Purpose

miRNAs have been suggested as biomarkers of embryo viability; however, findings from preliminary studies are divergent. Furthermore, the presence of other types of small RNA molecules remains to be investigated. The purpose of this study was to perform a comprehensive analysis of small non-coding RNA levels in spent and unconditioned embryo culture media, along with miRNA levels in blastocoelic fluid samples from human embryos.

Methods

miRNAs in unconditioned culture medium from 3 different manufacturers, along with miRNA from day 5 conditioned culture medium, control medium, and corresponding blastocoel fluid from 10 human blastocysts were analyzed with array-based q-PCR analysis. Subsequently, deep sequencing of total and small RNA in day 5 spent culture medium from 5 human blastocysts and corresponding controls was performed.

Results

In spite of using state-of-the-art sensitive detection methods, no miRNAs were found to be reliably present in the spent culture medium or the blastocoel fluid. C_t values were above the recommended limit for detection in the array-based analysis, a finding that was confirmed by deep sequencing. The majority of miRNAs identified by deep sequencing were expressed in all samples including control media and seem to originate from sources other than conditioned IVF media.

Conclusions

Our findings question the use of miRNAs as a reliable biomarker and highlight the need for a critical methodological approach in miRNA studies. Interestingly, tiRNA fragments appear to be overexpressed in conditioned IVF media samples and could potentially be a novel biomarker worthy of investigation.

中文翻译：

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人胚胎培养基和囊胚腔液中可溶性 RNA 的综合分析。

目的

miRNAs 已被建议作为胚胎活力的生物标志物；然而，初步研究的结果各不相同。此外，其他类型的小 RNA 分子的存在仍有待研究。本研究的目的是对用过的和未调节的胚胎培养基中的非编码小 RNA 水平以及来自人类胚胎的囊胚液样本中的 miRNA 水平进行综合分析。

方法

来自 3 个不同制造商的无条件培养基中的 miRNA，以及来自第 5 天的条件培养基、对照培养基和来自 10 个人类囊胚的相应囊胚液中的 miRNA，通过基于阵列的 q-PCR 分析进行了分析。随后，对来自 5 个人类囊胚和相应对照的第 5 天用过的培养基中的总 RNA 和小 RNA 进行深度测序。

结果

尽管使用了最先进的灵敏检测方法，但未发现用过的培养基或囊胚腔液中可靠地存在 miRNA。在基于阵列的分析中，C _t值高于推荐的检测限，这一发现已通过深度测序得到证实。通过深度测序鉴定的大多数 miRNA 在包括对照培养基在内的所有样品中表达，并且似乎来自条件 IVF 培养基以外的来源。

结论

我们的研究结果质疑使用 miRNA 作为可靠的生物标志物，并强调在 miRNA 研究中需要一种关键的方法学方法。有趣的是，tiRNA 片段似乎在有条件的 IVF 培养基样本中过度表达，可能是一种值得研究的新型生物标志物。