Summary Human embryos generated in vitro have a high incidence of chromosomal abnormalities that negatively affect pregnancy rate. Embryos generated in vitro secrete extracellular vesicles (EVs) into the culture medium that could be used potentially as indicators of embryo competence. This research aimed to evaluate the concentration and size of EVs and their gDNA content as an indicator of developmental competence in human embryos. Human embryos generated by intracytoplasmic sperm injection (ICSI) were classified morphologically as of either TOP, FAIR or POOR quality. Culture medium and developmentally arrested embryos (which were not able to be used for embryo transfer) were collected. Microvesicles, exosomes (MV/Exo) and apoptotic bodies (ABs) were isolated from culture medium. Nanoparticle tracking analysis (NTA) and array comparative genomic hybridization (aCGH) analysis were performed to evaluate EVs and their gDNA content. From NTA, the diameter (mean) of MVs/Exo from TOP quality embryos was higher (112.17 nm) compared with that of FAIR (108.02) and POOR quality embryos (102.78 nm) (P < 0.05). aCGH analysis indicated that MVs/Exo and ABs carried gDNA with the presence of 23 chromosome pairs. However, when arrested embryos were compared with their respective MVs/Exo and ABs, the latter had an increased rate of chromosomal abnormalities (24.9%) compared with embryos (8.7%) (P < 0.05). In conclusion, the size of EVs from culture medium might be an alternative for evaluating competence of human embryos, however more studies are needed to validate the use of gDNA from EVs as an indicator of embryo competence.
During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo–maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells; miRNAs participate in embryo–maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5–5). Individual culture media from in vitro–produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8–16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs; bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.
The embryo-maternal interaction occurs during the early stages of embryo development and is essential for the implantation and full-term development of the embryo. In bovines, the secretion of interferon Tau (IFNT) during elongation is the main signal for pregnancy recognition, but its expression starts around the blastocyst stage. Embryos release extracellular vesicles (EVs) as an alternative mechanism of embryo-maternal communication. The aim of the study was to determine whether EVs secreted by bovine embryos during blastulation (D5-D7) could induce transcriptomic modifications, activating IFNT signaling in endometrial cells. Additionally, it aims to assess whether the EVs secreted by embryos produced in vivo (EVs-IVV) or in vitro (EVs-IVP) have different effects on the transcriptomic profiles of the endometrial cells. In vitro- and in vivo-produced bovine morulae were selected and individually cultured for 48 h to collect embryonic EVs (E-EVs) secreted during blastulation. E-EVs stained with PKH67 were added to in vitro-cultured bovine endometrial cells to assess EV internalization. The effect of EVs on the transcriptomic profile of endometrial cells was determined by RNA sequencing. EVs from both types of embryos induced several classical and non-classical IFNT-stimulated genes (ISGs) and other pathways related to endometrial function in epithelial endometrial cells. Higher numbers of differentially expressed genes (3552) were induced by EVs released by IVP embryos compared to EVs from IVV (1838). Gene ontology analysis showed that EVs-IVP/IVV induced the upregulation of the extracellular exosome pathway, the cellular response to stimulus, and the protein modification processes. This work provides evidence regarding the effect of embryo origin (in vivo or in vitro) on the early embryo-maternal interaction mediated by extracellular vesicles.
Extracellular vesicles (EV) are currently considered a mechanism of cell communication. These are secreted from different cell types, including embryos, to serve as mediators of short and long distance signals. EV can be identified in vivo in different biological fluids, as well as in vitro embryo culture medium. Usually, media used for embryo in vitro culture are supplemented with serum or other protein sources that favour cell proliferation and development. Serum and protein sources contain EV, including microvesicles and exosomes that in principle can be internalized by embryonic cells. The aim of this study was to evaluate if serum-derived EV are internalized by the embryo at different stages of the early development, and if EV from the serum are required for in vitro bovine embryo development. For that, it was first evaluated if EV depleted culture media affect embryo development up to the blastocyst stage; oocytes were in vitro matured for 22 to 23h and in vitro fertilized for 18h. Posteriorly, presumptive zygotes were in vitro cultured in groups (25 embryos/well in 4-well plates) in SOF or SOF depleted of EV for 8 days. To evaluate EV internalization, culture media was supplemented with labelled EV and confocal imaging was performed. The EV were obtained by ultrafiltration (centrifugal filter devices 100 kDa, Amicon; Millipore, Billerica, MA, USA) for 15min at 3000 rpm. Then, EV were stained with PKH67 dye and washed 3 times with PBS by ultrafiltration to remove excess dye. The EV labelled with PKH67 were resuspended in SOFaa depleted of EV (3×109 particles per 500µL) and supplemented for 24h at the 1-cell stage (Day 1 post IVF), 16 cells (Day 4 post IVF), and early blastocyst (Day 6 post IVF) in 5% CO2, 5% O2, and 90% N2. PBS with PKH67 dye was used as a control treatment. Hoechst 33343 was used to label the nuclei before washing with PBS and fixation with 0.4% paraformaldehyde. Images were acquired on a Zeiss (Zeiss, Jena, Germany) LSM 780 confocal microscope. There were no statistical differences on blastocyst rate at Day 8 between embryos cultured in SOF depleted of EV (19.5%) and control group (SOF; 22.7%; P>0.05). We observed punctuated green fluorescence near the embryo nuclei in the 3 stages studied in embryos supplemented with EV but not in the control treatment, which indicates that EV from serum are uptaken by embryonic cells in early development. Therefore, we demonstrated uptake of EV from fetal calf serum added to culture media, although its absence does not affect embryo development. Research was supported by FONDECYT, Chile (1170310).
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