It is important to note that semen contains one of the highest reported concentrations of extracellular vesicles of any body fluid. These sEVs originate from the different organs of the male reproductive system: prostate (these sEVs, also known as prostasomes, represent 40% of semen sEVs), epididymis (epididysomes), seminal vesicles, and testicles ^[1][8].

Although it is developmentally mature, sperm exiting the seminiferous tubule still requires further modification. Changes in sperm morphology and post-testicular sperm function rely on sperm interaction with the intraluminal fluid during transit through the epididymis and vas deferens, suggesting that EVs in male reproductive biofluids may participate in intercellular communication after spermatogenesis. Additionally, in the female reproductive system, sperm acquisition of various functions including increased motility, transfer of cargoes, and ability to undertake the acrosome reaction is mediated through the interaction between the sperm and semen EVs derived from the organs of the male reproductive tract. Thus, semen EVs have a relevant physiological function related to the process of fertility ^[1][8] (point 1), but they are also involved in pathological states of the male reproductive tract such as the development and dissemination of prostate cancer ^[2][3][9,10] (point 2).

Seminal plasma (SP) sEVs have been proposed as a means of selectively transporting and delivering various regulatory molecules to the female reproductive system to facilitate conception and contribute to fertilization ^[1][8] and embryo implantation ^[4][11], the latter by targeting endometrial stromal cells. Strikingly, as a first step, semen sEVs transfer their content directly to the male germ cell in the acid medium of the vagina ^[5][12] to protect sperm and modulate their activity. The numerous enzyme systems, small signaling molecules, and neuroendocrine markers associated with semen sEVs suggest that these vesicles directly or indirectly play a complex role in the regulation of sperm viability and function ^[6][13]; (reviewed in ^[7][8][14,15]).

Tumor-derived sEVs have been described as being involved in the development and dissemination of PCa ^[2][3][9,10]. In the tumor environment, EVs derived from malignant cells carry genetic and proteinic messages to the target cells which reduce their immune response. In this way, they influence the homeostasis of the surrounding environment and the progression of the tumor in the donor tissue. Reproductive system sEVs are directly secreted to semen and some of them can also reach blood and plasma. In this way, through the bloodstream, they contribute to the formation of the premetastatic niche in other body tissues (metastasis). The EVs secreted by cancer cells into the blood have a cancer-specific molecule content ^[22][29] such as a high proportion of oncoproteins and RNAs ^[23][30]. These EVs not only intervene in tumor progression, but also in the communication between tumors and the immune system, contributing to the drug-resistant character of cancer cells.

sEVs are characterized by a high content of cholesterol and sphingomyelin, as well as by a very complex protein composition. EVs also carry RNA that can be transferred to other cells, modulating the function of the recipient cells ^[24][25][3,4]. This RNA can be found in the form of mRNAs and non-coding regulatory RNAs (including small and long ncRNAs) ^[24][3].

sEVs in semen contain a very important population of small ncRNAs (sncRNA, 20 to 100 nucleotides), which have a significant impact on the regulation of gene expression through a variety of epigenetic and post-transcriptional mechanisms ^[26][31]. RNA biotypes identified within sncRNAs include microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), and endogenous interfering RNAs (endo-siRNAs), which are critical regulators of germ cell development. There are also other more recently described molecules such as transfer RNA (tRNA)-derived small RNAs (tsRNAs) and ribosomal RNA (rRNA)-derived small RNAs (rsRNAs), whose role in reproduction and fertility is yet to be fully settled (reviewed in ^[27][32]). MiRNAs (21%) and tsRNAs (16%) represent the most abundant sEV sncRNAs in semen ^[1][8]. The sncRNA profile of sEVs in semen is unique and differs from the profile found in sEVs from other fluids ^[1][8].

Since sncRNAs are encapsulated inside sEVs, they remain stable and protected from RNAse. Due to these attributes, sEV sncRNAs are considered relevant for study as reliable biomarkers. Additionally, EVs contain molecules of the progenitor cell, so these EVs in the fluids can reflect the identity, characteristics, and health of the cell or tissue of origin. This is highly relevant in the context of the study of the content of sEVs as biomarkers. Specifically, in recent years, an increasing number of studies have been published that evaluate the miRNAs of sEVs in fluids as diagnostic biomarkers for physiological processes, such as the immune response, and pathological processes such as cancer, with promising results. The miRNAs participate in the post-transcriptional regulation of the expression of the genes: the specific binding of these small molecules of RNA to the transcripts affects their stability and activates transcript degradation. The tissue-specific expression profile and the high stability of these molecules mean that miRNAs, and other regulatory small RNAs, are becoming more and more relevant as biomarkers for the diagnosis or prognosis of different pathological conditions.

In recent years, it has been shown that ncRNAs play an important role in tumor progression and metastasis by their dissemination through fluids to regulate cancer cells and/or cancer stem cells, which suggests that extracellular ncRNAs in circulating sEVs can be used not only for diagnosis but also as prognostic biomarkers ^[28][33].

As previously mentioned, semen contains a heterogeneous composition of sEVs, which are released from the different organs of the male reproductive tract, and these sEVs contain molecules from their cell of origin. Levels of these molecules can reflect the features and the health of that original cell. Certain sncRNA subtypes, such as miRNAs, have been associated with gene deregulation in diseases affecting organs of the male reproductive tract, including prostate cancer ^[29][34] and spermatogenic disorders ^[30][35]. These attributes support the idea that the study of the small RNA content of semen sEVs can reflect the pathophysiological state of male reproductive organs. The small RNA population varies between tissues; therefore, it is essential to know the expression pattern of the small RNAs in each of the organs of the male reproductive tract to be able to correctly interpret semen sEV sncRNA expression behavior and use it as a potential biomarker of urogenital disease.

Spermatogenesis is a highly orchestrated developmental process which occurs in the testicular seminiferous tubules, and by which primordial germ-cells or spermatogonia develop into haploid spermatozoa. Production of sperm depends on precise developmental stage- and germ-cell type-specific gene expression. Thus, spermatogenesis is heavily dependent on post-transcriptional regulatory processes, and miRNAs have emerged as important regulators of these events ^[31][32][33][36,37,38]; miRNAs specifically expressed in the germline, such as miR-122, miR-449a, and miR-34b/c, are especially relevant ^[30][34][35][35,39,40]. In human testes, it is not only miRNAs that are highly abundant but also piRNAs, and tsRNAs at a lesser extent ^[36][41]. Interestingly, in physiological conditions, piRNAs are almost exclusively found in the male gonad, mainly in the germline ^[37][38][39][42,43,44].

The epididymis is a convoluted tube at the posterior part of the testis, where the sperm mature and are stored. When the sperm leaves the testis, it harbors a specific profile of small RNA, including all major RNA classes, with piRNAs being the most abundant sncRNAs. During its post-testicular maturation, the spermatozoon does not generate sncRNAs, but acquires them exogenously thanks to its fusion with extracellular vesicles ^{[40][41][42][43]}[45,46,47,48]. In the epididymal lumen, epididymal epithelial cells secrete epididymosomes that contain small RNAs and proteins. Epididymosomes can deliver miRNAs, tsRNAs, and other sncRNAs to sperm that are transported from the proximal to distal epididymal region (from the caput to the cauda), resulting in changes in sncRNA patterns in mature sperm, with a notable enrichment of tRNA fragments (tRFs) ^[42][47] and other miRNAs ^[44][49] and a global loss of piRNAs.

Functional studies of epididymal sncRNAs have revealed a relevant role in epididymal region-specific gene regulation and epithelium–sperm interactions. Analysis of epididymosomes revealed that their cargo contains most small RNA species, including miRNAs, tsRNAs, and RNAs derived from snRNAs, snoRNAs, and rRNAs ^[45][50]. Some semen EVs contained miRNAs such as the miR-888 family (located in epididymis-enriched cluster of chromosome X), which have been described as being specifically enriched in the distal region of epididymis ^[34][35][46][39,40,51].

Expression profiles of miRNAs obtained from malignant and benign prostate tissues differ significantly ^[47][48][49][52,53,54]. In recent years, PCa-related miRNAs have been identified and proposed as a tool for early and specific detection of the disease. Recent studies have revealed that abnormal expression of a large number of tsRNAs also occurs in PCa ^[50][51][55,56], contributing to DNA synthesis, cell viability, and cell proliferation.

Approximately 40% of semen is derived from prostatic tissue. At ejaculation, cauda epididymal spermatozoa are mixed with secretions from the reproductive tract accessory glands such as the prostate. The use of semen, rather than other fluids such as urine after prostate massage, is preferable as a non-invasive source of information of prostate health because semen represents a liquid biopsy from the whole prostatic gland whereas a sample is only obtained from the posterior part of the gland when urine is used ^[52][57].

4. Technical Issues to Consider for a Proper Interpretation of Results on Semen sEVs

4.1. Impact of Surgical Procedures for Contraception

The number of couples selecting male vasectomy as a contraceptive method has been increasing in recent years. The practice of vasectomy affects the concentration of certain semen sEV sncRNAs because the fluid from the testis and epididymis cannot reach semen, so whether the subject has undergone vasectomy should be taken into account for the accurate interpretation of results on semen for biomarker discovery/validation in urogenital diseases ^[53][69].

4.2. Impact of Extracellular Vesicle Isolation Methods and Preclinical Variables on Downstream sncRNA Analysis in Semen

Several exosome-EV isolation technologies have been optimized for their use in semen, and their EV purifying effectiveness (EV quantity, size, and transmembrane protein composition, as well as the quality of RNA they contain) and the impact on the down-stream analysis of miRNAs, compared against the standard use of ultracentrifugation, have been further evaluated ^[54][70]. A study provides evidence that the exosome-EV isolation method has a great impact on the analysis of the miRNAs they contain; it will be important to take this into consideration when implementing the use of semen exosomal/EV miRNAs as a diagnostic tool in the clinical laboratory. The observed over-representation of certain miRNAs from vesicles obtained by a particular isolation method suggests that some miRNAs are likely to be associated with specific vesicle sets enriched by this particular method of isolation. Therefore, the method used for EV isolation can produce variations in EV concentration as well as determine the composition of sEV subpopulations (such as microvesicles and exosomes) in nanovesicle preparation, introducing a bias for subsequent miRNA analysis. Accordingly, until clear markers for delineation between microvesicles and exosomes are established, the results of the analysis of miRNAs contained in EVs cannot be directly extrapolated between different EV isolation methods for clinical application due to the possibility of obtaining misleading results and conclusions.

4.3. Impact of Profiling Methods for Biomarker Testing

Due to the small size of sncRNAs and their low abundance in human fluids, sncRNA expression profiling is technically challenging. Since the discovery of sncRNA, many platforms have been developed for their expression quantification, specially tested for miRNAs, such as small RNA sequencing, microarray hybridization, and reverse transcription-quantitative PCR (RT-qPCR). Several studies in human biofluids, such as serum and plasma, have assessed platform performance in terms of reproductivity, sensitivity, accuracy, and specificity, and indicated that each method has its strengths and weaknesses, which can impact on differential expression analysis ^[55][56][71,72]. Other factors such as normalization and data imputation methods are of relevance to be considered, and will help for a standardized quantification of semen sEV sncRNAs for their clinical use as biomarkers for urogenital diseases.