评估卵母细胞质量和发育潜能的方法及生物标志物组合

本发明涉及用于评估哺乳动物卵母细胞质量和发育潜能的生物标志物及其在卵母细胞体外成熟培养技术中的应用，属于生物和生殖医学。

背景技术：

1、表观遗传学是研究基因的dna序列不发生改变，而基因的功能发生可遗传的改变的学科。表观遗传包含dna甲基化重编程、染色质开放状态变化、组蛋白修饰和微小rna水平变化等分子事件。dna甲基化(5mc)在哺乳动物的cpg二核苷酸中占主导地位，可在有丝分裂过程中被子细胞忠实地继承，并作为染色质结合蛋白和修饰因子的调控标记，影响基因表达和染色质结构。这种相对静态的结构会受到甲基胞嘧啶双加氧酶家族蛋白(tets)的局部干扰，5mc会被tet转化为连续的氧化衍生物，如5-羟甲基胞嘧啶(5hmc)、5-甲酰基胞嘧啶(5fc)和5-羧基胞嘧啶(5cac)。5hmc虽然只占所有5mc的极小一部分，但在体细胞中的含量仍然比5fc和5cac高得多，预示着5hmc可能在体细胞中发挥重要功能。已有报道证实dna羟甲基化在发育、衰老、神经退行性疾病、复杂疾病及肿瘤发生过程中起重要作用。dna羟甲基化可作为复杂疾病预后相关的潜在表观遗传标志物，用于肿瘤筛查、诊断、检测或预后评估。但未有报道将其应用于生殖生物学领域。

2、卵母细胞体外成熟培养技术(ivm)是指模拟体内卵母细胞成熟环境，从未经药物刺激或低剂量药物刺激的卵巢直接获取未成熟生殖泡期(gv)卵母细胞在体外培养为成熟的mⅱ的卵母细胞。ivm技术被用于辅助生殖技术、多囊卵巢综合征(pcos)的治疗、肿瘤患者的生育力保存等领域。ivm获得的成熟卵母细胞的质量决定了受孕率、后续胚胎的存活和发育潜能。目前评估卵母细胞质量的方法主要有形态学评估和代谢评估，这些评估手段可能存在主观偏差，而针对人卵母细胞的无偏的，全局性的，且可进一步通过遗传影响后代发育潜能的质量评估仍然欠缺。

技术实现思路

1、本发明的目的在于寻找无偏的、全局性的评估卵母细胞质量和发育潜能的方法和生物标志物。具体涉及dna羟甲基化检测和dna羟甲基化生物标志物组合在评估人卵母细胞质量和发育潜能中的应用。

2、本发明的研究发现，人成熟卵母细胞的基因组中存在大量的高dna羟甲基化修饰区域，数量是小鼠成熟卵细胞中高羟甲基化修饰区域的40倍。与小鼠卵细胞中高羟甲基化区域的分布模式不同的是，人卵细胞中高羟甲基化的区域主要分布在基因体、重复元件sine以及增强子(enhancer)区域。这些人卵细胞中高羟甲基化的区域的羟甲基化水平还与一些基因或重复元件在人早期胚胎中的表达呈正相关。人植入前胚胎的母本基因组从卵细胞中继承了一部分卵母细胞的高dna羟甲基化。

3、本发明的研究鉴定出了人卵母细胞高dna羟甲基化的位点，由于同一个体的卵母细胞dna羟甲基化状况基本一致，在卵母细胞体外成熟培养技术中会同时培养多个卵母细胞，因此可通过检测其中少数卵母细胞的dna羟甲基化状态来评估该个体或该批次体外成熟培养的卵母细胞的质量和发育潜能。此外，本发明的研究鉴定出的正常人卵母细胞中有dna羟甲基化修饰的核酸可作为生物标志物组合评估卵母细胞的质量和发育潜能。而且，由于人植入前胚胎的母本基因组从卵细胞中继承了一部分卵母细胞的高dna羟甲基化修饰，因此上述信息也可作为生物标志物评估体外获得的人类全能样干细胞或类囊胚模型与体内相应细胞或组织的相似度。

4、在本发明的第一方面，提出一种基于dna羟甲基化状态评估卵母细胞质量和发育潜能的方法，通过检测卵母细胞基因组的dna羟甲基化来评估卵母细胞的质量和发育潜能。

5、其中，检测dna羟甲基化的方法包括但不限于生物化学方法，例如dna斑点杂交实验(dna dot plot analysis)，超高效液相色谱-串联质谱法，基于click反应的荧光检测的方法。

6、检测dna羟甲基化的方法还可以是基于重亚硫酸盐处理或化学标记或酶法处理的测序方法，例如氧化亚硫酸氢钠测序的方法(oxidative bisulfite sequencing,oxbs-seq)，tet协同的亚硫酸氢钠法测序的方法(tet-assisted bisulfite sequencing,tab-seq)，化学辅助的dna羟甲基化测序的方法(chemical-assisted c-to-t conversion of5hmc sequencing，hmc-catch)，apobec偶联的表观测序的方法(apobec-coupledepigenetic sequencing,ace-seq)。

7、检测dna羟甲基化的方法还可以是基于特异性抗体捕获的测序方法，例如羟甲基化dna免疫共沉淀测序的方法(hmedip-seq)。

8、检测dna羟甲基化的方法还可以是特异位点的羟甲基化检测的方法，例如基于桥式pcr的dna羟甲基化高通量芯片检测的方法，或者结合亚硫酸盐的限制性消化分析方法(cobra)。

9、在本发明的另一方面，提供了一种生物标志物组合作为参比标志物，用于评估卵母细胞的质量和发育潜能。所述生物标志物组合是指基因组上的一系列具有dna羟甲基化修饰的核酸，它们在人类基因组上所在的位点如下所列，其中，这些位点的编码是基于人类参考基因组版本hg19，所列位点上的胞嘧啶c有羟甲基化修饰，在通过上述检测dna羟甲基化的方法检测后仍被检测为c；所述生物标志物组合位于下面所列的每一个位点。

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11、二号染色体(chr2)：673936,1017876,1041674,1113190,1130185,1370787,1496075,1571951,1603775,3056763,3103689,3111289,3147776,3169814,3199602,3254109,3428898,3581739,3953337,3968789,3974779,4046564,4982334,7058148,7202365,8009249,8208146,8208212,8372671,9588936,9623642,10005531,10865026,11686243,12027284,12047547,12057780,12175452,12698892,16130482,18248361,22379376,23759571,23767334,24136458,25036202,25070367,25071551,25082547,26088036,26327107,26451843,27576382,27576390,28833368,28846705,28848494,28984046,29299422,29300833,29309461,30829975,32302013,33637746,33637824,36997386,37257036,37622299,37638317,37662056,38365248,38373776,38480512,38924055,42080785,42144675,42146507,42804512,43190189,43209447,45501950,46154831,46571643,47329289,47329327,47334849,47336037,47345385,47639905,48089952,48576403,54501181,55028178,55108619,55108659,55834503,61535107,61546965,61549091,61618355,61628746,61676294,61916047,62139653,63552711,64153695,65335293,65949282,66157422,68036849,69471624,69580618,70683801,70749726,70848022,71343800,71777793,72289275,72300335,72337129,72339429,72339542,73283442,73283754,73327954,74124222,74155403,74185982,74300764,74350665,75541975,75548843,77241809,86726775,86726811,96889627,98488917,99755650,101478916,101545585,101625498,101691629,101873235,102379040,106380797,106514776,109080618,109157637,109173207,110357539,112441681,112444256,112801911,118756747,121022007,121061580,121063778,128129453,128129476,128132839,128418167,128611279,133198438,133409805,134922325,135201962,135219027,135295380,135299544,135308185,135320284,137265831,144517163,152271728,153022112,159585875,159857547,160093986,160098788,168638504,168666765,168722296,168760619,170425487,170948510,171184479,171487611,172293572,172612456,172623101,172741241,173453386,173980075,174076596,174128745,174147304,174147340,175702977,177350165,177661205,177661306,178260139,183546896,183566915,184021111,184375526,188816553,196953978,196955101,196955110,198292976,201460633,202984619,203255791,203357075,204183208,204202739,204273958,204368766,205444044,206422249,206456979,208136535,217766886,217807260,217877831,217896953,217914829,218015457,218047885,218162751,218515754,218595886,218599270,219043092,219593231,221016906,225086445,228875202,231439226,233517221,234061735,235156635,235156663,236477830,236511635,236641360,236643622,236721013,236827728,238003371,238966236,239319538,239992819,242513609,242513648,242558414,402403,3273360,3349456,3351416,3376052,3376076,3734573,3734617,5354333,8758092,10436617,11984941,12017579,12061861,12716840,17569894,23363748,23397967,25310372,25343885,30981564,30984971,32364285,32365308,33137026,33195988,33562130,33660907,33702234,34160369,34626485,34629241,34843462,35165851,35197229,37106718,37230200,39808068,39895765,40401093,40490171,41974626,42582828,42767744,43267486,44436360,44471018,44507213,45671431,45703646,45975018,46139045,46166483,46172532,46280600,46368177,46369045,46399392,46408499,47151735,47153175,47158303,47203137,47682539,47856084,47858890,47890391,48603904,49202297,49816943,50065096,50076649,50682797,50703305,50710968,50782682,51749007,51759739,51913972,52051938,52064341,52101009,53409083,54117942,55053309,55055962,55368933,55955365,57711447,58432851,58874609,60029866,60038841,60072946,60097755,60107090,60712776,60898048,62344516,62533739,62541788,19815549,27173796,34121567,34809319,34815181,35083368,35472689,36175838,37231172,37820001,37837835,38313238,38321676,38549317,38856369,38944904,39083697,39259113,40303393,40397700,40661766,42103747,42110664,42131170,44225792,44259311,44289652,44329874,44412271,45454565,45463753,46153702,47534231,47535446,47542433,47646297,47870592,47906273,17568622,17584084,18481065,20867878,21133791,21299647,22055412,22191209,23539063,25118508,25422819,25426269,25572417,25743248,25791644,25794642,28904460,29077992,29313113,29695913,30214217,30231498,30412395,31221343,31298649,31337533,31347213,31509022,31872650,32182305,32282004,32724538,34134325,34141620,34141628,34168115,34720240,35533299,35540539,35582103,35597202,35828520,40695521,40782004,40795828,40841612,40854803,40948117,40949324,41073918,41543511,41551294,41789485,41903492,41922979,42302398,42427761,42572750,42596390,42904095,42957628,42961898,43003269,43186205,43193363,43206362,43313675,44134821,44389898,44401634,44535893,44645464,44653816,44658939,45186892,45188015,45189749,45221079,45231455,45273037,45570309,46762987,46772428,46796426,47269621,47716014,47744447,47778738,47835329,47837046,49056345,49312892,49332316,49343803,49356603,50029615,50789759；

12、三号染色体(chr3)：2396146,4457266,4858179,4858318,4858363,4859633,6279893,7818724,8661699,8750998,10263311,10303370,10351773,10444200,10620615,11530052,11555243,11623525,12063362,12076951,12625746,12679244,12888590,12929757,13526947,14074039,15458700,16370127,16388483,16812494,17734349,21602230,23431853,23936124,24996760,25551122,29136290,31690950,31754981,31833465,31909213,31912038,31983714,31983722,32673261,33117852,33425684,37045123,37660147,38164511,40175694,41794621,41883180,41991763,41997789,42095281,42183508,42825305,43459683,44182799,44214736,44220974,44228797,44377920,44985066,45014682,45553092,47063504,47201935,47375157,47437948,47452046,47554176,47767414,47804242,47820086,47929130,48096205,48927577,49074962,49081026,49108646,49347648,49357766,49398282,49455677,50733204,52129145,53245587,53261999,53268231,53658427,53857682,54349404,54596028,54718133,54785017,55443012,55443374,56599700,57358644,57358670,57358910,59764029,60219067,60619515,61564665,61629219,62053132,62654827,63942579,65732802,66277874,66279836,66430556,66452062,66469312,68849021,71078104,71090579,72490525,73019905,73186282,73202743,110636157,115835424,119393140,119721310,119904931,120009123,122155488,124307301,124307309,124497322,124562491,125288660,125770636,126454134,126490343,126599169,126628775,128708209,129471465,129527863,129729349,129839890,129865018,129872372,129872379,132256064,132887597,134757652,136408752,136571221,136623916,138089420,138175602,139899638,141135734,141753768,141852589,142148970,148721908,148721921,150579308,154914430,155631985,159116345,169763073,170833126,171018649,171842172,172417052,172417632,176870188,176894722,179077268,183515043,183876624,183877352,183902653,185600326,186371455,186509573,187443172,188925363,192538478,192550355,194384970,194864810,194935525,195665114,196478025,196566982,197443934,197450861,197560115,197561742,197612251；

13、四号染色体(chr4)：758482,768282,768901,1143020,1886704,1957493,2474258,2621664,4052998,4394068,4540188,5166054,5204218,5225132,5225133,5500755,5677571,5681086,5722797,5819938,5863419,6125919,6428005,6474389,6474391,6474394,6474398,6802297,6913504,6915580,6934662,7312188,7312239,7316355,7773978,7902057,7925538,8421446,8594474,8594531,8594549,8594552,8594565,8594571,9539104,9626649,10176753,10203380,10214632,10298687,10347719,11559514,11704097,11808377,14816761,15133343,15133350,15142375,15600116,17643888,20811385,23153435,24284095,24498359,39362223,39719908,41136322,41262779,53583048,71579229,71582453,78153671,78167286,78241020,78279257,79645105,87955307,96098685,96696646,96709142,99653381,99933721,103530489,106187127,110616876,110622409,110627860,114693915,128919104,129187726,139608158,139625161,139943959,139946814,139946822,139952203,140362221,148991572,151192202,152157543,154553902,156538761,159579917,165588003,165660681,165676359,167384840,167638023,167638045,169774680,169991737,170926055,185548013,186738785；

14、五号染色体(chr5)：255262,442288,914598,1018441,6758384,6801525,7231116,10509937,13244152,13244182,14208614,14392512,14454914,14728183,14834531,15585621,16415057,16595711,16638512,16638799,16646884,16649275,16685841,18532721,43531721,56508991,66076330,66525803,68504724,68536643,68695126,70890246,73067241,73212365,73320944,73471837,73481814,76175639,78525867,78571205,78800975,79473505,80485561,80566069,81675969,82222462,82266994,86593997,94513777,94514601,95104731,102765627,108739939,109274909,109613644,111327509,112549313,112588128,115747004,117659329,117718320,117906753,124376377,130623739,131903886,131981268,132397104,132437421,133116663,133163038,133482304,133495652,133554550,133806938,134086510,134175794,134428455,134674405,135641638,136419168,136540546,136678802,136686135,136713709,137034892,137334533,138104016,138384485,138391204,138530344,138691836,141778324,141827941,141846563,154141043,154143633,154244944,154258876,154322729,155794836,156568014,156828775,156844560,156845859,156851699,156851758,157307630,158153872,160259996,167727837,167732264,167768982,168096495,168567125,168610043,168779897,169236495,170023923,171396896,171401947,172458546,172514437,173038780,174671125,174760212,174778922,175358312,175610694,175621477,176771861,177637312,177637626,179011963,179179199,179238227,179273839,179273972,179525069,179535263,179865126,179886179,179886181,179941313,180286133；

15、六号染色体(chr6)：1414683,1433879,1456679,2239958,3306454,4027256,4133058,4243014,4364024,4374815,4382587,4418518,4808661,4836401,5770339,6210926,6314831,10741512,10741522,10741551,10796495,10990031,12047856,13120042,13287495,14311349,14480573,15302515,15358642,15401054,15401067,15418487,15474332,15711905,18302994,19475291,22056683,23161549,23867574,24427934,24511809,25369902,27090667,27106355,30612475,30696331,34574581,35417120,36102619,36197075,36435501,38597813,39796987,39877096,39909197,39964904,39966627,39990492,40092661,42758494,42999130,43510248,53672665,74085671,74700367,74965649,79279555,88193081,90007869,91170601,107073719,116596701,124384187,129944102,130885682,131623612,135055853,135100551,135136699,135171312,135211549,136197454,136831380,137094411,137531131,137531299,148957285,150474074,151004169,151097115,151097229,151196338,151196381,155357436,155394398,155452376,156678553,156887125,158013515,158018168,158128794,158323040,158428000,158450528,159041791,159094706,159193875,160229866,161730465,164107807,165201240,166067861,166745891,167017287；

16、七号染色体(chr7)：846773,848705,863946,903276,913113,931078,1191897,1194873,1696568,1817341,1833188,1836917,1885160,1963124,1976634,2112000,2470468,2497215,2898649,3418343,3572075,3886771,3969714,3975680,4106474,4108645,5117957,5122682,5334823,5396733,5404812,5687305,5873754,5880376,5882543,5884757,6205919,6207948,6247004,6288037,6304767,6434341,9133442,12340905,17609972,21576850,21935592,21945353,21969504,23353581,23477921,23499886,24420987,27561178,27889056,27896920,27896921,28599120,29335897,29343183,29346574,29415086,29549615,30039583,30342857,32643904,34344282,36214000,36507493,36604257,37195848,43550946,43935870,44065136,44620008,44637693,50805302,51203935,51224662,55592435,66317484,66440943,69454965,71741636,71745312,71869180,72200705,72214297,72262044,72374528,72375457,72375471,72862629,72967067,74104417,75048168,75171101,75195922,75244637,75575680,75660012,75684692,75701288,76061606,76997611,77346425,86836633,96755488,96792545,97934238,97941751,97949229,98020399,98436857,98504097,98628256,98703578,98803114,99054492,99793692,99860624,99863173,99863284,100274961,101038757,101585844,101731682,103822049,104566479,104669583,104721723,105009058,105222960,105228195,105236284,106517097,107148991,116009313,116642944,117366032,129490527,129531982,129567800,129576773,129672020,129795866,130743486,131195254,131231255,131255313,131351780,131694586,132073400,133467494,135294212,135542572,135784448,136854299,137566953,137567313,138011324,138043073,138049777,138110630,138310811,139077522,139105022,140814463,141279396,141385648,148116443,148452290,148723353,148939409,149095426,150094483,150094487,151285735,151292162,151372391,151736574,152345988,152716685,152717008,153894503,153928811,154066805,156336057,156360047,158033180,158073343,158110456,158212484,158316376；

17、八号染色体(chr8)：433193,1117060,1164311,3825195,3840541,4188035,4311751,5534370,8567490,8634136,8686381,9649071,9969451,9974375,10050792,10128113,10357829,11005440,11352866,11660285,13034012,15390712,15635775,17146190,17512258,17587457,17605263,18275361,19268430,19304148,19491534,19528328,20517847,21893985,22307439,22307447,22445185,22449116,22917049,23167684,23216015,23225163,23237537,23903616,25564404,25799326,27177512,27787587,28234092,28236305,28483985,28908935,28996645,29100938,29105083,30130485,30257895,30257952,30621399,31086376,31107129,38417373,38659846,42734972,42966499,48253226,48284446,55015486,67605667,68113012,74859933,75256422,79358035,95127259,95133728,95660817,97810257,98709099,102962262,104013148,105731405,105755385,116171385,119611847,119629305,124238835,124899611,125071009,125072543,125918451,127559360,127977673,128004536,128440559,131578377,132266419,135351583,135931930,135941899,135957986,136062226,136094097,136790356,136870145,140243740,140257070,140552760,140559202,140563661,140596369,141296366,141348421,141350406,141597201,141626439,141761514,141901711,141987449,142002050,142303196,142307728,142321008,145265931,146060527,146067532；

18、九号染色体(chr9)：540194,581404,740901,745197,766876,767008,2155654,4253450,4299933,4367248,4370405,4389667,4711100,4721535,5175603,5177128,5634375,5779702,10885286,10886252,15576866,16277069,17296081,17594275,18877269,19493496,19505276,19545826,20274359,20316240,23770262,27240688,27268876,32710798,34196220,34340076,34989756,34989804,35594140,35594782,36480099,36482498,37580115,71950753,72026652,72060365,72111157,73113786,74016006,78765743,79078682,79250134,80104914,80286764,80533888,80533893,81320230,83278951,83584779,85495826,85957136,87032784,87133789,87142967,88475360,88647003,91086395,91088078,93879015,95017377,95119868,95259774,96320436,99098227,99273839,101033371,101232727,107891149,107917544,110388985,110589457,110597602,110601558,110610117,110612976,110616208,110686364,111292350,111563834,111788763,111794788,112250406,113155728,115160219,116138396,116470029,116515367,116537444,116671702,116822581,117178892,117179706,117241350,117241369,117255585,117392828,117400890,119873243,120079695,120107797,122573253,122600669,122725672,123263192,123320191,123536659,124474851,124552883,124964260,125357795,127898895,127898927,127928326,129287326,130189615,130275753,130736227,130737372,131381156,131488961,131657976,132390414,132593890,133954851,134210380,134211160,134274526,134278594,135053201,135484835,135554337,135981012,135981015,136191986,136212356,136819636,137740671,138701981,138706724,138761303,138791160,139756373,139817934,140440474；

19、十号染色体(chr10)：233302,369947,437506,480413,482906,499199,553514,611935,639847,640011,1265703,1727888,3133734,3182170,3183815,3212279,5100862,5101063,5144821,5264292,5264493,5284016,5284483,5286920,5935133,6304841,6488245,7077159,11129462,11130837,11227523,11255349,11265759,11336765,11612187,11907538,11907558,12243421,12951610,13081056,13859458,16319501,16849077,17586406,17586598,18828261,18887167,22287233,22826373,22886225,22892703,22981183,25679142,28539498,34926668,35056292,35264436,35275768,35755413,43900869,59964141,64944794,65250462,70628194,70685669,72893060,72907926,72931115,74137347,74515970,74753721,74837609,75123540,75123893,75160439,75317702,75317725,76236107,76606384,82481433,88253627,89372964,91770871,93999993,94073347,96174378,96174398,97021502,101627183,102043499,103124603,103124923,103952184,104547017,104764890,104766208,104766773,104766882,104813874,104816126,104816244,104819022,104848423,104893237,104936091,107684974,108724346,108755751,108759559,112367283,113142480,113810389,113857794,114783488,115095590,115352917,115996517,116802811,117760432,119219745,120442967,120932055,120932918,121510064,121686666,125204725,125226905,125261666,125277147,125522800,125545438,125625980,125990395,126415787,126424505,126728750,126792634,126808706,127603436,131270689,131289951,131326795,131386837,131437939,131536523,132177356,132195610,133739859,133857231,133859057,133860884,133871925,133882093,133905629,134104715,135198714；

20、十一号染色体(chr11)：837944,1519611,2481084,2499149,3526826,3561872,4474307,5417456,5433816,5623629,8857421,9271156,9527115,9527226,10598200,11282346,11593392,12088418,12263892,12342964,12412092,12472946,12504209,12914748,14112487,16895824,16920903,16930331,17135149,18651005,20048837,20281562,27667723,27699023,27699037,33170102,33499971,33704768,34148944,34228374,34249802,34260591,34288182,34334850,35861890,36689142,44149029,46606662,46762377,47329450,47519856,47539424,57083611,57550705,57632366,57648519,57679953,57701829,60920703,61651767,62705792,62706447,62766771,62773918,63751603,64806384,65051847,65057867,65307243,66207588,66326570,66906745,68383545,68388218,68911079,68938865,70741878,70880567,70973081,72010927,73060433,73584214,73675615,75136573,78117546,78640381,83087873,86019784,86237408,86237547,93200456,93870719,94891862,94893726,102097592,102282046,102314722,102799120,106168113,106267362,107010256,109265271,109270124,109566635,110405195,110436172,110919290,112501680,113741387,115131297,115461872,115605062,115751826,116332137,116915473,117208159,117217399,117320475,117389754,117522661,117618969,118623882,118850247,119506677,120805635,121526388,121821829,125092020,125455177,126026109,126250196,129595966,129633571,129811874,130120426,131129866,131333369,131338196,131374997,131391133,131404956,131601517,131738995,131851837,131909879,132188420,132203160,132244176,134110234,134110449；

21、十二号染色体(chr12)：802447,1173466,4550988,6318269,8488919,8883217,8895822,8920906,8938752,8947242,8947511,8947711,9827644,11818066,12035481,12382102,12572709,12640029,13690559,19614528,19862099,19949246,20973333,23076318,31231722,31713919,31723431,31883410,32131764,32132075,32858365,45712804,49095969,49661942,50486250,50628107,50693356,51258693,51270151,51327698,51343594,51370535,52361839,53663545,53841897,54546437,56207215,57044656,57468367,58113647,64799925,64971895,65094252,65128231,68175135,68244859,68329419,70159422,70161442,70903020,76690861,77298364,89877132,89885394,95454345,95951391,95969896,96525684,98615253,102215956,102336986,102915630,105407763,105585549,105622916,105622918,108484152,108487603,109191157,110025268,110157422,110185044,110467368,110781685,110810669,111111901,111529486,111602819,112202018,112904455,114494870,114496867,114577809,116337339,116688438,117468366,117511654,118281189,120149967,120881066,120885941,121148973,121171260,121531337,122106172,122186196,122595085,122837480,123243493,123726312,123891549,128793656,129279306,129305479,130167263,130255460,130488621,130978437,131017750,131031277,131098914,131107518,132367311,132676832,132678589,132696045,133340523,133387770,133415902,133421759,133437674；

22、十三号染色体(chr13)：20557407,24295308,28728829,29633711,33015688,41507862,43436643,45030707,45134073,45154425,46083234,47440433,47540448,48201616,48634376,49576143,50257479,51329766,51737399,60081086,60178722,60585131,66808279,77546869,79351987,79632041,80194760,95651106,95853897,95911451,95925590,96337424,96699723,97797030,98196492,98809763,98863626,98884237,98925702,98997446,99077045,99124086,99159510,99867954,99868094,99882592,100262189,100265901,107538715,110292927,110333761,111850587,111851339,111961529,112039015,112045020,112049750,112093380,112140784,112152829,112161365,113236005,113914639,113975364；

23、十四号染色体(chr14)：21693407,22015343,31672187,31727959,31731418,32599900,34360324,35220632,35305001,35308722,35559659,39611076,39797372,40254905,50405863,50989755,51196099,51196417,52478206,53228500,53573994,53598964,55542991,55835216,56260805,56306974,56992748,57100058,57106292,58021477,59825325,61603614,61706034,62292271,64171085,64350212,64420742,64462266,64585471,64613689,64666832,64666833,64740396,64740749,64751919,64756058,65117494,65235772,65545277,65816996,66323054,66395877,66482598,66571594,67928632,69610940,70083255,70171756,70778024,71625434,71649529,72230049,72239958,72580975,72580976,72640894,72744075,72881975,72882024,73582046,73712709,73712718,73966077,74029570,74030168,74142485,74393196,74821614,75171898,75553540,76103833,76103834,78013154,78437494,80309741,87885685,88082190,88127403,88201507,89238561,89715678,89799756,91752069,91766060,91800712,91903192,91903200,91926576,92397161,93094000,95636415,96425239,99459268,100285193,100860472,102484854,102741793,102759975,102814334,103250839,103279809,103503754,104094090,104427556,104703075,104703085,105726426,106013869,106023255；

24、十五号染色体(chr15)：23669036,23669052,26707601,26715045,26715857,33741200,33747701,33769842,33878258,33918545,36339195,39008529,39008679,39240497,39712001,39713415,39731328,39744160,41042764,41236486,41261933,41261950,41908998,41922251,42138255,42959200,43694297,44146082,44170880,44474230,44859480,45063462,45064463,45064495,45921483,50752541,50788474,52346550,52554227,53037296,58061797,58072097,58271062,58951342,59291911,59408310,59627948,59862080,59864483,59893251,59897009,59899996,60528524,60542070,61871790,62937782,63060315,63154001,63194785,63212993,63234858,63804721,63873153,64274513,64852744,64979932,65023687,65027470,66195617,66204614,66228097,66248794,66256192,66256261,66322870,66363038,66691815,66712594,66770205,66883286,66897236,68209143,68210485,68243323,68382598,68410716,68424704,68491360,68599534,68621185,68702963,68843361,69075353,69690499,70799565,70858219,70858511,70867671,70867693,72388648,72767072,72767075,72767078,72999560,73028228,73057551,73855003,75824633,76238846,76358164,76474622,76536303,76541620,76555709,76598692,76598753,76598795,77111294,77243222,77329662,77359476,78251441,78251823,78577330,78866577,78866581,78900230,78949918,79012275,79145940,79154165,80072793,81154386,83342767,83501835,83509383,83534866,85162178,85538107,85703726,86308935,86442653,87876798,88173712,88176058,88184475,88210358,88663779,88663792,88702030,89042579,89104062,89718586,89729522,89784530,89863380,90162525,90172900,91159394,91175992,91187171,91548778,91950034,92488723,92506103,92514421,92593780,92706073,92748257,93159434,93198790,93888334,93890947,96890875,96892228,98621699,99253214,99303306,99309346,99471133,99506100,99525746,100140625,100514184,100524043,100549867,100550229,100711410,100725110,100737542,100745011,100865670,101400271,101599471,101641902；

25、十六号染色体(chr16)：91873,269019,1863174,2996455,2996466,3675028,3748686,3862585,4517145,4768157,5367335,5577390,5990933,6033147,6565385,6754054,6774582,6963408,6974933,7038635,7039142,7269031,7325959,7384890,8820392,8834010,8834078,8998023,9005610,9144234,9221279,9482526,9796238,9812493,10174243,10224601,10380376,10578373,10578392,10579875,10814046,10891301,11060623,11202924,11615787,11622100,11625235,11916078,12182535,12227051,12246001,12274633,12293907,12301097,12322744,12378705,12399136,12412932,12424371,12425785,12462303,12498285,12527142,12557437,12581446,12607906,12634387,12648895,13055650,14369986,14689444,14690745,15762207,15785115,16056528,16056677,16064340,16068972,16076131,16114599,16142780,16152988,16177392,16181400,16214565,16231089,17108218,17117369,17163189,17165001,17232712,17312528,17345090,17345166,17457754,17463732,18925822,18933910,19715695,20866649,21065552,21203147,21273570,22223072,22225644,22344633,22351818,23468428,23493445,23524277,23524293,23573476,25313207,27331187,28204956,29623303,30356675,46962482,48399731,50277962,50294671,50321545,50350549,50351015,57899657,58216988,58655326,58829352,65401656,65682263,65715540,65763046,65809956,65898564,66250665,66368357,67595219,67755358,68350405,68576361,69200468,69761600,69810469,70230251,70233841,70466868,70647733,70662479,70669534,70704989,71482651,71968261,74312716,74397158,74470117,75221382,75254576,75647885,77672183,78029294,78418922,78436501,78451995,78553974,78601343,78667014,78667026,78670734,79041087,79245589,80289339,81769425,81837324,81861562,82692792,84055496,84062373,84067332,84068820,84133033,84207394,84298959,84348089,84363166,84415131,84540385,84550691,85652016,85667712,85686987,85980375,86265810,86282945,86296065,86749423,86765931,86790868,86967361,87009363,87035743,87047156,87057531,87506301,87678320,87723350,87723370,87774116,88791088,88802987,88803135,89329583,89581382,89595651,89620461,89805317；

26、十七号染色体(chr17)：13439,705207,1299712,1632760,1645086,1649481,1792981,2531307,2531419,2578832,3034963,3056337,4018448,4027822,4323838,4753308,4804103,4807095,5671220,5729269,6766498,6779419,6798271,8356142,9048081,9075781,9210180,9233659,9245049,9321079,9353925,10219316,12129031,12142403,12154386,12264622,12274176,17152786,18092428,18099447,25829919,25832365,25844426,25905446,26980937,26982289,27103164,27103165,28445868,29052381,29425444,30285757,30288225,31193793,32399289,32465294,37227161,38379995,38465655,41961997,43057072,43756506,44282978,46195708,47724966,47750740,49273361,49647737,49746811,49763218,49771281,57465556,58562094,58690938,59411900,60785826,64354984,64409869,64412744,64423337,64435691,64441552,64677250,64757355,65657122,65668237,65737270,66516100,70234897,70398782,70534424,70913509,71052833,71251741,71772034,72084962,72761648,72857470,72857480,72857534,72857537,72857588,72857681,72857683,72857686,73245654,74692801,74709468,74781914,74959041,76566627,76569098,76573640,76614697,76858500,76913753,78296503,78297360,78365354,78368706,78985652,80720748,80983640；

27、十八号染色体(chr18)：2810781,2816211,2921194,3006630,9165119,9174194,9231546,12670055,12680509,19286871,19286896,20712652,20840347,20846732,20855514,21107494,21547500,23968715,29231008,33358610,33770381,33889204,39546384,42688954,42688955,42796500,43242413,43741255,44440475,44464739,44848105,44938054,45217266,46062972,46424500,46946685,46947068,46978704,46978727,47098992,47171337,47281940,47378318,47433577,47439064,47460600,47516839,47655928,50404483,51347689,51526688,55310454,55724675,55731442,55810250,56360601,56438928,56545464,56655198,57135458,57232692,57338298,57348128,61050325,62338642,67379238,74153690,75119372,77346613,77461818,77461828,77687129,77739706,77768066,78001768；

28、十九号染色体(chr19)：311000,340069,885993,1466740,1534365,1615774,2420172,2833236,2984684,3945115,4222721,4361736,4369161,4527713,4914133,5222936,5228409,5279914,5293558,5516137,5709165,5740231,5768459,8301524,8589672,10294169,10294777,10314234,10473603,10479056,10666119,11428732,11436033,11787751,11800130,12112930,12122934,12131401,12131954,12326699,12335280,12359820,13304308,13319533,13380834,13748759,14601118,15496077,16539988,16545562,16660036,16730033,16781208,18467803,18467920,18611026,18615424,18615899,18618929,18618995,19049128,24235460,33651385,33933780,34001773,34914586,34961985,35159107,38371105,38414665,38520740,38759886,38760528,38767945,38928207,39042419,39222917,42720337,44042524,45856096,46524806,47188176,47591415,47642757,47814220,47821698,48118698,48250857,48257863,49091629,49735107,49746119,50072828,52479413,53976084,54316323,54327697,54331290,55494188,55523067,55566605,56232337,56241500,56383307,56402358,56520077,56611074,56679980,58811987；

29、二十号染色体(chr20)：402403,3273360,3349456,3351416,3376052,3376076,3734573,3734617,5354333,8758092,10436617,11984941,12017579,12061861,12716840,17569894,23363748,23397967,25310372,25343885,30981564,30984971,32364285,32365308,33137026,33195988,33562130,33660907,33702234,34160369,34626485,34629241,34843462,35165851,35197229,37106718,37230200,39808068,39895765,40401093,40490171,41974626,42582828,42767744,43267486,44436360,44471018,44507213,45671431,45703646,45975018,46139045,46166483,46172532,46280600,46368177,46369045,46399392,46408499,47151735,47153175,47158303,47203137,47682539,47856084,47858890,47890391,48603904,49202297,49816943,50065096,50076649,50682797,50703305,50710968,50782682,51749007,51759739,51913972,52051938,52064341,52101009,53409083,54117942,55053309,55055962,55368933,55955365,57711447,58432851,58874609,60029866,60038841,60072946,60097755,60107090,60712776,60898048,62344516,62533739,62541788；

30、二十一号染色体(chr21)：19815549,27173796,34121567,34809319,34815181,35083368,35472689,36175838,37231172,37820001,37837835,38313238,38321676,38549317,38856369,38944904,39083697,39259113,40303393,40397700,40661766,42103747,42110664,42131170,44225792,44259311,44289652,44329874,44412271,45454565,45463753,46153702,47534231,47535446,47542433,47646297,47870592,47906273；

31、二十二号染色体(chr22)：17568622,17584084,18481065,20867878,21133791,21299647,22055412,22191209,23539063,25118508,25422819,25426269,25572417,25743248,25791644,25794642,28904460,29077992,29313113,29695913,30214217,30231498,30412395,31221343,31298649,31337533,31347213,31509022,31872650,32182305,32282004,32724538,34134325,34141620,34141628,34168115,34720240,35533299,35540539,35582103,35597202,35828520,40695521,40782004,40795828,40841612,40854803,40948117,40949324,41073918,41543511,41551294,41789485,41903492,41922979,42302398,42427761,42572750,42596390,42904095,42957628,42961898,43003269,43186205,43193363,43206362,43313675,44134821,44389898,44401634,44535893,44645464,44653816,44658939,45186892,45188015,45189749,45221079,45231455,45273037,45570309,46762987,46772428,46796426,47269621,47716014,47744447,47778738,47835329,47837046,49056345,49312892,49332316,49343803,49356603,50029615,50789759；

32、x染色体(chrx)：3734552,6195678,9436044,9782941,9858278,9891846,10219385,13731167,13783126,13791201,16067412,18464346,18712168,19378921,19388189,19439406,19461884,21980115,22620088,30794034,44176897,44176946,44192527,46345306,48805106,54061213,54061703,106906786,118604898,121011524,121779628,129376109,138991481,146294321,150029345,153349120。

33、所述生物标志物组合作为参比标志物，用于评估人卵母细胞质量和发育潜能是指将待评估样品中检测到有dna羟甲基化的位点与上述每一个位点进行对比，获得覆盖到这些位点的比例，覆盖比例越高，则卵母细胞质量和发育潜能越好。

34、进一步的，在待评估样品全基因组dna羟甲基化水平维持在正常水平，即羟甲基化水平在0.057±0.002的基础上进行位点比对，如果待评估样品dna羟甲基化的位点覆盖到上述位点的比例越高，则卵母细胞质量和发育潜能越好。

35、所述生物标志物组合还可以用于评估体外获得的人类全能样干细胞或类囊胚模型与体内相应细胞或组织的相似度。

36、本发明的有益效果：

37、本发明提出的评估方法可以全局地、无偏地评估人类卵母细胞的质量和发育潜能，以及体外获得的人类全能样干细胞或类囊胚模型与体内相应细胞或组织的相似度。本发明提供的生物标志物组合可以作为评估人类卵母细胞的质量和发育潜能的参比标志物，帮助筛选具有高质量高发育潜能的卵母细胞进行体外受精，从而提高卵母细胞的受孕成功率以及后续胚胎的发育潜能，或者有助于构建更接近体内状态下的体外人类胚胎模型。