Expression of miR‐Let‐7a, miR‐15a, miR‐16‐1, and their target genes in fresh and vitrified embryos and its surrounding culture media for noninvasive embryo assessment

Abstract

microRNAs (miRNAs) play a critical role in implantation and development of mouse embryos. In this study, we aim to evaluate the possibility of miRNAs as potential biomarkers in the blastocyst culture to assess embryo quality. We also intend to investigate whether improved clinical outcomes of vitrified embryos agree with altered miRNA expressions. Mouse embryos from in vitro fertilization were vitrified at the two-cell stage. After thawing, the embryos were individually cultured and developed to the blastocyst stage. We used quantitative real-time polymerase chain reaction to evaluate miRNA expression levels in both vitrified and fresh groups, and culture medium (CM). The fibronectin binding assay was performed to examine for blastocyst attachment.The findings showed reduced expressions of miR-16-1 (0.2 ± 0.06) and miR-Let-7a (0.65 ± 0.1) after vitrification compared to fresh embryos. We observed significant upregulation of the target genes Vav3 (4.33 ± 0.25), integrin β-3 (Itg β3; 4.73 ± 0.2), and Bcl2 (2.29 ± 0.16) in the vitrified embryos compared to the fresh groups. Evaluation of blastocyst CM showed upregulation of miR-Let-7a (15.68 ± 0.89), miR-16-1 (16.18 ± 0.75), and miR-15a (13.36 ± 0.73) in the vitrified group in comparison to the fresh blastocysts (P < .05). The expression levels of miR-16-1 (3.28 ± 0.63), miR-15a (5.91 ± 0.38), and miR-Let-7a (9.07 ± 0.6) in CM of the vitrified blastocysts conducted on fibronectin were significantly higher than the fresh group (P < .05).This study showed that vitrification of embryos changes implantation and proliferation biomarkers. In addition, upregulated miRNAs in CM could be potentially used for noninvasive early assessment of embryo quality.

Murine Blastocysts Release Mature MicroRNAs Into Culture Media That Reflect Developmental Status

“…Extracellular miRNAs and extracellular vesicles (EVs) of embryonic origin are of particular interest owing to their potential application in the ongoing initiative aimed at improving embryo transfer outcomes during assisted reproduction [reviewed by Hawke et al (2020)]. Clinical studies are now reporting detection of nearly 150 miRNAs within spent media microdroplets used to culture human blastocysts (Russell et al, 2020); miRNAs have also been identified within the spent media used to culture bovine (Kropp et al, 2014;Kropp and Khatib, 2015;Gross et al, 2017b;Lin et al, 2019a,b) and murine (Heidari et al, 2019) pre-implantation embryos. Embryo-derived exosomes have been identified in the spent media conditioned with pre-implantation embryos of numerous species, including human (Saadeldin et al, 2014;Giacomini et al, 2017;Mellisho et al, 2017), and both miRNAs (Battaglia et al, 2019) and EVs (Battaglia et al, 2019;Simon et al, 2020) have also been identified within the blastocyst cavity.…”

Extracellular vesicles, microRNA and the preimplantation embryo: non-invasive clues of embryo well-being

“…Ailing blastocysts presenting degenerating morphology confirmed that implantation failure or even a history of vitrification release greater amounts and variety of miRNA sequences into the spent culture medium; however, the correlation with ploidy status remains unclear. miR-16-1 and let-7a, despite being downregulated in whole vitrified murine blastocysts compared with fresh blastocyst controls (∼5-fold and ∼1.5-fold, respectively), showed an increase in the concentration of both sequences (∼16-fold each) in the conditioned medium (Heidari et al, 2019). Given the transcriptional overlap between the secretome and the trophectoderm and even whole embryo, this may suggest that vitrified embryos have a much more active release mechanism, i.e.…”

“…media selection, additives, microdroplet sizes, embryo culture density and culture times. In all studies, human and animal models alike, data were presented as either unnormalized (Gombos et al, 2019), normalized to an exogeneous spike-in (Kropp et al, 2014;Borges Jr. et al, 2016;Abu-Halima et al, 2017;Gross et al, 2017;Cimadomo et al, 2019), normalized to an endogenous control: snU6 (Rosenbluth et al, 2014;Cuman et al, 2015;Heidari et al, 2019;Lin et al, 2019a), B2M (Heidari et al, 2019), SNORD96 (Sánchez-Ribas et al, 2019), GAPDH (Kropp et al, 2014), miR-16-2 (Abu-Halima et al, 2017 or miR-372 (Battaglia et al, 2019), or a combination of these strategies. The choice of normalization strategy may dictate the outcome of a secretomics study (Abu-Halima et al, 2017).…”

“…Clinical studies have sought to correlate the miRnome with pregnancy outcomes after transfer and association with ploidy status according to PGT-A data, whereas studies involving animal models have sought correlation with morphology, developmental pace, ability to form outgrowths (Heidari et al, 2019) and cryopreservation (TABLE 2). Ailing blastocysts presenting degenerating morphology confirmed that implantation failure or even a history of vitrification release greater amounts and variety of miRNA sequences into the spent culture medium; however, the correlation with ploidy status remains unclear.…”

Alterations of miR-16, miR-let-7a and their target genes expression in human blastocysts following vitrification and re-vitrification

“…MiR-let7 is among the first discovered microRNAs which is phylogenetically preserved as well. Previous studies indicate decreased levels of this microRNA in mouse embryos prior to implantation [ 9 , 10 ] In a study by Liu et al [ 11 ] it was shown that let-7a is involved in implantation process, partly via regulation of integrin-β3 expression. Integrins are transmembrane heterodimer glycoproteins and are expressed by trophectoderm during implantation and mediate embryonic adhesion through binding to an extracellular mediatory ligand [ 12 ].…”