CN113755571B - 用于胚胎着床成功率检测的生物标志物及应用 - Google Patents
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Abstract
本发明提供一种用于胚胎着床成功率检测的生物标志物及应用,主要解决了现有技术在进行胚胎能否成功着床的预测时,过程复杂、结论片面且有创伤的问题。该用于胚胎着床成功率检测的生物标志物是miR‑374a‑5p。利用本发明提供的标志物、应用该标志物的物质、设备等,可以微创、可靠的检测和/或诊断着床成功率,有效改善妊娠结局,并避免优质胚胎被浪费,减轻患者的经济压力与精神压力。
Description
技术领域
本发明属于分子诊断和分子生物学技术领域,具体涉及一种以miRNA作为胚胎着床成功率预测的生物标志物及应用。
背景技术
我国育龄夫妇的不孕不育发生率不断升高,已从20年前的2.5%-3%增长到12.5%-15%,人类辅助生殖技术已成为不孕患者的首选治疗方案。然而仍有很多患者在接受体外受精/卵细胞浆内单精子注射(in vitro fertilization/intracytoplasmic sperminjection,IVF/ICSI)治疗以后仍未成功妊娠。反复种植失败(recurrent implantationfailure,RIF)是指年龄小于40岁的不孕患者,接受三次或者三次以上IVF-ET,至少移植了4枚优质胚胎,仍未获得临床妊娠。其发生率在接受IVF/ICSI助孕治疗患者中达5%-10%。RIF的发生给临床医生与病人都带来了很大的压力,尤其是对于卵巢功能不好且优质胚胎数目较少的患者,倘若可以在胚胎移植前通过检测评估母体状态预测胚胎是否能够种植成功,不仅可以改善妊娠结局,还可以避免优质胚胎被浪费,减轻患者的经济压力与精神压力。因此对胚胎能否成功着床的预测是尤为重要的(特别是RIF患者),对子宫内膜进行诊刮来评估母体状态是片面的、有创的,故临床工作中尚需要一个简单易行、稳定可靠的预测方法。
目前已发表的用于预测RIF的技术主要包括:通过子宫内膜活检对子宫内膜容受性进行检测与评估,此方法是侵入性的、有创性的,在临床上实施较为困难(DOI:10.12688/f1000research.22403.1)。也有研究通过子宫内膜上差异表达的以及血清中差异表的miRNA对RIF进行诊断与预测,但是血清中的miRNA在提取过程中,易被环境中RNA酶所降解,从而造成结果的误差。
WOUS20015110公开了一种题为“用于预测生育治疗成功的子宫内膜液”(UTERINEENDOMETRIAL FLUID FOR PREDICTION OF SUCCESS IN FERTILITY TREATMENT)的方案,其采用的方案是“使来自患者的子宫内膜分泌物与试剂盒接触”,正如前文所述的,该方案具有侵入性和有创性。
US16498883公开了一种题为“评估妊娠结果的方法”(METHODS FOR ASSESSINGPREGNANCY OUTCOME)的方案,其采用的方案仍然是“开发了一种基于通过RT-QPCR对子宫内 膜活组织检查中的这些生物标记进行定量的测试”,该方案依然具有侵入性和有创性。
发明内容
本发明提供一种miRNA作为着床成功率预测的生物标志物及应用,主要解决了现有技术在进行胚胎能否成功着床的预测时,过程复杂、结论片面且有创伤的问题。
本发明的具体技术解决方案如下:
该用于胚胎着床成功率检测的生物标志物是miR-374a-5p。
该用于胚胎着床成功率检测的生物标志物来自胚胎种植窗口期的血清中,以来自胚胎种植窗口期的血清外泌体中为佳,其准确率和稳定性更高。具体理由是:胚胎种植窗口期的血清更能反应胚胎植入时母体的状态,非植入窗口期血清各种激素水平与植入窗口期有很大的差别,因此选择胚胎植入窗口期的血清能更佳的预测是否可以成功种植。其次,血清外泌体中miRNA的表达量较血清miRNA稳定,故选择血清外泌体miRNA更佳。
相对于健康人群,着床率较低的患者中miR-374a-5p表达为上调。表达上调选择标准即通过高通量测序筛选多种差异表达的miRNA(即P<0.05),进一步使用不同的样本来验证所筛选出的miRNA,选择同样差异表达的miRNA。
该标志物尤其适用于预测或辅助,诊断或辅助诊断不孕患者、RIF患者的胚胎着床成功率。
上述生物标志物表达水平的物质在制备胚胎着床成功率检测产品中的应用时,可以包括基于高通量测序方法和/或基于定量PCR方法和/或基于探针杂交方法检测上述生物标志物表达水平的物质。例如检测装置、试剂盒和设备,这类装置例如:如寡核苷酸探针或其集成、芯片基片或检测基板上的高通量miRNA检测芯片、以及微流控检测芯片等。
术语“探针”是指通常用于检测与探针的序列互补的靶RNA和/或RNA序列的单链寡核苷酸。探针与核苷酸序列由于探针与靶序列之间的互补性而允许核苷酸配对的单链核酸(DNA或RNA)杂交。探针的长度取决于预期用途以及所需的探针特异性。
术语“试剂盒”是指单独提供或提供在单个容器内的上述组分的集合。
利用上述物质制造的用于检测胚胎着床成功率的设备,包含:
1)分析单元,所述分析单元包含:用于确定对象的样品中生物标志物表达水平的检测剂,以及
2)包含有数据处理器的评估单元,所述数据处理器有形地嵌入有用于将通过所述分析单元确定的量与参考进行比较的算法,并且能够生成包含基于所述比较建立的诊断结果的输出文件。
术语“设备”涉及至少包含有效地互相联系以允许进行诊断的上述装置的装置系统,。例如,在应用用于自动化确定基因产物的甲基化状态或量的装置的情况下,可以通过例如计算机程序来处理由所述自动化操作装置获得的数据以建立诊断。
本发明的优点在于:
利用本发明提供的标志物、应用该标志物的物质、设备等,可以微创、可靠的检测和/或诊断着床成功率,有效改善妊娠结局,并避免优质胚胎被浪费,减轻患者的经济压力与精神压力。
附图说明
图1血清外泌体电镜鉴定图(bar=100nm);
图2血清外泌体粒径分析图;
图3血清外泌体Western-blot鉴定图;
图4差异miRNAs聚类性热图;
图5差异miRNAs的qRT-PCR验证图;
图6miR-374a-5p与miR-183-5p在RIF(n=20)与NC(n=20)中的表达图(与NC组相比,*P<0.05,**P<0.01);
图7ROC曲线评估外泌体miR-183-5p与miR-374a-5p的预测效能图;
附图说明:
图1、图2中:A为NC患者外泌体;B为RIF患者外泌体;
图7中:A为外泌体miR-183-5p,B为外泌体miR-374a-5p,C为外泌体miR-183-5p联合miR-374a-5p。
具体实施方式
首先需要说明的是,以下详细说明都是例示性的,旨在对本发明提供进一步的说明。除非另有指明,本发明使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本发明的示例性实施方式。当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。应理解,本发明的保护范围不局限于下述特定的具体实施方案;还应当理解,本发明实施例中使用的术语是为了描述特定的具体实施方案,而不是为了限制本发明的保护范围。
技术人员理解本发明中使用的术语例如“miRNA”和“miR”的变化形式,并且其涉及见于真核生物细胞和后生动物生物体的体液中的短核糖核酸(RNA)分子。miRNA包括人miRNA、成熟单链miRNA、前体miRNA(pre-miR)、及其变体,其可以是天然存在的。在一些情况下,术语“miRNA”还包括初级miRNA转录物(pri-miRNA)和双链体miRNA。除非另外指出,否则当在本发明中使用时,特定miRNA的名称是指成熟miRNA。miRNA前体可以由25至数千个核苷酸,通常40至130、50至120、或60至110个核苷酸组成。通常,成熟miRNA由5至100个核苷酸,通常10至50、12至40、或18至26个核苷酸组成。术语miRNA还包括最终进入RNA诱导沉默复合体(RNA-induced silencing complex,RISC)的“引导”链以及与其互补的“过客”链。
术语指标的水平“下调”、“降低”或“下降”是指与参考或参考样品相比,样品中这样的指标的水平降低。术语指标的水平“上调”、“升高”或“提高”是指与参考或参考样品相比,样品中这样的指标的水平更高。属于RIF是指反复种植失败,NC是指自然受孕。
以下结合实施例进行详述:
在本实施例中,通过miRNA转录组学测序技术对RIF组患者与不孕对照组患者胚胎种植窗口期母体血清外泌体miRNAs进行测序,筛选出差异表达的外泌体miRNAs,然后对差异性表达的外泌体miRNAs进行qRT-PCR验证,之后扩大样本量来评估差异性表达的外泌体miRNA在RIF中的预测价值。
(1)对血清外泌体进行提取与鉴定:所有参与者留取胚胎种植窗时期(正常月经周期第20-25天)静脉血5ml于非抗凝管中,常温静置1小时,然后4℃,3000rpm,离心10分钟,将上清转移至1.5mL无菌离心管中,4℃,3000g,离心10分钟,转移上清于新1.5mL无菌离心管,将血清样品储存于-80℃备用。根据外泌体提取试剂盒(加拿大,Norgen,57400)说明书进行操作。提取好的外泌体通过电镜鉴定、粒径分析以及Wester-blot鉴定。
结果:实验所得颗粒经鉴定为外泌体。
(2)对血清外泌体进行RNA提取与测序:根据外泌体RNA提取试剂盒(加拿大,Norgen,58000)说明书进行操作。外泌体miRNA测序主要包括制备文库与测序实验,小RNA测序文库的制备采用TruSeq Small RNA Sample Prep Kits(Illumina,San Diego,USA)试剂盒,测序实验使用lluminaHiseq 2000/2500,测序所读长度为单端1×50bp。数据分析使用联川生物公司自主研发的分析软件ACGT101-miR(LC Sciences,Houston,Texas,USA),将原始数据经过质控处理,得到clean reads。clean reads去除3’接头后进行筛选,保留碱基长度为18-26nt的序列。再将剩余序列与各种RNA数据库序列进行比对(不包括miRNA数据库),并进行过滤,最后获得的数据即为有效数据,用于后续miRNA数据分析。
结果:选取差异倍数∣log2(Foldchange)∣≥1且P<0.05为差异性miRNA,结果显示:RIF组与NC组相比,有251种miRNAs上调,66种miRNAs下调;在∣log2(Foldchange)∣≥1且P<0.01的条件下,RIF组与NC组相比,有110种miRNAs上调,20种miRNAs下调。在表达量为中高等水平(平均表达量>500)、∣log2(Foldchange)∣≥1、P<0.01、种属为人类的条件下,RIF组与NC相比,有27种miRNAs上调,2种miRNAs下调。
(3)miRNA反转录与实时荧光定量PCR:外泌体miRNA逆转录根据miRNA逆转录试剂盒(德国,Qiagen,218161)说明书进行操作。在逆转录体系中加入0.5μL 20μM的cel-miR-39作为外源性参照物。外泌体miRNA实时荧光定量PCR根据miRNA定量PCR试剂盒说明书(德国,Qiagen,218073)进行操作,以cel-miR-39与hsa-miR-26a-5p作为参照,比较RIF组与NC组血清外泌体miRNAs的表达水平。
结果:根据差异miRNAs筛选结果以及文献查阅,我们选择6种可能与RIF相关的miRNAs进行qRT-PCR验证,包括miR-30d-5p,miR-182-5p,miR-183-5p,miR-223-3p,miR-374a-5p,miR-451a。结果显示:miR-183-5p与miR-374a-5p在RIF组中表达量明显高于NC组,差异具有统计学意义(P<0.05),与测序结果一致;其余四种miRNAs在RIF组与NC组中表达量无显著性差异(P>0.05)。对miR-183-5p与miR-374a-5p扩大样本量(每组n=20)再次进行验证,结果显示:miR-183-5p与miR-374a-5p在RIF组中表达量明显高于NC组,差异具有统计学意义(P<0.05)。
(4)绘制外泌体miR-183-5p与miR-374a-5p用于预测RIF的ROC曲线并计算AUC。结果显示:外泌体miR-183-5p用于预测RIF的AUC为0.700(95%CI:0.532-0.868,P=0.031),cutoff值为0.733,敏感性为75%,特异性为70%。外泌体miR-374a-5p用于预测RIF的AUC为0.847(95%CI:0.729-0.966,P=0.0002),cutoff值为0.832,敏感性为80%,特异性为80%;外泌体miR-183-5p与miR-374a-5p联合用于预测RIF的AUC为0.850(95%CI:0.698-0.969,P=0.0006),cutoff值为4.909,敏感性为85%,特异性为75%。
表1研究样本的基本信息(筛查)
不孕诊断:0-原发性不孕,1-继发性不孕;不孕因素:1-输卵管因素,2-男方因素,3-不明原因;活产史/自然流产史:0-无,1-有。
表2研究样本基线资料(验证)
表3差异性miRNAs
在本实施例中,选外泌体的动机是:外泌体是一种纳米级别的细胞外囊泡,包裹多种活性分子包括蛋白质、miRNA等,参与细胞间通讯并发挥生物学功能。miRNA属于非编码RNA的子集,是一类长度约为22个核苷酸的内源性的具有调控功能的小RNA,miRNA可以作为无创性分子标志物对疾病进行预测与诊断。近年来,研究发现外泌体中miRNA较血液中miRNA更稳定,外泌体miRNA将是临床诊断中最理想的生物标志物。
本实施例通过对RIF患者与不孕对照患者胚胎种植窗口期血清外泌体miRNAs转录组学测序,筛选出具有差异性表达的miRNAs,并进行验证。
本实施例的优点在于血清外泌体中miRNAs较血清中miRNAs更稳定,更适合作为预测分子标志物;
本实施例在胚胎植入窗口期对不孕患者进行评估,通过母体状态评估以达到预测胚胎是否可以种植成功的目的。
本实施例在可预期的在临床应用中,改善妊娠结局,并避免优质胚胎被浪费,减轻患者的经济压力与精神压力。
Claims (1)
1.检测来源于胚胎种植窗口期血清外泌体中的miR-374a-5p的物质在制备RIF患者的胚胎着床成功率检测产品中的应用。
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