69 DNase treatment of extracellular vesicles released by in vitro-produced bovine embryos increases accuracy of preimplantation genetic testing

Extracellular vesicles (EVs) are membrane-enclosed nanoparticles released from cells that are involved in intercellular communication. EV populations can be isolated from different biological fluids and are usually made up of three types: exosomes, microvesicles, and apoptotic bodies. EVs contain several bioactive molecules such as proteins, metabolites, mRNA, and microRNA. In the last decade, free DNA in cells’ secretome has been reported and it seems that this DNA is part of the EV cargo and/or is located on the outer surface of the EVs. The free DNA present in fluids has gained importance during the last year for diagnostic and prognostic purposes, becoming a type of liquid biopsy. In humans, DNA in embryo culture medium (eDNA) has been used for preimplantation genetic diagnosis (PGD), with almost 80% accuracy. However, a significant proportion of DNA in culture medium is of nonembryonic origin, including cumulus cells. In this sense, embryonic DNA within EVs could help to separate the contaminants and increase the accuracy of genetic diagnosis. In this work, we proposed DNase treatment of embryo-derived EVs. For this purpose, bovine embryos were produced in vitro by IVF. Embryos were cultured in groups up to Day 5 in synthetic oviductal fluid (SOF) medium. On Day 5, morulae were selected and cultured in EV-depleted SOF medium (SOFd) until Day 7.5. At this moment, conditioned culture medium (CCM) was collected and subjected to EVs separation. First, CCM was cleaned by serial centrifugation to remove cells and detritus and then EVs were isolated by density-gradient Optiprep™ (Iodixanol, Merck KGaA) and ultracentrifugation (OPUC) by 18 h. Isolated EVs were separated in two groups: (1) wild-type EVs (wtEVs), and (2) treated with 2 U of DNase I (M0303, New England Biolabs) (dEVs). The DNAse treatment was applied before lysing the EVs. Subsequently, the DNase was inactivated at 75°C for 10 min. The size, concentration, and zeta potential were measured by NanoSight NS300 and Zeta potential NS500 (Malvern Instruments Ltd.), respectively. EVs were lysed using the lysis buffer from the Cells-to-cDNA™ II Kit (ThermoFisher Scientific) and PCR performed to confirm the presence of DNA from EVs by the amplification of bovine-specific satellite sequence primer (SAT1: a multicopy gene). PCR products were visualised in a 2% agarose gel. In this culture system, 20% of embryos developed to blastocyst stage. The EV fraction isolated from OPUC had a mean size of 159.1 ± 2.1 nm and a concentration of 5 × 10⁷ particles mL⁻¹. A band corresponding to STA1 amplification was visualised in both groups. The zeta potential was significantly higher in dEVs (wtEVs: −16.7 mV and dEVs: −13.6 mV; P = 0.028), probably due to the removal of bound DNase treatment. We concluded that the DNase treatment of EVs secreted in culture medium might improve the accuracy of PGD analysis.

This research was supported by FONDECYT, Chile (11210334).