Intracellular miRNAs influence embryo viability
Embryo viability is one of the key factors affecting implantation. During their development from zygotes to blastocysts, mammalian embryos undergo multiple events, including cell division, proliferation, establishment of cell polarity, compaction and lineage differentiation [
38]. Unlike primates and rodents, whose embryos almost immediately attach to the endometrium after shedding from the zona pellucida, the embryo of ruminants and pigs experience a process termed ‘elongation’ where the embryo goes through morphological changes, developing from a spherical shape to an oval or tubular shape, and eventually to a filamentous form before attachment [
4]. In horses, the embryo undergoes an extended period of mobility, growing as an ovoid shaped conceptus without obvious morphological extension [
39].
Most of the embryonic changes can be related to activity of the genome [
40]. Studies in mice revealed that the genomic information within pre-implantation embryo experiences wave-like changes associated with degradation of maternal transcriptome and zygote genome activation (ZGA) [
41]. Though miRNA functions as a transcriptional regulator, its role in clearance of the maternal genome remains controversial. Studies have shown that miRNA function is generally suppressed in mouse oocytes and early pre-implantation embryos [
42]. MiRNA activity was inhibited prior to the 2-cell stage, which is consistent with the timing of large-scale maternal gene degradation. However, the suppression is relieved after 2-cell stage and the expression of miRNA is reactivated [
43].
Despite the repression of miRNA activity during ZGA, deletion of zygotic Dgcr8, which encodes an RNA-binding protein specifically required for miRNA processing, results in embryonic arrest prior to E6.5 [
42]. Knocking out other key enzymes in the miRNA biosynthesis pathway such as DICER [
44] and AGO2 [
45] also leads to embryonic death around gastrulation, suggesting an important role of miRNA in early embryonic development [
46]. Analysis of embryos at different developing stages revealed variable trends in miRNA expression, and the role of specific miRNAs in embryonic development has been studied in multiple species. MiR-29b might contribute to disruption of DNA methylation by regulating the expression of
DNMT3a/b, which leads to early embryonic developmental blockade in mice [
47]. Higher expression level of miR-130b was verified in the morula and blastocyst stages of bovine IVF produced embryos, while inhibition of this miRNA significantly reduced morula and blastocyst formation [
48]. By inducing embryonic stem cells to differentiate into trophectodermal cells, miR-297, miR-96, miR-21, miR-29c, let-7, miR-214, miR-125a, miR-424 and miR-376a were suggested to be involved in trophectoderm specification [
49]. MiR-519d, miR-378a-5p, miR-376, and miR-155 were reported to regulate the migration and invasion ability of human trophoblast cells [
50‐
53]. MiRNAs are also implicated in regulation of embryo elongation. Functional annotations for comparisons among porcine conceptuses collected on Day10 (spherical/ovoid shape), Day 12 (filamentous form), Day 16 (elongated shape), and Day 20 (presence of evident vascularization on embryonic tissues) revealed that the differently expressed miRNAs were associated with cell cycle, cellular development, tissue morphology, inflammatory response and organismal development [
54].
Environmental factors can regulate embryo viability by altering the expression of miRNAs. The mice embryo, when exposed to a progesterone-primed uterus, becomes metabolically dormant, and implantation is delayed. However, dormant embryos can be rapidly activated by a slight stimulus of estrogen and regain their implantation competency [
7]. Delayed implantation in mice can be artificially induced through progesterone injection, which provides an excellent model for investigating the environmental influence on embryo implantation. Liu et al. [
55] examined the miRNA profiles between dormant and activated mouse embryos and found that 45 miRNAs were differently expressed. Five of the let-7 family were down-regulated after activation. Further investigation revealed elevated let-7a reduced the number of implantation sites partly through targeting integrin beta 3. Another study in mice verified the relatively high level of let-7a in dormant embryos and found that this miRNA could also inhibit the expression of Dicer and prevent embryo implantation [
56]. Although there is no evidence that delayed implantation exists in human and domestic animals, studies in mice suggest that in vivo environmental signals alter the miRNA expression patterns and eventually influence the active status of the pre-implantation embryo.
An embryo can be produced in vitro. However, production methods affect the expression pattern of miRNA in pre-implantation embryos, which in turn, affect embryo viability. In vivo fertilized porcine embryos presented lower expression of miR-24 in the blastocyst stage compared with in vitro fertilized (IVF) embryos [
57]. A study in cattle revealed that elevated expression of miR-24 inhibited the development of embryo to the blastocyst stage [
58]. Considering miR-24 is highly conserved across mammalian species, it may serve as biomarker for embryo quality. Down-regulated miR-199a-5p was shown in IVF mouse embryos compared with in vivo produced embryos, leading to higher glycolytic rate and lower developmental potential of blastocyst as well as reduced number of survived fetuses [
59]. Together, these results reinforce the epigenetic modifications induced by the environmental factors on embryo development, since in vitro environment does not perfectly capitulate normal environment in maternal uterus [
59], clarifying the functional miRNAs may help to improve the IVF system by artificially adjusting the amount of specific miRNA expression.
Recent studies demonstrate that miRNAs exist not only within the embryo but can also be secreted by the embryo to the extracellular environment. MiRNAs have been detected in the culture media (CM) derived from IVF human and bovine embryos, with their unique expression profiles associated with the embryonic developmental and chromosomal status, sexual dimorphism, and the reproductive competence after transfer to the uterus.
Kropp et al. [
60] compared the expression of several miRNAs between IVF bovine blastocysts and degenerate embryos (IVF embryos failed to develop to the blastocyst stage) and found relatively higher levels of miR-181a2, miR-196a2, miR-302c and miR-25 in degenerate embryos. They further investigated the miRNA contents in the CM and found that miR-25 was only present in CM containing embryos but not in the control media (embryo free). This finding suggested that miRNAs might be secreted by the embryos. Additionally, the absence of miR-302 in all media indicated that the secretion of miRNAs was selective. In another study, Kropp and Khatib [
58] applied deep sequencing to characterize miRNA profiles in CM from IVF produced bovine blastocysts and degenerate embryos, and miR-24 was confirmed to be highly expressed in CM from degenerate embryos. Addition of miR-24 mimics to the CM from normal morula significantly reduced the development rate of embryos, partly through inhibiting cell proliferation by targeting
CDKN1b, a cell cycle inhibitor. The result indicates the significant effect of extracellular miRNAs on embryo development.
The presence of miRNAs has also been confirmed in the CM from IVF human embryos. Rosenbluth et al. [
61] found that miR-645 was only expressed in the control media and was found to be undetectable in CM from embryos. On the contrary, miR-372 and miR-191 were only detected in the spent CM. Moreover, the expressions of extracellular miR-372 and miR-191 were associated with IVF failure, since they were both highly detected in CM from failed IVF cycle embryos. Interestingly, a relatively higher level of miR-191 was discovered in CM from aneuploid embryos, indicating the possible role of miR-191 as a biomarker of embryo aneuploidy, a major cause of recurrent implantation failure.
Capalbo et al. [
62] conducted a comprehensive analysis of miRNA profiles between the spent blastocyst culture media (SBCM) collected from human euploid blastocyst that failed to implant and blastocyst that implanted. Two miRNAs, miR-20a and miR-30c, presented significantly higher expression in SBCM from implanted blastocysts. Intriguingly, the target genes (such as
PTEN, NRAS, MAPK1, APC, KRAS, PIK3CD, SOS1) of these miRNAs were predicted to be involved in endometrial cell proliferation, which suggested the potential of blastocyst secreting miRNAs as modulators of the uterine functions. However, this speculation was not verified in this study. The group also tested the CM from embryos at other developmental stages (cleavage and morula) and discovered that the analyzed miRNAs in SBCM were specific to the blastocyst stage, strengthening the point that during this particular stage the embryo may send signals to the environment in order to facilitate the subsequent implantation process.
A very recent study showed that embryos with different genders secreted different miRNAs [
63]. A relatively abundant amount of miR-22, miR-122 and miR-320a were detected in CM from female bovine embryos. Taking into account that male and female embryo apply different adaptations to the external environment, they may secrete different miRNAs into the maternal environment, inducing transcriptional response of the mother to create an appropriate environment for their development.
The detection of miRNAs in the CM from pre-implantation embryos shed new light for embryo screening in the IVF process. At present, the methods used for screening embryos can be categorized as non-invasive ways and invasive ways. Non-invasive screening is mainly based on the morphological observation and metabolic profiling of the CM in order to determine the development status of embryos [
64]. Although advanced technologies such as the time-lapse system have permitted keeping track of the development steps of an embryo under minimum variation of the culture environment [
65], chromosomal abnormalities - which contribute to repeated failure of implantation, taking up 44.9% of morphological normal embryos [
66] - cannot be ruled out. Invasive screening methods are able to identify the genomic information within the embryos. Novel technologies such as comparative genomic hybridization overcome the limitation of pre-implantation genetic screening and fluorescence in situ hybridization, promising a comprehensive analysis of chromosomes. However, the challenge of invasive screening remains regarding the damage to the embryos, and there is no definite conclusion that biopsy procedure will do no harm to the further development of embryos after they have been transferred to the uterus.
An ideal screening method should be non-invasive and accurate. Based on such consideration, miRNAs secreted by pre-implantation embryos may serve as potential biomarkers in the screening process because of their embryonic specificity, stability and easy access. However, how the secreted miRNAs are packaged might influence the judgement of embryo quality. Only one study has shown that embryonic secretion of miRNAs was carried through AGO1 [
23]. None of the studies mentioned above have tested the exact release form of these extracellular miRNAs. What should be noticed is that the composition of the CM (e.g. the addition of BSA [
60]) and the manipulation strategies (e.g. fertilization methods) [
61,
67] have certain influence on extracellular miRNA profiles. Further investigation should clarify the forms of extracellular miRNAs within the CM in order to improve the accuracy of selection. Moreover, whether the discussed miRNAs can be used to reflect embryo viability is defined by their relative expression levels, rather than their existence, even though the latter makes them more ideal indicators. Hence, repeated experiments are required to establish measurement standards of miRNA expressions before taking them as effective biomarkers.