Bladder cancer (BCa) is the tenth most prevalent malignancy of the urinary system diagnosed globally, impacting more men than women. In 2022, the incidence rate of BCa, with 614,298 cases reported, and its mortality, accounting for 220,596 deaths, highlight the significant global health impact of this disease, as reported by the Global Cancer Observatory (GCO) (gco.iarc.fr.) [1]. On the basis of clinical finding and malignancy features, BCa can be categorized into nonmuscle invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) [2]. Roughly 3-quarters of recently diagnosed individuals are afflicted with NMIBC, while a quarter experience MIBC or metastatic conditions [3]. Bladder tumors display varying clinical behaviors, spanning from low-grade to highly aggressive forms with recurrent progression risks [4]. The grade and stage of BCa have substantial prognostic significance, as they exhibit a high correlation with tumor recurrence [5], progression, and patient survival rates [6]. Noninvasive urothelial lesions exhibit a spectrum of morphologies, including flat, papillary, and inverted patterns, reflecting their relationship with the surrounding mucosa [7]. Hematuria is a pivotal clinical indicator for BCa. The standard and widely practiced method for detecting, diagnosing, and monitoring this condition continues to be the amalgamation of CT/MRI abdomen, urine cytology, and cystoscopy. Cystoscopy, though established as the gold standard for BCa diagnosis, presents challenges due to its invasive nature and relatively modest sensitivity (58%–68%), particularly when addressing flat lesions, satellite tumors, and nonpapillary tumors [8,9]. Urine cytopathology is a widely adopted and noninvasive method for the detection and assessment of BCa. Urine cytology demonstrates pronounced specificity (95%) when identifying high-grade malignancies, but its sensitivity (37%) is notably subpar, restricting its applicability in cases involving low-grade tumors [10,11]. The spectrum of survival rates over a 5-year period for BCa varies from 97% for carcinoma in situ to 8% for metastatic cases, emphasizing the vital significance of early detection in improving survival outcomes [12]. Enhancing long-term survival among BCa patients depends on early identification, precise risk assessment, timely intervention, and thorough monitoring. Hence, it is imperative to discern BCa-specific biomarkers that not only facilitate early and precise disease diagnosis but also enable effective tumor classification. Specifically, urinary biomarkers have the potential to serve as valuable diagnostic complements in BCa. As a rich reservoir of biomarkers, urine's content of exfoliated cells, proteins, cell-free DNA (cfDNA),cell-free RNA (cfRNA), extracellular vesicles (EVs), and a multitude of metabolites provides valuable insights into the health and functionality of the urinary system [13]. Malignant cells actively discharge EVs into various bodily fluids, including urine, and content of these EVs remain intact due to the protective lipid bilayer enveloping them. Urinary EVs are considered good source noninvasive biomarker development because of the existence of tumor-associated molecules within EVs and the convenient proximity of urine with the urinary bladder epithelium [14,15]. Consequently, the cargo of EVs is believed to play a crucial role in shaping the tumor microenvironment, fostering an environment conducive to primary tumor growth, and the establishment of distant metastases [16].

Extracellular vesicles (EVs) are double-layered lipid structures that are released by almost all cells into physiological fluids, like blood, breastmilk, and urine [17]. The cargo of EVs, comprising membrane receptors, proteins, and diverse RNA species, reflects the parent cell's molecular signature and when delivered to target cells, prompt protein translation, altering cellular function [18]. Following the size and biogenesis process, the primary EV subtypes include exosomes (30–100 nm), microvesicles (also known as ectosomes; 100–1,000 nm), apoptotic bodies/vesicles (100–5,000 nm), and large oncosomes (1–10 µm) [19]. The formation of EVs involves a process where the plasma membrane undergoes a double inward folding, leading to the generation of intraluminal vesicles housed within multivesicular bodies (MVBs) (Fig. 2) [20]. Exosomes, tiny bilayered vesicles, form through inward and reverse budding of MVBs endosomal membranes, ultimately released into the extracellular environment via fusion with the plasma membrane [21]. In contrast, cells employ budding and shedding processes to directly discharge microvesicles from their membranes. Apoptotic bodies are extruded from the cellular membrane as a result of the intricate process of programmed cell death [22]. Their release remains steady in normal physiological states and persists even in conditions like cancer progressions [23]. The interaction of EVs with the tumor microenvironment directly reflects the disease's state, unlocking the diagnostic and prognostic power of EVs and improving cancer care by enabling early detection, tailored treatment, and proactive disease monitoring.

MicroRNAs (miRNAs), which are short noncoding RNA consisting of around 20–22 nucleotides, play a crucial role in the regulation of genes after transcription. The transcribing of DNA sequences results in the formation of primary miRNAs (pri-miRNAs). The elongated pri-miRNAs originating from intronic regions of genomic DNA undergo modification through RNase type-III enzymes Drosha and Dicer endonuclease, resulting in the formation of precursor miRNAs (pre-miRNAs). The formation of mature miRNAs from these pre-miRNAs is facilitated by a protein complex that includes Argonaute and Helicase. Acting as negative regulators of protein synthesis, these small RNAs pair with the 3 untranslated region (UTR) of relevant messenger RNAs (mRNAs) through the assistance of the RNA-induced silencing complex. Playing a pivotal role, miRNAs regulate cellular differentiation pathways [24]. They act as oncogenes or tumor suppressor genes, with oncogenic miRNAs potentially overexpressed to target tumor suppressors, and tumor-suppressor miRNAs underexpressed to promote oncogene overexpression [25]. Urogenital tract-derived EVs contribute significantly to the complex composition of uEVs, with distinct subpopulations originating from kidneys, bladder, and prostate/utero-vaginal tract [26]. Encapsulated miRNA in EVs may also boost tumor invasiveness and the dissemination of metastatic cells through intercellular communication [27]. This review article evaluates the promising utility of uEVs and their miRNA payloads as biomarkers, while critically scrutinizing the latest progress in uEVs miRNAs biomarker research and high-throughput technologies.