In detail, two groups of 12 animals each were subjected to either a conventional diet or a supplemented diet with extruded linseeds as a source of omega-3 fatty acids and plant extracts as a source of phenolics antioxidant compounds. The digested meat was subjected to an untargeted metabolomics approach. Several metabolites deriving from lipid and protein digestion were detected. Our untargeted approach allowed for discriminating the two different meat cuts, based on their metabolomic profiles. Nonetheless, multivariate statistics allowed clearly discriminating between samples obtained from different animal diets. In particular, the inclusion of linseeds and polyphenols in the animal diet led to a decrease in metabolites generated from oxidative degradation reactions, in comparison to the conventional diet group. In the latter, fatty acyls, fatty aldehydes and oxylipins, as well as cholesterol and vitamin D3 precursors and derivatives, could be highlighted.Meat quality seems to be influenced by the dietary regimes applied for animal feeding. Several research studies are aimed at improving meat quality, preserving it from oxidative processes, by the incorporation of antioxidant components in animal feeding. The main part of these studies evaluates meat quality, determining different parameters directly on meat, while few research studies take into account what may happen after meat ingestion. To address this topic, in this study, an in vitro gastrointestinal digestion protocol was applied to two different pork muscles,  Meat quality, in particular in terms of fat composition, is affected by the animal feeding regimen. For this reason, the current trend is toward formulation of specific diets for animals, such as pigs, in order to obtain meat with specific and healthier characteristics. As an example, following the use of linseeds in feed formulation, the ratio between n-6 and n-3 polyunsaturated fatty acids, considered a risk factor for both cardiovascular disease and cancer, significantly decreases in pig meat . MoreoveMeat quality, both in terms of physico-chemical composition and rheological properties, could depend indeed on many factors: environmental and growth conditions, feeding, age of animals, slaughter conditions, genetic characteristics, post-mortem treatments, manufacturing processes, etc. . For insTo address the contribution of all the factors that can influence the overall quality of meat, “omics” techniques coupled with chemometric tools may be successfully used to explore such a large variability ,9. In paIn recent times, great attention has been given to the transformation of food components once entering the human body. In this context, the use of a consensus gastrointestinal simulation assay  has becolongissimus dorsi and rectus femoris—were sampled and subjected to further digestion and analysis. Results could then be related to potential positive activity in the human body and ultimately used to further finetune animal feeding and improve pork meat quality.Therefore, this study aims to investigate the profile of pork meat digests obtained from animals under different feeding regimens. In particular, a barley-based compound feed was used for the control group, while the same feed was enriched with extruded linseeds and plant extracts for treated pigs, in order to increase the intake of n-3 polyunsaturated fatty acids and antioxidant compounds. From the control and treated groups, two muscles—Untargeted UHPLC-HRMS analyses were performed on the digested meat samples gained from the in vitro gastrointestinal simulated digestion process.To perform sample classification, at first, all the chromatograms were independently aligned. This returned a primary dataset with 5728 features. The primary filtering step excluded the background peaks present in the blank digested sample. The second filtering step was performed by choosing all the molecular features present in at least 80% of the samples in one group, leaving 686 and 490 peaks for the two different diets, T1/T2, and the two meat cuts, RF/LD, comparison, respectively.At this point, the principal components analysis (PCA) models were built to investigate metabolome patterns and, therefore, relatedness/unrelatedness between the samples. The mechanism is based on the ability of the PC model to cluster samples in an unsupervised approach, since no information on group identity is used to build the model.The PCA score plots obtained are depicted in Overall, 3 out of 32 samples fell outside the 95% confidence ellipse, as it is shown in the PCA score plot . HoweverAt this point, two different supervised models were constructed on the basis of the different clusters observed in the PCA model. Orthogonal partial least-squares discriminant analysis (OPLS-DA) models were constructed to highlight key variables. The quality of both models was excellent see A,B, withOn the contrary, the OPLS-DA model built for meat cuts comparison B was infIn addition, to avoid the risk of overfitting, each generated model was validated by a cross-validation tool, using the leave-1/3-out approach. The percentage of total correct classification was 100% for both OPLS-DA models, with all the samples correctly predicted.Compounds annotation was achieved on the basis of the accurate mass spectra registered and using online available databases. Blank digested samples were also analyzed to exclude all the peaks present as background.Among the assigned metabolites, classified on the basis of their chemical nature, it was possible to mainly observe lipids and peptides derivatives. The compounds having the highest discrimination potential between samples derived from the different alimentary regimes (T1 and T2) are listed in The applied approach allowed for differentiating meat samples on the basis of the different feeding regimens, as well as on the basis of the diverse meat cuts.In general, both clusterizations observed are characterized by differences in small peptides profiles, mainly di- and tripeptides. This is probably due to a different proteolytic process occurring in the gastric step (GI) in the in vitro model. The composition, as well as the buffering capacity of the matrix, may actually affect protein hydrolysis in vitro. The adopted protocol  is indeeConcerning the effect of the diet on the metabolite profiles in digests—phospholipids, well-known constituents of cell membranes, are major contributors in the clusterization of T1 and T2 samples. They are actually more accumulated in T1 control samples, with the only exception phosphatidylglycerol PG (22:4) . These dAs an additional confirmation of the higher lipid oxidation that occurred in control samples, four secosteroids involved in the vitamin D3-related pathway are more accumulated in the control than in treated samples.The degradation of lipids by reactive oxygen species can be delayed or inhibited by the presence of antioxidant compounds, such as polyphenols. In the last years, the use of phenolic-rich plant and herbs ingredients or extracts as meat preservatives has become more and more studied to prevent or reduce lipid oxidation ,21. A reInterestingly, compounds deriving from the metabolism of cholesterol, such as azacholesterol and dehydrocholesterol, were more abundant in T1 digests than in T2. This could be ascribed to a sort of protection due to the presence of polyphenols in the alimentary regimen applied to T2 animals. Two additional compounds derived from oxidation reactions and linked to cholesterol metabolism were found in higher amount in T1 digests, dihydroxy-cholenoic acid and hydroxycholenoic acid. Cholenoic acids, pertaining to the class of steroid derivatives and more in the detail of bile acids, could be intermediates of the cholesterol metabolism and, in particular, of the synthesis of cholic and deoxycholic acids . MoreoveBesides lipids, control samples (T1) were also characterized by a higher amount of 4-hydroxyphenylglyoxylate, deriving from the degradative oxidation of phenylalanine . Among arectus femoris and 2.02% for longissimus dorsi [In regard to meat cuts, those selected for this study differ in the content of heme-iron, which is lower in LD and higher in RF. In addition, total fat content may be different for RF and LD. Data recorded for different pork meat cuts showed an average fat content of 1.54% for us dorsi .Our data clearly suggest a strong involvement of the lipid fraction, and more in details of the compounds originated by the lipoxidation cascade, in the classification of meat cuts. It has been reported that a higher content of iron in RF compared to LD samples may significantly affect the oxidative degradation via Fenton’s reaction . Iron, hSimilarly to data reported for T1 versus T2, phospholipids also contributed to the classification of meat cuts. In particular, the two groups are discriminated by the trend of several diacylglycerophosphoserins (PSs), diacylgrlycerophospholycerols (PGs) and diacylglycerophosphoinositols (PIs), likely according to fatty acyl residues distribution . HomocarTaken altogether, our results pinpointed that the use of an extruded linseeds and phenols-enriched diet leads to a reduction in pork meat of compounds related to oxidative degradation. This trends could be different for diverse meat cuts, as shown for those considered in the present study. The results suggested that the addition of antioxidants in animal feeding may lead to obtaining a higher quality meat, and may inhibit or delay oxidation processes. This may turn into a reduction of preservatives normally used in meat products as ascorbate, sodium nitrites and/or nitrates . Overallv/v)—were purchased from Sigma-Aldrich . Enzymes as α-amylase from porcine pancreas, pepsin from porcine gastric mucosa, bile extract porcine, and pancreatin from porcine pancreas were all obtained from Sigma-Aldrich . LC-grade methanol was purchased from Sigma-Aldrich , while bi-distilled water was produced in our laboratory by a Millipore Alpha Q purification system .All the salts and reagents used in the digestion protocol—potassium chloride, potassium dihydrogen phosphate, sodium bicarbonate, sodium chloride, sodium hydroxide, ammonium carbonate, magnesium chloride hexahydrate, calcium chloride dehydrate and hydrochloric acid  to slaughter (about 150 kg l.w.), the animals were allotted to two subgroups of 4 subjects each and fed two experimental diets: a standard diet for growing–finishing pigs, barley-based (T1), and a standard diet enriched with extruded linseed (5% of feed) and plant extract  from grape skin  and oregano  (T2). In particular, the linseed fatty acid composition was characterized by the presence of α-linolenic acid . Moreover, the addition of linseed to animal feeding led to an increase in the α-linolenic content in the dietary fatty acid fraction from 5.2% (T1) to 25.4% (T2) .longissimus dorsi “LD” and rectus femoris “RF”. The two muscles were collected immediately after pigs slaughtering, and the meat samples were minced, maintaining the two cuts separately in order to evaluate the effect of the diet as the contribution given by a white (LD) and a red (RF) cut separately. In this way, 4 samples of LD and 4 samples of RF were finally obtained for the T1 and T2 group separately.At the end of the trial, the animals were slaughtered, balanced for feeding regimen, on two different days of slaughter (replicates). Two muscles were selected for the experiments: The meat samples were stored at −80 °C until performing gastrointestinal digestion experiments and analyses. All the simulated digestion processes were repeated twice on each meat sample, so 32 different digests were characterized.The in vitro gastrointestinal digestion of pork meat samples, and the preparation of the artificial digestive juices , was according to Minekus et al. 2014 , with sl2 solution and 487.5 µL of distilled water. Gastric digestion was started by adding 0.8 mL of pepsin solution , 3.75 mL of gastric juice, 2.5 µL of CaCl2 solution, 0.3475 mL of distilled water and an opportune volume of HCl 1 M to reach a pH value of 3. For this step, the samples were maintained in the same conditions (37 °C and 200 strokes/minute) for 2 h. The duodenal digestion was then started using 2.5 mL of pancreatin solution (800 U/mL), 5.5 mL of the duodenal juice, 1.25 of bile juice (160 mM), 20 µL of CaCl2 solution, 0.655 mL of distilled water and an opportune volume of NaOH 0.1 M to reach a pH value of 7. This step was extended for 2 h under the same conditions.Briefly, to simulate oral mastication, 2.5 g of minced meat were exactly weighted into a 50 mL plastic tube and digested with 0.25 mL of amylase solution (1500 U/mL) at pH = 7 on a thermostated shaker at 37 °C and 200 strokes/minute for exactly 2 min, adding 1.75 mL of salivary juice, 12.5 µL of CaClAt the end of the digestion process, the samples were centrifuged at 2688 g at room temperature for 10 min, and then, 2 mL of supernatant was collected and added with 200 µL of methanol in order to stop the enzymatic activity. The obtained samples were stored at −80 °C.Before the UHPLC-HRMS analyses, 200 µL of the stock digested sample was transferred to a 1.5 mL Eppendorf tube and extracted with 400 µL of cold methanol by mixing the solution on a vortex for 1 min. Then, the mixture was centrifuged for 30 min at 16,800 g at 4 °C. The supernatant (500 µL) was finally collected and transferred to a vial. At the same time, two quality control samples (QCs) were prepared by mixing 10 µL of each sample extract. In addition to that, blank samples were prepared by subjecting 2.5 g of bi-distilled water to the same digestion protocol used for meat samples, as to the extraction procedure applied for all the other samples. Blanks, quality controls and extracted meat digested samples were then subjected to the UHPLC-HRMS analyses and statistic elaborations. All the samples were injected twice.Digested samples were then screened through a hybrid quadrupole-time-of-flight mass spectrometer coupled to an UHPLC chromatographic system. A 1290 liquid chromatograph system, equipped with a binary pump and coupled to a G6550 iFunnel quadrupole-time-of-flight mass spectrometer detector , was used. Jetstream electrospray was used as the ionization source and run as previously set up for untargeted profiling . Brieflym/z range, in positive polarity, at a rate of 0.8 spectra/s. The MS was set in “extended dynamic range” mode, with a nominal mass resolution of 30,000 FWHM. Mass accuracy was ensured by direct infusion of lock masses (121.0509 and 922.0098 m/z) through a separate electrospray.Chromatographic separation was achieved in reverse-phase mode, using a Waters Zorbax eclipse plus C18 column  and under a binary elution mixture, made of water (A) and methanol (B) with ammonium formate (5 mM) and 0.1% formic acid. The injection volume was 3 μL, the flow was 0.3 μL/min, and gradient elution started from 10% B to 90% B in 35 min, as previously described . The mashttp://cosmos-fp7.eu/msi (accessed on 10 January 2023)). Expansion of values for chromatograms extraction was set to 10 ppm, whereas single-ion identification was excluded. Data pre-processing  was conducted in Profinder B.07. Compounds were filtered by abundance and by frequency , normalized at the 75th percentile and baselined to the median of each compound in all samples.Deconvolution and compound annotation from raw data were carried out using the software Profinder B.07 , based on the ‘molecular features extraction’ algorithm and the small-molecules isotopic model. Annotation was achieved based on the database exported from Metlin, Phenol-Explorer 3.6, and using the entire isotopic profile , with a maximum mass accuracy of 5 ppm for each centroid mass in the isotopic cluster. Based on the strategy applied, identification was carried out according to Level 2 (putatively annotated compounds), as set out by the COSMOS Metabolomics Standards Initiative , Pareto scaled, and the presence of outliers was investigated according to Hotelling’s T2 , using 95% and 99% confidence limits for suspect and strong outliers, respectively.The quality of the models was evaluated by the goodness-of-fit parameter (R2X), the proportion of the variance of the response variable that is explained by the model (R2Y) and the predictive ability parameter (Q2), which was calculated by a seven-round internal cross-validation of the data using a default option of the SIMCA software. R2X and R2Y represent the fraction of the variance of the X-matrix and Y-matrix, respectively, while Q2 suggests the predictive accuracy of the model. R2X, R2Y and Q2 values close to 1 indicate an excellent model, and thus, values higher than 0.5 indicate the good quality of OPLS-DA models. In order to select the most significant and reliable variables, variable importance in the projection (VIP) was used. This parameter summarizes the importance of the X-variables, both for the X- and Y-models.In this research, VIP with the threshold >1 was used for selection of the most significant markers. To avoid the risk of overfitting, as the results found after Multivariate Data Analysis (MVDA) are sensitive to chance correlations, statistical models have to be validated. For this reason, supervised models, i.e., OPLS-DA, were validated by cross-validation, using the leave one-third out approach. The dataset was divided into three parts, and one-third of samples were excluded to build a model with the remaining two-thirds of samples. Excluded samples, one-third of samples, were then predicted by this new model, and the procedure was repeated until all samples had been predicted at least once. Each time, the percentage of correctly classified samples was calculated by generating a misclassification table.rectus femoris and longissimus dorsi, and two different dietary regimes, a barley-based diet and one supplemented with extruded linseed and plant extracts (grape skin and oregano). To the best of our knowledge, this study represents the first attempt to classify different pork meat cuts derived from pigs fed with different diets on the basis of the metabolic profile of their corresponding digested samples. No studies, indeed, aimed to relate the metabolic composition of meat digests to meat quality, evaluating these parameters on the basis of meat derived from animals subjected to different dietary regimes.The present study was focused on the application of an untargeted UHPLC-HRMS approach to evaluate the possible differences between pork meat digests belonging to two different cuts, The results obtained from UHPLC-HRMS analyses and subsequent statistical elaborations returned a clear clusterization of the digested samples on the basis of the dietary regimen applied for feeding animals. Among all the detected metabolites, lipid and protein derivatives significantly influenced sample clusterization. In particular, differences in the lipid metabolism, degradation and oxidation products were observed, such as the accumulation of fatty acyls, fatty aldehydes, oxylipins, cholesterol and vitamin D3 precursors and derivatives. The presence of these oxidative derivatives was observed especially in digests from control diet meat cuts, while the dietetic addition of linseed and polyphenols led to an inhibition of the oxidative degradation and, therefore, to a lower accumulation of oxidation products. On the other hand, different meat cut digests showed a diverse metabolic profile in terms of phospholipids, homocarnosine and pentylbenzene, relating to their different iron and lipid contents.It is thus possible to conclude that the diet supplementation with components rich in omega 3 and polyphenols may exert a positive influence on the meat quality, leading to a reduced oxidative degradation of lipids and proteins, in respect to a conventional diet. Moreover, characterizing digested samples, rather than extracts of pork meat cuts, allowed for investigating which metabolites may enter in contact with the gastrointestinal tract when different quality meat is consumed. This research, therefore, represents a first attempt to investigate the relationship between the meat metabolites released upon digestion and the initial meat quality. More studies on different pork meat cuts, as to other kinds of meat, should be performed in this sense.


Background: Splenic injuries are common solid organ injuries resulting from blunt abdominal trauma in road traffic accidents. Very often, splenic injuries can be life-threatening. Earlier, splenic injuries were often dealt with surgical intervention, such as splenectomy. With the recognition of the immunological function of the spleen and possible complications of splenectomy surgery, such as overwhelming post-splenectomy infections (OPSI), there has been a recent trend for non-operative management (NOM).Objective: To study the variables predicting failure of NOM in blunt abdominal trauma patients with splenic injury.Methods: This is a retrospective study that includes 235 patients who presented to the Safdarjung Hospital emergency room  with blunt trauma abdomen and splenic injuries with or without associated injuries between January 2019 and December 2021. The data was entered in a Microsoft Excel spreadsheet . Categorical variables were expressed as frequencies and percentages. Pearson's chi-square test of association was used to determine if there is a relationship between two variables. A p-value of <0.05 was considered statistically significant.Results: Out of 235 patients with blunt abdominal trauma and splenic injuries, 82 were hemodynamically unstable despite resuscitation and were taken up for emergency laparotomy. The remaining 153 patients, who were either hemodynamically stable or stabilized after adequate resuscitation, were managed on the lines of NOM. The number of patients with splenic injury in AAST grades 1, 2, 3, 4, and 5 was 36, 50, 40, 24, and three, respectively. Out of 153 patients, 130 (85%) were successfully managed by NOM, while eight (5%) had to discontinue NOM as they required surgical intervention. The failure of NOM (fNOM) is seen mostly with grade 5 injuries , followed by grade 4  and grade 3 . The mean age in fNOM was 58.3 years, as compared to 42.2 years in the success of NOM (sNOM). All eight patients had multiple concomitant injuries, with femur fracture being the most common association in up to six patients (p<0.01), followed by liver injury in four patients. There were 15 mortalities, irrespective of AAST severity grade. All of these patients had associated concomitant injuries, with intracranial bleeding  being the most common association, followed by femur fracture  and liver injury . Also, the cause of death was unrelated to splenic trauma (p = 0.67), with pulmonary embolism  being the most common cause, followed by brain stem herniation .Conclusion: Non-operative management is a safe and efficient method for treating patients with splenic injuries who are hemodynamically stable or stabilized. The factors associated with fNOM include elderly age, a higher American Association for the Surgery of Trauma (AAST) grade of splenic injury, and associated concomitant injuries. Femur fracture was the most common concomitant injury present in cases where NOM failed, followed by liver injury. The presence of intracranial bleeds in these patients was a common association with mortality, irrespective of the grade of splenic injury. A splenic injury from abdominal trauma is one of the most fatal solid organ injuries ,2. Non-oMany decades ago, the majority of splenic trauma was managed by splenectomy. In recent times, with the introduction of newer resuscitation guidelines, advancements in imaging techniques, and angioembolization techniques, major surgical interventions like splenectomy and splenorrhaphy can be avoided. There has been an increasing trend towards spleen salvage procedures and the NOM of splenic injury patients, recognizing the immunological role of spleen in humans, erythrogenesis, and the possible complications after splenectomy, like overwhelming post-splenectomy infections (OPSI). Consequently, NOM has evolved into the preferred standard of care for hemodynamically stable or stabilized cases of splenic damage in institutions with adequate diagnostic facilities and intensive care units -5.There are studies where 80% of traumatic spleen injury patients were successfully treated with NOM -7. The fA retrospective observational study was conducted to include over 235 patients who presented to the emergency room of Safdarjung Hospital, New Delhi, India, with blunt trauma abdomen and splenic injuries with or without associated injuries between January 2019 and December 2021 . Statistical testing was conducted with SPSS Statistics version 28.0 . Continuous variables were presented as mean (SD) or median (IQR) for non-normally distributed data. Categorical variables were expressed as frequencies and percentages. Pearson’s chi-square test, or the chi-square test of association, was used to determine if there is a relationship between two variables. A p-value of <0.05 was considered statistically significant. A wide range of parameters were taken into consideration, such as the age of the patient, systolic blood pressure and heart rate at presentation, and concomitant injuries at the time of presentation in the emergency room. The fNOM of splenic trauma in eight patients was analyzed in detail to deduce the possible factors that led to it. The 15 mortalities were also studied in detail to analyze the possible association between concomitant injuries and the cause of death.The number of patients with splenic injury in AAST grades 1, 2, 3, 4, and 5 was 36, 50, 40, 24, and three, respectively. Of the 153 patients, 130 (85%) were successfully managed by NOM. For 8 patients (5%), NOM had to be discontinued as they were intervened surgically. Grade 5 injuries  mostly resulted in fNOM, followed by grades 4  and 3 . No fNOM) was reported in patients with splenic injuries of AAST grades 1 and 2 . Retrospectively, seven out of eight patients under fNOM had to be stabilized by adequate resuscitation in the emergency room. The mean systolic blood pressure and heart rate at admission in fNOM were 88.5 mmHg and 108.8 beats per minute, respectively, as compared to 100.2 mmHg and 97.2 beats per minute, respectively, in sNOM , followed by liver injury in four patients Figure , Table 1, followeThere were 15 mortalities (10%), irrespective of AAST severity grade. All of these patients had associated concomitant injuries (p<0.01); intracranial bleeding n = 10, 32%, p<0.01) was the most common association, followed by femur fracture n = 6, 20%) and liver injury  (Figure , 20% and%, p<0.01The spleen is the intra-abdominal organ that suffers blunt abdominal damage most frequently in cases of automobile crashes. Gruber et al. initially recognized the link between the spleen and immunological function in 1951 when they reported that an infant receiving therapy for idiopathic thrombocytopenic purpura encountered severe sepsis following splenectomy . ZarrabbFew studies point out that multisystemic trauma, significant brain damage, related injuries such as diaphragmatic injury and hollow viscus injury, age >55 years, and sick spleen are relative contraindications for NOM -14. Low According to Peitzman et al. , NOM proAn investigation of 129 isolated splenic injuries from 2009 to 2016 was done retrospectively by Bagaria et al. in 2019. Patients were successfully treated non-operatively in 71.3% of cases. A splenectomy was later performed on three patients in the NOM arm, giving NOM an overall success rate of 96.8% . SimilarElderly age, higher grades of splenic injury, and concomitant injuries are often associated with fNOM. The limitations of this study are its small sample size and the need to eliminate confounding factors before declaring the association of certain injuries with fNOM.Hemodynamically stable or stabilized patients with splenic injuries can be managed safely and effectively with NOM. Factors such as elderly age, a high AAST grade of splenic injury, and associated concomitant injuries are associated with fNOM. Femur fractures followed by liver injuries were the most common concomitant injuries present in cases where NOM failed. The presence of intracranial bleeding in these patients was a common association with mortality, irrespective of the grade of splenic injury.


Saccharomyces cerevisiae var. boulardii (LSB), one of the most studied yeasts, has been used throughout the world for the prevention and treatment of a variety of gut inflammatory diseases. In our study, in order to obtain insights into the potential beneficial effects of this yeast as a probiotic, the dietary inclusion of an LSB strain (CNCM I-1079) was evaluated, at increasing concentrations  for 90 days, on the welfare and health status of juvenile European sea bass, an important fish species in Mediterranean aquaculture. Our results indicate that LSB has an immunomodulatory action, and the observed lowering of pro-inflammatory gene transcripts in the gut suggests a possible anti-inflammatory action of this probiotic strain on the gut immune system of sea bass, with a possible enhancement of fish disease prevention. LSB also exerted a low, albeit insignificant, stimulating effect on the hematological and immunological parameters.In animal science, dietary supplementation with probiotics offers several advantages, in terms of both environmental sustainability and production, and probiotic supplementation in aquatic animals is known to enhance stress tolerance, disease prevention, and growth, as has already been documented for several fish species. Among the various probiotics, the yeast-based ones have been stimulating a great deal of interest because of their ability to produce polyamines, which participate in numerous biological processes, and Saccharomyces cerevisiae var. boulardii (LSB) administered at increasing concentrations  for 90 days, on the health conditions of European sea bass. The main zootechnical parameters, histological and morphological analyses, innate immunity response parameters  were measured as fish welfare parameters. LSB did not impair either growth parameters or the morphometric indexes. LSB down-regulated interleukin-1β transcription in the distal gut of fish treated with 5.4 × 105 CFU g−1 (LSB100) for 21 days. The interleukin-6 mRNA level decreased significantly in the proximal gut for both doses of yeast, after 21 days of feeding; the gene expression of interleukin-6 was significantly lower in the sea bass fed 10.81 × 105 CFU g−1 (LSB300) probiotic. The levels of TNF-α mRNA were not influenced by probiotic supplementation. Increases, although not significant, in the hematological and immunological parameters were also recorded. The data collected in the present study suggests that an LSB-supplemented diet acts on the gut immune system of sea bass by modulating the expression of the key inflammatory genes.The present study has been aimed at evaluating the effects of the dietary inclusion of the live yeasts,  Probiotics, according to the definition currently adopted by the Food and Agriculture Organization of the United Nations (FAO), are “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” ,2,3,4. PDietary supplementation with probiotics offers many advantages in terms of both environmental sustainability and production. It allows the limitations and side effects of antibiotics or other drugs to be overcome, resulting in enhanced growth and disease prevention ,4,9. ProHowever, in fact, it is still necessary to consider other variables, such as selective ingestion, manipulation in the intestinal tract, microbial interactions, and/or nutritional environment. The effect of probiotics can vary, according to several variables, including the form of supplementation , the level of dosage, and the duration of application, as well as the length of the delivery period .il-1β; il-6; il-21; tnf-α; and tlr-1, 3, and 4  × 100iiiFeed Conversion Ratio (FCR): total supplied feed as DM (g)/WG (g), (where DM indicates Dry Matter)ivProtein Efficiency Ratio (PER): WG (g)/total protein fed (g)vFeeding rate (FR) (%): (ingested feed (g)/days of feeding) × 100i.Viscerosomatic index (VSI): (viscera weight/BW) × 100iiHepatosomatic index (HSI): (liver weight/BW) × 100iiiCoefficient of fatness (CF):  × 100iv3) × 100Condition factor (K): : intestinal length/BWWhile the morphometric indices were calculated using the formulae:Tissue samples were fixed in Bouin’s solution (Bio-Optica) (pH 1.75 at room temperature) for 48 h, processed for paraffin embedding, and cut into thin sections (3–5 μm) for microscopic observation. Sections were stained with hematoxylin-eosin (H&E) and examined using a light microscopy (Leica DMR) equipped with a camera (DFC295) and with image management software (Leica IM1000).Histological analyses were carried out to evaluate the following intestinal morphological indexes: villi length (VL); villi thickness (VT); microvilli length (MT); number of goblet cells (GC); thickness of the lamina propria (LP), serous layer (SL), muscular layer (ML), and submucosa layer (SML).The measurements and observations adopted in this study were based on a combination of criteria previously suggested by different authors ,61, taki2) . The Kruskal–Wallis test was used for heterogeneous variances. PAST4 Colony Forming Units (CFU)/g, ranging from 7.80 × 103 to 4.30 × 104 CFU/g in the sea bass fed with LSB 100, and from 1.73 × 104 to 1.03 × 105 CFU/g in the fish fed LSB 300  elongation of the length of the villi was observed in the LSB300 group, compared with the LSB100 group, after feeding for 21 days. Statistical differences were also observed in the SML thickness (SML). The SML thickness of the LSB100 group was significantly higher than the control after 45 days of feeding. The length of the microvilli showed significant differences between the probiotic-supplemented diets (LSB100 and LSB300) and the control after feeding for 90 days.The analysis of the histological parameters of the proximal gut  revealedThe analyses on the distal portion of the intestine did not show any significant differences, except the presence of yeast colonizing the mucosa detected with SEM.S. cerevisiae var. boulardii.The histological study of the liver, which considered the number of hepatocytes per area, was performed as described in S. cerevisiae was absent in the anterior intestine but it was instead concentrated in the posterior tract , where the SH50 values were 1.72 and 2.27 times higher than the control values after 21 and 45 days of dietary supplementation with LSB, respectively. However, the differences between the three experimental groups were not significant. After 45 days of feeding, a slight decrease was recorded in the hemolytic activity values, which then increased after 90 days.S. cerevisiae var. boulardii, although the increase was not significant. On the other hand, contrasting results, characterized by the absence of variations (at T45) or by opposite trends to the previous ones, i.e., with a depressive effect on the hemagglutinating activity for an increase in the probiotic concentration, and a peak in the control group were observed for the successive samplings.The effect of LSB dietary supplementation on the hemagglutinating titer values was already evident after 21 days of feeding for the highest dose of Respiratory burst activity, which was measured as the production of superoxide anion, highlighted that LSB induced a stimulating effect on the sea bass fed the LSB300-supplemented diet, with values that increased 4 and 11 times compared to the control values at 45 and 90 days after feeding with LSB. This effect, although not significant, appeared to be delayed, compared to the other parameters of the immune response.The peroxidase activity values measured in the serum depicted a variable pattern, which initially showed an insignificant increase in the sea bass fed with the diet containing LSB 100 and reached almost triplicate values (>0.24 U/mL), after feeding for 90 days, in those fed with LSB 300.The results of the assay on the antibacterial activity of the serum, kidneys, intestinal, and skin mucus against the target pathogenic bacteria were expressed in terms of the percentage of the total number of examined samples that showed a positive reaction .S. epidermidis; only after feeding for 90 days was a positive reaction against P. aeruginosa detected in the LSB300 group. No activity was observed against E. coli or S. aureus.The serum samples showed antibacterial activity almost exclusively against E. coli, while no activity against S. epidermidis was observed. A high percentage of samples was positive after feeding for 21 days, even in the control group; no clear correlation was observed between the number of positive reactions and the dose of the probiotic contained in the diet at T45 or T90.High percentages of the kidney samples showed antibacterial activity toward P. aeruginosa. A low percentage of samples also exhibited activity against A high antibacterial activity against all the tested bacteria was observed for all the skin mucus samples. Greater antibacterial activity was recorded for T21, T45, and T90, probably in relation to the higher temperatures of the summer months than those of the spring season (T0 sampling), while no differences were found among the various experimental groups.P. aeruginosa and S. epidermidis, while the activity against E. coli was lower. A positive response was not found for S. aureus, except for the control group after feeding for 45 days. In general, there was more activity at 90 days; no significant relationship was highlighted, in relation to the diet, between the expression of the antibacterial activity and the levels of probiotic supplementation.The intestinal mucus samples showed a high bactericidal activity against all the tested pathogens, and in particular against il-1β, il-6, and tnf-α genes were examined to evaluate whether the probiotic treatment had an effect, at the gut mucosal level, on cytokine mRNA expression, using a real-time PCR method. The expression of the cytokine genes is reported in il-1β expression in the proximal gut between the probiotic-supplemented groups and the control, at any of the sampling times, whereas the level of il-1β mRNA was significantly higher in the LSB100 group after 45 days than in the LSB300 group. Instead, the level of il-1β mRNA was significantly lower in the distal portion of the gut in the LSB100 group after 21 days of feeding.The il-6 was down-regulated in both probiotic groups. After 21 days of feeding, the LSB100 and LSB300 groups both showed a significant difference from the control and between each other. At this time of sampling, the expression of the il-6 gene was significantly lower in the fish fed the LSB300 probiotic. After 90 days, the expression of the il-6 gene in LSB300 was significantly higher than in the control and LSB100 groups. No significant differences in expression were observed for the distal gut, except after 45 days, when the LSB100 il-6 gene expression was significantly higher than the LSB300 group. The levels of TNF-α mRNA do not seem to have been influenced by probiotic supplementation. No significant differences between groups were observed, either over time or in the different portions of the gut.The gene expression level in the proximal gut of S. cerevisiae var. boulardii in sea bass. Although the beneficial effects of diets supplemented with this yeast species have been documented for mammals [The comprehensive dataset collected in our study can be considered a first contribution to knowledge on the effects of dietary supplementation with  mammals ,70, and  mammals , the pot mammals .Although several studies have reported on the use of probiotics in aquaculture nutrition, caution should be taken in the interpretation of the biological data, because both zootechnical parameters and morphometric indices can vary to a great extent in relation to different variables, such as the stocking density , and no S. cerevisiae var. boulardii had positive effects on the growth performance, health, and survival rate of the reared fish, especially when the probiotic was added at a concentration of 2.5% of body weight [S. cerevisiae enriched diets [S. cerevisiae administered at a dose of 15 × 109 CFU/g, thus suggesting that the response to administration could change depending on the yeast concentration [As far as the biometric measurements are concerned, none of the zootechnical parameters or morphometric indices examined in our study showed significant differences related to the administered experimental diets, as expected, considering the results published in the literature. The supplementation with y weight . An enhaed diets ,13,75. Fntration . A signintration .S. cerevisiae has been shown to accelerate digestive system maturation and increase nutrient digestibility as a result of improvements in the integrity of the intestinal mucosa and the density of the intestine villi [The dietary inclusion of ne villi .S. cerevisiae, have shown a significant increase in villi length [Nile tilapia fed with probiotics, in particular i length ,43,75. TIn addition, in our study, the microvilli thickness was found to increase, which resulted in an improved absorption, without any events of necrosis or cell disruption, thus confirming the results obtained for the same species by Rawling et al. .The histological examination results are in agreement with previous results on the same species , where tNutritional status is recognized as an important indicator of the resistance of fish to diseases ; the relSander lucioperca protected by Lactobacillus brevis against Aeromonas hydrophila [S. cerevisiae var. boulardii, administered at a dosage of 300 mg/kg (after 21 days of feeding), is able to protect the intestinal mucosa of gilthead seabream against V. anguillarum infections.Among the beneficial effects of the use of probiotics in fish, those related to the modulation of the immune system are well known. Probiotics are effective in modulating the inflammatory response and in increasing disease resistance to bacterial infections, as demonstrated in drophila . Moreovedrophila  demonstrS. cerevisiae, administrated as whole cell or cell walls, may provide very important non-nutritive compounds, including various immunostimulating compounds such as glucose polymers (β glucans), mannan oligosaccharides [Enhancement of the immune response following dietary supplementation with probiotics has been widely documented ,82. Probcharides , nucleiccharides ,32.S. cerevisiae var. boulardii has been reported to act as a pathogen antagonist and immunomodulator in mammals [S. cerevisiae var. boulardii has also been reported in rats and mice [S. cerevisiae is known to increase the growth performance, immune responses, and disease resistance of several species of fish, such as gilthead seabream [D. labrax [ mammals ,70; an iand mice ,87. S. cseabream ,88,89, Nseabream ,90, Chanseabream , and raiseabream ,92. The seabream  and in DS. cerevisiae seems to depend on the used strain [The effects on the non-specific immune parameters of dietary supplementation with probiotics can in general be affected to a great extent by the fish species, the stage of development, the strain of the probiotic, dosage, feeding duration, feed composition, mode of supplementation and/or certain environmental-rearing conditions, such as water temperature fluctuations . With red strain , and a dd strain ,19. In ad strain ,97.S. cerevisiae var. boulardii CNCM I-1079 was not detected in the gut of the juvenile sea bass, and a low level of colonization was found, even after 9 months; the same strain did not seem to affect the intestinal bacteria of rainbow trout.In the present study on sea bass, the viability of the probiotic administered with the diet was assessed by evaluating the abundance of yeast in the feces; the amount of viable yeast was gradually increased during the experiment. However, only a study through electron microscopy can provide information on the type of yeast present in the gut, i.e., can reliably indicate whether the flora has colonized the intestinal mucosa transiently or actively. The presence of S. cerevisiae var. boulardii on the lysozyme content, slight and insignificant increases in the values of this parameter were observed during the present study. Rodriguez et al. [Sparus aurata, 2–4 weeks after dietary supplementation with a mutant strain (fks-1) of S. cerevisiae as a probiotic. Song et al. [Miichtys miiuy fed a mixture of microorganisms, that is, both bacteria and yeasts , which would seem to suggest that stimulation of the immune system had occurred. Supplementation with S. cerevisiae var. boulardii CNCM I-3799 enhanced the lysozyme, the complement activities, and the Ig level of rainbow trout [S. cerevisiae has also been reported in other species, such as Pangasianodon hypophthalmus [S. cerevisiae supplementation of the diet [Regarding the effects of z et al.  reportedg et al.  recordedow trout . The enhhthalmus . The lysthe diet .Pangasianodon hypophthalmus fingerlings fed for 8 weeks on diets supplemented with a mixture of yeast (S. cerevisiae) and bacteria  [Micropterus salmoides) fed for 8 weeks with Bacillus subtilis, Lactobacillus plantarum, and S. cerevisiae) [An improved immunological response  has recently been recorded for faecium) . Similarevisiae) .Epinephelus coioides) fed a diet supplemented with S. cerevisiae at concentrations of 105 and 107 CFU/kg of body biomass, compared to the control diet (without any probiotic). A significant increase in ACH 50 activity  has been observed in striped catfish  in the presence of S. cerevisiae enriched diets (106 and 108 CFU S. cerevisiae g−1 diets) after an experimental period of 120 days [The increases in the hemolytic activity recorded in the examined sea bass, although not significant, were related to the beneficial effects produced by probiotics on the hematology parameters, which are commonly referred to as indicators of the physiological state of fish, in agreement with what Mohapatra et al. reported . Chiu et120 days .Epinephalus corioides fed diets containing S. cerevisiae at 105 and 107 CFU/kg were significantly higher than those of fish fed a lower probiotic concentration (103 CFU/kg).Chiu et al.  found thOreochromis niloticus fed with different forms of S. cerevisiae  and as a mixture of the whole cell and its extract).Abu-Elala et al.  found a S. cerevisiae var. boulardii were quite variable, and they therefore did not allow us to correlate any effect with the administration of the probiotic. Furthermore, the reduction in the hemagglutinating activity measured during the last sampling could also be associated with a depression in the activity of the complement, as previously observed by Rodriguez et al. [Sparus aurata after 6 weeks of feeding with the S. cerevisiae mutant strain fks-1.The patterns described for the hemagglutination titer in sea bass fed with z et al.  in SparuS. cerevisiae had a positive effect on both the respiratory burst and the complement activities.Sakai  reportedS. cerevisiae has been shown to have a marked effect on the respiratory burst activity of Nile tilapia, where the supplementation of 2, 4, 6, and 10 g of S. cerevisiae/kg diet was found to determine the linear and quadratic enhancement of respiratory burst and lysozyme as well as superoxide dismutase, catalase, and glutathione peroxidase [S. cerevisiae as a probiotic (107 CFU/g) significantly affects the respiratory burst activity, especially after 130 days of treatment [Uronema marinum in olive flounder  supplemented with S. cerevisiae, with a significant increase in lysozyme and superoxide anion production after 4–8 weeks of feeding. Significant improvements in respiratory burst activity have also been reported in tilapias fed diets supplemented with Bacillus subtilis, S. cerevisiae and Aspergillus oryzae as probiotics (p < 0.05), compared to control fish without any probiotics [roxidase . The finrobiotic  CFU/g sireatment  found anobiotics .S. cerevisiae in the diet of rainbow trout over 130 days led to increased respiratory burst activity and an increased number of blood cells  [A single inclusion of nocytes) .S. cerevisiae, particularly at the skin mucus level. However, the results of the antibacterial activity assay did not allow a clear stimulating action on the bactericidal activity following the probiotic supplementation to be observed. Conversely to our findings, Taoka et al. [Oreochromis niloticus after 15 and 30 days of feeding with such probiotics as Bacillus subtilis, Lactobacillus acidophilus, Clostridium butyricum, and S. cerevisiae. Moreover, significant antimicrobial activity against several pathogens, such as E. coli, Shigella, Salmonella, V. cholerae, C. difficile, and Candida albicans, was recorded following a dietary supplementation with S. cerevisiae boulardii CNCMI-745 in several animal models [Regarding the antibacterial activity of the serum, kidneys, and skin and intestine mucus, a discrete activity was found in sea bass fed with a et al.  reportedl models .S. cerevisiae had a stimulating, although somewhat delayed, effect on this parameter, as observed at the last sampling. This result was in contrast with the depression of peroxidase activity reported in the serum of S. aurata by Rodriguez et al. [S. cerevisiae (mutant strain fks-1).The patterns of peroxidase activity obtained during the present experiment suggest that z et al. , 6 weeksPagrus major) fed with S. cerevisiae or B. subtilis, while, surprisingly, the catalase activity decreases [S. cerevisiae also seemed to enhance the peroxidase activity in Atlantic salmon receiving dietary supplementation with Kluyveromyces marxianus. The same results were obtained in Nile tilapia (Oreochromis niloticus) fed diets supplemented with S. cerevisiae [S. cerevisiae enhances the peroxidase activity in European sea bass, with a simultaneous increase in the lysozyme and other antioxidant enzyme contents [It has also been shown that peroxidase activity increases significantly in red sea bream  fingerlings after feeding with prebiotics and probiotics  for 16 weeks, followed by 8 weeks of feeding with a control diet without any pre- or probiotics supplementation.In short, the differences in our experimental trial in the hematological and immune response parameters induced by the LSB administration were not significant. This result is consistent with the absence of the differences reported by Munir et al.  in snakeS. boulardii dietary supplementation significantly modulated the expression of inflammatory interleukin genes in the gut of sea bass. The considered dosages both had a positive effect on the expression of the interleukins in the gut, but the most evident benefit was observed after 21 days of feeding, especially in the proximal gut .In the present study, il-1β transcript in the probiotic groups than in the controls after 21 days of feeding. This result is in further agreement with the literature that has reported the downregulation of IL-1β in Micropterus salmoides [Epinephelus coioides [Oncorhynchus mykiss [IL-1β is one of the first pro-inflammatory cytokines to have been expressed . In our almoides , Epinephcoioides , and Oncs mykiss  when a dMoreover, again regarding IL-6, our studies noted a downregulation of transcription at day 21 in the groups fed on a supplemented diet, in a dose-dependent manner, compared to the control group.il-6 gene in the gut has never been explored in fish, especially under normal conditions in the absence of damage of any kind.IL-6 is a potent, pleiotropic cytokine produced by various cells to regulate hematopoiesis, inflammation, immune responses, and bone homeostasis . It playS. boulardii could be interpreted as a positive signal of “gut health” in D. labrax.In our study, the down-regulation of both interleukins in response to the administration of tnf-α was not influenced by the administration of the probiotic. The behavior observed in TNF-α may be the result of a combination of factors, such as the organ in which the expression was analyzed, the fish species, and the administered probiotic.TNF-α is an important component during the onset of early inflammatory events. TNF-α is synthesized by various cell types upon stimulation with endotoxin, inflammatory mediators, or cytokine, such as IL-1, and, in an autocrine manner, upon stimulation by TNF itself. In our study, the expression of S. cerevisiae usually determines the up-regulation of TNF- α, which is coupled with a histological variation, such as an increase in goblet cells in different species [6) [ species ,90. Our  species , or for cies [6) ,42.S. boulardii in particular, modulate the immunological function by decreasing the levels of kinases and pro-inflammatory molecules [In conclusion, as also documented for other animals, yeasts, and olecules .D. labrax, thus suggesting a possible anti-inflammatory effect of this probiotic strain.The present results may indicate that a similar immunomodulatory action takes place in the gut of D. labrax at the tested concentrations because it did not impair either the growth parameters and morphometric indexes, or the intestinal mucosa structure, and it positively modulated the expression of the key inflammatory genes of the sea bass gut immune system. LSB, at a dosage of 300 mg/kg, lowered the expression of the interleukin-6 gene, when it was administrated over short time periods. Indeed, Maricchiolo et al. [S. cerevisiae var. boulardii as a probiotic for sea bass.In conclusion, LSB was found to have a positive effect on o et al.  recommen


L. crispatus (M247) could increase pregnancy and live birth rates in women undergoing assisted reproductive technology procedures. Enrolled women (N = 160) were divided into two groups: treated (N = 80) or untreated (N = 80) with the probiotic strain. The odds ratio (OR) for a treated woman to have a clinical pregnancy (CP) was 1.56. In women aged 30–40 years, M247 increased the probability of a CP in correlation with the progressive rise in BMI, reaching 47% (35% in controls) with a BMI of 35 (OR: 2.00). The CAID statistics showed that in a woman of the blastocyst subgroup, below 43 years, with a BMI over 18.6, treatment with M247 increased the chance of a CP from 28.4% to 44.5% . Considering live births, the rate of the probiotic group was 12.5% versus 7.5% (OR: 1.76). Considering only the blastocyst subgroup, the treatment increased the number of live births by 200% . As confirmed also by statistical indices NNT, NNH, and LHH, the use of M247 demonstrated a risk-benefit ratio to the full advantage of the benefits.The aim of our study was to retrospectively evaluate whether the oral administration of  Cutibacterium and Anaerobacillus) could be a predictor of RPL in the absence of an aneuploid karyotype and that the cervicovaginal microbiota may be a useful area of investigation into possible causes of RPL [Lactobacillus spp. dominance [L. crispatus dominance (CST I) correlate with a low obstetric-gynaecological risk. Those characterized by high richness and poor Lactobacillus dominance (CST IV) correlate mostly with vaginal discomfort and/or obstetric-gynaecological diseases [Infertility, defined as a disease by the WHO , is an is of RPL . Vaginalominance . Communidiseases .Lactobacillus content and female infertility [L. crispatus pre-pregnancy dominance with a better chance of falling pregnant within 12 months [Lactobacillus-dominated vaginal microbiota; among the Lactobacillus-dominated consortia, L. crispatus was reported to be the least common [L. crispatus in women achieving a positive pregnancy test, clinical pregnancy, and live birth compared with those who did not [Lactobacillus-dominated vaginal microbiota [L. crispatus [Lactobacillus and particularly by the species L. crispatus in a similar way to what is observed in cervicovaginal samples, would reduce the endometrial inflammatory phenomena, favouring the onset of pregnancy [Regarding fertility, systematic review and meta-analysis have identified a negative correlation between vaginal microbiota with high ertility . A recen2 months . Two welt common ,14,15,16t common . Similar did not . Moreovecrobiota . Again, rispatus . Despiterispatus . The corregnancy ,23,24,25regnancy ,27,28,29Lactobacillus-dominated composition by means of a probiotic prior to fertility treatment failed [L. crispatus strains would have a better chance ofsuccess. In fact, the trial was performed using a probiotic product containing strains of L. gasseri and L. rhamnosus. A double-blind, placebo-controlled, multicentre trial to evaluate the reproductive outcomes of IVF patients with abnormal vaginal microbiota treated with the likely most investigated strain of L. crispatus (strain CTV-05) is currently ongoing [To our knowledge, the only attempt to positively affect the vaginal microbiota to restore a t failed . As a po ongoing .L. crispatus in women undergoing IVF [L. crispatus in favoring clinical pregnancy and live birth rates, we have retrospectively compared the results obtained with the probiotic treatment with those obtained before its introduction (January 2018–December 2019) at the U.O.S.D. PMA of Conegliano Hospital .In January 2020, our department started to use the well-documented M247 strain of oing IVF . This sthttps://clinicaltrials.gov./ (accessed on 23 May 2023) (identifier: NCT05871242) and approved by the local (Treviso and Belluno) Ethics Committee for Clinical Trials, with approval number 1111, on 5 June 2023. Informed written consent was obtained from all 160 women who took part in the study. The aim of the study was to retrospectively evaluate the role played by the oral administration of a nutraceutical, containing as a single active ingredient the probiotic L. crispatus M247, in women undergoing ART procedures. The retrospective analysis of the results obtained with the use of the probiotic strain concerned women  managed in our hospital department from January 2020 to December 2021. The retrospective evaluation of the control group (N = 80) concerned women managed in the same hospital department from January 2018 to December 2019. All women of the probiotic group declared they had used not less than 95% of the probiotic doses prescribed. The women of both groups were aged between 18 and 45, suffered from primary infertility and had not had any pregnancies. Women suffering from uterine myomas and/or malformations, endocrinological, metabolic, cardiovascular, and genetic diseases were excluded from enrolment. The primary outcome of our analysis was to evaluate to what extent the oral administration of L. crispatus could affect the clinical pregnancy rate, defined as number of pregnancies  or definitive clinical signs of pregnancy [This retrospective, controlled, observational, and open-label study was carried out at the U.O.S.D. PMA of Conegliano Hospital , registered on regnancy . SecondaTo reduce any possible bias in our retrospective analysis, we compared the results of women who had received the same ovarian synchronization with estrogen-progestogen, using the same endometrial preparation technique (estrogen and subsequently estrogen and progesterone). The same criteria were also used to evaluate the endometrium (trilaminar aspect with a thickness of at least 7 mm) and blood progesterone value (before progesterone administration) not exceeding 1.5 mg. For the 80 women of the two groups, the same medical personnel were involved and used the same type of catheter (for embryo transfer). The same laboratory staff  handled the two groups of women. In addition, regarding frozen-thawed cycles, we compared women whose oocytes or embryos were cryopreserved using the same vitrification/warmed protocol. To verify the rate of overlap between the two groups of women, we have then evaluated the following features: age, BMI, anamnestic factor of infertility, duration and type of infertility, basal hormones, vaginal-rectal swab prior to ovarian synchronization treatment, antibiotic and/or anti-fungal therapy, stimulation protocol, ovulation induction, number of retrieved and mature oocytes, endometrial preparation (hormone therapy), β-hCG, ultrasonographic visualization of clinical pregnancy, and number of live births.As shown in As regards to patient stimulation protocols, in our study we adopted four different schemes (A–D) according to their characteristics. Scheme A: induction was achieved usingof recombinant follitropin  at a dose of 150 IU/day injected subcutaneously (s. c.). Furthermore, one vial/day of cetrotide acetate (0.25 mg) was added from a 14 mm folliculometry until the moment of ovulation induction (trigger) 34–36 h before the oocyte retrieval. Scheme B: induction was achieved usingof a recombinant follitropin  at a dose of 150 IU/day s. c. Furthermore, one vial/day of cetrotide acetate (0.25 mg) was added from a 14 mm folliculometry until the moment of ovulation induction (trigger) 34–36 h before the oocyte retrieval. Scheme C: induction was obtained with a recombinant follitropin (Elonva 150 µg) in a single dose s. c., with the addition (s. c.) of a vial of urinary follitropin (Meropur 75 IU) from the fifth day until the day of ovulation trigger. Scheme D: induction was achieved with a urinary follitropin (Meropur 75 IU) at a dose of 150 IU/day until the trigger day. Furthermore, one vial/day of cetrotide acetate (0.25 mg) was added from a 14 mm folliculometry until the moment of ovulation induction (trigger) 34–36 h before the oocyte retrieval. In all women of both groups, the induction of ovulation (trigger) was carried out 34–36 h before the surgical retrieval of the oocytes (pick-up) by administering chorionic gonadotropin  at a dose of 5000 IU administered s. c. in a single dose. Transvaginal ultrasound and serum oestradiol concentrations were used to monitor the cycle. Oocyte retrieval was performed under vaginal ultrasound guidance 34–36 h after hCG trigger.When the fresh embryo transfers were performed, patients were administered with progesterone ovules (Progeffik 200), 3–4 times/die starting from the pick up day according to the plasmatic progesterone value, the endometrial thickness, and the patient’s clinical conditions. Frozen embryo transfers (FET) were performed after hormone replacement therapy-based protocol. In the hormone replacement therapy-based protocol, oral oestradiol valerate  was administered three times a day from the second or third day of the cycle. When the endometrial thickness reached at least 7 mm, 600 mg/day of micronized progesterone was administered (Day 0). ET was performed after four and six days of progesterone supplementation according to cleavage or blastocyst stage, respectively. Positive pregnancy test was defined as a β-hCG (human chorionic gonadotropin) ≥ 50 IU/L on day 10 after embryo transfer. β-hCG was measured 11 or 14 days after cleavage or blastocyst stage FET, respectively. Positive pregnancy test was defined as a β-hCG (human chorionic gonadotropin) ≥ 50 IU/L on day 10 after embryo transfer. Clinical pregnancy was defined as the presence of a gestational sac with fetal heartbeat. A clinical pregnancy loss earlier than the 20th gestational week was considered a miscarriage .2 and 5% O2), the oocytes were denuded in HEPES-buffered medium  supplemented with 5% human serum albumin  and containing 20 IU/mL of hyaluronidase  and ICSI was conducted immediately after denudation as previously described [® medium  in Petri plates covered with mineral oil  in a controlled atmosphere . Vitrification was conducted using VT801 medium  or a Freeze Kit  and thawing using VT802 medium  or a Vitrification Thaw Kit  according to the previously published protocol [After 2 h of incubation in a controlled atmosphere  was formulated in sachets. Each sachet contained not less than 20 billion colony forming units (CFU) of L. crispatus M247 (LMGP-23257) [The probiotic product used in our clinical study (CrispactP-23257) . The prop < 0.05. Continuous variables were analyzed using descriptive statistics and multivariate analysis. Nominal or ordinal variables were evaluated using the chi-square technique. All data processing and statistical analysis procedures were performed on a Macintosh iMac computer. All statistical calculation and evaluations are available upon reasonable request.The study sample was assessed using methods of descriptive statistics. The ART procedures , vaginal-rectal swab and endometrial therapy were analyzed using the SAS categorical data analysis (JMP14.3 software). The probability of a positive pregnancy test was investigated using the multiple logistic regression model and prediction profiles involving the following variables: participant’s response to therapy , treatment (or not) with the probiotic, age, BMI, vaginal-rectal swab, hormonal therapy. The same variables were employed in a segmentation model (CHAID) to assess relationships with the response variable. Regarding live births, Pearson and Fisher’s exact test and odds ratio were evaluated. The chosen level of significance for all analyses performed was Our retrospective analysis was conducted on 160 infertile women. The mean age of the investigated sample was 37.55 ± 4.79 years . Twenty-five percent of all participants were younger than 35 years, 75% did not exceed the age of 41 and 90% were not older than 43 years. The modal ages were 41 (11.9% of the sample), 35 (10%), 31 (7.5%), and 36 (7.5%). For further details, see L. crispatus M247 (probiotic group) and the other group was untreated (control group). Probiotic treatment was well-tolerated and adverse events were almost superimposable in both groups for type , incidence , severity (mild and transient), and duration (2 to 3 days each).The enrolled women were divided in two groups of 80 women ; one grop = 0.0787 versus control) while in the control group the same outcome was achieved in 8 of 37 women (21.62%). The clearest difference concerned women under the age of 40. The clinical pregnancy rate in this age bracket was 13 out of 29 (44.8%) in the probiotic group versus 7 out of 29 (24.1%) in the control group (p = 0.0974). This suggests that age and probiotic treatment could positively influence the chances of pregnancy. Indeed, the odds ratio of falling pregnant was 7.45 (95% CI = 1.65–33.57) for a woman aged between 30 and 40 years, independently by treatment, and 1.56 (95% CI = 0.69–3.49) for a woman treated with the probiotic independently by age  and also to investigate the role played by BMI in such an outcome, we analyzed the role played by these three prediction profilers using the multiple logistic model. As regards to ages, we considered women between 20 and 30 years (3 in the control group and 5 in the treated one), women between 30 and 40 (44 in the control group and 42 in the treated one), women between 40 and 42 (16 in the control group and 19 in the treated one) and women over 42 (17 in the control group and 14 in the treated one). For example, considering the control group and setting the age and BMI values by default respectively as 20–30 and 22, the CPR was 9.9% . When thp < 0.05), while for the women in the control group undergoing the same procedure and with same age and BMI, the probability dropped to 28.4%. No significant result was observed in any of the other subgroups.After analyzing the possible effects on CPR of the simpler prediction profilers , we tried to integrate into our statistical approach the different fertilization and transfer procedures used in our study. Since segmentation analysis allows an exploration of the relationships between the different variables by progressively dividing the initial sample into groups that are increasingly homogeneous, we used the regression tree model to identify which parameters significantly modified the probabilities of a positive pregnancy test. According to our data, the most important predictor in this case was the parameter age, with a cut-off of 43. This predictor in fact divided the 160 analyzed women in two groups characterized by different probabilities of returning a positive pregnancy test. One group, all aged below 43, had an average probability of a positive pregnancy test of 25%; a second group, all aged over 43, had a probability of 0.07%. As shown in p = 0.2059; Since the women involved in the control group were selected from women managed in our hospital department earlier, in contrast to those actually treated with the probiotic, we tried to evaluate the rate of comparability between the two groups. The mean ages were not significantly different. In fact, the age of the women of the probiotic group was 37.53 ± 5.12; and that of the control group was 37.56 ± 4.63. Similarly, regarding BMI, the two group values were not significantly different, 22.14 ± 3.13 and 21.81 ± 3.09 and 17.8–37.3 and 17.51–36.05 being the BMI and the BMI ranges of the probiotic group and of the control group, respectively. Basal hormonal assets, evaluated by analysing FSH, AMH and estradiol, were not significantly different between the two groups either . RegardiThe results of our study showed that the use of the probiotic strain M247 significantly affected, in some subgroups of women, live birth rates. Indeed, regarding live births , out of p = 0.05), a positive role exerted by the probiotic is indicated. Considering the mother-child dyad, no significant difference between the two groups was observed concerning the mother’s age and BMI, gestational time, child’s weight and sex, and mode of delivery.We then analyzed the relationship between the live births in the two groups and the ART applied. As shown in Finally, to evaluate the global clinical meaning of our results, we used three statistical indices obtained by using the live birth rates calculated from 80 women per group ,39,40. TLactobacillus-dominated or, more precisely, not L. crispatus-dominated—has been proposed as a possible additional factor [Lactobacillus dominated), or with CST III (that is L. iners dominated) or with CST II (L. gasseri dominated), had a lower ART success rate than women with the L. crispatus-predominant vaginal microbiota, that is, CST I [Female causes of infertility include sexually transmitted infections, tuboperitoneal abnormalities, endometriosis, uterine anatomical abnormalities, as well as autoimmune, genetic, and endocrine disorders ,42. As il factor . ART is l factor ,45. Howes, CST I .L. crispatus could affect the success of ART, we retrospectively analyzed results routinely obtained in our hospital department over two years in which we treated 80 unfertile women with M247 orally, using a well-documented and safe strain of L. crispatus described to increase, after oral administration, the vaginal content of L. crispatus and also clinically capable of exerting an anti-HPV role [2), respectively. Within this range of age and with a BMI of 35, treatment with the probiotic increased the chance of a positive pregnancy test by 34%, versus an identical control independent of the ART procedure adopted. Besides age and BMI, the ART procedure adopted also demonstrated a favorable outcome. In fact, a woman subjected to embryo transfer with a D5- blastocysts, below 43 years, with a BMI over 18.6, and treated with the strain M247, had a significantly higher chance of a clinical pregnancy, with an increase of 66.3% versus an identical control.To analyze whether the treatment with a probiotic containing the species HPV role ,49. Our L. crispatus M247 may significantly increase the chance of pregnancy, they do not help us to understand exactly why. Of course, our assumption is that the probiotic could improve the woman’s vaginal environment, enriching and/or restoring an eubiotic  vaginal bacterial community. A trial on HPV-infected women and a very recent clinical case report hasin fact demonstrated the capability of the strain M247 to effectively restore a CST I [While these results seem to demonstrate that the use of  a CST I . Moreove a CST I .Lactobacillus-dominated. Undeniably, having available data from which to deduce that the administered strain was able to improve vaginal eubiosis in women treated with the probiotic, with particular reference to those in whom a clinical pregnancy was subsequently demonstrated, could have allowed us to confirm some recent results which should seem to have demonstrated, in a very preliminary manner, a causative and anti-pathological role of the L. crispatus species. Indeed, in the few cases of vaginal microbiota transplantation performed so far, in which CST IV women were transplanted with vaginal secretions from CST I women, the authors clearly demonstrated a shift of CST, from CST IV to CST I, together with the resolution of the “problem,” be it an intractable bacterial vaginosis condition or an infertility condition [Being a retrospective analysis of data obtained in our clinical routine and since the sampling and investigation of the vaginal microbiota are not routinely carried out either before or after treatment with the probiotic, it is impossible for us to demonstrate both the possibility of an effective colonization of the strain and/or the possible restoration of a vaginal microbiota classifiable as CST I or at least ondition ,52. Simiondition ,54,55. Sondition ,57,58. ILactobacillus-dominated vaginal microbiota also increases [The analysis of the data obtained clearly indicates that maternal age is decisive in favoring, or not, a clinical pregnancy. It is well recognized that increasing age contributes to difficulties in becoming pregnant. Fertility rates begin to decline gradually at the age of 30, more so at 35, and markedly at 40 . At thisncreases . A similncreases ,64,65. Oncreases . It may Regarding the ART method adopted, our analysis indicates blastocysts transfer as the method in which the probiotic seems to determine the greatest clinical success. One might wonder whether, as in the case of age and BMI, this procedure is the one favoring the least positive outcome and therefore the one in which the probiotic could show its greatest effects in restoring a correct vaginal eubiosis. However, we can also assume that the effect clearly identified in this subgroup is linked to a numerical issue, to the extent that any other method is so weakly represented in our study as to likely fail to demonstrate any possible therapeutic effect.The number of women enrolled in our analysis is the first among the many limitations of our study. Indeed, in addition to the known limitations of non-prospective, non-randomized and non-blind studies, the results of which have maybe a lower predictive value in general terms, 160 women is perhaps too few in number to distinguish the effect of a probiotic in relation to the different procedures adopted. Having said that, our approach has been focused on obtaining the most controlled data possible, to the best of what can be done in a retrospective study. All the analyses performed indeed showed us that the two groups were extremely superimposable and therefore the data obtained with our analysis could be considered of sufficient quality.In the attempt to understand how comparable the two groups were, we discovered a single difference between the two groups: the method of administration of the progesterone. Progesterone was mainly administered orally in the control group and mainly by injection in the probiotic-treated group. Statistical analysis, however, did not show any influence of this difference on the final result. Similarly, our study demonstrated no influence of other parameters such as the vaginal-rectal swab results or the antibiotic and/or antifungal therapy adopted.A further limitation intrinsic to our retrospective analysis is the lack of information regarding the ploidy of the implanted embryos. This aspect would have allowed a better interpretation of the results obtained.L. crispatus during the adoption of ART methods should in any case be considered safe and potentially advantageous to the extent that it would seem to increase the possibility of a clinical pregnancy by about 50%, regardless of age, BMI, and procedure adopted. This chance is further increased in women between 30 and 40 years of age and with a BMI greater than 22 and would further increase as the BMI increases, at least up to a value of 35. It is also possible that of all methods, the ART method using a 5-day blastocyst may highlight a greater success for the probiotic. In our study, in women under 43 years of age and with a BMI of at least around 20, this success was found to be significant (p < 0.05) with a net doubling of the chances of a clinical pregnancy. Larger, randomized, controlled, prospective, and double-blind studies are urgently needed to confirm the validity of what we have observed.Within the framework of the obvious caution necessary when considering the results of non-blind, open-label, and retrospective studies, our analysis would seem to show that the administration of Of the women in our study who had a clinical pregnancy, we can report 10 and 6 live births from a total of 19 and 14 women in the probiotic group and control group, respectively. Despite the fact that these results are non-significant, the calculation of the odds ratio demonstrated an increase  a NNT value indicating that few patients need to be treated to achieve positive results; (ii) a NNH value suggesting that the treatment is less likely to cause harm compared to control; and (iii) a LHH value showing a higher likelihood of benefits compared to harms associated with the treatment. Taken together, these results indicate that there is a good overall probability that women undergoing ART may benefit from oral treatment with L. crispatus M247.Finally, using specific statistical indices capable of extrapolating the risk-benefit ratio deriving from treating, or not, a woman with the


Patients with sepsis exhibited lower BMI values  and poorer glycemic control ; (4) Conclusions: This hospital-based data analysis in Mongolia highlights sepsis as the primary cause of mortality among diabetes patients in tertiary hospitals regardless of age, gender, or diabetes type while also indicating a potential association between a lower BMI, poor glycemic control, smoking, and the risk of sepsis.(1) Background: Given the growing global diabetes crisis, this study examined the causes of mortality in diabetic patients at a Mongolian tertiary care hospital. (2) Between 2017 and 2021, data from 100 individuals with diabetes  were reviewed. (3) Results: The predominant cause of mortality was sepsis, accounting for 65.0% of cases and emerging as a contributing factor in 75.0% of instances. Renal failure constituted the second leading cause of death, accounting for 19.0% of mortalities. Other contributing factors included chronic liver disease (6.0%) and ARDS (3.0%). Regarding sepsis, the individuals affected were relatively younger (57.5 ± 11.2 vs. 61.7 ± 11.2,  Diabetes is undeniably one of the most rapidly increasing diseases on a global scale . AccordiAs diabetes continues to pose a growing threat, even in developing countries such as Mongolia, where its prevalence has tripled in the last two decades, understanding the main causes of mortality in diabetic patients becomes paramount. The prevalence of diabetes increased from 3.2% in 1999 . CardiovIn this study, a comprehensive dataset was collected from the First State Central Hospital Mongolia, a prominent tertiary healthcare institution established in 1965. Throughout its history, the hospital has played a crucial role, serving as the largest medical facility in Mongolia until the year 2000, and it continues to be one of the country’s leading hospitals, providing medical services to patients from across the nation. The hospital, operating at a third-tier level, specializes in managing complex cases referred from district or provincial hospitals, excluding trauma and burn cases, which are directed to specialized centers. In Mongolia, there are dedicated cancer and stroke centers. The hospital provides comprehensive care for a diverse range of diseases, offering services in both outpatient and inpatient settings. This includes managing conditions, such as conducting diabetic follow-ups, and addressing critical cases such as stroke or cancer. The hospital is well-equipped with specialized units and facilities, including a dedicated stroke unit and emergency rooms.To assemble the necessary data for analysis, this study specifically concentrated on hospital discharge information for cases in which diabetes was a significant factor contributing to the patient’s condition. This study covered a substantial period, ranging from 1 January 2017 to 31 December 2020, to capture a comprehensive snapshot of diabetes-related mortality within the hospital. Ethical approval for the study was obtained from the Medical Ethical Committee of the Mongolian National University of Medical Sciences (METc2020/03), and the study was conducted in accordance with the Declaration of Helsinki.Through systematic and detailed data categorizations, this study aimed to gain valuable insights into both the primary and secondary causes of mortality among diabetic patients admitted to the First State Central Hospital Mongolia over a four-year period. This comprehensive analysis sought to shed light on the critical aspects of diabetes-related mortality, ultimately contributing to the improvement of healthcare strategies and patient outcomes in managing diabetes in Mongolia and beyond.The data were categorized Into two main variables: “Single Cause” and “Contributory Causes”. Under the “Single Cause” category, the primary cause of death was recorded for each patient. In cases in which individuals had multiple causes of death, they were classified under “Multiple Cause” cases within the “Single Cause” category. The research team conducted a thorough translation of the International Classification of Diseases, Tenth Revision (ICD10), codes into the specific causes listed in To further explore the nuances of diabetes-related mortality, the investigators developed a second variable named “Contributory Causes”. This variable involved a detailed breakdown of the multiple causes found within the “Single Cause” category. By segregating these multiple causes and identifying specific factors contributing to mortality, the study aimed to discern patterns and associations that might shed light on the complexities of diabetes-related deaths. For instance, if an infection presented as secondary cause of death along with other conditions, it was classified as one of multiple causes in the “Single Cause” category. Subsequently, the study team added these cases to the infection category within the “Contributory Cause” variable separately. This approach allowed for a more comprehensive understanding of the role infections play in diabetes-related mortality, providing valuable insights into their impact as both primary and contributory factors to patient outcomes.After conducting a detailed analysis, sepsis was identified as the primary cause of mortality. To further understand the underlying factors contributing to sepsis, we categorized cases into different types, including respiratory , abdominal , urinary , and skin and soft tissue infections , as well as cardiovascular  and central nervous system infections . It is important to note that due to the absence of bacterial, viral, and fungal infections, these were not considered in this analysis.t-test and an ANOVA, were applied to normally distributed continuous variables. For categorical variables, Pearson’s chi-squared test was employed.The general characteristics of the study population were expressed as means with standard deviations (SDs) or means with standard errors of the mean (SEMs) based on the distribution of variables, and as numbers with percentages (%) for categorical variables. To compare the differences between groups, parametric and non-parametric tests were used. Parametric tests, such as Student’s p < 0.05 for all tests.All statistical analyses were performed using IBM SPSS V.28.0 , and the level of statistical significance was set at During the research period (2017–2021), a total of 100 people were registered. Among these, 53.0% (n = 53) were male. The descriptive statistics for the variables are as follows: the mean age was 58.5 years ± 11.3, ranging from 32 to 84 years, and the duration of diabetes ranged from 0 to 37 years, with a mean of 9.6 years. The data analysis revealed that mortality could occur at any age and following any duration of diabetes, with no specific age group or duration of diabetes showing immunity to mortality risks .However, among the study participants, the most prevalent age group was found to be individuals aged 51 to 67 years, covering the 25th to 75th percentile range. Similarly, the duration of diabetes was most commonly found to be between 2 and 13 years, also encompassing the 25th to 75th percentile range. The body mass index (BMI) values ranged from 17.3 to 43.8, with a mean of 27.1 ± 4.3. Glycated hemoglobin (HbA1c) data were available for 97 participants, showing HbA1c levels ranging from 4.6 to 26.0, with a mean of 9.7 ± 3.6%. In the treatment of diabetes, 15% of the patients relied on multiple daily doses of insulin, while 36% used oral glycemic drugs; the remaining individuals utilized a combination of both approaches. Among the prescribed medications, sulfonylureas were the most commonly used, followed by biguanides, incretin-based drugs, and sodium–glucose cotransporter-2 inhibitors SGLT-2I, . Notablyp-values of 0.022 and 0.009, respectively. Other parameters, such as the duration of diabetes, the type of diabetes, glycated hemoglobin levels, diabetes treatment, hypertensive status, and length of hospital stay, showed no significant differences between the two groups.We conducted a comparative analysis between male and female patients, as outlined in The study delved into the various factors influencing mortality among patients with diabetes, shedding light on both primary (single) and contributing causes, as well as distinctions based on gender . NotablyIn this study, 11 participants (11.0%) had T1DM, while the majority, 89.0%, had T2DM. Notably, the analysis revealed no significant disparities in terms of contributing factors to mortality causes. However, it is worth noting that sepsis demonstrated a slightly higher occurrence in individuals with T1DM compared to those with T2DM. Renal failure emerged as a common factor across both types of diabetes, implying that it contributed to mortality in both cases. Notably, diabetic ketoacidosis (DKA) was more prevalent in participants with T1DM. Additionally, the study noted that the participants with T2DM had higher incidences of various other miscellaneous factors contributing to mortality .p = 0.026). Additionally, patients with chronic renal failure were significantly older, with an average age of 66.3 years, compared to those without chronic renal failure, who had an average age of 58.4 years (p < 0.001).When examining the causes of mortality and their relationship to age and the duration of diabetes, patients with coronary artery disease had a longer diabetes duration of 17.0 years compared to those without coronary artery disease, who had an average diabetes duration of 9.4 years (p < 0.05). Additionally, the sepsis group exhibited a higher frequency of smokers (p = 0.024). Age and diabetes duration showed no significant differences between the sepsis and non-sepsis groups. However, the sepsis patients tended to be younger, with shorter diabetes durations and a higher proportion of males. These differences were not statistically significant , hinting at a potential connection to the risk of sepsis  were attributed to skin and soft tissue infections, making it a significant contributor . AdditioDiabetes has emerged as a pressing global health concern, with its prevalence on a steady incline, even in regions such as Mongolia, where it has tripled over the last two decades ,10. HoweOur findings underscore the pivotal role of infections in the mortality of the study participants, both as contributing factors and sole causes. In addition, our analysis reveals other noteworthy contributors to these fatalities. Notably, chronic renal failure, acute respiratory distress syndrome (ARDS), and chronic liver disease emerged as significant contributory causes. Furthermore, malignancy and cerebral vascular accidents (strokes) were identified as both contributory factors and primary causes. Interestingly, research conducted in tertiary medical facilities in India echoed similar patterns, highlighting infections and sepsis 40.9%), alongside chronic renal failure 33.6%), as the primary culprits behind the mortality of diabetic patients . Corresp0.9%, alo%, as theOn a broader scale, our data imply that the distribution of the contributory causes of mortality generally aligns between participants with T1DM and T2DM, with minimal disparities across most causes. It is imperative, however, to approach these findings cautiously due to the relatively limited sample size for certain causes , which could impact the statistical reliability of our analysis. The relatively higher prevalence of chronic renal failure in T2DM patients may be attributed to the longer duration of diabetes in this group. Additionally, the prevalence of chronic liver failure in both T1DM and T2DM patients could be associated with Mongolia-specific conditions, possibly linked to a heightened incidence of viral liver infections in the country .Expanding our investigation, we explored potential correlations involving variables such as age, gender, diabetes duration, smoking, and BMI status. Unfortunately, this exploration did not yield definitive outcomes. Nevertheless, a descriptive analysis hinted at the possibility that females might experience diabetes-related mortality at an older age despite having a shorter disease duration. This aligns with existing research highlighting gender disparities which often render females more susceptible to increased severity and mortality ,25. WhilWe initially anticipated observing elevated mortality rates in Mongolia attributed to hypoglycemia and DKA. However, intriguingly, our findings did not identify any instances of mortality linked to hypoglycemia. Moreover, the mortality associated with DKA was lower than expected, indicating potential enhancements in the Mongolian healthcare system. This trend aligns with global observations from other studies, which demonstrate a decreasing trend in mortality related to acute diabetic complications on a worldwide scale ,32It is important to acknowledge the limitations of our study when interpreting the results. Firstly, the sample size was constrained due to the reliance on data from a single tertiary care hospital in Mongolia, potentially affecting the generalizability of our findings. Nevertheless, it is worth highlighting that this hospital is a significant institution catering to patients from both rural and urban areas, functioning as a nearly fourth-level institution in the country’s healthcare hierarchy. Additionally, the retrospective study design, which is reliant on pre-existing data, introduces inherent biases and potential gaps in information. Furthermore, the lack of multicenter data could restrict our ability to capture variations in mortality causes across diverse healthcare centers or regions. Notably, the recent establishment of a hospital unit focused on cerebrovascular accidents might have led to the omission of patients redirected there, potentially overlooking cases that were not within the scope of our study. Despite these limitations, our study boasts notable strengths. It leveraged a comprehensive dataset spanning four years, facilitating a thorough analysis of mortality causes in diabetic patients. Moreover, its relevance to developing countries like Mongolia offers insights that can steer targeted enhancements in healthcare strategies for the management of diabetes in analogous contexts. Through the implementation of a hospital registration system, our findings can furnish valuable insights which are applicable to developing nations, catalyzing improvements in diabetes management and healthcare systems, thereby curbing mortality rates on a broader scale.In Mongolia, this analysis of hospital-based data underscores sepsis as the leading cause of mortality among diabetic patients admitted to tertiary hospitals, irrespective of age, gender, or the type of diabetes. Notably, a potential link between a lower BMI, inadequate glycemic control, smoking, and the risk of sepsis emerged. To reinforce these findings, there is a pressing need for comprehensive studies across all hospitals in Mongolia, emphasizing the necessity of establishing a nationwide diabetes registration system.