Characterization of endometrial fluid-derived extracellular vesicles.

(A-I) Analysis of ABs, MVs, and EXOs isolated from human EF samples: morphology by TEM (A, D, and G), size distribution by DLS (B, E, and H), and protein marker expression by Western blotting (C, F, and I). TEM images obtained using two different protocols for an external (deposition processing, upper images) or internal (ultrathin slide processing, lower images) view of EVs. Size distribution analyzed in a single EF sample by DLS during the receptive phase for (B) ABs, (E) MVs, and (H) EXOs. Graphs show the average size distribution and percentage of total particles contained within the populations. Specific protein markers analyzed by Western blotting for (C) ABs, (F) MVs, and (I) EXOs. Analyzed markers (and associated molecular mass) were calnexin (90–100 kDa), calreticulin (60 kDa), VDAC1 (31 kDa), ARF6 (18 kDa), CD9 (24 kDa), CD63 (30–60 kDa), and TSG101 (45–50 kDa). (J and K) Particle concentration and size distribution measured by NTA for (J) MVs and (K) EXOs secreted throughout the menstrual cycle. One-way ANOVA and Kruskal-Wallis rank sum tests performed to compare the distinct menstrual cycle phases - no significant differences were observed.

DNA sequencing analysis and coding sequence comparisons of human endometrial fluid-derived extracellular vesicle populations.

(A and B) Volcano plots comparing DNA sequence enrichment between ABs, MVs, and EXOs. Only MVs show significant sequence enrichment compared to ABs and EXOs. (C) Specific gene ID DNA sequences encapsulated within MVs compared to ABs and EXOs, which are mainly mitochondrial DNA.

Quantification of mitochondrial DNA in human endometrial tissues and human endometrial fluid-derived microvesicles.

(A) Relative mtDNA/nDNA ratio calculated from endometrial biopsies from donors undergoing HRT in pre-receptive (P+2), receptive (P+5), and post-receptive (P+8) periods. (B) Gene expression analysis of endometrial biopsies for nuclear genes coding for mitophagy- and mtDNA packing-related proteins (upper panel) and for genes coding for proteins related to mitochondrial function (lower panel). (C) Quantification of relative mtDNA copy number packed into MVs isolated from the EF in pre-receptive, receptive, and post-receptive periods. One-way ANOVA and Kruskal-Wallis rank sum tests performed to compare the distinct periods - no significant differences were observed.

Internalization of endometrial extracellular vesicle-derived DNA by cells of the murine embryo.

Confocal images show hatched embryos after co-culture with EdU-tagged ABs, MVs, and EXOs isolated from Ishikawa cell supernatants. Embryo membranes were visualized with Wheat germ agglutinin (WGA) in red, embryo nuclei with DAPI, and EdU-tagged transferred DNA in green. Zoomed images taken from the areas demarcated by white boxes in merge images. Cell-free DNA and residual small-sized EVs were used as control conditions (Neg). Scale bar in zoom = 20 µm

Mitochondrial function in embryos incubated with human endometrial fluid-derived extracellular vesicles.

(A) Murine embryo ATP content after overnight co-incubation with the EF-derived EV populations (phase IV or receptive phase of the natural menstrual cycle). “All EVs” indicates a combination of ABs, MVs, and EXOs. Embryos not incubated with EVs used as a control condition (Cnt). (B) OCR was recorded on a Seahorse instrument before and after drug injection (timing indicated on the graph). Blocked embryos used as an additional negative control (Neg Cnt). (C) Basal respiration [(Last rate measurement before the first injection)-(minimum rate measurement after Rotenone/antimycin A injection)] and (D) Maximal respiration [(Maximal rate measurement after FCCP injection)-(minimum rate measurement after Rotenone/antimycin A injection)] shown for each condition. One-way ANOVA and Tukey comparison post-hoc performed - no significant differences between conditions were observed (excluding the Neg Cnt condition).