GENERA, Centre for Reproductive Medicine, Clinica Valle Giulia, Rome, Italy; GENETYX, Molecular Genetics Laboratory, Vicenza, Italy.
GENERA, Centre for Reproductive Medicine, Clinica Valle Giulia, Rome, Italy; GENETYX, Molecular Genetics Laboratory, Vicenza, Italy.
Fertil Steril. 2016 Jan;105(1):225-35.e1-3. doi: 10.1016/j.fertnstert.2015.09.014. Epub 2015 Oct 9.
To assess whether extracellular microRNAs (miRNAs) can be accurately profiled from spent blastocyst culture media (SBM) and used as embryonic biomarkers.
Prospective cohort study.
Private and academic in vitro fertilization centers.
PATIENT(S): Inner cell mass-free trophectoderm (TE) samples and their relative SBM from five good-quality human blastocysts.
INTERVENTION(S): Protocol for miRNA purification and analysis based on quantitative polymerase chain reaction set and validated on human embryonic stem cells (hESCs) and on SBM with and without biological variability.
MAIN OUTCOMES MEASURE(S): Analysis of miRNAs in culture media in relation with TE cells and comparison of miRNA profiles between implanted and unimplanted euploid blastocysts.
RESULT(S): Culture media from embryos in the cleavage, morula, and blastocyst stages were collected to investigate the presence of miRNAs. The SBM were prospectively collected from euploid implanted (n = 25) and unimplanted blastocysts (n = 28) for comparison. We hypothesized that human embryos secrete miRNAs in culture media that can be used as biomarkers. The comparative analysis of TE and SBM samples revealed that 96.6% (57 of 59; 95 CI, 88.3-99.6) of the miRNAs detected in the SBM were expressed from TE cells, suggesting a TE origin. The culture media collected from cleavage and morula stage embryos showed a pattern similar to blanks, suggesting that miRNAs profiling from spent culture media applies only for blastocysts. MicroRNAs analysis of SBM from euploid implanted and unimplanted blastocysts highlighted two miRNAs (miR-20a, miR-30c) that showed increased concentrations in the former and were predicted in silico to be involved in 23 implantation-related pathways.
CONCLUSION(S): MicroRNAs secreted from human blastocysts in culture media can be profiled with high reproducibility, and this approach can be further explored for noninvasive embryo selection.
评估胚胎培养液（SBM）中是否可以准确地分析细胞外微小 RNA（miRNA），并将其用作胚胎生物标志物。
前瞻性队列研究。
私人和学术体外受精中心。
来自五个优质人类囊胚的无内细胞团滋养外胚层（TE）样本及其相对 SBM。
基于定量聚合酶链反应（qPCR）方案的 miRNA 纯化和分析方案，并在人类胚胎干细胞（hESC）和具有和不具有生物学变异性的 SBM 上进行了验证。
分析与 TE 细胞相关的培养介质中的 miRNA，并比较植入和未植入整倍体囊胚的 miRNA 谱。
在卵裂、桑椹胚和囊胚阶段收集胚胎培养液以研究 miRNA 的存在。前瞻性收集来自植入的整倍体囊胚（n = 25）和未植入囊胚（n = 28）的 SBM 进行比较。我们假设人类胚胎在培养物中分泌 miRNA，可以用作生物标志物。TE 和 SBM 样本的比较分析显示，在 SBM 中检测到的 miRNA 中有 96.6%（57 个中的 59 个；95%CI，88.3-99.6）来自 TE 细胞，提示来源于 TE。从卵裂和桑椹胚期胚胎收集的培养液显示出与空白相似的模式，表明仅适用于囊胚的耗竭培养物中 miRNA 分析。来自植入的整倍体囊胚和未植入囊胚的 SBM 的 miRNA 分析突出了两个 miRNA（miR-20a，miR-30c），它们在前一种情况下浓度增加，并在计算机上预测与 23 个植入相关途径有关。
从培养物中分泌的人类囊胚的 miRNA 可以进行高重复性的分析，并且可以进一步探索这种方法用于非侵入性胚胎选择。
