人类不同时期未成熟卵母细胞玻璃化冷冻后发育潜能的比较

目的:探索未成熟卵子的最佳冷冻时期。方法:收集卵胞质内单精子显微注射-胚胎移植(ICSI-ET)周期中未成熟的卵母细胞,按其成熟度分为生发泡期(GV组)卵子179枚和第一次减数分裂中期(MI组)卵子323枚,所有卵子均经玻璃化冷冻,解冻后行体外成熟(IVM)培养,ICSI受精,观察比较GV组和MI组解冻后存活、体外成熟、受精及胚胎发育情况。结果:GV组复苏存活率显著高于MI组(83.24%vs 75.54%,P=0.045),MI组体外成熟率、受精率、卵裂率、优质胚胎率均略均高于GV组,但无统计学差异(P0.05),MI组可利用胚胎率显著高于GV组(78.67%vs 60.53%,P=0.041)。结论:超促排卵周期中未成熟卵母细胞先玻璃化冷冻保存,再行体外培养是可行的。GV期卵母细胞复苏存活率高于MI期卵母细胞,但MI期卵母细胞冻融后发育潜能优于GV组卵母细胞。     

不同冷冻方法对小鼠不同成熟时期卵母细胞纺锤体的影响

目的:探讨不同冷冻方法对小鼠成熟期(MⅡ期)及生发泡期(GV期)卵母细胞的纺锤体及胚胎发育的影响。方法:收集GV期和有纺锤体的MⅡ期小鼠卵母细胞,随机分为3组:慢速冷冻-快速复温组、超高速玻璃化冷冻组和对照组(未冷冻组)。Polscope观察解冻0、3、6h后存活的MⅡ期及体外培养成熟GV期卵母细胞的纺锤体,有明显纺锤体的行卵胞浆内单精子显微注射受精,评价胚胎发育。结果:(1)超高速玻璃化GV组的存活率、卵裂率均显著高于慢冻GV组(P<0.05);(2)两冷冻MⅡ组解冻后0、3及6h纺锤体出现率和优质胚胎率均显著低于对照MⅡ组(P<0.05);(3)超高速玻璃化GV组体外成熟后纺锤体的出现率、优质胚胎率均显著高于超高速玻璃化MⅡ组(P<0.05)。结论:慢速冷冻-快速复温法对小鼠不同成熟时期卵母细胞的纺锤体损伤较大;超高速玻璃化冷冻对小鼠生发泡期卵母细胞纺锤体的影响则较小,是一种简便、快捷、高效的冷冻方法。     

《国外医学计划生育／生殖健康分册》2006年第3期重点内容

人自体胚胎干细胞创建技术的研究进展，体细胞单倍体化的正确性，卵母细胞体外成熟调节的研究进展，未成熟卵母细胞体外培养的临床应用研究进展。影响体外受精胞浆内单精子注射结局的相关因素研究进展，冷冻卵巢组织应用的研究进展．人类囊胚玻璃化冷冻研究进展，辅助生殖技术出生儿童的生长发育，     

冻融体外受精-胚胎移植周期人未成熟卵母细胞的体外成熟及纺锤体结局

目的:探讨冻融的不同状态人未成熟卵母细胞体外成熟后纺锤体状态与受精率的关系。方法:随机收集本中心108个体外受精-胚胎移植周期(IVF)中183枚不同状态废气的成熟卵母细胞,分为:卵丘-卵母细胞复合物组,48枚;裸卵组,135枚,其中第一次减数分裂中期(MI)65枚,生发泡期(GV)70枚。玻璃化冷冻保存,经解冻、体外培养成熟后,应用Polscope成像系统观察纺锤体,然后行卵胞浆内单精子显微注射受精,记录各指标情况。结果:①卵丘-卵母细胞复合物组与裸卵组的存活率、体外成熟率、纺锤体出现率、受精率比较,均无统计学差异(P>0.05);②GV组的存活率显著高于MI组(P<0.05),而前者的体外成熟率显著低于后者(P<0.05);③各组体外成熟后有纺锤体出现的卵母细胞受精率均显著高于无纺锤体组(P<0.05,P<0.01)。结论:冻融IVF周期不同状态的人未成熟卵母细胞都有一定发育潜能;有纺锤体出现的冻融人未成熟卵母细胞质量较高。     

玻璃化冷冻和程序化冷冻方法对人类卵母细胞骨架及其发育潜能的影响

目的探讨玻璃化冷冻和程序化冷冻对人卵母细胞纺锤体定位、细胞骨架及其发育潜能的影响。方法将第2日发育为M_II卵母细胞随机分为对照组、程序化冻融组、玻璃化冻融组(解冻0 h、1 h、3 h)。应用液晶偏振光显微镜(Polscope)成像系统观察卵母细胞纺锤体与第一极体(Pb)的夹角、表面积、卵透明带内层光阻值和外层光阻值。采用扫描电子显微镜和透射电子显微镜观察卵母细胞的表面和内部超微结构。统计2种冻融方法对卵母细胞发育潜能的影响。结果对照组、程序化冷冻解冻培养3 h组和玻璃化冷冻解冻后培养0 h、1 h、3 h组中的纺锤体可见率分别为92.4%、56.4%、11.2%、24.8%、61.1%。与程序化冻融组相比,玻璃化冷冻解冻培养3 h后卵母细胞中纺锤体与Pb的夹角更小(37.3°与68°,P=0.023)。对照组、程序化冻融组和玻璃化冻融后培养3 h组中卵母细胞的纺锤体面积、卵透明带内层光阻值和透明带外层光阻值差异无统计学意义(P0.05)。与程序化冻融组相比,玻璃化冻融后培养3 h组中卵母细胞表面突起丰富,微绒毛形态较为正常,倒伏在细胞表面,卵透明带边界较为清晰,与对照组较为接近。程序化冻融组的正常受精率(65.7%)明显低于对照组(79.2%,P=0.041),而卵裂率和囊胚形成率与对照组和玻璃化冻融组差异无统计学意义(P0.05)。玻璃化冻融后培养3 h组中正常受精率、卵裂率、囊胚形成率与对照组相比,差异无统计学意义(P0.05)。结论相比程序化冷冻,玻璃化冷冻对卵母细胞纺锤体和卵透明带的损伤及对卵母细胞的发育潜能的影响都较小,可以作为卵母细胞冷冻的一种有效方法。     

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。     

不同冷冻法对小鼠未成熟与成熟卵母细胞的成熟及胚胎发育的影响

本研究观察应用慢速冷冻．快速复温(常规冷冻法)与玻璃化冷冻法，对小鼠生殖泡期未成熟及第二次减数分裂中期(metaphageⅡ，MⅡ)成熟卵母细胞冷冻后的解冻、体外成熟、受精及早期胚胎发育的影响。     

人卵丘细胞线粒体膜电位与卵母细胞成熟度相关关系的研究

目的:通过检测人类卵母细胞周围卵丘细胞的线粒体膜电位(△Ψm),研究其与卵母细胞成熟度的关系。方法:收集卵母细胞质内单精子注射(ICSI)的166个卵母细胞周围的卵丘细胞,根据卵母细胞的成熟度及受精情况分为3组:不成熟卵母细胞组23例;成熟卵母细胞未受精组18例;成熟卵母细胞受精组103例。分别测定线粒体膜电位。用透射电镜观察3例未成熟卵母细胞、3例成熟卵母细胞周围卵丘细胞线粒体的变化。结果:未成熟卵母细胞组、成熟卵母细胞未受精组、成熟卵母细胞受精组线粒体膜电位分别为1．5±0．43,3．9±1．68,5．8±2．24,两两比较差异有统计学意义。透射电镜观察发现未成熟卵母细胞周围卵丘细胞质内线粒体肿胀、嵴减少、嵴消失、空泡化的发生率较成熟卵母细胞周围卵丘细胞高。结论:卵丘细胞线粒体膜电位与相应卵母细胞的成熟度相关。     

成熟卵母细胞玻璃化冷冻时机及解冻方法对辅助生殖结局的影响

目的 探讨玻璃化冷冻成熟卵母细胞过程中不同冷冻时机及解冻方法对辅助生殖结局的影响.方法 回顾性分析郑州大学第一附属医院生殖医学中心2007年5月-2009年5月实施辅助生殖周期中因不同原因接受成熟卵母细胞冷冻及解冻的不孕症患者30例的临床资料,其中女方双侧输卵管梗阻21例,男方无精症9例.将30例患者按照成熟卵母细胞的冷冻时机和解冻方法不同分为3组:A组共5例,取卵后4～5 h冷冻且采用常规方法解冻;B组共9例,取卵后2 h内冷冻且采用常规方法解冻;C组共16例,取卵后2 h内冷冻且采用改良法解冻.所有卵母细胞均采用玻璃化冷冻.冻存2～12个月解冻,存活的卵母细胞行卵母细胞胞质内单精子注射(ICSI)后进行胚胎移植.观察并比较3组患者的辅助生殖结局及孕期随访情况.结果 (1)B组和C组患者的卵母细胞存活率[(65±33)%、(72±23)%]、移植周期率(9/9、16/16)均明显高于A组[(16±17)%、1/5],差异均有统计学意义(P=0.001、0.021);B组与C组分别比较,差异均无统计学意义(P>0.05).C组的平均胚胎种植率[(33±38)%]、临床妊娠率(9/16)均明显高于B组[(4±11)%、1/9],差异均有统计学意义(P=0.033、0.040).(2)C组内采用自身胚胎移植、赠卵胚胎移植及供精胚胎移植者的平均年龄[分别为(28.6±2.1)、(28.0±4.6)、(28.1±3.4)岁]、卵母细胞存活率[分别为(73±25)%、(88±10)%、(66±25)%]、受精率[分别为(84.6±0.9)%、(79.3±2.0)%、(82.8±15.0)%]、胚胎种植率[分别为(20.0±44.7)%、(33.0±0.1)%、(41.6±41.7)%]及临床妊娠率(分别为1/5、3/3、5/8)之间比较,差异均无统计学意义(P>0.05).(3)A组1例患者行胚胎移植,但未妊娠;B组1例临床妊娠,2个月后胚胎停止发育;C组9例临床妊娠,其中1例妊娠4个月流产,8例已顺利分娩5男婴、4女婴,新生儿染色体及发育均正常,每解冻卵母细胞活产率为5.9%(8/135),平均孕周为(39.4±0.9)周,平均新生儿出生体质量为(3574±569)g.结论 取卵后2 h内玻璃化冷冻及改良法解冻成熟卵母细胞,可以提高胚胎质量及改善临床妊娠结局.     

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。     

Vitrification of human immature oocytes before and after in vitro maturation: a review

The use of immature oocytes subjected to in vitro maturation (IVM) opens interesting perspectives for fertility preservation where ovarian reserves are damaged by pathologies or therapies, as in PCO/PCOS and cancer patients. Human oocyte cryopreservation may offer some advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation and postponing childbirth. It also eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In addition, a successful oocyte cryopreservation program could eliminate the need for donor and recipient menstrual cycle synchronization. Recent advances in vitrification technology have markedly improved the oocyte survival rate after warming, with fertilization and implantation rates comparable with those of fresh oocytes. Healthy live births can be achieved from the combination of IVM and vitrification, even if vitrification of in vivo matured oocytes is still more effective. Recently, attention is given to highlight whether vitrification procedures are more successful when performed before or after IVM, on immature GV-stage oocytes, or on in vitro matured MII-stage oocytes. In this review, we emphasize that, even if there are no differences in survival rates between oocytes vitrified prior to or post-IVM, reduced maturation rates of immature oocytes vitrified prior to IVM can be, at least in part, explained by underlying ultrastructural and biomolecular alterations.     

Highly efficient vitrification method for cryopreservation of human oocytes

Two experiments were performed to develop a method to cryopreserve MII human oocytes. In the first experiment, three vitrification methods were compared using bovine MII oocytes with regard to their developmental competence after cryopreservation: (i) vitrification within 0.25-ml plastic straws followed by in-straw dilution after warming (ISD method); (ii) vitrification in open-pulled straws (OPS method); and (iii) vitrification in <0.1 microl medium droplet on the surface of a specially constructed fine polypropylene strip attached to a plastic handle (Cryotop method). In the second experiment, the Cryotop method, which had yielded the best results, was used to vitrify human oocytes. Out of 64 vitrified oocytes, 58 (91%) exhibited normal morphology after warming. After intracytoplasmic sperm injection, 52 became fertilized, and 32 (50%) developed to the blastocyst stage in vitro. Analysis by fluorescence in-situ hybridization of five blastocysts showed that all were normal diploid embryos. Twenty-nine embryo transfers with a mean number of 2.2 embryos per transfer on days 2 and 5 resulted in 12 initial pregnancies, seven healthy babies and three ongoing pregnancies. The results suggest that vitrification using the Cryotop is the most efficient method for human oocyte cryopreservation.     

Cryopreservation of immature and in-vitro matured human oocytes by vitrification

The objective of this study was to determine the efficacy of vitrification of human oocytes before and after in-vitro maturation (IVM). The immature oocytes recovered (n = 472) were divided into two groups: (i) immature oocytes (n = 219) vitrified at the germinal vesicle (GV) stage; and (ii) immature GV-stage oocytes (n = 253) that were firstly matured in vitro (MII-stage oocytes; n = 178), then vitrified (n = 79). The remaining oocytes (n = 99), which were not vitrified, were processed as controls. After warming, the oocyte survival, maturation and fertilization rates, as well as embryonic development, were compared. The results showed no significant difference between the survival rates of the oocytes vitrified at GV stage and those vitrified at MII stage (85.4% versus 86.1%). However, oocyte maturation rates were significantly reduced (P < 0.05) when oocytes were vitrified at immature GV stage followed by IVM (50.8%) in comparison with the control group (70.4%). Following insemination by intracytoplasmic sperm injection, there was no difference in the fertilization (62.1% versus 58.8%), cleavage (69.5% versus 67.5%) and blastocyst development (0.0% versus 0.0%) rates between these two groups. However, these results were significantly lower (P < 0.05) than those achieved in the control group. This suggests that better results can be achieved by vitrifying mature oocytes rather than immature oocytes.     

The presence of 1 mM glycine in vitrification solutions protects oocyte mitochondrial homeostasis and improves blastocyst development

Purpose

Embryos generated from oocytes which have been vitrified have lower blastocyst development rates than embryos generated from fresh oocytes. This is indicative of a level of irreversible damage to the oocyte possibly due to exposure to high cryoprotectant levels and osmotic stress. This study aimed to assess the effects of vitrification on the mitochondria of mature mouse oocytes while also examining the ability of the osmolyte glycine, to maintain cell function after vitrification.

Methods

Oocytes were cryopreserved via vitrification with or without 1 mM Glycine and compared to fresh oocyte controls. Oocytes were assessed for mitochondrial distribution and membrane potential as well as their ability to fertilise. Blastocyst development and gene expression was also examined.

Results

Vitrification altered mitochondrial distribution and membrane potential, which did not recover after 2 h of culture. Addition of 1 mM glycine to the vitrification media prevented these perturbations. Furthermore, blastocyst development from oocytes that were vitrified with glycine was significantly higher compared to those vitrified without glycine (83.9 % vs. 76.5 % respectively; p?<?0.05) and blastocysts derived from oocytes that were vitrified without glycine had significantly decreased levels of IGF2 and Glut3 compared to control blastocysts however those derived from oocytes vitrified with glycine had comparable levels of these genes compared to fresh controls.

Conclusion

Addition of 1 mM glycine to the vitrification solutions improved the ability of the oocyte to maintain its mitochondrial physiology and subsequent development and therefore could be considered for routine inclusion in cryopreservation solutions.     

Defective oocytes: a new subgroup of unexplained infertility.

OBJECTIVE: To define a new category of unexplained infertility and its potential treatment. DESIGN: Normal infertile couples underwent prospectively, cross-fertilization attempts in which the wife's oocytes were inseminated by the husband and donor semen. After recurrent failure of fertilization, cross insemination of donor oocytes was attempted with the husband sperm. SETTING: In vitro fertilization unit at a teaching hospital. PATIENTS: Three couples who were diagnosed as suffering of unexplained infertility and treated by in vitro fertilization (IVF). RESULTS: The female partner of these couples produced morphologically normal oocytes that were demonstrated to be functionally defective and failed to fertilize in vitro with both husband and donor sperm. Donated oocytes inseminated by the husband's sperm were fertilized in all patients, demonstrating the normal fertilizing ability of the husbands' semen. One patient conceived and delivered after an oocyte donation. CONCLUSIONS: Conclusive diagnosis of defective oocytes as a cause of infertility may be made only after IVF and oocyte donation.     

Vitrification negatively affects the Ca2+-releasing and activation potential of mouse oocytes,but vitrified oocytes are potentially useful for diagnostic purposes

Research questionTo what extent does vitrification affect the Ca²⁺-releasing and activation potential of mouse oocytes, which are commonly used to determine the oocyte activation potential of human spermatozoa?DesignThe effect of mouse oocyte vitrification on Ca²⁺ dynamics and developmental competence after oocyte activation was assessed and compared with fresh mouse oocytes. Moreover, the Ca²⁺ store content of the endoplasmic reticulum was determined at different time points during the vitrification–warming procedure. Finally, the Ca²⁺ pattern induced by cryoprotectant exposure was determined.ResultsAfter human sperm injection into mouse oocytes, Ca²⁺ dynamics but not fertilization rates were significantly altered by vitrification warming (P < 0.05). Ca²⁺ dynamics in response to SrCl₂ or ionomycin were also altered by oocyte vitrification. In contrast, activation and blastocyst rates after SrCl₂ exposure were not affected (P > 0.05), whereas activation rates after ionomycin exposure were significantly lower in vitrified–warmed oocytes (P < 0.05); blastocyst rates were not affected (P > 0.05). Cryoprotectant exposure was associated with a strong drop in endoplasmic reticulum Ca²⁺ store content. Oocytes rapidly recovered during warming and recovery in Ca²⁺-containing media; a threshold area under the curve of Ca²⁺ dynamics to obtain activation rates above 90% was determined.ConclusionsVitrified–warmed mouse oocytes display reduced Ca²⁺-releasing potential upon oocyte activation, caused by cryoprotectant exposure. With adapted classification criteria, these oocytes could be used for diagnosing oocyte activation deficiencies in patients. Evaluating the Ca²⁺-signalling machinery in vitrified–warmed human oocytes is required.     

Cryopreservation of immature and mature human oocytes

Cryopreservation of oocytes facilitates the long-term storage of oocytes for patients in danger of losing ovarian function. It also alleviates many of the ethical concerns associated with embryo cryopreservation. Problems associated with metaphase II oocyte cryopreservation include zona pellucida hardening and spindle damage. The cryopreservation of germinal vesicle-stage oocytes has been undertaken as a means of circumventing the problem of spindle damage in mature oocytes. One of the main disadvantages of immature oocyte cryopreservation is the fact that in vitro maturation is required post-thaw. The majority of live births from oocyte cryopreservation have involved the use of 1,2-propanediol and slow freezing protocols. Various methods have been used in an attempt to improve survival rates. These include vitrification and use of novel cryopreservatives. Future areas of concentration should include in vitro maturation, vitrification, and alternate cryopreservatives.     

Timed IVM followed by ICSI in a patient with immature ovarian oocytes

A complete failure of meiotic maturation occasionally occurs following human chorionic gonadotrophin administration during IVF-intracytoplasmic sperm injection (ICSI) cycles. ICSI on day 1 is commonly used to allow maturation in culture. However, if the oocytes become mature in the evening soon after their recovery but ICSI is delayed until the next day, then subsequent ageing of matured oocytes may be unfavourable for fertilization and development. To avoid the deterioration associated with oocyte ageing, the timing of polar body extrusion was checked every 3 h and rescue in-vitro maturation (IVM)-ICSI was performed shortly after the polar body extrusion was confirmed. This report describes a successful pregnancy and birth of a healthy baby in a patient who had no mature oocytes at the time of oocyte retrieval, and illustrates the value of extra monitoring for IVM and ICSI in cases where only immature oocytes are available.