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  • One other study used PCR arrays to identify miR expression

    2018-10-25

    One other study used PCR arrays to identify miR expression profiles in BCM (Rosenbluth et al., 2014). They identified two miRs solely expressed in BCM, miR-372 and miR-191 that were not present in control media. miR-372 was expressed in both euploid and aneuploid BCM and was higher in BCM from embryos that failed to implant when correlated with the use of ICSI only embryos. These results are consistent with our finding that miR-372 was detected only in non-implanted BCM samples. miR-191 was not included in the array panel used in our study. We however identified a large number of miRs that were differentially secreted into BCM from implanted compared to non-implanted BCM. The differences in the miRs detected between our study and the previous studies are likely due to differences in the experimental methods between the studies including differences in RNA extraction, cDNA synthesis, miR array panels used for detecting miRs in BCMand, differences in embryo culture media. Specifically, in our study we cultured embryos for 48h compared to 24h, and used different media for culture compared to a previous study (Rosenbluth et al., 2014). This may have affected differences in the miRs detected in BCM between the two studies. In addition, we used a miR array system that required less BCM compared to a previous study suggesting that the array system we used was highly sensitive which may have contributed to differences in detection of specific miRs between the present and other studies (Rosenbluth et al., 2014). In addition, a previous study used embryos that had been frozen at the pronuclear stage, thawed and cultured to histamine-1 receptor antagonist stage and arrays undertaken on BCM collected at the blastocyst stage (Rosenbluth et al., 2014). By comparison, in our study we did not freeze/thaw the embryos which may have affected the pattern of miR secretion between the present and a previous study (Rosenbluth et al., 2014). Extracellular miRs are released from cells in membrane bound vesicles (such as exosomes), bound to RBC proteins (Ago1 and Ago2) or attached to high density lipo-proteins. miRs encapsulated in membrane bound vesicles or attached to proteins, protect miRs from RNase activity (Arroyo et al., 2011).Our study demonstrates a mechanism by which human blastocysts secrete miR-661 and transport it for uptake by the primary endometrial epithelial cells. We demonstrated that extracellular miR-661 was bound to the RBC Ago 1 and not Ago 2 or in vesicles, a finding that has not been previously identified in any cell type. Our study however, does not rule out whether primary HEEC take up other miRs or any other factors present in BCM. Our data however does demonstrate that the increased miR-661 expression in the primary HEEC occurs primarily via uptake from the media and not via stimulation of miR-661 expression in HEEC from factors present in the media. It is unknown if miR transport mechanisms for specific miRs, remain the same in all cell systems or whether a specific cell transports most miRs via one or multiple modes. While this study shows a mechanism of miR transport from human trophectoderm cells in vitro, it remains to be investigated whether this is a generalised phenomenon for most miRs secreted by human blastocysts. Studies investigating the expression of miRs secreted by the endometrium are limited to the capture of exosomes and the miR carried in their cargo (Kresowik et al., 2014; Ng et al., 2013). To date no study, has examined the role Ago proteins play in the communication between the blastocyst and the endometrium. Studies of the miRs in the endometrium are limited to expression studies, comparing the expression of miRs in receptive with non-receptive phase endometrium (Altmae et al., 2013; Kresowik et al., 2014; Kuokkanen et al., 2010), or in endometrium from fertile, infertile and repeat implantation failure (RIF) women (Dior et al., 2014; Revel et al., 2011; Zhao et al., 2012), with the aim of identifying endometrial receptivity biomarkers. To date only one recent study, has investigated a functional role of miRs in human endometrial cells in vitro, specifically miR-145. miR-145, is a previously identified miR with high expression in endometrium from women with repeat implantation failure (RIF) compared to normal fertile women (Revel et al., 2011). miR-145 overexpression in a human endometrial carcinoma cell line, was shown to inhibit mouse embryo adhesion to the cells (Kang et al., 2015). Our study however, provides evidence of direct uptake of a miR from human BCM by HEEC and demonstrates a functional effect on adhesion.