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The role of micro-ribonucleic acids and extracellular vesicles in In-Vitro Fertilization

ABSTRACT: BACKGROUND: Molecular signaling in in vitro fertilization procedures seems to play a crucial role for embryonic development. In a prospective blinded cohort study, we investigated the role of microRNAs (miRNA) and extracellular vesicles (EV) as potential carriers of miRNAs in embryonic culture media. METHODS: Embryonic culture media from 56 female partners undergoing infertility treatment. miRNA microarray and RT-qPCR assays in combination with bioinformatics were used for the screening, validation and analysis of identified miRNAs. Nanoparticle tracking analysis was applied to measure EV in culture media. RESULTS: The overall miRNA repertoire in culture media was astonishingly complex: 621 miRNAs were present in embryonic culture media. Samples with positive outcome had an overall decreased miRNA repertoire, with the largest effects for miR-29c-3p. RT-qPCR measurements were largely consistent with the microarray results. Comparison to human spermatozoa profiles highlighted that a large part of spermatozoa miRNAs (101 miRNAs) are also present in culture media following embryonic transfer. As potential carrier molecules that could be of relevance for the decreased miRNA repertoire in samples with positive outcome we investigated EV in a small proof-of-concept study. While the size distribution was similar between both cohorts, the total number of EV in pure culture media was zero, in culture media of patients with positive outcome 3.8 billion per ml and in culture media of samples with negative outcome 7.35 billion per ml, correlating well with the results for the miRNAs. CONCLUSIONS: Embryonic culture media of pre-implantation embryos contains a stable population of highly specific to ubiquitous miRNAs that are correlated to embryogenesis and spermatogenesis processes. These miRNAs are potentially harbored by EV.

ORGANISM(S): Homo sapiens

PROVIDER: GSE93810 | GEO | 2020/01/09

REPOSITORIES: GEO

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Project description:Extracellular vesicles (EVs) are involved in intercellular communication in health and disease and affect processes including immune and antiviral responses. We have previously demonstrated that ultracentrifuged serum is depleted of EVs and, when used in cell culture media, is associated with declines in growth and viability of numerous cultured cell types. Although EVs had been reported to enhance or interfere with HIV-1 infection, depending on the setting, the effects of EVs on HIV-1 production and infectivity of released virions were unknown. In this study, we examined the effects of EV-depleted serum on HIV-1 replication in primary cells and cell lines, including two HIV-1 latency models. Cell culture media were prepared with EV-replete fetal bovine serum (FBS) or serum depleted of EVs via ultracentrifugation or a proprietary method (ThermoFisher/Gibco). T-cell and myeloid-lineage cell lines, including ACH-2 and U1 HIV-1 latency models, and primary cells were grown in 10% FBS-based culture media. Cell counts, viability, and proliferation were assessed throughout. HIV-1 production and infectivity were measured by p24 ELISA and luciferase reporter cell lines, respectively. Flow cytometry, Seahorse assays, and miRNA and mRNA expression arrays were done to assess cellular responses to EV-depleted conditions. Significant increases in HIV-1 production were observed in EV-depleted conditions, along with, in some cases, morphology changes and decreased cell viability. Add-back of pelleted EVs reduced HIV-1 production almost to baseline. Primary cells appeared to be less susceptible to EV depletion. ACH-2 and U1 latency models also produced more HIV-1 under EV-depleted conditions. Virus produced under EV-depleted conditions was more infectious. Changes in cellular metabolism and gene expression were associated with EV-depleted culture. The EV environment of HIV-1 infected cells appears to have a significant effect on virus production and infectivity. In cell lines of HIV-latency, significantly higher concentrations of p24 were observed in those cells cultured in EVD conditions. EV-dependence of cell cultures should be examined carefully prior to examining additional experimental variables. However, we also sound a cautionary note that direct actions of EVs may be accompanied by the effects of other, closely associated factors.

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The role of micro-ribonucleic acids and extracellular vesicles in In-Vitro Fertilization

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