CN118033008A - 用于卵巢储备功能减退诊断的生物标志物和试剂盒及使用方法 - Google Patents

用于卵巢储备功能减退诊断的生物标志物和试剂盒及使用方法 Download PDF

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CN118033008A
CN118033008A CN202410434417.2A CN202410434417A CN118033008A CN 118033008 A CN118033008 A CN 118033008A CN 202410434417 A CN202410434417 A CN 202410434417A CN 118033008 A CN118033008 A CN 118033008A
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齐新宇
乔杰
丁澍
李文燕
司曼飞
熊湘蕾
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Abstract

本发明公开了一组用于卵巢储备功能减退诊断的生物标志物和试剂盒及使用方法,所述标志物包括胆酸、脱氧胆酸、鹅去氧胆酸、石胆酸和熊去氧胆酸。通过本发明的标志物和试剂盒,可以相对准确的及早诊断卵巢储备功能减退并恢复其卵巢功能,精准靶向解决生育力下降的“瓶颈问题”。

Description

用于卵巢储备功能减退诊断的生物标志物和试剂盒及使用 方法
技术领域
本发明属于生物检测技术领域,具体涉及卵巢储备功能减退诊断检测用的生物标志物和试剂盒领域。
背景技术
卵巢储备功能减退(Diminished Ovarian Reserve, DOR)是指卵母细胞数量和(或)质量的下降,导致卵巢功能不足。40岁以后发生的DOR通常是生理性的,而女性早期经历DOR则会导致其生殖功能过早下降,引起生育力不足。DOR最常见的临床表现为月经紊乱、内分泌紊乱、卵巢刺激反应不良和不孕,如果不及时治疗,很可能在几年内进展为卵巢早衰。DOR在女性卵巢疾病中占比约为20%,在不孕人群中的发生率约为10%,其发病率逐年上升,且患者年龄呈现低龄化趋势,严重影响了大量女性的生殖健康和生活质量。由于病因复杂、起病隐匿、预防困难,且缺乏早期筛查标志物,故治疗效果有限,目前仍是不孕症临床治疗的巨大挑战之一。因此如何及早识别并恢复DOR患者卵巢功能、精准靶向解决生育力下降的“瓶颈问题”,是有关人类福祉且亟待解决的问题。
目前DOR主要通过窦卵泡数(Antral follicle counting, AFC)和抗苗勒管激素(Anti-Müllerian hormone, AMH)的减少以及血清基础卵泡刺激素(Follicle-stimulating hormone, FSH)水平的升高来诊断。但目前并没有公认统一的诊断标准,通过上述方法诊断出的DOR患者卵巢储备功能已经严重下降,已有的诊断方法并不能及早识别并进行干预。以上问题使得DOR的诊断与治疗已经成为妇科和生殖医学领域共同关注的热点和难点问题。因此,找到一种易于检测的无创性生物标记物诊断方法,来预测和诊断卵巢储备功能减退,对于卵巢储备功能减退的早期诊断和干预有很大的意义。目前,市场上还没有针对卵巢储备功能减退的快速高效诊断试剂盒问世,影响到卵巢储备功能减退早期识别及治疗。
近几年发现,胆汁酸代谢对卵巢功能存在明显的影响,能够通过刺激雄激素分泌进而降低卵巢内总睾酮与胰岛素水平,改善卵巢形态,减少囊性和闭锁性卵泡,但若积累的胆汁酸含量过高,就会通过破坏线粒体功能、诱发氧化应激而影响卵巢微环境以及卵泡的生长,进而导致排卵障碍,损害卵巢的功能。因此,胆汁酸在体内的丰度变化有望为卵巢储备功能减退的诊断提供新的路径。
发明内容
本发明提供一组可用于检测卵巢储备功能减退的生物标志物,进一步提供包含该等生物标志物的用于检测卵巢储备功能减退的试剂盒及其使用方法。
本发明的一个方面,提供了一组可用于卵巢储备功能减退诊断的生物标志物,所述生物标志物包括多种代谢物,所述的代谢物选自胆汁中的胆酸组分,其在至少一种靶血浆、血清或卵泡液中与在一种对照血浆或血清中差异表达。所述生物标志物可用于受试体卵巢储备功能减退的诊断。
在一种实施方案中,本发明生物标志物指受试体生物样本中存在的代谢物成分,受试体可以是人类或哺乳动物。生物样本可选自来源于受试体的血浆、血清或卵泡液。优选的,所述生物样本为血清。
在一种实施方案中,所述代谢物选自下列代谢物中一种或多种: 胆酸、脱氧胆酸、鹅去氧胆酸、石胆酸和熊去氧胆酸。
在一种实施方案中,所述的生物标志物用于制备测试受试体卵巢储备功能减退的试剂盒的用途,所述试剂盒的使用方法包括,可通过测试受试体血浆、血清或卵泡液中代谢物的水平,测试受试体卵巢储备功能减退。
本发明的另一方面,提供包含胆酸、脱氧胆酸、鹅去氧胆酸、石胆酸和熊去氧胆酸五种代谢物的可用于卵巢储备功能减退诊断试剂盒,所述试剂盒包含胆酸、脱氧胆酸、鹅去氧胆酸、石胆酸和熊去氧胆酸的标准溶液和内标标准溶液,所述内标标准溶液指同位素标记的胆酸、脱氧胆酸、鹅去氧胆酸、石胆酸和熊去氧胆酸,同位素标记方式可以是2H或13C。
在一种实施方案中,所述试剂盒还包含萃取液,萃取液由甲醇和乙腈组成,甲醇和乙腈的体积比为1:1-5:1。进一步地,所述试剂盒还包含700μL的96孔板、350μL的V型96孔板、封板硅胶、96孔密封铝膜。
本发明的另一方面,提供上述试剂盒用于诊断卵巢储备功能减退的使用方法,具体为,通过测定受试体的血清、血浆或卵泡液中代谢物胆酸、脱氧胆酸、鹅去氧胆酸、石胆酸和熊去氧胆酸的水平,并将这些测定值输入随机森林模型得到分值截断值进行判定。
在一种实施方案中,上述试剂盒用于诊断卵巢储备功能减退的使用方法,包括下列步骤:a) 配制不同浓度的代谢产物标准品溶液:分别以胆酸、脱氧胆酸、鹅去氧胆酸、石胆酸和熊去氧胆酸的标准溶液配制不同浓度的溶液,与空白对照一起置于离心管,于台式离心机4000-10000转下离心10-30分钟;向各离心管加入新鲜制备的去离子水200微升,剧烈振荡,在800-1200转/分钟振荡溶解10-15分钟,静置,备用;b) 内标标准品溶液配制:取3毫升的甲醇作内标稀释液,加入到内标准品溶液中,盖上盖子,剧烈振荡,静置约15分钟溶解。稀释内标溶液,内标溶液加到96孔微孔板;c) 血清、血浆或卵泡液样本的制备:取出试剂盒提供的700微升微孔板,在A1到A8孔中依次加入5微升标准品1到标准品7和空白对照,在其他孔中加入5微升血清(或血浆、卵泡液)样品或5微升低、中、高浓度质控品。在各孔中加入25微升的内标准品溶液,盖上硅胶盖,1000rpm振荡10min。2000g离心2分钟。轻轻取下硅胶盖,避免微孔板中液体溅出,妥善放置硅胶垫防止污染,备用;30℃ 1450rpm反应60分钟,小心取下硅胶垫,妥善放置硅胶垫防止污染,备用;各孔加入350微升上样缓冲液,盖上硅胶盖剧烈振荡;-20℃放置20分钟,2000g离心20分钟;取下硅胶盖,小心吸取150微升上清至干净的V型底微孔板中,盖上铝箔封套,放入自动进样器中;d) 将c)中制得的样本以液相色谱和质谱进行测定,计算样本中代谢产物的浓度;e) 将d)中计算得到的生物标志物的浓度输入随机森林模型进行计算,按照分值对受试体的卵巢储备功能减退进行判定。
在一种实施方案中,本发明应用的随机森林模型可以是市场上可提供的软件,这些软件也可以任选地作为上述试剂盒的一部分。作为优选方案,软件包作为试剂盒的一部分。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。在附图中:
图1:胆汁酸组合的诊断价值的ROC曲线图。
图2:诊断模型在外部验证集中的ROC曲线图。
具体实施方式
为更好地理解本发明,下面通过以下实施例对本发明作进一步具体的阐述,但不可理解为对本发明的限定,对于本领域的技术人员根据上述发明内容所作的一些非本质的改进与调整,也视为落在本发明的保护范围内。
实施例中未注明具体条件的实验方法,通常按照常规条件,例如教科书和实验指南中所述的条件,或按照制造厂商所建议的条件。
实施例1:差异性生物标志物的筛选
本发明中的试验样本得到本地伦理委员会的批准并获得所有受试者的知情同意书。本发明共入组 182名受试者,使用超高效液相色谱串联质谱技术分别检测了91例卵巢储备功能正常(NOR)的患者和91例经证实的卵巢储备功能减退(DOR)患者卵泡液标本中胆汁酸的含量。检测方法如下所述。检测结果见表1。
1. 卵泡液收集
在接受辅助生殖技术治疗患者取卵的当日,从DOR患者和NOR患者中收集含有卵母细胞(直径>1.4cm)的优势卵泡的卵泡液,并将其分离并储存在-80℃下直至使用。
2. 卵泡液样本的胆酸检测
样本制备:在4℃下缓慢解冻卵泡液样品,取100μL卵泡液于1.5mL的离心管中,加入150μL甲醇(含胆汁酸内标)。漩涡震荡混匀10分钟,静置10分钟,然后4度13500转离心20分钟,取上清液进行UPLC-TQMS(超高效液相色谱-三重四级杆质谱)分析。
分析仪器测试:UPLC-TQMS:采用沃特斯超高效液相色谱系统(沃特斯公司,美国),配备二元溶剂控制器和样本控制室。采用沃特斯 XEVO三重四级杆质谱仪(沃特斯公司,美国),配备双电喷雾离子源。
色谱条件:采用UPLC BEH C18色谱柱(100mm x 2 .1mm,1.7m);柱温 45℃;流动相A:水 (0.1%甲酸),B:乙腈(0.1%甲酸,);流速为0.4mL/min;进样量为5uL;梯度洗脱条件:0-1min(5%B),1-5min(5-25%B),5-15.5min(25- 40%B),15.5-17.5min(40-95%B),17.5-19min(95%B), 19-19.5min(95-5% B),19.6-21min(5%B)。
质谱条件:电喷雾离子源采用负离子扫描模式(ESI-),具体条件如下:毛细管电压1.2kV,锥孔电压55V,萃取锥孔电压4V,离子源温度150C,脱溶剂气温度550C,反向锥孔气流50L/h,脱溶剂气650L/h,低质量区分辨率4.7,高质量区分辨率15,多反应检测模式采集数据。
待测诊断标志物浓度的确定:以待测诊断标志物的标准品溶液的浓度与对应的待测诊断标志物以及与待测诊断标志物一样的稳定同位素内标面积比绘制标准曲线,采用同位素内标定量测定。同时通过样品添加同位素内标对样品检测过程进行质量控制。
表1. 卵泡液胆汁酸检测指标
编号 CA TCA GUDCA GCA TUDCA UDCA GCDCA GDCA TCDCA TDCA CDCA DCA TLCA LCA GLCA THDCA
NOR-1 64.398 36.279 32.190 109.036 25.593 2.029 500.846 248.547 92.913 51.186 12.880 133.410 24.302 61.273 54.575 5.575
NOR-2 150.000 23.895 94.051 86.145 16.832 11.855 521.151 38.983 66.063 33.664 238.610 1752.304 5.725 69.818 4.110 3.964
NOR-3 604.712 3.837 38.321 46.024 3.133 77.818 303.553 260.756 1.606 6.265 586.100 1230.415 5.725 69.091 36.164 0.096
NOR-4 52.382 0.886 26.715 65.060 0.542 11.127 338.071 425.581 15.433 1.083 87.568 649.770 2.856 74.909 77.589 0.926
NOR-5 128.796 11.494 9.296 65.060 8.655 18.182 382.403 259.884 28.819 17.310 13.390 218.088 2.856 90.727 103.233 1.729
NOR-6 1091.623 14.860 233.212 177.711 5.788 217.818 497.462 924.419 20.472 11.575 762.162 2847.926 4.284 86.727 96.986 1.228
NOR-7 106.806 5.110 169.708 45.904 5.867 97.818 467.005 8.625 20.472 11.735 463.320 3.664 5.725 47.636 0.000 1.228
NOR-8 36.675 3.994 95.474 37.410 4.248 24.909 419.628 286.919 9.606 8.496 130.656 306.221 0.000 61.818 46.685 0.576
NOR-9 45.157 35.233 431.387 212.048 31.062 80.000 1086.294 230.233 105.512 62.124 247.876 212.903 0.000 56.000 14.038 6.331
NOR-10 13.743 47.616 26.058 405.422 27.345 8.109 1519.459 4238.372 95.276 54.690 71.120 1023.041 8.581 102.364 345.205 5.717
NOR-11 140.576 5.494 717.153 62.651 11.973 261.455 883.249 24.767 42.205 23.947 711.197 1061.060 0.000 57.091 3.081 2.532
NOR-12 362.042 28.256 828.832 76.506 20.018 429.091 781.726 113.372 72.992 40.035 641.699 280.300 8.581 106.182 0.000 4.380
NOR-13 49.712 31.395 647.080 271.084 13.593 222.909 1093.063 243.314 3.118 27.186 317.375 294.816 4.284 83.455 6.510 0.187
NOR-14 125.654 6.087 71.606 59.398 4.540 29.855 277.496 457.849 16.063 9.080 171.429 1074.885 0.000 91.455 61.808 0.964
NOR-15 220.681 25.640 374.453 276.506 32.920 96.364 1827.411 20.669 119.685 65.841 903.475 394.009 0.000 84.364 3.781 7.181
NOR-16 58.586 12.297 83.978 74.699 15.345 10.727 433.164 574.709 53.780 30.690 141.776 535.714 14.279 70.364 93.041 3.227
NOR-17 45.079 64.012 109.380 219.880 22.274 13.127 598.985 867.733 75.276 44.549 87.799 601.382 20.046 66.727 97.644 4.517
NOR-18 24.974 19.884 48.394 149.398 12.080 2.084 663.283 968.023 38.976 24.159 8.571 490.783 5.725 61.455 115.397 2.339
NOR-19 226.178 12.541 117.153 79.518 7.593 72.000 446.701 476.163 30.394 15.186 347.490 905.530 0.000 66.000 15.748 1.824
NOR-20 235.602 34.012 984.307 183.133 23.575 345.091 795.262 2049.419 81.102 47.150 479.537 715.438 0.000 77.273 0.000 4.866
NOR-21 43.194 6.523 177.372 71.687 5.973 114.182 578.680 739.535 22.362 11.947 135.521 1064.516 0.000 90.545 50.630 1.342
NOR-22 207.330 37.326 155.474 166.265 10.965 88.364 551.607 552.035 39.370 21.929 259.459 850.230 18.535 97.818 57.863 2.362
NOR-23 23.717 0.999 96.788 45.301 5.973 12.182 588.832 959.302 19.370 11.947 188.108 1178.571 10.009 61.636 140.384 1.162
NOR-24 26.623 11.442 101.387 174.699 4.673 36.727 571.912 248.547 18.346 9.345 82.239 285.829 0.000 91.455 16.438 1.101
NOR-25 204.188 13.831 162.044 125.301 7.699 109.091 751.269 1029.070 2.764 15.398 495.753 1741.935 0.000 138.364 58.849 0.166
NOR-26 54.188 6.488 237.591 95.783 11.336 92.364 876.481 2258.721 10.551 22.673 282.625 421.659 0.000 98.545 0.000 0.633
NOR-27 399.738 64.360 317.518 503.012 36.106 139.636 2267.343 222.384 125.197 72.212 1301.931 1942.396 17.162 121.273 0.000 7.512
NOR-28 190.838 12.663 247.445 166.265 7.540 94.909 548.223 85.029 26.457 15.080 217.066 1506.912 4.284 187.273 90.740 1.587
NOR-29 75.393 17.163 71.825 168.072 7.487 24.873 477.157 373.256 26.457 14.973 77.606 425.115 4.284 122.909 0.000 1.587
NOR-30 190.838 8.791 916.423 20.602 21.212 385.455 1925.550 303.488 76.929 42.425 1338.996 294.816 8.581 102.000 22.586 4.616
NOR-31 482.984 2.773 188.321 9.699 7.381 178.182 514.382 451.744 3.969 14.761 877.992 1786.866 5.712 113.818 23.441 0.238
NOR-32 151.571 81.802 635.036 570.482 28.938 171.273 1800.338 169.186 105.512 57.876 294.208 2.755 0.000 108.545 0.000 6.331
NOR-33 33.455 5.372 249.635 53.434 6.053 60.000 436.548 595.640 20.945 12.106 84.324 867.512 0.000 125.636 59.836 1.257
NOR-34 67.461 25.465 81.350 151.205 1.750 11.818 500.846 427.326 46.063 3.499 102.857 355.991 11.437 103.636 30.805 2.764
NOR-35 102.880 11.407 213.504 179.518 5.549 149.091 680.203 411.628 21.181 11.097 298.842 857.143 5.725 167.818 14.367 1.271
NOR-36 27.251 35.581 116.058 135.542 27.080 2.764 538.071 425.581 100.787 54.159 30.116 147.926 11.437 88.727 41.096 6.047
NOR-37 14.843 15.855 280.292 142.169 14.814 34.582 700.508 1159.884 53.465 29.628 178.378 1112.903 14.279 108.182 206.137 3.208
NOR-38 97.382 14.860 144.526 145.181 8.336 25.782 632.826 532.849 28.189 16.673 238.610 760.369 10.009 109.091 100.274 1.691
NOR-39 89.529 10.901 424.818 48.976 17.655 112.364 785.110 409.884 60.394 35.310 359.073 646.313 2.856 123.636 30.477 3.624
NOR-40 120.157 9.017 206.934 189.759 9.027 94.545 155.668 6.358 30.709 18.053 361.390 15.449 0.000 112.182 0.000 1.843
NOR-41 295.288 7.552 82.007 198.795 7.726 48.364 1143.824 2947.674 27.638 15.451 831.660 143.779 2.856 96.727 0.000 1.658
NOR-42 185.340 9.140 1041.241 143.373 13.540 636.364 1279.188 39.506 50.000 27.080 634.749 397.465 0.000 71.636 35.836 3.000
NOR-43 83.246 8.215 639.416 71.687 12.027 265.818 720.812 10.378 43.465 24.053 440.154 33.491 0.000 94.182 0.000 2.608
NOR-44 29.293 9.349 377.737 37.831 8.230 178.909 429.780 361.919 30.709 16.460 110.039 487.327 0.000 140.000 28.077 1.843
NOR-45 113.874 65.756 1959.854 662.651 30.531 538.182 2253.807 130.814 105.512 61.062 688.031 1026.498 4.284 107.273 0.000 6.331
NOR-46 157.068 5.634 866.058 10.241 10.354 410.909 758.037 15.349 38.425 20.708 465.637 694.700 0.000 207.273 0.000 2.306
NOR-47 36.754 32.965 10.949 228.313 12.345 27.964 978.003 1412.791 47.795 24.690 121.853 1264.977 28.558 79.091 204.493 2.868
NOR-48 180.628 12.558 1054.380 107.229 20.655 643.636 954.315 2459.302 75.984 41.310 722.780 1078.341 5.725 96.545 0.000 4.559
NOR-49 1162.304 10.360 300.000 193.976 7.885 174.909 751.269 575.581 27.559 15.770 769.112 1593.318 0.000 82.364 51.616 1.654
NOR-50 43.115 18.488 51.460 154.217 5.920 20.145 330.964 741.279 2.724 11.841 49.575 912.442 0.000 78.727 102.575 0.163
NOR-51 61.571 14.599 797.080 164.458 26.018 162.545 1729.272 493.605 88.189 52.035 442.471 293.779 0.000 87.091 7.529 5.291
NOR-52 539.529 6.924 716.058 91.566 8.655 647.273 612.521 43.953 31.969 17.310 903.475 1347.926 0.000 100.000 0.000 1.918
NOR-53 188.482 7.360 513.504 71.084 15.345 436.364 730.964 38.721 53.386 30.690 1121.236 185.945 12.865 87.273 4.438 3.203
NOR-54 47.120 41.860 662.409 219.880 9.319 310.545 379.019 6.828 32.126 18.637 111.660 42.166 0.000 70.364 2.055 1.928
NOR-55 895.288 6.209 107.956 79.518 5.549 120.727 419.628 709.884 20.236 11.097 887.259 3352.535 2.856 73.273 139.068 1.214
NOR-56 95.026 14.163 17.737 104.217 9.398 9.236 439.932 357.558 30.866 18.796 90.347 425.115 7.153 79.273 31.167 1.852
NOR-57 467.277 14.128 90.766 161.446 10.381 65.455 605.753 762.209 37.402 20.761 428.571 1503.456 0.000 75.818 101.918 2.244
NOR-58 120.942 57.384 260.584 530.723 14.257 11.127 1086.294 1927.326 48.661 28.513 97.066 1182.028 20.046 176.727 226.521 2.920
NOR-59 292.147 4.221 687.591 35.904 11.814 476.364 703.892 57.645 40.945 23.628 762.162 1137.097 5.712 79.273 0.000 2.457
NOR-60 56.623 17.791 733.577 92.771 15.425 360.364 734.349 402.907 52.992 30.850 284.942 291.359 27.185 64.909 5.819 3.180
NOR-61 743.717 6.872 132.482 146.386 6.876 80.364 473.773 478.779 21.732 13.752 676.448 1220.046 8.581 121.818 22.586 1.304
NOR-62 38.246 11.599 736.861 80.120 17.310 200.727 886.633 28.256 60.551 34.619 294.208 1.980 20.046 83.455 0.000 3.633
NOR-63 515.183 25.291 86.168 175.301 17.097 32.073 741.117 624.419 64.094 34.195 324.324 760.369 12.865 60.000 36.164 3.846
NOR-64 82.461 2.948 152.190 65.060 10.460 49.091 1289.340 238.081 34.488 20.920 398.456 199.424 7.153 64.182 51.945 2.069
NOR-65 258.377 37.849 104.343 237.349 28.673 74.182 1025.381 1281.977 95.276 57.345 238.610 981.567 11.437 80.727 38.137 5.717
NOR-66 456.283 5.023 258.394 19.940 9.345 143.273 605.753 968.023 35.118 18.690 634.749 2398.618 5.725 86.364 182.466 2.107
NOR-67 731.152 20.930 596.715 292.771 21.000 197.455 1560.068 101.163 72.362 42.000 861.776 1.690 5.725 86.909 0.000 4.342
NOR-68 75.000 24.070 463.139 217.470 32.124 25.964 1576.988 994.186 107.874 64.248 86.409 45.968 15.789 116.000 32.351 6.472
NOR-69 266.230 12.471 139.051 137.349 13.752 27.891 818.951 484.884 47.165 27.504 247.876 373.272 2.856 84.909 55.890 2.830
NOR-70 47.592 17.965 1313.869 213.855 23.735 443.636 1340.102 44.826 89.764 47.469 250.193 373.272 0.000 122.545 0.000 5.386
NOR-71 629.843 7.953 270.438 131.928 6.956 121.455 839.255 859.012 23.228 13.912 555.985 1337.558 5.725 79.091 122.301 1.394
NOR-72 208.115 15.244 396.350 169.277 8.602 81.455 673.435 1002.907 30.000 17.204 227.027 1029.954 11.437 180.909 63.123 1.800
NOR-73 201.047 23.721 1521.898 277.711 18.850 233.091 1225.042 474.419 68.425 37.699 245.560 230.530 4.284 112.000 2.739 4.106
NOR-74 119.372 15.820 81.350 178.313 7.221 26.800 483.926 572.965 25.669 14.442 203.398 708.525 0.000 74.727 15.058 1.540
NOR-75 40.759 7.308 37.336 62.651 9.186 8.618 251.438 199.709 30.000 18.372 37.066 135.829 5.712 58.545 53.589 1.800
NOR-76 30.628 6.070 88.358 40.000 9.000 80.727 737.733 2.817 31.890 18.000 363.707 542.627 0.000 52.727 0.000 1.913
NOR-77 44.372 5.110 1065.328 65.663 20.204 292.727 1377.327 525.000 77.638 40.407 428.571 320.737 5.725 63.273 28.767 4.658
NOR-78 131.937 43.605 561.679 266.867 23.336 150.909 1201.354 4.814 78.583 46.673 345.174 154.147 4.284 76.909 17.819 4.715
NOR-79 570.157 18.663 101.277 141.566 16.434 15.164 761.421 755.233 56.299 32.867 766.795 1475.806 8.581 67.636 77.589 3.378
NOR-80 21.518 25.465 110.584 203.614 7.540 30.945 419.628 690.698 24.803 15.080 96.139 739.631 5.712 41.091 76.603 1.488
NOR-81 58.194 12.890 502.555 175.301 19.938 154.182 1353.638 258.140 69.291 39.876 312.741 144.470 4.284 50.182 0.000 4.157
NOR-82 98.168 23.547 331.752 181.325 16.619 35.964 994.924 724.709 55.591 33.239 82.008 224.654 4.284 67.636 54.247 3.335
NOR-83 439.005 4.744 186.131 65.663 3.664 181.455 368.866 431.686 5.630 7.327 650.965 1534.562 0.000 51.091 47.671 0.338
NOR-84 102.880 36.628 69.197 149.398 17.310 15.091 467.005 165.698 58.425 34.619 55.367 55.991 2.856 38.364 23.967 3.506
NOR-85 40.681 2.459 1020.438 35.904 11.947 192.000 710.660 3.724 40.472 23.894 192.973 287.903 4.284 45.273 0.000 2.428
NOR-86 252.094 12.000 131.387 54.217 10.805 95.636 416.244 1186.047 40.630 21.611 298.842 2163.594 15.789 55.636 31.167 2.438
NOR-87 105.236 13.064 116.058 50.120 8.230 9.345 234.856 219.767 29.449 16.460 49.112 169.700 7.153 28.545 47.671 1.767
NOR-88 34.005 20.233 188.321 198.193 12.000 26.655 680.203 1421.512 42.835 24.000 52.124 649.770 17.162 50.545 82.521 2.570
NOR-89 27.958 5.041 67.117 84.337 6.664 9.236 355.330 443.023 24.252 13.327 66.950 279.263 0.000 27.636 10.948 1.455
NOR-90 150.785 7.047 522.263 93.976 4.858 262.182 426.396 89.826 15.433 9.717 250.193 153.456 0.000 38.545 0.000 0.926
NOR-91 1.720 1.408 82.226 17.711 1.755 30.291 161.760 470.930 2.520 3.510 18.463 327.650 0.000 34.364 60.164 0.151
DOR-1 177.487 22.674 300.000 130.723 25.062 74.545 609.137 1116.279 87.402 50.124 268.726 1589.862 24.302 26.545 98.959 5.244
DOR-2 20.576 15.558 99.745 130.723 10.407 17.891 812.183 941.860 38.346 20.814 74.595 521.889 8.581 38.000 305.425 2.301
DOR-3 16.963 57.209 248.540 283.735 22.832 45.091 707.276 1247.093 79.528 45.664 140.386 839.862 9.295 31.636 160.438 4.772
DOR-4 84.031 7.012 80.255 35.241 8.655 9.855 622.673 740.407 31.102 17.310 65.792 466.590 14.279 47.091 55.562 1.866
DOR-5 141.361 103.605 251.825 309.639 52.301 54.182 1011.844 2607.558 174.803 104.602 236.293 122.350 0.000 32.545 0.000 10.488
DOR-6 2.615 9.052 60.438 74.096 4.088 0.935 190.186 208.430 14.016 8.177 31.042 151.728 0.000 20.727 9.238 0.841
DOR-7 244.241 25.640 1872.263 300.000 34.779 329.091 1698.816 126.453 112.598 69.558 430.888 642.857 8.581 27.273 0.000 6.756
DOR-8 134.293 38.721 436.861 457.831 23.018 85.455 1411.168 1203.488 74.882 46.035 192.278 729.263 8.581 24.182 115.726 4.493
DOR-9 16.178 8.285 167.518 106.024 5.257 52.000 446.701 790.988 20.079 10.513 39.151 794.931 7.153 29.091 17.458 1.205
DOR-10 138.220 17.145 339.416 257.831 26.389 36.218 1231.810 78.750 88.189 52.779 379.923 566.820 4.284 35.818 0.000 5.291
DOR-11 15.942 9.157 174.088 84.940 6.637 10.473 260.914 218.895 2.079 13.274 8.595 31.210 0.000 25.273 7.200 0.125
DOR-12 74.136 19.012 159.854 233.735 14.602 3.596 609.137 815.407 51.654 29.204 63.475 411.290 0.000 39.818 79.233 3.099
DOR-13 36.911 8.826 1047.810 296.988 12.425 193.091 1634.518 20.494 42.126 24.850 204.788 305.530 0.000 28.182 0.000 2.528
DOR-14 64.476 7.605 70.620 83.735 10.142 6.145 463.621 3.907 36.457 20.283 76.216 113.710 0.000 15.400 0.000 2.187
DOR-15 30.942 6.419 153.285 162.048 8.071 7.309 754.653 633.140 8.898 16.142 35.907 2132.488 7.153 24.727 22.948 0.534
DOR-16 26.859 34.012 1138.686 136.747 28.938 20.400 419.628 270.349 95.276 57.876 47.490 244.009 0.000 29.636 0.000 5.717
DOR-17 465.707 59.651 250.730 310.241 28.673 64.364 967.851 1735.465 99.213 57.345 284.942 1365.207 10.009 28.000 88.438 5.953
DOR-18 33.534 36.628 96.679 116.265 37.965 6.618 477.157 218.023 118.898 75.929 36.834 104.032 12.865 26.000 21.041 7.134
DOR-19 292.932 1.988 483.942 50.904 4.646 165.455 494.078 1273.256 16.693 9.292 347.490 518.433 5.725 22.000 0.000 1.002
DOR-20 15.157 16.169 118.248 189.759 11.867 9.345 690.355 568.605 42.126 23.735 5.050 206.682 5.712 26.364 102.904 2.528
DOR-21 47.042 122.616 284.672 855.422 42.212 17.018 1576.988 1421.512 148.031 84.425 41.236 310.714 5.725 37.091 72.658 8.882
DOR-22 6.377 12.349 233.212 225.904 13.779 3.560 937.394 858.140 46.142 27.558 22.819 47.005 2.856 19.818 35.507 2.769
DOR-23 365.969 16.657 51.898 220.482 7.991 6.218 558.376 470.058 29.134 15.982 187.876 497.696 0.000 32.364 75.945 1.748
DOR-24 214.398 8.616 182.847 192.771 7.434 39.636 714.044 985.465 7.701 14.867 131.351 746.544 5.712 31.636 210.411 0.462
DOR-25 453.141 24.419 69.745 176.506 13.619 9.418 548.223 608.721 5.197 27.239 187.413 770.737 5.725 52.545 73.315 0.312
DOR-26 162.565 5.843 111.679 93.373 11.204 45.455 876.481 27.122 40.000 22.407 326.641 64.977 4.284 19.273 0.000 2.400
DOR-27 15.079 13.901 263.869 101.205 10.938 38.182 575.296 284.302 41.260 21.876 47.954 171.774 0.000 25.636 14.203 2.476
DOR-28 24.581 89.826 492.701 277.711 41.416 66.182 842.640 1438.953 151.969 82.832 24.556 566.820 14.279 36.182 20.186 9.118
DOR-29 37.225 22.500 187.226 76.506 21.159 22.873 439.932 1133.721 68.583 42.319 65.097 97.120 4.284 46.182 0.000 4.115
DOR-30 62.513 22.674 127.007 203.012 14.416 27.636 395.939 258.140 51.575 28.832 78.301 95.737 0.000 93.455 0.000 3.094
DOR-31 185.340 12.105 96.460 131.928 6.584 41.455 497.462 1351.744 24.173 13.168 216.834 1679.724 8.581 38.000 76.932 1.450
DOR-32 91.099 9.227 110.584 133.133 6.398 2.273 514.382 568.605 20.709 12.796 74.826 376.728 5.725 22.909 78.575 1.243
DOR-33 104.450 0.000 150.000 96.386 8.363 20.182 1025.381 716.860 32.835 16.726 199.459 400.922 4.297 24.000 244.932 1.970
DOR-34 54.581 30.000 360.219 281.325 19.115 29.418 866.328 976.744 62.441 38.230 41.004 362.903 11.437 28.545 72.329 3.746
DOR-35 12.173 15.471 174.088 134.337 13.301 16.800 626.058 1046.512 45.354 26.602 36.602 425.115 10.009 29.091 146.301 2.721
DOR-36 158.639 18.837 222.263 125.301 14.867 44.000 676.819 642.733 51.339 29.735 166.100 508.065 10.009 19.636 91.397 3.080
DOR-37 281.937 21.105 241.971 252.410 15.425 21.345 937.394 756.977 51.024 30.850 224.247 352.535 0.000 40.727 33.205 3.061
DOR-38 28.822 16.692 621.898 117.470 16.646 97.455 802.030 84.767 57.953 33.292 120.695 49.424 0.000 24.364 3.419 3.477
DOR-39 28.429 28.081 77.628 40.241 17.257 1.855 531.303 852.907 56.614 34.513 24.324 250.922 22.929 43.636 120.658 3.397
DOR-40 856.021 19.884 143.431 171.687 10.195 41.091 666.667 404.651 37.559 20.389 428.571 466.590 10.009 32.364 97.644 2.254
DOR-41 958.115 35.407 194.891 362.048 16.301 11.527 957.699 1438.953 56.220 32.602 389.189 1486.175 11.437 42.909 140.712 3.373
DOR-42 21.990 2.512 70.839 66.867 2.312 7.091 247.716 539.826 7.677 4.625 26.178 316.935 0.000 19.091 14.729 0.461
DOR-43 25.052 16.919 82.336 125.904 15.876 3.818 510.998 427.326 51.969 31.752 36.834 188.710 11.437 30.909 13.512 3.118
DOR-44 13.665 11.128 374.453 112.651 18.425 52.727 1120.135 770.058 65.039 36.850 119.537 525.346 0.000 29.636 94.356 3.902
DOR-45 27.173 1.003 55.401 36.205 6.531 10.509 795.262 17.180 21.811 13.062 158.456 51.843 0.000 22.182 7.858 1.309
DOR-46 8.089 3.244 40.839 10.843 3.558 1.949 259.898 7.491 3.228 7.115 8.085 12.062 0.000 14.982 0.000 0.194
DOR-47 98.168 12.401 129.197 213.253 12.080 18.509 798.646 915.698 43.228 24.159 49.344 345.622 4.284 15.491 11.803 2.594
DOR-48 8.717 16.116 99.854 106.627 27.611 0.644 741.117 641.860 92.913 55.221 19.274 169.700 7.153 31.091 59.507 5.575
DOR-49 13.665 2.302 48.394 116.265 6.664 1.400 571.912 1203.488 8.661 13.327 65.560 618.664 11.437 28.182 180.493 0.520
DOR-50 488.482 24.244 198.175 389.759 13.513 48.727 998.308 692.442 44.173 27.027 294.208 549.539 0.000 47.636 78.247 2.650
DOR-51 7.241 11.843 204.745 11.145 15.903 35.164 483.926 395.930 53.701 31.805 38.919 171.083 11.437 31.455 97.644 3.222
DOR-52 244.241 17.616 153.285 312.651 17.947 18.400 896.785 518.023 61.181 35.894 118.147 233.986 5.712 18.909 0.000 3.671
DOR-53 22.853 20.233 107.847 139.759 12.558 19.018 389.171 620.930 40.315 25.115 23.398 483.871 10.009 8.000 30.115 2.419
DOR-54 173.560 9.628 79.599 99.398 11.681 12.618 385.787 337.500 32.835 23.363 113.745 288.249 7.153 17.109 10.258 1.970
DOR-55 2.780 20.233 345.985 225.301 19.991 16.836 822.335 28.605 67.008 39.982 6.788 10.127 5.725 21.455 0.000 4.020
DOR-56 131.937 11.703 643.796 169.277 21.823 70.909 1292.724 560.756 70.709 43.646 191.351 317.627 8.581 16.545 35.507 4.243
DOR-57 286.649 19.360 347.080 293.976 11.071 62.545 1109.983 859.884 41.181 22.142 212.664 521.889 5.725 17.673 36.164 2.471
DOR-58 185.340 4.762 13.577 125.904 4.673 9.309 286.294 279.070 13.937 9.345 44.479 221.889 0.000 16.091 34.521 0.836
DOR-59 144.503 11.686 505.839 166.867 20.655 10.436 862.944 613.081 72.126 41.310 138.069 337.673 14.279 17.109 43.068 4.328
DOR-60 29.372 20.058 85.292 151.205 0.417 25.782 212.183 189.244 18.189 0.834 35.907 196.659 8.581 17.091 16.767 1.091
DOR-61 114.660 21.453 385.401 317.470 18.000 33.891 998.308 497.965 59.291 36.000 76.680 199.424 0.000 21.091 29.096 3.557
DOR-62 12.330 15.645 78.942 69.277 14.761 7.709 295.093 383.721 48.976 29.522 13.367 116.475 0.000 14.364 5.819 2.939
DOR-63 1.225 0.945 556.204 68.675 3.398 60.364 433.164 3.497 4.063 6.796 53.977 80.530 5.712 16.909 0.000 0.244
DOR-64 435.079 62.093 194.891 469.880 12.796 42.545 517.766 968.023 45.354 25.593 183.707 777.650 5.725 14.509 59.836 2.721
DOR-65 43.822 9.820 404.015 101.205 9.212 33.455 548.223 624.419 32.520 18.425 63.707 306.912 17.162 14.400 48.658 1.951
DOR-66 41.937 200.581 626.277 873.494 41.947 62.909 1028.765 290.407 148.031 83.894 42.625 23.675 0.000 15.636 0.000 8.882
DOR-67 91.885 64.884 107.956 825.301 17.257 8.327 1062.606 858.140 63.465 34.513 39.382 228.456 18.535 16.945 14.729 3.808
DOR-68 72.565 8.965 172.993 80.120 10.327 9.855 385.787 296.512 34.331 20.655 49.807 151.037 0.000 20.000 33.863 2.060
DOR-69 63.613 5.703 488.321 114.458 9.053 73.818 1177.665 769.186 30.157 18.106 162.394 304.839 0.000 28.000 2.739 1.809
DOR-70 55.602 56.860 86.168 301.807 14.814 2.611 379.019 662.791 45.512 29.628 6.069 327.995 8.581 6.691 37.808 2.731
DOR-71 89.529 16.012 163.139 222.289 18.478 21.309 1258.883 290.407 58.425 36.956 187.645 153.111 0.000 14.127 34.521 3.506
DOR-72 98.168 3.488 205.839 59.518 4.699 35.273 338.409 71.163 15.118 9.398 70.656 85.714 0.000 15.200 10.619 0.907
DOR-73 113.089 9.785 361.314 74.096 8.655 47.636 460.237 5.721 28.346 17.310 149.653 46.659 0.000 1.855 0.000 1.701
DOR-74 66.047 7.326 397.445 114.458 10.221 34.109 639.594 470.930 36.378 20.442 105.869 192.166 0.000 13.073 2.055 2.183
DOR-75 51.047 4.919 324.088 39.518 10.513 40.000 412.860 29.302 4.583 21.027 71.120 106.106 0.000 14.364 0.000 0.275
DOR-76 75.864 0.000 42.044 1.916 3.106 8.509 316.751 120.349 9.528 6.212 154.981 146.198 0.000 13.218 22.258 0.572
DOR-77 14.058 0.431 90.766 32.590 2.788 15.418 157.699 210.174 8.031 5.575 18.139 95.737 2.856 22.545 15.058 0.482
DOR-78 4.005 4.727 130.292 32.048 4.673 18.909 147.208 109.884 13.937 9.345 13.737 79.839 0.000 9.964 11.310 0.836
DOR-79 5.599 34.186 49.489 171.084 22.673 6.000 578.680 112.500 74.567 45.345 23.629 11.578 0.000 14.855 0.000 4.474
DOR-80 16.963 4.552 393.066 55.120 9.106 64.000 480.541 172.674 33.386 18.212 65.792 100.230 2.856 16.236 9.929 2.003
DOR-81 59.607 0.000 60.876 30.241 2.196 8.945 221.658 442.151 6.354 4.391 40.077 332.143 0.000 26.909 0.000 0.381
DOR-82 32.670 10.308 14.672 100.000 6.690 4.000 351.946 572.965 22.362 13.381 17.537 199.078 0.000 13.327 14.729 1.342
DOR-83 18.298 13.988 72.482 106.024 17.257 0.607 534.687 361.919 60.157 34.513 18.139 97.465 7.153 14.055 66.740 3.609
DOR-84 52.853 6.942 67.336 50.542 5.124 4.000 224.704 238.081 16.378 10.248 25.019 118.894 0.000 5.564 17.129 0.983
DOR-85 1.147 3.157 0.000 2.633 3.611 0.491 58.545 29.302 1.646 7.221 6.000 12.373 0.000 11.964 0.000 0.099
DOR-86 120.942 16.552 148.905 128.313 14.389 4.509 707.276 97.674 46.850 28.779 109.807 38.364 0.000 11.236 2.739 2.811
DOR-87 23.325 2.093 9.230 5.831 0.308 2.273 77.496 15.349 7.362 0.616 14.826 4.631 0.000 14.436 0.000 0.442
DOR-88 89.529 8.215 258.394 98.193 6.053 62.545 302.538 779.651 20.945 12.106 55.367 14.309 4.284 12.855 2.397 1.257
DOR-89 86.387 14.826 215.693 143.373 8.549 18.036 602.369 41.512 27.559 17.097 76.911 8.779 0.000 18.545 1.713 1.654
DOR-90 10.681 10.570 192.701 118.072 10.619 12.764 554.992 351.453 30.079 21.239 32.201 116.129 2.856 18.727 17.819 1.805
DOR-91 64.005 3.174 127.007 55.602 3.398 25.382 412.860 491.860 4.811 6.796 81.081 338.018 0.000 26.000 26.696 0.289
为了区分卵巢储备功能正常患者和卵巢储备功能减退患者,我们使用Mann-Whitney U检验来挑选和鉴定候选的生物标记物,结果见表2。我们选择了两组之间具有显著差异的五种胆汁酸亚型作为候选变量,并用随机森林模型来评价候选变量并建立模型。
表2:NOR组和DOR组卵泡液中胆汁酸的差异
随机森林模型建立方法及相关参数选择:相关模型建立在R语言的R-Studio环境下进行。首先对原始数据进行清洗预处理,并且按照1:1的比例划分数据,建立训练集和测试集。进一步, 选择两组之间具有显著差异的胆汁酸-胆酸、脱氧胆酸、鹅去氧胆酸、石胆酸和熊去氧胆酸,作为构建随机森林模型的输入特征,建立基于randomForest函数(Ntree =500和mtry = 2)的诊断模型。pROC用于绘制受试者工作曲线(ROC)并评估曲线下面积(AUCs)的95%置信区间(CI),测量随机森林在训练集和外部验证集中的预测性能。
91例卵巢储备功能正常(NOR)的患者(正常人)和91例经证实的卵巢储备功能减退(DOR)患者,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有卵巢储备功能减退的可能性,并通过ROC分析中的约登最优点找到最佳截断值,可评估该模型对于区分卵巢储备功能减退患者和健康人的总能力。结果如图1所示:ROC曲线下面积为0.947,最佳截断值为1.500,最佳截断值处的灵敏度和特异性百分比分别为97.7%和91.7%。如所述对象测定标志物水平,将这些测定值带入随机森林模型后,如果个体得分阈值大于1.500,表明此个体具有较高的患卵巢储备功能减退风险;如果个体得分阈值小于1.500,表明此个体具有较低的患卵巢储备功能减退风险。
为了进一步验证上述模型的诊断能力,我们进一步纳入20例DOR患者和20例正常人,分别用本发明的试剂盒检测卵泡液中的胆汁酸浓度,检测结果见表3。将这些测定值带入诊断模型后,结果如图2所示:ROC曲线下面积为1.000。检测结果符合我们的预期,诊断模型在外部验证集里依旧能够极好得区分DOR患者和正常人。
表3. 试剂盒检测结果
编号 CA UDCA CDCA DCA LCA
DOR-1 84.031 39.636 152.201 313.479 44.000
DOR-2 253.665 19.309 147.104 205.991 10.655
DOR-3 40.681 83.273 85.251 127.189 16.782
DOR-4 54.581 29.418 41.004 362.903 28.545
DOR-5 43.272 23.927 71.815 418.203 30.182
DOR-6 415.445 29.491 345.174 225.000 20.364
DOR-7 175.131 2.575 55.135 229.493 32.000
DOR-8 135.079 36.727 106.100 622.120 18.182
DOR-9 48.377 169.455 97.297 50.461 14.636
DOR-10 645.550 60.727 437.838 684.332 15.182
DOR-11 108.377 0.858 51.892 200.115 24.909
DOR-12 97.382 11.527 45.405 157.258 20.000
DOR-13 41.937 10.218 46.332 165.207 14.364
DOR-14 165.707 13.164 122.548 221.544 11.455
DOR-15 56.466 20.945 69.961 362.903 31.273
DOR-16 54.503 14.545 78.069 134.101 24.545
DOR-17 116.230 5.236 103.089 96.083 17.873
DOR-18 8.089 0.000 28.031 182.143 15.200
DOR-19 125.654 189.818 117.452 48.041 19.455
DOR-20 7.115 29.964 43.089 366.359 25.455
NOR-1 185.340 11.200 312.741 362.903 72.182
NOR-2 430.366 89.091 386.873 864.055 48.727
NOR-3 80.105 59.273 86.873 566.820 192.727
NOR-4 387.173 34.364 236.293 514.977 66.727
NOR-5 210.471 69.455 889.575 1327.189 78.182
NOR-6 465.707 96.727 662.548 584.101 65.273
NOR-7 197.120 39.273 221.467 1078.341 68.545
NOR-8 302.356 68.364 481.853 725.806 47.455
NOR-9 755.497 304.364 1661.004 438.940 46.727
NOR-10 226.178 262.182 474.903 708.525 115.091
NOR-11 292.147 71.273 275.676 359.447 46.364
NOR-12 36.440 15.964 8.595 185.253 120.909
NOR-13 179.843 7.382 147.336 404.378 64.909
NOR-14 11.152 8.000 37.529 250.922 58.909
NOR-15 2.458 37.818 28.263 394.009 106.364
NOR-16 11.545 13.527 30.347 291.359 62.364
NOR-17 16.806 15.127 18.927 226.728 61.455
NOR-18 130.366 41.091 92.664 546.083 55.091
NOR-19 47.356 2.084 65.792 355.991 54.727
NOR-20 5.317 10.545 68.108 129.263 61.091
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。

Claims (4)

1.一种用于预测卵巢储备功能减退的试剂盒,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、脱氧胆酸、鹅去氧胆酸、石胆酸和熊去氧胆酸,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、血清或卵泡液;所述内标标准溶液为经同位素标记的所述生物标志物,同位素标记方式为2H或13C标记。
2.如权利要求1所述的试剂盒,其特征在于,所述试剂盒还包含萃取液,萃取液由甲醇和乙腈组成,甲醇和乙腈的体积比为1:1-5:1。
3.如权利要求1所述的试剂盒的使用方法,其特征在于,通过测定受试体的血清、血浆或卵泡液中代谢物胆酸、脱氧胆酸、鹅去氧胆酸、石胆酸和熊去氧胆酸的浓度,并将这些测定值输入随机森林模型得到截断值进行判定。
4.如权利要求3所述的诊断试剂盒的使用方法,包括下列步骤:
a) 配制不同浓度的生物标志物标准品溶液;
b) 配制内标标准品溶液配制;
c) 制备受试体血清、血浆或卵泡液样本;
d) 将c)中制得的样本以液相色谱和质谱进行测定,计算样本中代谢产物的浓度;
e) 将d)中计算得到的生物标志物的浓度输入随机森林模型,按照截断值对受试体是否患有卵巢储备功能减退进行判定。
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