Monitoring and understanding the composition of biofluids from the reproductive tract is essential for advancing not only human reproductive health, but also practices in agriculture and conservation. For instance, biomarkers could be used to fine-tune windows for assistive reproductive technologies (ART) in humans and animals, thereby improving ART success rates. The same could be said for the identification of biofluid components that benefit oocytes, embryos or spermatozoa, especially if such components are suitable additives for ART. ART is important not just for human fertility and in agriculture, but also for conservation where suitable partners may not be able to physically breed due to separation by space or even time. Additionally, these biofluids could be key to disease diagnosis and in understanding the pathogenesis of reproductive tract diseases.
One of the most versatile and complex components of biofluids are extracellular vesicles (EVs). EVs are nanoscale, lipid-bilayer encapsulated vesicles containing a variety of proteins, nucleic acids and small molecules from the cell in which they originated. This makes them attractive biomarker candidates and effective mediators of cell-cell communication between the cell of origin and target cells into which EVs are internalised. In this article, we will discuss considerations for isolating EVs and highlighting research for EVs from the follicular fluid, oviduct (fallopian tube) fluid, uterine fluid and vaginal fluid.
The composition of reproductive tract fluids
If your goal is to isolate EVs, it is useful to be aware of general differences between fluids of the reproductive tract and plasma. As plasma is the most common biofluid for EV isolation, differences in fluid composition may necessitate workflow changes. Unfortunately, detailed biochemical composition is not available for most biofluids discussed here, limiting comparisons to total protein, glucose and sodium (Table 1).
As a key potential contaminant of EV isolates, total protein is an important component to consider. Whilst oviduct, uterine and vaginal fluids all have approximately half the total protein concentration of plasma, EV concentration and the EV-to-protein ratio have not been fully characterised in these biofluids.
Considerations for isolating EVs from reproductive tract fluids
To date, isolation methods for reproductive tract fluids have not been studied extensively; there is currently insufficient data to recommend isolation methods for vaginal fluid. However, one 2021 study compared EV isolation methodologies for uterine fluid9 and determined that size exclusion chromatography (SEC) using qEV columns (qEVoriginal) performed the best out of the options considered, with qEV isolates giving clean electron micrographs, low protein contamination and good enrichment for EV markers CD9, TSG101 and flotillin-1.9 Another study of follicular and oviductal fluid EVs compared qEV columns with density gradient ultracentrifugation (DG-UC) and found that despite no difference in EV size or concentration, there was improved quality of blastocysts cultured in DG-UC-isolates than with qEV isolates.10 However, this study did not determine whether EVs or co-isolated proteins were causing this difference, precluding this readout as a measure of isolation success. This is particularly important as size exclusion chromatography is known to isolate EVs in a functionally active state, making it unlikely that differences in blastocyst quality are due to differences in EV functionality and more likely that a specific subpopulation of EVs or a non-EV contaminant isolated by DG-UC was responsible for this difference.11 Situations such as this are the reason for MISEV guidelines which recommend the testing of EV-depleted fractions for functionality to ensure that any activity seen with EV-enriched fractions is due to EVs and not other components.12 In order to truly gauge the most effective EV isolation method, it is recommended that you use EV size, purity (i.e., low protein contamination) and morphology as measures of isolation success.
Functional biology and biomarker potential of reproductive tract EVs
Follicular fluid is the fluid contained in ovarian follicles within which oocytes mature. The contents of EVs isolated from follicular fluid has been shown to change during follicle development from a small follicle containing an immature oocyte, to a large follicle containing a mature oocyte (See Figure 1).13-15 This is tied to an increased ability to promote granulosa cell (hormone-producing cells surrounding the oocyte) proliferation in EVs from small follicles, perhaps reflecting their role in follicle development.14
From a medical and agricultural viewpoint, follicular EVs may be of use in ART, as EVs isolated from follicular fluid improve meiotic resumption of oocytes which have been thawed following cryopreservation.16 This could also be of particular importance in conservation, where oocytes may have to be cryopreserved for long periods until suitable sperm is identified for fertilisation.
Oviductal fluid is the fluid of the fallopian tube through which the oocyte travels to meet with spermatozoa and where fertilisation and early embryonic development take place. Across the estrous cycle, EV contents change – as has been reported for RNA, protein and small molecules.17,18 Part of this change is due to increased EV energy substrates and enzymes involved in energy production, which could be delivered to embryos to aid in their growth.17 For ART – including ART for agriculture, conservation and human fertility – oviductal fluid EVs may be of particular importance. Oviductal fluid EVs improve in vitro embryo quality and development19-22, and can even improve birth rates in mice.20 For spermatozoa, which traverse the oviduct to reach the oocyte, oviductal EVs have been shown to improve capacitation (i.e., a stage of spermatozoan maturation required for fertilisation), viability and mobility.23-26 This suggests that oviductal fluid EVs may be of particular use in ART where physiological conditions are not present and need to be simulated.
Interest in uterine fluid EVs mostly concerns the steps of embryo maturation leading up to and including implantation. This phase concerns bidirectional communication between the embryo and the endometrium (i.e., uterine lining). EVs isolated from uterine fluid may be involved in the recognition of pregnancy, with embryo-derived CAPG (a recognition of pregnancy signal27) being present in uterine fluid EVs on days 15-17 of pregnancy.28 In vitro, uterine EVs have been shown to increase blastocyst formation and hatching (i.e., a required step for implantation).29 In contrast, uterine fluid EVs from cows with endometritis inhibited blastocyst development.30 Other studies have shown that uterine fluid EVs may aid in embryo implantation, perhaps via mechanisms involving interferon tau.31-34 Collectively, these studies suggest that uterine fluid EVs are important for bidirectional communication during late embryo development and implantation, suggesting possible uses in ART.
Vaginal fluid EVs are recovered from either one or both of the liquid and mucus components following vaginal flushing. Interestingly, these EVs may be helpful in the search for diagnostic tests for endometriosis, a disease which, on average, takes seven years to diagnose.35 An early study in a macaque with spontaneously presenting endometriosis identified markedly reduced EV concentrations in vaginal fluid.36 Though this is not yet sufficient for a diagnostic test, it does provide hope that vaginal EVs could be altered in endometriosis, thus providing an accessible diagnostic test.
Vaginal EVs could also be of use in cervical cancer diagnosis as two miRNAs – miR-21 and miR-146a – were found to be increased in vaginal fluid EVs of cervical cancer patients.37 Changes to vaginal fluid EVs are also seen in viral infection, with EV miRNA in particular being altered in response to the causative infection for cervical cancer, Human Papilloma Virus (HPV), and a relative of HIV which primarily affects non-human primates, Simian Immunodeficiency Virus (SIV).38,39 In SIV and its human equivalent HIV, vaginal EVs were also found to have antiviral effects, reducing viral replication.39,40 This may have implications for the development of preventative treatments for sexually acquired HIV infections, and requires further study.
Key questions for reproductive tract fluid EV research
Several key questions remain to be addressed to help develop the field of reproductive tract EVs.
1. What is the biochemical composition of reproductive tract fluid?
Characterisation of these fluids still falls well short of characterisation of most biofluids. This information will be useful in the optimisation of EV isolation procedures, particularly when scaling for diagnostics or therapeutics. This is of particular importance for vaginal fluid where viscosity may impact on EV recovery.
2. How do reproductive tract EVs compare between anatomical locations?
Are potentially diagnostic changes in uterine EVs reflected in vaginal EVs, for example? If so, this could reduce the invasiveness of diagnostic tests.
3. Are EV-based tests for uterine receptivity and pregnancy recognition practical for agriculture?
To allow rapid diagnostics for uterine receptivity and pregnancy recognition, tests would most likely need to be performed at the same location. This would require a user friendly, standardised, commercial EV isolation and downstream diagnostic test which would minimise user error. A system like the qEV Isolation platform, which includes optimised columns paired with the automation of the Automatic Fraction Collector (AFC), would be ideal for such an application.
4. Can localised EVs perform above and beyond blood tests for diseases of the reproductive tract?
EVs from reproductive tract fluids have shown promise as potential sources of biomarkers for disease, but it is as yet unknown whether these could outperform potential circulating biomarkers for the same diseases.
Research in this field shows just how valuable reproductive tract fluid EVs may be in a variety of fields, from medicine to farming and conservation. This makes research into this area not only possibly revolutionary to human health, but also potentially highly valuable for worldwide agriculture.
