CN116334260B - 用于诊断多囊卵巢综合征不孕的代谢生物标记物及其应用 - Google Patents
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Abstract
本发明公开了一种用于诊断多囊卵巢综合征不孕的生物代谢标记物及其应用，所述生物标记物包括丁酸菌属和瘤胃球菌属Ruminococcaceae_UCG‑014。本发明首次发现了丁酸菌属和瘤胃球菌属Ruminococcaceae_UCG‑014与复发性流产相关，丁酸菌属和瘤胃球菌属Ruminococcaceae_UCG‑014在多囊卵巢综合征不孕患者中丰度显著增加，说明丁酸菌属和瘤胃球菌属Ruminococcaceae_UCG‑014可作为检测靶标应用于多囊卵巢综合征不孕的诊断和预测。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种用于诊断多囊卵巢综合征不孕的代谢生物标记物及其应用。
背景技术
多囊卵巢综合征(Polycystic ovary syndrome，PCOS)影响6-8％的生殖年龄妇女，是最常见的内分泌疾病之一(Shi等，2012)。PCOS目前被定义为高雄激素性疾病，具有一定的体征和症状，其中必然包括排卵功能障碍(ovulatory dysfunction)和/或多囊卵巢形态(polycystic ovarianmorphology)(Azziz等，2006)。PCOS也与代谢紊乱，包括胰岛素耐受，肥胖和糖尿病有关(Sam，2015)。虽然肥胖不是PCOS表型的一部分，但它加剧了葡萄糖耐受不良，血脂异常和妊娠并发症(Legro，2012)。据估计，30％-75％的PCOS患者超重或肥胖(Diamanti-Kandarakis，2007)，但超重感染和PCOS的关联仍然未知。
目前关于多囊卵巢综合征的分析方法主要有三种：
1、鹿特丹共识(2003版)，其要求至少满足以下三个条件中的两个：高雄激素血症、多囊性卵巢和月经失调；
2、美国国立卫生研究院的标准；
3、国际高雄激素和多囊卵巢综合征协会的标准。
以上标准具有异质性、侵入性和价格昂贵等缺点，极大阻碍了多囊卵巢综合征发病机制、精确诊断及个性化治疗的研究。
为了解决上述技术问题，本领域的技术人员试图开发出能够诊断多囊卵巢综合征不孕患者的标志物。例如，中国授权专利CN111830169B公开了一种用于多囊卵巢综合征诊断中的化合物，所述化合物为雄甾酮葡糖苷酸或鞘磷脂(d17:1/24:1)中的一种，或者所述化合为雄甾酮葡糖苷酸与鞘磷脂(d17:1/24:1)的组合。该发明通过超高压液相串联飞行时间质谱进行多囊卵巢综合征尿液的非靶标代谢组学研究，获得雄甾酮葡糖苷酸、鞘磷脂脂(d17:1/24:1)及他们的组合对多囊卵巢综合征有较好的诊断价值。实现了简单快速诊断多囊卵巢综合征的目的，同时降低了检测成本。
再例如，中国授权专利CN112763727B公开了一种用于多囊卵巢综合征诊断的试剂盒，其特征在于，包含检测生物标志物表达水平的试剂，所述生物标志物为酪氨酸、天门冬酰胺、瓜氨酸、苏氨酸和肌氨酸。
再例如，中国授权专利CN112229937B公开了一种用于多囊卵巢综合征诊断的试剂盒，其特征在于，
包含生物标志物的标准溶液和内标标准溶液，所述生物标志物包括胆酸、甘氨去氧胆酸和牛磺熊去氧胆酸，并且至少一种生物标志物在受试体生物样本中与对照样本中差异表达；所述受试体生物样本选自血浆或血清；所述内标标准溶液为经同位素标记的所述生物标志物溶液，同位素标记方式为2H或13C标记；所述生物标志物任选地与临床指标结合用于诊断受试体多囊卵巢综合征亚型。
虽然现有技术中公开了一些用于诊断多囊卵巢综合征不孕患者的标志物，但是，这些标志物大多需要侵入性的取样，导致患者的接受程度较低。
发明内容
为了弥补现有技术的不足，本发明的目的在于发现与多囊卵巢综合征不孕相关的肠道菌群，通过检测所述菌群的丰度，可以为疾病的诊断提供新的思路。为了实现本发明的目的，拟采用如下技术方案：
本发明提供了一种用于诊断多囊卵巢综合征不孕的生物标记物，所述生物标记物包括丁酸菌属和瘤胃球菌属Ruminococcaceae_UCG-014。
在本发明的一个优选实施方式中，所述标记物为肠道生物标记物。
本发明另一方面涉及一种用于诊断多囊卵巢综合征不孕的试剂盒，所述试剂盒包括检测丁酸菌属和瘤胃球菌属Ruminococcaceae_UCG-014的丰度的试剂。
在本发明的一个优选实施方式中，所述试剂包括检测丁酸菌属和瘤胃球菌属Ruminococcaceae_UCG-014的特异性的引物、探针、反义寡核苷酸、适配体或抗体。
在本发明的一个优选实施方式中，所述特异性引物为能够扩增丁酸菌属和瘤胃球菌属Ruminococcaceae_UCG-014的16SrRNA的引物。
本发明另一方面还涉及上述生物标记物或试剂盒在制备诊断多囊卵巢综合征不孕的诊断试剂中的应用。
本发明首次发现了丁酸菌属和瘤胃球菌属Ruminococcaceae_UCG-014与复发性流产相关，丁酸菌属和瘤胃球菌属Ruminococcaceae_UCG-014在多囊卵巢综合征不孕患者中丰度显著增加，说明丁酸菌属和瘤胃球菌属Ruminococcaceae_UCG-014可作为检测靶标应用于多囊卵巢综合征不孕的诊断和预测。
附图说明
图1是丁酸菌属和瘤胃球菌属Ruminococcaceae_UCG-014在多囊卵巢综合征不孕患者中的丰度图。
图2:临床指标的诊断价值的ROC曲线图。
具体实施方式
为了进一步理解本发明，下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
如无特殊说明，本发明实施例中所涉及的试剂均为市售产品，均可以通过商业渠道购买获得。
实施例1：
1、样本的采集
选取多囊卵巢综合征不孕患者56例作为实验组，选取同期妊娠妇女20例作为对照组，后者经过严格匹配。所有标本的使用均由患者或其委托人签署知情同意书，获得了伦理委员会的审核与批准。
2、16S rRNA测序
2.1DNA的提取
使用DNA提取试剂盒自阴道分泌物中提取细菌DNA，操作步骤按说明书进行。
2.2DNA样本纯度及浓度测定
利用1％琼脂糖凝胶电泳检测基因组DNA。
2.3PCR扩增及产物纯化
采用TransGen AP221-02(TransStart Fastpfu DNA Polymerase)进行PCR扩增反应，全部样本按照正式实验条件进行，每个样本3个重复，同一样本的PCR产物混合后用2％琼脂糖凝胶电泳检测，使用AxyPrepDNA凝胶回收试剂盒(AXYGEN公司)切胶回收PCR产物，Tris-HCl洗脱；2％琼脂糖电泳检测。
2.4荧光定量
将PCR产物用QuantiFluorTM-ST蓝色荧光定量系统(Promega公司)进行检测定量，之后按照每个样本的测序量要求，进行相应比例的混合。
2.5Miseq文库构建
使用TruSeqTM DNA Sample Prep Kit进行文库的构建，具体步骤按照说明书进行。
2.6Miseq测序
以DNA片段为模板，PCR合成目标待测DNA片段，cBot上进行桥式PCR扩增，生成DNA簇，Hiseq4000测序平台，进行2*150bp测序。
3、数据分析
3.1数据预处理
使用FLASH、Trimmomatic等软件对Miseq测序得到的PEreads根据overlap关系进行拼接，同时对序列质量进行质控和过滤；使用Usearch软件进行聚类操作，将序列按照彼此的相似性归为许多OUT，采用RDP classifier贝叶斯算法在97％的相似水平下的OTU进行生物信息统计分析，并使用Silva数据库进行比对。
3.2肠道菌群物种差异分析
使用LEfSe多级物种差异判别分析进行差异物种的筛选，首先使用non-parametric factorial Kruskal-Wallis(KW)sum-rank test(非参数因子克鲁斯卡尔-沃利斯秩和检验)检测具有显著丰度差异特征，并找到与丰度有显著性差异的类群。最后，采用线性判别分析(LDA)来估算每个组分(物种)丰度对差异效果影响的大小。筛选标准LDA>2且P<0.05。
4、结果
结果显示，与对照组相比，丁酸菌属和瘤胃球菌属Ruminococcaceae_UCG-014的丰度在多囊卵巢综合征不孕患者中显著增加，差异具有统计学意义(P＜0.05)，提示丁酸菌属和瘤胃球菌属Ruminococcaceae_UCG-014可以作为生物标记物应用于多囊卵巢综合征不孕患者的诊断。
如实施例1所述，56例多囊卵巢综合征不孕患者和20例健康人，利用在生物标记物组合随机森林模型，可输出上述受试者患多囊卵巢综合征不孕的可能性，ROC曲线结果如图2所示，AUC为0.875。
以上描述了本发明优选实施方式，然其并非用以限定本发明。本领域技术人员对在此公开的实施方案可进行并不偏离本发明范畴和精神的改进和变化。
Claims (4)
1.生物标记物在制备诊断多囊卵巢综合征不孕的诊断试剂中的应用，所述生物标记物包括丁酸菌属和瘤胃球菌属Ruminococcaceae_UCG-014。
2.根据权利要求1所述的应用，所述生物标记物为阴道生物标记物。
3.试剂盒在制备诊断多囊卵巢综合征不孕的诊断试剂中的应用，所述试剂盒包括检测丁酸菌属和瘤胃球菌属Ruminococcaceae_UCG-014的特异性的引物、探针、反义寡核苷酸、适配体或抗体。
4.根据权利要求3所述的应用，所述特异性引物为能够扩增丁酸菌属和瘤胃球菌属Ruminococcaceae_UCG-014的16SrRNA的引物。
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