人类未成熟卵母细胞玻璃化冷冻研究

目的:探讨玻璃化冷冻未成熟卵母细胞的有效性。方法:根据有无颗粒细胞将实施玻璃化冷冻的GV期卵母细胞分为含颗粒细胞(非裸卵)组和不含颗粒细胞(裸卵)组;将部分GV期卵母细胞体外培养至MⅡ期卵母细胞实施玻璃化冷冻,比较非冷冻IVM组与MⅡ卵玻璃化冷冻组间、裸卵组与非裸卵组间的存活率、成熟率、受精率、卵裂率及囊胚形成率。结果:非裸卵组的成熟率大于裸卵组(P<0.05),而存活率、受精率、2-细胞形成率、>2-细胞形成率之间均无统计学差异(P>0.05)。另外,非冷冻IVM组与GV玻化组间成熟率、受精率、卵裂率均存在显著性差异(P<0.05);非冷冻IVM组与MⅡ期卵玻化组间成熟率、受精率、卵裂率间均存在统计学差异(P<0.05);GV玻化组与MⅡ玻化组间存活率、成熟率、受精率、卵裂率间均无统计学差异(P>0.05)。结论:玻璃化冷冻未成熟卵母细胞需要保留颗粒细胞,同时初步构建了人GV期卵的玻璃化冷冻联合IVM技术的雏形。     

卵母细胞玻璃化冻融21例临床分析

目的:探讨玻璃化冷冻方法在冷冻保存卵母细胞中的临床应用。方法:选择2007年10月～2009年1月在我中心行卵母细胞玻璃化冷冻,并于2008年1月～2009年4月行卵母细胞玻璃化解冻的21例移植周期,对其复苏率、受精率及临床妊娠率进行统计。结果:共冷冻203枚卵母细胞,解冻后存活137枚(67.5%,137/203),对其中98枚成熟卵母细胞行单精子卵浆内显微注射,76枚(77.6%,76/98)正常受精,8例(38.1%,8/21)获得临床妊娠,其中6例单胎妊娠,2例3胎妊娠,并进行了减胎术。目前,1例已成功分娩,1例早期流产,6例正在妊娠中。结论:玻璃化冻融卵母细胞是一种方便可行的方法,可以获得较高的临床妊娠率。     

冻融液的温度对人卵母细胞玻璃化冻融效果的影响

目的:探讨冻融液的操作温度对成熟阶段(MII)人卵母细胞的玻璃化冻融效果的影响.方法:收集体外受精-胚胎移植(IVF-ET)患者中受精失败的成熟卵母细胞,根据放置在冻融液操作温度的不同分为A组、B组、C组.解冻后存活卵母细胞及对照组(未冷冻组,D组)卵母细胞固定后进行免疫荧光染色.然后用Nikon CISI激光扫描共聚焦显微镜观察纺锤体、染色体形态.结果:A组与对照组卵的纺锤体和染色体形态正常率分别为13.0%和13.0%、18.2%和18.2%,相互比较差异均无统计学意义(P＞0.05);A组与对照组卵的纺锤体缺失率(Ⅳ型)为65.2%与39.4%,相互比较差异无统计学意叉(P＞0.05);B组卵的染色体形态正常率及C组卵的纺锤体和染色体形态正常率均为0,与对照组比较差异均有统计学意义(P＜0.05);B、C组卵的纺锤体缺失率(Ⅳ型)为78.2%、90.0%,与对照组比较差异有高度统计学意义(P＜0.01).结论:降温前放置在冷冻液的合适操作温度可以减少冻融对卵子纺锤体和染色体的损伤程度.     

人黄体期卵母细胞的体外成熟及玻璃化冷冻的实验研究

目的:探讨未成熟卵母细胞体外成熟(IVM)技术联合玻璃化冷冻保存黄体期卵母细胞对某些女性肿瘤患者的生育能力的保存情况。方法:采集因妇科肿瘤等行卵巢切除手术过程中穿刺获取的256枚未成熟卵母细胞,按取卵时患者的月经周期分为卵泡期组(143枚)与黄体期组(113枚),每组再随即分为新鲜对照组及玻璃化冷冻组。分别进行IVM后行新鲜卵胞质内单精子注射(ICSI)授精和玻璃化冻融卵ICSI授精,比较各组间IVM后MII卵率、受精率、卵裂率、优质胚胎率。结果:①卵泡期与黄体期卵母细胞的IVM率差异无统计学意义;而组间复苏存活率(68.0%vs 48.1%)有统计学差异(P<0.05);卵泡期与黄体期卵母细胞新鲜组间受精率、卵裂率、优质胚胎率无统计学差异,冷冻组间差异亦无统计学意义。②与新鲜组相比,玻璃化冷冻使卵泡期与黄体期卵母细胞的受精率均降低(P<0.01)。结论:黄体期未成熟卵母细胞可以体外成熟并有继续发育为优质胚胎的能力;玻璃化冷冻使卵母细胞受精率、卵裂率下降。IVM和冻融后体外授精是某些女性肿瘤患者保存生育能力的一种有临床应用前景的方式。     

冷冻环玻璃化冷冻对小鼠成熟卵母细胞DNA的损伤

卵母细胞冷冻是辅助生育技术(assistel repro-ductive technology,ART)领域的研究热点之一.1986年全世界第一例通过冻存卵母细胞获得试管婴儿成功降生,然而由于卵母细胞体积大、细胞膜与细胞浆比例小等自身生物学特点,卵母细胞冷冻发展较慢,目前临床上尚未广泛应用,仅局限于实验研究[1].我院冷冻环玻璃化法冻存小鼠成熟卵母细胞获得了较好效果[2],本研究通过脱氧核糖核苷酸     

人胚胎的玻璃化冷冻进展

人类胚胎冷冻技术已成为生殖助育的重要组成部分。其意义在于：合理限制胚胎移植数目，有效降低多胎妊娠率；为新鲜移植周期失败或流产患者提供再次移植机会，降低费用，提高单次取卵累积成功率；促排卵周期出现卵巢过度刺激综合征（OHSS）或移植过程中出现意外的患者，将胚胎冻存供以后使用；胚胎捐赠。目前，临床上广泛使用的胚胎冷冻方法为慢速冷冻法。该方法需高精密的程序冷冻仪，且操作复杂，全程约需2．5～3h，耗液氮量多。     

两种玻璃化冷冻载体对小鼠自然周期及超排卵周期卵母细胞冻融结局的影响

目的:建立优化玻璃化冷冻卵母细胞的方法,了解超排卵后小鼠卵母细胞的复苏率及体外受精率,为改善人类卵母细胞玻璃化冷冻技术提供依据。方法:建立小鼠超排卵及自然周期模型,比较开放式拉细麦管(open pulled straw,OPS)及叶片两种玻璃化冷冻载体冷冻小鼠卵母细胞的复苏率及体外受精率。结果:(1)叶片法玻璃化冷冻卵母细胞复苏率高于OPS法(自然周期组:75.0%vs55.6%,P=0.0003,超排卵周期组:65.3%vs46.3%,P=0.0000);(2)自然周期组卵母细胞的冷冻复苏率(OPS组:55.6%vs46.3%,叶片组:75.0%vs65.3%),体外受精率(OPS组:53.5%vs40.5%,叶片组:52.8%vs39.0%)均高于超排卵周期组。结论:叶片载体玻璃化法优于OPS载体玻璃化法;自然周期组小鼠卵母细胞玻璃化冷冻复苏率及体外受精率均高于超排卵周期组。     

玻璃化冷冻成熟卵母细胞经解冻精液单精子注射受精并胚胎冻融及移植妊娠成功

冷冻精子及胚胎技术,在国内外生殖医学中心已广泛应用于临床,但卵母细胞冷冻,尤其是快速冷冻,近年来才在国内外开展.冷冻精子、冷冻卵母细胞、冷冻胚胎（即“三冻”）技术用于1例患者最终成功妊娠并分娩活婴,于2004年为意大利首次报道.本文报道了北京大学第三医院生殖医学中心应用“三冻”技术,成功获得世界第2例、我国第1例“三冻”婴儿,反映了我国目前在辅助生育技术方面的发展,已经达到了国际水平.当然,卵母细胞冷冻技术仍需进一步完善,对出生的婴儿还应随访.同时,应强调必须遵守相应的法规和伦理.[编者按]     

人卵巢组织玻璃化冷冻技术研究进展

卵巢组织冷冻是女性生育力保存的重要方式,程序化慢速冷冻一直是卵巢组织冷冻的主流方法.随着玻璃化冷冻技术在辅助生殖领域的成功应用,玻璃化冷冻技术在卵巢组织冷冻领域中的应用也越来越广.本文概括性地对卵巢组织玻璃化冷冻的发展现状、实验室技术及临床应用效果做一综述,以期为临床和实验研究提供借鉴和参考.     

玻璃化冷冻人早期胚胎的效果观察

目的探讨玻璃化冷冻人早期胚胎的临床应用价值。方法采用由5．5mol／L乙二醇及1．0mol／L蔗糖制成的冷冻保护液,于0．25ml冷冻麦管中,冷冻经体外受精一胚胎移植第2天或第3天后剩余的胚胎;对新鲜周期胚胎移植后未妊娠的胚胎,至少3个月或3个月以上施行胚胎复苏,观察胚胎复苏后的临床妊娠效果。结果共行胚胎复苏219例,957个胚胎,胚胎复苏后存活691个,胚胎存活率为72．2％（691／957）;移植胚胎178例,临床妊娠35例,临床妊娠率为19．7％（35／178）。35例中,分娩16例,流产6例,继续妊娠13例。结论玻璃化冷冻法简便,胚胎存活率高,是一种可用于临床的人早期胚胎冷冻的方法。     

A Quantitative and Morphological Analysis of Oocytes Collected During 438 IVF Cycles

Purpose: Our purpose was to determine if the number ofretrieved oocytes, oocyte maturity, morphology, and otherembryological parameters are related to the outcome oftreatment. Methods: This retrospective study on 438 IVF cyclesanalyzes the numbers of retrieved oocytes, fractured zonaoocytes, germinal vesicle-stage oocytes, normally andabnormally fertilized oocytes, pregnancy rate, age of femalepartner, ovarian stimulation protocol, day of hCG injection,and serum estradiol concentration. Results: (1) Pregnancy rate increases with an increase inthe number of retrieved oocytes, (2) a high incidence offractured zona pellucida oocytes has a negative effect onfertilization rate but none on pregnancy rate, (3) a highincidence of immature oocytes is associated with improvedfertilization and pregnancy rates, and (4) an inverserelationship between the presence of immature oocytes and oocyteswith fractured zona pellucida is suggested. Conclusions: Precise oocyte assessment in IVF cyclesprovides informations useful for the analysis and improvementof ovarian stimulation protocols.     

有无卵丘细胞对玻璃化冷冻后小鼠卵母细胞细胞骨架及体外发育能力的初步研究

目的:探讨卵丘细胞在玻璃化冷冻中对小鼠卵母细胞发育潜能及细胞骨架的影响。方法:采用玻璃化冷冻技术,保存带卵丘细胞或完全剥除卵丘细胞的小鼠MⅡ期和GV期卵丘复合体/裸卵(COC/DO),复苏且GV卵体外培养成熟后分别作体外受精或免疫荧光标记检查纺锤体和染色体的完整性。结果:MⅡ-COC和GV-COC的复苏率均显著高于MⅡ-DO和GV-DO(分别为86.49%vs60.92%和85.94%vs64.93%,P<0.01)。GV-COC组的受精率、囊胚率均高于GV-DO组,且MⅡ-COC组和GV-COC组的纺锤体和染色体均正常率均分别高于MⅡ-DO组和GV-DO组,但无显著性差异(P>0.05)。结论:卵丘细胞在玻璃化冷冻卵母细胞中能有效减少冷冻对细胞骨架的损伤,并改善卵子复苏及胚胎发育潜能。     

激光皱缩法在囊胚玻璃化冷冻中的应用价值

目的：探讨激光打孔使囊胚腔皱缩在体外受精周期囊胚玻璃化冷冻中的应用价值。方法：将常规取卵后第3日移植、冷冻后剩余的形态学评分较差的胚胎发育而来的囊胚,采用或不采用激光打孔使囊胚腔皱缩后冷冻。分析606例解冻囊胚周期,比较采用和未采用激光皱缩2种方法冻存囊胚的效率,分析激光皱缩在囊胚玻璃化冷冻中的价值。结果：激光皱缩组解冻247例,4例（1．62％）取消移植,移植243例;未皱缩组359例,24例（6．69％）取消移植,移植335例。移植患者的年龄、不孕原因、排卵日内膜厚度、平均移植胚胎数组间均无统计学差异（P〉0．05）。激光皱缩组冻融取消率和流产率均显著低于非皱缩组（P〈0．05）,生化妊娠率（45．68％vs35．82％）、种植率（22．54％vs16．56％）和继续妊娠率（87．50％vs75．56％）显著高于非皱缩组（P〈0．05）,临床妊娠率激光皱缩组略高于非皱缩组（32．92％Ⅷ26．87％）,但无统计学差异（P〉0．05）。结论：囊胚冷冻前采用激光打孔皱缩可提高解冻后的胚胎存活率,并能显著降低流产率。     

人卵母细胞在不同培养液下成熟与凋亡的比较性研究

为了解卵母细胞体外成熟与凋亡过程,进而提高卵母细胞体外成熟率.本文采自手术标本中卵巢及卵巢组织.吸取4～10mm卵泡.获得未成熟卵母细胞.用正交设计.于培养液中加入不同量组合的人绝经期促性腺激素(hMG)、卵泡液、颗粒细胞.在37℃.CO_2 5%培养箱中培养,并应用形态学观察和原位DNA片段末端标记(Tunel)检测卵母细胞的凋亡状况.结果:在本研究体外培养条件下能使未成熟卵母细胞成熟、受精,胚胎发育至早期胚泡(Blastocyst);以含hMG 0.15IU/ml卵泡液40%,颗粒细胞10~2/ml组合的培液培养卵母细胞的成熟率、受精率及印裂率最高,而凋亡率最低(P<0.05,P<0.005).结论:未成熟卵母细胞可在体外成熟、受精,并形成早期胚泡.     

Effect of vitrification on mitochondrial membrane potential in human metaphase II oocytes

Objective

The aim of this study was to evaluate the impact of vitrification on mitochondrial membrane potential (ΔΨm) in human metaphase II (MII) oocytes, and the changes of ΔΨm on thawed MII oocytes.

Methods

MII oocytes were obtained from clinical IVF cycles when the oocytes were failed to fertilization within 24 h after insemination. All oocytes were randomly divided into 4 groups: non-frozen (fresh group), cultured for 0 h (0 h group), 2 h (2 h group) and 4 h (4 h group) after vitrification/thawing. All oocytes were stained with the ΔΨm-specific probe JC-1 and detected by laser scanning confocal microscope (LSCM) for mitochondrial analysis.

Results

The ΔΨm of oocytes was significantly decreased in 0 h and 2 h groups when compared with fresh group (0.93, 1.09 vs 1.34, P < 0.05), but similar between 4 h group and fresh group (1.30 vs 1.34, P > 0.05).

Conclusion

In the vitrification/thawing process, the ΔΨm of MII oocytes could have temporally dynamic changes within 2 h after thawing but would be fully recovered after 4 h culture.     

Vitrification of human blastocysts previously cryopreserved by slow controlled-rate freezing at the cleavage stage

Purpose

The objective of this study was to evaluate the efficiency of vitrified blastocysts derived from frozen-thawed cleavage stage embryos in terms of morphological survival and re-expansion status of the blastocoelic cavity.

Results

After warming 162 blastocysts derived from fresh embryos (= control group) and 90 blastocysts from frozen-thawed cleavage stage embryos (= study group) and after 2–3 h of in vitro culture the percentage of blastocysts with morphological survival was not different between the two groups. After 24 h of in vitro culture, the percentage of fully expanded, hatching or hatched blastocysts was not different between both groups.

Conclusion(s)

The results show that blastocysts derived from frozen-thawed cleavage stage embryos can be cryopreserved successfully a second time by vitrification method. Re-cryopreservation by vitrification still needs to be approached with some caution because little data on long term safety of multiple freezing is available.     

A live birth from vitrified-warmed oocytes in a Philadelphia chromosome-positive acute lymphoid leukemia patient 5 years following allogenic bone marrow transplantation and after a magnitude 9.0 earthquake in Japan

Purpose

To report a live birth from vitrified-warmed oocytes for a Philadelphia chromosome-positive acute lymphoid leukemia (Ph-ALL) patient.

Methods

A 20-year-old single woman with Ph-ALL requested oocyte cryopreservation at a private fertility clinic using assisted reproduction technology (ART). In cases of leukemia, there is a very short time before chemotherapy, follwed shortly by total body irradiation (TBI), and although she had already received the chemotherapy, ten oocytes were vitrified and stored for 59 months before warming. Soon after the oocyte cryopreservation, she received TBI and bone marrow transplant (BMT). During the storage, a magnitude 9.0 earthquake occurred making oocyte transport necessary. The embryo transfer was planned in a hormone replacement cycle, and intracytoplasmic sperm injection (ICSI) was performed on the vitrified-warmed oocytes. On day 3, two embryos were transferred.

Results

The patient became pregnant and delivered a healthy girl after ICSI using vitrified-warmed oocytes.

Conclusions:

Oocyte cryopreservation is the best option for fertility preservation of young single women with leukemia. Oncologists and gynecologists who conduct ART should cooperate to improve the quality of life of cancer patients.     

Supplementation with low concentrations of melatonin improves nuclear maturation of human oocytes in vitro

Purpose

Studies in bovine and porcine have indicated that melatonin (MT) could induce meiotic maturation of immature oocytes in vitro. The object of the current study was to investigate if MT could ameliorate human oocytes maturation during rescue in vitro maturation (IVM).

Methods

Two hundred seventy eight germinal vesicle (GV) oocytes and 451 (MI) metaphase I oocytes were vitrified, thawed and then matured in vitro. All the oocytes were randomly allocated into six groups in which the oocytes were cultured in medium supplemented with different concentrations of MT (0, 10⁻², 1, 10², 10⁴, 10⁶ nM) and nuclear maturation was evaluated at 6 h, 12 h, 18 h, 24 h and 48 h of culture.

Results

The optimal MT concentration for both GV and MI oocytes was 1 nM. At 24 h of culture, nuclear maturation rate of MI oocytes cultured in 1 nM MT medium was significantly higher than other groups (P < 0.05); Nuclear maturation rate of GV oocytes cultured in 1 nM MT medium was also significantly higher than the control group (P < 0.05). On the other hand, decreased nuclear maturation rate was observed in the high MT concentration group (10⁶ nM).

Conclusions

The current study demonstrated that low concentration of exogenous MT could ameliorate nuclear maturation of human oocyte during rescue IVM, while high concentration of MT presented negative effects.