Search Results (8,981)

The beta-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is a promising and rational target for Alzheimer’s disease (AD), but current clinical trials have been disappointing. Consequently, utilizing the intrinsic regulatory mechanisms of BACE1 during AD pathogenesis might provide valuable insights into the treatment of this devastating disease. In this study, we proposed a combination of AAV delivery and microRNA therapeutics targeting AD at its root by sustained and spatial inhibition of BACE1 with a single therapeutic injection. We demonstrate that upregulation of BACE1 is correlated with downregulation of miR-143-3p in the hippocampus of individuals with AD, and miR-143-3p can directly target BACE1 to inhibit Aβ generation. In the brains of 5×FAD model mice, BACE1 levels are found to be elevated with age in the cornu ammonis 1 (CA1) subfield of the hippocampus. AAV-mediated miR-143-3p restoration in the hippocampal CA1 subfield of AD mice can improve cognitive performance, attenuate BACE1 expression, reduce Aβ levels, induce microglia polarization toward the anti-inflammatory phenotype, modulate neural-related genes including Gal3, and promote synaptic functions. Collectively, the AAV-mediated microRNA gene therapy approach developed for spatial suppression of BACE1 can effectively enhance cognitive performance in AD model mice, offering an attractive therapeutic option for AD treatment with long-lasting efficacy. Full article

Sickle cell disease (SCD) is a globally distributed hereditary red cell disorder with still high mortality. Growing evidence indicates that sickle cell related cardiovascular disease contributes to the early death of adults with SCD. Here, we show that humanized SCD mice developed an age-dependent cardiomyopathy characterized by (i) increased circulating Th17 lymphocytes, Th17 heart infiltration associated with increased plasma IL-17; (ii) collagen deposition and activation of both platelet derived growth factor-B (PDGF-B) and transforming growth factor-β1 (TGF-β1) canonical pathways; (iii) overactivation of heart NF-κB associated with up-regulation of NLRP3 and of inflammatory vasculopathy markers. We then used colchicine (CLC) that acts as anti-inflammatory drug with immunomodulatory effects. In humanized SCD mice, we demonstrated the protective role of low-dose CLC treatment against chronic inflammation and preserving myocardial performance. Of note, we found CLC also attenuating sickle cell related lung damage, suggesting a multiorgan effect of CLC in SCD mice. Our data generate the rationale to further explore CLC as new therapeutic tool to treat early stages of sickle cell cardiomyopathy. Full article

Objectives: Celiac disease (CD) is an immune-mediated disorder triggered by gluten ingestion. Although a gluten-free diet (GFD) is the only effective treatment, patients may still experience inflammation, nutritional imbalances, and reduced quality of life. Physical activity (PA) has demonstrated anti-inflammatory benefits; however, its role in CD management remains unclear. This systematic review aimed to synthesize evidence on the associations of PA and the outcomes of exercise interventions in individuals with CD, focusing on metabolic, nutritional, clinical, and functional outcomes. Methods: Following PRISMA guidelines, we included peer-reviewed observational and experimental studies assessing PA in individuals with CD across all age groups, without language or date restrictions. Searches were conducted in PubMed, Scopus, Web of Science, EMBASE, and SPORTDiscus. The risk of bias was evaluated using JBI and Cochrane RoB 2 tools. Results: Fourteen studies (17 publications) were included, the vast majority of which were cross-sectional, along with one quasi-experimental study and two randomized controlled trial cohorts reported across five publications. Data from these predominantly observational studies suggested possible, yet inconsistent, links between higher PA levels and better profiles in body composition, inflammatory and oxidative markers, quality of life, and psychological outcomes. Positive associations were also observed in some studies regarding gastrointestinal symptoms and adherence to the GFD. However, findings on metabolic markers and bone mineral density were inconsistent and linked to dietary factors. Conclusions: While PA represents a potential adjunct in the comprehensive management of CD, particularly in relation to functional and inflammatory outcomes. However, the current evidence remains highly preliminary, limited, and inconsistent, which restricts the strength of any definitive conclusions. Therefore, high-quality, longitudinal studies and well-designed clinical trials are needed to confirm its long-term benefits, especially in the pediatric population, and to establish specific recommendations. Full article

Background: Opportunistic salpingectomy is widely used in gynecological surgery as a preventive measure against ovarian carcinoma. Questions have been raised about the potential impact of salpingectomy on ovarian function, given the possibility of ovarian vascularity disruption through the utero-ovarian arch located in the mesosalpinx. The aim of this study was to evaluate the short-term effects of bilateral salpingectomy on ovarian reserve. Methods: This prospective cohort study included forty premenopausal women, aged 34–50 years, who underwent hysterectomy with bilateral salpingectomy and ovarian preservation for benign uterine conditions. Ovarian reserve was evaluated before surgery and three months postoperatively, using serum anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH) and antral follicle count (AFC). Health-related quality of life was assessed using the Women’s Health Questionnaire (WHQ). Results: Serum FSH levels increased significantly after surgery, while AMH levels decreased significantly (both p < 0.05). No significant postoperative changes were observed in AFC. The overall WHQ score increased significantly, indicating a mild deterioration in health-related quality of life. The greatest postoperative changes were observed in the depressive, somatic and sexual domains, where menstrual symptoms improved following surgery. No significant correlation was found between WHQ scores and hormonal or ultrasonographic markers of ovarian reserve. Conclusions: Bilateral salpingectomy performed during hysterectomy was associated with significant short-term hormonal changes, while AFC remained stable. Although patient-reported quality of life slightly worsened after surgery, these changes were not correlated with hormonal or ultrasonographic markers of ovarian reserve. Larger controlled studies with longer follow-up are required to determine the long-term clinical significance of these findings. Full article

Hepatitis B surface antigen (HBsAg) signal-to-cutoff (S/CO) values and isolated anti-HBc positivity represent two significant diagnostic challenges in hepatitis B virus (HBV) serology. This retrospective study, conducted in Karabük between 2021 and 2025, aimed to evaluate the correlation between these profiles and HBV DNA positivity to determine the optimal S/CO thresholds for confirmatory testing and clarify the clinical relevance of isolated anti-HBc reactivity. Among 17,356 patients screened for anti-HBc, isolated anti-HBc positivity was identified in 506 patients (2.9%), of whom 3.2% had detectable HBV DNA. Positivity was significantly higher in patients older than 50 years and in the Gastroenterology department. Separately, 213 specimens with HBsAg S/CO values between 1.0 and 10.0 underwent HBV DNA confirmation testing. ROC analysis yielded an excellent area under the curve of 0.937, with an S/CO threshold of ≥3.04, achieving 100% sensitivity for HBV DNA detection. All specimens with S/CO values between 1.0 and 3.0 were HBV DNA-negative. In this study population, no serum HBV DNA positivity was detected among samples with S/CO values below 3.04, suggesting that HBV DNA testing may help avoid unnecessary confirmatory work-up in this range. Anti-HBc screening should be prioritized in older patients. S/CO-based confirmatory algorithm using HBV DNA testing may improve diagnostic accuracy and reduce unnecessary clinical interventions in routine hepatitis B screening programs. Full article

Background/Objectives: Sensory nerves, as essential peripheral nerves, innervate bone and release various neuroactive substances—including neurotransmitters, neuropeptides, and neurocrine factors—that participate in bone growth, remodeling, and metabolism. Interleukin-1 receptor antagonist (IL1RN), an endogenous anti-inflammatory mediator, is a key regulatory molecule in the pathogenesis of inflammatory diseases such as osteoarthritis and rheumatoid arthritis. However, its role as a sensory neurocrine factor in the regulation of bone tissue has rarely been investigated. This study aimed to explore the regulatory effects of sensory nerve–derived IL1RN on bone tissue. Methods: A dorsal root ganglion (DRG)-targeted delivery system was developed using DNA origami technology to load IL1RN protein or IL1RN-targeting siRNA and was functionalized with a DRG-homing peptide. Bone defect and age-related bone loss models were established in C57BL/6 mice to preliminarily investigate the regulatory role of IL1RN secreted from sensory nerve endings in bone tissue. Results: IL1RN suppressed bone resorption and promoted new bone formation at defect sites. In the age-related bone loss model, IL1RN preserved the integrity of the growth plate. These findings indicate that sensory nerve–derived IL1RN may participate in the regulation of bone repair and skeletal homeostasis. Conclusions: IL1RN may serve as a potential therapeutic target for DRG-mediated regulation of bone repair. These findings suggest that DRG-targeted modulation of IL1RN may represent a potential approach for investigating and regulating sensory nerve–associated bone repair. Full article

Over the past decade, the incidence of infectious syphilis has been on the rise in the general Chinese population. Consequently, Treponema Pallidum (TP) testing has been proposed as a surrogate marker for sexually transmitted pathogens and for monitoring risky sexual behaviors among blood donors globally. In addition, sexual contact with individuals chronically infected with hepatitis B virus (HBV) is recognized as one of the primary routes of HBV transmission. Blood donors may acquire HBV infection through sexual contact with chronically infected partners, particularly with occult hepatitis B infections (OBIs), which are characterized by intermittent and extremely low viral loads. Therefore, the prevalence of OBIs among syphilis-positive blood donations and the corresponding risks to blood safety require further investigation. This study aimed to investigate the prevalence of OBIs among syphilis-positive blood donors and assess the surrogate value of TP testing for evaluating OBI-related risks to blood supply. After routine screening using serological assays and nucleic acid testing (NAT), blood donation samples with positive anti-TP enzyme-linked immunosorbent assay (ELISA) results were collected and further confirmed by the Treponema Pallidum Particle Agglutination Assay (TPPA). For blood donations confirmed positive for syphilis, further tests were performed to characterize whether the donations had HBV co-infection, including electrochemiluminescence immunoassay (ECLI) for the detection of hepatitis B surface antigen (HBsAg), anti-hepatitis B surface antibody (anti-HBs), hepatitis B e antigen (HBeAg), anti-hepatitis B e antibody (anti-HBe), and anti-hepatitis B core antibody (anti-HBc). Additionally, quantitative real-time polymerase chain reaction (qPCR) was used for HBV DNA quantification, and nested PCRs for the S and basal core promoter/precore (BCP/PC) region were conducted in combination with high-volume nucleic acid extraction. Subsequently, molecular characterization of HBV DNA in these co-infected samples was carried out by DNA sequencing to analyze the viral genetic features. Of 252 anti-TP ELISA+ donations screened from 64,871 blood samples, 138 (138/250, 55.2%) donations were confirmed syphilis-positive but NAT−, among which 78 (78/138, 56.5%) were anti-HBc-positive, and 88 (88/138, 63.7%) had anti-HBs. Notably, seven donations (7/138, 5.1%) were diagnosed as OBI co-infections, and available sequence analysis revealed that three cases were genotype B and one case was genotype C. In addition, several mutations in the S region of the HBV genome were identified, including Q101R, K122R, Q129H, T131N, M133T, G145R, and Y161F mutations. Furthermore, nucleotide mutations such as T1719G, A1752T, G1896A, and A1762T/G1764A in the BCP/PC regions were also detected in these OBI donations. These mutations may contribute to the extremely low HBV viral loads and/or failure in HBsAg detection, collectively leading to OBIs. These data indicate that syphilis screening of blood donors has potential to serve as an additional safeguard measure for excluding donations co-infected with OBIs. The high prevalence of undetected OBIs in syphilis-positive blood donors further supports that syphilis screening has the potential to serve as a surrogate marker for HBV-related risks in the blood supply. Full article

Background and objective: A huge part of cosmetics research is devoted to the identification of new molecules and new ingredients which could prevent skin aging. Crithmum maritimum has attracted interest in the cosmetic field because it has been used since antiquity in traditional medicine and is traditionally consumed in Mediterranean regions. However, data about demonstrated efficacy on living cells remain sparse. To better understand the potential of this plant for the cosmetics industry, we aimed to investigate in vitro some key properties in line with anti-aging skin care. Methods and results: Molecular results demonstrated an impressive antioxidant effect which was confirmed in vitro on living keratinocytes with a decrease of 50% of oxidative stress measured with the fluorescent CM-H₂DCFDA probe. Additionally, an inhibitory effect around 20% was observed on isolated collagenase and elastase, highlighting the ability of C. maritimum to preserve the skin matrix. Lastly, anti-inflammatory properties were explored at protein levels by ELISA method, indicating a clear decrease of six inflammatory mediators, IL-6, IL-8, IL-18, CXCL9, CXCL10, and CCL5, on stressed keratinocytes. These observed decreases span from 20% up to 75% in the case of the IL-18. Conclusions: All together, these results reveal how efficient C. maritimum extract could be to alleviate signs of aging. Full article

Cutaneous co-infection with Mycobacterium tuberculosis (MTB) and Mycobacterium avium complex (MAC) is extremely rare and easily missed due to overlapping histopathological features. We report a previously healthy, HIV-negative middle-aged woman who presented with a progressive destructive mass in the left inguinal-perineal region. Imaging revealed sinus tract formation, osteolytic bone lesions, and chronic inflammation in the right middle lobe of the lung. Initial metagenomic next-generation sequencing (mNGS) detected 3756 reads of the Mycobacterium tuberculosis complex (MTBC) and 111 reads of Mycobacterium intracellulare (M. intracellulare); the latter was interpreted as possible colonization or contamination because of its low abundance. Empirical anti-tuberculosis therapy produced only transient partial improvement, followed by paradoxical worsening, local recurrence, and new bone destruction. After a high suspicion of mixed infection, a MAC-directed combination regimen (including azithromycin and a short course of amikacin) was added, leading to complete clinical cure; subsequent repeat cultures confirmed the presence of MAC. This is the first report of cutaneous MTB-MAC co-infection in the inguinal-perineal region of an adult without overt immune abnormalities, accompanied by disseminated bone lesions. This case highlights that in regions where nontuberculous mycobacteria (NTM) are co-endemic, atypical destructive skin lesions with paradoxical worsening despite initial response to anti-tuberculosis therapy should raise suspicion of MAC co-infection. The combination of mNGS and conventional culture facilitates identification of mixed infections and guides precision therapy, but mNGS results must be interpreted cautiously in the clinical context. Full article

Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are chronic inflammatory diseases resulting from complex interactions between host genetics, environmental factors, immune dysregulation, and the gut microbiome. Among environmental exposures, antibiotics have emerged as important factors of IBD risk and disease course because of their profound effects on intestinal microbial communities. This review synthesizes current evidence on the class-specific effects of antibiotics on IBD, integrating epidemiological, mechanistic, and clinical studies to examine how different antibiotic classes influence disease susceptibility, progression, and microbiome recovery. Current evidence indicates that antibiotic-associated IBD risk varies according to antibiotic class, cumulative exposure, age at exposure, and antimicrobial spectrum, with broad-spectrum and anti-anaerobic agents showing the strongest associations. Mechanistically, antibiotics promote dysbiosis by depleting beneficial commensal bacteria, disrupting microbial metabolite production, expanding pathobionts and the intestinal resistome, and impairing epithelial barrier integrity and immune homeostasis. The review also discusses microbiome-preserving and microbiome-restorative approaches, including antimicrobial stewardship, fecal microbiota transplantation, prebiotics, probiotics, synbiotics, postbiotics, and dietary interventions, as potential strategies to mitigate antibiotic-associated dysbiosis. Overall, the evidence highlights the class-specific effects of antibiotics in IBD and underscores the importance of microbiome-informed antimicrobial stewardship and precision therapeutic strategies to optimize patient outcomes while minimizing long-term disruptions of host–microbiome homeostasis. Full article

Dietary protein is essential for poultry performance, influencing egg production, growth, immunity, and physiological functions. However, the high cost and environmental impact of soybean meal has encouraged the development of reduced crude protein (CP) diets supplemented with crystalline amino acids (AAs), coupled with the reduced cost and improved availability of synthetic AA. This strategy enables precise AA balancing, improves nitrogen utilization, and reduces nitrogen excretion and ammonia emissions. Among these strategies, supplementation with arginine (Arg), an indispensable AA in chickens, is particularly important due to its critical roles in protein synthesis, reproductive function, and eggshell formation. In addition, functional Arg precursors, such as guanidinoacetic acid (GAA) and citrulline (Cit), can further enhance Arg availability while supporting energy metabolism and hormonal regulation. Despite these benefits, excessive protein reduction without adequate AA balance can impair laying performance, particularly in aged hens. Furthermore, nutrient digestibility remains a key limitation in reduced protein (RP) diets, as non-starch polysaccharides present in cereal-based feeds can reduce nutrient availability. The inclusion of exogenous enzymes, such as xylanase and β-glucanase, can improve digestibility, while phytase supplementation further enhances nutrient utilization by increasing phosphorus availability and mitigating the anti-nutritional effects of phytate; however, responses are often inconsistent. Another critical factor is maintaining an optimal energy-to-protein ratio, as imbalances may lead to increased fat deposition and reduced productive performance. While supplementation of key limiting AAs (lysine, methionine, and threonine) supports protein reduction, other AAs, such as Arg, isoleucine, and valine, may become limiting in RP diets. Hence, this review explores developments toward RP diets by highlighting three key strategies: enzyme supplementation to enhance nutrient digestibility, inclusion of functional Arg sources to improve performance and bone quality, and optimization of the energy–protein ratio in RP diets through precise AA formulation, with the overall aim to improve the sustainability of the poultry industry. Full article

Accumulating data indicate a role for the dysregulation of the cell cycle, autophagy and apoptosis in diabetic kidney disease. We aimed to evaluate the mediators of the PI3K/Akt and MAPK/ERK signaling pathways in the kidney of db/db mice, a model of type 2 diabetes, treated by the SGLT2 inhibitor empagliflozin, the DPP4 inhibitor linagliptin, and metformin. Eight-week-old male db/db mice were randomly assigned to treatment by these agents or vehicle for 8 weeks. Age-matched db/+ mice acted as controls. AMPKα1 and PI3Kp110β were evaluated in the renal cortex and medulla by Western Blot. Phosphorylated forms of principal molecules involved in the PI3K/Akt and MAPK/ERK pathways were assessed by multiplex analysis. Db/db mice had decreased PI3Kp110β, increased phospho-PTEN, HSP27 and MEK1 in the renal cortex and medulla, BAD in the renal cortex and decreased phospho-rpS6 in the renal medulla. Empagliflozin prevented the changes in the levels of cortical PI3Kp110β, phospho-MEK1, and medullar phospho-PTEN. Linagliptin restored PI3Kp110β levels. Both agents further decreased medullar phospho-rpS6. Metformin upregulated cortical AMPKα1, medullar PI3Kp110β, phospho-GSK-3α/β and MEK1, and increased phospho-HSP27 in the renal cortex and medulla. The data may provide further explanation of the mechanism underlying the development of diabetic kidney disease, as well as the renal protective effect of anti-diabetic agents. Full article

Background/Objectives: Thrombotic microangiopathies are rare, life-threatening hematological disorders characterized by microangiopathic hemolytic anemia, thrombocytopenia, and end-organ injury. This study was conducted in a setting where ADAMTS13 activity testing became available only from 2015 onward and complement-targeted therapy (eculizumab) had limited accessibility throughout most of the study period, conditions that shaped both diagnostic classification and treatment outcomes. Their clinical presentation, treatment response, and prognosis vary according to etiology, making early recognition and subtype classification clinically important. This study aimed to evaluate the etiological distribution, clinical features, treatment responses, and outcomes of adult patients with thrombotic microangiopathy at a tertiary-center real-world cohort. Methods: This retrospective cohort study included 47 adult patients (≥18 years) hospitalized with thrombocytopenia and microangiopathic hemolytic anemia (MAHA) in a nine-year period. Patients were classified as thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), or secondary TMA based on clinical and laboratory evaluation. Demographic characteristics, clinical manifestations, laboratory parameters, treatments, and outcomes were analyzed. Results: The mean age was 45.3 ± 15.2 years, and 72.3% of patients were female. Primary thrombotic microangiopathy accounted for 74.5% of cases, including thrombotic thrombocytopenic purpura in 53.2% and hemolytic uremic syndrome in 21.2%; secondary thrombotic microangiopathy accounted for 25.5%. Hemodialysis was required in all patients with hemolytic uremic syndrome compared with 16% of those with thrombotic thrombocytopenic purpura. The complete response rate was 74.5%, and in-hospital mortality was 25.5%. In multivariable Cox regression analysis, treatment non-response and reduced post-treatment estimated glomerular filtration rate independently predicted mortality. Conclusions: Adult TMAs are characterized by considerable etiological and clinical heterogeneity, which makes differential diagnosis challenging, particularly in settings where access to contemporary diagnostic tests and targeted treatments is limited. In this cohort, in the absence of ADAMTS13 testing, TTP was the most frequent subtype, while treatment non-response and renal impairment emerged as the main factors associated with mortality. These findings emphasize the need for early clinical recognition and careful subtype-based differential diagnosis, which will reduce morbidity and mortality by permitting rapid initiation of pathophysiology-based appropriate interventions, i.e., PEx, immune suppression and caplacizumab for immune TTP and anti-complement therapy for aHUS, and limiting the inappropriate use of PEx with its complications, including sepsis. Full article

Protocatechuic acid (PCA) is a simple natural phenolic acid widely distributed in plants. It is recognized as an active constituent of numerous traditional herbal medicines and serves as an important metabolic intermediate of polyphenolic compounds such as anthocyanins and proanthocyanidins. At present, PCA production relies on plant extraction and microbial fermentation. Among these, microbial fermentation has emerged as an attractive approach for industrial development owing to its process controllability, environmentally benign nature, and potential for high productivity. This review systematically summarizes the major pharmacological properties of PCA, including its antioxidant, anti-inflammatory, antimicrobial, antiviral, anti-aging, neuroprotective, and hepatoprotective activities. It further highlights the therapeutic potential of PCA in the prevention and management of various chronic diseases, such as cancer, diabetes, Alzheimer’s disease, and hypertension. Furthermore, this review summarizes the representative microbial biosynthetic pathways for PCA and discusses recent progress in metabolic engineering strategies aimed at enhancing its microbial production. These strategies include reinforcing precursor supply, redirecting metabolic flux toward the shikimate pathway, blocking PCA degradation routes, relieving intracellular feedback regulation, improving host tolerance, and optimizing fermentation processes to achieve higher PCA productivity and yield. Finally, the major bottlenecks limiting PCA biomanufacturing are discussed, and prospective directions for future research are proposed. Full article

Background: Buffaloes have a significant share in the agricultural economies of many developing countries; however, the industry is not well-established because of the low reproductive efficiency of buffaloes. In this study, we designed a novel strategy to enhance buffalo reproductive efficiency and examined the significance of intramuscular AMH-INH-RFRP DNA vaccine immunization on reproductive performance. Methods: Allocated into four groups (n = 30/group), female buffaloes (aged 5–8 years, n = 120) were randomly enrolled in the study. Treatment groups received 10 mL of AMH-INH-RFRP DNA vaccine intramuscularly, once daily for three consecutive days, at the following concentrations: T1—3 × 10⁸ CFU/mL, T2—3 × 10⁹ CFU/mL, and T3—3 × 10¹⁰ CFU/mL. All vaccinated groups received a booster vaccination two weeks later, while the control group received 10 mL of phosphate-buffered saline (PBS) intramuscularly on the same schedule. Serum antibody titers against anti-Müllerian hormone (AMH), inhibin (INH), and RF-amide-related peptide (RFRP) were quantified using indirect ELISA on days 14 (post-primary immunization) and 28 (post-booster immunization). Results: Following primary immunization, antibody titers against AMH, INH, and RFRP were significantly higher in the T3 group than in the T1 group (p < 0.05), and booster immunization further increased antibody positivity rates. Serum concentrations of IL-4, IFN-γ, and E₂ were significantly elevated in the T2 and T3 groups compared to the control group (p < 0.05), while P₄ levels showed no significant differences among groups. Ultrasonography revealed that the ovulatory follicle diameter and dominant follicle growth rate were significantly increased in the T2 and T3 groups compared to the control and T1 groups (p < 0.05). The estrus rate was significantly higher in the T3 group than in the control group (76.67% vs. 40.00%, p < 0.05), and ovulation rates were significantly higher in the T2 (83.33%) and T3 (86.67%) groups compared to the control group (56.67%) (p < 0.05). However, conception rates did not differ significantly between vaccinated and control groups in the primary dose comparison (p > 0.05). In exploratory post hoc analyses, antibody-positive (Ab+) buffaloes exhibited higher E₂ concentrations, larger ovulatory follicle diameters, accelerated dominant follicle growth rates, and higher estrus and ovulation rates compared to antibody-negative (Ab−) buffaloes. Furthermore, gestating buffaloes were monitored until calving, revealing no significant effect of the vaccine on newborn calf weight or body size. Conclusions: In total, intramuscular immunization with the AMH-INH-RFRP DNA vaccine, particularly at the highest dose, enhances reproductive performance in buffaloes by stimulating E₂ secretion and follicular development. Full article