女性生育力保存方法探索：ICSI周期中未成熟卵母细胞的再利用研究
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摘要
目的探讨低氧（5%O_2）环境及个体化体外成熟方案对控制性超排卵周期中人未成熟卵母细胞IVM结果的影响。
方法共收集ICSI周期中成熟失败人卵母细胞556枚，并随机分为两组：常氧组（20%O_2）319枚未成熟人卵母细胞，行IVM；低氧组（5%O_2）237枚未成熟人卵母细胞。经体外培养，成熟后卵母细胞行ICSI。ICSI后16~18h观察第二极体排出及原核形成情况，挑选出正常受精卵继续培养至扩张囊胚，期间观察和记录IVM后卵母细胞的成熟、2PN、2PN卵裂、D3-优质胚胎、囊胚、优质囊胚数目并计算相应的比率。根据未成熟卵体外成熟方案，常氧组（20%O_2）和低氧组（5%O_2）又分别被随机分成两组：常规IVM组未成熟人卵母细胞入IVM液，在37℃、6%CO_2、20%O_2及饱和湿度的条件下培养，36h后体视显微镜下挑选出发育至MII期的卵母细胞，随后行ICSI；个体化IVM组人卵母细胞在相同条件下培养4h后，每间隔2h在倒置显微镜下观察卵母细胞第一极体排出情况，挑出排出第一极体的卵母细胞入受精液中，同一条件下培养4h后行ICSI，剩余未成熟卵重复以上的观察和处理，整个试验中未成熟卵的最长体外成熟时间限定为36h。ICSI后16~18h观察第二极体排出及原核形成情况，挑选出正常受精卵继续培养至扩张囊胚，期间观察和记录IVM后卵母细胞的成熟、2PN、2PN卵裂、D3-优质胚胎、囊胚、优质囊胚数目并计算相应的比率。试验中所有的优质囊胚均行两轮FISH共8种染色体（13，15，16，18，21，22，X，Y）进行检测。第1轮FISH13、21、22、16、18探针分别被红、绿、金、青、蓝荧光所标记，第2轮FISH15、X、Y探针分别被绿，橙，青荧光所标记。
结果常氧组（20%O_2）：GV期+MI期卵母细胞319枚，IVM成熟258枚，ICSI后受精227枚，双原核（2PN）201枚，2PN卵裂184枚，D3-优质胚胎30枚，囊胚22枚，其中优质囊胚4枚；低氧组（5%O_2）：GV期+MI期卵母细胞237枚，IVM成熟194枚，ICSI后受精179枚，双原核（2PN）162枚，2PN卵裂153枚，D3-优质胚胎53枚，囊胚37枚，其中优质囊胚9枚。两组间比较：成熟率、受精率、2PN率、2PN卵裂率及优质囊胚率经统计学检验无显著性差异（P＞0.05），D3-优质胚胎率及囊胚率呈极显著性差异（P＜0.01，x2＝15.13; P＜0.01，x2＝8.65）。20%O_2培养条件下，常规IVM组卵母细胞112枚，IVM成熟94枚，ICSI后受精84枚，双原核（2PN）76枚，2PN卵裂70枚，D3-优质胚胎10枚，囊胚6枚，未获得优质囊胚；个体化IVM组卵母细胞207枚，IVM成熟164枚，ICSI后受精143枚，双原核（2PN）125枚，2PN卵裂114枚，D3-优质胚胎20枚，囊胚16枚，其中优质囊胚4枚。两组间比较：成熟率、受精率、2PN率、2PN卵裂率、D3-优质胚胎率、囊胚率及优质囊胚率经统计学检验无显著性差异（P＞0.05），但个体化IVM组的囊胚率及优质囊胚率明显高于常规IVM组（14.0%Vs8.57%；25.0%Vs0）。5%O_2培养条件下，常规IVM组卵母细胞100枚，IVM成熟78枚，ICSI后受精72枚，双原核（2PN）68枚，2PN卵裂65枚，D3-优质胚胎10枚，囊胚6枚，其中优质囊胚2枚；个体化IVM组卵母细胞137枚，IVM成熟116枚，ICSI后受精107枚，双原核（2PN）94枚，2PN卵裂88枚，D3-优质胚胎43枚，囊胚31枚，其中优质囊胚7枚。两组间比较：成熟率、受精率、2PN率、2PN卵裂率及优质囊胚率经统计学检验无显著性差异（P＞0.05），D3-优质胚胎率及囊胚率呈极显著性差异（P＜0.01，x~2＝18.51; P＜0.01，x~2＝13.78）。试验中共获得13枚优质囊胚，均成功固定，低氧组（5%O_2）获得9枚优质囊胚，非整倍体胚胎4枚，非整倍体发生率44.4%。常氧组（20%O_2）获得4枚优质囊胚，异常2枚，异常率50%。两组异常胚胎率相当，差异没有统计学意义（P＞0.05）。
结论低氧（5%O_2）的体外培养环境及有利于ICSI周期人未成熟卵母细胞的体外成熟、受精及所获胚胎的发育；个体化的体外成熟方案有利于人未成熟卵母细胞体外成熟及受精后所获胚胎的发育；ICSI周期中人未成熟卵母细胞通过IVM，ICSI及早期胚胎培养能获得八种染色体正常的优质囊胚。总之，控制性超促排卵周期中人未成熟卵母细胞在5%O_2浓度的体外培养条件下，经过个体化方案的体外成熟，受精及早期胚胎培养能获得较为满意的IVM结果，实现了未成熟卵母细胞的再利用。
一、冷冻方式对ICSI周期人未成熟卵母细胞冻融后发育潜力的影响研究
目的冷冻方式对ICSI周期中人未成熟卵母细胞冻融后存活、成熟、受精、胚胎发育的影响。
方法共收集ICSI周期中成熟失败人卵母细胞454枚，并随机分为两组。慢速冷冻组：192枚未成熟卵母细胞，其中GV70和MI122枚，行人未成熟卵冻融；玻璃化冷冻组：262枚卵母细胞（GV92枚，MI170枚）。未成熟卵冻融后，挑选出存活的卵母细胞行体外成熟培养，成熟后卵母细胞行ICSI，ICSI后16~18h观察第二极体排出及原核形成情况，挑选出正常受精卵继续培养至扩张囊胚，期间观察和记录卵母细胞的存活、成熟、2PN、2PN卵裂、D3-优质胚胎、囊胚、优质囊胚数目并计算相应的比率。
结果慢冷组卵母细胞（GV+MI）冻融后的总存活率显著低于玻璃化组（62.0%Vs82.4%），且经统计学检验呈显著性差异（P＜0.01,2＝23.99）。慢冷组GV，MI卵母细胞存活率分别为85.7%和48.4%，玻璃化组分别为93.5%和76.5%。综合两组，GV期卵母细胞冻融后存活率显著高于MI期卵母细胞（90.1%Vs64.7%），且经统计学检验呈显著性差异（P＜0.01，X~2＝34.75）。慢冷组冻融后GV,MI期卵母细胞IVM的成熟率分别是16.7%（10/60）和50.8%（30/59），而玻璃化组分别是24.4%和55.4%。综合两组，冻融后总的GV期卵母细胞经过36h IVM培养获得21.2%的体外成熟率，显著低于总的MI的体外成熟率（54.0%），差异有显著性（P＜0.01，x~2＝36.87）；慢冷组119枚存活的卵母细胞（包括GV+MI）中，40枚发育成熟；玻璃化组216枚中，93枚成熟。成熟率方面：玻璃化组的43.1%（93/216）高于慢冷组的33.6%（40/119），但两者之间经统计学检验无显著性差异（P＞0.05，X~2＝1.26）。慢冷组10枚冻融后GV期卵母细胞IVM成熟后ICSI，没有获得受精；而玻璃组21枚GV期卵母细胞经IVM成熟后获得受精卵4枚，受精率为19.0%。冻融后MI期卵母细胞经IVM成熟后ICSI，其中，慢冷组12枚受精，受精率为36%（12/30），玻璃化组44枚受精，受精率是61.1%（44/72），但两组MI期卵母细胞经IVM后ICSI，受精率经统计学检验无显著性差异（P＞0.05）。综合两组，冻融后MI期卵母细胞经IVM成熟后ICSI，其受精率（58.8%）显著高于GV期卵母细胞经IVM后ICSI的受精率（12.9%），差异呈极显著性（P＜0.01，X~2＝8.61）。在玻璃化组，冻融后GV期卵母细胞最终未获得卵裂期胚胎。在慢冷组，冻融后MI期卵母细胞获得12枚受精卵，体外培养后最终获得2枚卵裂期胚胎，但均发育阻滞于2细胞阶段；而在玻璃化组，冻融后MI期卵母细胞行IVM后ICSI及体外培养后获得22枚卵裂期胚胎，并最终获得5枚囊胚。提示MI期卵母细胞IVM后行ICSI所获得的胚胎卵裂率，玻璃化组显著高于慢速冷冻组（50.0%Vs16.7）。
结论冻融人未成熟卵母细胞，玻璃化冷冻法优于慢速冷冻法，且GV期卵母细胞比MI期卵母细胞更能耐受低温和冷冻保护剂的影响，但MI期卵母细胞玻璃化冻融后经IVM、ICSI及胚胎培养能获得较为满意的胚胎。
二、乙醇激活/海藻糖应用与ICSI周期人未成熟卵母细胞冻融后发育潜力
目的乙醇激活及海藻糖应用对ICSI周期中人未成熟卵母细胞冻融后存活、成熟、受精、胚胎发育及胚胎非整倍体发生的影响
方法纳入本试验中所有成熟失败人卵母细胞被随机分为：蔗糖玻璃化组，274枚未成熟卵使用含蔗糖为冷冻保护剂的冻融液进行冻融研究；海藻糖玻璃化组，245枚未成熟卵使用含海藻糖为冷冻保护剂的冻融液进行冻融；对照组（新鲜组），291枚未成熟卵未经冷冻直接行体外成熟培养。随后又根据体外成熟卵ICSI后是否进行乙醇激活，将以上三个研究组进一步分成六组，分别为：蔗糖玻璃化未激活组，蔗糖玻璃化激活组；海藻糖玻璃化未激活组，海藻糖玻璃化激活组，新鲜未激活组及新鲜激活组。期间观察和记录冻融后卵母细胞的存活、成熟、2PN、2PN卵裂、D3-优质胚胎、囊胚、优质囊胚数目并计算相应的率。试验中所有的优质囊胚均行两轮FISH共8种染色体（13，15，16，18，21，22，X，Y）进行检测。第1轮FISH13、21、22、16、18探针分别被红、绿、金、青、蓝荧光所标记，第2轮FISH15、X、Y探针分别被绿，橙，青荧光所标记。
结果新鲜未激活组（GV+MI）：112枚卵直接行IVM方案，94枚成熟，76枚受精，70枚卵裂，优质胚胎10枚及形成囊胚6枚，未获优质囊胚；新鲜激活组（GV+MI）：179枚未成熟卵，IVM成熟152枚,，ICSI后行乙醇激活，激活后受精130枚,卵裂率124枚,优质胚胎39枚，形成囊胚25枚及优质囊胚14枚。蔗糖玻璃化未激活组（GV+MI）：未成熟卵母细胞128枚，解冻后存活110枚，IVM成熟100枚，ICSI后受精82枚,卵裂48枚，未获优质胚胎及囊胚；蔗糖玻璃化激活组（GV+MI）：未成熟卵母细胞146枚，解冻后存活124枚，IVM后成熟104枚，ICSI卵激活后受精86枚,56发生卵裂,形成优质胚胎16枚，形成囊胚8枚，其中优质囊胚4枚。新鲜激活组与新鲜未激活组比较：成熟、受精及卵裂率之间无差异（P＞0.05），优质胚胎率经统计学检验呈显著性差异（31.5%Vs14.3%，P＜0.01， X~2＝6.98），囊胚率及优质囊胚率间差异有统计学意义（20.2%Vs8.6%，P＜0.05，X~2＝4.48；56.0%Vs0，P＜0.05， X~2＝6.13）；玻璃化未激活组与玻璃化激活组比较，优质胚胎率及囊胚率分别经统计学检验呈显著性差异（28.6%Vs0%，P＜0.01，X~2＝16.21；14.3%Vs0，P＜0.01，X~2＝7.43），且激活组获得4枚优质囊胚。新鲜激活组与玻璃化激活组相比：成熟、受精，优质胚胎，囊胚及优质囊胚率之间无差异（P＞0.05），但新鲜组卵裂率（95.4%）显著高于玻璃化组（65.1%），经统计学检验呈显著性差异（P＜0.01，X~2＝34.14）；新鲜未激活组和玻璃化未激活组相比：新鲜组卵裂率，优质胚胎率及囊胚率分别高于玻璃化组（92.1%Vs58.5%；14.3%Vs0；8.6%Vs0），差异分别有统计学意义（P＜0.01，X~2＝23.51；P＜0.01，X~2＝7.49；P＜0.05，X~2＝4.33）。
海藻糖玻璃化组：125枚未成熟卵，冻融后108枚存活，IVM后98枚成熟，ICSI后82枚受精，培养后45枚发生卵裂，5枚发育为优质胚胎，3枚发育至囊胚，但未获得优质囊胚；海藻糖玻璃化激活组：未成熟卵母细胞120枚，解冻后存活100枚，IVM后成熟87枚，ICSI卵激活后受精70枚,38枚发生卵裂,形成优质胚胎13枚，囊胚形成10枚，其中优质囊胚6枚。海藻糖玻璃化组与蔗糖玻璃化组相比：存活、成熟、受精，卵裂、优质胚胎，囊胚及优质囊胚率之间无差异（P＞0.05），同样海藻糖玻璃化组与蔗糖玻璃化激活相比各参数间也无差异（P＞0.05）。
新鲜激活组和玻璃化激活组共获得X~24枚优质囊胚，均成功固定，新鲜组14枚优质囊胚，异常胚胎5枚，异常胚胎率35.7%；蔗糖玻璃化激活组4，异常3枚，非整倍体发生率75%；海藻糖玻璃化激活组6枚，3枚被检测出为非整倍体，发生率50%。分别与新鲜组相比，非整倍体率相当，无差异（P＞0.05）。结论ICSI周期中人未成熟卵母细胞在冻融过程中遭受损伤，降低其形成胚胎的潜力，人工辅助激活不能促进胚胎的形成，但有利于所获胚胎后期更好的发育；在人未成熟卵母细胞的冻融过程中，与蔗糖相比其冻融效果相当。
目的通过共聚焦显微镜观察人未成熟卵母细胞经IVM获得成熟卵子冻融后超微结构的状态，旨在分析此类卵子在冻融过程中的冷冻损伤。
方法共收集62枚新鲜人未成熟卵母细胞，行IVM，51枚发育至MII期，选出42枚形态正常的MII卵随机分为两组：IVM-蔗糖组，20枚卵经过含蔗糖的玻璃化液冻融后行共聚焦显微镜观察，分析纺锤体结构变化；IVM-海藻糖组，22枚应用海藻糖玻璃化液冷冻；对照组，共收集X~20枚IVF周期中形态正常的MII卵（患者鉴定知情同意，同意赠卵）：2枚直接行固定和随后的纺锤体观察，另18枚，9枚行蔗糖，9枚行海藻糖玻璃化冻融及纺锤体观察。
结果IVM成熟卵母细胞42枚，20枚应用蔗糖玻璃化液冷冻，解冻后，存活17枚（IVM-蔗糖组）；22枚应用海藻糖玻璃化液冷冻，解冻后，存活19枚（IVM-海藻糖组）；IVF-蔗糖组成活8枚，IVF-海藻糖组存活8枚。四组间的存活率经统计学检验无显著性差异（P＞0.05），四组存活的卵母细胞分别固定染色体，有效固定的卵母细胞数分别为：IVM-蔗糖组10枚，IVM-海藻糖组11枚，IVF-蔗糖组6枚及IVF-海藻糖组7枚，染色体与纺锤体形态结构均正常分别有3枚（30.0%）、4枚（36.4%）、4枚（66.7%）和5枚（71.4%）。四组间纺锤体及染色体正常形态率经统计学检验无显著性差异（P＞0.05）。
结论应用海藻糖作为非渗透性保护剂行玻璃化冻融人成熟卵母细胞可能有利于保护纺锤体形态结构；人卵母细胞玻璃化冻融过程中，体外成熟卵和体内成熟卵冻融后均存在更严重的结构损伤，但体内成熟卵母细胞似乎具有更强的抗冻性。
Objective: To explore the effect of low oxygen（5%O_2）and individual protocol onin-vitro maturation of immature human oocyte from ICSI cycle.
Methods: Totally556immature human oocytes were collected in ICSI cycles，and wererandomly divided into two groups: conventional group（20%O_2）,319immatureoocytes for IVM; Low oxygen group（5%O_2）,X~237immature oocytes. All the immatureoocytes in the two groups were cultured in vitro, and then in-vitro matured oocytes wereperformed with ICSI,16~18h later, the status of fertilization of the injected oocyteswere identified on the basis of the appearance of the second polar body and formation ofpronuclear, subsequently, normally-fertilized oocytes were picked up and cultured toblastocyst stage, at the same time, observing and recording the rates of maturation, fertilization, cleavage, D3high-quality embryos, blastocyst and high-quality blastocysts.According to in-vitro maturation protocol of immature oocytes, conventional group（20%O_2）and low oxygen（5%O_2）group were also randomly divided into two groups,respectively：conventional IVM group, all of the immature oocytes were placed intoIVM medium to cultured in37℃,6%CO_2,20%O_2and100%humidity condition,36hlater, the oocytes developing to metaphase II（MII）stage were conducted with ICSI;Individualized IVM group, all of the oocytes in this group were cultured for4h in thesame condition as the former, then observing the appearance of the first polar bodyevery two hours under the inverted microscope, picking up the oocyte with the firstpolar body and placing them into fertilization medium for a further4hours of in-vitrocuture,4h later, ICSI and embryo culture was conducted. The remaining immatureoocytes repeated the previous procedure until the longest36hours of in-vitromaturation time. In the process of the experiment, the rates of maturation, fertilization,cleavage, D3high-quality embryos, blastocyst and high-quality blastocysts wererecorded. Furthermore, All of the formed high-quality blastocysts were analysed for8pairs of chromosomes：13，15，16，18，21，22，X and Y through two rounds of FISH.In the first round of FISH,13,21,22,16and18pairs chromosomes were analyzed witha corresponding probe marked with red, green, gold, aqua and blue fluorescent; In thesecond round of FISH,15, X and Y chromosomes were diagnosed, and thecorresponding probes were respectively marked with green, orange and aquafluorescent,.
Results: conventional group（20%O_2）：319GV+MI oocytes were collected，258oocyteswere matured with IVM，227matured oocytes were aquired and fertilized with ICSI，1622PN embryos were obtained，in which153oocytes were cleavaged，formed53good quality embryos （D3），37blastocysts including9high-quality blastocysts wereobtained. Comparison between the two groups showed no significantly difference inmaturation rate, fertilization rate,2PN rate,2PN cleavage rate and high-quality blastocyst rate（P＞0.05）. However, D3-good quality embryos rate and blastocyst rateshowed highly statistical difference（P＜0.01，X~2＝15.13; P＜0.01，X~2＝8.65）. Underthe condition ofX~20%O_2,conventional IVM group contained112oocytes,94oocytesmatured after IVM，84oocytes fertilized after ICSI，containedX~2PN embryos76，inwhich702PN embryos cleavaged，10D3-good quality embryos，6blastocysts wereformed, no high-quality blastocysts were obtained. Individualized IVM group contained207oocytes,164oocytes matured after IVM，143oocytes fertilized after ICSI，125embryos（including2PN），in which1142PN embryos cleavaged，20D3-good qualityembryos，16blastocysts were formed which included4high-quality blastocysts.Comparison between the two groups, there was no significantly difference in maturationrate, fertilization rate,2PN rate,2PN cleavage rate and high-quality blastocyst rate（P＞0.05），D3-good quality embryos formation rate and blastocyst formation rate showedhighly statistically difference（P＜0.01，X~2＝18.51; P＜0.01，X~2＝13.78）.13high-quality blastocyst were obtained，all of them were fixed successfully for thefollowing FISH diagnosis,9high-quality blastocysts in the low oxygen group（5%O_2）,4showed aneuploid and aneuploid rate was44.4%; For4high-quality blastocysts in theconventional group（20%O_2）,2showed abnormal after FISH diagnosis and abnormalrate50%. No statistical difference in abnormal embryonic rate was found between thetwo groups（P＞0.05）.
Conclusion Low oxygen（5%O_2）is better to improve in-vitro maturation of immatureoocyte, fertilization and the development of the formed embryos; individualized IVMprotocol improves the development of the resulting embryos; high-quality blastocystscan be obtained from the immature oocytes followed by IVM，ICSI and early embryoculture. In a word，in the controlled ovarian hyperstimulation cycle，immature oocytesfrom ICSI cycle can achieve a better IVM result through an individualized IVMprotocol in a low oxygen（5%O_2） of in-vitro culture condition.
i. The style of Cryopreservation of human immature oocytes followed by in vitromaturation: slow-freezingVs.Vitrification.
Objective: To explore the survival rate, maturation rate, the fertilization rate andembryonic development of immature human oocytes from ICSI cycle afterslow-freezing and vitrification style.
Methods: A total of454immature oocytes [germinal vesicle（GV）and metaphase I（MI）stages] were collected and randomly divided into a slow-freezing group [1.5mol/L-1,2-propanediol（PROH）+0.2mol/l sucrose] and vitrification group [20%PROH+20%ethylene glycol（EG）+0.5mol/l sucrose].
Results: The vitrification protocol yielded a better survival rate than the slow freezingprotocol at each maturation stage assessed. Regardless of the maturation stage（GV+MI）, the slow freezing protocol had a significantly lower survival rate than thevitrification protocol（P＜0.001）. In addition, a significantly difference was found inthe survival rates between GV and MI oocytes totally in two groups（90.1Vs.64.7%,respectively;P＜0.01）. We also found that the maturation rates of GV and MI oocytesfrom the slow freezing and vitrification groups were16.7Vs.24.4%and50.8Vs55.4%,respectively. The GV oocytes had significantly lower viability than MI oocytes after36h of in vitro maturation（21.2Vs54.0%, respectively;P＜0.01）totally in two groups.In addition, the GV and MI oocytes in the slow freezing group had a markedly lowermaturation rate than those in the vitrification group（33.6Vs.43.1%, respectively）, butno statistical difference was found between the two groups（P＞0.05）. For the GV-matured oocytes, no fertilized eggs were obtained in the slow-freezing group, whilea19.0%（4/21）fertilization rate was obtained in the vitrification group. For theMI-matured oocytes, fertilization rates in the slow-freezing and vitrification group were36%and61.1%, respectively, but no significant difference was found between the twogroups（P＞0.05）. In the GV vitrification group, no embryo formed; however, for MIoocytes, in slow-freezing group,12oocytes were fertilized, but only two cleavageembryos had been got and were subsequently blocked at the2-cell stage. In vitrificationgroup, a total of22embryos were obtained from MI oocytes, five embryos developed tothe blastocyst stage.
Conclusions: Vitrification is superior to the slow-freezing method in terms of thesurvival and developmental rates for the cryopreservation of human immature oocytes.In addition, GV oocytes appeared to be more tolarete than MI oocytes to the lowtemperature and cryopreservation cryoprotectant.
ii. Effect of ethanol activation/application of trehalose on the developmental potential offrozen-thawed immature human oocytes from ICSI cycle
Objective: To observe the survival rate, maturation rate, the fertilization rate andembryonic development and the aneuploid rate of the immature oocytes after ethanolactivation/application of trehalose.
Methods: All the immature oocytes were randomly divided into three groups：thesucrose virification group，274immature oocytes were frozen and thawed using mediumcontaining sucrose as cryoprotectant；the trehalose virification group，245immatureoocytes were used trehalose; the control group（fresh group），291fresh immatureoocytes were directly conducted with IVM culture. all of the in-vitro matured oocytesfrom the three groups were used with ICSI fertilization, after ICSI, these oocytes inthree groups were further divided into six subgroups according to using ethanolactivation or not: no-activation sucrose virification group and sucrose virification groupwith activation, no-activation trehalose virification group and trehalose virification group with activation, no-activation fresh group and fresh group with activation. Therates of maturation, fertilization, cleavage, D3high-quality embryos, blastocysts andhigh-quality blastocysts were recorded. Furthermore, All of the formed high-qualityblastocysts were analysed with8chromosomes：13，15，16，18，21，22，X and Y throughtwo rounds of FISH. In the first round of FISH,13,21,22,16and18chromosomeswere analyzed with a corresponding probe marked with red, green, gold, aqua and bluefluorescent; in the second round of FISH,15, X and Y chromosomes were diagnosed,and the corresponding probes were respectively marked with green, orange and aquafluorescent.
Results: In the no activation fresh group（GV+MI）：112oocytes were cultrued withIVM，94matured oocytes was obtained,76of them were fertilized，70of themcleavaged，10D3-good quality embryos and6blastocyst were obtained，but nohigh-quality blastocyst formed. In fresh group with activation（GV+MI）：179oocyteswere cultured with IVM and152matured, after ICSI, the injected oocytes wereexposed to7%ethanol for6minutes, subsequently cultured,130fertilized,124cleavaged, and39D3high-quality embryos,25blastocysts and14high-qualityblastocysts were obtained. In the no-activation sucrose virification grou（pGV+MI）:128immature oocytes were obtained.110survived after thawing,100matured after IVM,82fertilized after ICSI,48cleavaged，no high-quality embryos and blastocysts formed;In the sucrose virification group with activation（GV+MI）：146immature oocytes wereobtained，124survived after thawing,104matured after IVM,86fertilized,56cleavaged,16high-quality embryos and8blastocysts including4high-qualityblastocysts formed. Comparison between the fresh group with activation andno-activation, no statistical difference was found in maturation rate, fertilization rateand cleavage rate（P＞0.05）, and a significant statistical difference for high-qualityembryos rate （31.5%Vs14.3%，P＜0.01，X~2＝6.98）, blastocysts formation andhigh-quality blastocysts rate （20.2%Vs8.6%，P＜0.05，X~2＝4.48；56.0%Vs0，P ＜0.05，X~2＝6.13）. Comparison between the no-activation vitrification group and withactivation, there was a significant statistically difference in high-quality embryos andblastocysts formation rate （28.6%Vs0%，P＜0.01，X~2＝16.21；14.3%Vs0，P＜0.01，X~2＝7.43）, and4high-quality blastocysts were obtained in the activation group.Comparison between the fresh group with activation and vitrification group withactivation,no significant statistically difference was found for maturation, fertilization,high-quality embryo, blastocyst and high-quality blastocyst rates（P＞0.05）, butcleavage rate in the fresh group （95.4%）was significantly higher than vitrificationgroup（65.1%）（P＜0.01，X~2＝34.14）. Comparison between the no-activation freshgroup and no-activation vitrification group, cleavage rate, high-quality embryo rate andblastocyst rate in fresh group were significantly higher than those in the vitrificationgroup respectively（92.1%Vs58.5%;14.3%Vs0;8.6%Vs0）（P＜0.01，X~2＝23.51；P＜0.01，X~2＝7.49；P＜0.05，X~2＝4.33）. In the trehalose virification group, immatureoocytes was125, the survived108were obtained after thawing,98matured after IVM,82fertilized after ICSI,45cleavaged, and5D3high-quality embryos,3blastocystswere obtained, but no high-quality blastocyst. In the trehalose virification group withactivation, totally120immature oocytes，100survived after thawing,87matured afterIVM,70fertilized after ICSI,38cleavaged, subsequently cultured,13D3high-qualityembryos,10blastocysts including6high-quality blastocysts were obtained. Comparisonbetween trehalose virification group and sucrose virification group, no statisticallydifference was founded for survival rate, maturation rate, fertilization rate, cleavage rate,high-quality embryo rate, blastocyst rate and high-quality blastocyst rate （P＞0.05）.Also, the parameters in trehalose virification group with activation and sucrosevirification group with activation O_2no difference（P＞0.05）.Totally24high-quality blastocyst were obtained in fresh group with activation and virificationgroup with activation. All of them were fixed successfully for the following FISHdiagnosis,14high-quality blastocyst in fresh group,5abnormal embryos were founded （abnormal rate35.7%）; for4high-quality blastocysts from the sucrose virificationgroup with activation,3abnormal embryos were founded （the aneuploid rate75%）;6embryos in the trehalose virification group with activation,3O_2aneuploidy（aneuploid rate50%）. no difference was found in aneuploid rate between the freshgroup and virification group（P＞0.05）.
Conclusions oocytes suffer damage in the freezing-thawing process and reduce theformation of embryos; Artificial activation can not improve the formation of embryos，but their development; Trehalose as impermeable protectant appears not to improve thefreezing–thawing effect.
Objective: Aim to analyse freezing-thawing damage by using co-focal microscopy toobserve ultramicrostructure of frozen-thawed matured oocytes derived from immatureoocytes from ICSI cycle.
Methods:62immature oocytes with IVM and51matured,42of them with a normalshape were picked up and randomly divided into two groups: IVM-sucrose group，20oocytes were observed using co-focal microscopy after thawing with vitrificationmedium containing sucrose to analyze the structure change of the spindles. IVM-trehalose group，22oocytes using vitrification medium containing trehalose. The controlgroup，20MII oocytes with normal shape were collected from IVF cycles（patients hadsigned informed consent and agreed to oocyte donation）,2of them were directly fixedand then observed the configuration of their spindles, for the other18oocytes，9of themwere frozen and thawed using vitrification medium containing sucrose, and the other9using trehalose, subsequently, all the survived oocytes were fixed to be observed the configuration of their spindles through co-focal microscope.
Results: A total of42mature oocytes was frozen,,20of them were used withvitrification medium containing sucrose, after thawing,17survived （IVM-sucrosegroup）, the other22with trehalose medium and19survived after thawing（IVM-trehalose group）. In IVF-sucrose and IVF-trehalose groups, respectively8survived. There was no significantly difference for surviving rate among four groups（P＞0.05）. all of the survived oocytes were included in the four groups were fixed. InIVM-sucrose group,10were successfully fixed,11in IVM-trehalose group,6IVF-sucrose group and7IVF-trehalose group had been fixed seccessfully. The oocyteswere observed under co-focal microscope, the normal morphology both inchromosomes and spindle morphology of the oocytes were l3（30.0%）,4（36.4%）,4（66.7%） and5（71.4%）, respectively. There were no significantly difference innormal morphology rate of spindle and chromosomes among four groups （P＞0.05）.
Conclusions Application of trehalose as an impermeable protectant during vitrificationof human MII oocytes can achieve an acceptible results. In the freezing-thawing process,both in-vivo and in-vitro mature oocytes suffer serious damage, but in-vivo matureoocyte appears to be more tolerate to cryopreservation.
方法共收集ICSI周期中成熟失败人卵母细胞556枚，并随机分为两组：常氧组（20%O_2）319枚未成熟人卵母细胞，行IVM；低氧组（5%O_2）237枚未成熟人卵母细胞。经体外培养，成熟后卵母细胞行ICSI。ICSI后16~18h观察第二极体排出及原核形成情况，挑选出正常受精卵继续培养至扩张囊胚，期间观察和记录IVM后卵母细胞的成熟、2PN、2PN卵裂、D3-优质胚胎、囊胚、优质囊胚数目并计算相应的比率。根据未成熟卵体外成熟方案，常氧组（20%O_2）和低氧组（5%O_2）又分别被随机分成两组：常规IVM组未成熟人卵母细胞入IVM液，在37℃、6%CO_2、20%O_2及饱和湿度的条件下培养，36h后体视显微镜下挑选出发育至MII期的卵母细胞，随后行ICSI；个体化IVM组人卵母细胞在相同条件下培养4h后，每间隔2h在倒置显微镜下观察卵母细胞第一极体排出情况，挑出排出第一极体的卵母细胞入受精液中，同一条件下培养4h后行ICSI，剩余未成熟卵重复以上的观察和处理，整个试验中未成熟卵的最长体外成熟时间限定为36h。ICSI后16~18h观察第二极体排出及原核形成情况，挑选出正常受精卵继续培养至扩张囊胚，期间观察和记录IVM后卵母细胞的成熟、2PN、2PN卵裂、D3-优质胚胎、囊胚、优质囊胚数目并计算相应的比率。试验中所有的优质囊胚均行两轮FISH共8种染色体（13，15，16，18，21，22，X，Y）进行检测。第1轮FISH13、21、22、16、18探针分别被红、绿、金、青、蓝荧光所标记，第2轮FISH15、X、Y探针分别被绿，橙，青荧光所标记。
结果常氧组（20%O_2）：GV期+MI期卵母细胞319枚，IVM成熟258枚，ICSI后受精227枚，双原核（2PN）201枚，2PN卵裂184枚，D3-优质胚胎30枚，囊胚22枚，其中优质囊胚4枚；低氧组（5%O_2）：GV期+MI期卵母细胞237枚，IVM成熟194枚，ICSI后受精179枚，双原核（2PN）162枚，2PN卵裂153枚，D3-优质胚胎53枚，囊胚37枚，其中优质囊胚9枚。两组间比较：成熟率、受精率、2PN率、2PN卵裂率及优质囊胚率经统计学检验无显著性差异（P＞0.05），D3-优质胚胎率及囊胚率呈极显著性差异（P＜0.01，x2＝15.13; P＜0.01，x2＝8.65）。20%O_2培养条件下，常规IVM组卵母细胞112枚，IVM成熟94枚，ICSI后受精84枚，双原核（2PN）76枚，2PN卵裂70枚，D3-优质胚胎10枚，囊胚6枚，未获得优质囊胚；个体化IVM组卵母细胞207枚，IVM成熟164枚，ICSI后受精143枚，双原核（2PN）125枚，2PN卵裂114枚，D3-优质胚胎20枚，囊胚16枚，其中优质囊胚4枚。两组间比较：成熟率、受精率、2PN率、2PN卵裂率、D3-优质胚胎率、囊胚率及优质囊胚率经统计学检验无显著性差异（P＞0.05），但个体化IVM组的囊胚率及优质囊胚率明显高于常规IVM组（14.0%Vs8.57%；25.0%Vs0）。5%O_2培养条件下，常规IVM组卵母细胞100枚，IVM成熟78枚，ICSI后受精72枚，双原核（2PN）68枚，2PN卵裂65枚，D3-优质胚胎10枚，囊胚6枚，其中优质囊胚2枚；个体化IVM组卵母细胞137枚，IVM成熟116枚，ICSI后受精107枚，双原核（2PN）94枚，2PN卵裂88枚，D3-优质胚胎43枚，囊胚31枚，其中优质囊胚7枚。两组间比较：成熟率、受精率、2PN率、2PN卵裂率及优质囊胚率经统计学检验无显著性差异（P＞0.05），D3-优质胚胎率及囊胚率呈极显著性差异（P＜0.01，x~2＝18.51; P＜0.01，x~2＝13.78）。试验中共获得13枚优质囊胚，均成功固定，低氧组（5%O_2）获得9枚优质囊胚，非整倍体胚胎4枚，非整倍体发生率44.4%。常氧组（20%O_2）获得4枚优质囊胚，异常2枚，异常率50%。两组异常胚胎率相当，差异没有统计学意义（P＞0.05）。
结论低氧（5%O_2）的体外培养环境及有利于ICSI周期人未成熟卵母细胞的体外成熟、受精及所获胚胎的发育；个体化的体外成熟方案有利于人未成熟卵母细胞体外成熟及受精后所获胚胎的发育；ICSI周期中人未成熟卵母细胞通过IVM，ICSI及早期胚胎培养能获得八种染色体正常的优质囊胚。总之，控制性超促排卵周期中人未成熟卵母细胞在5%O_2浓度的体外培养条件下，经过个体化方案的体外成熟，受精及早期胚胎培养能获得较为满意的IVM结果，实现了未成熟卵母细胞的再利用。
一、冷冻方式对ICSI周期人未成熟卵母细胞冻融后发育潜力的影响研究
目的冷冻方式对ICSI周期中人未成熟卵母细胞冻融后存活、成熟、受精、胚胎发育的影响。
方法共收集ICSI周期中成熟失败人卵母细胞454枚，并随机分为两组。慢速冷冻组：192枚未成熟卵母细胞，其中GV70和MI122枚，行人未成熟卵冻融；玻璃化冷冻组：262枚卵母细胞（GV92枚，MI170枚）。未成熟卵冻融后，挑选出存活的卵母细胞行体外成熟培养，成熟后卵母细胞行ICSI，ICSI后16~18h观察第二极体排出及原核形成情况，挑选出正常受精卵继续培养至扩张囊胚，期间观察和记录卵母细胞的存活、成熟、2PN、2PN卵裂、D3-优质胚胎、囊胚、优质囊胚数目并计算相应的比率。
结果慢冷组卵母细胞（GV+MI）冻融后的总存活率显著低于玻璃化组（62.0%Vs82.4%），且经统计学检验呈显著性差异（P＜0.01,2＝23.99）。慢冷组GV，MI卵母细胞存活率分别为85.7%和48.4%，玻璃化组分别为93.5%和76.5%。综合两组，GV期卵母细胞冻融后存活率显著高于MI期卵母细胞（90.1%Vs64.7%），且经统计学检验呈显著性差异（P＜0.01，X~2＝34.75）。慢冷组冻融后GV,MI期卵母细胞IVM的成熟率分别是16.7%（10/60）和50.8%（30/59），而玻璃化组分别是24.4%和55.4%。综合两组，冻融后总的GV期卵母细胞经过36h IVM培养获得21.2%的体外成熟率，显著低于总的MI的体外成熟率（54.0%），差异有显著性（P＜0.01，x~2＝36.87）；慢冷组119枚存活的卵母细胞（包括GV+MI）中，40枚发育成熟；玻璃化组216枚中，93枚成熟。成熟率方面：玻璃化组的43.1%（93/216）高于慢冷组的33.6%（40/119），但两者之间经统计学检验无显著性差异（P＞0.05，X~2＝1.26）。慢冷组10枚冻融后GV期卵母细胞IVM成熟后ICSI，没有获得受精；而玻璃组21枚GV期卵母细胞经IVM成熟后获得受精卵4枚，受精率为19.0%。冻融后MI期卵母细胞经IVM成熟后ICSI，其中，慢冷组12枚受精，受精率为36%（12/30），玻璃化组44枚受精，受精率是61.1%（44/72），但两组MI期卵母细胞经IVM后ICSI，受精率经统计学检验无显著性差异（P＞0.05）。综合两组，冻融后MI期卵母细胞经IVM成熟后ICSI，其受精率（58.8%）显著高于GV期卵母细胞经IVM后ICSI的受精率（12.9%），差异呈极显著性（P＜0.01，X~2＝8.61）。在玻璃化组，冻融后GV期卵母细胞最终未获得卵裂期胚胎。在慢冷组，冻融后MI期卵母细胞获得12枚受精卵，体外培养后最终获得2枚卵裂期胚胎，但均发育阻滞于2细胞阶段；而在玻璃化组，冻融后MI期卵母细胞行IVM后ICSI及体外培养后获得22枚卵裂期胚胎，并最终获得5枚囊胚。提示MI期卵母细胞IVM后行ICSI所获得的胚胎卵裂率，玻璃化组显著高于慢速冷冻组（50.0%Vs16.7）。
结论冻融人未成熟卵母细胞，玻璃化冷冻法优于慢速冷冻法，且GV期卵母细胞比MI期卵母细胞更能耐受低温和冷冻保护剂的影响，但MI期卵母细胞玻璃化冻融后经IVM、ICSI及胚胎培养能获得较为满意的胚胎。
二、乙醇激活/海藻糖应用与ICSI周期人未成熟卵母细胞冻融后发育潜力
目的乙醇激活及海藻糖应用对ICSI周期中人未成熟卵母细胞冻融后存活、成熟、受精、胚胎发育及胚胎非整倍体发生的影响
方法纳入本试验中所有成熟失败人卵母细胞被随机分为：蔗糖玻璃化组，274枚未成熟卵使用含蔗糖为冷冻保护剂的冻融液进行冻融研究；海藻糖玻璃化组，245枚未成熟卵使用含海藻糖为冷冻保护剂的冻融液进行冻融；对照组（新鲜组），291枚未成熟卵未经冷冻直接行体外成熟培养。随后又根据体外成熟卵ICSI后是否进行乙醇激活，将以上三个研究组进一步分成六组，分别为：蔗糖玻璃化未激活组，蔗糖玻璃化激活组；海藻糖玻璃化未激活组，海藻糖玻璃化激活组，新鲜未激活组及新鲜激活组。期间观察和记录冻融后卵母细胞的存活、成熟、2PN、2PN卵裂、D3-优质胚胎、囊胚、优质囊胚数目并计算相应的率。试验中所有的优质囊胚均行两轮FISH共8种染色体（13，15，16，18，21，22，X，Y）进行检测。第1轮FISH13、21、22、16、18探针分别被红、绿、金、青、蓝荧光所标记，第2轮FISH15、X、Y探针分别被绿，橙，青荧光所标记。
结果新鲜未激活组（GV+MI）：112枚卵直接行IVM方案，94枚成熟，76枚受精，70枚卵裂，优质胚胎10枚及形成囊胚6枚，未获优质囊胚；新鲜激活组（GV+MI）：179枚未成熟卵，IVM成熟152枚,，ICSI后行乙醇激活，激活后受精130枚,卵裂率124枚,优质胚胎39枚，形成囊胚25枚及优质囊胚14枚。蔗糖玻璃化未激活组（GV+MI）：未成熟卵母细胞128枚，解冻后存活110枚，IVM成熟100枚，ICSI后受精82枚,卵裂48枚，未获优质胚胎及囊胚；蔗糖玻璃化激活组（GV+MI）：未成熟卵母细胞146枚，解冻后存活124枚，IVM后成熟104枚，ICSI卵激活后受精86枚,56发生卵裂,形成优质胚胎16枚，形成囊胚8枚，其中优质囊胚4枚。新鲜激活组与新鲜未激活组比较：成熟、受精及卵裂率之间无差异（P＞0.05），优质胚胎率经统计学检验呈显著性差异（31.5%Vs14.3%，P＜0.01， X~2＝6.98），囊胚率及优质囊胚率间差异有统计学意义（20.2%Vs8.6%，P＜0.05，X~2＝4.48；56.0%Vs0，P＜0.05， X~2＝6.13）；玻璃化未激活组与玻璃化激活组比较，优质胚胎率及囊胚率分别经统计学检验呈显著性差异（28.6%Vs0%，P＜0.01，X~2＝16.21；14.3%Vs0，P＜0.01，X~2＝7.43），且激活组获得4枚优质囊胚。新鲜激活组与玻璃化激活组相比：成熟、受精，优质胚胎，囊胚及优质囊胚率之间无差异（P＞0.05），但新鲜组卵裂率（95.4%）显著高于玻璃化组（65.1%），经统计学检验呈显著性差异（P＜0.01，X~2＝34.14）；新鲜未激活组和玻璃化未激活组相比：新鲜组卵裂率，优质胚胎率及囊胚率分别高于玻璃化组（92.1%Vs58.5%；14.3%Vs0；8.6%Vs0），差异分别有统计学意义（P＜0.01，X~2＝23.51；P＜0.01，X~2＝7.49；P＜0.05，X~2＝4.33）。
海藻糖玻璃化组：125枚未成熟卵，冻融后108枚存活，IVM后98枚成熟，ICSI后82枚受精，培养后45枚发生卵裂，5枚发育为优质胚胎，3枚发育至囊胚，但未获得优质囊胚；海藻糖玻璃化激活组：未成熟卵母细胞120枚，解冻后存活100枚，IVM后成熟87枚，ICSI卵激活后受精70枚,38枚发生卵裂,形成优质胚胎13枚，囊胚形成10枚，其中优质囊胚6枚。海藻糖玻璃化组与蔗糖玻璃化组相比：存活、成熟、受精，卵裂、优质胚胎，囊胚及优质囊胚率之间无差异（P＞0.05），同样海藻糖玻璃化组与蔗糖玻璃化激活相比各参数间也无差异（P＞0.05）。
新鲜激活组和玻璃化激活组共获得X~24枚优质囊胚，均成功固定，新鲜组14枚优质囊胚，异常胚胎5枚，异常胚胎率35.7%；蔗糖玻璃化激活组4，异常3枚，非整倍体发生率75%；海藻糖玻璃化激活组6枚，3枚被检测出为非整倍体，发生率50%。分别与新鲜组相比，非整倍体率相当，无差异（P＞0.05）。结论ICSI周期中人未成熟卵母细胞在冻融过程中遭受损伤，降低其形成胚胎的潜力，人工辅助激活不能促进胚胎的形成，但有利于所获胚胎后期更好的发育；在人未成熟卵母细胞的冻融过程中，与蔗糖相比其冻融效果相当。
目的通过共聚焦显微镜观察人未成熟卵母细胞经IVM获得成熟卵子冻融后超微结构的状态，旨在分析此类卵子在冻融过程中的冷冻损伤。
方法共收集62枚新鲜人未成熟卵母细胞，行IVM，51枚发育至MII期，选出42枚形态正常的MII卵随机分为两组：IVM-蔗糖组，20枚卵经过含蔗糖的玻璃化液冻融后行共聚焦显微镜观察，分析纺锤体结构变化；IVM-海藻糖组，22枚应用海藻糖玻璃化液冷冻；对照组，共收集X~20枚IVF周期中形态正常的MII卵（患者鉴定知情同意，同意赠卵）：2枚直接行固定和随后的纺锤体观察，另18枚，9枚行蔗糖，9枚行海藻糖玻璃化冻融及纺锤体观察。
结果IVM成熟卵母细胞42枚，20枚应用蔗糖玻璃化液冷冻，解冻后，存活17枚（IVM-蔗糖组）；22枚应用海藻糖玻璃化液冷冻，解冻后，存活19枚（IVM-海藻糖组）；IVF-蔗糖组成活8枚，IVF-海藻糖组存活8枚。四组间的存活率经统计学检验无显著性差异（P＞0.05），四组存活的卵母细胞分别固定染色体，有效固定的卵母细胞数分别为：IVM-蔗糖组10枚，IVM-海藻糖组11枚，IVF-蔗糖组6枚及IVF-海藻糖组7枚，染色体与纺锤体形态结构均正常分别有3枚（30.0%）、4枚（36.4%）、4枚（66.7%）和5枚（71.4%）。四组间纺锤体及染色体正常形态率经统计学检验无显著性差异（P＞0.05）。
结论应用海藻糖作为非渗透性保护剂行玻璃化冻融人成熟卵母细胞可能有利于保护纺锤体形态结构；人卵母细胞玻璃化冻融过程中，体外成熟卵和体内成熟卵冻融后均存在更严重的结构损伤，但体内成熟卵母细胞似乎具有更强的抗冻性。
Objective: To explore the effect of low oxygen（5%O_2）and individual protocol onin-vitro maturation of immature human oocyte from ICSI cycle.
Methods: Totally556immature human oocytes were collected in ICSI cycles，and wererandomly divided into two groups: conventional group（20%O_2）,319immatureoocytes for IVM; Low oxygen group（5%O_2）,X~237immature oocytes. All the immatureoocytes in the two groups were cultured in vitro, and then in-vitro matured oocytes wereperformed with ICSI,16~18h later, the status of fertilization of the injected oocyteswere identified on the basis of the appearance of the second polar body and formation ofpronuclear, subsequently, normally-fertilized oocytes were picked up and cultured toblastocyst stage, at the same time, observing and recording the rates of maturation, fertilization, cleavage, D3high-quality embryos, blastocyst and high-quality blastocysts.According to in-vitro maturation protocol of immature oocytes, conventional group（20%O_2）and low oxygen（5%O_2）group were also randomly divided into two groups,respectively：conventional IVM group, all of the immature oocytes were placed intoIVM medium to cultured in37℃,6%CO_2,20%O_2and100%humidity condition,36hlater, the oocytes developing to metaphase II（MII）stage were conducted with ICSI;Individualized IVM group, all of the oocytes in this group were cultured for4h in thesame condition as the former, then observing the appearance of the first polar bodyevery two hours under the inverted microscope, picking up the oocyte with the firstpolar body and placing them into fertilization medium for a further4hours of in-vitrocuture,4h later, ICSI and embryo culture was conducted. The remaining immatureoocytes repeated the previous procedure until the longest36hours of in-vitromaturation time. In the process of the experiment, the rates of maturation, fertilization,cleavage, D3high-quality embryos, blastocyst and high-quality blastocysts wererecorded. Furthermore, All of the formed high-quality blastocysts were analysed for8pairs of chromosomes：13，15，16，18，21，22，X and Y through two rounds of FISH.In the first round of FISH,13,21,22,16and18pairs chromosomes were analyzed witha corresponding probe marked with red, green, gold, aqua and blue fluorescent; In thesecond round of FISH,15, X and Y chromosomes were diagnosed, and thecorresponding probes were respectively marked with green, orange and aquafluorescent,.
Results: conventional group（20%O_2）：319GV+MI oocytes were collected，258oocyteswere matured with IVM，227matured oocytes were aquired and fertilized with ICSI，1622PN embryos were obtained，in which153oocytes were cleavaged，formed53good quality embryos （D3），37blastocysts including9high-quality blastocysts wereobtained. Comparison between the two groups showed no significantly difference inmaturation rate, fertilization rate,2PN rate,2PN cleavage rate and high-quality blastocyst rate（P＞0.05）. However, D3-good quality embryos rate and blastocyst rateshowed highly statistical difference（P＜0.01，X~2＝15.13; P＜0.01，X~2＝8.65）. Underthe condition ofX~20%O_2,conventional IVM group contained112oocytes,94oocytesmatured after IVM，84oocytes fertilized after ICSI，containedX~2PN embryos76，inwhich702PN embryos cleavaged，10D3-good quality embryos，6blastocysts wereformed, no high-quality blastocysts were obtained. Individualized IVM group contained207oocytes,164oocytes matured after IVM，143oocytes fertilized after ICSI，125embryos（including2PN），in which1142PN embryos cleavaged，20D3-good qualityembryos，16blastocysts were formed which included4high-quality blastocysts.Comparison between the two groups, there was no significantly difference in maturationrate, fertilization rate,2PN rate,2PN cleavage rate and high-quality blastocyst rate（P＞0.05），D3-good quality embryos formation rate and blastocyst formation rate showedhighly statistically difference（P＜0.01，X~2＝18.51; P＜0.01，X~2＝13.78）.13high-quality blastocyst were obtained，all of them were fixed successfully for thefollowing FISH diagnosis,9high-quality blastocysts in the low oxygen group（5%O_2）,4showed aneuploid and aneuploid rate was44.4%; For4high-quality blastocysts in theconventional group（20%O_2）,2showed abnormal after FISH diagnosis and abnormalrate50%. No statistical difference in abnormal embryonic rate was found between thetwo groups（P＞0.05）.
Conclusion Low oxygen（5%O_2）is better to improve in-vitro maturation of immatureoocyte, fertilization and the development of the formed embryos; individualized IVMprotocol improves the development of the resulting embryos; high-quality blastocystscan be obtained from the immature oocytes followed by IVM，ICSI and early embryoculture. In a word，in the controlled ovarian hyperstimulation cycle，immature oocytesfrom ICSI cycle can achieve a better IVM result through an individualized IVMprotocol in a low oxygen（5%O_2） of in-vitro culture condition.
i. The style of Cryopreservation of human immature oocytes followed by in vitromaturation: slow-freezingVs.Vitrification.
Objective: To explore the survival rate, maturation rate, the fertilization rate andembryonic development of immature human oocytes from ICSI cycle afterslow-freezing and vitrification style.
Methods: A total of454immature oocytes [germinal vesicle（GV）and metaphase I（MI）stages] were collected and randomly divided into a slow-freezing group [1.5mol/L-1,2-propanediol（PROH）+0.2mol/l sucrose] and vitrification group [20%PROH+20%ethylene glycol（EG）+0.5mol/l sucrose].
Results: The vitrification protocol yielded a better survival rate than the slow freezingprotocol at each maturation stage assessed. Regardless of the maturation stage（GV+MI）, the slow freezing protocol had a significantly lower survival rate than thevitrification protocol（P＜0.001）. In addition, a significantly difference was found inthe survival rates between GV and MI oocytes totally in two groups（90.1Vs.64.7%,respectively;P＜0.01）. We also found that the maturation rates of GV and MI oocytesfrom the slow freezing and vitrification groups were16.7Vs.24.4%and50.8Vs55.4%,respectively. The GV oocytes had significantly lower viability than MI oocytes after36h of in vitro maturation（21.2Vs54.0%, respectively;P＜0.01）totally in two groups.In addition, the GV and MI oocytes in the slow freezing group had a markedly lowermaturation rate than those in the vitrification group（33.6Vs.43.1%, respectively）, butno statistical difference was found between the two groups（P＞0.05）. For the GV-matured oocytes, no fertilized eggs were obtained in the slow-freezing group, whilea19.0%（4/21）fertilization rate was obtained in the vitrification group. For theMI-matured oocytes, fertilization rates in the slow-freezing and vitrification group were36%and61.1%, respectively, but no significant difference was found between the twogroups（P＞0.05）. In the GV vitrification group, no embryo formed; however, for MIoocytes, in slow-freezing group,12oocytes were fertilized, but only two cleavageembryos had been got and were subsequently blocked at the2-cell stage. In vitrificationgroup, a total of22embryos were obtained from MI oocytes, five embryos developed tothe blastocyst stage.
Conclusions: Vitrification is superior to the slow-freezing method in terms of thesurvival and developmental rates for the cryopreservation of human immature oocytes.In addition, GV oocytes appeared to be more tolarete than MI oocytes to the lowtemperature and cryopreservation cryoprotectant.
ii. Effect of ethanol activation/application of trehalose on the developmental potential offrozen-thawed immature human oocytes from ICSI cycle
Objective: To observe the survival rate, maturation rate, the fertilization rate andembryonic development and the aneuploid rate of the immature oocytes after ethanolactivation/application of trehalose.
Methods: All the immature oocytes were randomly divided into three groups：thesucrose virification group，274immature oocytes were frozen and thawed using mediumcontaining sucrose as cryoprotectant；the trehalose virification group，245immatureoocytes were used trehalose; the control group（fresh group），291fresh immatureoocytes were directly conducted with IVM culture. all of the in-vitro matured oocytesfrom the three groups were used with ICSI fertilization, after ICSI, these oocytes inthree groups were further divided into six subgroups according to using ethanolactivation or not: no-activation sucrose virification group and sucrose virification groupwith activation, no-activation trehalose virification group and trehalose virification group with activation, no-activation fresh group and fresh group with activation. Therates of maturation, fertilization, cleavage, D3high-quality embryos, blastocysts andhigh-quality blastocysts were recorded. Furthermore, All of the formed high-qualityblastocysts were analysed with8chromosomes：13，15，16，18，21，22，X and Y throughtwo rounds of FISH. In the first round of FISH,13,21,22,16and18chromosomeswere analyzed with a corresponding probe marked with red, green, gold, aqua and bluefluorescent; in the second round of FISH,15, X and Y chromosomes were diagnosed,and the corresponding probes were respectively marked with green, orange and aquafluorescent.
Results: In the no activation fresh group（GV+MI）：112oocytes were cultrued withIVM，94matured oocytes was obtained,76of them were fertilized，70of themcleavaged，10D3-good quality embryos and6blastocyst were obtained，but nohigh-quality blastocyst formed. In fresh group with activation（GV+MI）：179oocyteswere cultured with IVM and152matured, after ICSI, the injected oocytes wereexposed to7%ethanol for6minutes, subsequently cultured,130fertilized,124cleavaged, and39D3high-quality embryos,25blastocysts and14high-qualityblastocysts were obtained. In the no-activation sucrose virification grou（pGV+MI）:128immature oocytes were obtained.110survived after thawing,100matured after IVM,82fertilized after ICSI,48cleavaged，no high-quality embryos and blastocysts formed;In the sucrose virification group with activation（GV+MI）：146immature oocytes wereobtained，124survived after thawing,104matured after IVM,86fertilized,56cleavaged,16high-quality embryos and8blastocysts including4high-qualityblastocysts formed. Comparison between the fresh group with activation andno-activation, no statistical difference was found in maturation rate, fertilization rateand cleavage rate（P＞0.05）, and a significant statistical difference for high-qualityembryos rate （31.5%Vs14.3%，P＜0.01，X~2＝6.98）, blastocysts formation andhigh-quality blastocysts rate （20.2%Vs8.6%，P＜0.05，X~2＝4.48；56.0%Vs0，P ＜0.05，X~2＝6.13）. Comparison between the no-activation vitrification group and withactivation, there was a significant statistically difference in high-quality embryos andblastocysts formation rate （28.6%Vs0%，P＜0.01，X~2＝16.21；14.3%Vs0，P＜0.01，X~2＝7.43）, and4high-quality blastocysts were obtained in the activation group.Comparison between the fresh group with activation and vitrification group withactivation,no significant statistically difference was found for maturation, fertilization,high-quality embryo, blastocyst and high-quality blastocyst rates（P＞0.05）, butcleavage rate in the fresh group （95.4%）was significantly higher than vitrificationgroup（65.1%）（P＜0.01，X~2＝34.14）. Comparison between the no-activation freshgroup and no-activation vitrification group, cleavage rate, high-quality embryo rate andblastocyst rate in fresh group were significantly higher than those in the vitrificationgroup respectively（92.1%Vs58.5%;14.3%Vs0;8.6%Vs0）（P＜0.01，X~2＝23.51；P＜0.01，X~2＝7.49；P＜0.05，X~2＝4.33）. In the trehalose virification group, immatureoocytes was125, the survived108were obtained after thawing,98matured after IVM,82fertilized after ICSI,45cleavaged, and5D3high-quality embryos,3blastocystswere obtained, but no high-quality blastocyst. In the trehalose virification group withactivation, totally120immature oocytes，100survived after thawing,87matured afterIVM,70fertilized after ICSI,38cleavaged, subsequently cultured,13D3high-qualityembryos,10blastocysts including6high-quality blastocysts were obtained. Comparisonbetween trehalose virification group and sucrose virification group, no statisticallydifference was founded for survival rate, maturation rate, fertilization rate, cleavage rate,high-quality embryo rate, blastocyst rate and high-quality blastocyst rate （P＞0.05）.Also, the parameters in trehalose virification group with activation and sucrosevirification group with activation O_2no difference（P＞0.05）.Totally24high-quality blastocyst were obtained in fresh group with activation and virificationgroup with activation. All of them were fixed successfully for the following FISHdiagnosis,14high-quality blastocyst in fresh group,5abnormal embryos were founded （abnormal rate35.7%）; for4high-quality blastocysts from the sucrose virificationgroup with activation,3abnormal embryos were founded （the aneuploid rate75%）;6embryos in the trehalose virification group with activation,3O_2aneuploidy（aneuploid rate50%）. no difference was found in aneuploid rate between the freshgroup and virification group（P＞0.05）.
Conclusions oocytes suffer damage in the freezing-thawing process and reduce theformation of embryos; Artificial activation can not improve the formation of embryos，but their development; Trehalose as impermeable protectant appears not to improve thefreezing–thawing effect.
Objective: Aim to analyse freezing-thawing damage by using co-focal microscopy toobserve ultramicrostructure of frozen-thawed matured oocytes derived from immatureoocytes from ICSI cycle.
Methods:62immature oocytes with IVM and51matured,42of them with a normalshape were picked up and randomly divided into two groups: IVM-sucrose group，20oocytes were observed using co-focal microscopy after thawing with vitrificationmedium containing sucrose to analyze the structure change of the spindles. IVM-trehalose group，22oocytes using vitrification medium containing trehalose. The controlgroup，20MII oocytes with normal shape were collected from IVF cycles（patients hadsigned informed consent and agreed to oocyte donation）,2of them were directly fixedand then observed the configuration of their spindles, for the other18oocytes，9of themwere frozen and thawed using vitrification medium containing sucrose, and the other9using trehalose, subsequently, all the survived oocytes were fixed to be observed the configuration of their spindles through co-focal microscope.
Results: A total of42mature oocytes was frozen,,20of them were used withvitrification medium containing sucrose, after thawing,17survived （IVM-sucrosegroup）, the other22with trehalose medium and19survived after thawing（IVM-trehalose group）. In IVF-sucrose and IVF-trehalose groups, respectively8survived. There was no significantly difference for surviving rate among four groups（P＞0.05）. all of the survived oocytes were included in the four groups were fixed. InIVM-sucrose group,10were successfully fixed,11in IVM-trehalose group,6IVF-sucrose group and7IVF-trehalose group had been fixed seccessfully. The oocyteswere observed under co-focal microscope, the normal morphology both inchromosomes and spindle morphology of the oocytes were l3（30.0%）,4（36.4%）,4（66.7%） and5（71.4%）, respectively. There were no significantly difference innormal morphology rate of spindle and chromosomes among four groups （P＞0.05）.
Conclusions Application of trehalose as an impermeable protectant during vitrificationof human MII oocytes can achieve an acceptible results. In the freezing-thawing process,both in-vivo and in-vitro mature oocytes suffer serious damage, but in-vivo matureoocyte appears to be more tolerate to cryopreservation.
引文
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