CD40 gene overexpression was correlated with improved five-year overall survival (OS) (p < 0.001), while increased BAFFR and LTβR mRNA levels were associated with worse OS in patients with adenocarcinomas . Similarly, patients with adenocarcinomas exhibited a negative correlation between membranous BAFFR protein expression in carcinoma cells and three- and five-year survival  as well as between BAFFR protein overexpression in cancer-associated fibroblasts (CAFs) and two-year survival . Patients with increased LTβR nuclear protein staining or stage II patients with lower cytoplasmic LTβR protein expression had worse five-year OS . Moreover, CD40 protein expression in tumor infiltrating lymphocytes (TILs) and CAFs was positively associated with metastatic spread while BAFFR protein expression in CAFs was negatively associated with bone metastasis (p = 0.041). Our data suggests that CD40, BAFFR, RANK and LTβR play an important role in NSCLC and further supports the role of NF-κB alternative pathway in NSCLC.An increasing number of studies implicates the NF-κB (Nuclear Factor of kappa light chain gene enhancer in B cells) alternative pathway in non-small-cell lung cancer (NSCLC). We assessed the clinical significance of CD40 , BAFFR (B-cell activating factor receptor), RANK (Receptor activator of NF-κB) and LTβR (lymphotoxin β receptor) receptors, which activate the alternative pathway of NF-κB, in NSCLC. Evaluation of CD40, BAFFR, RANK and LTβR expression was performed based on the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) datasets, while protein expression was assessed by immunohistochemistry in specimens from 119 operated NSCLC patients.  The Nuclear Factor of kappa light chain gene enhancer in B cells (NF-κB) remains one of the most studied transcription factors in cancer biology due to its pivotal role in many cellular functions such as inflammation, metastasis, angiogenesis, metabolism, epithelial-mesenchymal transition and tumor cell survival . In addiNF-κB2 and BCL3 are related to lung cancer risk and OS [NF-κB signaling is mediated through NF-κB family members, mainly through the activation of two pathways, which are termed classical and alternative . During k and OS ,9.Although the NF-κB alternative pathway can be activated by viruses such as Epstein-Barr Virus (EBV) and the human T-cell leukemia-lymphoma virus (HTLV) , the priThe purpose of this study was to evaluate alterations in the expression (in protein and mRNA level) of the major surface receptors of NF-κB alternative pathway, namely CD40, BAFFR, RANK and LTβR, and to investigate their clinical significance and prognostic value in NSCLC patients.Patients with NSCLC that had undergone curative resection of a lung tumor in the University Hospital of Patras between 2005 and 2010 were serially and retrospectively selected from the electronic database of the Department of Pathology of the University Hospital of Patras. Patients enrolled in this study were in a good performance status (PS = 0) and no data was available regarding driver mutations status. Patients who relapsed during the period of observation were mainly managed with chemotherapy. Ethical guidelines of the Helsinki Declaration (2013) were followed for the designation of this study . The curThe clinical data, including medical history, relapse and OS were retrieved from the archive of the Division of Oncology of the University Hospital of Patras. When data were missing from the clinical files, the patients were personally contacted. OS outcome was defined after an observation period of 60 months. CD40, BAFFR, RANK and LTβR, data were analyzed from 483 lung cancer adenocarcinomas, 486 squamous-cell carcinomas cases and 347 normal cases provided by the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) projects. The dot Box Plots were generated to compare the CD40, BAFFR, RANK and LTβR mRNA expression levels between the tumor and normal tissues of NSCLC patients. The Gene Expression Profiling Interactive Analysis (GEPIA) interactive tool was used for TCGA and GTEx data processing and visualization in this study [CD40, BAFFR, RANK and LTβR genes from stages I-III NSCLC patients . To assess the gene expression changes of is study . Kaplan The expression of CD40, BAFFR, RANK and LTβR was assessed by immunohistochemistry (IHC), as previously described . ConditiEach slide was scored blindly by an experienced pathologist (VT). Evaluation of the immunohistochemical staining was performed as described previously . HistoloThe intensity of staining was also evaluated using a three-tiered scale. A total score was calculated by multiplying the percentage of positive cells by the intensity of staining (range 0–300). Other components of the tumor microenvironment (tumor-infiltrating lymphocytes (TILs), tumor-associated macrophages (TAMs), endothelial cells) were also evaluated and scored as positive or negative, based on the presence or absence of any staining. Microphotographs were obtained by Lumenera’s INFINITY HD digital camera  mounted on an Olympus BX41 microscope . t test was used for continuous variables with normal distribution. Analysis for ordinal or continuous data was performed by using Kruskal-Wallis or the Mann-Whitney tests. Spearman’s correlation was used to assess associations between variables. The Kaplan-Meier curves and the log-rank test were used for plotting and comparison of survival rates, respectively. Multivariate analysis of the studied molecules was assessed by Cox regression analysis. The X-tile software was used in order to provide the best cut-off points [p < 0.05 was considered statistically significant for all comparisons. Statistical analysis was performed by using the Statistical Package for Social Sciences version 17 . Categorical nominal variables were evaluated using the Chi-square test or Fisher exact test. The f points . Best cuClinical and pathological characteristics of the cohort of 119 patients are presented in CD40, BAFFR, RANK and LTβR provided by the TCGA and GTEx public databases were analyzed. [CD40, BAFFR, RANK and LTβR had similar patterns in the LUSC (lung squamous cell carcinoma) and LUAD (lung adenocarcinoma) subgroups analyzed and both were similar to control samples  in most of the cases. CAFs also showed faint cytoplasmic staining. Immunostaining was very rare in scattered lymphocytes within the tumor. However, lymphoid aggregates within the tumor or adjacent non neoplastic parenchyma were strongly positive. Positive staining was also found in the cytoplasm of vascular muscle and endothelial cells as well as in the cytoplasm of macrophages a–c. Regarding BAFFR expression, neoplastic cells had a finely dot-like cytoplasmic staining. Membranous staining was also noted in a few cases. CAFs showed cytoplasmic staining. BAFFR staining was intensely positive in lymphoid aggregates and mantle zone lymphocytes (in lymphoid follicles formed in adjacent tissue and in lymph nodes). In addition, staining in lymphocytes was detected in both the cytoplasm and membrane, while in macrophages staining was restricted to the cytoplasm. Furthermore, BAFFR was expressed in the cytoplasm of vascular muscle and in endothelial cells d–f.Regarding RANK, the staining was mainly cytoplasmic with a coarse dot-like pattern in neoplastic cells and CAFs. In some cells, dots were lined close to the cell membrane. In lymphocytes, the localization of staining was cytoplasmic and membranous, while in macrophages staining was limited to the cytoplasm. Similarly to the other markers, RANK was expressed in vascular muscle and endothelial cells g–i.LTβR signal had a coarse dot-like cytoplasmic staining in neoplastic cells. Membranous staining was seen in some cases. However, interestingly, nuclear staining was seen in most of the cases. Cytoplasmic and rare membranous immunostaining was detected in vascular muscle and endothelial cells. Lymphocytes, macrophages and CAFs showed cytoplasmic staining in all specimens j–l.p = 0.005 and p < 0.001, respectively). Immunohistochemical staining for CD40 in the cytoplasm and membrane of the tumor cells was noted in 99% and 49% of the cases, respectively b–c. Exprp = 0.001), while the cytoplasmic staining was similar between tumor and adjacent tissues (p = 0.689).BAFFR expression was detected in the cytoplasm of the tumor cells in all cases, while expression in the tumor cell membrane was noted only in 26.7% of cases e–f. Furtp = 0.289 and p = 0.400, respectively).Immunostaining for RANK in the cytoplasm and the membrane of tumor cells was positive in 100% and 64.1% of cases, respectively h–i. No dp = 0.211, p = 0.602, p = 0.638, respectively).Membranous, cytoplasmic and nuclear staining for LTβR was observed in 34.6%, in 100%, and in 86.5% of cases, respectively, and no difference was observed in the levels of expression between cancer cells and adjacent non-neoplastic cells (p < 0.001). Moreover, CD40 staining in fibroblasts was positively correlated with its expression in TILs and macrophages . Furthermore, cytoplasmic and membrane CD40 expression of carcinoma cells was positively correlated with each other (p = 0.017). CD40 staining was positive in 5, 96.6 and 97.5% of the cases in TILs, TAMs and CAFs, respectively. Cytoplasmic and membranous CD40 expression in carcinoma cells was positively associated with expression in CAFs (both p < 0.001). No other association regarding BAFFR expression in the cells of the tumor microenvironment was observed.TAMs and CAFs were positive for BAFFR in all samples, whereas TILs expressed BAFFR in 83% of the cases. BAFFR cytoplasmic expression in tumor cells was positively correlated with stromal BAFFR expression (p < 0.001) was noted.Regarding RANK, TILs, TAMs and CAFs, they were found positive for BAFFR in all cases. A negative association between cytoplasmic and membrane RANK expression in tumor cells .With regard to LTβR, all tissue specimens were positive for LTβR expression in CAFs, TILs and TAMs. Cytoplasmic LTβR expression was negatively correlated with membranous and nuclear expression in neoplastic cells (n = 1926) with squamous cell carcinomas and adenocarcinomas .Survival analysis using KM-plotter for the JetSet probe set alone and best cut-off option for all available datasets combined, revealed that elevated CD40 gene expression was correlated with improved five-year OS of NSCLC patients (n = 1145), showed that BAFFR expression over the median was significantly associated with worse OS (p = 0.001). Further analysis by histological subtype stratification revealed that this relation is limited to patients with adenocarcinomas (p < 0.001) and not in the cases of squamous cell carcinomas (p = 0.32). Evaluation of BAFFR expression in relation to OS, using the median as a cutoff in NSCLC patients . On the contrary, upon further stratification with histology, no correlation was found between RANK gene expression and OS in patients with squamous-cell carcinomas or adenocarcinomas . Additionally, a statistically significant positive association was observed between LTβR expression, patients with adenocarcinomas and high LTβR expression had poor clinical outcome after a five-year observation period (p < 0.001). On the contrary, OS was not affected by LTβR expression in patients with squamous cell carcinomas . With regard to p = 0.007 and p = 0.022, respectively). Furthermore, multivariate analysis with Cox regression models and age, gender and primary location as coefficients, further supported the previous findings . No similar correlation was observed for three- and five-year survival outcome in patients with squamous cell carcinomas .With regard to membranous BAFFR, patients with adenocarcinomas and no BAFFR expression had better three- and five-year OS compared to patients whose tumors expressed BAFFR (p = 0.042), but not with three- and five-year OS . This finding persisted after multivariate analysis with age, gender, stage and primary location as coefficients .On the other hand, increased BAFFR expression in CAFs was correlated with worse two-year survival (p = 0.008). This finding remained significant after performing a Cox regression analysis . In addition, LTβR nuclear staining was associated with five-year OS (p = 0.039), with increased expression being correlated with worse survival. In addition, univariate analysis revealed that lower cytoplasmic expression levels of LTβR in tumor cells of stage II patients were associated with decreased five-year OS (p = 0.258). Interestingly, increased expression of RANK in the CAFs was marginally associated with poor two-, but not three- and five-year survival outcome .No statistically significant association was found between RANK expression in the cancer cell cytoplasm and five-year survival, although the separation of the two curves in the Kaplan Meier plot after the second year of observation suggests a possibly better five-year OS for patients with higher expression after an initial two-year period .CD40 expression in the cytoplasm or on the membrane was not significantly associated with five-year OS, although a trend was noted for a relation between high CD40 levels and improved five-year OS, compared to patients with lower expression rates (p = 0.003), liver (p < 0.001) and brain (p = 0.048). Furthermore, CD40 expression in CAFs was also positively related to liver metastasis (p = 0.018). In addition, higher BAFFR expression in CAFs was associated with lower rate of bone metastasis (p = 0.041), while no correlation was found between BAFFR expression in tumor cells, TILs, TAMs and distal or nodal metastatic spread.CD40 and BAFFR protein expression were correlated with metastasis development during a two-year follow up. In particular, CD40 immunodetection in TILs was positively associated with metastatic spread in adrenal glands (p = 0.040). Expression of CD40 and LTβR in CAFs was also correlated with disease stage . In addition, squamous cell carcinomas had lower expression levels of membranous RANK and higher levels of nuclear LTβR compared to adenocarcinomas and large cell carcinomas . Membranous RANK and nuclear LTβR expression also correlated with gender. Female patients displayed increased RANK and decreased LTβR expression compared to male patients . Moreover, RANK expression was lower in the centrally located better differentiated cells compared to the peripherally located less differentiated ones, in the tumor nodules of squamous cell carcinoma. No association was detected between CD40, BAFFR, RANK and LTβR expression and other clinicopathological characteristics.Patients with higher pathological disease stage had lower expression of CD40 in neoplastic cells . Our findings are in agreement with published reports in other types of cancer. In particular, LTβR expression has been noted in ovarian cancer cells and their CAFs  while ac−/−), having a significant reduction in lung metastases after injection with melanoma cells [Another intriguing finding is the association of CD40 expression in TILs and in CAFs with the development of metastatic disease . Supportive to our finding is the association of CD40 expression with metastatic spread of human lung cancer cells in vitro . Moreovema cells ,57. In ama cells , while, ma cells .BAFFR tumor or stromal expression was negatively correlated with OS in agreement with transcriptomic data analysis for patients with adenocarcinomas and the well-established knowledge that activation of BAFFR leads to a potent survival signal ,61. AddiLTβR gene expression in patients with adenocarcinomas. In addition, LTβR protein expression was related to OS too, with LTβR localization dependency. A negative association with OS was noted for nuclear staining and a positive association was observed for cytoplasmic expression in stage II patients. This finding might reflect a compartment-dependent mechanism for LTβR. Supportive to this is the observation that internalization of LTβR is associated with the activation of the alternative, but not the classical, NF-κB pathway [A positive association with OS also emerged for  pathway . In addi pathway . Despite our promising results, we have to acknowledge some weak points. In the current study, patients with stage I, II and III NSCLC, who were surgically managed, were exclusively enrolled, limiting the investigation to non-metastatic disease. In addition, a larger cohort would be required to achieve more robust results. Furthermore, although IHC is a widely used technique to assess protein expression, it would be reasonable to have data from multiplex techniques. Moreover, double staining could help in discrimination of the different types of cells, which was based on morphological characteristics.In conclusion, this study shows that although mRNA expression levels of the immune system-related transmembrane receptors CD40, BAFFR, RANK and LTβR do not differ, protein levels of CD40 and BAFFR were lower in NSCLC while no change was observed in protein levels of RANK and LTβR. Moreover, CD40 and BAFFR protein expression in microenvironment cells were correlated with development of metastasis. In addition, our data provide strong evidence that the expression of these receptors in tumor, immune or stroma cells have prognostic significance influencing the clinical outcome of operated NSCLC patients, while CD40 and BAFFR protein expression was correlated with development of distant metastases.


Sustained hyperglycaemia leads to the development of haematological alterations which, if left untreated, is associated with cardiovascular complications. Insulin is the mainstay drug in type 1 diabetes mellitus (T1D); however, the use of insulin is associated with haematological alterations that could further worsen cardiovascular complications. Therefore, the aim of the study was to investigate the haematological effects of oleanolic acid (OA) in streptozotocin- (STZ-) induced diabetic rats. μg/kg, s.c), metformin , and OA . OA was administered orally twice a day. Thereafter, the animals were sacrificed, and blood and tissues were collected for haematological, hormonal, and oxidative status analysis.  The animals were separated into five groups; the nondiabetic group (ND), the diabetic control group (DC), and the treatment groups of insulin , oxidative stress, and a reduced erythropoietin (EPO) concentration when compared to ND rats. However, administration of OA attenuated hyperglycaemia, HbA1c, and EPO concentrations compared to DC rats. The reduction of blood glucose concentration, HbA1c, and improved EPO concentrations was further associated with a notable increase in red blood cell (RBC) count and other RBC indices. We also observed an increase in the antioxidant status of the RBCs with a concomitant decrease in oxidative stress.  These findings suggest that OA improves diabetes-induced haematological changes caused by hyperglycaemia and attenuates the progression of cardiovascular complications in DM individuals. OA was isolated from ratories .n = 6) which were bred and housed in the Biomedical Research Unit (BRU) in the University of KwaZulu-Natal. Standard laboratory conditions included constant temperature (22 ± 2°C), CO2 content of <5000 p.m., relative humidity of 55 ± 5%, and illumination (12 h light/dark cycles) with noise levels of less than 65 decibels. The animals were given rat chow daily for the 5-week experimental period. The institutional guidelines of the University of KwaZulu-Natal (AREC\041\018M) were used for the conduction of animal care. The animals were acclimatized for 5 days in metabolic cages before commencement of the study.We used 30 male Sprague-Dawley rats  following a previously described protocol [Type 1 DM was induced by a single intraperitoneal injection of 60 mg · kgprotocol .μg/kg s.c), serving as positive controls, respectively. Over the period of 5 weeks, blood glucose concentrations were monitored where blood was collected via the tail prick method every 3rd day at 09:00 am for the duration of experimental period using the Elite® glucometer (Elite (Pty) Ltd., Health Care Division, South Africa). EDTA anticoagulated blood was collected into the capillary tubes, which were then sealed at one end with plasticine, and centrifuged at 3000g for 5 min, after which RBC indexes in the capillary tubes were read using a microhaematocrit reader.The short-term effects of OA were monitored for haematological parameters in separate groups of untreated and treated STZ-induced diabetic male Sprague-Dawley rats for 5 weeks using a previously validated standard protocol that has been reported from our laboratories . Briefly−1) for 3 minutes . Thereafter, precooled heparinized containers were used for the collection of blood via cardiac puncture and centrifuged  at 4°C, 503g for 15 minutes. In addition, the remaining RBCs were stored in separate Eppendorf tubes at -80°C in a BioUltra freezer until use for flow cytometry analysis. Kidney tissues were removed and weighed before freezing in liquid nitrogen. For further biochemical analysis, the kidneys were stored at −80°C in the BioUltra freezer .Blood and Tissue: at the end of the 5-week experimental period, all animals were sacrificed via exposure to halothane via an anaesthetic gas chamber . The absorbance from these wavelengths was used to calculate the concentration of MDA using Beer's Law. To investigate oxidative stress, 50 mg kidney tissues were homogenized in 500 SOD, GPx, EPO, and HbA1c concentrations were analyzed using separate specific ELISA kits  that use the Sandwich-ELISA method, strictly following manufacturer's instruction.To acquire data, the BD FACS Canto-II flow cytometer and BD FACSdiva software  were used following a previously described protocol .Detector settings: for small particle detection, forward scatter (FS) and side scatter (SS) parameters were set at a log scale . For optp < 0.05 indicate statistical significance.Data is expressed as means ± standard error of means (SEM). Statistical analysis was conducted using GraphPad Prism InStat Software . One-way and two-way analysis of variance (ANOVA) followed by the Tukey-Kramer post hoc test was used to analyze differences between the controls and the experimental groups. Values of p < 0.05; OA, MET, and INS vs. DC).p < 0.05; OA, MET, and INS vs. DC).p < 0.05, DC vs. ND). This was associated with a decrease in RBC count in diabetic rats . Diabetic rats treated with OA significantly decreased MDA concentration in both plasma and kidney tissues by comparison with plasma and kidneys of untreated diabetic animals . This was associated with an improvement of plasma SOD and GPx concentration by comparison with untreated diabetic rats.p < 0.05). The administration of OA, together with insulin and metformin, significantly decreased HbA1c concentration by comparison with the diabetic controls (p < 0.05).p < 0.05). The administration of OA however significantly increased RBC membrane annexin-V percentage by comparison with the diabetic control group (p < 0.05). This was also correlated with a significant increase in MCV as shown in p < 0.05).r = 1; p = 0.7) and the diabetic control group  of the STZ-induced diabetic animals. A significant decrease in annexin-V as seen in Figures r = 1; p = 0.7) and standard drugs, MET , and INS  of the STZ-induced diabetic animals. The OA group together with the standard drugs showed a significant increase in annexin-V and a significant increase in MCV in comparison with the diabetic control group.Figures The present study investigated the haematological effects of OA in STZ-induced diabetic rats. STZ is widely used to establish diabetic animal models that mimic human diabetes . In our 12 and folic acid, since vitamin B12 and folic acid play an essential role in red blood cell maturation [12 for proliferation during their differentiation (39). Deficiency of both vitamin B12 and folic acid inhibits purine and thymidylate syntheses, impairs DNA synthesis, and causes erythroblast apoptosis further resulting in anaemia [12 and folic acid [Erythrocytes play an essential role in the delivery of oxygen to body tissues throughout the circulatory system . RBCs caturation . Erythro anaemia . The anilic acid .2+-dependent phospholipid-binding protein, has high affinity for PS, which can be detected by FITC fluorochrome-labeled annexin-V using flow cytometry [OA also improved the RBC indices possibly by improving the antioxidant status since previous studies have shown the ability of OA to improve SOD and GPx in STZ-induced diabetic rats . SOD andytometry . Our curytometry . Decreasytometry . Our curRenal dysfunction also caused by ROS through hyperglycaemia has been shown to be associated with a decrease in EPO concentration as interstitial fibroblasts responsible for EPO secretion are often destroyed during the development of diabetic nephropathy . HoweverCurrent drugs of DM are associated with cardiovascular complications through haematological alterations. There have been ongoing studies on the therapeutic beneficial effects of medicinal plants in individuals with cardiovascular complications. The current study demonstrates the haematological effects of OA in STZ-induced diabetic rats and therefore the improvement of cardiovascular complications with prolonged use. Our results showed that OA reduces hyperglycaemia, further improving erythrocyte function possibly through the increase in EPO secretion, improvement of antioxidant status, and decrease of glycated haemoglobin and by improving cell survival for the whole lifespan of the erythrocyte.


EMS. Excessive ROS accumulation and ER stress are most likely the major factors limiting the multipotency of these cells. However, we observed autophagic flux during differentiation as a protective mechanism that allows cells to maintain homeostasis and remove dysfunctional mitochondria.Mesenchymal stem cells (MSCs) are frequently used in both human and veterinary medicine because their unique properties, such as modulating the immune response and differentiating into multiple lineages, make them a valuable tool in cell-based therapies. However, many studies have indicated the age-, lifestyle-, and disease-related deterioration of MSC regenerative characteristics. However, it still needs to be elucidated how the patient's health status affects the effectiveness of MSC differentiation. In the present study, we isolated mesenchymal stem cells from adipose tissue ) from horses diagnosed with equine metabolic syndrome (EMS), a common metabolic disorder characterized by pathological obesity and insulin resistance. We investigated the metabolic status of isolated cells during adipogenic differentiation using multiple research methods, such as flow cytometry, PCR, immunofluorescence, or transmission and confocal microscopy. The results indicated the impaired differentiation potential of ASC Mesenchymal stem cells (MSCs) can be isolated from multiple sources, including bone marrow and adipose tissue , 2. MSCsRecent alarming data suggest that age , 10–12, α) leads to local and systemic inflammation in EMS horses [What is more, it was shown that the adipose tissue (AT) of EMS horses is characterized by hyperplasia and hypertrophy . IncreasS horses . The proEMS) were seriously limited [EMS. Moreover, osteoblast and chondroblast precursors exhibited the impaired formation of extracellular matrix and the reduced expression of the following transcripts: bone morphogenetic protein 2 (BMP-2), vimentin, decorin, and Sox9. Obtained data suggested that under insulin resistance and EMS conditions, mitophagy is a predominant form of autophagy during chondrogenic differentiation of ASCEMS, and serves as a protective mechanism that allows maintaining multipotency. Increased autophagy and autophagic flux were also observed in the adipose tissue of obese humans and rodents [EMS remains to be further defined. In the current study, we hypothesized that selective mitophagy may become a critical process in adipogenic differentiation.Our previous study showed that the potential for both chondrogenic differentiation and osteogenic differentiation of ASC isolated from EMS horses  stress induces selective mitophagy and autophagy to protect ASCEMS against cell death. Moreover, these factors directly contributed to the decreased differentiation potential of ASCEMS.The effectiveness of autologous ASC application in the course of EMS treatment strongly relies on their cytophysiological status, including viability, senescence, and accumulation of oxidative stress factors. EMS per se may be caused by profound changes in ASC physiology. Therefore, in this study, we investigated the impact of oxidative stress factors and mitochondrial deterioration in ASCAll reagents used in this experiment were purchased from Sigma-Aldrich (Poland), unless indicated otherwise.n = 6) and the control group, consisting of animals in good physical condition (n = 6). The division was done on the basis of detailed interviews with owners and clinical parameters such as body weight, body condition score, cresty neck score, combined glucose-insulin test, leptin concentration, and insulin levels. Comprehensive information about animals qualified to the experiments described in this paper are listed in The study involved twelve mixed-sex, age-matched  horses, divided into two groups: a group of individuals suffering from EMS  supplemented with 1% of a penicillin, streptomycin, and amphotericin B (PSA) solution. Afterwards, the biological material was fragmented and digested with collagenase type I (1 mg/ml) for 40 minutes at 37°C. Cell suspension was centrifuged at 1200 ×g for 10 minutes at 23°C, then pellets were resuspended in the culture medium and transferred to the culture flasks.2. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 4500 mg/l glucose supplemented with 10% of fetal bovine serum (FBS) and 1% of a penicillin, streptomycin, and amphotericin B (PSA) solution. Medium was changed every two days. After reaching 80–90% of confluence, the cells were passaged using a trypsin solution .Cultures were maintained under aseptic conditions at 37°C, 95% humidity, and 5% COTo determine the phenotype of the cells, the expression of the following surface markers was investigated: CD44, CD45, and CD90. The cells were prepared for flow cytometry as follows: ASCs were treated with TrypLE Express solution, then extensively washed with HBSS, and resuspended at a total of 5 × 105 cells/ml. Afterwards, cell suspension was incubated with antibodies preconjugated with peridinin chlorophyll protein (PerCP) and fluorescein isothiocyanate (FITC)  or appropriate isotope control antibodies at 4°C for 20 minutes. The analysis was performed using Becton Dickinson FACS Calibur Flow Cytometer. The results were analyzed by CellQuest Pro software .Additionally, cell cycle analysis was performed using Muse Cell Cycle Assay Kit with Muse Cell Analyzer (Merck) according to the manufacturers' instruction.4 cells per cm2. The proliferation rate was determined at the 1st, 2nd, and 5th day of the experiment. Absorbance levels were measured spectrophotometrically  at a wavelength of 690 nm as a reference wavelength and 600 nm for resazurin. Additionally, the population-doubling time (PDT) was estimated using the formula presented below:The viability of cultured cells was evaluated using the resazurin-based assay kit (TOX8). To perform the experiment, cells were seeded on a 24-well plate at a concentration of 1 × 10The synthesis of DNA in ASC cultures was evaluated by the detection of 5-bromo-2-deoxyuridine incorporation into cellular deoxyribonucleic acid. Cell proliferation rates were measured twice—after 24 and 120 hours of culture. The experiment was conducted using bromodeoxyuridine (BrdU) assay (Abcam). The absorbance was measured with a spectrophotometer microplate reader at a wavelength of 450/550 nm.2. After 5 days, biological material was fixed with 4% ice-cold paraformaldehyde and stained with pararosaniline. Colonies consisting of 50 or more cells were considered as colony forming units (CFU). The experimental procedure was performed in triplicate. The efficiency of CFU was calculated according to the following formula:For the clonogenic assay, cells were seeded on 6-well culture plates at a density of 110 cells per cmAll experimental procedures included in the proliferation assay were performed in accordance with the manufacturer's instructions.ug/ml of insulin, 60 uM of indomethacin, 50 ug/ml of gentamicin, 10% of FBS, and Dulbecco's modified Eagle's medium (DMEM) with Nutrient F-12 Ham. The cells were seeded at a density of 15 × 103 cells per cm2 on 24-well plates, and the culture media were changed every second day. Cells cultured in standard growth medium were used as a control for the experiment and allowed to establish differentiation effectiveness. Stimulation toward adipogenic lineage lasted 10 days. To evaluate the effects of the adipogenesis stimulation, the cells were fixed with 4% PFA. Then, Oil Red O staining was used for the detection of intracellular lipid droplets. The results obtained during the staining procedure were analyzed using an Axio Observer A1 inverted microscope , while the photographic documentation was made using a Canon PowerShot digital camera. The percentage of the Oil Red O stained area was calculated using Image J.Cell differentiation was induced using an adipogenic differentiation medium containing 0.5 mM of 1-methyl-3-isobutylxantine (IMBX), 0.5 mM of dexamethasone, 6.25 Cell morphology was evaluated using an inverted epifluorescent microscope  as described elsewhere . In ordeDetailed morphological analyses were conducted using a scanning electron microscope  and focused ion beam . For scanning electron microscopy analysis, the cell cultures were fixed with 2.5% glutaraldehyde for 1 h at room temperature, then dehydrated in graded ethanol-water mixtures, air-dried for 30 minutes at room temperature, and coated with gold . Prepared samples were captured using a SE1 detector at 10 kV filament tension. For transmission electron microscopy analysis, the samples were fixed overnight at 4°C in 2.5% glutaraldehyde. Afterwards, the cells were rinsed several times with PBS and incubated for 2 h with 1% osmium tetroxide. The biological material was then contrasted with lead citrate and uranyl acetate, dehydrated in a graded series of acetone, and embedded using an Agar Low Viscosity Resin Kit . Ultrathin sections were obtained using an ultramicrotome (Leica EM UC7) and collected on copper grids. The cells were observed with a TEM detector at 20 kV filament tension. The cells for FIB imaging were centrifuged and fixed with 2.5% glutaraldehyde. The procedure of preparing the biological material for FIB was conducted as previously described by Marycz et al. [In order to perform the assay, cultures were fixed with 4% PFA for 40 minutes. After fixation, cells were extensively washed with HBSS. Washing step was repeated three times. Then, the cell membranes were permeabilized for 20 minutes at room temperature using 0.5% Triton X-100 and rinsed with HBSS once again. After this procedure, unspecific binding sites were blocked with blocking buffer composed of HBSS supplemented with 10% goat serum and 0.2% Tween-20 for 35 minutes. Then, the cells were incubated overnight with specific primary antibodies against Ki67 (Abcam) diluted 1 : 200 in HBSS with 1% goat serum and 0.2% Tween-20. Following procedure, the biological material was washed with HBSS and incubated with mouse anti-rabbit secondary antibodies conjugated with atto-594 and atto-488 (dilution 1 : 200) for 1 hour in the dark. Nuclei were counterstained by incubation with DAPI for 5 min.To image 5-methylocytosine (5-MC) and 5-hMC, cells were fixed with 4% PFA for 40 minutes and permeabilized with 0.5% Triton X-100 in HBSS. Afterwards, the cells were incubated with 4 N HCl for 15 min, then with blocking buffer containing HBSS with 10% goat serum and 0.2% Tween-20 for 45 minutes. Incubation with primary antibodies  diluted 1 : 200 in HBSS supplemented with 1% of goat serum and 0.2% Tween-20 was conducted overnight. After three washes with HBSS, the cells were treated with specific secondary antibodies  conjugated with atto-488 diluted 1 : 200 for 1 hour.Immunofluorescent staining of investigated cells was also performed using anti-LAMP2 and anti-PARKIN antibodies . The cells were observed and imaged using confocal microscopy .β-galactosidase activity was performed using the Senescence Cells Histochemical Staining Kit. The number of viable and dead cells were estimated with a Cellstain Double Staining Kit. The dead cells' nuclei were stained with propidium iodide, while the nuclei of viable cells were dyed with Calcein-AM. Images of colored cell organs were captured using a Canon PowerShot camera. All procedures were carried out based on the protocols provided by the manufacturers.The procedure of oxidative stress level quantification was performed after 10 days of adipogenesis stimulation comparing with control, noninduced cell cultures. Superoxide dismutase (SOD) activity analysis was conducted using a SOD Assay Kit, and the level of nitric oxide (NO) was assessed with a Griess reagent kit , whereas reactive oxygen species (ROS) concentration was evaluated in two ways: with a 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate  solution and using a Muse® Cell Cycle Assay Kit with a Muse Cell Analyzer (Merck). Detection of senescence-associated μl of accurate matrix in a total volume of 20 μl. Primer concentration was 0.5 μM. Sequences of the primers used in the quantitative real-time PCR are listed in After 120 hours of culture, cells were rinsed gently using HBSS and homogenized with 1 ml of TRI Reagent. Total RNA was isolated from cells after culture, and tissue samples were collected directly from experimental animals according to a single-step method described previously by Chomczynski and Sacchi  and RT-Pt-test. Statistical analysis was performed with GraphPad Prism 5 software . Differences were considered statistically significant for p < 0.05.The results were evaluated based on measurements obtained in subsequent repetitions. All experiments were performed at least in triplicate. Differences between groups were determined using the nonparametric Isolated cells presented typical for the ASC profile of surface antigens . They exEMS proliferate at a significantly lower rate in comparison to control cells (p < 0.001). Furthermore, we evaluated the rate of proliferating cells in culture by the analysis of BrdU incorporation after 24 and 120 hours of culture (CTRL was characterized by a significantly increased proliferation rate only after 24 hours of cultivation (p < 0.05). PDT was estimated based on the number of cells in each day of experiment (EMS required more time to double their number in comparison to control cells (p < 0.001). The ability of cells to form colonies originating from single cells was performed using the CFU-F assay. For that reason, colonies were stained with pararosaniline . Cell morphology was evaluated on the last (5th) day of the experiment using staining for f-actin and ER .The viability and proliferation rate of investigated cells were established during a five-day lasting culture period. The number of metabolic-active cells was established with a resazurin-based assay (TOX8) in accordance with the manufacturer's protocol. Cells from both investigated groups proliferated at a similar rate during the experiment. Only on the last day did the ASC culture . ASCCTRLperiment . The ressaniline  and counlgorithm , revealin and ER . An enlavaluated . Data frvaluated . ObtaineCTRL, although these were smaller in size. Lipid droplets were also visualized by a TEM microscope. Ultrathin sections by TEM showed large, electron-dense lipid droplets in ASCEMS, which sometimes were associated with mitochondria. ASCCTRL had lipid droplets that resembled those in ASCEMS, however, these were much less electron dense. Those differences in lipid droplet features may indicate a distinct lipid composition and thus, the energy status of the cells. Using SEM, secretion of MVs by cells was analyzed but no visible differences were noted. Oil Red O staining was quantified using Image J (γ (EMS (p < 0.05). Analysis of DNA content revealed an increased number of cells in the G0/G1 phase in the control group (p < 0.001) .Proliferation of cells was established by the BrdU incorporation assay . However Image J , howeverage J  . HoweverEMS (p < 0.001), while the amount of ROS (p < 0.001) was upregulated. Interestingly, the amount of NO was decreased in ASCEMS (p < 0.001). Representative photographs of β-galactosidase staining are shown on EMS senescence. Moreover, the accumulation of intracellular ROS was analyzed using a Muse Cell Analyzer (EMS (p < 0.01). Expression of apoptotic-related genes was established using RT-PCR. The mRNA levels of p21 (EMS (p < 0.001) while BCL-2 was upregulated (EMS (p < 0.05) (p < 0.01).After the last (10th) day of adipogenesis, the SOD , ROS Fi, and NO Analyzer  indicatis of p21  were upregulated . Express < 0.05) . Express < 0.05) . SimilarEMS were characterized by the loss of heterochromatin amount at the nuclear periphery. Furthermore, DNA methylation status was analyzed by immunofluorescence staining for 5-mC and 5-hmC  confirming staining results. Expression of DNMT-1 was upregulated (p < 0.05) in ASCEMS.Nuclei of cells were visualized using a TEM microscope . ASCEMS nd 5-hmC . Obtaine of TET2  and TET3 of TET2  genes waEMS. Localization of LAMP2 in cells was established using immunofluorescence staining and confocal microscope (EMS. Expression of autophagy-related genes was investigated using RT-PCR: Beclin (EMS (p < 0.05), confirming confocal microscope data. Interestingly, no differences in LAMP2 expression in adipose tissue of horses were noted   .EMS which indicates its decreased activity and/or impairment. TEM photographs confirmed ER deterioration in ASCEMS as it was fragmented and swollen  in ASCEMS while the remaining transcripts were upregulated. Furthermore, expression of both CHOP .Using fluorescence staining we evaluated the ER distribution in cells . Decreas swollen . On the of eIF2α , BIP  has been considered only as an energy storage, but in fact it is an active endocrine organ, playing a crucial role in EMS development . At the elopment . Moreoveelopment . Thus, tEMS under adipogenic differentiation conditions. We found that ASCEMS accumulated smaller lipid droplets, and the expression of adipogenesis-related genes was decreased in those cells in comparison to the control group. Moreover, ASCEMS showed the following characteristics during adipogenic differentiation: (i) senescent phenotype, (ii) increased aging, (iii) excessive accumulation of oxidative stress factors, and ultimately, (iv) apoptosis.Furthermore, we investigated growth kinetics, mitochondrial dynamics, autophagy, and apoptosis during adipogenic differentiation of ASCs in order to assess metabolic changes that occur during this process. Impaired proliferative activity along with decreased viability and enhanced apoptosis were also observed in ASCγ). Here, we observed that ASCEMS under adipogenic conditions exhibited lower proliferative and metabolic activity when compared to healthy ASCs. This can be explained by the observed downregulation of genes involved in early events of adipocyte differentiation—STAT5A and SREBP1c. It was found that the expression of STAT5A and SREBP1c in subcutaneous adipose tissue was significantly reduced in type 2 diabetic patients [γ in adipose tissue of diabetic mice versus healthy wild type donors [γ, and type 2 diabetic patients had decreased expression of both PPARγ and STAT5A genes [EMS, which indicated a different mechanism underlying the deterioration of stem cells caused by metabolic syndrome from those observed in adipose tissue. The obtained data are consistent with the findings of other groups [CTRL and ASCEMS. We have observed that ASCEMS accumulates lipid droplets (LD) of various sizes under adipogenic conditions in contrast to ASCCTRL where lipid droplet sizes were similar. Interestingly, Yang et al. have recently shown that adipocyte size variation along with the presence of large adipocytes are predictors of insulin resistance and type 2 diabetes [Adipogenesis is a well-orchestrated process, in which preadipocyte proliferation, differentiation, and lipid biosynthesis are regulated by a set of factors that include signal transducer and activator of transcription 5A (STAT5A), sterol-regulatory-element-binding protein 1c (SREBP 1c), and peroxisome proliferator-activated receptor gamma .Furthermore, it is tempting to speculate, that selective mitophagy is a key mechanism that allows the removal of defective mitochondria and helps sustain proper cellular bioenergetics in ASCi et al. , murine i et al.  have shoEMS were characterized by increased methylation status and decreased expression of demethylation-related proteins, TET 2 and TET3. Progression from stem cells to differentiated cells requires certain changes in the gene expression profile, and epigenetic mechanisms are crucial in the process of differentiation [EMS is responsible for silencing genes involved in adipogenic differentiation, thereby affecting its effectiveness. However, further research must be carried out to confirm our presumption.During adipogenic differentiation, ASCntiation . We hypoα-subunit of eukaryotic translational initiating factor 2 (eIF2α) in adipose tissue [α [. In contrast, in the presented research, we found that IL-6 expression is decreased in ASC during adipogenic differentiation. Data regarding the role of IL-6 in MSC differentiation exists, however, in relation to osteogenesis. Research conducted by Huh and Lee [ER stress in adipose tissue is responsible for the development of inflammation and strongly affects the adipokine secretion profile. Recently, ob/ob diabetic mice have been shown to overexpress ER stress markers, such as BiP, phosphorylated PERK, and the phosphorylated e tissue . Here, wissue [α . In cont and Lee  indicate and Lee  showed tEMS are characterized by increased oxidative stress, senescence, and apoptotic phenotype. Moreover, for the first time we observed a high level of mitochondrial and ER stress caused by excessive ROS. Finally, we have discovered that triggering of selective mitophagy may serve as a rescue mechanism that allows ASCEMS to maintain multipotency and survive in an unfavorable, proinflammatory microenvironment of adipose tissue. We thus, speculate that targeting mitophagy in ASCEMS might become a novel therapeutic approach in the future, aimed at improving mitochondrial biogenesis and metabolism, thereby affecting cell fate.The prevalence of obesity, metabolic syndrome, and diabetes has dramatically risen in recent decades. Excessive hypertrophy and hyperplasia of adipocytes are decisive determinants of abnormal adipose tissue growth, which contributes to the development of the abovementioned disorders. In the present study, we have shown that ASC


Cannabis sativa (hemp) causes male gonadotoxicity by inducing hyperprolactinemia, down-regulation of hypothalamic-pituitary-testicular axis, and oxidative stress. Benin republic hemp has been preferred by illicit users in Nigeria but its effect on male fertility is not understood. This study determined and compared the compositions of Benin republic hemp ethanol extract (BHE) and Nigerian hemp. The effects of BHE on semen parameters, reproductive hormones, and anti-oxidant status, and the possibility of bromocriptine (prolactin inhibitor) to abolish hemp-induced toxicities in rats were also investigated.Nigerian n = 6 each). Groups I (control) and II received normal saline and bromocriptine (3 mg/kg) respectively. Groups III and IV received 2 mg/kg of BHE alone and in combination with bromocriptine respectively, while groups V and VI received 10 mg/kg BHE alone and in combination with bromocriptine respectively. Comparisons among the groups were done by one-way analysis of variance, followed by post-hoc Tukey multiple comparison test. Statistical significance was considered at p < 0.05.Thirty-six male Wistar rats were blindly randomized into 6 oral treatment groups , luteinizing hormone, estradiol, malondialdehyde and glutathione peroxidase. The 10 mg/kg BHE or bromocriptine+BHE (both doses) increased total anti-oxidant capacity and catalase.The BHE improves semen parameters by reducing plasma prolactin and enhancing plasma anti-oxidant status. Its pro-fertility potential might be associated with its deficiency in the widely known gonadotoxic phytocannabinoids. Cannabis sativa (hemp), the most widely abused recreational drug worldwide  and follicle stimulating hormone [FSH]) and testosterone secretion. This prolactin-induced down-regulation of HPT axis ultimately leads to spermatogenic arrest, altered sperm quality, impaired sperm motility, and later produces secondary hypogonadism and male infertility. In addition to this indirect effect, hyperprolactinemia also directly influences steroidogenesis and spermatogenesis by acting on prolactin receptors present in Leydig and Sertoli cells in the testes to produce primary hypogonadism and male infertility [Hyperprolactinemia has been reported to have a definite role in male infertility. It causes infertility in about 11% of oligospermic males  by inhibertility .There have been evidences that the hyperprolactinemia-induced infertility is reversible –18. A siThe National Drug Law Enforcement Agency (NDLEA) of Nigeria, through series of online and print media publications, has recently observed that most of the illegal hemp users in Nigeria have been preferentially obtaining hemp from Benin republic. The NDLEA also observed that users now compress this imported hemp and soak it with ethanol prior to its use; and that the “high” and euphoria felt by users of Nigerian and Benin republic hemp are comparatively different. It is not yet known whether there is difference in the quality of both hemp samples or not. The present study determined and compared the compositions of Benin republic hemp ethanol extract (BHE) and Nigerian hemps.The effects of BHE on semen parameters, reproductive hormones, and anti-oxidant status were also investigated in rats. Bearing the prolactin-mediated gonadotoxic effect of Nigerian hemp  in mind,Thirty-six (36) adult male Wistar rats (weight range: 160–180 g) were obtained from the Department of Biochemistry, University of Ilorin, Nigeria. They were housed in wooden cages maintained under standardized conditions , and were acclimatized in the laboratory for 2 weeks before the commencement of the study. The rats were fed with standard pelletized rodent diet  and water ad libitum. All the animals were well-catered for according to the criteria outlined in the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the National Academy of Science and approved by the Ethical Research Committee of the University of Ilorin, Nigeria.Mixture of hemp seeds and leaves (75%:15% respectively), both of Benin republic and Nigeria origin, were kindly donated by the National Drug Law Enforcement Agency (NDLEA), Nigeria, for research purpose only. About 200 g of each of these samples was subjected to extraction with 98% ethanol in Soxhlet apparatus for 4–8 h as described earlier [A gas chromatography (GC) from AgilentCo. USA, hyphenated to a mass spectrometer  with triple axis detector equipped with an auto-injector (10 μl syringe) was used. Helium gas was used as a carrier gas. All chromatographic separation was performed on capillary column having the following specifications: length- 30 m, internal diameter- 0.25 μm, thickness- 250 μm, treated with phenylmethylsilox. Other gas chromatography mass spectrometry (GC-MS) conditions are: ion source temperature (EI)- 250 °C, interface temperature- 300 °C, pressure- 16.2 psi, cut time- 1.8 min, 1.0 μL injector in splitless mode with split ratio 1:50 and injection temperature of 300 °C. The column temperature started at 35 °C for 5 min and changed to 150 °C at the rate of 4 °C/min for 2 min. The temperature was raised to 250 °C at the rate of 20 °C/min and held for 5 min.The MS scanning was performed from M/Z85 to M/Z380. The GC-MS solution software provided by supplier was used to control the system and to acquire the data. The separation of the injected samples was carried out on an HP capillary column (HP-5MS). Identification of the compounds was carried out by comparing the mass spectra obtained with those of the standard mass spectra from National Institute for Standards and Technology (NIST) library (NIST II). Identification was based on the molecular structure, molecular mass and calculated fragments. The fatty acids and other organic compounds were identified by comparing their retention times with those of the standards. The quantity of each compound present was expressed as percentage of the total compounds.n = 6 per group). Groups I (control) and II received normal saline and bromocriptine (3 mg/kg) respectively. Groups III and IV received 2 mg/kg of BHE [Since the hemp sample of Nigerian origin has been repeatedly used in the series of our previous studies , 11, 13,g of BHE , 11 along of BHE  respectig of BHE  alone anim). Thereafter, they were dissected in order to collect blood by cardiac puncture. Whole blood for the determination of the biochemical markers were collected in heparinized sample bottles which were centrifuged at 3500 rpm for about 10 min at -4 °C using a cold centrifuge . The separated plasma samples were collected into separate plain bottles and were stored at -20 °C prior to the biochemical analyses.Animals were anaesthetized a day after the last treatment with sodium pentobarbital  while the total number of all the sperm cells counted was recorded as (T). Sperm motility (%) was calculated using (N/T × 100%).The principle for determination of sperm morphology was based on the ability of morphologically normal sperm to appear white in color as the plasma membrane will prevent the dye to enter, while abnormal sperms take up the dye and stain dark color. The microscope slides and the eosin stain were pre-warmed to room temperature. One milliliter of the sperm suspension – normal saline solution was transferred to a test tube and 2 drops of 1% eosin were added and mixed gently for agitation. This was incubated for 45–60 min to allow its proper staining and then re-suspended with a Pasteur pipette. A clean grease-free glass slide was used. Potential damage to the sperm cells should be avoided. One or 2 drops of the stained sperm were placed approximately 1 cm from the end of the slide lying on a flat surface. A second slide was held with the slide’s long edge gently touching across the width of the sperm slide and pulled across to produce a sperm smear. After air-drying the slide, using a microscope at 100X objective, the sperm cells were examined. The sperm along the periphery was normally excluded from the examination because there is a greater tendency for artifacts to occur in these regions. At least, five fields were viewed covering the whole slide. Examples of morphological abnormalities are double-headed, elongated head, pyriform head, bent head, bent tail, bent mid-piece, coiled tail, double tail, headless, tailless, etc. All those with normal morphology were recorded as N while the total number of the counted spermatozoa was recorded as T. The percentage sperm morphology was calculated as (N/T × 100%).Diagnostic kits for the determination of malondialdehyde (MDA), total anti-oxidant capacity (TAC), superoxide dismutase (SOD), glutathione Peroxidase (GPx), and catalase were obtained from Fortress Diagnostics Limited, United Kingdom. In addition, analytic kits for the determination of GnRH, prolactin, FSH, LH, testosterone, and estradiol were procured from Elabscience Biotechnology Company Ltd. Wuhan, Hubei, China. The analyses were done with spectrophotometer  according to the manufacturers’ instructions.post-hoc Tukey multiple comparison test. Statistical significance was considered at p < 0.05Statistical analyses were done using statistical package for social sciences (SPSS) version 16.0 . Values were expressed as Mean ± S.E.M. of the variables measured. Comparisons among the groups were done by one-way analysis of variance (ANOVA), followed by The BHE is rich in stearic acid (27.98%), followed by benzoic acid 14.43%), oleic acid (11.51%), benzeneacetic acid (10.72%), and benzophenone azine (9.96%). It has intermediate quantity of aniline (3.18%), cyclohexanecarboxylic acid (4.75%), and stearic glycidol (3.81%) but lower quantity of 2,3,4- Trimethoxyphenylethylamine (0.35%), Benzamide (0.29%), palmitic acid (1.09%), and cannabinol (1.20%). The acetone fraction from the ethanol extract of Nigerian hemp has its highest constituent as cannabinol (36.5%), followed by tetrahydrocannabinol , α-linolenic acid (12.13%), palmitic acid (8.91%), oleic acid (5.94%), linoleic acid (4.87%), cannabichromene (1.7%), and stearic acid (1.42%). However, the n-hexane fraction from the ethanol extract of Nigerian hemp has its highest constituent as THC (49.11%), followed by cannabinol (20.25%), cannabichromene (5.58%), and linoleic acid (4.31%)  when compared to control. Neither bromocriptine nor any of the doses of BHE affected the plasma testosterone, but their co-administration significantly increased the plasma testosterone level Table .Table 2ETreatment of rats with bromocriptine and/or BHE caused increase in the plasma SOD activity but no significant change in the plasma MDA and GPx when compared to control. Rats that received 10 mg/kg of BHE and those that received combination bromocriptine and either dose of BHE had higher TAC and catalase when compared to control Fig. a-e.Fig. Hemp seeds have been developed into numerous products for food, cosmetic, therapeutic, functional food and nutraceutical industries . The preBecause about 35% of the available oil has been reported to remain in the seed cake following pressing process while supercritical carbon dioxide extraction has been documented as a better alternative to pressing or organic solvent methods for extraction of lipids such as PUFAs from fish lipids and vegetable oils , 29, theWe have consistently shown that ethanol extract of hemp leaves of Nigeria origin causes gonadotoxicity in male rats by eliciting hyperprolactinemia, down-regulation of HPT axis, oxidative stress, and endocrine disruption , 11, 13.Telfairia occidentalis leaves in rats [The beneficial effect of lipids on semen parameters has been well-documented. Cholesterol has been reported to increase cell membrane stability and decrease the transition temperature from the fluid to the gelatinous phase . Phospho in rats . On the  in rats , 36–38.  in rats . The imp in rats , 11, 13.The release of prolactin from adenohypophysis is under tonic inhibition of dopamine, which is delivered to adenohypophysis from the hypothalamus via the hypothalamic-hypophyseal portal system. Dopamine decreases prolactin secretion by binding to the dopamine D2 receptors on the surface of lactotroph and diminishing intracellular cyclic adenosine monophosphate (cAMP) level. We have previously shown that increase in the plasma prolactin level is one of the mechanisms through which Nigerian hemp induces male gonadotoxicity . TherefoApart from hyperprolactinemia and down-regulation of HPT axis, we have further previously shown that the gonadotoxic effect of Nigerian hemp is also mediated through oxidative stress , 11. In In conclusion, this study shows that hemp sample from Benin republic is deficient in cannabinoids. It also shows that this hemp sample improves semen parameters by reducing plasma prolactin and enhancing plasma anti-oxidant status. It was opined that the pro-fertility effect of the Benin republic hemp sample could be due to its deficiency in the widely known gonadotoxic cannabinoids. This study has cautioned us from making a generalized claim that hemp is gonadotoxic. It shows that the origin of hemp samples might determine their composition and their ethno-pharmacological effects on the body functions.


Low cardiac output syndrome (LCOS) is a severe condition which can occur after cardiac surgery, especially among patients with pre-existing left ventricular dysfunction. Dobutamine, its first-line treatment, is associated with sinus tachycardia. This study aims to assess the ability of intravenous ivabradine to decrease sinus tachycardia associated with dobutamine infused for LCOS after coronary artery bypass graft (CABG) surgery.In a phase 2, multi-center, single-blind, randomized controlled trial, patients with left ventricular ejection fraction below 40% presenting sinus tachycardia of at least 100 beats per minute (bpm) following dobutamine infusion for LCOS after CABG surgery received either intravenous ivabradine or placebo (three ivabradine for one placebo). Treatment lasted until dobutamine weaning or up to 48 h. The primary endpoint was the proportion of patients achieving a heart rate (HR) in the 80- to 90-bpm range. Secondary endpoints were invasive and non-invasive hemodynamic parameters and arrhythmia events.P = 0.04). Median times to reach target HR were 1.0 h in the ivabradine group and 5.7 h in the placebo group. Ivabradine decreased HR  while increasing cardiac index (P = 0.02), stroke volume (P <0.001), and systolic blood pressure (P = 0.03). In the placebo group, these parameters remained unchanged from baseline. In the ivabradine group, five patients (36%) developed atrial fibrillation (AF) and one (7%) was discontinued for sustained AF; two (14%) were discontinued for bradycardia.Nineteen patients were included. More patients reached the primary endpoint in the ivabradine than in the placebo group (13 (93%) versus 2 (40%); Intravenous ivabradine achieved effective and rapid correction of sinus tachycardia in patients who received dobutamine for LCOS after CABG surgery. Simultaneously, stroke volume and systolic blood pressure increased, suggesting a beneficial effect of this treatment on tissue perfusion.EudraCT 2009–018175-14. Registered February 2, 2010.European Clinical Trials Database: The online version of this article (10.1186/s13054-018-2124-8) contains supplementary material, which is available to authorized users. The low cardiac output syndrome (LCOS) is an acute circulatory disorder that may occur in 2% to 10% of patients after cardiac surgery . When prf channel, which regulates the diastolic depolarization slope, therefore inducing a decrease in HR without affecting the conduction times (atrioventricular and intraventricular) or ventricular repolarization  for the ivabradine group and 5.7 h [5.7–5.9] for placebo (P = 0.13).The primary endpoint was reached in 13 (93%) out of 14 patients of the ivabradine group and in 2 (40%) out of 5 of the placebo group. The introduction of ivabradine was followed by a quick and sustainable HR reduction from 112 [105–120] to 86 [78–96] bpm (P <0.05); SV ; CO , CI , and LVSWI  , and LVSWI (+10.6 [3.7–17.6]). Values for these variables at H3 were comparable to H2 , SV (+19 [9–35] mL), CO (+0.6 [− 0.1–1.7] l/min), CI (+0.4 [0–0.8] l/min/mn = 8)  with persisting LCOS in the ivabradine group as opposed to none in the placebo group. The resulting hemodynamic variations were similar in the subgroup of patients in whom dobutamine regimen remained unchanged (% with peP = 0.18). This difference was due mostly to a difference in treatment duration: 23.7  hours in the ivabradine group versus 13.8  hours in the placebo group (P = 0.18). In the ivabradine and placebo groups, eight (57%) and two (40%) patients were treated with epinephrine, and four (29%) and one (20%) were treated with norepinephrine, respectively.Cumulative dose of dobutamine was comparable between the two groups  developed POAF in the ivabradine group as opposed to none in the placebo group. One episode of bradyarrythmia occurred during the administration of ivabradine, requiring its cessation. The other four episodes occurred after the cessation of ivabradine infusion. In addition, ivabradine was associated with three cases (21%) of HR decrease below the threshold of 75 bpm, of which two (14%) required study drug cessation. Finally, one patient presented with several episodes of sustained ventricular tachycardia prior to and after ivabradine administration.Troponin levels remained stable in both groups Table . One patIn this phase II exploratory multi-center randomized placebo-controlled trial involving patients with LCOS treated with dobutamine after CABG surgery, we observed that i.v. ivabradine infusion was associated with (1) a quick, durable, and significant control of HR in the target range of 80 to 90 bpm, (2) a significant increase in SV and SBP, and (3) a higher incidence of POAF and bradycardia.Like its oral counterpart, i.v. ivabradine was found to effectively reduce HR but with a shorter delay of action after treatment onset. This reduction was durable and allowed for a strict control of the HR in a pre-specified target range. In patients with LCOS, dobutamine is commonly used to improve oxygen delivery but induces tachycardia , 26. It These results confirm preliminary animal studies in which dobutamine combined with ivabradine allowed for a simultaneous contractile enhancement and prolongation of diastole, allowing for optimal filling and enhanced CO, in spite of a decrease in HR .Because of the greater SV, SBP increased in the ivabradine group. MBP and DBP remained unaltered, indicating that systemic and left ventricular perfusion pressures were preserved. Overall, LVSWI increased and atrial pressures (RAP and PCWP) tended to decrease, suggesting a beneficial effect on biventricular end-diastolic congestion. The association of increased systemic blood flow, preserved mean arterial pressure, and reduced venous congestion in the systemic and pulmonary veins attests to an improved hemodynamic status. This corroborates the observations that renal function was maintained and that serum lactate did not increase significantly. We can assume that oxygen delivery was adequate and end-organ function was preserved, indicating that LCOS management was effective. These findings suggest that ivabradine did not interfere with a compensatory tachycardia but rather corrected the unwanted dobutamine-induced acceleration in HR.These results were consistent and confirmed when assessing hemodynamic parameters at H2 and H3 after treatment initiation when most ivabradine patients reached target HR, emphasizing the causal role of ivabradine rather than the effect of time (as observed in the placebo group).Indeed, ivabradine represents one of a few i.v. HR-decreasing agents without negative effects on inotropism, allowing its use in post-operative LCOS. Other existing therapeutic agents include digoxin and magnesium. Intravenous digoxin has the major drawback of presenting dose-dependent toxicity, including potential lethal arrhythmias, hyperkaliemias, and vasoplegic shock. Intravenous magnesium, though less deleterious, has a less significant effect on sinus tachycardia. Beta-blockers are part of the routine treatment for most patients with heart failure  but, owing to their adverse effect on contractility and their antagonism with dobutamine, can rarely be administered in the post-operative period. Thus, ivabradine appears as a very promising tool offering an original alternative to control HR, even in patients with severely impaired left ventricular function.Epinephrine and norepinephrine usage was similar in the two treatment arms. Dobutamine regimen was increased by attending physicians in six ivabradine patients who remained in LCOS. Although we cannot rule out that controlling tachycardia might have reduced CO in this subgroup, we believe that HR reduction facilitated dobutamine up-titration. The fact that changes in HR, MAP, and SV were similar in the subgroup of patients in whom dobutamine was not increased supports the role of i.v. ivabradine in hemodynamic improvement independently from other concomitant treatments.As previously described for oral ivabradine in the SHIFT (Systolic Heart Failure Treatment with the I(f) Inhibitor Ivabradine Trial) , the i.vThe small number of patients is the main limitation to the validity of our observations. The inclusion criteria of this study were very restrictive in order to include a homogeneous population of patients and to avoid confounding factors. As a consequence, the number of inclusions was small but nevertheless appropriate to reach the primary objective: demonstrating the reduction of HR in the ivabradine-treated group. Inferential statistics were performed while taking into account the small sample size, allowing us to observe trends in the secondary hemodynamic endpoints. Consequently, adjusted statistical analysis could not be performed to rule out the confounding effects of concomitant catecholamine administration on hemodynamic changes. However, ad-hoc analyses showed that associated catecholamine administration was similar in the two treatment arms. However, even if subgroup comparison among ivabradine patients (a) with dobutamine increase and (b) without dobutamine increase showed similar results in hemodynamic variations, it is not possible to prove that the effect of ivabradine was independent from dobutamine dosage modifications.Regarding the primary endpoint, we choose to make comparisons between baseline and the time of study drug cessation rather than at a fixed time point. The timing of study drug interruption differed among patients, attesting to the variability in LCOS severity. Indeed, many factors can influence LCOS , which cannot be controlled for. Thus, we deliberately choose to make the comparisons between baseline and the time when LCOS was considered over in each patient (dobutamine weaning). This pragmatic approach allowed us to minimize the administration of study drug, which was important from a safety point of view, and to compare patients when they had reached a similar hemodynamic situation. However, it can be noted that changes between H0 and H2  were similar to changes between H0 and the time of study drug cessation. Finally, because investigators were not always available when eligible patients underwent surgery, those who were included in this cohort are not strictly consecutive patients.This exploratory study showed that in a specific population of patients, those who had dobutamine-induced tachycardia after elective CABG, the administration of i.v. ivabradine significantly and quickly reduced HR without impairing CO and arterial blood pressure. Larger studies would be necessary to better assess the overall hemodynamic effects of i.v. ivabradine alone or associated with dobutamine.Additional file 1:Figure S1. Study flow chart. Abbreviations: HR heart rate, LCOS low cardiac output syndrome. (PPTX 53 kb)Additional file 2:Figure S2. Hemodynamic variations in the ivabradine group (n = 14) between time of dobutamine initiation (LCOS), ivabradine initiation (H0), and 2 and 3 h after ivabradine initiation (H2 and H3). Bold lines represent median values, and dotted lines indicate quartiles 1 and 3. P values are from Wilcoxon signed-rank test. (DOCX 98 kb)


Non-immune hydrops fetalis (NIHF) is a complex condition with a high mortality and morbidity rate. Here we report the etiology and outcome of 1004 fetuses with NIHF, in a large single Maternal and Children’s hospital of Southern China, since the year of 2009 to 2016. Among these 1004 fetuses with NIHF, the etiology was identified prenatally in 722 of them (72%). The most common ones were hematologic diseases and chromosomal abnormalities. There were eight spontaneous abortions, 18 intrauterine fetal demise, 672 pregnancy terminations and 87 were lost to follow-up. 219 of the 1004 fetuses were live-born and the overall survival rate was 21.8% at this point. After birth 16 perinatal or early neonatal deaths were encountered and five lost to follow-up. Of the remaining 198 newborns, 153 thrived without apparent morbidity. The most significant factors associated with mortality were prematurity and low birthweight. In conclusion, we described the largest report of underlying causes and outcome of NIHF in Southern China. Etiologies were identified for 72% of 1004 fetuses with NIHF. And two poor prognostic factors for survival are preterm birth at less than 36.5 weeks and birthweight lower than 2575 g respectively. According to the clinical guideline from the Society for Maternal-Fetal Medicine4, HF is defined as the presence of at least two abnormal fluid collections in the fetus, including ascites, pericardial effusion, pleural effusions, and generalized skin edema (skin thickness >5 mm). Polyhydramnios and placental thickening are also frequent sonographic findings6. Generally, HF could be classified into two categories: iso-immune HF (IHF) and non-immune HF (NIHF). IHF is caused by fetal hemolysis, an process that is mediated by circulating maternal antibodies to antigens of fetal red blood cells. The incidence of IHF has markedly declined due to the use of anti-D gamma globulin prophylaxis. At present, about 90% of cases of hydrops are NIHF7, with prevalence reported as 1 in 1700–3000 pregnancies10.Hydrops fetalis (HF) refers to a condition in which an excess of fluid accumulates in fetal soft tissues and serous cavities11. Despite many improvements in diagnosis and management, however, the mortality rate of NIHF remains high during either fetal or neonatal period, and predicting survival still poses a challenge17. The prognosis for NIHF varies based on the underlying etiologies and gestational ages at diagnosis and at delivery19, thus clarifying the primary etiology is crucial for genetic counseling and future pregnancy management. Up to date, there have been limited studies into the prevalence and etiology of NIHF in China20, and the association between the etiology and either perinatal or developmental outcome has not been fully determined. The aim of this study was to describe the etiologies and outcome of 1004 NIHF cases in Southern China, and evaluate the relationship among the etiology, clinical characteristics, and outcome.NIHF may occur at various gestational ages with different types of etiologies, including hematologic, cardiovascular, chromosomal, infections, syndromic, thoracic, inborn errors of metabolism, lymphatic dysplasia, urinary tract malformations, extra thoracic tumors, gastrointestinal, twin-to-twin transfusion syndrome (TTTS)-placental, miscellaneous, and idiopathic causesIn this study, 1004 cases of NIHF were diagnosed in a total of 126,856 pregnancies (incidence of 7.9/1000) by antenatal ultrasound scanning. The median maternal age was 28 years (IQR 25–32 years), and the median gestational age (GA) at diagnosis of NIHF was 23 weeks (IQR 17–26 weeks).n = 285, 28.4%) and chromosomal abnormalities , followed by lymphatic anomalies , cardiovascular disorders , twin-to-twin transfusion syndrome (TTTS) – placental/cord problems , urinary tract malformations , intrauterine infections , thoracic malformations , miscellaneous causes , gastrointestinal disorders  and syndromic diseases .Table Cytomegalovirus, 9 cases with Hepatitis B virus, 3 cases with Parvovirus B19, 2 cases with Herpes simplex virus and 1 case with Syphilis. See Appendix Table In 285 cases of hematologic diseases, 267 cases were caused by typical Bart’s hemoglobin disease, 17 were caused by hemoglobin H disease associated with α-globin gene Quong Sze mutation, Constant Spring mutation and Westmead variant, and one case by α compound β thalassemia gene mutations. Among 199 cases of chromosomal abnormalities, there were 183 cases of chromosomal number abnormalities, 5 cases of chromosomal structure abnormalities and 11 cases of copy number variations, involving mostly chromosomes X, 21, 18 and 13. Among the group of intrauterine infections, there were 11 cases with P = 0.0002), GA at diagnosis (P < 0.0001) and placental thickness (P < 0.0001)  with appropriate sampling size, including maternal age (P < 0.000 and placn = 109), lymphatic anomalies (n = 11), intrauterine infections (n = 7) or urinary tract malformations (n = 6)  and 672 pregnancy terminations. Eighty seven pregnancies were lost to follow-up. A total of 219 of the 1004 fetuses (21.8%) survived till birth with a median GA at delivery of 38 weeks (IQR 37–40 weeks) and a mean birth weight of 2938 ± 554 g. Six percent of infants (14 of 219) were premature and thirty-six percent (79 of 219) were delivered via cesarean section. Five cases were lost to follow-up after birth. In the remaining 214 cases with follow-up, there were 16 perinatal deaths (PND) or early neonatal deaths (END). Overall, a total of 198  infants survived without mortality , idiopathic , urinary tract  and lymphatic defects  causes, and lowest among neonates with syndromic , chromosomal  and hematologic  diseases (Table P = 0.0002) and lower birthweight (P = 0.0046). The median GAs at delivery in the group of intact survivors, co-morbidity survivors, and fatal cases were 39 weeks (IQR 38–40 weeks), 38 weeks (IQR 37–39 weeks) and 34 weeks (IQR 28–39 weeks) and the mean birthweight were 2998 ± 468 g, 2878 ± 642 g and 1997 ± 903 g respectively. By using ROC curve analysis, the area under the curve is 0.76 for GAs at delivery and 0.86 for birthweight, respectively. The cutoff values of GA at delivery and birthweight are 36.5 weeks  and 2575 g , respectively.The overall survival rate was highest among neonates with gastrointestinal  of fetal NIHF could be well explained by the differences of regions and GAs at diagnosis21.In this study, the causes of NIHF could be identified in 72.0% of cases prenatally. The most common diagnoses associated with NIHF were hematologic disease (28.4%) and chromosomal abnormalities (19.8%) followed by unexplained NIHF (28.0%), lymphatic (7.8%), cardiovascular (4.1%), TTTS – placental/cord problems (3.0%), urinary tract malformations (2.9%), intrauterine infections (2.6%), thoracic malformations (1.7%), miscellaneous causes (0.8%), gastrointestinal disorders (0.7%) and syndromic diseases (0.2%). The high proportion of fetuses with hematologic disease and chromosomal abnormalities were caused by the regional difference and inclusion of pregnancies in the first trimester, as is the case with high incidence of NIHF in this area. We didn’t identify any cases with inborn errors of metabolism or extra thoracic tumors due to certain limitations of our diagnostic workflow. The maternal age and GA at the diagnosis of NIHF in the group of chromosomal abnormalities is very different from other groups, as expected23. The mortality rate was highest among neonates with syndromic (100%), chromosomal (99.5%) and hematologic (98.9%) diseases. In two cases with syndromic diseases, one with thanatophoric dysplasia were selectively terminated by the parents and one with acardiac twins sequence was delivered through premature labor and ended with perinatal death. The high mortality rate in the group of chromosomal and hematologic diseases were much in accordance with a high prenatal mortality rate . In our study, the parents opted to terminate the pregnancy without a guaranteed good prognosis, especially when a related genetic cause has been identified20. The intact survival rate was 71.5%, slightly higher than other reports17. This may be due to a lack of formal assessment of neurodevelopmental outcome after patient discharge from the hospital. Instead, information from parents and records of physical evaluation at birth describing the status of the baby as healthy was relied upon. Thus, it is less likely that severe developmental delay would be classified as healthy.Although early diagnoses have been achieved in many cases allowing for the option of antenatal therapy, the overall survival rate was 21.8% and the prognosis depended on the cause, similar to previous reports24. Among all these factors, GA at delivery has been identified as a prognostic factor in several findings21. In our study, preterm GA at delivery and lower birthweight were remarkably associated with the risk of postnatal deaths, whereas other factors including maternal age, GA at diagnosis, delivery mode and gender, were non-significant.Various prognostic indicators at antenatal and postnatal stages have been described in studies of NIHF, including prenatal diagnosis of NIHF, GA at delivery, Apgar scores at 1 and 5 minutes, intubation and chest compression in the delivery room, the presence of pleural effusion, ≥2 cavity effusions, low serum albumin concentration, and need for thoracentesis26.In conclusion, we retrospectively summarized the etiology and outcome of 1004 cases with NIHF in Southern China. Although the prevalence and etiologic causes vary greatly among different country regions and different studied populations, our study could thereby provide much specific information for genetic counseling of NIHF in this region of Southern China. The incidence of fetal NIHF in this area is relatively high (7.9/1000). Every effort should be made to determine the underlying etiology, as it not only associates with the disease prognosis but also affects the recurrence risk. Based on our study, hematologic disease (28.4%) and chromosomal abnormalities (19.8%) are the two major causes of NIHF in this population. The mortality rate of NIHF remains unfortunately high, of which preterm birth at less than 36.5 weeks and birthweight lower than 2575 g are two poor prognostic factors for survival. The management of NIHF thus continues to be a challenge in clinics, despite of increased diagnostic rate with advanced testing technologiesIn this study, we performed a retrospective review of 1004 cases with NIHF encountered in the Maternal and Children’s Hospital of Guangxi Zhuang Autonomous Region, one of the tertiary fetal medicine centers in the Southern region of China from January 2009 to June 2016. All diagnosed cases underwent detailed structural and cardiac ultrasound examinations. Trans-abdominal scans were applied by ultrasound equipment consisting of 3–5 MHz convex sector probe and GE Voluson 730 Expert , Toshiba Aplio , Aloka SSD-4000  and Philips iU22 . HF is defined according to clinical guideline from the Society for Maternal-Fetal Medicine, and NIHF was defined as fetuses with HF but in the absence of isoimmunization.et al.1. Our diagnostic workflow was listed as follows: 1) If both parents were found to be carriers for α or β thalassemia, genetic testing for fetal amniocentesis or cordocentesis will be offered. 2) If structural anomalies were detected and likely related to chromosomal abnormalities, fetal karyotyping and/or SNP-array would be offered and performed upon the parents’ request. 3) If other causes of hydrops than genetic factors were determined, such as congenital cystic adenomatoid malformation, the etiology was then confirmed after delivery. 4) If the cause could not be identified by scan, invasive approach for fetal karyotyping and/or SNP-array was provided. Meanwhile, the maternal blood samples were also collected and sent for hemoglobin electrophoresis examination as well as for microbiology testing to exclude parvovirus B19 and TORCH . If the result of α-thalassemia or infection turns out to be positive, fetal blood sample was then collected to confirm the diagnosis. Based on these testing results, the decision of whether or not to continue the pregnancy was made by the couples after appropriate counseling.In this work, maternal demographic features including maternal age, family history of thalassemia, parity, GA at diagnosis, NIHF etiology, delivery mode, pregnancy outcome, perinatal complications, perinatal survival, and one-year outcome were analyzed. The etiology of NIHF was grouped into 14 categories as suggested by Bellini P-value of < 0.05 was defined as statistically significant. All the variables remarkably associated with mortality were included in the receiver operating characteristics (ROC) curve analysis. Youden index (YI) was calculated as (specificity + sensitivity) − 1.Data are displayed as median with interquartile range (IQR), mean ± standard deviation, or frequency (%). Kruskal-Wallis test was utilized for multiple means comparison, and proportions were compared by chi-square test. A http://www.nature.com/srep/policies/index.html#experimental-subjects).This work was approved by the Medical Ethics Committee of Guangxi Maternal and Children’s Hospital, and the informed consent was obtained from all participating families. All methods were performed according to the guidelines and regulations (Appendix Table 1 and Table 2


We developed a novel plasma amino acid profile-based index (API) to detect ovarian, uterine, cervical, and endometrial cancers. In this study, we aimed to evaluate whether abnormal API values could be normalized after curative treatment in patients with gynecological malignant tumors.Patients with gynecological cancer with abnormal API values were included in this study. Pre-operative absolute API values were compared with those after curative treatment. The normalization rates of API values in patients negative for the expression of three well-known tumor markers  were also evaluated. In addition, related amino acid profiles in healthy controls and patients under pre- and post-treatment conditions were analyzed.Among 94 patients with abnormal pre-operative API values, the median API value was decreased from 9.52 to 2.17 after treatment . The decreased ranges were similar in patients with adenocarcinoma  and squamous carcinoma . In 93.5% (43/46) of patients negative for tumor markers prior to operation, API values were normalized after the successful treatment. In addition, some pre-operative abnormal amino acid profiles, including Ile, Trp, and His, were reversibly normalized after treatment.The API is a promising tumor marker in gynecological malignancies for the diagnosis of remission, particularly in patients negative for general tumor markers. Further studies are needed to explore the mechanisms related to the normalization of abnormal amino acid profiles.The online version of this article (10.1186/s12885-018-4875-7) contains supplementary material, which is available to authorized users. According to the 2016 cancer registry and statistics, the screening rate of cervical cancer in Japan is still as low as 42.3%, and the incidences of endometrial and ovarian cancer are in increasing trends [CA125 was first reported as a tumor marker to detect ovarian tumors in the 1980s . MoreoveThe plasma amino acid concentrations of healthy individuals are usually controlled in a certain range by the homeostatic function of the body; however, the levels of various amino acids are altered in patients with different types of cancer, including cervical , endometFor ovarian, cervical, and endometrial cancers, alterations in plasma amino acid profiles are similar; therefore, a single API was developed to include all of these cancers (AICS (uterine/ovarian)) , 7. UsinFor patients with gynecological cancer whose API value exceeds the cutoff value, it is not yet clear how API values change after treatment. Therefore, in this study, we examined changes in pre-operative and post-operative API values in patients with cervical cancer, uterine endometrial cancer, and ovarian cancer who underwent surgery and achieved complete remission after primary therapy. The aim of this study was to evaluate whether abnormal API values could be normalized after curative treatment in patients with gynecological malignant tumors with the cutoff value established in our previous study .We included patients who underwent surgery for gynecologic cancers, including epithelial ovarian cancers, cervical cancers, and uterine endometrial cancers, at the Department of Obstetrics and Gynecology of Yokohama City University Hospital between November 2006 and December 2012 and whose pre-operative API was 6.0 or more. Patients continued to visit our hospital as outpatients, and we performed follow-up blood tests to assess changes in API values from February 2013 to December 2014. In this study, an API of 6.0 or more was defined as an abnormal API according to a previous study [We used control group data obtained in a previous study  for receAfter an overnight fast, blood samples (5 mL) were collected in tubes containing ethylenediaminetetraacetic acid disodium salt  and immediately cooled with ice. Plasma was separated by centrifugation at 2000×g and 4 °C for 15 min and stored at − 80 °C until analysis. After thawing, the plasma samples were deproteinized using acetonitrile at a final concentration of 80% before measurement of amino acid concentrations by high-performance liquid chromatography electrospray ionization/mass spectrometry with precolumn derivatization.The API was originally developed as AminoIndex Cancer Screening , and AICS (uterine/ovarian) is a screening marker for cervical, endometrial, and ovarian cancers based on a multivariate analysis of the plasma free amino acid concentration, comparing patients with cancer to healthy controls. The formula for API includes the following amino acids: Trp, His, Cit, Val, Ile, and Gly. API values were measured by applying concentrations of amino acids as previously described  at the ITo determine the significance of differences in API values before and after surgery, Wilcoxon signed rank tests were used. Chi-squared tests were used to compare API normalization rates in the three gynecological cancers. ROC curve analysis of each amino acid was used to express their capabilities to discriminate between patients with cervical, endometrial, or ovarian cancer and healthy controls, and the area under the curve (AUC) was calculated. All statistical analyses were performed using R version 3.3.0  and GraphPad Prism version 6 .Of the 94 cases in this study, 22 (23.4%) cases were cervical cancers, 53 (56.4%) cases were endometrial cancers, and 19 (20.2%) cases were ovarian cancers. An average of 54.8 ± 23.1 months (range: 10–95 months) had passed since the operation at the time of postoperative blood tests.The characteristics of the patients are shown in Table Table p < 0.0001), and the median difference was 6.43  for all patients. A reversible decrease in API value after surgery was observed independent of histology, and the median differences in API before and after surgery were 6.28 (95% CI: 5.43–6.95) for adenocarcinoma and 7.44 (95% CI: 3.04–8.46) for SCC.Figure Changes in API before (pre) and after (post) complete surgery for patients with negative tumor markers before surgery are shown in Table We performed ROC curve analysis to determine whether each amino acid concentration in patients was distinguishable from that in healthy controls, and examined the change in each amino acid before and after complete surgery  and after (post) complete surgery. ROC curve analysis was performed for each plasma amino acid concentration in healthy controls and patients before and after surgery. The same healthy control group was used for analysis of all patients (a), cervical cancer patients (b), endometrial cancer patients (c), and ovarian cancer patients (d). The AUC was calculated and is indicated in each graph. (PDF 765 kb)