Abstract STUDY QUESTION Do extracellular vesicles (EVs) from human Fallopian tubes exert an influence on early embryo development in vitro? SUMMARY ANSWER Human Fallopian tube EVs carrying miRNAs increase murine embryo viability in vitro. WHAT IS KNOWN ALREADY 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. STUDY DESIGN, SIZE, DURATION 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. PARTICIPANTS/MATERIALS, SETTING, METHODS 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. MAIN RESULTS AND THE ROLE OF CHANCE 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. LARGE SCALE DATA Data are available from Gene Expression Omnibus: Accession number: [40]GSE225122. LIMITATIONS, REASONS FOR CAUTION 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. WIDER IMPLICATIONS OF THE FINDINGS 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. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the National Key Research and Development Project of China (2021YFC2700603). No competing interests are declared. Keywords: extracellular vesicles, Fallopian tube, miRNA, embryo development, in vitro culture __________________________________________________________________ WHAT DOES THIS MEAN FOR PATIENTS? 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. Introduction Over recent decades, ART has given thousands of infertile couples hope to conceive their own children ([41]Faddy et al., 2018). In vivo, two-cell embryos develop into a morula within the oviduct (the Fallopian tube in humans), where the first maternal–embryo crosstalk happens, and the oviductal fluid contains essential molecules involved in such communications ([42]Besenfelder et al., 2012). In contrast, the environment for embryos fertilized in vitro is the culture medium, mainly consisting of glucose, organic acid, amino acids, electrolytes, human serum albumin, etc. ([43]Morbeck et al., 2014). Notably, studies on human and animal embryos discovered that in vitro fertilized embryos, bypassing the gamete/embryo–oviductal communications, show genomic imprinting alterations or poorer developmental potential ([44]Rizos et al., 2002, [45]2008; [46]Lazaraviciute et al., 2014; [47]Duranthon and Chavatte-Palmer, 2018; [48]Kleijkers et al., 2014). Furthermore, the quality of embryos cultured in vitro is better when they are cocultured with oviduct epithelial cells ([49]Ellington et al., 1990). These results suggested that the current medium used in in vitro embryo culture is suboptimal, and oviduct–embryo interaction is pivotal for early embryo development. Extracellular vesicles (EVs) are bilayer-membrane vesicles secreted by all cells as part of their normal physiology and during pathologic conditions ([50]Kalluri and LeBleu, 2020). Cells selectively package EVs with bioactive molecules, including proteins, RNAs, and lipids, and as they are secreted to the fluidal environment and subsequently ingested by other cells, their bioactive cargo is transferred, which can regulate the activities of recipient cells ([51]Théry et al., 2018). Researchers have recognized the important role of EVs in cell-to-cell communication associated with immune responses, cardiovascular diseases, cancer progression, and pregnancy ([52]Tannetta et al., 2014; [53]Machtinger et al., 2016; [54]Simon et al., 2018; [55]Bridi et al., 2020). EV-mediated communications between the maternal oviduct and gametes/early embryos have received significant attention ([56]Almiñana and Bauersachs, 2019). Several studies showed that oviductal EVs (oEVs) can be internalized by oocytes, sperm, and embryos and subsequently regulate their function in vitro ([57]Almiñana et al., 2017; [58]Lange-Consiglio et al., 2017; [59]Lopera-Vasquez et al., 2017; [60]Bathala et al., 2018; [61]Ferraz et al., 2019; [62]Lee et al., 2020). Previous studies reported that adding oEVs to the culture media benefits embryo development ([63]Lopera-Vásquez et al., 2016; [64]Alminana et al., 2017; [65]Lopera-Vasquez et al., 2017; [66]Qu et al., 2019). When co-cultured with oEVs, bovine blastocysts have a lower apoptosis rate ([67]Lopera-Vasquez et al., 2017; [68]Bauersachs et al., 2020) and increased mitochondrial activity ([69]Sidrat et al., 2020). Previous studies have proposed that embryos obtained from IVF have increased DNA methylation levels than their in vivo counterparts ([70]Wright et al., 2011; [71]Lazaraviciute et al., 2014). Furthermore, when co-cultured with oEVs, murine embryos appear to have lower 5-methylcytosine levels, which reflects lower DNA methylation levels, and the overall blastocyst rates are increased ([72]Qu et al., 2020). These results suggest that animal-derived oEVs are important for preimplantation embryo development. However, whether human oEVs have similar effects on early embryo development needs exploration. Previous research found the cargos in EVs are transferrable between species ([73]Woith et al., 2019), and that oEVs are conserved in humans ([74]Bathala et al., 2018). In the current study, we hypothesized that the addition of human oEVs may influence murine embryo development in vitro. We isolated oEVs from the secretions of human Fallopian tubes and used high-throughput sequencing to identify the micro RNA (miRNA) profiles of the oEVs. Furthermore, we investigated the influence of oEVs on early embryo development using a co-culture system involving human oEVs and two-cell murine embryos. Materials and methods Ethics approval, participants, and sample collection The sample collection process in the current study was approved by the Institute Review Board of Tongji Hospital (No. TJ-IRB20210838). Fallopian tubes were obtained from 23 premenopausal women (34–42 years old) undergoing hysterectomy. Luminal fluids were collected immediately after the uterus and the Fallopian tubes were resected, by flushing the lumen of the Fallopian tubes with 50 ml sterile PBS, as previously described ([75]Bathala et al., 2018). EV isolation EVs were isolated using the ultracentrifugation method described previously ([76]Lopera-Vásquez et al., 2016; [77]Mazzarella et al., 2021). In brief, luminal fluid was delivered on ice after collection and was then centrifuged twice at 1500×g for 15 min at room temperature for cell removal. The supernatant was then centrifuged at 16 000×g for 30 min at 4°C to remove cell debris. Debris-free supernatant was filtered through a 0.22-μm filter. The fluid was ultracentrifuged at 120 000×g for 90 min at 4°C. The pellet was resuspended with PBS and ultracentrifuged at 120 000×g for 90 min at 4°C. The pellets were resuspended in 30 μl PBS, and after the evaluation by nanoparticle tracking analysis (NTA), the EVs were stored at −80°C. Western blot analysis EVs or tissue samples were analyzed for the content of EV protein markers using immunoblotting. The protein concentration was measured using a BCA Protein Assay Kit (Servicebio, Wuhan, China) according to the manufacturer’s instructions. Antibodies used for immunostaining were anti-CD9 (Abcam, Cambridge, UK), anti-ALIX (Cell Signaling Technology, Danvers, MA, USA), and anti-TSG101 (Abclonal, Woburn, MA, USA). Transmission electron microscopy A 10 μl aliquot of EVs was placed on a carbon-coated electron microscopy grid with extra fluid removed, as described ([78]Liu et al., 2020). EVs samples were negatively stained using 2% uranyl acetate. Subsequently, the EV-coated grids were observed and photographed using a transmission electron microscope (Carl Zeiss, Oberkochen, Germany). Nanoparticle tracking analysis The particle content within each sample was characterized with a NTA instrument, zetaview (Particle Metrix, Inning am Ammersee, Germany). EVs samples were diluted with PBS at a ratio of 1:1000 to reach the concentration recommended for the measurement. The particle concentration and sizes were recorded. RNA isolation, small RNA library construction, and deep sequencing A total of eight oEVs samples (isolated from the Fallopian tubes of eight women) were used for total RNA extraction, using the exoRNeasy Maxi Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The quality of the RNA samples was examined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and standard denaturing agarose gel electrophoresis. Small RNA library preparation was performed using TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, CA, USA). The quality-ensured RNA-seq libraries were then sequenced using Illumina Hiseq2000/2500. Identification of known miRNAs (mapped to the miRbase database) and read counting were processed using ACGT101-miR (LC Sciences, Houston, TX, USA). A modified normalization was used to correct copy number among different samples, as described previously ([79]Li et al., 2016), and a miRNA was considered present when the normalized read count was >0 in all the samples. A heatmap was constructed using the normalized read counts of the known miRNAs in each oEV sample, using R (R version 4.0.3) with a heatmap via a custom written R script. Target gene prediction and pathway enrichment analysis TargetScan (v5.0) ([80]Agarwal et al., 2015) and miRanda (v3.3a) ([81]Betel et al., 2010) were used to predict the target genes of miRNAs. The target genes predicted were screened according to the scoring criteria of each software. The TargetScan algorithm removes target genes whose context score percentile is less than 50, and the miRanda algorithm removes target genes whose max energy is greater than −10. The intersection of these two databases was taken as the target genes of the miRNA. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of these target genes was annotated. Animals and treatment The animal experiment was approved by the ethical committee of Tongji Hospital (TJ-202111004). Eighty female (6–8 weeks of age) and ten male (8–10 weeks of age) Institute of Cancer Research (ICR) mice were used in the study. Female mice were injected i.p. with 10 IU pregnant mare’s serum gonadotrophin, followed by 10 IU hCG 48 h later. After the injection of hCG, two female mice were mated with one male mouse, for one night. Thirty-six hours after the hCG injection, the pregnant mice were identified and killed by cervical dislocation, and their Fallopian tubes were removed for two-cell embryo collection. Culture of mouse embryos The basic embryo culture medium used here was CZB (AIBI bio, Nanjing, China), which mainly consists of organic acids, electrolyte, glucose, glutamine, and bovine serum albumin (BSA). Embryos at the two-cell stage were cultured with oEVs. oEVs were suspended in PBS after isolation, and a small volume of this primed PBS was mixed with CZB medium to obtain final concentrations of 1×10^9, 1×10^10, and 1×10^11 particles/ml. CZB medium treated with the same volume of PBS was used as a negative control. The embryo culture media were prepared and equilibrated for 2 h before the embryos were loaded. Embryos were cultured at 37°C under an atmosphere of 5% CO[2], 5% O[2], and 90% N[2]. Endocytosis of EVs by embryos To investigate whether oEVs were taken up by embryos, 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO, Beyotime, Shanghai, China) was used to dye the membrane of oEVs ([82]Liu et al., 2020). In brief, oEVs-primed CZB or negative control were incubated with DiO for 30 min, and then dyed oEVs were resuspended in PBS and ultracentrifuged at 120 000×g for 80 min. Labeled oEVs or negative control were co-cultured with the two-cell embryos for 4 h. oEV uptake was observed under a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Immunofluorescence After co-culture with oEVs for 72 h, blastocysts were fixed in 4% paraformaldehyde for 30 min. The fixed blastocysts were then permeabilized with 0.1% Triton X-100 (Servicebio, Wuhan, China) for 20 min, blocked in 3% BSA for 1 h, and then incubated with primary OCT3/4 antibody (Santa Cruz, Dallas, TX, USA) overnight at 4°C. Secondary antibody labeled with cyanine-3 was then used to mark the primary antibody. DNA was stained with 4 μg/ml Hoechst 33258 dye solution (Servicebio, Wuhan, China) for 10 min. The blastocysts were then mounted onto glass slides and observed under a confocal microscope (Nikon, Tokyo, Japan). ROS level measurement To visualize reactive oxygen species (ROS) levels in embryos by fluorescence, a stock solution of 2′,7′-dichlorodihydro-fluorescein diacetate (DCHF-DA, Sigma, St. Louis, MO, USA) was diluted to 10 μM with CZB medium, and blastocysts in each group (n = 10–15) were dyed using the resultant solution at 37°C for 20 min. The blastocysts were then washed three times in CZB and immediately observed under a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). All images were analyzed using Image J software (National Institute of Health, Bethesda, MD, USA). The fluorescence intensity value for each blastocyst was measured using the mean grey value of the field, subtracting out background signal. TUNEL assay The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed on blastocysts using the TMR (red) Cell Apoptosis Detection Kit (Servicebio, Wuhan, China) according to the manufacturer’s instructions. Blastocysts mounted on glass slides were observed under a confocal microscope (Nikon, Tokyo, Japan). The percentage of apoptotic cells was calculated as the TUNEL-positive cell number divided by the total cell number of the blastocyst. Real-time RT-PCR Blastocysts, treated with or without 1×10^10 particles/ml oEVs, were reverse transcribed directly using a Single-Cell Sequence Specific Amplification Kit (Vazyme, Nanjing, China) according to the manufacturer’s instruction. Real-time PCR was performed on a Roche Light Cycler 480 Instrument II PCR System with TB Green^® Premix Ex Taq™ (Tli RNaseH Plus) (TaKaRa, Kusatsu, Japan). The mRNA expression level of each gene was normalized by the housekeeping gene, Gapdh, and was calculated using the 2^−△△Cq method. Ten blastocysts per group were processed for each reaction, in triplicate. Primer information is listed in [83]Supplementary Table SI. Statistical analysis Experiments were performed at least three times. Quantitative variables are shown as mean±SEM. Blastocyst rates and hatching rates were analyzed using the chi-square test. To determine statistical differences in total cell numbers, inner cell mass cell proportions, relative expression levels of ROS, apoptotic cell proportions, and gene expression, one-way ANOVA was first used to detect any differences among groups. Student’s t-tests were then performed to determine significant differences between pairs. Differences were considered to be significant when P < 0.05. Statistical tests were performed using Graph Pad Prism 8.0 (Graphstats Technologies, San Diego, CA, USA). Results Characterization of EVs from human Fallopian tubal fluid In the current study, 23 women aged between 34 and 42 years old were recruited (clinical characteristics in [84]Supplementary Table SII). The morphology and distribution of oEVs from human tubal fluid were characterized using transmission electronic microscopy (TEM) and NTA. The NTA profiles of the oEVs samples showed peaks at around 137.4 nm, ranging from 124.5 to 152.6 nm ([85]Fig. 1A and B). The particle concentration of the oEVs sample was provided by NTA results, ranging from 1.2×10^10 to 3.80×10^11 particles/ml ([86]Fig. 1C). The western blotting analysis confirmed the presence of classic EV protein markers, including ALIX (apoptosis-linked gene 2-interacting protein X), TGS 101 (tumor susceptibility gene 101), and CD9 ([87]Fig. 1D). Tissues from human Fallopian tubes were used as control samples, and EV protein markers were almost undetectable in these samples. The TEM images showed the typical bilayer structure of the EVs, at the expected size, which confirmed the presence of EVs within human Fallopian tubes ([88]Fig. 1E). Figure 1. [89]Figure 1. [90]Open in a new tab Characterization of oviductal extracellular vesicles. (A) The size of oEVs was assessed using NTA. (B) Peak particle diameters of the samples are presented, and the mean value is shown. (C) The concentration of the oEVs samples. (D) The expression of EV protein markers was evaluated in the oEVs, and tissues from Fallopian tubes were used as controls. Western blotting showed that oEVs expressed ALIX (apoptosis-linked gene 2-interacting protein X), TSG 101 (tumor susceptibility gene 101), and CD9. (E) Representative TEM images of oEVs. (B, C) Results are expressed as least-square mean with range. oEVs, oviductal extracellular vesicles; NTA, nanoparticle tracking analysis; TEM, transmission electron microscopy. Target and functional analysis of the miRNAs derived from oEVs By mapping to the miRbase database, we identified 617 known miRNAs in oEVs ([91]Supplementary Table SIII), among which 79 miRNAs were commonly expressed in all eight oEVs samples ([92]Supplementary Fig. S1). The top 30 miRNAs are listed in [93]Fig. 2A. The target genes of the 79 miRNAs present in oEVs were predicted by TargetScan and miRanda. The results showed that 6554 overlapped genes were targeted by the 79 miRNAs present in oEVs. For these target genes, GO and KEGG pathway analyses were performed ([94]Fig. 2B and C). The main enriched GO terms include cell differentiation, cell cycle, apoptotic process, DNA-templated transcription, ion transport, metabolic process, extracellular exosome, and ATP binding. The enriched KEGG pathways include the p53 signaling pathway, Ras signaling pathway, phosphatidylinositol 3–kinase (PI3K)/protein kinase B (AKT) signaling pathway, and the Hippo signaling pathway. These results showed that miRNAs in oEVs are involved in the regulation of many biological events in embryos. Figure 2. [95]Figure 2. [96]Open in a new tab Top 30 known micro RNAs and pathway enrichment of the 79 known miRNAs in oviductal extracellular vesicles. (A) Box plots of the top 30 known miRNAs in oEVs. Results are expressed as Min to Max, and the vertical line in the box plot indicates the median. (B) GO enrichment of the 79 known miRNAs in oEVs. The blue bar represents the biological processes. The green bar represents the cellular component. The red bar represents the molecular function. (C) Enriched KEGG pathways. oEVs, oviductal extracellular vesicles; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes. Uptake of oEVs by two-cell embryos We assessed oEV uptake using fluorescence microscopy. oEVs were labeled with DiO for 30 min, and two-cell embryos were co-cultured with fluorescence-labeled oEVs, or PBS (control) for 4 h. Green fluorescence signals were observed within embryonic cells in the oEVs-treated group, whereas they were hardly seen in the control group ([97]Fig. 3A). The results suggest that oEVs were internalized by embryos. Figure 3. [98]Figure 3. [99]Open in a new tab Effect of oviductal extracellular vesicles on mouse blastocyst formation rate, hatching rate, and total cell number of blastocysts. (A) Images of two-cell embryos cultured with oEVs labeled by DiO (green) or with PBS (control). Green fluorescence was hardly detected around the zona pellucida, which confirmed the internalization of oEVs inside embryos, excluding the possibility that the oEVs could be attached to the membrane. (B) Blastocyst rate and hatching rate. The number of two-cell embryos and blastocysts in each group is provided. (C) Representative confocal microscopy images of dyed blastocysts. Blastocysts were stained with Hoechst 33258, and the total cell numbers were calculated. ICM cells were analyzed using immunostaining with anti-OCT3/4 antibody, a specific marker of ICM (red). (D) The total cell numbers were compared among groups, and each dot represents one blastocyst. Proportions of ICM cells are presented. Data of blastocyst rates and hatching rates were analyzed by chi-square tests. Total cell numbers were firstly analyzed by one-way ANOVA to detect any differences among groups, and Student’s t-tests were then performed to determine significant differences between pairs. Data were expressed as mean±SEM. Different superscripts per column (a, b) represent statistically significant differences (P < 0.05) among groups. oEVs, oviductal extracellular vesicles; DiO, 3,3′-dioctadecyloxacarbocyanine perchlorate; ICM, inner cell mass; OCT, octamer-binding transcription factor. oEVs have a positive effect on the development of embryos In the oEV-treated groups, the blastocyst rates in the 1×10^9 particles/ml (84.8%, 112/132), 1×10^10 particles/ml (85.3%, 116/136), and 1×10^11 particles/ml (83.8%, 109/130) groups were significantly increased compared with the control group (73.4%, 102/139). However, no significant difference was observed among the oEV-treated groups ([100]Fig. 3B). Hatching rates also increased in the oEV-treated groups compared with the control group, and significant increases were observed in the 1×10^10 and 1×10^11 particles/ml groups. To further investigate the quality of the blastocysts, we first analyzed the total cell number. In the oEV-treated groups, the total cell numbers in the 1×10^9 particles/ml (86.9), 1×10^10 particles/ml (87.4), and 1×10^11 particles/ml (86.0) groups were significantly increased compared with the control group (78.5) ([101]Fig. 3C and D). Furthermore, immunoblotting with OCT3/4 showed that oEVs treatment did not change the proportion of the inner cell mass cells ([102]Fig. 3D). The blastocysts were examined for ROS levels, and the fluorescence intensity was presented as the fold change relative to the control group. The results showed that ROS levels were significantly decreased in oEVs-treated groups (77.3%, 66.8%, 78.0% in the 1×10^9 particles/ml, 1×10^10 particles/ml, and 1×10^11 particles/ml groups, respectively versus 100% in the control group) ([103]Fig. 4A and B). Figure 4. [104]Figure 4. [105]Open in a new tab Effect of oviductal extracellular vesicles on the relative level of reactive oxygen species, apoptotic cell numbers, and relative expression of development-related genes in mouse embryos. (A) Fluorescence images of ROS. (B) The relative levels of ROS (fluorescence intensity value). (C) TUNEL assay of blastocysts. (D) The percentage of apoptosis-positive cells (red) was compared among groups. (E) Relative mRNA expression levels of Actr3 (actin-related protein 3), B3gnt5 (beta-1,3-N-acetylglucosaminyltransferase 5), Cdx2 (caudal type homeobox 2), Eomes (eomesodermin), and Wnt3a (Wnt family member 3A). Data were firstly analyzed by one-way ANOVA to detect any differences among groups, and then were analyzed by Student’s t-tests. Results were expressed as mean ± SEM. Different superscripts per column (a, b) represent statistically significant differences (P < 0.05) among groups. oEVs, oviductal extracellular vesicles; ROS, reactive oxygen species; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. The TUNEL assay showed a significant decrease in the number of apoptotic cells in blastocysts treated with 1×10^9 particles/ml and 1×10^10 particles/ml oEVs, compared to the control, as presented in [106]Fig. 4C and D. Transcript levels after embryo co-culture with oEVs We detected the mRNA levels of genes associated with embryo development (Actr3 (actin-related protein 3), B3gnt5 (beta-1,3-N-acetylglucosaminyltransferase 5), Cdx2 (caudal type homeobox 2), Eomes (eomesodermin), and Wnt3a (Wnt family member 3A)) ([107]Parks et al., 2011). For the oEV-treated group, 1×10^10 particles/ml were added the culture media. Each of these genes was expressed in every sample, as determined by quantitative RT–PCR and relative to the housekeeping gene, Gapdh. Relative expression level analysis showed significant up-regulation in levels of Actr3, Eomes, and Wnt3a mRNAs in embryos after co-culture with oEVs, whereas B3gnt5 and Cdx2 did not show differential expression between groups ([108]Fig. 4E). Discussion This study provides the first evidence of the beneficial effect that human oEVs exert on early embryo development. By deciphering miRNA content in human oEVs and co-culturing oEVs with two-cell murine embryos in vitro, we demonstrated that after being internalized by embryos, oEVs may affect various biological events in blastocysts, including improving total cell numbers and reducing ROS and apoptosis levels. These results indicate an important role of oEVs in reproductive processes. The oviduct (or the Fallopian tube in humans) is where early reproductive events occur, including gamete maturation and capacitation, fertilization, and early embryo development. Crosstalk between oviduct and gametes/embryos is indispensable for the success of all these events. Co-culturing embryos with oviductal epithelial cells could improve the embryo quality cultured in vitro ([109]Ellington et al., 1990), and [110]Lopera-Vásquez et al. (2016) showed oEVs can improve embryo quality in vitro to the same extent as co-culture with oviductal epithelial cells. Accumulating evidence in animals has confirmed that oEVs from in vivo sources can be taken up by gametes/embryos in vitro, and the cargos are transported to the gametes/embryos, which consequently exert an influence on their functions, suggesting critical roles for oEVs in the feto-maternal information exchange during early embryonic development ([111]Almiñana et al., 2017; [112]Lopera-Vasquez et al., 2017). Animal-derived oEVs have shown great potential in promoting embryo viability in vitro ([113]Lopera-Vásquez et al., 2016; [114]Almiñana et al., 2017; [115]Lopera-Vasquez et al., 2017; [116]Qu et al., 2019, [117]2020; [118]Sidrat et al., 2020). Notably, oEVs are evolutionarily conserved in humans ([119]Bathala et al., 2018) and transferrable between species, i.e. EVs produced in one species can be taken up by other species and function in the recipient cells ([120]Woith et al., 2019).This evidence justified the construction of a co-culture model using human oEVs and murine embryos to explore the potential modulatory role of human oEVs on embryo development. In the current study, we isolated EVs contained in the luminal fluid from human Fallopian tubes and determined their morphology and concentration, as previously documented ([121]Bathala et al., 2018). Our data showed that the isolated oEVs were positive for EV protein markers, had the classic bilayer-membrane structure, and the size range was 120–160 nm ([122]Fig 1A–E), consistent with previous literature ([123]Kalluri and LeBleu, 2020). Several studies have recognized RNA and protein content encapsulated within oEVs ([124]Almiñana et al., 2017, [125]2018), and these RNA cargos might act as mediators of the oviduct-gamete/embryo communication. It has been extensively reviewed that several important reproductive events are regulated by miRNAs carried by oEVs, including gamete maturation, fertilization, and early embryo development ([126]Capra and Lange-Consiglio, 2020). Researchers comprehensively analyzed the miRNAs in the bovine oEVs and their target genes and showed that the biological processes enriched by these target genes involve embryonic development, embryonic morphology, and implantation ([127]Almiñana et al., 2018). In the current study, we identified miRNAs within human oEVs using high-throughput sequencing and analyzed their potential roles in recipient cells. Through mapping to the miRbase database, we found 617 known miRNAs ([128]Supplementary Table SIII), among which 79 miRNAs were detected in all the eight oEVs samples ([129]Supplementary Fig. S1). The expression frequency of these miRNAs was variable across samples, and this may reflect individual differences. We validated the expression level of randomly selected miRNAs using quantitative RT–PCR, and the Cq values were between 33 and 45 ([130]Supplementary Fig. S2). Among the known miRNAs detected in oEVs, several have been validated to be EV-derived and are beneficial for embryo development. miR-375 affects early embryonic development by regulating cell metabolism ([131]Hinton et al., 2010; [132]Almiñana et al., 2018); miR-21 was reported to lower the percentage of arrest of embryo development ([133]Lv et al., 2018); miR-141 and miR-27b were expressed at a higher level in euploid embryos than in aneuploid embryos ([134]Rosenbluth et al., 2013). One of the top 10 miRNAs detected in human oEVs, miR-378a-3p, is also reported to be derived from EVs secreted by blastocysts, and was proven to enhance embryo quality, especially embryo hatching rates, by upregulating cell division, differentiation, and embryo development ([135]Pavani et al., 2022). Interestingly, the same study also showed that EV-encapsulated miRNAs are shielded from degradation and thus are more stable than the total RNA in the biofluids. These results indicate that EVs, as promising carriers of biomolecules, are worthy of investigation for their potential in clinical application, such as for targeted drug delivery or for biomarker establishment. The target genes of the 79 known miRNAs were predicted, and further pathway analysis revealed enrichment in cell differentiation, cell cycle, apoptotic process, metabolic process, extracellular exosome, and ATP binding ([136]Fig. 2B). Enriched KEGG pathways include the p53 signaling pathway, Ras signaling pathway, PI3K-Akt signaling pathway, and Hippo signaling pathway, which are functional in pre-implantation embryos ([137]Riley et al., 2005; [138]O’Neill et al., 2012; [139]Meinhardt et al., 2020). Our data show that miRNAs within oEVs are regulating genes involved in various biological processes, and are associated with embryo development, which is generally in line with previous studies involving oEVs ([140]Almiñana et al., 2018; [141]Capra and Lange-Consiglio, 2020; [142]Lee et al., 2021). To further investigate the influence oEVs exert on early embryos, we co-cultured murine two-cell embryos with different concentrations (0, 1 × 10^9, 1 × 10^10, and 1 × 10^11 particles/ml) of human oEVs in vitro. After confirming that oEVs of human origin could be taken in by two-cell murine embryos, we evaluated the quality of the blastocysts formed. Results showed that supplementation of medium with oEVs significantly increased the blastocyst rates, the hatching rates, and the total cell number of blastocysts, which were generally consistent with previous studies using animal-derived oEVs ([143]Lopera-Vásquez et al., 2016; [144]Qu et al., 2020), while the inner cell mass proportion was not influenced. We also observed that oEVs significantly decreased apoptotic cell proportions in blastocysts ([145]Fig. 4C and D), and these results suggest that oEVs increase total cell numbers by improving the competency of embryos to resist apoptosis. Moreover, excess ROS concentration is often connected to developmental impairment ([146]Yoon et al., 2014), and our results showed that oEVs decrease ROS levels in blastocysts in vitro, which accorded with previous studies ([147]Alminana et al., 2017; [148]Qu et al., 2020). However, these differences were not oEV concentration-dependent, and this phenomenon might be explained by the fact that oEVs concentration in vivo is fluctuating over quite a large range. Additionally, we assessed relative expression levels of genes associated with blastocyst developmental competence. The results showed that Actr3, Eomes, and Wnt3a mRNA expression levels were significantly upregulated in the oEVs-treated group, whereas B3gnt5 and Cdx2 were not ([149]Fig. 4E). Wnt3a, Eomes, and B3gnt5 were reported to characterize the success of implantation ([150]Parks et al., 2011). Actr3 and Cdx2 are associated with trophectoderm development and differentiation ([151]Strumpf et al., 2005; [152]Vauti et al., 2007). Overall, in this study, we showed that supplementing the culture media with human oEVs can benefit murine embryo development in vitro. The beneficial influence of oEVs on embryos is consistent with previous literature ([153]Almiñana and Bauersachs, 2019). As reviewed by [154]Gervasi et al. (2020), the positive effects of animal oEVs on oocyte maturation, sperm capacitation, and early embryo development, suggest great potential for oEVs for improving ART efficiency. Also, oEVs are known to transfer exogenous nucleic acids into preimplantation embryos, which suggests their application in gene-editing research, although many challenges still need to be overcome ([155]Fu et al., 2020). One possible limitation of the study is that the women underwent hysterectomy because of uterine fibroids. This pathological condition might alter the characteristics of oEVs. However, considering that hysterectomy would only be conducted when there are indications for surgery, it is unlikely that human Fallopian tube samples can be collected under physiological conditions. We tried our best to exclude known factors that may influence embryo quality, such as endometriosis or pelvic inflammation pathology, to minimize the risk of alterations in oEVs. Furthermore, concerning ethics and other limitations, the current study was limited by being based on in vitro experiments using murine, instead of human, embryos. Whether oEVs exert a similar influence on human embryos needs further exploration. In summary, the current study revealed the miRNA profiles of human oEVs and showed that human oEVs exert positive effects on murine embryos cultured in vitro. Our results call for a better understanding of the mechanisms mediating oviduct–embryo communication in vivo; these investigations would improve our understanding of the complex oviductal environment, which is beneficial in terms of constructing optimal systems for gamete maturation, fertilization, and early embryo development in vitro. These new developments would more than likely improve the success of ART in the future. Supplementary Material hoad006_Supplementary_Tables_SI_SII [156]Click here for additional data file.^ (16.5KB, docx) hoad006_Supplementary_Table_SIII [157]Click here for additional data file.^ (105.7KB, xlsx) hoad006_Supplementary_figures [158]Click here for additional data file.^ (394.8KB, docx) Acknowledgements