Table 2.

Summary of the studies in embryonic tissues.

Reference	Location	Age (years)	Population Studied	Study Design	Outcome and Aim	Analysis and Normalization Methods	Principal Conclusion	Quality Score
[41] (Capalbo et al., 2016)	Italy	ND	ICM-free TE samples and their relative SBM from 5 good-quality human blastocysts	Prospective	To identify miRNAs secreted by human embryos in culture media, which can be used as biomarkers of embryo quality during IVF cycles.	Analysis: qRT-PCR screening and qRT-PCR validation. Normalization: RNU44 and RNU48.	The comparative analysis of TE and SBM samples revealed that 96.6% (57/59) of the miRNAs detected in the SBM were derived from TE cells. MiRNAs analysis of SBM from euploid implanted and unimplanted blastocysts highlighted two miRNAs (hsa-miR-20a and 30c) that showed increased concentrations in the former.	7/14
[42] (Rosenbluth et al., 2014)	USA	ND	28 tested blastocysts SBM, from 13 couples	Prospective	To determine whether human blastocysts secrete miRNAs into culture media and whether these reflect embryonic ploidy status and can predict IVF outcomes.	Analysis: qRT-PCR screening and qRT-PCR validation. Normalization: U6.	Ten miRNAs (hsa-miR-106b, 191, 30c, 372, 376a, 548a-3p, 548c-3p, 548d-3p, 576d-3p, and 603) were consistently detected in the spent IVF culture media, but only two miRNAs (hsa-miR-372 and 191) were confirmed by later single assay qRT-PCR. Hsa-miR-191 was more highly concentrated in media from aneuploid embryos, and hsa-miR-191, 372, and 645 were more highly concentrated in media from failed IVF/non-intracytoplasmic sperm injection cycles.	7/14
[36] (Dominguez et al., 2014)	Spain	Screening: EP: 30.75 ± 1.78, VTOP: 26 ± 3.17. Validation: EP: 30.81 ± 1.55, VTOP: 24.62 ± 1.86.	Cases: 23 patients suffering from tubal EP (8 in screening and 15 in validation). Controls: 29 patients with VTOP (8 in screening and 21 in validation).	Case-control	To investigate the miRNA profile of embryonic tissues in EP and controlled abortions VTOP	Analysis: Microarray screening and qRT-PCR validation. Normalization: SNORD96A.	Four miRNAs (hsa-miR-196b, 30a, 873, and 337-3p) were found to be downregulated in EP versus healthy pregnancy tissues, and three miRNAs (hsa-miR-1288, 451, and 223) were upregulated in EP compared to control pregnancy tissue samples. Validation confirmed the differentially expression of the miRNAs hsa-miR-196 and 223.	7/12
[40] (Lozoya et al., 2014)	Spain	EP: 30.9, VTOP: 21.1	Cases: 17 patients suffering from tubal EP. Controls: 23 patients with VTOP.	Case-control	To determine the expression of the elements of the Lin28/Let-7 system, and related miRNAs in early stages of human placentation and ectopic pregnancy	Analysis: qRT-PCR. Normalization: RNU6.	LIN28B mRNA was barely detectable in embryonic tissue from early stages of gestation and sharply increased thereafter to plateau between gestational weeks 7–9. In contrast, expression levels of Let-7, mir-132 and mir-145 were high in embryonic tissue from early gestations (≤6-weeks) and abruptly declined thereafter, especially for Let-7. Opposite trends were detected for mir-323-3p. Embryonic expression of LIN28B mRNA was higher in early stages (≤6-weeks) of ectopic pregnancy than in normal gestation. In contrast, Let-7a expression was significantly lower in early ectopic pregnancies, while miR-132 and miR-145 levels were not altered. Expression of mir-323-3p was also suppressed in ectopic embryonic tissue.	6/12
[37] (McCallie et al., 2010)	USA	ND	Cryopreserved blastocysts (n = 22). Cases: blastocysts from MF infertility alone (n = 6), and blastocysts from PCOS women (n = 6). Controls: Oocyte donor cycles with no known MF infertility (n = 10)	Case-control	To examine human blastocyst miRNA expression (11 probes) in correlation with human infertility.	Analysis: qRT-PCR. Normalization: RNU48.	Morphologically similar blastocysts derived from patients with PCOS or MF infertility exhibited a significant decrease in the expression of six miRNAs (hsa-let-7a, miR-19a, 19b, 24, 93 and 94) in comparison with donor fertile control blastocysts. Annotation of predicted gene targets for these DE-miRNAs included gene ontology biological processes involved in cell growth and maintenance and transcription.	6/12
[39] (McCallie et al., 2014)	USA	Chromosomally normal blastocystsfrom young, OD (26.4 years), Chromosomallynormal blastocysts from women in their forties (40–44 years)	Cases: blastocysts produced from women in their forties (n = 5). Controls: young oocyte donor derived blastocysts (n = 5)	Case-control	To determine miRNA expression in human blastocysts relative to advanced maternal age and chromosome constitution.	Analysis: qRT-PCR. Normalization: MammU6.	42 DE-miRNAs. miR-93 was exclusively expressed in blastocysts from women in their forties and further up-regulated with an abnormal chromosome complement. Up-regulated miR-93 resulted in an inverse down-regulation of targets like SIRT1, resulting in reduced oxidative defense.	5/12
[38] (Rosenbluth et al., 2013)	USA	ND	Screening (Cases: 5 aneuploids. Controls: 4 male, 5 females euploid.). Validation (Cases: 9 aneuploids. Controls: 7 male, 11 females euploid.).	Case-control and Descriptive	To determine the most highly expressed miRNAs in human blastocysts and to compare miRNAs in euploid versus aneuploid embryos and in male versus female embryos.	Analysis: qRT-PCR screening and qRT-PCR validation. Normalization: snRNA U6.	The most highly expressed miRNA in euploid embryos was miR-372. Screening identified 39 miRNAs that were differentially expressed between euploid (n = 9) and aneuploid (n = 5) embryos, and 21 miRNAs that were differentially expressed between male (n = 4) and female (n = 5).	Case-control: 5/12. Descriptive: 6/9.

Abbreviations: DE-miRNAs, differentially expressed miRNAs; EP, ectopic pregnancies; ICM, inner cell mass; IVF, in vitro fertilization; MF, male factor; ND, no data; OD, oocyte donors; PCOS, polycystic ovary syndrome; qRT-PCR, quantitative real-time PCR; SBM, spent blastocyst culture media; TE, trophectoderm; VTOP, voluntary termination of pregnancy.