CN118033008B - 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
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- Investigating Or Analysing Biological Materials (AREA)
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
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 (3)
1.一种生物标志物在制备用于预测卵巢储备功能减退的试剂盒中的应用，其特征在于，所述试剂盒包含生物标志物的标准溶液和内标标准溶液，所述生物标志物为胆酸、脱氧胆酸、鹅去氧胆酸、石胆酸和熊去氧胆酸，并且所述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为卵泡液；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，所述试剂盒的使用方式为：通过测定受试体的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。
2.如权利要求1所述的应用，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或2所述的应用，其特征在于，所述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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