Background: Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder in women of reproductive age, the prevalence of which ranges from 8 to 13%. It is characterized by metabolic, reproductive, and psychological alterations. PCOS prevalence is related to body mass index (BMI). Women with BMI < 25 kg/m2 have a prevalence of 4.3%, whereas women with BMI > 30 kg/m2 have a prevalence of 14%. Moreover, women with PCOS have a risk of type 2 diabetes mellitus (T2DM) two-fold higher than controls, independently of BMI. Both PCOS and T2DM are also consequences of lower serum sex-hormone-binding globulin (SHBG) levels, which is currently considered a biomarker of metabolic disorders, in particular T2DM. Aim: To evaluate the effect of the very-low-calorie ketogenic diet (VLCKD) on markers suggested to be predictive of metabolic and ovulatory dysfunction. These comprehend SHBG, anti-Mullerian hormone (AMH), and progesterone levels on day 21 of the menstrual cycle in a cohort of obese non-diabetic women with PCOS and regular menses. Methods: Twenty-five patients (mean age 25.4 ± 3.44 years) with obesity and PCOS underwent VLCKD for 12 weeks. Each of them underwent measurements of anthropometric parameters  and blood testing to evaluate serum levels of SHBG, AMH, and progesterone before and after 12 weeks of VLCKD. Results: At enrollment, all patients had high BMI, WC, and AMH, whereas SHBG and progesterone levels were low. After VLCKD, the patients showed a significant reduction in BMI, WC, and HOMA index. In particular, 76% of patients (19/25) switched from obesity to overweight, and the HOMA index normalized, reaching values lower than 2.5 in 96% (24/25) of patients. In addition, serum AMH levels significantly decreased, and progesterone and SHBG significantly increased after VLCKD. Conclusions: This is the first study documenting the effects of VLCKD on ovarian reserve and luteal function in women with PCOS. VLCKD could be used to improve metabolic and ovulatory dysfunction in women with PCOS. Further studies are needed to understand the reasons for the AMH reduction. Weight loss could be the key to improving the metabolic profile and reducing the endocrine dysfunction responsible for infertility. Nutrition, in fact, could play an important role in improving reproductive outcomes .Several studies have demonstrated the benefits of the Mediterranean diet (MetD)  on the female reproductive system. In particular, it seems that MetD is associated with a reduction in the insulin resistance index and an improvement in metabolic parameters , an incrMissmer and colleagues, in a cohort study including 17,544 women planning pregnancy, found that there is a correlation between a pro-fertility diet  and a low risk of infertility due to ovulatory disorders ,3.In contrast to the MetD, there is the Western-style diet, characterized by high consumption of simple sugars and products with a high glycemic index, saturated fats, red meat, and a low intake of fresh fruit, vegetables, fish, fiber, and unrefined grains. Several studies have shown that a diet based on a high content of sugars, saturated fats, and animal proteins negatively impacts fertility and in particular is responsible for alterations in the menstrual cycle, with reduced production of progesterone and anti-Müllerian hormone (AMH), a greater number of antral follicles, and fewer blastocysts .As explained above, insulin resistance is one of the factors that aggravate oocyte quality and maturation and damages female fertility. The food intake with a high glycemic index is associated with a higher concentration of fasting glucose, androgens, and insulin growth factor-1 (IGF1) and can worsen insulin resistance and, therefore, ovulatory outcomes; not only that, it can be the cause of increased glucotoxicity and, therefore, of increased oxidative stress, which further damages ovarian function ,5. SeverAs far as fatty acids are concerned, a diet with a high lipid content can be associated with an alteration in the menstrual cycle and hormonal values, as well as an altered oocyte quality . The defRegarding the high trans fatty acids (TFA) content, there are conflicting studies. Chavarro and colleagues, in the previously described study, conducted in the USA from 1991 to 1995, showed that even a 2% increase in TFAs is responsible for an increased rate of infertility for ovulatory disorders . HoweverConversely, Mono Unsaturated Fatty Acids (MUFAs) bind to the PPAR-γ reducing inflammation, increasing insulin sensitivity, improving fertility, and reducing the time to pregnancy. The World Health Organization has recommended a reduction in the consumption of foods with TFAs, which must represent less than 1% of the total daily intake. Moreover, TFAs increase risk of type 2 diabetes, PCOS, and endometriosis [A defensive role in fertility seems to be played by omega 3 fat acids (FAs), which we find mainly in fish oil. They have an anti-inflammatory effect and act directly on steroidogenesis, increasing the concentration of progesterone; promoting the maturation of the oocyte and improving the quality of the embryo; reducing insulin resistance; and improving the lipid profile. Furthermore, it has been shown that a higher omega 3 content is associated with a higher rate of live births after ART .Finally, again, the study by Chavarro and colleagues shows how a low consumption of dairy products  is responsible for a reduced fertility rate. This is probably due to the fact that dairy products contain a high content of estrogen, fat-soluble vitamins, and trans-palmitoleic acid, which improves insulin sensitivity . HoweverThe role that proteins play within reproductive mechanisms is not yet clear. Mumford and colleagues showed that excess animal protein intake correlates with decreased testosterone levels . The ChaAccordingly, infertile obese women have worse outcomes in the case of assisted reproductive technique (ART), as they need a higher dose of gonadotropins, have a greater risk of ovarian hyperstimulation, increased cycle cancellation rate, and lower oocyte recovery, and increased abortion rate. Live birth rate is the most significant parameter when using ART. A systematic review and meta-analysis conducted by Sermondade and colleagues in 2019 evaluated the association between female obesity and the probability of live birth rate following ART . This meSeveral studies have demonstrated the benefits of the Mediterranean diet (MetD)  on the female reproductive system. In particular, it seems that MetD is associated with a reduction in the insulin resistance index and an improvement in metabolic parameters ; an incrFrom the studies conducted to date, it seems that a diet with low glycemic index foods, rich in mono and polyunsaturated fats, and which favors vegetable proteins is the most suitable for obese and overweight women who wish to become pregnant, both to improve endocrine dysfunctions of the female reproductive system and to ensure a better outcome of the maternal–fetal aspects of pregnancy.However, there are no specific guidelines regarding the type of diet to be carried out to lose weight in obese women who wish to become pregnant .Today, a particular nutritional protocol that is becoming increasingly popular to promote weight loss and improve fertility outcomes is the ketogenic diet (KD). KD is a nutritional protocol that mimics fasting through a marked restriction of daily carbohydrate intake  and a proportional intake of fat (about 44%) and protein (about 43%). It is not a high-protein diet, in fact, daily protein intake is 1.2–1.5 g/kg of ideal body weight [Ketone bodies  are produced in hepatocyte mitochondria in some specific condition, in which there is reduced availability of glucose, such as fasting, intensive physical activity, and very-low-calorie ketogenic diet (VLCKD) . Ketone Simulating fasting  has specific effects on numerous energy-dependent pathways, including the inhibition of the pathways mediated by IGF-1 and mTOR. Furthermore, 3-β-hydroxybutyrate, in addition to being an energy substrate, also represents a molecule with signaling and epigenetic functions, with modulatory effects on inflammation and oxidative stress. Moreover, KD promotes changes in the metabolic structure and in some molecular processes with numerous advantages: increasing insulin sensitivity, inhibiting hydroxymethylglutaryl-CoA reductase, reducing lipid synthesis, reducing inflammatory pathways, protecting from oxidative stress, and inducing autophagy and mitochondrion genesis. Therefore, it finds application in some conditions of female hormonal imbalance in the presence of ovulatory disorders or overweight/obesity. In fact, it appears to be particularly effective in overweight and obese women with PCOS, in which it was highlighted that after KD there is a 12% reduction in total weight, a 54% reduction in insulin levels, a 36% reversal of the luteinizing hormone (LH)/follicle-stimulating hormone (FSH) ratio, and a 22% reduction in free testosterone. In addition, about 24 weeks after the study ended, 40% of patients who had completed the KD protocol became pregnant . One of The aim of this study was to evaluate the 3-month-long impact of VLCKD on AMH levels , progesterone levels on the 21st day of menstrual cycle , and SHBG levels (prognostic metabolic risk marker for type 2 diabetes mellitus (T2DM) development) in a cohort of obese non-diabetic women with PCOS and regular menstruation. To accomplish this, we retrospectively reviewed the clinical charts of the patients attending the Endocrine Division, University-Teaching hospital Policlinico “G. Rodolico—San Marco”, for obesity and infertility.2) and underwent VLCKD, from January 2021 to January 2022, were consecutively enrolled in this study. We consecutively recruited twenty-five Caucasian women older than 18 years (mean age 25.4 ± 3.4 years). PCOS was diagnosed using at least two criteria of the 2003 Rotterdam Consensus  [This is a retrospective study performed on women who consulted the Division of Endocrinology, Metabolic Disease and Nutrition, University of Catania, for body weight increase. All PCOS patients that had first-degree obesity (BMI 30–34.9 kg/mulation) , family Anthropometric and biochemical parameters were evaluated at enrollment and at the end of the second phase (after 12 weeks from the beginning of VLCKD). ß-hydroxybutyrate levels were measured weekly through a reflex metric detection system and kept between 0.5 and 0.7 mmol/L. Body weight was measured in fasting conditions, using the same calibrated scale, without shoes, and with an empty bladder.Each woman underwent blood testing for the measurement of AMH, SHBG, and progesterone levels on the 21st day of the menstrual cycle, before and after 12 weeks of VLCKD. The hormones were analyzed immediately, and the samples were not frozen.The hormone evaluation was performed by Cobas Elecsys for AMH (measurement range: 0.000001–23 ng/mL) and SHBG (measurement range: 32.4–128 nmol/L) and by Cobas Elecsys III for Progesterone (measurement range: 1.83–23.9 ng/mL).The VLCKD protocol uses high-biological protein derived from milk, peas, and whey. Each preparation, which has about 100–150 kcal, contains 18 g of protein, 4 g of carbohydrate, and 3 or 4 g of fat. It is based on 3 stages: active stage, which represents the state of VLCKD  and includes the first three phases; re-education stage, which represents the state of LC diet  and includes from phases 4 to phases 6; and maintenance stage, that is, the balanced diet  and includes the seventh and last phase. Each phase had a duration of 4 weeks.In the first phase, there is a total replacement of natural protein with protein preparation , with the possibility of consuming low-glycemic-index vegetables during lunch and dinner.In the second phase, one protein preparation is replaced by natural protein food (lunch or dinner), such as meat, egg, or fish . In the third phase, both lunch and dinner protein preparations are replaced with natural proteins. During the active stage, it is recommended to integrate with micronutrients, such as vitamins , minerals  and omega-3 fatty acid. The re-education stage is a low-calorie diet during which carbohydrates are progressively re-integrated according to their increasing glycemic index: fruit and dairy products in the fourth phase; legumes in the fifth phase; and bread, pasta and cereal in the last phase, with a daily intake between 800 and 1500 kcal. Finally, during the maintenance stage, there is an eating plan balanced in macro- and micronutrients with a daily intake of between 1500 and 2000 kcal/day, depending on the individual .During VLCKD, patients must be closely and periodically monitored  Table 1Table 1 tContinuous variables are reported as mean ± SD or median (interquartile range) according to their distribution. Normality was evaluated using the Shapiro–Wilk test. Analysis of data was hence performed by the Student’s 𝑡-test or the Wilcoxon test, according to their distribution.p-value of less than 0.05 was accepted as statistically significant. A trend was assumed for p-values ranging from 0.05 to 0.099.Statistical analysis was performed using MedCalc Software Ltd. (Version 19.6–64 bit) Ostend, Belgium. A All patients enrolled in the study completed the VLCKD protocol, and no drop-out was registered. Baseline parameters are detailed in 2, respectively. The values of HOMA index were 5.6 ± 0.8, indicating the presence of insulin resistance. Furthermore, AMH and progesterone levels on the 21st day of the menstrual cycle were 7.8 ± 3.0 ng/mL and 3.4 ± 1.1 ng/mL, respectively. The pre-VLCKD SHBG serum levels were 5.9 ± 4.6 nmol/L.At enrollment, the mean waist circumference (WC) and body mass index (BMI) were 88.4 ± 3.2 cm and 32.8 ± 1.0 Kg/mAfter the 3-month-long VLCKD, the patients showed a significant reduction in the WC A, BMI FB, and HOThe present study was conducted to evaluate the effects of VLCKD on metabolic and ovulatory disorders in a cohort of obese non-diabetic women with PCOS and regular menstruation, family history of T2DM (mother or father), and male partners with normozoospermia. The diagnosis of PCOS was made using the criteria established by the 2003 Rotterdam Consensus  .2 have a prevalence of 4.3%, whereas women with BMI > 30 kg/m2 have a prevalence of 14%. Moreover, PCOS women have a risk of T2DM two-fold-higher than controls, independently of the BMI. This condition is also exacerbated by the reduction in SHBG, which is currently considered a biomarker of metabolic disorders, in particular T2DM. To date, in fact, several studies have demonstrated how SHBG also represents a biomarker of metabolic disorders, such as de novo lipogenesis. This is the formation of fatty acid from non-lipid precursors, such as glucose, and is one of the principal pathways involved in the accumulation of intrahepatic lipids. SHBG, by downregulating de novo lipogenesis, reduces intrahepatic lipids, which increase in the presence of T2DM, obesity, and PCOS [PCOS is the most common endocrine disorder in women of reproductive age and the most frequent cause of anovulatory infertility. It is characterized by metabolic, reproductive, and psychological alterations. The prevalence of PCOS is correlated with BMI. Women with BMI < 25 kg/mand PCOS . ConversOur study highlights that 3-month-long VLCKD had a significant reduction in the BMI and a significant improvement in the SHBG levels, therefore supporting the presence of an improvement in metabolic parameters, which is also evident with the reduction in BMI, waist circumference, and the HOMA index.SHBG gene) is associated with the risk of developing T2DM. These findings suggest that SHBG probably has a direct physiological role in glucose homeostasis [For a long time, it was thought that the common denominator of reduced SHBG synthesis was hyperinsulinemia. More recent studies, however, have shown that the transmission of SHBG-altering polymorphisms  . Today, The reduction in the AMH levels found in the present study may possibly indicate an improvement in the ovulation rate, due to both the increase in SHBG (with reduction in bioavailable androgens) and the reduction in insulin levels (and consequent improvement in LH pulsatility and further reduction in androgens). To date, however, there are no studies that demonstrate that weight loss has beneficial effects on serum levels of AMH, but above all, it is not certain that normalization can be a sign of better ovulatory outcome or a lower rate of ovarian hyperstimulation syndrome in the course of ART. In fact, AMH is a negative prognostic factor during ovarian stimulation cycles: the higher it is, the greater the probability of incurring in a hyperstimulation syndrome (AMH > 3.4 ng/mL).It is known that an adequate production of progesterone in the luteal phase is necessary to improve the secretory phase of the endometrium, which plays a relevant role for the implantation of the embryo. In our study, the patients enrolled had a regular cycle but low levels of progesterone in the mid-luteal phase. After 3 months of VLCKD, we achieved a significant increase in progesterone levels on day 21 of the menstrual cycle, obtaining an improvement in ovulatory dysfunction.To date, there is no specific definition of luteal phase insufficiency. Previous investigations have suggested cutoffs ranging from 10 to 15 ng/mL to differentiate low from normal mid-luteal progesterone levels . The AMIThis is probably due to the fact that higher progesterone levels are necessary to induce appropriate endometrial receptivity. The negative predictive value of a low mid-luteal progesterone cycle was 94.9% .Lower progesterone levels were more common in women with higher BMI, and they were also associated with longer follicular phases than in normal progesterone levels, as if they reflected a lower follicular count or an altered granulosa cell function. Moreover, it has been shown that progesterone also intervenes in the acrosomal reaction of the spermatozoa, playing a crucial role in the intracellular signaling cascade. The acrosome reaction is a calcium-dependent reaction, fundamental in fertilization . It alloWomen with PCOS and regular cycles can undergo anovulatory cycles. In these cases, the measurement of mid-luteal serum progesterone may help as an additional screening test . In factThis is the first study that correlates weight loss with a qualitative and quantitative improvement in a mid-luteal phase marker. Not having a control group with another nutritional protocol, it cannot be said that the increase in progesterone is exclusively due to VLCKD, but it can certainly be deduced that weight loss can improve not only metabolic parameters but also ovulatory ones.This is the first study documenting the effects of VLCKD on ovarian reserve and luteal function in obese women with PCOS. VLCKD can be considered a safe dietary approach and can be included among the therapeutic strategies for the metabolic and ovulatory improvement of women with PCOS. Despite this, having no control group, we cannot be sure that the benefits obtained are due to the ketogenic diet or weight loss itself. Further controlled studies on a larger sample size are needed to confirm our findings. Certainly, one of the advantages of VLCKD is that the metabolic and ovulatory improvement is achieved in a relatively short time, and this should be taken into consideration in obese women who have to undergo ART.


Immediately after a fracture occurs, a fracture hematoma (fxH) is formed. This fxH plays an important role in fracture healing and, under normal circumstances, aids in generating an environment in which a wide variety of cells orchestrate processes involved in fracture healing. MicroRNAs (miRNAs) may influence these processes. The aim of this study was therefore to determine the miRNA expression signature of human fxH in normal fracture healing and examine the potential influence of clinical parameters on these expression levels. during fracture surgery. miRNAs were isolated, transcribed and pooled for qPCR array analysis and validation. Qiagen fibrosis- and inflammation qPCR arrays were used based on an extensive literature study related to fracture healing and osteogenesis. Additionally, miRNA targets were determined.fxH was harvested from 61 patients From the array data, a selection of the twenty most regulated miRNAs, 10 up- and 10 down regulated, was validated in the study population. The expression levels of seven out of these twenty miRNAs were correlated to several clinical parameters. The time interval between trauma and surgery showed to influence the expression of three miRNAs, three other miRNAs were expressed in a patient age dependent manner and one miRNA was associated with the severity of trauma.In silico target analysis revealed 260 mRNA targets for 14 out of the 20 validated miRNAs.This study portrayed the role and importance of miRNAs in human fxH, linked to key processes in fracture healing. Seven miRNAs showed to be involved in multiple processes that are important in the fracture healing cascade, such as angiogenesis, mineralisation and cellular differentiation. These data broaden our view on the potential diagnostic and therapeutic implications of miRNAs in fracture healing. Physiological fracture healing can be divided into four main phases: the inflammatory, soft callus, hard callus, and remodelling phases . The fraDuring the soft and hard callus phases, a more regenerative microenvironment is required for fracture healing to take place, in which chondrocytes and osteoblasts form a granulation tissue called callus. Collagen formation and mineralisation of the granulation tissue are among important cellular processes in normal fracture healing. As time progresses, the callus gets ossified and the fracture gap is bridged. This process depends on the adequate supply of nutrients and cellular activity, which greatly relies on angiogenesis .Failing angiogenesis, or poor vascularization in the tissues surrounding the fracture can therefore also result in impaired fracture healing . The amoDisturbance of the multiphase fracture healing process can result in fracture healing impairments such as delayed unions or non-unions . These fin vitro studies have shown that miRNAs can influence processes that are involved in fracture healing, such as osteogenesis, angiogenesis, and inflammation [In vivo work by Seeliger et al. revealed 5 differentially expressed miRNAs linked to osteoporotic fractures in human serum- and bone tissue samples [MicroRNAs (miRNAs) are small, non-coding RNA molecules consisting of approximately 22 nucleotides. They are important post-transcriptional regulators that play key roles in both inter- and intracellular signalling . MiRNAs ammation ,9. In vi samples . In addi samples . For exa samples . Further samples . In a st samples . AlthougTo the best of our knowledge, no research has been done on miRNA expression in human fxH. Given the association of miRNAs with bone regeneration, it is important to investigate the miRNA expression and its regulation in human fxH and analyse the role of these miRNAs in the different processes within the fracture healing cascade. In particular, the influence of patient characteristics and clinical parameters on miRNA expression must be evaluated to assess the potential clinical application of miRNAs. The aim of this study is therefore to determine the miRNA expression signature of the human fxH in normal fracture healing, and to examine the potential relationship between clinical parameters and these expression levels. MiRNAs can have multiple mRNA targets which affect different cellular processes. Determining these mRNA targets is therefore required to clarify the physiological functions of miRNAs in the different phases of the fracture healing cascade.22.1Patients with a long bone fracture who were admitted to the division of traumasurgery at the Maastricht University Medical Center (MUMC+) were screened for inclusion in the study. Inclusion criteria were age ≥18 years and a fracture of long bones requiring open reduction and internal fixation (ORIF). Exclusion criteria were pathological fractures, fractures that occurred more than 21 days before fracture surgery, contaminated open fractures, irrigation of the fracture site prior to fxH harvesting and active bleeding at the fracture site if this prohibited accurate fxH harvesting.To accurately harvest fxH, the surgeon performed this during ORIF fracture surgery according to a standard surgical procedure using a non-traumatic technique. Harvesting fxH was only performed directly from, or near the fracture site; harvesting surrounding tissue, as well as harvesting from the medullary cavity, was avoided since this may introduce other tissue or bone marrow to the sample.For analysis, the following patient characteristics were obtained: fracture healing outcome , gender, age, smoking (pack years), number of days between trauma and primary surgery, fracture occurred during multi-trauma event (yes/no), diabetes mellitus (yes/no), osteoporosis (yes/no), hypertension (yes/no), and arthritic diseases (yes/no). An important parameter in this study is the time interval between trauma and primary surgery, as this allows for miRNA expression analysis at different phases of fracture healing. Normal fracture healing was defined as adequate consolidation based on X-ray imaging and examination throughout a clinical follow up after 6 weeks, 3 and 6 months.This study was approved by the local ethical committee of the MUMC+ . The study was performed according to the Declaration of Helsinki in its most recent version.2.2Immediately after harvesting, the fxH was transferred to 2 ml microcentrifuge tubes  using sterile tweezers after which it was snap frozen in liquid nitrogen. Prior to RNA extraction, the fxH was disrupted by adding Trizol Reagent  and using a Qiagen TissueLyser LT . The homogenate was snap frozen in liquid nitrogen and subsequently lysed for 5 min. Trizol Reagent was added in a 0.7:1 ratio, based on fxH sample volume.RNA extraction was performed by chloroform phenol extraction. The amount and purity of the RNA isolates were analysed by spectrophotometry . For samples to be included in this study, RNA purity cut off values of 1.7 and 1.8 were chosen for the A260/A230 and A260/A280 ratios respectively. Per patient, 250 ng template RNA were used for the transcription of miRNA to cDNA using the miScript II RT kit  according to the manufacturer's instructions.2.3For miRNA array analysis, patient cDNA samples were pooled by using equal amounts of cDNA from each sample. Two arrays for miRNAs were used, that being the inflammatory response and autoimmunity array  and the fibrosis array . The miRNA signature of the pooled sample group was determined by these two qPCR miRNA arrays containing primers for 84 individual miRNAs each, excluding housekeeper genes and controls. Since the two arrays showed overlap with regard to the miRNA primers that they contained, the total amount of miRNAs that were investigated was 145. SNORD61, SNORD68, SNORD72, SNORD95, SNORD96A, RNU6-6p were used as housekeeper genes. Further controls included the miRNA reverse transcription control, positive PCR control and the spike in control cel-miRNA-39-3p.The expression levels of the miRNAs from the array were determined by the cycle number (Cq) via qPCR using the CFX96 Real-Time PCR system . A ΔCq ≥2 was chosen as a cut-off value to define up- or downregulation. To assess the quality of the qPCR array reactions, melting curve analyses were performed in combination with a chosen cut off Cq value of 35, above which miRNAs were not considered for further analysis due to minute or absent expression.From each of the two arrays, the top 5 upregulated and top 5 downregulated miRNAs were selected for further individual validation within the study population. This resulted in a total of 20 miRNAs for further validation in the study population.2.4The selected 20 miRNAs were validated in each individual sample. For miRNA validation in the individual samples, SNORD96A was chosen as housekeeper gene since its expression approximated the mean expression of the six housekeeper genes provided in the array the most. The regulated miRNAs and the housekeeper gene were validated in duplicates for all patients using the miScript SYBR Green PCR Kit  and the CFX96 Touch Real-Time PCR system . The expression of each miRNA was determined by the Cq value, which was normalised to the average of SNORD96A expression by means of the ΔCq method. To assess the quality of the single patient qPCR reaction, melting curve analyses were performed in combination with a chosen cut off Cq value of 38, above which the miRNA was not considered to be present in the sample.2.5Qiagen's Ingenuity Pathway Analysis (IPA) was used to identify miRNA targets of the validated miRNAs, which expression levels showed to be dependent on clinical parameters. The IPA software groups miRNA targets based on known scientific data, level of evidence, and cell & tissue type. For this study, experimentally observed and highly predicted human miRNA targets were incorporated into the analysis. The following cell types were included in the analyses: osteoblasts, chondrocytes, mesenchymal stem cells, stromal cells, fibroblasts, neutrophils, and bone marrow cells.2.6All analyses were performed with GraphPad Prism version 9.1.1 . Data are presented as mean or number, accompanied by standard deviation or percentage as appropriate. Gender, smoking, age, and number of days between trauma and primary surgery were recorded as continuous variables, the remaining variables were dichotomised.Descriptive statistics were performed on the obtained patient characteristics and miRNA expression data. Subsequently, a stepwise multiple linear regression model was made to examine potential correlations between miRNA expression and patient characteristics. The following parameters were incorporated: gender, age, smoking (pack years), number of days between trauma and primary surgery, fracture occurred during multi-trauma event, diabetes mellitus, osteoporosis, hypertension, and arthritic diseases.Multiple linear regression, Mann–Kendall test, two samples t-test, and simple linear regression analyses were applied as appropriate to determine the effect of clinical parameters on miRNA expression. For this study, an α of 0.05 was considered statistically significant.33.1The study population consisted of 61 patients . Patients underwent surgery 7 ± 5 days after trauma. All patients showed uneventful fracture healing after surgery. Thirty-one percent of patients smoked; no data could be collected on smoking for the remaining 30% of patients. Twenty-one percent of the patient population suffered from a fracture that occurred during a multi-trauma event and 34% of patients suffered from one or multiple comorbidities . No sign3.2A total of 145 miRNAs were analysed. In the inflammatory response and autoimmunity array, 76 miRNAs were detected in the fxH samples, out of which 43 were up- or down-regulated . In the From each of the two arrays, the top 5 upregulated and top 5 downregulated miRNAs, 20 in total, were selected for further individual validation within the study population .3.3Each of the 20 miRNAs was validated in the study population, validation data were consistent with array data . All 20 3.4Multiple linear regression analyses showed that expression levels from seven of the 20 validated miRNAs were dependent on the following clinical parameters: patient age, number of days between trauma and primary surgery, or fracture in multi-trauma. For all miRNAs that showed expression based on patient age, or the number of days between trauma and primary surgery, Mann–Kendall tests showed significant differences . Neither comorbidities nor smoking influenced miRNA expression levels in fxH in this study population.For three miRNAs, expression was correlated on the time interval between trauma and primary surgery: miRNA-21-5p, miRNA-216a-5p, and miRNA-223-3p , Fig. 5.Three other miRNAs showed patient age-dependent expression: let-7a-5p, miRNA-20a-5p, and miRNA-126-3p , Transforming Growth Factor Beta Receptor 2 (TGFBR2), Tumor Necrosis Factor (TNF), Vimentin (VIM), Nuclear Factor 1A (NFIA), Signal Transducer and Activator of Transcription 3 (STAT3), and Vascular Endothelial Growth Factor A (VEGFA) , Fig. 6.4in silico target prediction of their mRNA targets in the normal fracture healing cascade. MiRNAs are important regulatory molecules in fracture healing, this study can therefore potentially function as a benchmark for future research on miRNAs in fxH, both in normal as well as impaired fracture healing.To the best of our knowledge, the present study is the first to report on the miRNA involvement in human fxH, correlations between miRNA expression in the human fxH and clinical parameters, and 4.1in vivo. This is in line with the results from our in silico target analysis which, among others, identified PTEN, TGFBR2, TNF, VIM and NFIA as mRNA targets for miRNA-21-5p [The time interval between trauma and primary surgery showed to influence the expression of three miRNAs, miRNA-21-5p, miRNA-223-3p, and miRNA-216a-5p. In this study, miRNA-21-5p was upregulated and increasingly expressed over time. MiRNA-21-5p is a mechanosensitive, anti-inflammatory miRNA whose expression increases in inflammatory conditions. It has been shown to enhance osteogenic differentiation, angiogenesis, mineralisation, and osteoclastogenesis NA-21-5p . FurtherNA-21-5p . This isMiRNA-223-3p, upregulated and decreasingly expressed over time, is an important regulator of the immune response. It shows high expression by hematopoietic cells and is involved in, among others, macrophage and neutrophil functioning. It dampens the inflammatory response and, like miRNA-21-5p, enhances M2 macrophage polarisation . Furtherin vivo in osteoporosis [MiRNA-216a-5p, downregulated and increasingly expressed over time, has shown to enhance osteogenic differentiation and bone formation oporosis . The ostoporosis ,18. Furtoporosis . The expPutting the expression levels of these three time dependently expressed miRNAs in perspective, it can be stated that the increasing expression of miRNA-21-5p over time matches physiological fracture healing since it dampens the immune response and enhances multiple processes that are involved in osteogenesis. The gradual decline of miRNA-223-3p expression over time could be attributed to the physiological fracture healing cascade in which the inflammatory phase transiently progresses towards a more stable, regenerative phase where soft callus is formed [4.2Three miRNAs were expressed in an age dependent manner, all of which were upregulated and higher expressed in younger patients. One of the three, let-7a-5p, is among the first miRNAs discovered and has numerous functions within eukaryotic cells, such as the augmentation of inflammation and apoptosis, whilst reducing cellular proliferation . InteresMiRNA-20a-5p has shown to reduce inflammation and apoptosis, whilst enhancing osteogenic differentiation and modulating angiogenesis ,27,28. Tin vivo [Lastly, miRNA-126a-3p has shown to promote chondrocyte migration and proliferation whilst reducing inflammation and apoptosis in vivo . Furtherin vivo ,32. Howein vivo .Placing the age dependent decline in expression levels of the above-mentioned miRNAs and their targets into perspective offers novel insights. The pro-inflammatory characteristics and seemingly contradictory effects of let-7a-5p on osteogenesis point out the requirement for more specific research in the field of bone regeneration in order to make solid claims about its function therein. On the other hand, both miRNA-20a-5p and miRNA-126a-3p reduce inflammation and apoptosis whilst enhancing osteogenesis and regulating angiogenesis. However, research on the mechanisms behind this regulation is lacking, underlining the need for future research to examine their angiogenic characteristics in the context of fracture healing.4.3The expression of miRNA-141-3p was dependent on the severity of the trauma; mono-vs. multi-trauma. MiRNA-141-3p showed decreased expression in the fxH of multiply traumatised patients as compared to the fxH of mono-trauma patients. The immune system might play a role in this expression pattern since the severity of the trauma greatly influences the immunological response . MiRNA-1This study has several limitations, the first being a lack of normal control samples. Unfortunately, due to the nature of the samples, controls are inexistent as a fracture hematoma only exists after a fracture occurs. Secondly, the number of patients in some subgroups, such as the different comorbidities, is low. Due to the prevalence, and multi-faceted pathophysiology of impaired fracture healing, investigating the influence of altered miRNA expression on cellular pathways in fracture healing disorders will require a larger sample size.in vitro application of microRNAs in both 2D, as well as 3D models, is required to gain more insights into their involvement in biological pathways.This study is amongst the first to investigate the expression of miRNAs in patients on this scale and instigates to further investigate the pathophysiology of impaired fracture healing. Collaborations between different research centers can be of great importance therein to enable a more rapid gathering of samples from different disease categories/conditions. Moreover, to accurately map potential tissue specific microRNA expression, it is important to research their expression in other tissues related to fracture healing, such as bone itself, periosteum, or soft tissues surrounding a fracture site. Finally, we believe that the 5In conclusion, this study profiled the miRNA expression signature of the human fxH in normal fracture healing and examined the potential relationship with clinical parameters on these expression levels. Eighty-eight miRNAs showed to be up- or downregulated. The expression levels of seven out of the twenty validated (most regulated) miRNAs were correlated to the number of days between trauma and surgery, as well as to patient age or the severity of trauma.In silico target analysis revealed 124 mRNA targets for these seven miRNAs, which all showed to be involved in multiple processes that are important in the fracture healing cascade, such as angiogenesis, mineralisation, and cellular differentiation. Furthermore, several miRNAs showed to influence the same mRNA targets and can therefore operate in a synergistic manner. This study portrayed the regulatory role and importance of miRNAs in fracture healing. Furthermore, this study may serve as a reference for future studies that investigate miRNA expression in impaired fracture healing, that is a non- or poorly healing patient cohort with possible infection, chronic inflammation, poor vascularisation, osteoporosis, high dose corticosteroid treatment, diabetes, or other comorbidities present. Future research should therefore focus on how aberrant miRNA expression could be correlated to impaired fracture healing, for which larger cohorts are necessary due to the lower prevalence of this condition. This should also be associated with concomitant changes in target mRNA expression and protein translation. Molecular mechanisms that have been elucidated in this study can serve as a means for future research in the field of fxH.N.A.There are no conflicts of interest to declare.


Eleutherine plicata, using the micronucleus test and the impact of structural alterations on toxicity and molecular docking (topoisomerase II and DNA complex). The extract was obtained by maceration and fractionation in a chromatography column. The genotoxicity was evaluated by the micronucleus test in human hepatoma cells (HepG2). Isoeleutherin was the starting molecule in the search for analogues by structural similarity, using the ZINC and e-Molecules databases. Isoeleutherin and analogues were subjected to in silico toxicity prediction, and compounds free of toxicological risks  were selected for molecular docking in Topoisomerase II (PDB: 1ZXM). In the micronucleus test, isoeleutherin was less genotoxic. Among the 22 isoeleutherin analogues there were variations in the toxicity profile. Molecular docking studies showed that the compounds have good complementarity in the active site with important hydrogens bonds. Therefore, the structural changes of isoeleutherin led to the obtaining of a molecule with a lower mutagenic potential, and the CP13 can be considered a prototype compound for the development of new molecules with pharmacological potential.This study evaluated the genotoxicity of Ethanol Extract (EEEp), Dichloromethane Fraction (FDCMEp) and isoeleutherin isolated from  Eleutherine plicata Herb. (Iridaceae) is a native American plant, with reports of occurrence in other tropical countries and regions such as Southern Africa, the eastern Mediterranean, and Central and South America. In Brazil, it is popularly known as marupari, marupazinho, marupá-piranga, palmeirinha, lírio-folha-de-palmeira and wáro. It presents itself in the form of a clump with red bulbs similar to onions, features whole, simple and pleated leaves with colorful flowers from white to pink. This species is widely used in Brazilian popular herbal medicine [medicine ,2.Eleutherine plicata Herb. has as heterotypic synonym Eleutherine bulbosa (Mill.) Urb, Cipura plicata (Sw.) Griseb, Eleutherine americana (Aubl.) Merr. ex K. Heyne, Eleutherine anomala Herb, Eleutherine longifolia Gagnep, Eleutherine subaphylla Gagnep, Ferraria parviflora Salisb, Galatea americana (Aubl.) Kuntze, Galatea plicata (Sw.) Baker, Galatea vespertina Salisb, Ixia americana Aubl, Marica plicata (Sw.) Ker Gawl, Moraea plicata Sw, Sisyrinchium americanum (Aubl.) Lemée, Sisyrinchium capitatum Pers, Sisyrinchium elatum Seub. ex Klatt, Sisyrinchium latifolium Sw, Sisyrinchium plicatum (Sw.) Spreng, Sisyrinchium racemosum Pers, Bermudiana bulbosa (Mill.) Molina, Galatea bulbosa (Mill.) Britton and basionym Sisyrinchium bulbosum Mill [sum Mill .E. plicata has popular claims as an anthelmintic, a menstrual regulator and abortifacient, and for disorders of the gastrointestinal tract. Scientifically, antimicrobial, healing, purgative, abortive, cardiovascular and respiratory system activities have been proven [The species is widely used in popular medicine in almost all regions of Brazil, with the heaviest usage in the Amazon region, due in large part to the ethnopharmacological dimensions of indigenous societies to which its first uses are related. n proven .E. plicata include anthraquinones , organic acids, alkaloids, aurones, coumarins, chalconam flavonoids and, primarily, naphthalene, anthraquinones and naphthoquinones. These metabolites are listed in the List of Medicinal Plants of Interest to SUS–Brazilian Unified Health System (RENISUS) [The classes of metabolites isolated from RENISUS) .E. plicata against malaria parasites [This species has been shown to be an important source of chemicals such as naphthoquinones, a class of natural products with various biological activities. The following compounds have already been isolated from this species: isoeleutherin A, eleutharasites  and as aarasites  and antiarasites . Pharmacarasites ,10.E. plicata were submitted to the micronucleus assay and we observed that the fractionation of the ethanol extract from E. plicata bulbs (EEEp) led to a fraction with higher toxic potential (Dichloromethane Fraction (FDCMEp)). Eleutherin and isoeleutherin were isolated from this fraction and proved to be less toxic than FDCMEp. The mutagenic potential test of eleutherin and isoleutherin in the Allium cepa model demonstrated that eleutherin has a higher mutagenic potential [Samples of otential . Anotherotential . In ordeotential .In another study, the isoeleutherin and eleutherin ability to stabilize the DNA-topoisomerase II complex was evaluated and the stabilization of the complex was confirmed . This stin silico study of isoeleutherin analogues, evaluating the impact of structural changes in toxicity and molecular docking in the stabilization of topoisomerase II and DNA (TOPO II) complex.The present study evaluated the genotoxicity of EEEp, FDCMEp and isoeleutherin using the micronucleus test. In addition, we performed an E. plicata (yield = 8.65%) the dichloromethane fraction was obtained (yield = 15.66%), in which the presence of naphthoquinone was detected by thin layer chromatography (TLC). The fractionation of FDCMEp led to the isolation of three subfractions , with S2 being identified as isoeleutherin. Other studies of this species, carried out by the same group, also isolated and identified isoeleutherin [From the ethanolic extract of eutherin ,8,10.E. plicata and are associated with the biological activities described for the species. They are quinones associated with the naphthalene system, a general group characterized by the presence of two carbonyl groups formed together by a conjugated system of two double bonds (C-C). Naphthoquinones are associated with the cytotoxic potential linked to the species, especially isoeleuterine through the inhibition of transcription factors [Naphthoquinones are a major group found in  factors ,7,8,10.EEEp, FDCMEp and Isoeleutherin were evaluated by micronucleus frequency using human hepatoma cells (HepG2). The fractionation of EEEp contributed to the increase in frequency. FDCMEp presented higher rates than EEEp and isoeleutherin with lower toxicity .However, a different study evaluated the genotoxicity of these samples using the comet assay and the same cell line, with the highest rate of damage observed in cells treated with isoeleutherin .50= 28.71 μg/mL + 1.054. The FDCMEp presented CC50= 26.19 μg/mL + 1.670. When comparing the results obtained, an increase in cytotoxicity was observed, suggesting a higher concentration of naphthoquinones in the FDCMEp. The FDCMEp subfractions resulted in CC50= 14.36 μg/mL + 1.12 (S1) and CC50= 18.67 μg/mL + 1.25 (S2), indicating that the fractionation contributed positively to the cytotoxic activity [Studies that evaluated the contribution of fractionation of the EEEp extract in relation to cytotoxicity in VERO cells obtained results of CCactivity .Allium cepa method, showed the rate of chromosomal aberrations caused by isoeleutherin was lower than the positive control, colchicine [Another study, using the lchicine . In summlchicine  while thlchicine . In ordeStudies aimed at predicting the toxicity of drug candidates are an important phase of development and cannot be excluded. Many drugs are withdrawn from the market due to toxic metabolites generated by these substances . Over thIsoleutherin and analogues were submitted to toxicity prediction studies in different models, including: Algae, Daphnia, Medaka, Minnow, cytotoxicity, mutagenicity, carcinogenicity and HeRG. The structural alterations of isoeleutherin do not seem to change the toxicity , suggestE. plicata, have been related to a redox potential of these constituents. Free radicals have one or more unpaired electrons, making them unstable and highly reactive. Examples of free radicals are: the hydrogen atom, superoxide (O2•-), hydroxyl (OH•) and nitric oxide (NO•). Oxidation is a fundamental part of the aerobic pathway and metabolism; thus, free radicals are produced naturally. These free radicals, whose unpaired electron is centered on oxygen or nitrogen atoms, are called reactive oxygen species (ROS), or reactive nitrogen species (RNS) [Biological activities of naphthoquinones, present in extracts and fractions of es (RNS) ,23, respes (RNS) .E. plicata, E. americana and Cipura paludosa are described in the literature as an important source of naphthoquinones. These species present high levels of this class of natural products and diverse biological activities. Aqueous extract obtained from dried bulbs of these herbs has been described as generating oxidative stress by inducing the deleterious endogenous formation of a bioactive oxygen-derived species that promotes apoptosis, inhibits inflammation and has important anticancer properties in different cancer cell lineages, such as glioma (U-251), breast (MCF-7), ovary (NCI/ADR-RES), kidney (786-0), non-small cell lung (NCI-H460), colon (HT-29), HepG2 cells and leukemia (K562) [Species such as a (K562) ,25.Regarding acute oral toxicity, some compounds were classified as class 3 , others as class 4  and some do not have data available to allow such analysis  in the TOPO II enzyme (PDB code: 1ZXM). This target was selected because isoeleutherin stabilizes the DNA-TOPO II complex and this could be involved in cell death mechanism . The valBased on the toxicity prediction studies, we selected CP13, CP14, CP17 and isoeleutherin to perform the TOPO II molecular docking study. From the values of the scores obtained in the GOLD program, it was observed that the compound CP13 presented the highest value (59.27), followed by CP14, CP17 and isoeleutherin, which presented values of 52.76, 48.45 and 44.15, respectively.From E. plicata bulbs were collected in Vila Fátima, a municipality of Traquateua in the state of Pará, Brazil  The botanical identification was deposited in the Herbarium João Murça Pires, of the Museu Paraense Emílio Goeldi under the registration MG. 202631. Subsequently, the bulbs were washed in running water, cut into smaller pieces and subjected to the drying process in a circulating air oven. After drying, they were ground in knife mills to obtain the powder and subjected to maceration in 96° GL ethanol. Then, the extractive solution was concentrated, resulting in the EEEp [The the EEEp .The EEEp was subjected to fractionation in column chromatography with silica gel as the stationary phase, resulting in 4 fractions: hexane, dichloromethane, ethyl acetate and methanol. The dichloromethane fraction (FDCMEp) was subjected to fractionation by preparative thin layer chromatography, obtaining 3 subfractions . The S2 fraction was identified by NMR as isoeleutherin.The reference molecule in this work, isoeleutherin, was used to search for analogues through structural similarity in the ZINC  and e-MoA combination of online tools (PreADMET and ProTox-II) was used to predict various toxicity outcomes such as acute toxicity, hepatotoxicity, cytotoxicity, carcinogenicity, mutagenicity and immunotoxicity. These tools use the structure of the ligand to correlate with extant drugs and toxic compounds and with known toxicity. In this study, the predictions evaluated the similarities for mutagenic activity, toxicological dosage and the level for different tissues in different models. Pharmacologically relevant properties of the compounds were predicted. The models employed here, PreADMET and ProTox-II, are tools based on the Quantitative Structure–Activity Relationship (QSAR) where the molecular structure is related to the effects on ecosystems and principally on human or microbiological metabolism. To this end, the QSAR model performs two processes. First, it correlates the chemical structure to a biological activity from existing products and then predicts values related to possible action through mathematical regression calculations. Calculations are based on variable predictions (X) and variable potential responses (Y). Among the primarily analyzed molecular properties, understood here as descriptors, are the physical–chemical and topographical characteristics of the molecules that can be applied in 2D or 3D, being used by us for the 2D model. The speed afforded by these tools allowed us to characterize what was observed in the in vitro study, whether isoeleutherin and its analogues hypothetically presented toxicity in the studied models and whether their use could lead to unwanted organic dysfunctions based on the relationship between their chemical structure and known toxic groups. The method is widely used to select molecules with an accurate potential to transform into drugs .Molecular docking was used to explore the possible conformations of the ligand with the binding receptor, estimating the strength of protein–ligand interaction . Isoeleu5 cells/mL in RPMI-1610 medium supplemented with 10% fetal bovine serum at a temperature of 37 °C and a humidified atmosphere containing 5% CO2 for 20h. After this period, the cells were treated with the following concentrations of samples: EEEp , FDCMEp  and isoeleutherin . The negative control consisted of cells and culture medium and the positive control was composed by cells treated with doxorubicin (0.02 µg/mL), due to its previously known genotoxicity. After a period of 24h, 3 µg/mL of cytochalasin-B was added and 24 h later the cells were trypsinized and centrifuged at 1000 rpm/5min, the supernatant was discarded and 5 mL of ice-cold hypotonic solution (0.075 M KCl) was added. Later, they were fixed with methanol and acetic acid for the preparation of slides, dried at room temperature and stained with 5% Giemsa [The micronucleus test was performed with HepG2 cells, distributed in a 12-well plate at a concentration of 2 × 10% Giemsa . The anaFor micronucleus analysis, 1000 binucleated cells were selected with intact nuclei and membrane. Those chosen were of identical morphology to the main nuclei, with a diameter between 1/16 to 1/3 of the main nuclei. The micronuclei could not have refringence or be connected to one main nuclei; they should have the same coloring as the nuclei; and could be adjacent to, but not superimposed on the nuclei. The Nuclear Division Index (NDI) was calculated by the following formula:fied (N) .Isoeleutherin was the compound that showed the lowest cytotoxicity in the micronucleus assay; therefore, it did not present toxicological risks. The structural changes suggest an increase in affinity with the TOPO II enzyme, observed in the increase in the amount of hydrogen bond interactions performed with amino acid residues of the active site. Using an integrative approach of in vitro and in silico studies to better understand the genotoxicity of isoeleutherin, we obtained molecular results from docking that suggest a good association with the biological activity observed for TOPO II, demonstrating that these compounds can be a good starting point in the search for new drugs for anticancer therapy.


The goal of this study was to establish an optimized metabolic panel by combining serum and urine biomarkers that could reflect the malignancy of cancer tissues to improve the non-invasive diagnosis of esophageal squamous cell cancer (ESCC).Urine and serum specimens representing the healthy and ESCC individuals, together with the paralleled ESCC cancer tissues and corresponding distant non-cancerous tissues were investigated in this study using the high-resolution 600 MHz 1H-NMR technique.We identified distinct 1H NMR-based serum and urine metabolic signatures respectively, which were linked to the metabolic profiles of esophageal-cancerous tissues. Creatine and glycine in both serum and urine were selected as the optimal biofluids biomarker panel for ESCC detection, as they were the overlapping discriminative metabolites across serum, urine and cancer tissues in ESCC patients. Also, the were the major metabolites involved in the perturbation of “glycine, serine, and threonine metabolism”, the significant pathway alteration associated with ESCC progression. Then a visual predictive nomogram was constructed by combining creatine and glycine in both serum and urine, which exhibited superior diagnostic efficiency (with an AUC of 0.930) than any diagnostic model constructed by a single urine or serum metabolic biomarkers.Overall, this study highlighted that NMR-based biofluids metabolomics fingerprinting, as a non-invasive predictor, has the potential utility for ESCC detection. Further studies based on a lager number size and in combination with other omics or molecular biological approaches are needed to validate the metabolic pathway disturbances in ESCC patients. Esophageal cancer (EC) is the sixth leading cause of cancer death and the eighth most common cancer worldwide, with a 5-year survival rate of only 15%-25% . ESCC of1H NMR)-based metabolomics provides simple, efficient and inexpensive technical support to discover potential cancer metabolic biomarkers for ESCC detection, mainly focusing on the analysis of urine, serum and tissue samples  with signed informed consent. ESCC patients were matched with HCs according to age and gender. The experimental group included 50 ESCCs patients who underwent esophagectomy and provided their esophageal cancer tissues (ECT), distant non-cancerous tissues , along with preoperative serum and urine samples. Control serum and urine samples were obtained from 50 HCs with no history of associated gastrointestinal problems. As for the validation group, serum and urine obtained from 20 ESCCs and 20 HCs were collected for verifying the predictive ability of the metabolic model in the experimental group. All samples were stored in a low-temperature refrigerator (−80°C) until further metabolic extraction. ESCC patients who received radiotherapy or preoperative chemotherapy were excluded. 2O stock solution (50 μL). After centrifugation of the mixture for 5 mins at 10,000 rpm, 500 μL of the supernatant was transferred to an NMR tube (5 mm in size) for 1H NMR spectroscopy. As for frozen serum and urine samples preparation, they were thawed at 25°C, respectively. After adding 200 μL PBS/D2O buffer to 400 μL of each urine and serum sample, respectively, the mixtures were then swirled for 60 seconds and centrifuged at 10,000 rpm for 5 minutes. Finally, 1H NMR analysis was carried out by using 500 μL of the serum supernatant. While for urine, 50 μL of TSP/D2O stock solution was added to the supernatant before 1H NMR spectral acquisition.Esophageal tissue, serum and urine were pre-processed according to our previous publications , 17. Bas1H NMR spectra of all samples were acquired by using a Bruker Avance NMR spectrometer  operating at 600MHz and 298K. NOESYPR1D pulse sequence was used to detect the 1H-NMR spectrum of esophageal tissues and urine samples, and the acquisition parameters were as follows: 90° Pulse Width, 14.1 μs; Relaxation Delay (RD), 4.0 s; Echo Time (TE), 70 ms; Spectral Width (SW), 12335 Hz; number of points (TD), 32,768; number of scans (NS), 64; acquisition time (AQ), 2.66 s. By using the 1D CPMG pulse sequence, serum 1H NMR spectra were recorded, and the acquisition parameters were as follows: 90° pulse width, 12.2μs; RD, 4.0 s; TE, 70 ms; SW, 12019 Hz; TD, 32,768; NS, 64 and AQ, 2.73 s. Irradiating a peak of water between RD and tm to achieve water suppression.Before Fourier Transformation, all FIDs were multiplied by a 1 Hz line-broadening to enhance the SNR. The spectrum was then manually phase adjusted, followed by baseline correction and frequency alignment with reference to the 0.0 ppm TSP signal. In order to minimize the complexity of the spectrum data, the spectral regions ofδ9.0-0.5 were divided into buckets with 0.004 ppm width, along with the removal of the residual water (4.5 to 5.5 ppm). Each bucket was normalized to the total integral of the spectrum to reduce concentration differences between samples while improving sample comparability.2Y values (ranging from 0 to 1) and Q2 values (ranging from negative to 1) were used to assess the model quality. A value of R2Y equal to 1 indicates that the model explains 100% of the variance, and a value of Q2 close to 1 means a reliable prediction in cross-validation. By using a permutation test of 400-times, the quality of OPLS-DA model was validated, where R2Y represents the degree of fit between the model and the data, and Q2 indicates how strong the predictive power of the model is, with a Q2 greater than 0.5 considered “good” and a Q2 greater than 0.9 deemed “excellent”. The peak selection coefficients were the VIP (variable importance of projection) from the OPLS-DA model. By integrating the area under the peak, the relative levels of the chosen peaks with VIP greater than 1 were quantified. The major metabolites in the profile were identified according to the available literature  to perform pattern recognition based on previous publications , 17, to terature –21 and dhttps://xenabrowser.net/datapages/). The expression of key genes related to the important metabolic pathway in ESCC cancer tissues were extracted from TCGA-ESCA  and GTEx databases. The expression of RNAseq data in TPM (transcripts per million reads) format and log2 transformation was compared between samples.The RNAseq data in TCGA and GTEx TPM format were processed uniformly by Toil process from UCSC XENA  corrections. False discovery rate (FDR) corrections were calculated to account for the multiple comparisons that often occur in metabolomics studies. In order to evaluate the diagnostic performance of metabolic biomarkers, ROC analysis was conducted and the area under the curve (AUC) was calculated using SPSS 16.0, with AUC≥0.8 indicating good diagnostic ability. Spearman correlation analysis was used to assess the association of potential biomarkers in the urine, serum and cancer tissues of ESCC patients using correlation coefficient (r). The cutoff value of the correlation coefficient r was determined according to the sample size. Correspondingly, |r|≥0.28 indicates a significant correlation between two kinds of metabolites, based on the sample size of 50 in the experimental group.The normality test was conducted for all data and the corrected 1H-NMR spectra of ECT and DNT, as well as those of serum and urine samples from ESCC patients and HCs. In the pattern recognition of the processed 1H NMR spectra from tissue extracts, serum and urine in ESCC patients and those from their corresponding controls, the score plots revealed a differential clustering alongside the first principal component direction , a number of potential metabolite biomarkers in the tissue, serum and urine in ESCC patients were observed, as shown in Under such criteria  value≥2. As can be seen in According to the analysis of TCGA-ESCA data, we found that the expression of serine hydroxymethyltransferases 1, 2  and glycine decarboxylase (GLDC), which catalyze the conversion of serine to glycine, were upregulated in ESCC cancer tissues compared with normal tissues  is mainly based on the clinical symptoms and invasive pathological examination, which lags far behind the disease progression and has limited role in cancer early diagnosis. Non-invasive, simple and accurate biomarkers may be more suitable for population screening. In addition to genetic and proteomic alterations, ESCC development is in association with cellular metabolic alterations that can give discernment into disease pathogenesis –24. In tAs can be seen in Cancer cells typically exhibit high rates of anabolic metabolism, by which they absorb large amounts of nutrients to promote TCA cycling and oxidative phosphorylation. Thus, in addition to energy metabolism, tumor cells exhibit changes in nutrients to meet the increased amino acid metabolism for proliferation. Therefore, the amounts of amino acids, such as isoleucine, valine, leucine, arginine, lysine, and asparagine in ESCC cancer tissues, were relatively higher than those in the distant non-cancerous tissues, implying an active protein synthesis and amino acid metabolism in tumor tissues. This is because they are branched-chain amino acids that can be synthesized by condensation of the corresponding keto acids to promote protein biosynthesis. In addition, they can enter the tricarboxylic acid cycle through succinyl-CoA to promote energy metabolism , 34. TheGlycine and creatine were overlapping discriminatory metabolites across serum, urine and tissue samples in ESCC patients in this study. Besides, they were the main metabolites involved in the perturbation of glycine, serine and threonine metabolism, the most significant pathway alteration, suggesting that the serine/glycine synthesis and one-carbon unit metabolism pathways were disturbed, so as to maintain the malignant hyperplasia of the tumor. One-carbon unit is a key metabolic pathway for cell proliferation, mainly derived from the metabolism of glycine, serine, histidine and tryptophan. Its main function is to participate in the synthesis of purine-pyrimidine, choline and epinephrine, as well as the synthesis and modification of DNA and RNA , 36. TheCreatine and glycine in both serum and urine were selected as the optimal biomarker panel for ESCC detection based on the following criteria: (i) they were the overlapping discriminative metabolites across serum, urine and tissue samples in ESCC patients; (ii) they were the major metabolites involved in the disturbance of “glycine, serine, and threonine metabolism”, the important metabolic pathway perturbation associated with ESCC progression; and (iii) changes in their biofluid levels were associated with changes of key genes expression involve in “glycine, serine, and threonine metabolism” in ESCC cancer tissues. Then, a predictive nomogram model was constructed by combining creatine and glycine in both serum and urine, which improved the diagnostic efficiency of ESCC compared to individual serum or urine metabolic biomarkers.Nomogram is a mapping prediction model that comprehensively analyzes multiple quantitative and qualitative variables to predict the occurrence of a specific event. It can use intuitive mapping to assess the risk of individual patients. The model can be based on Logistic regression model and Cox regression model, and its results can be visualized. Specifically, the scoring standard is formulated according to the size of the model regression coefficient, and a score is then assigned to each value of each independent variable to calculate the total score of each patient. Subsequently, the probability of outcome occurrence of each patient can be calculated through the conversion function between the score and the probability of outcome occurrence. Its axis structure and risk points reflect the impact and importance of each variable on the prediction results. Nomogram is a practical and convenient tool for clinical application, which has been widely used in the research in tumor prediction, lymph node metastasis, prognosis, and efficacy judgment –44. The This study has limitations, including a small sample size, a limited number of precancerous lesions and tumors at each stage, lack of genomics-derived molecular features or validation of the metabolic pathway disorders at other systemic biological levels, which need to be studied and analyzed in the near future.Our paralleled investigation observed a considerable number of changed metabolites in ESCC cancer tissue, serum and urine, demonstrating the networks for metabolic pathway disturbance in ESCC subjects. The changes in urine and serum metabolism in esophageal cancer could reflect the metabolic disorders of the cancer tissues, highlighting that NMR-based biofluids metabolomics fingerprints, as non-invasive predictors, have the potential utility for ESCC detection. The visual predictive nomogram model based on creatine and glycine in both serum and urine could improve the diagnostic efficiency of esophageal cancer. Further studies based on a larger number size and in combination with other omics or molecular biological approaches are needed to validate the metabolic pathway disturbances in ESCC patients.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 Ethical Review Board of Shantou University Medical College. The patients/participants provided their written informed consent to participate in this study.TO: Investigation, Data curation, Writing- Original draft preparation. CM: Investigation, Methodology, Resources. YZ: Investigation, Methodology, Resources. WY: Investigation. JZ: investigation. RC: Investigation. HZ: Investigation, Resources. PZ: Investigation. YL: Conceptualization, Methodology, Writing- Reviewing and Editing. The work reported in the paper has been performed by the authors, unless clearly specified in the text. All authors contributed to the article and approved the submitted version.


This cohort study evaluates the bidirectional, longitudinal associations between disturbed sleep and depressive symptoms in children and adolescents using cross-lagged models. What are the bidirectional, longitudinal associations between disturbed sleep and depressive symptoms in children and adolescents?In this cohort study including data on 1689 children and 1113 adolescents, cross-lagged analyses showed significant cascade processes  throughout childhood. In adolescence, significant bidirectional associations were found between ages 10 and 12 years, but after age 13 years, no significant cross-lagged estimates were found.These findings suggest disturbed sleep in childhood is an important target for early identification and intervention programs for depression. Understanding the longitudinal, bidirectional associations between disturbed sleep and depression in childhood and adolescence is crucial for the development of prevention and intervention programs.To test for bidirectional associations and cascade processes between disturbed sleep and depressive symptoms covering both childhood and adolescence and to test for the moderating processes of sex and pubertal status in adolescence.A prospective cohort study using the Québec Longitudinal Study of Child Development . QLSCD’s objective is to identify early childhood factors associated with long-term psychosocial and academic adjustment. Data were collected across 8 waves between ages 5 years (2003) and 17 years (2015). Associations were tested through cross-lagged models in childhood , and in adolescence . Data were analyzed from February to October 2021.Primary outcomes were disturbed sleep and depressive symptoms. Disturbed sleep was parent-reported and included sleep duration, time awake in bed, daytime sleepiness, sleep talking, sleepwalking, night terrors, and nightmares. Depressive symptoms were parent-reported in childhood , and self-reported in adolescence .Data on 1689 children  and 1113 adolescents  were included in the analyses. In childhood, significant bidirectional associations between depressive symptoms and disturbed sleep at all time points were found, indicating cascade processes . In adolescence, significant bidirectional associations from depressive symptoms to disturbed sleep  and vice versa  between 10 and 12 years were found. Between 12 and 13 years, depressive symptoms were modestly associated with disturbed sleep  but the reverse association was not significant. Cross-lagged estimates were nonsignificant after 13 years. The associations did not vary as a function of either sex or puberty-by-sex.These findings suggest that disturbed sleep is associated with the consolidation of depressive symptoms starting in childhood, which, in turn, is associated with ongoing sleep problems. It is possible that timely and appropriate interventions for incipient disturbed sleep and depression prevent spiraling effects on both domains. Modifiable risk factor management has proven effective in the prevention of various diseases15 and may be a complementary strategy to bolster depression prevention programs.Prevention programs for depression in children and adolescents that rely on cognitive, coping, and emotional management skills are effective.17 Previous research used a wide range of definitions for disturbed sleep, including insomnia symptoms,23 chronotype,24 daytime sleepiness,25 and broadly defined sleep disturbances, which include both insomnia and parasomnias .38 A recent meta-analysis39 showed that the pooled estimates of the association between disturbed sleep and depression did not differ significantly between insomnia and broadly defined sleep disturbances.Disturbed sleep is one of the many modifiable risk factors for depression in children and adolescents.41 Significant longitudinal, unidirectional associations have also been reported. A recent meta-analysis39 reported significant pooled estimates of the association between disturbed sleep and later depression controlling for baseline depression. Treating disturbed sleep has been effective in decreasing later depression in adolescence.42 However, whether analog prevention approaches in childhood will result in similar, or enhanced, outcomes is still unknown. There is also evidence of the prospective, unidirectional association between depression and later disturbed sleep in children and adolescents,44 although this finding is mixed, with some studies reporting nonsignificant associations.45Children and adolescents often report both disturbed sleep and depression, with co-occurrence rates equally distributed between boys and girls.46 with a minority testing 3 or more time points, therefore potentially looking at developmental cascades. Cascade processes occur when bidirectional links are carried forward across time, above and beyond within-time covariation and within-construct continuity.47 As of this writing and to our knowledge, 6 studies51 have tested 3 or more time points, and developmental cascades were found in adolescence only.48 Only 2 studies51 focused on childhood, and no studies covered both childhood and adolescence, which hinders a full understanding of the dynamics at play throughout these developmental stages.Several studies focused on bidirectional associations between disturbed sleep and depression in children and adolescents,47Understanding the longitudinal, bidirectional associations and possible developmental cascades between disturbed sleep and depression has the potential to inform risk identification and prevention programs. The fact that disturbed sleep may lead to depression and, potentially, vice-versa, can trigger an unhealthy cycle. If set in motion in childhood, such a cycle would have time to amplify, resulting in developmental cascades that consolidate the 2 problems .52 and the different assessment tools used in these developmental periods, we analyzed childhood and adolescence separately. In addition, given sex-related and puberty-related prevalence differences in both depression and disturbed sleep in adolescence,53 we conducted 2 multigroup analyses to examine the possible moderating role of sex and puberty in the cross-lagged model in adolescence. According to previous literature,51 we expected no sex difference. Given the lack of prior literature, the second multigroup analysis was considered exploratory.The primary goal of the current study was to replicate and expand this previous research by testing bidirectional associations and developmental cascades between disturbed sleep and depression using cross-lagged models and by extending the developmental window of investigation to cover both childhood and adolescence. We used data from the Québec Longitudinal Study of Child Development (QLSCD) collected across 8 waves between ages 5 and 17 years. Given the differences in risk between child-onset and adolescent-onset depression, particularly for girls,STROBE) reporting guideline for observational studies.54 The QLSCD is a representative birth cohort of infants born between 1997 and 1998 to mothers living in the province of Québec, Canada. Attrition rate and detailed cohort characteristics are reported in eMethods 1 in the 55 The study was approved by the ethics review boards of the Institut de la Statistique du Québec, the Centre Hospitalier Universitaire Sainte-Justine, the Louis-Hippolyte Lafontaine Hospital, and the Université de Montréal, Faculty of Medicine, and written informed consent was obtained from families at each assessment.55This cohort study follows the Strengthening the Reporting of Observational Studies in Epidemiology  5, 7, 8, and 10 years; (2) 12, 13, 15, and 17 years; and (3) 5, 7, 8, 10, 12, 13, 15, and 17 years. Participants were included if they had 2 or fewer missing values for depression or disturbed sleep across all time points. For childhood assessments, mothers were asked to report on their child’s sleep and depression, whereas for adolescence, mothers reported on their child’s sleep, and depression was self-reported by adolescents.For childhood analyses, data collected at ages 5, 7, and 8 years were considered, whereas for adolescent analyses, data at 10, 12, 13, 15, and 17 years were used. Of note, in the current study, we considered childhood the period between 5 and 9 years of age, and adolescence the period between 10 and 17 years.57 in the QLSCD cohort. A total score was computed, and the score was rescaled to range 0 to 10. The higher the score, the more disturbed the sleep. For comparability with previous research,23 cross-lagged analyses were repeated using an insomnia index—that is, the total score included sleep duration, time awake in bed, and daytime sleepiness.For a wider scope, we opted for a broad definition of disturbed sleep, including both parasomnias and insomnia. Sleep was assessed through a questionnaire of 7 items that assessed sleep duration, time awake in bed, daytime sleepiness, sleep talking, sleepwalking, night terrors, and nightmares .Sex, pubertal status, socioeconomic status (SES), and maternal depression were selected according to previous research.65 Cross-lagged models were conducted using Mplus statistical software version 7 (Muthén & Muthén).66 Missing data were treated through full information maximum likelihood, which uses maximum likelihood to estimate model parameters using all available raw data.68 Overall model fit was tested by considering together the comparative fit index (CFI), the Tucker-Lewis index (TLI), the root mean square error of approximation (RMSEA), the χ2 statistic and the ratio of χ2 to degrees of freedom.69 A nonsignificant χ2 value, a CFI and a TLI value of 0.90 or higher, an RMSEA value below 0.06, and a ratio of χ2 to degrees of freedom less than 3 were considered indicators of good fit.70 Secondary analyses used a multigroup approach to test whether model estimates differed across sex or puberty-by-sex using a χ2test. The unconstrained model was compared against a model in which across-time, within-time, and cross-lagged paths were constrained to be equal across levels of the moderator. A deterioration of model fit would suggest that the associations may be different across levels of the moderator. All P values were 2-sided. P < .05 was considered to be significant. Data were analyzed from February to October 2021.Descriptive statistics and Pearson correlations were computed using SPSS statistical software version 19.0 for Windows (SPSS Inc.). Missing data were examined by the missing value analysis module and the Little Missing Completely at Random test.A total of 1689 children  and 1113 adolescents  were included in the study. Missing data patterns for outcome variables in childhood and adolescence are reported in eResults 1, eTable 2, eTable 3, eTable 4, and eTable 5 in the t2115 = −6.55, P < .001; and χ21 = 6.34, P = .011, respectively). Descriptive statistics across time points for the study variables are shown in In the childhood analysis, 1689 children  were included . Particir = 0.18 at 5 years, r = 0.09 at 7 years, and r = 0.16 at 8 years). Cross-lagged path estimates showed that depressive symptoms were significantly associated with later disturbed sleep, and disturbed sleep was associated with later depressive symptoms at all time points , suggesting a developmental cascade throughout childhood. Also, there were significant associations between depressive symptoms at 5 and 8 years , and between disturbed sleep at 5 and 8 years . Among covariates, only paths between maternal depression and both disturbed sleep and depressive symptoms were significant .Bivariate correlations between depressive symptoms and disturbed sleep are reported in eTable 6 in the t2115 = −7.11; χ21 = 18.17; P < .001). Descriptive statistics across time points for the study variables are shown in r range, 0.07 to 0.10). Cross-lagged path estimates were significant from depressive symptoms to disturbed sleep  and vice versa  between 10 and 12 years. Between 12 and 13 years, depressive symptoms were modestly associated with disturbed sleep  but the reverse association was not significant. Cross-lagged estimates were nonsignificant after 13 years. Among covariates, significant paths were found between maternal depression and both disturbed sleep and depressive symptoms , and between SES and depressive symptoms . Compared with the unconstrained model, the fit of either the sex-invariant or the puberty-by-sex-invariant model was not deteriorated , indicating that the associations between depressive symptoms and disturbed sleep did not vary as a funciton of either sex or puberty-by-sex. Cross-lagged paths were identical when listwise deletion was used as an alternative missing data strategy, in both childhood and adolescence.In the adolescent analysis, 1113 individuals were included  . ParticiCross-lagged paths using the insomnia index are reported in eResults 2, eFigure 1, and eFigure 2 in the To our knowledge, this cohort study is the first to report developmental cascades between disturbed sleep and depressive symptoms throughout childhood and early adolescence. Our finding suggests that disturbed sleep makes a significant contribution to the stability over time of depressive symptoms across childhood and early adolescence, which, in turn, is associated with ongoing disturbed sleep.51 that we know of have tested cross-lagged associations over 3 or more time points in childhood and developmental cascades were not reported. Quach et al50 found that disturbed sleep is consistently associated with increased depression from preschool to 12 years, and bidirectionality was found only from 6 to 7 years. Although the study sample and measurements were similar to ours, analyses were not controlled for any covariates, which could plausibly account for the discrepancy of the results. Foley et al51 found no bidirectional effects but only a unidirectional association from disturbed sleep to depression between preschool and grade 1. Differently from our study, the authors tested the reciprocal effects of disturbed sleep, depression, and social competence, therefore adding 1 construct in the cross-lagged model, which could partly explain the discrepancy of the results. In adolescence, we found no developmental cascades, which was in line with 349 previous studies but differed from a fourth study,48 and a bidirectional association between 10 and 12 years in line with 1 previous study.37 Starting at 13 years, cross-lagged associations were not significant at any time points. Finally, effects held across sexes in line with previous results51 and were unaffected by including pubertal status as a moderator in the model.To date, 2 studies71 and yielded small effect sizes .42 Starting prevention efforts in childhood may have a similar, or even greater impact on depression compared with treatment initiated in adolescence,71 because it would reduce the risk before depression consolidates. To date, we are not aware of any randomized clinical trials of sleep interventions specifically addressing depressed children. To expand our understanding, randomized clinical trials with long-term follow-up and in children with different levels of baseline depression are needed. According to our findings, a sample of 100 individuals would be adequately powered for a sleep intervention for the prevention of depression in a children community sample.Taken together, our findings suggest that there might be a sensitive window between 5 and 12 years for sleep interventions to mitigate the emergence and/or growth of later depressive symptoms. Up to now, randomized clinical trials of sleep interventions in depressed adolescents have targeted mid adolescence 39 and with etiological models of depression. Indeed, multiple risk factors, each with a small effect, underlie the vulnerability to depression,72 and this may partly explain why sleep interventions, albeit having a significant effect, are limited in preventing depression in adolescents.71 The potential for a larger impact could be achieved by simultaneously addressing multiple risk factors of depression. Disturbed sleep often co-occurs with other lifestyle risk behaviors associated with depression, including poor diet,73 poor physical activity,74 and excessive recreational screen time.75 Future research should examine whether a multiple health behavior change approach could be a viable option for depression prevention.76The magnitude of the cross-lagged estimates that we found was overall small , but quite in line with previous reports77 are heritable4 and share common genetic risk factors.78 Both disturbed sleep79 and depression80 have been associated bidirectionally with inflammatory processes, suggesting that inflammation may be one mechanism driving the spiraling process. Interestingly, treating disturbed sleep and depression attenuates inflammation,82 and using anti-inflammatory medications to treat depression seems promising in adults.83 Future research is needed to determine whether patterns of proinflammatory responses may be one of the biological mechanisms through which disturbed sleep accelerates depression and whether it can be targeted for depression prevention in children and adolescents.Understanding the biological mechanisms underlying the developmental cascade linking disturbed sleep and depression is of paramount importance. Key outstanding questions include whether an early sensitive window of vulnerability can be identified, which could explain why cascade processes concentrate in early childhood and fade in adolescence. Both conditions84 Results may need to be replicated with objective measurements and/or validated questionnaires. However, self-reports remain the measure of choice in community surveys. Additionally, for disturbed sleep during childhood, there was a slight difference in the pattern of missing data related to time since enrollment. Although the analytical strategy that was used to deal with missing data mitigates the effects of attrition, we cannot exclude that missing data may have affected the results in the childhood analysis.There are some limitations in this study. First, participants were from a higher SES and more often girls than nonparticipants, which potentially limits the generalizability of our results. Second, the questionnaire for depression did not rate symptoms’ severity. Furthermore, depressive symptoms were parent-reported in childhood and self-reported in adolescence, which may have accounted for the differences found between childhood and adolescent findings. However, the high stability of scores suggests that both adolescent and parent-report measures tapped into the same constructs. Third, in childhood, parents reported on both disturbed sleep and depressive symptoms, which might have implied perception bias. Fourth, the questionnaire for disturbed sleep was not validated. Furthermore, it was parent-reported, which have been shown to be less accurate compared with self-report and objective measurements of sleep.In conclusion, bidirectional and developmental cascades linking disturbed sleep and depressive symptoms in childhood and early adolescence emphasize the importance of identifying and treating incipient disturbed sleep as early as possible to prevent spiraling effects on depression. More randomized clinical trials testing interventions possibly targeting common pathways are needed to bolster depression prevention. Because of the reliance on self-report data, results should be interpreted with caution.