The exosome: a review of current therapeutic roles and capabilities in human reproduction.

Important aspects to consider when discussing exosome cargo loading are the therapeutic viability of loaded exosomes and the possibility of cargo targeted delivery to the intended site of action, thereby avoiding off-target effects and reducing overall side effects. A method to examine the viability of loaded exosomes may be to test the effect of which exosomes loaded with therapeutic cargo would have on cells versus only the cargo or exosomes [ 142 ]. One study utilised drug-resistant A2780/DDP cells to test the effectiveness of using cisplatin-loaded exosomes against the medication by itself to determine if therapeutic viability had been increased [ 142 ]. The study found that by using the cisplatin-loaded exosomes, the cytotoxicity of cisplatin was increased by a factor of 3.3 in the drug-resistant cells and a factor of 1.4 in the drug-sensitive cells versus the medication by itself [ 142 ]. Another study tested therapeutic viability through loading blood-derived exosomes with dopamine for the treatment of PD [ 143 ]. It was found that there was a > 15-fold increase in the bioavailability of dopamine for distribution in the brain through utilising exosomes as delivery platforms [ 143 ]. As previous research has shown, delivering loaded exosomes can prove to be efficient in maintaining drug therapy, potentially avoiding factors such as first-pass metabolism, limited bioavailability, and moving through barriers such as the BBB [ 8 , 142 , 143 ]. Considering the clinical importance of exosome cargo loading, it becomes necessary to question the viability of targeted delivery to the intended site of action. Currently, some strategies for manufacturing targeted exosomes includes isolating unmodified exosomes from the specific tissue of interest and taking advantage of their intrinsic alignment to the organ or tissue in question [ 144 ]. Other methods may involve modifications of the exosomal surface such as the removal or addition of certain adhesion proteins such as integrins [ 145 ]. One study investigated the effect of overexpressing an exosomal membrane protein (Lamp2b) to increase the targeting rate towards integrin αvβ3-positive anaplastic thyroid carcinoma cells for enhanced doxorubicin delivery [ 145 ]. The study found that the overexpression of Lamp2b increased the cargo delivery to the target cells and resulted in a significant reduction in tumour size in 8505C xenograft mouse models, with minimal side effects [ 145 ]. Relatedly, another study explored targeted delivery for anti-inflammatory effects after cerebral ischemia [ 146 ]. The authors isolated exosomes from a human neural progenitor cell line (ReN cells) and attached specific targeting ligands to the exosomal surface by designing a recombinant fusion protein [ 146 ]. The result was the significant inhibition of site-specific inflammation in mouse models and was shown to induce minimal off-target effects [ 146 ]. Thus, while exosomal loading is important, targeted delivery is also crucial for optimising therapeutic outcomes by reducing off-target side effects and increasing bioavailability at the intended site of action.

Exosomes contain cellular cargo relating to the physiological state of the donor cell–they may be ideal as therapeutic tools for diagnosing and treating complications during reproduction in both males and females [ 58 ]. Recent studies have found that the concentration of exosomes in peripheral blood increases over time until the final stages of pregnancy and is further increased before and during relevant pathological complications [ 58 , 59 ]. Considering this, exosomes may be utilised for the early detection of many pregnancy-related complications, and therefore may assist providing early interventions through a timely and directed approach. An example from recent literature included the use of circulating exosomal miRNA species for the early diagnosis of foetal ventricular septal defects (VSDs) [ 60 ]. The study explained that certain exosomal miRNA species (hsa-miR-186-5p, hsa-miR-199a-3p, hsa-miR-146a-5p, hsa-miR-181a-5p, and hsa-miR-3158-3p) were dysregulated in VSD cases from early in pregnancy, signifying that these miRNAs may act as potential biomarkers [ 60 ]. Furthermore, it has been reported that placental-derived exosomes can suppress certain immune responses through increasing lymphocyte apoptosis and reducing CD3 expression [ 61 ]. It has also been found that exosomes can regulate the NKG2D receptor on NK, CD8(+), and gamma delta T cells, which has resulted in a reduction of cytotoxicity in vitro [ 61 ]. The clinical applicability of exosomes can therefore relate to their utilisation to treat a multitude of disorders during pregnancy, including impaired foetal growth and inflammation (refer to Tables 2 and 3 for a summary). Table 2 Exosomes as therapeutic tools in various reproductive pathology Reproductive pathology Exosomal sources Methods of isolation Potential biomarker molecule Potential therapeutic outcomes References PCOS Serum plasma, follicular fluid, adipose tissue Ultracentrifugation, differential centrifugation, ultrafiltration, precipitation miR-373, miR-640, miR-654-5p, hsa-miR-1299, has-miR-6818, miR-145-5p, miR-192, miR-590-3p, miR-27a-5p, DENND1A.V2, CYP11A, -19A, HSD17b1 Possible early detection of PCOS, silencing defective proteins [ 62 , 66 – 71 ] Reproductive inflammation Serum plasma, placental fluid, amniotic fluid Differential centrifugation, ultrafiltration, SEC hsa-miR-126-3p, hsa-miR-23a-3p, COX-2, GMCSF, IL-6, IL-8 Assist in early detection of inflammation, may help in detecting exact area and cause [ 34 , 72 – 74 ] Endometriosis Serum plasma, follicular fluid, endometrial stromal cells Ultracentrifugation, differential centrifugation miR-134-5p, miR-197-5p, miR-22-3p, miR-320a, miR-494-3p, miR-939-5p, PRDX1, H2A type 2-C, ANXA2, ITIH4, Tα Easier and faster diagnostics, leading to quicker therapeutics [ 75 – 78 ] POF Bone marrow–derived mesenchymal stem cells, granulosa cells Ultracentrifugation, differential centrifugation miR-144-5p, miR-127-5p, YY2, CP, CC3, fibrinogen, SHBG Easier and faster diagnostics, avoid long-term ovarian damage [ 62 , 66 – 68 ] GDM Serum plasma, urine, blood from the umbilical vein Ultracentrifugation, silicon carbide ultrafiltration miR-516-5p, miR-517-3p, miR-518-5p, miR-222-3p, miR-16-5p, miR-125b, miR-144, S100A9, DAMP Quicker therapeutics, may assist in avoiding long-term complications and early-onset type 2 diabetes mellitus, protect the growing foetus from complications [ 69 – 72 ] Pre-eclampsia Maternal circulation blood samples, HEK293T cell line Ultracentrifugation, differential centrifugation, SEC miR-153, miR325-3p, miR122-5p, miR2605-3p, miR2113, miR-374c-5p, miR505-3p, versican, biglycan, PZP Pre-emptive diagnostics, protect the mother and foetus from ongoing complications [ 34 , 73 – 76 ] Table 3 The different reproductive disorders in relation to exosomal isolation, pathogenesis, and limitations Reproductive disorder Exosomal isolation method Mechanism of pathogenesis Limitations of related research References Polycystic ovarian syndrome Serum filtration through 0.22-μM filters, membrane-based affinity binding, ultracentrifugation steps. Small RNA sequences in follicular fluid, altered miRNA expression, driver genes. Low patient study number. [ 62 , 66 , 67 , 76 , 78 ] Reproductive inflammation Ultracentrifugation, differential centrifugation. Parturition through foetal endocrine signalling–immune driven, preterm premature membrane rupture, inflammatory cytokines. Unknown paracrine mediators. [ 34 , 72 , 73 ] Endometriosis Ultracentrifugation, differential centrifugation. Inappropriate miRNA-related proliferation, growth, differentiation, and apoptosis. Limited patient samples and quantification of miRNA species, including validation. [ 75 , 76 ] Premature ovarian failure Ultracentrifugation, differential centrifugation. Enzyme autoimmunity, gene-driven miRNA overexpression, drug-induced, previous infections. miRNA characterisation extent, animal models may be inaccurate when transferred to human disease specifications. [ 79 – 82 , 84 ] Gestational diabetes mellitus Differential centrifugation, spin column chromatography. Genetic gene mutation, β-cell dysfunction, miRNA up/downregulation, gene dysregulation causing miRNA species imbalances. Not suitable for larger molecules, damage to the exosomal wall is often irreversible, may disrupt the cargo. [ 85 – 88 ] Pre-eclampsia Differential centrifugation. Hypoxia-inducible factor 1-alpha, miRNA up/downregulation. Rigour in assay utilisation, limited patient numbers, detection, and quantification of exosomal miRNA. [ 74 , 90 – 94 ] Exosomes as therapeutic tools in various reproductive pathology The different reproductive disorders in relation to exosomal isolation, pathogenesis, and limitations The reproductive cycle is a complicated mix of highly regulated processes, meaning that problems may occur relating to immunological function, cellular signalling, and nutrition malabsorption–thus, irregular placental and foetal development may ensue [ 62 ]. As previously mentioned, exosomes can mediate cell-to-cell communication and carry different cargo based upon the physiological state of the donor cell; this means that specific cargos such as miRNA and proteins may be useful in understanding the pathology behind reproductive disorders [ 38 , 39 , 58 ]. Exosomal function in obstetric pathology can be extended to syndromes such as PCOS, acute or chronic inflammation, endometriosis, POF, GDM, and pre-eclampsia [ 52 ]. Furthermore, the relevant pathology also becomes important when discussing male fertility/infertility as to how exosomes contribute to the progression of various functions such as sperm production and maturation, and therefore assist in healthy reproduction, as discussed below [ 63 – 65 ]. Although this review is mainly focussed on reproductive pathology within females, it is important to highlight certain aspects surrounding exosomes in the male reproductive system. Exosomes play a considerable role in the male reproductive system, more specifically in relation to fertility and infertility, being highly involved in sperm maturation, acrosome reactions, capacitation, and fertilisation [ 63 ]. These exosomes have been found to originate from within the prostate (prostasomes), testis, seminal fluid, and epididymosomes [ 63 ]. Due to their supportive role in various seminal functions, they have been associated with the progression of normal reproductive cycles and are implicated within the transportation of various regulatory proteins and nucleotides [ 63 ]. Recently, a study focussed on the exosomal profiles of azoospermia patient semen samples and found that several types of RNA (miRNA, piRNA Y RNA, rRNA, and tRNA) were present in the related exosomes, with miRNA displaying the most differential profile compared to other bodily fluids [ 63 ]. Considering, the study found that many miRNA levels were dysregulated in seminal plasma exosomes, including species which were germ-cell specific [ 63 ]. Importantly, a high predictive accuracy was found in certain RNA species (miR-205-5p, miR-31-5p, and germ cell piR-58527) with a diagnostic efficiency of AUC > 0.95 specifically for miR-31-5p and a related high level of sensitivity and specificity [ 63 ]. While more research needs to be established, miRNA-based exosomal profiles may assist in building an efficacious diagnostic model for azoospermia and related possible causes. The possibility of diagnostic models has been found in exosome-associated proteins, which are important for the maturation of spermatozoa and can thus also be implicated as potential biomarkers for male infertility [ 64 ]. One study described that exosomal proteins annexin A2 (ANXA2), semenogelin-1 (SEMG1), transferrin (TF), and kinesin-1 heavy chain (KIF5B) were dysregulated in male patients with varicocele–a condition characterised by the formation of varicose veins within the scrotum which can often lead to infertility [ 64 ]. As these proteins are variable in their expression, it may be possible to utilise them as biomarkers to detect early signs of infertility in males [ 64 ]. Another similar study compared seminal exosomal protein levels in fertile versus infertile men and distinguished that within the infertility group, many proteins were differentially expressed, including the upregulation of ANXA2 and the downregulation of KIF5B [ 65 ]. The same study found that 47 seminal plasma proteins were dysregulated in unilateral varicocele patients in comparison to controls–it was further deducted that dysregulated proteins were correlated to androgen receptors YB1 and NRF2 [ 65 ]. Overall, exosome-associated proteins may be useful in the early diagnostics of male infertility and may also point towards the relevant pathology involved within the progression of reproductive disorders [ 64 , 65 ]. In relation to female reproduction, many of the pathways involved in the pathological progression of reproductive disorders are connected to exosomes and their cargo, such as miRNA species and proteins (refer to Table 2 ). While there has been relevant research in this field, many studies have had various limitations relating to smaller sample sizes, and narrow or inconclusive results, with much research not being replicable to an acceptable standard. Further, research gaps can be identified relating to various exosomal biomarkers and how these may be used in a clinical setting, such as for diagnostic or interventional purposes. Hence, it becomes important to further explore as to how exosomes may be of viable clinical applicability when discussing reproductive pathology [ 38 , 39 ]. PCOS is a common reproductive endocrine disorder affecting approximately 8–10% of women of child-bearing age in which pathological changes occur in relation to abnormal follicular granulosa cell proliferation, abnormal apoptosis, and hyperandrogenism [ 62 , 66 ]. PCOS can be phenotypically characterised through affected individuals experiencing symptoms relating to hair loss, hair overgrowth, obesity, amenorrhea, and menstrual irregularities [ 66 ]. As exosomes are involved in follicular development through cellular signalling and communication, specific exosomal biomarkers may indicate towards the progression of PCOS, which ultimately may lead to an early intervention. Recent studies have found that cargo from follicle-derived exosomes includes various PCOS-specific miRNAs such as miR-373, miR-640, and miR-654-5p, with newer biomarkers being hsa-miR-1299, hsa-miR-6818-5p hsa-miR-192-5p, and hsa-miR-145-5p [ 66 , 67 ]. These specific miRNA biomarkers have been found to be overexpressed in PCOS patients and may possibly be used as diagnostic tools [ 67 ]. Furthermore, another recent study found that serum exosomes from PCOS patients expressed highly elevated levels of miR-590-3p and miR-27a-5p [ 62 ]. Functionally, miR-27a-5p in PCOS-derived exosomes was found to promote proliferation and migration in endometrial cancers, which may be linked with the progression of PCOS through the driver gene SMAD4 [ 62 ]. This pathway was found to occur through the targeting of the SMAD4 gene by miR-27a-5p, in which the gene is upregulated, and migration and proliferation are promoted [ 62 ]. Conversely, one study found that exosomal miR-323-3p derived from mesenchymal stem cells both promoted proliferation and inhibited apoptosis in PCOS, which resulted in the condition somewhat improving [ 68 ]. Additionally, another recent study explained that PCOS follicular fluid–derived exosomes carrying miR-424-5p upregulated granulosa cell senescence through the direct targeting of the CDCA4 gene, which resulted in downregulated cell proliferation [ 69 ]. Research has also found that the DENND1A.V2 protein was higher in PCOS theca cells compared to that in controls, which may have an indirect downstream effect on insulin and luteinising hormone through the RAB5B system [ 70 ]. Similarly, one study indicated that significantly higher levels of mRNA expression relating to the proteins CYP11A, CYP19A, and HSD17b1 were found in follicular fluid of PCOS patients compared to study controls [ 71 ]. The dysregulation of such constituents can also point to the strategy of loading these particles into exosomes to act as therapeutic cargo for targeted delivery, where specific types of molecules such as miRNA or proteins could mediate dysregulated pathways. Thus, the miRNA exosomal markers may have the potential to provide a basis for the early diagnosis of PCOS and understanding precipitating factors which may also promote the development of endometrial cancer from PCOS [ 62 , 66 – 71 ]. Exosomes carry inflammatory mediators which differ among cellular pathways depending on their site of origin and can signify the unique properties and disease state of their original environment [ 72 ]. One study explains that exosomal inflammatory mediators were observed in mice in gestation days E5 to E19 and that increased inflammatory markers can be responsible for causing early inflammatory activation in maternal gestational cells [ 72 ]. Important inflammatory markers in reproduction are cyclo-oxyenase-2 (COX-2), granulocyte–macrophage colony-stimulating factor (GMCSF), interleukin 6 (IL-6), and interleukin-8 (IL-8) [ 73 ]. These markers have the potential to provide information on the state of the host cell and how some inflammatory processes may progress within the reproductive cycle [ 73 ]. Research has found that the pro-inflammatory cytokines GMCSF, IL-6, and IL-8 were abundant in exosomes which were exposed to increased levels of oxidative stress (OS), and in turn contributed to a state of inflammation in various tissues [ 73 ]. Additionally, foetal-derived exosomes were found to exhibit differential characteristics and were distinct in promoting an inflammatory state in uterine cells, which was associated with the initial development of parturition [ 73 ]. Exosomes may have modulatory roles in various inflammatory pathways, and potentially be involved in the up- and downregulation of pro-inflammatory cytokines. Adding to this, a recent study explained that exosomes derived from amniotic fluid can project the current inflammatory status of the uterine environment through their specific miRNA and protein contents [ 74 ]. Furthermore, miRNA biomarkers are of particular interest in distinguishing maternal systemic inflammation [ 34 ], for example, the upregulation of hsa-miR-126-3p and hsa-miR-23a-3p, which are both involved in pathways relating to vascular cell adhesion molecule 1 (VCAM1) inhibition, limiting leukocyte cell adhesion, and targeting ATG12-mediated autophagy [ 34 ]. Such exosomal biomarkers could possibly indicate the development of abnormal pregnancies and provide specific information about uterine health, including any imminent risks and predisposing factors contributing to a potential miscarriage [ 74 ]. Endometriosis can be defined as the presence of endometrial tissue around the outside of the uterine cavity and affects approximately 10–15% of women of reproductive age [ 75 ]. The main symptoms are presented as pelvic pain, infertility, heavy bleeding, and ovulatory pain [ 75 ]. Exosome-derived miRNA species may be considered potential diagnostic biomarkers for various reproductive disorders, including endometriosis. Research has found that exosomes derived from follicular fluid have shown differences in the contents of their cargo between control and PCOS patients, specifically relating to miRNA species [ 76 ]. It was explained that the differences in exosomal cargo could alter processes relating to the development and progression of endometriosis, indicating that exosomal cargo may be involved in regulatory pathways [ 76 ]. Relatedly, a recent study explored the use of endogenous exosomal miRNA for the early diagnosis of endometriosis through attempting to identify multiple dysregulated miRNAs in serum exosomes derived from patients [ 75 ]. Some notable targets were namely miR-134-5p, miR-197-5p, miR-22-3p, miR-320a, miR-494-3p, and miR-939-5p–the main miRNA biomarkers which were highly upregulated in endometriosis patients were found to be miR-22-3p and miR-320a [ 75 ]. These miRNA targets were found to have a high specificity towards the progression of endometriosis and could potentially increase the diagnostic sensitivity relating to screening for and treating endometriosis early and effectively [ 75 ]. Similarly, another recent study found various exosomal biomarkers relating to the progression of endometriosis [ 77 ]. The notable biomarkers circular RNA_0026129, miRNA-15a-5p, and the genomic marker ATP6V1A were highly related to the endometriosis-associated exosomal competing endogenous RNA network [ 77 ]. Overall, the study found these markers to be differentially expressed between endometriosis patients and related healthy controls, indicating reliability in sequencing and their possible use as specific biomarkers for diagnosis or targeted treatment [ 63 ]. Hence, these may indicate the different stages and processes in the progression and development of endometriosis during pregnancy. Relatedly, another recent study described dysregulated proteins in endometriosis patients compared to healthy control patients, of which includes PRDX1, H2A type 2-C, ANXA2, ITIH4, and the tubulin α-chain (Tα) [ 78 ]. These proteins may have endometriosis-specific roles and in which case would assist with early detection of related pathology, although further research would be required to confirm uniqueness [ 78 ]. Premature ovarian failure (POF) is a disease of women’s reproductive health which results in the premature cessation of ovarian function before the age of 40, and its prevalence is largely genetically linked [ 79 ]. Individuals will typically present with symptoms of reduced oestrogen levels, amenorrhea, infertility, reduced mature follicles, and high gonadotropin levels [ 79 ]. POF accounts for approximately 1% of infertility occurrences in females, and as fertility is key to reproduction, it becomes pertinent to screen for POF as early as possible [ 80 ]. Overall, recent literature has stated that the follicle-derived exosomal miRNA component miR-144-5p has been used to identify and treat chemotherapy-induced ovarian failure in animal models, indicating promise towards a potential POF biomarker [ 81 ]. Furthermore, another recent study found that the transcription factor Yin Yang 2 (YY2) is significantly reduced in patients with POF [ 82 ]. The study focused on exosomes derived from peripheral blood from patients with POF and denoted a positive correlation between progesterone/oestradiol levels and YY2, as these levels are usually diminished in patients with POF [ 82 ]. Hence, YY2 was found to be related with fluctuating hormonal levels during disease progression in POF, making it a possible target for early diagnostics and therapeutics [ 82 ]. Additionally, another recent study found that exosomal miR-127-5p, a miRNA involved in downregulation pathway of DNA repair mechanisms, was overexpressed in patients with POF [ 83 ]. Furthermore, a similar study noted the presence of multiple up/downregulated proteins in patients with POF [ 84 ]. These included proteins involved in the reproductive process such as ceruloplasmin (CP), complement C3 (CC3), fibrinogen, and sex hormone binding globulin (SHBG) [ 84 ]. These protein biomarkers were described to be increased twofold within POF patients compared to the control group [ 84 ]. Their relevance can be linked to specific functions, such as CP being important for transporting copper throughout the body, especially during pregnancy [ 84 ]. Relatedly, CC3 is involved in the complement system, which is a part of the regulation cycle of immune system and phagocytosis [ 84 ]. Finally, fibrinogen plays a pivotal role in clotting factors to stop bleeding and SHBG is attached to androgens and estrogens and is usually increased in postmenopausal women [ 84 ]. Hence, dysregulation of these proteins may help in understanding a variety of disease factors before, during, and after POF. Gestational diabetes mellitus (GDM) affects roughly 14% of worldwide pregnancies and is a complication in which patients without a previous history of diabetes begin to develop clinically significant and chronic levels of hyperglycaemia during gestation [ 85 ]. The development of GDM is linked to the impairment of glucose tolerance in relation to pancreatic β-cell dysfunction and is usually more common in women with pre-existing risk factors [ 85 ]. Such can include being overweight, previous family history of diabetes, and an advanced maternal age [ 85 ]. Exosomes may improve both early diagnostics assist in tailoring therapy for the management of GDM, which in turn may act as a type of prevention and treatment for both the foetus and the mother. A recent study explored potential exosomal miRNA biomarkers which were downregulated in GDM and during the 3rd trimester of gestation (miR‑516‑5p, miR‑517‑3p, miR‑518‑5p, miR‑222‑3p, and miR‑16‑5p) [ 86 ]. The downregulated exosomal miRNAs were linked to various metabolic pathways associated with the development and progression of GDM. This indicates that exosomes and their cargo may be pivotal in cellular pathways relating to inflammation, energy production, and insulin mobilisation [ 86 ]. Furthermore, the cellular mechanisms of the related miRNA correspond to pathways in stress responses and variations in circulating blood glucose levels [ 86 ]. Another recent study found that circulating exosome release was higher in GDM patients compared to non-GDM patients, and that exosomal miRNA may affect pathways relating to lipid metabolism, glucagon signalling, and glucose homeostasis [ 87 ]. The research suggests that exosomes which express specific contents could modulate various metabolic pathways and alter processes in which normal metabolism may be dysregulated through the stages of pregnancy. Specific miRNA species could possibly be used as both biomarkers and therapeutic targets and further indicate as to which metabolic pathways may be dysregulated before and during GDM [ 87 ]. Another study which focused on exploring different potential exosomal miRNA biomarkers found that miR-125b was consistently downregulated in GDM while miR-144 was found to be consistently upregulated [ 88 ]. The authors further performed AUC models for both miR-125b and miR-144 and obtained results of 0.898 and 0.875 respectively, indicating favourable diagnostics [ 88 ]. These findings detail the dysregulation of miR-125b and miR-144 within GDM and outline their potential use as diagnostic tools. Additionally, another research study explained the presence of protein biomarkers through their dysregulation within GDM patient samples, specifically S100 calcium binding protein A9 (S100A9) and damage associated molecular patterns, which are involved in cell cycle progression/differentiation and the innate immune response system, respectively [ 89 ]. The study found that a more specific increase in S100A9 protein numbers correlated to maternal obesity in GDM patients and increased the chances of macrosomia in newborns [ 89 ]. Hence, a mixture of such miRNA and protein biomarkers may be more efficient in determining treatment options in GDP patients and providing better long-term patient outcomes [ 89 ]. Pre-eclampsia can be a common and serious complication during pregnancy, in which 5–8% of pregnancies are affected and the exact pathological cause remains an unknown area, with higher incidences in at-risk populations, such as smoking, obesity, and a family history of hypertension [ 90 ]. The disorder is characterised by symptoms such as hypertension and multiple organ injury stemming from placental malperfusion in which various disease-promoting factors are released into maternal circulation [ 90 ]. As no medication has been shown to completely alleviate the progression of the condition, early diagnostics may help with the initial management and timing during the pregnancy to optimise foetal and maternal outcomes. Considering, exosomes may help in understanding the pathologies involved in pre-eclampsia and how this condition may develop, including a multitude of other reproductive disorders. A recent study showed that isolated exosomes from patients with pre-eclampsia contain several miRNA makers specific to the progression of pre-eclampsia [ 91 ]. The identified exosomal miRNA species included miR-153 and miR-325-3p, which were both significantly upregulated in pre-eclampsia [ 91 ]. Literature has found both miR-153 and miR-325-3p to be associated with reduced tube formation in primary human umbilical vein endothelial cells and endothelial cell dysfunction, respectively [ 92 , 93 ]. Considering the high amount of upregulation of these miRNA species, they may be utilised as potential biomarkers and provide more information regarding pre-eclampsia during the different stages of pregnancy [ 91 ]. Similarly, another study explored related miRNA exosomal biomarkers which could describe the stages of pre-eclampsia relating to late-onset and early-onset, such examples included miR-122-5p, miR-3605-3p, miR-2113, miR-374c-5p, and miR-505-3p, which confirms that a multitude of miRNA species may be involved in the pathogenesis of pre-eclampsia (see Fig. 5 ) [ 74 ]. The relevance of miRNA species is related to the variation between patients with and without pre-eclampsia–however, further research is required to ascertain exact pathogenesis [ 74 ]. A recent study looking at therapeutic targets found that miRNA (miR-18b-3p) obtained from human umbilical cord mesenchymal stem cell–derived exosomes inhibits the development of pre-eclampsia through targeting the leptin protein [ 94 ]. The study found that inhibition of leptin through miR-18b-3p lowered systolic blood pressure and proteinuria in pre-eclampsia rat models. With the result of leptin inhibition, it would be possible to load this miRNA cargo into exosomes for targeted delivery into patients who have or are at risk of pre-eclampsia and gestation hypertensive issues (refer to Table 3 ). Furthermore, one study found that exosomal PLAP levels was increased in women who developed pre-eclampsia, which could potentially differentiate between exosomal content useful in determining biomarkers [ 95 ]. Another recent study identified increased levels in pre-eclampsia-associated proteins as being the glycocalyx-associated proteins, versican and biglycan [ 96 ]. Both proteins have been implicated in endothelial dysfunction and eventually pre-eclampsia [ 96 ]. The study also found that women with pre-eclampsia displayed reduced levels of the pregnancy zone protein (PZP), which is involved in the inhibition of misfolded protein aggregates [ 96 ]. The reduced PZP levels may be associated with a high incidence of protein aggregates in patients with pre-eclampsia [ 96 ]. However, other barriers exist towards therapeutic progression–such as efficient exosomal cargo loading, targeted delivery, and the translation of exosome therapeutics from the laboratory setting to a clinical setting. These barriers must be overcome so factors such as cargo loading, and delivery may be efficiently and safely implemented. Fig. 5 The exosome within the structure and vessels of the placenta, including the various exosomal markers which may indicate the early diagnosis of different reproductive diseases. This summary includes–polycystic ovarian syndrome (PCOS) [ 62 , 66 – 69 , 76 , 78 ], uterine and reproductive inflammation [ 34 , 73 , 74 ], endometriosis [ 75 , 76 ], premature ovarian failure (POF) [ 79 – 82 , 84 ], gestation diabetes mellitus (GDM) [ 85 – 88 ], and pre-eclampsia [ 74 , 90 – 94 ]. These exosomal markers are possible milestones of the pathological responses which are undergone in these reproductive diseases and indicates that early diagnosis may indeed be a possibility The exosome within the structure and vessels of the placenta, including the various exosomal markers which may indicate the early diagnosis of different reproductive diseases. This summary includes–polycystic ovarian syndrome (PCOS) [ 62 , 66 – 69 , 76 , 78 ], uterine and reproductive inflammation [ 34 , 73 , 74 ], endometriosis [ 75 , 76 ], premature ovarian failure (POF) [ 79 – 82 , 84 ], gestation diabetes mellitus (GDM) [ 85 – 88 ], and pre-eclampsia [ 74 , 90 – 94 ]. These exosomal markers are possible milestones of the pathological responses which are undergone in these reproductive diseases and indicates that early diagnosis may indeed be a possibility

Considering the promising applicability of exosomes in diagnosing and treating reproductive disorders, various challenges remain for the translation from the laboratory to the clinic. Firstly, there are limitation surrounding vesicle isolation and further determining whether the isolated exosomes would be viable for clinical use. This can be due to the inconsistencies related to the isolated particle number, relevant morphology, and the source of isolated exosomes. In example, exosome characterisation may need to be completed using exosomes from both non-malignant and malignant cell lines for acceptable comparison if utilising exosomes for cancer therapy related research [ 147 ]. Further, techniques such as electron microscopy and western blotting may be useful for confirmation of presence and composition, although they provide no quantitative information [ 147 ]. Relatedly, nanoparticle tracking analysis is useful for estimating the number of exosomes present in a sample, although it does not provide information regarding the cell of origin and may also be hindered by limitations in measuring Brownian motion over a set period [ 148 ]. Another prominent challenge is the issue of particle aggregation following certain loading methods such as electroporation and sonication. This may result in less efficient cargo delivery if particles are aggregated together, and it may render some compounds inactive, therefore making them inappropriate for therapeutic delivery. Despite the issue of particle aggregation, both electroporation and sonication have been shown in previous research as highly efficient loading methods [ 94 , 97 , 102 , 122 ]. Due to the rapid changes on the exosomal surface while exosomes are near each other, particles can align together and fuse during the reformation of the exosomal wall. Various efforts to mitigate particle aggregation have not been explicitly addressed in recent literature. However, it has been noted that various incubation protocols following loading could separate membranes alongside utilising less intense voltages and sound waves, depending on the desired cargo [ 77 ]. Another prominent limitation is the variability in exosomal yield and purity related to isolation techniques. While some isolation techniques provide higher yield and purity than others, inconsistencies in methodologies, equipment, and human error prove it difficult to quantify which method is most appropriate to use [ 149 , 150 ]. For example, the ultracentrifugation method is efficient for exosome isolation although often produces mixed results with protein contaminants and modifications to exosome structural integrity [ 149 , 150 ]. Method optimisations would be required to infer the optimal conditions for different types of isolations [ 149 , 150 ]. Alongside this is the inconsistency in cargo loading efficiencies of various methods such as electroporation, sonication, and extrusion, including the methods for measuring cargo loading efficiency. The differences in parameter outputs (electroporation, sonication) and the possible differences in equipment and technique (extrusion), along with inconsistencies in determining loading efficiencies, may be ongoing issues for the reproducibility of reliable results in future literature. However, the type of cargo also plays a role in determining loading efficiencies. For example, the variance in zeta-potential of the cargo and the exosome in response to loading treatment may hinder encapsulation, such as when attempting to load certain drug molecules (PTX) [ 36 ]. Moreover, the zeta-potential can be a determinant of the colloidal stability of exosomes and their actions at target cells, meaning that if significantly altered, detrimental effects may be experienced at target sites [ 36 ]. Relatedly, it has been reported that a positive surface charge (zeta-potential) can change the immunogenicity profile of exosomes, while a negative surface charge increases compatibility and the efficient delivery into target cells [ 130 ]. To address this potential issue, it may become beneficial to tailor parameters and methodologies relative to the specific physicochemical properties of the desired cargo, such including the zeta-potential and other factors relating to size, lipophilicity, and compatibility. Considering, testing various parameters against loading efficiencies has not been actively included in recent literature–which highlights the importance of method optimisations in response to experimental reproducibility.

It is evident that exosomes are important in various physiological states, including during the reproductive cycle. The potential of exosomal research pertaining to human reproduction is extensive, although still faces many different questions in which future research must clarify. Such includes the challenge of utilising exosomes as therapeutic tools in many of the complications during pregnancy, both as diagnostic tools and as therapeutic treatment options. The question also remains as to how exosomes can be efficiently loaded and made clinically viable on a consistent basis, thereby allowing mass production for use in various clinical settings. The notion of isolating exosomes, therapeutic cargo loading, and further using exosomal biomarkers as early diagnostic and clinical tools for the detection and possible treatment of reproductive diseases is both exciting and perplexing. However, it is still unknown as to (1) how exosomes may be utilised effectively as diagnostic tools, both from a time-critical, ease-of-access, and financial point of view; (2) how loading exosomes with specific cargo can be clinically viable in terms of using exosomal markers to target sites of disease action; and (3) whether exosomal contents (such as miRNA, proteins, etc.) are different in the various stages of reproduction and if any differences could affect exosomal markers when considering targeted delivery. Furthermore, an enduring challenge in exosome research remains the unanswered questions relating to the reproducibility of research and the optimisation of loading methods to ensure higher loading efficiencies. Following these challenges, the potential towards a solid groundwork for establishing exosomes as fast and efficient diagnostic tools and therapeutic delivery vehicles in pregnancy disorders and other clinical settings may be in reach. In turn, optimising reproducible methods in future research may help in paving the pathway towards the eventual use of exosomes in multiple areas of clinical research and practice. Thus, it becomes important to examine not only exosomal cargo loading, but also the various mechanisms relating to exosomal isolation, cellular uptake, and their function as biomarker carriers. While the field of reproductive extracellular vesicle research is abundant with potential, it is important to pursue the different hypotheses and extend the field further into real-world clinical applicability.