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申请/专利权人：中国科学院动物研究所

摘要：本发明公开了一种评估卵母细胞质量和发育潜能的方法及生物标志物组合，以应用于卵母细胞体外成熟培养技术。所述生物标志物组合包括基因组中的一系列DNA羟甲基化位点，通过获得这些位点上的5hmC的覆盖度可以全局地、无偏地评估人类卵母细胞的质量和发育潜能，通过比较这些位点上的5hmC的覆盖度可以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。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。

主权项：1.一种生物标志物组合作为参比标志物在非诊断非治疗方法中的应用，用于评估人卵母细胞的质量和发育潜能，或者，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，其特征在于，所述生物标志物组合包括下述基因组DNA羟甲基化位点：一号染色体chr1：785223,1039428,1039468,1318591,1389660,1400697,1401561,1813244,2010902,5857024,5857529,6409530,6426369,6698215,7948726,8746230,9864398,9869333,10186250,11267391,12253899,14546859,14609316,15470603,15517141,18146451,18150592,18732323,19415053,19804096,20971703,22346347,22358881,24754558,24765255,25586245,26053121,26056401,26173154,26223665,26858674,27033380,31005607,31014660,32388330,32788160,32915328,32990634,33224227,33634194,33774513,34356119,34504952,35572743,35602890,35625018,36610173,36887609,37875984,38146786,38365093,41161681,44013985,44017252,45216369,45972570,46076332,47797150,51292051,51352223,51385261,51796338,51805690,52624715,52645411,52738457,53002290,53195676,54382540,54835753,55327125,55368406,55801682,55911489,62411010,68568081,68683393,75192338,78458926,83500586,87458125,92368756,92491531,109967476,112948760,149954811,150242950,150589358,151298666,151752558,151752565,153831093,154152797,154452745,155870793,156458967,162249087,165814386,173687834,173842059,177346740,178707204,179273819,179808442,181216807,181516733,181941926,186133626,187761440,193161865,193161964,200150070,200169381,201842065,203629880,204080370,204094625,205532815,205561740,205575976,206865175,209618776,210536390,210538990,210638732,211086675,211535275,212532628,220211971,220637386,223196735,223205320,224018730,224320374,224381406,225787968,225809392,225820960,225973597,225979164,225979701,227468944,227662232,229525814,235279650,235459993,235551059,235632054,235639501,236129611,236131115,236285502,236330098,237317642,237331855,238047940,240299081,240328537,242224730,243661752,243970703,244845589,244862089,244885831,244890360,244890377,245374756,245815220,245866670,245866733,246048209,246974601,246974644,247527394,233302,369947,437506,480413,482906,499199,553514,611935,639847,640011,1265703,1727888,3133734,3182170,3183815,3212279,5100862,5101063,5144821,5264292,5264493,5284016,5284483,5286920,5935133,6304841,6488245,7077159,11129462,11130837,11227523,11255349,11265759,11336765,11612187,11907538,11907558,12243421,12951610,13081056,13859458,16319501,16849077,17586406,17586598,18828261,18887167,22287233,22826373,22886225,22892703,22981183,25679142,28539498,34926668,35056292,35264436,35275768,35755413,43900869,59964141,64944794,65250462,70628194,70685669,72893060,72907926,72931115,74137347,74515970,74753721,74837609,75123540,75123893,75160439,75317702,75317725,76236107,76606384,82481433,88253627,89372964,91770871,93999993,94073347,96174378,96174398,97021502,101627183,102043499,103124603,103124923,103952184,104547017,104764890,104766208,104766773,104766882,104813874,104816126,104816244,104819022,104848423,104893237,104936091,107684974,108724346,108755751,108759559,112367283,113142480,113810389,113857794,114783488,115095590,115352917,115996517,116802811,117760432,119219745,120442967,120932055,120932918,121510064,121686666,125204725,125226905,125261666,125277147,125522800,125545438,125625980,125990395,126415787,126424505,126728750,126792634,126808706,127603436,131270689,131289951,131326795,131386837,131437939,131536523,132177356,132195610,133739859,133857231,133859057,133860884,133871925,133882093,133905629,134104715,135198714,837944,1519611,2481084,2499149,3526826,3561872,4474307,5417456,5433816,5623629,8857421,9271156,9527115,9527226,10598200,11282346,11593392,12088418,12263892,12342964,12412092,12472946,12504209,12914748,14112487,16895824,16920903,16930331,17135149,18651005,20048837,20281562,27667723,27699023,27699037,33170102,33499971,33704768,34148944,34228374,34249802,34260591,34288182,34334850,35861890,36689142,44149029,46606662,46762377,47329450,47519856,47539424,57083611,57550705,57632366,57648519,57679953,57701829,60920703,61651767,62705792,62706447,62766771,62773918,63751603,64806384,65051847,65057867,65307243,66207588,66326570,66906745,68383545,68388218,68911079,68938865,70741878,70880567,70973081,72010927,73060433,73584214,73675615,75136573,78117546,78640381,83087873,86019784,86237408,86237547,93200456,93870719,94891862,94893726,102097592,102282046,102314722,102799120,106168113,106267362,107010256,109265271,109270124,109566635,110405195,110436172,110919290,112501680,113741387,115131297,115461872,115605062,115751826,116332137,116915473,117208159,117217399,117320475,117389754,117522661,117618969,118623882,118850247,119506677,120805635,121526388,121821829,125092020,125455177,126026109,126250196,129595966,129633571,129811874,130120426,131129866,131333369,131338196,131374997,131391133,131404956,131601517,131738995,131851837,131909879,132188420,132203160,132244176,134110234,134110449,802447,1173466,4550988,6318269,8488919,8883217,8895822,8920906,8938752,8947242,8947511,8947711,9827644,11818066,12035481,12382102,12572709,12640029,13690559,19614528,19862099,19949246,20973333,23076318,31231722,31713919,31723431,31883410,32131764,32132075,32858365,45712804,49095969,49661942,50486250,50628107,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148944,34228374,34249802,34260591,34288182,34334850,35861890,36689142,44149029,46606662,46762377,47329450,47519856,47539424,57083611,57550705,57632366,57648519,57679953,57701829,60920703,61651767,62705792,62706447,62766771,62773918,63751603,64806384,65051847,65057867,65307243,66207588,66326570,66906745,68383545,68388218,68911079,68938865,70741878,70880567,70973081,72010927,73060433,73584214,73675615,75136573,78117546,78640381,83087873,86019784,86237408,86237547,93200456,93870719,94891862,94893726,102097592,102282046,102314722,102799120,106168113,106267362,107010256,109265271,109270124,109566635,110405195,110436172,110919290,112501680,113741387,115131297,115461872,115605062,115751826,116332137,116915473,117208159,117217399,117320475,117389754,117522661,117618969,118623882,118850247,119506677,120805635,121526388,121821829,125092020,125455177,126026109,126250196,129595966,129633571,129811874,130120426,131129866,131333369,131338196,131374997,131391133,131404956,131601517,131738995,131851837,131909879,132188420,132203160,132244176,134110234,134110449；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；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；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；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；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；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；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；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；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；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；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；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；其中，以上所列位点的编码基于人类参考基因组版本hg19；通过获得这些位点上的胞嘧啶C羟甲基化修饰的覆盖度来评估人卵母细胞的质量和发育潜能；或者，通过比较这些位点上的胞嘧啶C羟甲基化修饰的重合度来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。

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