Reproductive disorders, including intrauterine adhesion (IUA), premature ovarian insufficiency (POI), and polycystic ovary syndrome (PCOS), are great threats to female reproduction. Recently, mesenchymal stem cells derived–extracellular vesicles (MSC-EVs) have presented their potentials to cure these diseases, not only for the propensity ability they stemmed from the parent cells, but also for the higher biology stability and lower immunogenicity, compared to MSCs. EVs are lipid bilayer complexes, functional as mediators by transferring multiple molecules to recipient cells, such as proteins, microRNAs, lipids, and cytokines. EVs appeared to have a therapeutic effect on the female reproductive disorder, such as repairing injured endometrium, suppressing fibrosis of endometrium, regulating immunity and anti-inflammatory, and repressing apoptosis of granulosa cells (GCs) in ovaries. Although the underlying mechanisms of MSC-EVs have reached a consensus, several theories have been proposed, including promoting angiogenesis, regulating immunity, and reducing oxidate stress levels. In the current study, we summarized the current knowledge of functions of MSC-EVs on IUA, POI, and PCOS. Given the great potentials of MSC-EVs on reproductive health, the critical issues discussed will guide new insights in this rapidly expanding field.

Do extracellular vesicles (EVs) from human Fallopian tubes exert an influence on early embryo development in vitro? Human Fallopian tube EVs carrying miRNAs increase murine embryo viability in vitro. Oviductal EVs (oEVs) are recently identified key players in embryo-oviduct interactions that contribute to successful pregnancy in vivo. Their absence in current in vitro systems may partly explain the suboptimal embryo development observed, therefore further knowledge is needed about their impact on early embryos. The oEVs were isolated from the luminal fluid of human Fallopian tubes using ultracentrifugation. We cocultured oEVs with murine two-cell embryos until the blastocyst stage. The study was conducted between August 2021 and July 2022. A total of 23 premenopausal women were recruited for Fallopian-tubes collection, and the oEVs were isolated. The micro RNA (miRNA) contents were detected using high-throughput sequencing and their target genes and effects were analyzed. After in vitro culture with or without oEVs, the blastocyst and hatching rates were recorded. Furthermore, for the blastocysts formed, we assessed the total cell number, inner cell mass proportion, reactive oxygen species (ROS) level, number of apoptotic cells and mRNA expression levels of genes involved in development. EVs were successfully isolated from the human Fallopian tubal fluid and the concentrations were evaluated. A total of 79 known miRNAs were identified from eight samples that had been sequenced, all involved in various biological processes. The blastocyst rate, hatching rate, as well as total cell number of blastocysts were significantly increased in the oEVs-treated groups (p < 0.05 versus untreated), while the proportion of inner cell mass showed no significant difference between groups. ROS levels and apoptotic cell proportions were decreased in the oEVs-treated groups (p < 0.05 versus untreated). The genes, Actr3 (actin-related protein 3), Eomes (eomesodermin), and Wnt3a (Wnt family member 3A) were upregulated in blastocysts in the oEVs-treated group. Data are available from Gene Expression Omnibus: Accession number: GSE225122. The Fallopian tubes in the current study were collected from patients with uterine fibroids (the reason they underwent hysterectomy), and this pathological condition may affect the characteristics of EVs in luminal fluid. Also, owing to restrictions for ethical reasons, an in vitro co-culture system using murine embryos was used instead of human embryos, and the findings may not be transferable. Deciphering miRNA contents in human oEVs and providing new evidence that oEVs benefit embryo development in vitro will not only increase our knowledge on embryo-oviduct communication but also potentially improve ART outcomes. This study was supported by the National Key Research and Development Project of China (2021YFC2700603). No competing interests are declared. One of the steps in assisted reproduction involves growing the early embryo in the laboratory. Previous studies have suggested that the composition of the solution that embryos are grown in may have a long-term impact on the embryos produced in IVF/ICSI cycles, and that the culture media used now needs improving. Communication between the embryos and the oviduct (or Fallopian tube in humans) involves numerous factors that are essential for optimal embryo development. Extracellular vesicles are very small particles that are released by almost every type of cell in the body. In our study, we isolated extracellular vesicles from human Fallopian tubes (along which eggs travel from the ovaries to the uterus) and showed that they may positively influence various biological processes in embryos. This finding backs up previous research that showed these vesicles are beneficial for egg maturation, the correct functioning of sperm and early embryo development when studied in the laboratory. Fallopian tube vesicles are therefore showing great potential for improving ART efficiency, although many methodological and ethical challenges are still to be overcome.

Objective: To investigate the influence of oviductal extracellular vesicles from patients with endometriosis on early embryo development.Design: In vitro experimental studySetting: University-affiliated hospital.Patients: Women with and without endometriosis who underwent hysterectomy (n = 27 in total).Interventions: None.Main outcome measures: Oviductal extracellular vesicles from patients with endometriosis (oEV-EMT) or without endometriosis (oEV-ctrl) were isolated and co-cultured with two-cell murine embryos for 75 hours. Blastocyst rates were recorded. RNA sequencing was used to identify the differentially expressed genes in blastocysts cultured either with oEV-EMT or with oEV-ctrl. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed to identify potential biological processes in embryos that oEV-EMT affects. The functions of oEV on early embryo development were determined by reactive oxygen species (ROS) levels, mitochondrial membrane potentials (MMP), total cell numbers, and apoptotic cell proportions.Results: Extracellular vesicles were successfully isolated from human Fallopian tubal fluid, and their characterizations were described. The blastocyst rates were significantly decreased in the oEV-EMT group. RNA sequencing revealed that oxidative phosphorylation was down-regulated in blastocysts cultured with oEV-EMT. Analysis of oxidative stress and apoptosis at the blastocysts stage showed that embryos cultured with oEV-EMT had increased ROS levels, decreased MMP, and increased apoptotic index. Total cell numbers were not influenced.Conclusion: Oviductal extracellular vesicles from patients with endometriosis negatively influence early embryo development by down-regulating oxidative phosphorylation.

Background: Recurrent implantation failure (RIF) presents a crucial obstacle to in vitro fertilization success. Previous research has shown that small extracellular vesicles (EVs) from endometrial RIF patients hinder embryo development, yet the underlying mechanism and potential solutions remain largely unexplored. In this study, we aimed to investigate the effectiveness of miR-218-5p as a molecular factor in RIF-EVs. Our findings revealed that miR-218-5p disrupted mouse embryo development, and this effect could be reversed by engineered extracellular vesicles (E-EVs) containing anti-miR-218-5p. Methods: The percentage of blastocyst development and hatching rates, embryo morphology, and the total cell number were measured. RNA-sequencing was used to analyze transcriptional changes in embryos post miR-218-5p agomir treatment. The abnormal segregation genes of trophectoderm (TE) and inner cell mass (ICM) were visualized via qRT-PCR and immunofluorescence staining. The E-EVs were using the EVs derived from Human Umbilical Cord Mesenchymal Stem Cells (HUMSCs). Characteristics of the EVs were measured using Western blotting, nanoparticle tracking analysis, and transmission electron microscopy. EVs internalization was visualized using BODIPY TR ceramide staining. Results: Mouse embryos were arrested at the morula stage and demonstrated reduced blastocyst and hatching rates following miR-218-5p agomir treatment (P < 0.001). Essential transcription factors for TE and ICM, such as Cdx2, Yap1, Sox2, Nanog, Tead4, were reduced at the mRNA level in the miR-218-5p treated morula. This was accompanied by decreased Cdx2 protein levels at the 8– 16-cell stage (P < 0.001) and disruption of co-localization of Yap1 and Cdx2. The blastocyte rate was increased by anti-miR-218-5p-encapsulated E-EVs compared with miR-218-5p group (P < 0.001). Conclusion: This study offers valuable insights into the potential role of miR-218-5p in RIF and presents. The utilization of engineered vesicles containing anti-miR-218-5p may present a promising avenue for patients facing challenges with RIF. Plain Language Summary: The phenomenon of recurrent implantation failure (RIF) is a common occurrence in patients undergoing the assisted reproductive technology (ART) process. It intensifies the strain on patients, but as of now, there is no universally accepted definition, cause, or therapeutic strategy. RIF can result from various factors, and recently, the impact of small vesicles released from cells called extracellular vesicles (EVs) has garnered increasing attention in the disease process of RIF. However, there is still a lack of mechanistic evidence and therapeutic recommendations. In this study, we discovered that miR-218-5p is one of the functional molecules inside the RIF-EVs, and it disrupts the development of the embryo into a blastocyst. Furthermore, we found a deeper mechanism related to the distribution of the formation of the trophectoderm (the embryo’s outer cell). This provides a plausible explanation for one form of embryonic disorder associated with RIF. Most significantly, our work introduces a novel therapeutic approach, employing engineered EVs encapsulating anti-miR-218-5p, which effectively mitigates the deleterious effects of miR-218-5p. This research provides a novel and non-invasive insight into potential therapeutic strategies for RIF.