 46% and 38%, respectively, in the control city, while in the intervention city, these values were 44% and 45%. Consequently, there was a significantly higher prevalence of severe vitamin D deficiency in the intervention city . Severe vitamin D deficiency prevalence at delivery was reported at 1.5% and 45% in the intervention and control cities , respectively. The prevalence of cesarean section in the case and control groups were 29% and 37%, respectively .Table Figure Vitamin D level at delivery was a function of vitamin D level at the start of the study (baseline) and the city (intervention city vs control city) where females lived. This conditional relationship adjusts the results for baseline status and receiving interventions. The vitamin D level at delivery was related to the mode of delivery, preeclampsia, and gestational age. Finally, the gestational age and GDM determined the birth weight of newborns.P-values < 0.001. The most frequent causes of cesarean section were prolonged labor 44.86 (95% CI: 42.46–47.26), malpresentation 29.35 (95% CI: 27.15–31.55), and CPD 11.78% (95% CI: 10.22–13.34).Conditional probabilities in BN revealed that knowledge about vitamin D level at delivery informed us about the pregnancy outcomes and type of delivery. The type of delivery was conditionally independent of vitamin D status at baseline, given the vitamin D status at the time of delivery. Therefore, in Fig. Four different logistic models were fitted in Table Model 1 had an AUC of 67% 95% CI: 62.2–71.7), with a sensitivity of 39.22%, specificity of 8.82%, and accuracy of 70.71%. These metrics suggest superior performance compared to the other models in Table .2–71.7, 42 as well as the capacity to handle complex problems with uncertainties and associations43. Based on our investigation, we report a significant association between vitamin D levels and the mode of delivery. Specifically, our findings indicate that women with moderate and severe vitamin D deficiency had increased odds of undergoing cesarean section compared to those with normal levels of this essential nutrient. Furthermore, our analysis revealed an indirect effect of vitamin D status on pregnancy outcomes, as serum concentrations of 25(OH)D at delivery were positively correlated with the incidence of preeclampsia and gestational age. Importantly, we identified two critical factors that impact 25(OH)D values at delivery: baseline vitamin D concentrations and receiving vitamin D supplementation during pregnancy. Additionally, we found that the type of residence (house vs apartment) was associated with differences in vitamin D levels. Taken together, our results demonstrate the importance of monitoring and maintaining optimal vitamin D levels during pregnancy for optimal maternal and fetal health outcomes.In the present study, we explored various influential factors related to the association between serum concentrations of 25(OH)D at delivery and mode of delivery using a BN approach. This method has several advantages, including the ability to manage missing data and collinear data effectively32. Many studies have utilized regression models with and/or without adjustment for potential confounding variables, documenting an increased incidence of cesarean section in mothers with vitamin D deficiency17. For example, after controlling for factors such as race, age, education level, insurance status, and alcohol use, a cross-sectional study on 253 American women revealed that participants with 25(OH)D levels less than 37.5 nmol/liter had almost four times the odds of cesarean delivery compared to those with levels of 25(OH)D 37.5 nmol/liter or greater12. Another cross-sectional study on 200 Indian pregnant women with singleton pregnancies showed a higher prevalence of hypovitaminosis D among mothers who underwent cesarean delivery compared to those who delivered vaginally (92% of women with vitamin D deficiency and 6% with insufficiency versus 85.6% with deficiency and 10.8% with insufficiency)14. A case–control study of 60 Danish women demonstrated a significant association between serum 25(OH)D levels and an increased risk of emergency cesarean section due to dystocia, highlighting the importance of vitamin D in the preparation of the uterus muscles and smooth muscle contractility during normal vaginal delivery44. Similarly, another study on 2,251 low-income pregnancies demonstrated that after adjusting for energy intake, other nutrients, and other potentially confounding variables, women with vitamin D deficiency had a two-fold increased risk of cesarean delivery due to prolonged labor compared to those with sufficient serum concentrations45. Additionally, a cross-sectional study of Iranian women with singleton pregnancies aged 15–45 years suggested that vitamin D deficiency was associated with a higher rate of cesarean delivery and other adverse maternal and neonatal outcomes13. Data from a multi-ethnic Asian cohort study of 940 women showed that Chinese and Indian women with 25 (OH) D inadequacy had higher odds ratios of emergency cesarean Sect.  than those with sufficient values. Although this study reported an inverse association between maternal 25 (OH)D concentrations and fasting plasma glucose, there was no association between vitamin D insufficiency and risk of GDM in the overall cohort or within any ethnic group16. A prospective study of 995 singleton pregnancies found that after adjusting for maternal age, BMI, racial origin, smoking, method of conception and season of blood testing, first-trimester maternal serum levels of 25(OH)D were similar between women who delivered vaginally and those required elective or emergency caesarean delivery29. However, it is important to note that although levels of 25(OH)D in the first trimester were similar among all participants, those with declining vitamin levels during pregnancy may have a higher risk of cesarean delivery than those who maintain their vitamin D levels more effectively. Data from a prospective cohort study of 1153 low-income and minority gravidae revealed that after adjustment for age, parity, ethnicity, smoking, BMI, gestation, and season at entry, the cesarean delivery risk was significantly increased for women with vitamin D deficiency, but not for those with vitamin D insufficiency. The authors found that vitamin D deficiency was associated with a two-fold increased risk of cesarean delivery due to prolonged labor17. However, some investigations report no significant association between 25(OH)D and mode of delivery32. For example, a study on 461 Nigerian pregnant women found no significant differences between study groups based on 25(OH)D levels  regarding the rate of cesarean delivery or other pregnancy complications such as preeclampsia, GDM, and preterm delivery30. Similarly, a cross-sectional study of 154 Turkish pregnant women showed no significant difference in the rate of cesarean delivery between women with serum vitamin D levels < 15 ng/ml compared to those with values > 15 ng/ml31. Conflicting results among studies could be attributed to limitations such as small sample size, lack of adjustment for potential confounders, and measurement of vitamin D in the first trimester.Previous studies have evaluated the relationship between maternal vitamin D status and mode of delivery using various statistical methods, yielding inconsistent findings46, which can lead to proximal muscle weakness47. and suboptimal muscle function and strength in individuals with vitamin D deficiency47. Additionally, calcium levels, which are regulated by 25(OH)D, play a critical role in smooth muscle performance during labor initiation18. Adequate levels of serum calcium are necessary for initiating labor, and vitamin D deficiency may negatively impact both skeletal muscle and smooth muscle strength, leading to specific etiologies of cesarean delivery such as CPD or failure to progress12. Vitamin D may affect myometrial contractility via two pathways: involving the intracellular vitamin D receptor and changes in the calcium metabolism44. Invitro studies have demonstrated that vitamin D regulates contractile proteins in myometrial cells48. Our analysis predicted that 44.86% of pregnant women will undergo cesarean section due to prolonged labor. Thus, maternal vitamin D deficiency at delivery might increase the probability of cesarean section by reducing the ability to push and more complicated and extended labor.The mechanisms underlying the association between 25(OH)D deficiency and an increased risk of cesarean section are complex and require further investigation. One potential explanation for this association is the presence of vitamin D receptors in skeletal muscle9. A recent systematic review and meta-analysis reported a strong link between maternal vitamin D deficiency and severe preeclampsia, and vitamin D supplementation may be beneficial in preventing this pregnancy complication49. Vitamin D deficiency is also associated with many risk factors for endothelial dysfunction and vascular health impairment19. On the other hand, adequate vitamin D intake can maintain calcium homeostasis, which is inversely correlated with blood pressure50 and may directly suppress the proliferation of vascular smooth muscle cells. Vitamin D may also play a critical role in blood pressure control by regulating the renin-angiotensin system51. Additionally, vitamin D can modulate the synthesis of adipokines related to endothelial and vascular health52. Vitamin D concentration can affect glucose homeostasis via various mechanisms, such as improving the insulin sensitivity of target cells , improving and protecting β-cell function, and reducing insulin resistant through immunoregulatory and anti-inflammatory effects21. Women with vitamin D deficiency might be at risk for GDM22. Moreover, vitamin D can influence the pathophysiology of preterm delivery through inflammation and immunomodulation processes; pregnant women with vitamin D deficiency might be susceptible to infection, which is an important predisposing factor for preterm delivery10. A meta-analysis of 10 studies including 10,098 participants showed that maternal serum concentrations of 25(OH)D levels less than 20 ng/mL were significantly associated with an increased risk of preterm delivery53. Our study based on the structure of BN revealed that moderate and severe vitamin D deficiencies at delivery significantly increase the probability of pregnancy complications like cesarean section, preeclampsia, and preterm delivery, but not GDM. Nevertheless, the absence of a significant relationship between 25(OH)D status and GDM in our study could be due to the conditionally dependent approach of the model. Therefore, no significant relationship between vitamin D concentrations and GDM given the city does not necessarily imply that the relationship does not exist overall. Our findings indicated that fetal distress contributed to approximately 4.3% of cesarean section cases. Fetal distress can partly reflect maternal medical conditions, such as preeclampsia, gestational diabetes, and preterm delivery, which are indirectly associated with cesarean Sect.25. Additionally, previous studies have demonstrated that GDM can increase fetal weight and macrosomia, leading to an elevated risk of elective cesarean section and emergency cesarean due to CPD and dystocia28.Moreover, maternal vitamin D deficiency during pregnancy and at delivery has been implicated in the pathogenesis of some maternal medical conditions, including preeclampsia, GDM, and preterm delivery, which can be associated with a higher risk of cesarean Sect.We utilized logistic regression models, the most commonly used statistical approach in medical research, to estimate the OR of cesarean section in women with maternal vitamin D deficiency after adjusting confounders. Our results from logistic regression provided similar to those obtained from the BN. In particular, our unadjusted logistic regression model (model 1) showed higher OR for cesarean section in pregnant women with moderate and severe vitamin D deficiency compared with those with normal vitamin D levels. This finding remained significant even after adjusting for other factors such as vitamin D at baseline and city (intervention with vitamin D supplementation) (model 2), vitamin D at baseline, city, and residence type (model 3), and vitamin D at baseline, city, residence type, education, and age at current pregnancy (model 4). Additionally, we found that the AUC of all these models was comparable to that of the BN approach.43.To the best of our knowledge, this is the first population-based study globally to illustrate the relationship between the association between 25(OH)D concentrations at delivery and mode of delivery using both BN and logistic regression models. Our study has several strengths, including its population-based design, measurement of 25(OH)D at both baseline and delivery, and the use of appropriate statistical methods for data analysis. These methods have been shown to be superior to traditional regression analysis in various fields of epidemiological studies54. Secondly, there were variations in vitamin D supplementation intake among the study population. However, our results suggest that vitamin D was the critical determinant of the mode of delivery, regardless of vitamin D at baseline and intervention. Additionally, we had no data on UVB exposure during pregnancy trimesters, calcium intake, sartorial habits, and skin types. Thirdly, our study population was limited to two cities mainly composed of the Persian ethnic group, thus generalizing our results to other ethnicities should be approached with caution. Fourthly, we could not measure myometrial dysfunction by contraction frequency or Montevideo units due to a lack of data. Finally, since the cesarean rate is very high in many countries, including Iran, our findings may not be generalizable to populations with a lower cesarean rate.However, several limitations should be kept in mind when interpreting our findings. Firstly, we could not apply the liquid chromatography technique as the gold standard method for measuring the circulating levels of 25(OH)D, due to inaccessibility. Nevertheless, the ELISA technique is reliable when performed by expert staffIn conclusion, our study highlights the adverse effects of maternal vitamin D deficiency on mode of delivery, both directly and indirectly, through maternal complications such as preeclampsia and preterm delivery, leading to a higher probability of cesarean section. Therefore, screening all pregnant women for serum concentrations of 25(OH)D can help identify mothers at risk of adverse pregnancy outcomes.


Notocyamus hydrophobus gen. nov. et sp. nov., which is now the oldest and most complete fossil record of Nelumbonaceae. In addition, it displays a unique mosaic of ancestral and derived macro- and micromorphological traits that has never been documented before in this family. This new Brazilian fossil-species also provides a rare illustration of the potential morphological and anatomical transitions experienced by Nelumbonaceae prior to a long period of relative stasis. Its potential plesiomorphic and apomorphic features shared with Proteaceae and Platanaceae not only fill a major morphological gap within Proteales but also provide new support for their unexpected relationships first suggested by molecular phylogenies.Dating back to the late Early Cretaceous, the macrofossil record of the iconic lotus family (Nelumbonaceae) is one of the oldest of flowering plants and suggests that their unmistakable leaves and nutlets embedded in large pitted receptacular fruits evolved relatively little in the 100 million years since their first known appearance. Here we describe a new fossil from the late Barremian/Aptian Crato Formation flora (NE Brazil) with both vegetative and reproductive structures,  Although the fossil record may help to reduce morphological gaps between living taxa, it is inherently incomplete and may thus miss important evolutionary transitions. Such gaps in evidence for documenting plant evolution can be interpreted either as a consequence of a period with higher evolutionary rates over a short timescale or lower rates of evolution and relative stasis over a long timescale2. However, the scarcity of earliest and succeeding forms in fossil records can be also caused by preservational biases, undersampling and low intensity of investigation. Numerous studies of middle paleolatitude floras have significantly reduced the knowledge gaps on Early Cretaceous angiosperms, while those from paleotropical areas, where allegedly flowering plants started to diversify, are still in need of more investigations, especially regarding macrofossils of eudicots, the most diverse angiosperm clade4. The knowledge gaps on macrofossils in the Early Cretaceous tropics contrast with the substantial and comparatively well-studied microfossil record6.The origin and early radiation of flowering plants are still unclear and remain one of the most enigmatic themes concerning the evolutionary history of vascular plants as a whole7. These reports are mainly based on their distinctive tricolpate pollen7, but also on macro- and mesofossils thought to be related to basal lineages9. Among the pieces of evidence, some records are related to Ranunculales and Proteales9, which are the two earliest diverging lineages of eudicots according to molecular analyses10. However, in contrast to the extant families of Ranunculales, there are huge differences and remarkable gaps in gross morphologies between those of Proteales, i.e., Sabiaceae, Nelumbonaceae, Platanaceae, and Proteaceae9. Their close phylogenetic relationship was thus not suspected before advances in molecular phylogenetics11, and potential morphological synapomorphies supporting their shared common ancestry started to be ascertained thereafter13. In addition, all these families occur in Cretaceous floras and Nelumbonaceae, Platanaceae, and Proteaceae particularly stand out by their relative abundance and richness in the fossil record9. Unfortunately, few intermediate forms linking the morphologies of these families have been found, and it seemed unlikely that they would be found since Nelumbonaceae and Platanaceae have undergone a long period of relative morphological stasis15.The first reports of eudicots date back to the middle Early Cretaceous from circumtethyan areas, i.e., northern Gondwanan and southern Laurasian regions, and suggest that their origin and center of diversification were in northern GondwanaNelumbites Berry is found in the middle Albian of Kazakhstan16, the upper Albian of Siberia17, and the middle and upper Albian Potomac Group of Virginia (USA)19, and the latter was confirmed its nelumbonaceous relationship by a cladistic analysis19. There are also Exnelumbites Estrada-Ruiz, Upchurch, Wolfe et Cevallos-Ferriz from the Campanian to the Maastrichtian of Mexico20, and Nelumbago McIver et Basinger and Paleonelumbo Knowlton documented from the Paleocene of Canada21 and Colorado (USA)22, respectively. Although numerous reports coming from Africa, Asia, Europe, and North and South America are attributed to Nelumbo Adans., most of them do not bear synapomorphies of the crown group and need a taxonomic reevaluation25. These fossils suggest that, mainly from the Late Cretaceous onwards, Nelumbonaceae, including Nelumbo, have had a nearly worldwide distribution, mostly in the Northern Hemisphere. Today, the genus has a relictual disjunct distribution with two extant species, i.e., wild populations of N. lutea (Willd.) Pers. in southern North America and N. nucifera Gaertn. in southern Asia to Australasia26.Representatives of Nelumbonaceae are thought to have remained more or less similar in terms of their gross morphologies from the late Early Cretaceous to the present day, and they are relatively diverse not only through geologic time but also across the world. For instance, In this paper, we describe a new fossil-genus and fossil-species of Nelumbonaceae from the Crato Formation of Brazil. This new macrofossil-taxon from the middle Early Cretaceous not only represents the earliest occurrence of this family known to date but also displays a mosaic of traits finally bridging the morphological gap between Nelumbonaceae and their woody sister clade consisting of Platanaceae and Proteaceae.28. The Crato Formation mainly represents a lacustrine paleoenvironment documented by laminated limestones29. In these lacustrine rocks, especially the stratigraphically youngest C6 carbonate unit, countless fossils of vertebrates, invertebrates, and plants are found29. Such abundance of fossils and their often remarkable preservation have led to the designation of the Crato Formation as a Konservat-Lagerstätte28. Its scientific value is demonstrated by the fact that it contains one of the currently best records for understanding mid-Early Cretaceous low-latitude paleo-ecosystems30. The age of the Crato Formation has usually been considered to be upper Aptian32, but there is no longer a consensus since new age estimates for the Santana Group, together with an updated concept and position for the international chronostratigraphic unit Aptian, suggest that at least a lowermost Aptian age (ca. 121 Ma) is more plausible (see in Methods)36. Furthermore, the usual lack of information on the detailed lithostratigraphic positions of fossils collected from the C6 unit of the Crato Fossil Lagerstätte raises the question of whether particular organisms existed simultaneously or not since the laminated limestone section represents a substantial time interval . The latter fact complicates reconstructions of the paleoecological context of certain fossil biotas from the lagerstätte.The sedimentary Araripe Basin (NE Brazil) includes several lithological units among which the Santana Group stands out in yielding fossil lagerstätten, especially that from the Crato Formation30. Angiosperms show a notably high taxonomic diversity and wide paleoecological spectrum even though not all fossil elements have been formally described yet37. Mesangiosperms make up the bulk of described fossils, including magnoliids41, monocots42, and eudicots8. By contrast, only a few records were assigned to the ANA grade, specifically Nymphaeales44. In addition, the angiosperm pollen records are mainly represented by monocolpate and tricolpate forms and may support the presence of chloranthoid, magnoliid, monocot, and eudicot taxa37.The macrofossil assemblage of the Crato Formation includes a few spore-bearing plants but it is dominated by seed plants Angiospermae Lindley (P.D.Cantino et M.J.Donoghue).Eudicotyledoneae M.J.Donoghue, J.A.Doyle et P.D.Cantino.Order Proteales Juss. ex Berchtold et J.Presl.Family Nelumbonaceae A.Rich.Notocyamus Gobo, Coiffard, Bachelier, L.Kunzmann, Conceição et Iannuzzi, gen.nov.Aquatic angiosperm with herbaceous rhizomatous growth habit, with adventitious roots and long-petiolate, alternate simple leaves. Leaf with marginal petiole attachment, unlobed and untoothed margin, and distinctly papillate epidermis with anomocytic and (brachy-) paracytic stomata on both sides. Primary venation palinactinodromous, distally bifurcating, with agrophic veins. Major secondary venation festooned brochidodromous. Fruit solitary, terminal, and aggregate, derived from an apocarpous gynoecium with fruitlets embedded in an enlarged ellipsoidal receptacle, attached to a long woody peduncle. Fruitlets inserted in pits and free from the receptacle.Noto is derived from notos from Ancient Greek νότος (south) and cyamus from Ancient Greek κύαμος (bean), in reference to the Egyptian “bean” described by Theophrastos of Eresos, which was probably a lotus seed.PFN003134Notocyamus hydrophobus Gobo, Coiffard, Bachelier, L. Kunzmann, Conceição et Iannuzzi, sp. nov.Notocyamus gen. nov. are the palinactinodromous leaf venation, in which lateral primary veins branch successively rather than from one point, and the secondary xylem in the peduncle, which are distinct from any living Nelumbo or any fossil-genus in the family.The main diagnostic characters of Notocyamus hydrophobusGobo, Coiffard, Bachelier, L. Kunzmann, Conceição et Iannuzzi, sp. nov.Lamina noto- to mesophyllous in size with L/W ratio ca. 1, chartaceous, obovate to elliptical, with obtuse and rounded to slightly cordate base and obtuse and rounded to straight-sided apex. Interior secondaries and intersecondary veins present. Tertiary venation mixed percurrent, predominantly opposite. Quaternary venation mixed percurrent, mostly alternate. Quinternary veins regular reticulate. Areolation five- to six-sided. Freely ending veinlets absent. Marginal ultimate venation looped.hydrophobus is derived from Ancient Greek ὑδροφόβος meaning fearing water, in relation to the epidermis structure similar to the superhydrophobic epidermis of living lotus.MB.Pb. 2002/1047 , Fig. SMF SM.B 16.522) , Fig. 2 (reposiPFN003135Opencast pit(s) southwest of Nova Olinda, Ceará, Brazil. Lower Cretaceous (uppermost Barremian?/lowermost Aptian), C6 limestone horizon, Crato Formation, Santana Group, Araripe Basin.Vegetative structures are preserved as reddish-brown replacement fossils and/or impressions with or without a thin iron oxide covering layer. Tissues of the peduncle are also replaced by iron oxide, while the receptacle is a mold with two casts of fruitlets. Clastic material is especially abundant on the rhizome of the holotype, and also in the petiole of the paratype.The holotype is about 50 cm long and 25 cm wide and consists of a rhizome bearing about 30 roots, 13 leaves, and a single aggregate fruit with two visible fruitlets embedded in an enlarged receptacle .The leaves are petiolate, alternate and simple. Petioles are 12–20 cm long by 0.5–1 cm width. The position of the lamina attachment to the petiole is marginal. Laminar sizes are noto- to mesophyllous, 60 to 95 mm long, and about 60 to 100 mm wide. Laminar shapes are obovate to elliptical, slightly medially asymmetrical to symmetrical, with a length/width (L/W) ratio of about 1. The lamina texture is likely chartaceous. Leaves are unlobed with untoothed margins, obtuse and rounded to slightly cordate bases, and obtuse and rounded to straight-sided apices. The primary venation is palinactinodromous with five primary veins running decurrently basally and bifurcating once or twice distally Figs. B. The ar−2 adaxially, and 100–120 mm−2 abaxially. The stomata are usually 30–35 µm long and 8–30 µm wide, with 15–25 µm long apertures randomly oriented. When the outer periclinal walls of the epidermis are abraded, their guard cells are often kidney-shaped  xylem without growth rings, and is surrounded by a  discontinuous layer interpreted as the remains of the cambial zone Figs.  and 6.The pith is ovate and 0.6–1.5 mm in diameter  xylem bundles, with cells that are unevenly isodiametric and have a 13 to 60 µm wide diameter and a 4–10 µm thick wall Fig. B. Walls The homoxylic wood (secondary xylem) consists predominantly of cells interpreted as fibers separated by 19 parenchymatous, homocellular, and usually multiseriate (rarely uniseriate) rays that are about 30–120 µm wide Fig. A. In conThe cambial zone is very poorly preserved. However, it comprises compressed fusiform and elongated cells outside the secondary xylem, from which arise new vascular increments with tracheary elements characterized by scalariform-like pits, including fibers with simple pits Figs. F,G. ThesThe anatomy of the petiole also includes vessel elements, which are 65–75 µm in diameter and have 5 µm thick secondary walls with scalariform pits and scalariform perforation plates  125 linked to the Functional Feeding Group (FFG) Galling was found in 46 and Coiro et al.47 with the D&E backbone from Endress & Doyle48. Our results show that the single most parsimonious position of Notocyamus is within Proteales, as sister to Nelumbo (Nelumbonaceae), and supported by synapomorphies of the two taxa within Proteales, such as the lack of freely ending veinlets (CH14), solitary flowers (CH43), and the presence of pits in receptacle bearing individual carpels (CH52). The herbaceous habit (CH1) and anomocytic stomata (CH37) are also shared between them and could equally be synapomorphies among Proteales since all other taxa (including Sabiaceae) are woody and predominantly possess paracytic and laterocytic stomata (modified to anomocytic in some Platanaceae and Sabiaceae)50. However, the point on the tree at which these shared character states arose is ambiguous. Chloranthoid teeth (CH36) are a plesiomorphic character widespread in basal eudicots12, and their loss is a synapomorphy of Proteales (modified to platanoid teeth in Platanaceae). However, Notocyamus and Nelumbo still differ in the scoring of cambium (CH6), vessel perforation (CH09), leaf shape (CH30), and base of blade (CH33), and by the presence of paracytic stomata (CH37). In Proteales, loss of cambium (CH6) and evolution of peltate leaves (CH33) are autapomorphies of Nelumbo.Our cladistic analysis Fig.  was perfNotocyamus and Nelumbo in having freely ending veinlets (CH14) (lacking only in Lauraceae), pinnate major venation (CH31) , and in lacking pits in the receptacle (CH52).The position sister to Magnoliales and Laurales is one step less parsimonious and supported by the fiber pitting (CH10), and the presence of solitary flowers (CH43) . However, they differ markedly from Notocyamus sister to Platanaceae + Proteaceae. This position is supported by mixed scalariform and simple vessel perforation plates (CH9), but it requires two origins of freely ending veinlets (CH14), solitary flowers (CH43), and receptacle with carpel-bearing pits (CH52), or one origin of these features followed by a reversal in Platanus and Proteaceae.We also found an alternative two step less parsimonious position in Proteales with Notocyamus sister to either Piperales or Galbulimima in Magnoliales, and monocot. The alternate position as sister to Piperales is supported by palmate venation (CH31) but requires one more step in each of these characters  than the position with Nelumbo. The position sister to Galbulimima is supported by vessel perforation (CH9) and fiber pitting (CH10) but also requires one more step in several characters, in this case due to the woody habit (CH01), vessel grouping (CH11), freely ending veinlets (CH14), pinnate major venation (CH30) and the absence of pits in the receptacle (CH51). The position as sister to monocot is supported by leaf shape (CH30) but requires one more step in each of these characters  than the position with Nelumbo.Three additional two step less parsimonious positions were retrieved among magnoliids, with Notocyamus is described from the late Barremian?/Aptian of Brazil and is thus older than the previously oldest representative of its clade, i.e., Nelumbites extenuinervis from the middle to upper Albian of USA19, but also than isolated leaves of Nelumbites from Kazakhstan16. Its older age is consistent with the fact that several of its morphological characters are more plesiomorphic than those of Albian Nelumbites and extant Nelumbo. In addition, molecular dating indicates that the divergence time of Nelumbonaceae and Platanaceae is around 83 to 123 Ma53. These older age estimates are very close to the first pieces of evidence from the fossil record of this family even though their divergence time could still be earlier54.Nelumbo in Laurasian mid-latitudes25. Also, Ramírez-Barahona et al.55 suggest that the ancestral biome of Nelumbonaceae is most likely coupled with more temperate than tropical climates. However, whether their initial evolutionary history is linked to Laurasian mid-latitude areas and ancestral temperate biomes55, or not, remains unclear. Our report preliminarily suggests that the initial evolution of Nelumbonaceae may have occurred in northern Gondwanan low-latitude areas under a seasonal tropical climate since Notocyamus is currently the oldest record of this family and displays traits interpreted as ancestral for the family. This new Brazilian fossil also adds data to postulate that the paleoequatorial tropical areas were an important center of early diversification of basal eudicots in the Early Cretaceous before reaching mid-latitude areas, where they became widespread from the Albian onwards. This hypothesis was discussed by Doyle5 based on fossil pollen records and is currently being reinforced by an increasing number of macrofossils of basal eudicots described from the Crato Formation8, but also by evidence from tricolpate pollen already reported in this same lithostratigraphic unit32.Our findings also raise questions on the paleogeographic origin of the family since until our study, distributions of all previous fossil records suggested an origin of Notocyamus resembles living Nelumbonaceae and implies a paleoecology adapted to similar aquatic conditions  paracytic stomata and distinct epidermal papillae on both sides, whereas those of Nelumbo are exclusively anomocytic and almost entirely epistomatic with papillae present only on their adaxial (upper) surfaces67. It suggests the development of emergent leaves in Notocyamus and subsequent exaptation of the Lotus effect in peltate leaves of Nelumbonaceae. Such emergent and amphistomatic leaves, despite being rare in Nelumbo, are also well-documented in other aquatic plant lineages59. In addition, the relatively high leaf vein density (ca. 9 mm−1) in Notocyamus may support a higher water supply capacity leading to a better leaf hydraulic conductance and consequently higher photosynthetic and transpiration rates70. As in living Nelumbo species, it would probably also allow establishment in sunny habitats under conditions of high temperatures.The leaf epidermises of ata Fig. D,F with ies Fig. , produciNotocyamus and living Nelumbonaceae also display a typically enlarged floral receptacle that develops into an aggregate fruit with several nutlets embedded in pits71  are unknown in Notocyamus, but both have similar palinactinodromous venation and marginal lamina attachment 74, suggesting that these traits were homologous in all these groups. This similarity is also seen in some Cretaceous platanoid leaves assigned to Credneria Zenker and Platanus, which also display palinactinodromous venation75. Notocyamus often shares the obovate to elliptical leaf shapes  and derived (anomocytic stomata) anatomical traits.The presence of paracytic and anomocytic stomata in Notocyamus is considered to be plesiomorphic for the family, Notocyamus can be interpreted as a first evolutionary step towards Nelumbo. Nelumbites18, from the Albian, can be interpreted as a second evolutionary step. Its leaf blade is already peltate, but eccentric, its primary venation is actinomorphic but comprises only a few primaries (< 10), and its lamina is orbicular, displaying a basal indentation reminiscent of the sometimes-cordate leaf bases of Notocyamus. Exnelumbites20, from the Campanian–Maastrichtian, which can be interpreted as a third evolutionary step, is already centrally peltate but displays 12–13 primaries and surprisingly bears chloranthoid teeth. Paleonelumbo, from the Paleocene, is another genus with distinct leaf architecture: although its leaves are centrally peltate, they are quite distinct from those of Nelumbo in being lobed and having 10–15 primary veins20. Nelumbago, also from the Paleocene, is, however, very similar to extant and extinct species of Nelumbo in terms of foliar architecture and can therefore be interpreted as the next to last evolutionary step in having centrally peltate and entire-margined leaves with > 20 bifurcating primary veins but still no central disk20. All extinct and extant Nelumbonaceae cited above suggest that peltation of their leaves did not evolve until a certain time after the divergence of Nelumbonaceae.If the leaf architecture of Notocyamus exhibits a special similarity to Nelumbo in lacking the freely ending veinlets. This leaf character is rare in basal eudicots and occurs only in Nelumbonaceae in Proteales. In addition, Notocyamus and Nelumbo share the same entire-margined leaf blades, bifurcating primary venation, festooned brochidodromous secondary venation, tertiary and quaternary veins with mixed course, and six-sided areolae20.Despite differing significantly in its leaf architecture by having a distinct primary venation type, Notocyamus and other Nelumbonaceae both produce single flowers sharing an apomorphic feature: carpel-bearing pits in the receptacle. The shape of the floral receptacle appears to have evolved from ellipsoidal in Notocyamus, to slightly conical in Nelumbites extenuinervis and Nelumbo puertae23, culminating in the well-known obconical shape in Nelumbo. Although the reproductive organs of both Notocyamus and Nelumbo are borne on long peduncles  nonconductive cells  occur . In addition, the fossils show that the living Platanus and Nelumbo are similar to some of their Early Cretaceous members, and that these clades thus likely experienced early rapid morphological evolution followed by a long period of relative stasis.Fossils of Proteales show that members of this group already inhabited contrasting environments during the Early Cretaceous, with terrestrial woody Platanaceae and aquatic herbaceous Nelumbonaceae. Unfortunately, macrofossils of Proteaceae and Sabiaceae are sporadic in the Cretaceous and better recognized and described in Cenozoic florasNotocyamus as a stem-relative of Nelumbonaceae, the fossils not only push back the minimum age of the appearance of the lotus lineage to at least the early Aptian but also shed light on the morphological evolution of its vegetative and reproductive traits. Despite the putative stasis previously reported in this family, their stem lineages may still have undergone considerable morphological changes throughout geological time. In addition, our report surprisingly provides new lines of evidence that the divergence between Nelumbonaceae and other members of Proteales may have occurred in a stepwise fashion. Compared to other extinct nelumbonaceous fossils, Notocyamus displays a new and unique mosaic of ancestral states shared with Platanaceae and Proteaceae, and derived states shared with Nelumbo, and clarifies important evolutionary steps of Nelumbonaceae and Proteales as a whole. Thus, Notocyamus ideally fills the morphological gap between aquatic Nelumbonaceae and their most recent common ancestor with woody Proteales, and can therefore be seen as one of those “missing links” that are rarely preserved in the fossil record.Besides our results from cladistics supporting the position of Notocyamus exhibit anatomical details preserved by iron oxide replacement of tissues. These details were observed in a high vacuum with a Scanning Electron Microscope (SEM) . For SEM analyses, pieces of leaves, petiole and peduncle were removed from the specimen, mounted on stubs, and coated with gold for four minutes with a Polaron SC7640 sputter coater. Photographs of fossils were edited with Adobe Photoshop software.The fossils were studied using a Leica Wild M10 microscope equipped with a Leica DFC 425 camera. Overview images were taken with a Canon 250D camera. Vegetative structures  of 45.The description of leaf architecture follows Ellis et al.Notocyamus (holotype) were inferred from leaves with at least half the lamina preserved (including folded leaves), multiplying the distance from the midvein to the lamina margin by two supposing a symmetrical lamina shape. They were used to estimate the laminar size, L:W ratio, and leaf shape.The leaf width measurements of 2) of Notocyamus was calculated based on an SEM image .The vein density  on leaves of 3, following Tiffney82.The volume of fruitlets was estimated based on the formula V = 4/3πr83). Slides were analyzed and photographed using a Zeiss light microscope (Axioscope 5) coupled to a Canon 60D camera.Petrographic thin sections of the peduncle were made in the three standard sections used for studying wood anatomy: transversal, radial, and tangential  in each alternative tree was recalculated. All characters were unordered and equally weighted.The phylogenetic analysis was performed by adding the fossil as a new taxon to the morphological data set from Kvaček et al.32. By contrast, new results of Melo et al.34, Barreto et al.36 and Arai & Assine85 consistently revealed an upper Aptian age for the Romualdo Formation, the uppermost unit of the Santana Group, based on relative and absolute dating methods. In addition, the Ipubi Formation, which underlies the Romualdo Formation, contains two important layers for age estimation, the black shale at its base and the evaporite layer soon above. Lúcio et al.33 dated this evaporite layer by Re–Os isotope studies as 123 ± 3.5 Ma (126.5 to 119.5 Ma). Besides, isotope and organic geochemistry of this black shale provides strong evidence that it was deposited during the global Ocean Anoxic Event OAE-1a35. The numerical age of the base of OAE-1a, called the Selli event, is ca. 119–120 Ma86. Thus, the Crato Formation, underlying the Ipubi Formation, is at least older than 120 Ma, which is not far above the base of the Aptian, or even older if there was a hiatus between the formations27.The Crato Formation, the second lithostratigraphic unit from the base of the Santana Group, has been repeatedly dated as upper Aptian age based on palynological evidence87.Since 2022, the updated international chronostratigraphic chart shows a new age for the base of the Aptian (121.4 Ma instead of 125 Ma), which is correlated with a short-lasting magnetostratigraphic zone29, the C6 carbonate unit of the Crato Formation could represent a period of up to 0.5 Ma. Altogether, the Crato limestones C1–C6 could likely represent several million years, which would extend its absolute age range towards the upper Barremian. However, such a fundamental change in the age of the Crato Formation needs to be supported by additional unambiguous evidence in future publications.According to Neumann et al.Supplementary Information.


Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is a common clinical phenotype of citrin deficiency in infants. Its phenotype is atypical, so genetic testing is quite necessary for the diagnosis.We report 4 patients with jaundice and low body weight. Furthermore, the biochemical examination of all showed abnormal liver function and metabolic changes. DNA samples of the patients were extracted and subjected to genetic screening. All candidate pathogenic variants were validated by Sanger sequencing, and CNVs were ascertained by qPCR. The genetic screening revealed 6 variants in 4 patients, and all patients carried compound heterozygous variants of SLC25A13. Importantly, 3 variants were newly discovered: a nonsense mutation in exon17 (c.1803C > G), a frameshift mutation in exon 11(c.1141delG) and a deletion of the whole exon11. Thus, four NICCD patients were clearly caused by variants of SLC25A13. Biochemical indicators of all patients gradually returned to normal after dietary adjustment.Our study clarified the genetic etiology of the four infants, expanded the variant spectrum of SLC25A13, and provided a basis for genetic counseling of the family. Early diagnosis and intervention should be given to patients with NICCD. Citrin deficiency (CD) is an autosomal recessive disease caused by SLC25A13 gene mutation. It includes three age-dependent clinical phenotypes: neonatal intrahepatic cholestasis caused by citrin deficiency(NICCD), adult-onset type II citrullinemia (CTLN2) and failure to thrive and dyslipidemia caused by citrin deficiency (FTTDCD) between NICCD and CTLN2 stages  from non-consanguineous families were enrolled in this study. They were all full-term, and some patients  showed low birth weight and/or low birth length. They all appeared jaundiced with normal color stool about 3 days after birth. They were given blue light irradiation, ursodeoxycholic acid, liver protection drugs and other symptomatic treatment, but there was no significant improvement in their symptoms. Patients 1, 2, and 3 were admitted to our hospital for jaundice, and patient 4 for jaundice and abnormal liver function. All of them were underweight and had no signs of chubby faces. Yellow staining of the skin and sclera was observed. Their livers were palpable 2–3 cm under the rib cage. Laboratory tests are shown in Genomic DNA was extracted from the peripheral blood of all probands. Genetic screening was performed for each proband: proband 1; proband 2 ; proband 3 ; proband 4 . All sequencing data were mapped with human reference genome(hg19), and variants were classified according to ACMG guidelines. All candidate pathogenic variants were validated by Sanger sequencing, and CNVs were validated by quantitative real-time polymerase chain reaction (qPCR).Compound heterozygous variants from SLAC25A13 were detected in all four probands; all variants were classified as likely pathogenic or pathogenic according to ACMG guidelines : PatientAll four patients adjusted their diets: breastfeeding was stopped and it was replaced by a lactose-free and MCT-rich formula. The clinical manifestations improved significantly and none of them suffered long-term complications  in protein synthesis, and according to ACMG guidelines, pathogenicity analysis is PVS1 + PM2 + PP4, it is classified as likely pathogenic, so far as we know, which has not been reported previously. While in our study, it was found in two patients and two family members. After a detailed medical history we found no consanguinity between the two families; however, they all came from Hubei Province, China, which spans the Yangtze River and belongs to the central city in China. The previous large-scale studies on NICCD were generally in the south and north of China, with few central cities. As we all know, there is a significant difference in the mutation carrier rate of SLC25A13 gene between North and South populations along the boundary of 30° North latitude in China. The results of our study suggested that the carrier rates of c.1803C > G may be frequent in the central cities of China, and there are differences in the distribution of the mutation spectrum in different geographical areas. As for the relationship between genotype and phenotype, although some studies declared that there were likely to be phenotype–genotype correlations in the specific genotypes , c.70–86in situ hybridization (FISH)  , and qPCn (FISH)  can helpAfter the dietary adjustment, the liver function such as ALT, GGT, TB, DB, TBA, ALB, AFP returned to normal at the last review . The patIn summary, our study reviewed the clinical manifestations, genetic test results, and prognosis of NICCD patients from 4 unrelated families. There are 6 variants of SLC25A13 were identified, and three novel variants were found: c.1803C > G, c.1141delG, and exon 11 del. This result clarified their genetic etiology and provided a strong basis for the clinical diagnosis of patients. At the same time, the detection of the novel variants enriched the variant spectrum of the SLC25A13, which further laid the foundation for the correlation study of the genotype and phenotype of NICCD.


Kaplan–Meier curves were used to analyze the relationship between tumor size and overall survival (OS). Univariate Cox analysis was applied to assess the factors associated with the prognosis of mCRC patients in the training cohort, and then multivariate Cox analysis was used to construct a nomogram model. The area under the receiver-operating characteristics curve (AUC) and calibration curve were used to evaluate the predictive ability of the model. Patients with larger tumors had a worse prognosis. While brain metastases were associated with larger tumors compared to liver or lung metastases, bone metastases tended to be associated with smaller tumors. Multivariate Cox analysis revealed that tumor size was an independent prognostic risk factor , in addition to the other ten variables . The 1-, 3-, and 5-year OS nomogram model yielded AUC values of more than 0.70 in both the training and validation cohorts, and its predictive performance was superior to that of the traditional TNM stage. Calibration plots demonstrated a good agreement between the predicted and observed 1-, 3-, and 5-year OS outcomes in both cohorts. The size of primary tumor was found to be significantly associated with prognosis of mCRC, and was also correlated with specific metastatic organ. In this study, we presented the first effort to create and validate a novel nomogram for predicting 1-, 3- and 5-year OS probabilities of mCRC. The prognostic nomogram was demonstrated to have an excellent predictive ability in estimating individualized OS of patients with mCRC.Given the poor prognosis of metastatic colorectal cancer (mCRC), this research aimed to investigate the correlation between tumor size and prognosis, and develop a novel prediction model to guide individualized treatment. Patients pathologically diagnosed with mCRC were recruited from the Surveillance, Epidemiology, and End Results (SEER) database between 2010 and 2015, and were randomly divided (7:3 ratio) into a training cohort ( Among all malignant tumors, colorectal cancer (CRC) has the third-highest incidence rate and the second-highest mortality rate . DistantThe American Joint Committee on Cancer (AJCC) system of tumor (T), nodes (N), and metastases (M) is a widely accepted approach for classifying cancer risk in patients with colorectal cancer . ResecteEven though the majority of these studies employed small sample sizes, further research is necessary owing to the potential for tumor size to serve as an indicator of CRC survival. This study employed the largest cohort to date to explore the effect of tumor size on mCRC patients.https://seer.cancer.gov/seerstat/), sponsored by the National Cancer Institute, contains information on the incidence and survival characteristics of malignancies among 26% of the population and 18 cancer registries in the USA.After being granted access to study data files with the reference number 12271-Nov2019, information on patients with newly diagnosed CRC from 2010 to 2015 was taken from the Surveillance, Epidemiology and End Results (SEER) database. The SEER data collection  information was also collected, together with clinicopathological characteristics and pertinent therapy details. The following criteria were met by the patients: (1) patients had to be between the ages of 18 and 75; (2) they had to have stage IV CRC pathologically determined; and (3) CRC was the only primary cancer. Patients with insufficient staging, missing metastatic information, diagnoses made only through autopsy, and patients whose prognosis was unknown were also disqualified. In addition, the tumor size indicated in this study was the primary tumor size.χ2 test. The Cox regression model was used to analyze the survival difference, and then a nomogram model was created using the results of cox regression. The nomogram’s discrimination power was confirmed by the receiver-operator characteristic (ROC) curve, which also exhibited prediction and decision curve analysis (DCA) of nomogram for predicting patients’ OS at 1, 3, and 5 years. All statistical analyses were conducted bilaterally, and a P value of 0.05 or less was regarded as statistically significant.X-tile 3.6.1, SPSS 26.0, and R 4.1.1 were the application software used in this study’s statistical analysis. Baseline characteristics were shown. Categorical data were given as frequencies with percentages, while continuous variables were expressed as the median [IQR]. Categorical data were compared using a n = 5268) were male, and nearly third of fourths were white . More patients had tumor in left colon , while others were in right colon , transverse colon  and rectum . In this cohort, 72.6% (n = 5801) were histopathologically moderately differentiated and 94.6% (n = 7567) were adenocarcinoma. Radiation was given to 16.5% (n = 1321) of all patients, and 83.2% (n = 6652) of all patients accepted chemotherapy. 82.4% (n = 6591) of all patients showed CEA-positive results. Among all patients with distant metastases, 75.4% (n = 6031) of patients had liver metastasis, 20.2% (n = 1614) of patients had lung metastases, 3.5% (n = 280) had bone metastases, and 0.9% (n = 70) had brain metastases. The training cohort and validation cohort were then divided at a ratio of 7:3, and as there were no statistically significant differences between the 2 patient groups (P > 0.05), they were considered comparable . The results of multivariate Cox analysis showed that radiation was not an independent prognostic factor while other 11 factors were effective .According to univariate Cox analysis Table , age, raBased on the selected independent prognostic factors affecting patients’ OS, we constructed nomogram model to predict patient OS Fig. . In patiROC curve of training cohort’s nomogram was drawn to predict the OS of patients at 1, 3, and 5 years, and then calculated their AUC values to be 0.766, 0.726, and 0.746  was performed at 1, 3 and 5 years of OS in training cohort Fig. A and valCRC is one of the most common malignant tumors globally, accounting for 10% of newly diagnosed cases of malignancy –16. CompP value was over 0.05. This might be due to the fact that these patients have a poor prognosis themselves, for example, the prognosis of brain metastatic patients is a median survival time of 3–6 months upon diagnosis and no effective therapy [The distant metastasis of tumors, including CRC, is thought to comprise a multistep process of three phases: (1) Invasion phase: in situ tumor cells increase their invasiveness through epithelial–mesenchymal transition (EMT) processes, penetrating the surrounding tissues and migrating to the blood vessels or lymphatic vessels, creating circulating tumor cells (CTCs); (2) circulation phase: platelets adhere directly to the surface of CTCs, creating micro-thrombi structures that reduce the recognition and clearance of the immune system; (3) settlement phase: CTCs settle in distant organs, forming a pre-metastatic niche, a microenvironment characterized by immune suppression, enabled by the secretion of cytokines or exosomes from the primary tumor site . The cla therapy .The relationship between tumor size and prognosis of CRC patients has long been of great concern to clinicians. This study, based on a large-scale clinical data analysis, found that tumor size is an independent prognostic factor for mCRC patients. Nevertheless, certain limitations of the study could not be overlooked. Despite the large size of SEER database, our stratification by tumor size and metastatic site resulted in relatively small subgroups, reducing statistical power to detect small differences. This may help explain why we failed to detect significant associations between tumor size and metastatic site in the subgroups of bone and brain metastatic patients. In addition, information on missing molecular characteristics such as BRAF mutation status, microsatellite status, adjuvant therapy, or pathology techniques was not included in the SEER database. Furthermore, although this study was conducted in large populations, the proportion of Asian patients was still too small, thus necessitating further multicentral research to validate the results of this study.Our findings suggested that patients with mCRC who had lung or liver metastases have a poorer prognosis when their primary tumors are larger. We developed an accurate prognostic risk assessment model for such patients, allowing them to estimate their overall score via a nomogram and calculate their chances of survival. This plays a significant role in providing clinical guidance. Further prospective research is necessary to determine the role of tumor size in clinical staging models for management selection.