1. Introduction
It is well-established that embryos of differing developmental potential have different genomic, proteomic, and metabolomic profiles [
1]. These variations in expression are detectable intracellularly, and, more recently, in the spent culture media of in vitro fertilization (IVF) systems [
1,
2]. This presents an opportunity to optimize the embryo selection process and improve prognosis, as selection is crucial for successful implantation and live birth rates [
3]. The traditional morphological evaluation is still considered the gold standard for embryo selection [
4,
5]. Cleavage rates and blastocyst formation are often judged subjectively, so some selected embryos may fail to implant or miscarry from underlying chromosomal abnormalities [
6]. While pre-implantation genetic screening (PGS) can detect aneuploidies, this service requires an invasive embryo biopsy and can be costly [
7]. Therefore, there is a need to establish a non-invasive, highly accurate method of embryo selection. The analysis of small molecules found in the spent media (SM) has the possibility of revealing biomarkers related to intrinsic embryo physiology. Strong evidence suggests that array-based metabolomics and miRNAomic analysis are good candidates for adjunct assessment methods of embryo quality. Metabolomics and the miRNAomic profiling of embryo SM have revealed distinct signatures between embryos of different morphological appearance [
8], chromosomal status [
9], sex [
10], and implantation outcome.
Advancements in time-lapse imaging have also revealed the importance of a morpho-kinetic assessment in assessing embryo quality [
11,
12]. Time-lapse imaging allows the embryologist to visualize the embryos in the incubator in between assessments, while minimizing the negative effects of removing the embryos from the incubator. This is an important tool, given the status and grade of an embryo can change within hours [
13,
14]. This relatively new technique has shown the timing of the onset and duration of key morphological events, such as cleavage, compaction, and blastocyst formation, may indicate normal and aberrant embryo development. Since in vivo embryos develop faster than their in vitro counterparts, it is commonly accepted that faster-developing in vitro embryos are healthier [
15]. In fact, research has shown in vitro embryos that cleave earlier have higher blastocyst rates. The timing may be indicative of the stress experienced by an embryo. The absence or low levels of stress factors such as reactive oxygen species may mean embryos can develop faster as less time can be spent initiating repair pathways [
15].
However, other groups have presented a counter idea that the slower-growing embryo has more time to correctly initiate and choreograph the events of embryogenesis. Research by Market-Velker and colleagues compared slow-growing (SG) and fast-growing (FG) embryos with in vivo embryos on factors such as methylation status, expression of imprinted genes, embryo cell number, and morphology [
16]. Their findings showed that SG embryos were most similar to in vivo embryos on all parameters measured [
15]. Genomic imprinting and the expression of metabolic markers in SG embryos closely mirrored those of in vivo embryos. Market-Velker and colleagues postulate FG embryos may transition too rapidly during the first few embryonic stages, causing an inability to maintain epigenetic information [
16]. Thus, it appears embryos grow within a time range, whereby anything too slow may indicate a pathological condition, while anything too fast may signify an embryo erroneously rewired to move onto the next developmental stage.
To date, few studies have profiled SM conditioned with SG and FG embryos. One study that did so examined the metabolites present in the SM cultured with SG and FG embryos. Using nuclear magnetic resonance (NMR), Perkel and Madan were able to detect distinct metabolic signatures in the SM between SG and FG embryos at the 2-cell, 8-cell, 16-cell, and blastocyst stage of development [
17]. Specifically, their data showed distinct differences between media cultured with the 4-cell SG and FG embryos for pyruvate, and at the 16-cell stage for acetate, tryptophan, leucine/isoleucine, valine, and histidine. Four-cell SG embryos had a higher consumption of pyruvate, while 16-cell SG embryos released more acetate into the media, in comparison to their FG counterparts [
17]. Acetate is produced when there is an over-abundance of acetyl-CoA, such as in situations where the Krebs cycle or electron-transport chain is malfunctioning. Since both processes lie within the mitochondria, this observation suggests SG embryos exhibit some level of mitochondrial dysfunction, resulting in a metabolic disturbance.
The embryonic mitochondrial biogenesis analysis conducted in our lab revealed metabolic distress may be related to mitochondrial dysfunction. In this study, GLYCOX and OXPHOS gene expression were examined in SG and FG embryos at the 2-cell, 8-cell, morula, and blastocyst stage of development [
18]. GLYCOX and OXPHOS genes regulate the pathways in embryo energy production, whereby OXPHOS is a mitochondrial-dependent pathway and dominates in early embryo development, and GLYCOX dominates in late embryo development [
18]. The data indicate SG embryos had a higher expression of both OXPHOS and GLYCOX genes at all time-stages in comparison to FG embryos. This over-expression may serve as a compensatory mechanism for mitochondrial dysfunction occurring in SG embryos. Overall, the data from our lab suggest that metabolic assays are sensitive in detecting differences between embryos growing at different rates [
18].
Recent data from the Madan lab has also shown the miRNA expression can be detected in the SM of embryos cultured in vitro [
19]. This study clearly demonstrated that SM can be used to detect the miRNA expression from embryos harvested at timed stages of developmental. However, determining the global SM miRNA expression profiles between SG and FG embryos has not been attempted. Therefore, the objective of the present study was to globally profile, using a heterologous miRNA microarray, the miRNAs expression in the SM of SG and FG embryos at the 2-cell, 8-cell, and blastocyst stage of development.
4. Discussion
This study is pioneering in its global profiling of miRNAs in the SM conditioned with embryos growing at different developmental rates. Our results indicate distinct miRNA populations can be identified between SG and FG embryos. More importantly, these unique miRNA signatures can be detected at the early, mid, and late stages of pre-implantation embryo development. Our results suggest SG embryos, at all conditions examined (2-cell SM, 8-cell SM, and blastocyst SM), preferentially release miRNAs into the extracellular environment. Although no other study has reported this, metabolomics studies have detected distinct metabolites in the SM conditioned with embryos differing in developmental rate and viability. These studies suggest lower-quality embryos are metabolically more active than their higher-quality counterparts. Since metabolism is influenced by the genes expressed within the cell, it can be postulated that increases in embryonic metabolism are preceded by higher genetic activity. Perhaps miRNAs are used within the embryo to initiate and modulate gene expression influencing metabolic turnover. Evidence does suggest the extracellular miRNA population serves as a good indicator of intracellular miRNA expression. Thus, our result indicates increases in metabolic activity in non-viable and SG embryos may also be driven by increases in miRNA expression, which are detectable in SM.
Across the three conditions examined, only the miRNAs detected between blastocyst SG vs. blastocyst FG SM had some previous annotation in literature. Specifically, miR-320a and miR-24-3p, which were detected to be upregulated in blastocyst SG SM, have also been cited in previous SM studies. According to Kropp and Khatib, miR-24-3p was one of the five miRNAs they detected to be upregulated in SM conditioned with degenerate blastocyst [
8]. In a subsequent supplementation study, miR-24 was added to the plain media of morula stage embryos. Supplementation resulted in a 44-fold increase in expression of miR-24 in blastocyst cultured with miR-24 and a 27.3% decrease in blastocyst rates [
8]. Kropp and Khatib postulate miR-24 influenced the expression of CDKN1b, which is a cell-cycle regulator. Despite the differences in the classification of non-viable embryos, whereby we used the developmental rate and Kropp and Khatib examined arrested/degenerate embryos, both studies indicate miR-24 may serve as a biomarker of embryo viability at the blastocyst stage of development.
Another miRNA identified in this study and previously annotated in literature was miR-320a. A recent study by Berkhout and colleagues suggest that miR-320a is a pre-implantation marker secreted by embryos. Specifically, the researchers profiled the miRNome of SM conditioned with embryos either scoring low or high in morphological scores [
25]. It was determined that miR-320a was secreted by higher-quality embryos. Subsequently, miR-320a was supplemented in the culture media of human embryonic stem cells. Berkhout and colleagues reported miR-320a was able to stimulate the migration of decidualized human embryonic stem cells, with a downstream transcriptome analysis revealing miR-320a modulates genes regulating cell adhesion and cytoskeleton organization [
25]. Interestingly, our study found miR-320a to be upregulated in SM conditioned with SG embryos. Capalbo and colleagues have postulated embryos release miRNAs into the extracellular environment as a means of paracrine communication with endometrial tissue [
6]. Thus, the findings in our study suggest miR-320a may serve to inhibit implantation as it was found to be released by SG embryos. Although consensus about the role of miR-320a is mixed, it should be noted that our study and the one conducted by Berkhout and colleagues were carried out in different species and culture conditions were not identical. Thus, inter-species differences and environmental conditions may have influenced the findings of both studies. Perhaps, miR-320a may have both inhibitory and stimulatory effects on implantation as miRNAs have various targets within the genome.
Aside from miR-320a and miR-24, miR-615 and miR-17 have also been previously annotated in literature, albeit in cancer-related studies. Specifically, miR-615 has been characterized to play a role in angiogenic events influencing tumorigenesis. Icli and colleagues demonstrated that miR-615 has anti-angiogenic effects, whereby the expression of the miRNA significantly inhibited endothelial cell proliferation and migration [
26]. Similarly, miR-17 has been cited in literature to have anti-oncogenic effects. Hossain and colleagues reported miR-17 transfection in breast cancer tissue resulted in the translational repression of the breast-cancer-associated gene
A1B1 [
27]. The subsequent downregulation of
A1B1 decreased breast cancer proliferation. Thus, it seems the miRNAs upregulated in SG embryos at the blastocyst stage have roles in cancer development. Cancer and embryogenesis rely on similar pathways for growth and development. Therefore, it is interesting to see that cancer-related miRNAs are detectable in media conditioned with embryos growing at different rates. Perhaps these anti-proliferative miRNAs in cancer serve to inhibit growth and development in an embryo.
Aside from previously annotated miRNAs, GSEA analysis also revealed novel stage-specific miRNA biomarkers. Bta-miR-1343 and bta-miR-2443 were miRNAs upregulated in SM cultured with 2-cell SG embryos. GSEA analysis indicated the gene targets of the two miRNAs had roles in regulating transcription and cell proliferation. Specifically, the majority of gene targets had biological implications pertaining to the positive and negative regulation of transcription from RNA polymerase II promoter. This is an interesting finding as previous research suggests little to no transcription occurs at the 2-cell stage in bovine embryos. Prior to the 8-cell stage, the developing bovine embryo relies on parentally inherited transcripts (mRNA and miRNA) and proteins for survival. Therefore, it is unclear why the gene targets of miR-1343 and miR-2443 would cluster around promoting transcriptional events.
However, research by Vassena and colleagues does suggest embryos are capable of transcription prior to embryonic genome activation (EGA). Through the genomic-wide transcript analysis of human oocytes and embryos, Vassena and colleagues detected a series of successive waves of embryonic transcriptional initiation events beginning as early as the 2-cell stage [
28]. Therefore, their findings suggest transcriptional events in human embryos may begin as early as the 2-cell stage, and not at the 4–8 cell stage as previously thought. Although unexplored in bovine embryos, our results indicate transcriptional events may be occurring earlier than EGA. It can be hypothesized SG embryos may initiate transcriptional events earlier as a response to its delayed development. The early activation of the embryonic genome may serve as a repair mechanism to salvage an embryo during the pre-implantation period.
It should also be noted bta-miR-1343 and bta-miR-2443 had gene targets relating to spermatogenesis. Previous research profiling the origins of embryonic miRNAs has suggested the majority of miRNAs expressed prior to EGA are of maternal origin. However, researchers did discover that embryos can also inherit sperm-borne miRNAs. Although bta-miR-1343 and bta-miR-2443 have not been annotated in mammalian sperm, results from our study suggest that these miRNAs are of paternal origin, capable of influencing transcriptional events in an embryo.
It should also be highlighted that bta-miR-1343, bta-miR-760-5p, and bta-miR-450b were co-detected in more than one SM condition. Bta-miR-1343 was found to be upregulated in the SM of SG embryos at the 2-cell and 8-cell stage. With regard to bta-miR-760-5p and bta-miR-450bs, their expression was detected in media conditioned with the SG 8-cell and blastocyst embryos. Contrasting bta-miR-1343, both bta-miR-760-5p and bta-miR-450b were upregulated in 8-cell SG media, then were downregulated in blastocyst SG media. Although all three miRNAs have not been previously annotated in embryos or in SM, their consistent expression between 2-cell and 8-cell or 8-cell and blastocyst SM, respectively, suggest they may have functional roles in normal and aberrant embryo development.