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Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts (miRNA)

ABSTRACT: The current study is focused on Isolating EVs and their cargo content (miRNAs) from culture medium conditioned by individual embryos that can differentiate between the blastocyst and non-blastocyst embryos. We were able to isolate EVs from both blastocyst and non-blastocyst group with size exclusion chromatography (Izon’s™ qEV single column) and characterize them with Western blotting, nanoparticle tracking analysis, and Transmission electron microscopy. Further, we could isolate total RNA (including small RNAs) from the blastocyst and non-blastocyst EVs for miRNA sequencing.

ORGANISM(S): Bos taurus

PROVIDER: GSE197875 | GEO | 2022/03/10

REPOSITORIES: GEO

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Project description:The study of genomic imprinting in mammals started with analysis of parthenogenetic embryos. At the phenotypic level, embryos with two maternal genomes and no paternal genome proceed through early development unimpaired, and only begin to fail after implantation. The most recognizable early defect is reduced or non-existent trophoblast, the tissue that gives rise to the placenta. We applied the procedure for establishing Trophoblast Stem cells (TS cells) developed in the Rossant lab to parthenogenetic embryos, and were successful in making four different TS cell lines, three from MI oocyte derived blastocysts and one from MII derived blastocysts. Initial molecular characterization, including microarray analysis, indicates that these cells are indistinguishable from fertilized TS cells, with the single exception of null expression of the paternally expressed gene Snrpn. The only significant difference between parthenogenetic and fertilized TS cells was the frequency with which they could be derived. In our hands, fertilized blastocyst outgrowths produced TS cells at robust rates (10-12 colonies per blastocyst), while parthenogenetic blastocyst outgrowths produced only 4 colonies in 50 outgrowths, 100 times less frequently than fertilized embryos. This led us to hypothesize that those few TS cells that arose in parthenogenetic outgrowths were probably a result of very low frequency stochastic variation in the imprint status of a gene or genes required for either establishment or maintenance of stem cells, or both. The corollary to this hypothesis posits that the early failure of parthenogenetic embryos, and in particular parthenogenetic trophoblast, is a function of impaired stem cell function. This raises the intriguing possibility that microarray comparisons of parthenogenetic, fertilized and androgenetic blastocysts may reveal the identities of genes important for stem cell biology. To this end, my colleague Keith Latham, at the Fels Institute, Temple University, Philadelphia, made amplified cDNAs from pools of ten each of androgenetic, gynogenetic, or fertilized blastocysts. Three separate pools for each type of embryo were prepared for microarray analysis. The embryos were all produced by nuclear transfers between zygotes, a difficult technique that is Keith's special expertise. He used the Brady/Iscove protocol to generate quantitative 3' end biased cDNAs. We would like to compare the transcriptomes of these embryos using the MOE430 2.0 arrays. We expect that important insights into the biology of uniparental embryos in general, and stem cells in particular may be revealed. Keywords: other

Project description:Study question: Could extracellular vesicles (EVs) secreted by aneuploid embryos a) serve for development of RNA biomarkers for preimplantation genetic testing for aneuploidy (PGT-A) and b) elicit a relevant transcriptomic response in decidualised primary endometrial stromal cells (dESCs)? Summary answer: Aneuploid embryo EVs a) contain genes PPM1J, LINC00561, ANKRD34C and TMED10 in differential abundance from euploid EVs and b) induce up-regulation of MUC1 transcript in dESCs. What is known already: PGT-A is popular as it is thought to facilitate transfer of euploid embryos to increase implantation probability but the technology is controversial, as it requires an invasive embryo biopsy with an elusive long-term biosafety. The development of non-invasive methods to screen out aneuploid embryos is paramount. It is also critical to decode the embryo-endometrial dialog underlying implantation failure. We have previously reported that IVF embryos secrete EVs that can be internalised by ESCs, conceptualising that successful implantation to the endometrium is facilitated by EVs, which may additionally serve as biomarkers of ploidy status. Study design, size, duration: Embryos destined for biopsy on days 5-7 for PGT-A were grown under standard conditions. Spent media (30μl) were collected from euploid (n=175) and aneuploid (n=140)embryos at cleavage (days 1-3) stage and from euploid (n=187) and aneuploid (n=142) embryos at blastocyst (days 3-5) stage. Media samples from n=35 cleavage embryos were pooled in order to obtain five euploid and four aneuploid pools. Similarly, media samples from blastocyst were pooled to create one euploid and one aneuploid pool. ESCs were obtained from five women undergoing diagnostic laparoscopy. Participants/materials, setting, methods: EVs were isolated from pools of media derived from euploid and aneuploid day 3 embryos with differential centrifugation and EV-RNA sequencing was performed following the SMARTer Stranded Total RNA-Seq approach. ESCs were decidualised (E2:10nM, P4:1uM, cAMP:0.5 mM twice every 48 hours) and treated for 24 hours with 50 ng/ml euploid or aneuploid EVs extracted from blastocyst media. RNA sequencing was performed on ESCs following the Illumina Truseq RNAseq protocol. Main results and the role of chance: Aneuploid cleavage stage embryos secreted EVs that were less abundant in RNA fragments originating from the genes PPM1J (log2fc=-5.13, p=0.011), LINC00561 (log2fc=-7.87, p=0.010) and ANKRD34C (log2fc=-7.30, p=0.017) and more abundant in TMED10 (log2fc=1.63 p=0.025) compared to EVs from euploid embryos. Decidualisation per se induced downregulation of MUC1 (log2FC=-0.54, p=0.0028) in ESCs as prerequisite for the establishment of receptive endometrium. The expression of MUC1 transcript in decidualised ESCs was significantly increased following treatment with aneuploid compared to euploid embryo-secreted EVs (log2FC=0.85, p=0.0201). Limitations, reasons for caution: The findings of the study may require validation utilising a second cohort of EVs samples. Wider implications of the findings: The discovery that the RNA cargo of EVs secreted from aneuploid cleavage stage embryos is diverse from that of euploid embryos potentiates the development of non-invasive methodology for PGT-A. The upregulation of MUC1 in dESCs following aneuploid embryo EV treatment proposes a new mechanism underlying implantation failure. Funding: The study was supported by a MSCA fellowship awarded to SM by the European Commission (CERVINO grant agreement ID: 79620) and by a BIRTH research grant from Theramex HQ UK Ltd

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Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts (miRNA)

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