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Small noncoding RNAs (sncRNAs) are key regulators of the majority of human reproduction events. Understanding their function in the context of gametogenesis and embryogenesis will allow insight into the possible causes of in vitro fertilization (IVF) implantation failure. The aim of this study was to analyze the sncRNA expression profile of the spent culture media on day 4 after fertilization and to reveal a relationship with the morphofunctional characteristics of gametes and resultant embryos, in particular, with the embryo development and implantation potential. Thereto, cell-free, embryo-specific sncRNAs were identified by next generation sequencing (NGS) and quantified by reverse transcription coupled with polymerase chain reaction (RT-PCR) in real-time. Significant differences in the expression level of let-7b-5p, let-7i-5p, piR020401, piR16735, piR19675, piR20326, and piR17716 were revealed between embryo groups of various morphological gradings. Statistically significant correlations were found between the expression profiles of piR16735 and piR020401 with the oocyte-cumulus complex number, let-7b-5p and piR020401 with metaphase II oocyte and two pronuclei embryo numbers, let-7i-5p and piR20497 with the spermatozoid count per milliliter of ejaculate, piR19675 with the percentage of linearly motile spermatozoids, let-7b-5p with the embryo development grade, and let-7i-5p with embryo implantation. According to partial least squares discriminant analysis (PLS-DA), the expression levels of let-7i-5p (Variable Importance in Projection score (VIP) = 1.6262), piR020401 (VIP = 1.45281), and piR20497 (VIP = 1.42765) have the strongest influences on the implantation outcome.

Despite the improvements in biotechnological approaches and the selection of controlled ovarian hyperstimulation protocols, the resulting pregnancy rate from in vitro fertilization (IVF) protocols still does not exceed 30–40%. In this connection, there is an acute question of the development of a non-invasive, sensitive, and specific method for assessing the implantation potential of an embryo. A total of 110 subfertile couples were included in the study to undergo the IVF/ICSI program. Obtained embryos for transfer into the uterine cavity of patient cohort 1 (n = 60) and cohort 2 (n = 50) were excellent/good-quality blastocysts, and small noncoding RNA (sncRNA) content in the corresponding spent culture medium samples at the morula stage (n = 43) or at the blastocyst stage (n = 31) was analyzed by deep sequencing followed by qRT-PCR in real time. Two logistic regression models were developed to predict the implantation potential of the embryo with 100% sensitivity and 100% specificity: model 1 at the morula stage, using various combinations of hsa_piR_022258, hsa-let-7i-5p, hsa_piR_000765, hsa_piR_015249, hsa_piR_019122, and hsa_piR_008112, and model 2 at the blastocyst stage, using various combinations of hsa_piR_020497, hsa_piR_008113, hsa-miR-381-3p, hsa_piR_022258, and hsa-let-7a-5p. Protein products of sncRNA potential target genes participate in the selective turnover of proteins through the ubiquitination system and in the organization of the various cell cytoskeleton and nucleoskeleton structures, regulating the activity of the Hippo signaling pathway, which determines the fate specification of the blastomers. [ABSTRACT FROM AUTHOR]