Borges Edson, Setti Amanda Souza, Braga Daniela P A F, Geraldo Murilo V, Figueira Rita de Cássia S, Iaconelli Assumpto
Fertility Medical Group - São Paulo/SP - Brazil.
Instituto Sapientiae - Centro de Estudos e Pesquisa em Reprodução Assistida - São Paulo/SP - Brazil.
JBRA Assist Reprod. 2016 Dec 1;20(4):200-205. doi: 10.5935/1518-0557.20160039.
This study aims to find whether microRNAs (miRNAs) detected in the culture medium of embryos produced in vitro could be potential biomarkers of embryo implantation.
Culture media samples from 36 embryos, derived from patients undergoing intracytoplasmic sperm injection (ICSI) in a private university-affiliated IVF center, were collected between January/2015 and November/2015. Samples were collected on day three and embryo transfers were performed on day five and all embryos reached the blastocyst stage. Samples were split into groups according to the embryo implantation result: Positive-Implantation-Group (n=18) or Negative-Implantation-Group (n=18). For the first analysis, samples were pooled in three sets for each group (6-7 spent media per pool). MicroRNAs were extracted from spent media and cDNA was synthesized. C. elegans miR-39 was used as RNA spike-in to normalize the gene expression analysis. The expression of microRNAs into the spent media from the Positive-Implantation-Group was compared with those from the Negative-Implantation-Group. A set of seven miRNAs (miR-21, miR-142-3p, miR-19b, miR-92a, miR-20b, miR-125a and miR148a) selected according with the literature, was tested. To check whether miRNAs could be detected in individual samples of culture media, in a second analysis, ten more samples were tested for miR-21 and miR-142-3p.
From the sevens tested miRNAs, a significant increased expression of miR-142-3p could be noted in the Negative-Implantation-Group (P<0.001). For other three miRNAs (miR-21, miR-19b and miR-92a) a difference in expression was observed, however it did not reach a statistical significance. In addition, when ten non-redundant samples were tested to check if miRNAs could be detected in individual samples of culture media, the highly specific amplification of mature miRNAs, including miR-142-3p, could be noted.
Our findings suggest that miR-142-3p, previously described as a tumor suppressor and cell cycle inhibitor, may be a potential biomarker of blastocyst implantation failure. The identification of miRNAs on individual culture medium samples offers unique opportunities for non-invasive early diagnosis of blastocyst implantation.
本研究旨在探究体外培养胚胎的培养基中检测到的微小RNA（miRNA）是否可能成为胚胎着床的潜在生物标志物。
收集了一所私立大学附属医院体外受精中心36例接受卵胞浆内单精子注射（ICSI）患者胚胎的培养基样本，收集时间为2015年1月至2015年11月。在第三天收集样本，第五天进行胚胎移植，所有胚胎均发育至囊胚阶段。根据胚胎着床结果将样本分为两组：着床阳性组（n = 18）和着床阴性组（n = 18）。对于首次分析，每组样本分为三组（每组6 - 7份废弃培养基）。从废弃培养基中提取miRNA并合成cDNA。将秀丽隐杆线虫miR - 39用作RNA内参来标准化基因表达分析。比较着床阳性组和着床阴性组废弃培养基中miRNA的表达。根据文献选择了一组7种miRNA（miR - 21、miR - 142 - 3p、miR - 19b、miR - 92a、miR - 20b、miR - 125a和miR148a）进行检测。为了检查是否能在培养基的单个样本中检测到miRNA，在第二次分析中，又对10个样本检测了miR - 21和miR - 142 - 3p。
在检测的7种miRNA中，着床阴性组中miR - 142 - 3p的表达显著增加（P < 0.001）。对于其他3种miRNA（miR - 21、miR - 19b和miR - 92a），观察到表达有差异，但未达到统计学意义。此外，当检测10个非重复样本以检查是否能在培养基的单个样本中检测到miRNA时，可以观察到包括miR - 142 - 3p在内的成熟miRNA的高特异性扩增。
我们的研究结果表明，先前被描述为肿瘤抑制因子和细胞周期抑制剂的miR - 142 - 3p可能是囊胚着床失败的潜在生物标志物。在单个培养基样本中鉴定miRNA为囊胚着床的非侵入性早期诊断提供了独特的机会。
