食蟹猴体内自然成熟和体外培养成熟的卵母细胞对卵胞质内单精子显微注射(ICSI)结局的影响

目的:探讨食蟹猴体内自然成熟和体外培养成熟(IVM)的卵母细胞对卵胞质内单精子显微注射(ICSI)结局的影响。方法:选取有正常月经周期和生育功能的13只健康成熟的雌性食蟹猴,使用优化的促排卵方案进行促排卵后,B超下观察用药后的卵巢情况,手术取卵后对已成熟的MⅡ期卵母细胞进行ICSI,未成熟的GV及MⅠ期卵母细胞在IVM 16~38 h后发育为MⅡ期卵母细胞,再进行ICSI,观察IVM及体内自然成熟的卵母细胞的受精情况和胚胎发育情况。结果:IVM为76.1%±14.9%,受精率为56.79%,卵裂率为73.91%,优质胚胎率为44.12%;体内自然成熟的卵母细胞受精率为68.52%,卵裂率为94.59%,优质胚胎率为58.57%。在受精率和卵裂率方面,两者间均有统计学差异(P0.05和P0.01),而优质胚胎率无统计学差异(P0.05)。结论:食蟹猴体外培养成熟的卵母细胞对ICSI结局有较大影响。     

氨基双吡酮对小鼠卵母细胞成熟的影响

目的研究氨基双吡酮(氨力农)对体外和体内小鼠卵母细胞成熟的抑制作用,检测磷酸二酯酶(PDE)3抑制剂氨力农能否作为一个潜在的避孕药。方法将不同浓度药物作用于体外培养的不成熟小鼠卵母细胞,以及注射于小鼠体内,观察其对卵母细胞成熟的阻滞作用并找出合适的浓度。卵母细胞成熟后进行卵胞质内单精子注射(ICSI),并进行移植,观察其受精率、胚胎形成情况以及出生后代比较。并监测体内注射组小鼠的不良反应情况。结果 (1)随着浓度的增加,氨力农导致减数分裂阻滞呈剂量依赖性,体外有效浓度为1μmol/L,而在体内为300 mg/kg。(2)其作用是可逆的。药物去除后,减数分裂恢复,小鼠卵母细胞成熟,并呈正常染色体排列和纺锤体组织。(3)与对照组比较,ICSI受精后,卵母细胞表现出正常的形态、受精率、卵裂、囊胚形成。(4)其出生后代表现出相似的体质量和生育能力。(5)在体内,注入一定浓度药物后小鼠表现为不孕,停药后恢复。结论氨力农可以抑制体外和体内小鼠卵母细胞的成熟,并且是可逆的;交配实验证明氨力农可能可作为一种避孕药物。     

钙离子载体A23187联合嘌呤霉素激活行卵母细胞质内单精子注射后未受精卵母细胞的研究

目的观察钙离子载体A23187联合嘌呤霉素对卵母细胞质内单精子注射（ICSI）后未受精卵母细胞的激活作用。方法将体外成熟（IVM）-ICSI和常规ICSI后未受精卵母细胞,按行ICSI后体外培养的时间,分为IVM-ICSI 22h组（33个）、IVM—ICSI 44h组（18个）、ICSI44h组（37个）、ICSI68h组（25个）,分别采用钙离子载体A23187联合嘌呤霉素进行激活处理。应用荧光原位杂交（FISH）技术,对来源于双原核合并第二极体合子的激活胚胎进行性染色体分析。结果钙离子载体A23187联合嘌呤霉素能激活行ICSI后22—68h未受精的卵母细胞。其中IVM-ICSI22h组卵母细胞激活率为88％（29／33）、总卵裂率为62％（18／29）、4细胞阶段胚胎发育率为28％（5／18）,1个胚胎发育到桑椹胚阶段;而IVM-ICSI44h组、ICSI44h组、ICSI68h组的未受精卵母细胞激活率分别为56％（10／18）、65％（24／37）、52％（13／25）;总卵裂率分别为20％（2／10）、42％（10／24）、46％（6／13）,仅ICSI44h组有1个胚胎发育到4细胞阶段。FISH对激活胚胎的分析显示,4个胚胎为XX,9个胚胎为XY。结论钙离子裁体A23187联合嘌呤霉素能有效激活行ICSI失败的卵母细胞;行ICSI后22h内,是对未受精卵母细胞进行辅助激活较为理想的时机。激活的双原核合并第二极体胚胎中有雄原核的存在。     

卵母细胞激活技术在卵细胞质内单精子注射受精失败或低下患者中的应用

目的探讨卵母细胞激活技术在卵细胞质内单精子注射（ICSI）受精失败或低下患者和可疑受精失败或低下患者中的应用价值。方法选择2011年6月至2013年5月至少有1次ICSI受精失败或低下的患者lO例,再次接受助孕治疗时,实施卵母细胞激活技术（作为激活周期）;对可疑ICSI受精失败或低下患者3例,实施半数卵母细胞激活处理,将获得的67枚卵子分为常规ICSI组和激活组。观察卵母细胞激活前后的受精率、卵裂率和优质胚胎的变化及妊娠结局。结果在ICSI受精失败或低下患者中,既往周期的受精率为29．67％（27／91）,激活周期的受精率为72．58％（45／62）,两者比较,差异有统计学意义（P〈O．01）;既往周期和激活周期的卵裂率分别为85．19％（23／27）和95．56％（43／45）,两者比较,差异无统计学意义（P〉0．05）;优质胚胎量由既往IC$I周期的（0．25±0．45）个提高到激活周期的（1．18±1．33）个（P〈0．05）;5例患者获得临床妊娠。在可疑ICSI受精失败或低下患者中,常规ICSI组和激活组的受精率、卵裂率和优质胚胎量比较,差异均无统计学意义（P均〉0．05）。结论卵母细胞激活技术可提高ICSI受精失败或低下史患者的受精率,但是对于没有ICSI受精失败或低下史的患者效果不佳。     

冷冻保存人类未成熟卵母细胞对其发育潜能的影响

目的 评价冷冻保存人类未成熟卵母细胞对其发育潜能的影响。方法 收集常规胞质内单精子显微注射-胚胎移植（ICSI-ET）中不成熟的卵母细胞168个,根据成熟程度分为生发小泡期（GV）卵母细胞103个和第1次减数分裂中期（MI）卵母细胞65个,再将各期卵母细胞分别分为冷冻组和对照组,冷冻组细胞经慢速冷冻-快速融解（慢冻-速融）后在体外培养成熟,对照组直接进行体外培养成熟。最后进行免疫荧光染色并观察。结果 GV冷冻组和GV对照组卵母细胞之间体外成熟率（分别为69．1％、74．3％）比较,差异无统计学意义（P〉0．05）;GV冷冻组和GV对照组卵母细胞之间纺锤体形态正常率（分别为28．9％、53．9％）和染色体形态正常率（分别为23．7％、50．0％）比较,差异有统计学意义（P〈0．05）;MI冷冻组和MI对照组卵母细胞之间体外成熟率（分别为68．6％、88．0％）比较,差异无统计学意义（P〉0．05）。MI冷冻组和MI对照组卵母细胞之间纺锤体形态正常率（分别为20．8％、54．6％）和染色体形态正常率（分别为25．0％、63．6％）比较,差异有统计学意义（P〈0．05）;GV冷冻组和MI冷冻组之间各项结果比较,差异均无统计学意义（P〉0．05）。结论 应用常规慢冻．速融方案并不能很好地保存未成熟卵母细胞的体外成熟后的纺锤体形成能力,未成熟的卵母细胞的发育潜能从而受损。     

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。     

体内外成熟过程中小鼠卵母细胞纺锤体形态和皮质颗粒分布的比较

目的:了解体内外成熟过程中小鼠卵母细胞减数分裂进程、纺锤体形态、皮质颗粒分布的差异。方法:分别收集体内和体外成熟2h、4h、8h、16h的卵母细胞,用免疫荧光法标记微管,Hochest33258标记染色体,FITC-LCA标记皮质颗粒,分别观察卵母细胞减数分裂进程、纺锤体形态和皮质颗粒分布,并进行比较。结果:与体外成熟卵母细胞相比,体内成熟的卵母细胞减数分裂进程高度一致。体内成熟的卵母细胞减数分裂中期的纺锤体呈两端逐渐变细的"纺锤状",且靠近卵膜(100%);而大多数体外成熟的卵母细胞的纺锤体两端宽大,呈"桶状",在第一次减数分裂中期和第二次减数分裂中期各有34.29%和43.24%靠近卵膜。体外成熟卵母细胞在第一次减数分裂中期和第二次减数分裂中期出现无皮质颗粒区的比例(分别为12.5%和75%)明显低于体内成熟卵(均为100%)。结论:体内和体外成熟的卵母细胞在减数分裂进程、纺锤体的形态和运动、皮质颗粒分布等方面均存在巨大的差异。体外成熟的卵母细胞在这些方面的变化可能与其发育能力下降有关。     

体外成熟对卵母细胞纺锤体及染色体形态的影响

以往的研究发现,未成熟卵母细胞体外成熟培养及体外受精-胚胎移植后着床率低的原因之一,可能是体外成熟的卵母细胞纺锤体和染色体排列发生了异常,引起胚胎发育潜能降低.本研究通过观察未成熟卵母细胞体外成熟培养后,卵母细胞纺锤体与染色体的变化,探讨体外成熟培养对卵母细胞纺锤体与染色体的影响.     

多囊卵巢综合征妇女体外成熟卵母细胞胚胎慢速冷冻复苏移植结局分析

目的评价未成熟卵母细胞体外成熟（IVM）后形成的卵裂期胚胎经慢速冷冻一解冻后的发育能力。方法将2006年1月至2010年12月北京大学第三医院因多囊卵巢综合征（PCOS）合并不孕症行卵裂期胚胎复苏移植的385例患者分为两组：复苏胚胎来源于体外成熟的卵母细胞组（IVM组,46例）和复苏胚胎来源于常规体内成熟的卵母细胞组（IVF组,339例）。采用慢冻速溶法解冻移植后比较两组患者的临床结局。结果IVM组复苏胚胎243枚,复苏后存活162枚,复苏率为66．67％;IVF组复苏胚胎1605枚,复苏后存活1082枚,复苏率为67．41％,两组比较,差异无统计学意义（P〉0．05）。IVM组患者的临床妊娠率和着床率分别为19．30％（11／57）和10．61％（14／132）,明显低于IvF组临床妊娠率（45．45％,175／385）和着床率（26．14％,240／918;P均〈O．05）。结论体外成熟卵母细胞发育形成的卵裂期胚胎慢速冷冻后临床结局欠佳,可能与冻融前胚胎自身的发育潜力有关。     

未经促排卵的未成熟卵母细胞行体外成熟治疗不孕症的临床研究

目的探讨未经任何药物刺激的未成熟卵母细胞行体外成熟（IVM）治疗不孕症的临床价值。方法40例不孕患者接受54个IVM周期,其中多囊卵巢综合征（PCOS）不孕患者26例,经其他辅助生育技术失败14例。在未采用任何药物刺激的前提下,于月经周期的第9—12天,在超声引导下经阴道对两侧卵巢内直径≤10mm的卵泡进行穿刺取卵。对取出卵母细胞于体外培养24～48h,待第一极体出现后,进行卵母细胞质内单精于注射（ICSI）,18h后观察受精情况,继续培养24—48h,直至胚胎移植,移植前行激光辅助胚胎孵化。结果54个IVM周期中,有7个周期取消,取消率为13％;共移植周期47个,共获得未成熟卵母细胞857个,平均每周期18．2个。体外培养48h后,卵母细胞成熟率为73．7％（632／857）,正常受精率为75．3％（476／632）,卵裂率为91．2％（434／476）。移植日子宫内膜厚度平均为8．9mm,平均移植胚胎4．3个（2—6个）;1例生化妊娠,19例临床妊娠,每取卵周期的临床妊娠率为35％（19／54）,每移植周期的临床妊娠率为40％（19／47）。26例PCOS不孕患者共移植周期34个,1例生化妊娠,15例临床妊娠,每移植周期的临床妊娠率为44％（15／34）。结论未经促排卵药物刺激的卵母细胞行IVM用于治疗各种原因的不孕症,尤其是PCOS不孕患者,是一种有效的治疗方法。     

Visualization of meiotic spindle and subsequent embryonic development in in vitro and in vivo matured human oocytes

Purpose The aim of this study is to investigate the relationship between spindle location and embryonic development of in vivo and in vitro matured human oocytes. Methods The spindles of 134 in vivo matured, 105 in vitro matured oocytes were examined by Polscope at the time of ICSI. Results The spindles were visualized in 83.6 and 77.1% of in vivo and in vitro matured oocytes respectively. The rate of fertilization of in vivo matured oocytes with spindles beneath or adjacent to the first polar body (angle of 0–5°) was significantly higher (93.3%) than all other groups. The proportions of various spindle positions did not differ statistically in in vivo and in vitro matured oocytes. Conclusions Meiotic spindle location with regard to the first polar body appears to influence fertilization rate. In this study, Spindle locations did not show significant difference between in vivo and in vitro matured oocytes, but seemed to influence the rate of fertilization.     

Meiotic spindle visualization in living human oocytes

A computer-assisted polarization microscopy system (polscope) has made it possible to analyse the meiotic spindle of oocytes subjected to intracytoplasmic sperm injection (ICSI) without affecting their viability. It has been shown that the presence of a detectable birefringent meiotic spindle inside the oocyte cytoplasm of human metaphase II (MII) prepared for ICSI is an indicator of oocyte quality, such as fertilization and developmental ability. Meiotic spindle imaging has also shown that this structure, when detectable, is not always aligned with the first polar body (PB1) in fresh MII oocytes. The relationship between the degree of meiotic spindle deviation from the PB1 location and ICSI outcomes is discussed in this paper. When the meiotic spindle of in-vitro matured oocytes is analysed, it is always found to be aligned with the PB1, suggesting that the misalignment observed in the oocytes matured in vivo results from the PB1 displacement during the manipulations for the cumulus and corona removal. Furthermore, polscope analysis of meiotic spindle changes in living MII oocytes subjected to freezing and thawing procedures has shown that the current techniques of oocyte cryopreservation cause meiotic spindle destruction. The polscope system may assist in the selection of fresh and thawed oocytes for ICSI.     

Meiotic spindle presence and oocyte morphology do not predict clinical ICSI outcomes: a study of 967 transferred embryos

With a view to correlating oocyte morphology and meiotic spindle presence to clinical intracytoplasmic sperm injection (ICSI) outcomes, 967 oocytes that led to 967 transferred embryos in 404 embryo transfers were studied. No relationship was found between oocyte morphology (ooplasm texture, perivitelline space largeness, perivitelline space granulation absence/presence and the first polar body shape) or meiotic spindle presence or absence and clinical pregnancy per transfer and implantation rates after ICSI. It was concluded that oocyte morphology and meiotic spindle presence or absence can only predict fertilization, cleavage rates and embryo quality, as previously described in the literature, but do not help in daily ICSI practice in the choice of the metaphase II oocyte that will lead to the embryo that starts clinical pregnancy.     

超排卵周期未成熟卵体外培养的研究

目的:研究来源于超排卵周期中的未成熟卵在拆除卵丘细胞后进行体外成熟培养(IVM)的成熟、受精及胚胎发育能力,探讨IVM技术的临床应用。方法:选取46名体外受精/卵胞浆内单精子显微注射-胚胎移植(IVF/ICSI-ET)患者为研究对象,比较MI和GV期不成熟卵的体外成熟情况,并比较体内成熟卵和体外成熟卵进行ICSI后的正常受精、异常受精、卵裂和优质胚胎形成情况。结果:体外培养中69.8%的MI期卵和77.2%的GV期卵均在24小时内达到成熟,其24小时和48小时的成熟率、总成熟率均无明显差异(P>0.05)。体外成熟卵与体内成熟卵相比较,正常受精率、异常受精率和卵裂率均无明显差异(P>0.05),优质胚胎形成率较低,差异有显著性(P<0.05)。结论:常规超排卵周期中的未成熟卵在拆除卵丘细胞后能够继续体外发育成熟,具有与体内成熟卵相似的ICSI受精、卵裂能力。虽然优质胚胎的形成率低于体内成熟卵,但增加了可移植胚胎和冷冻胚胎数量,提高了助孕成功率。     

Fertilization and pregnancy using metaphase I oocytes in an intracytoplasmic sperm injection program

Purpose: In this study we investigated whether metaphase I oocytes collected in an intracytoplasmic sperm injection program could successfully be matured and fertilized by injecting aged (>20-hr) spermatozoa. Materials and Methods: Metaphase I oocytes aspirated were preincubated for 20 hr to allow the oocytes to reach meiotic maturity. Only metaphase II oocytes were injected. The original sperm sample processed on the day of aspiration was used in the microinjection process. Results: One hundred eighty-three oocytes were collected, of which 42 (23%) were metaphase I oocytes. These were incubated for 20 hr and microinjected with the original sperm sample. Thirty-one (74%) of the metaphase I oocytes reached meiotic maturity (extruded polar body); 67.7% showed two pronuclei 18 hr after injection and 61.3% embryo development 40 hr postinjection. No difference in fertilization and embryo development rate was found in metaphase II oocytes injected 6 hr postaspiration versus 20 hr postaspiration. An ongoing pregnancy was also achieved using only embryos obtained from matured metaphase I oocytes. Conclusions: Metaphase I oocytes can be successfully maturedin vitro and injected using aged (>20-hr) sperm samples. Matured metaphase I oocytes, if successfully injected, produce embryos able to induce pregnancy.     

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。     

Meiotic spindle size is a strong indicator of human oocyte quality

Purpose

To investigate the relationship between the meiotic spindle size in human metaphase II oocytes and embryo developmental potential after intracytoplasmic sperm injection (ICSI).

Methods

Analyzed were 1302 oocytes with a visible meiotic spindle from 281 patients aged under 40 years undergoing ICSI cycles. The meiotic spindle was imaged by using PolScope before ICSI. The oocytes were classified into three groups, according to spindle size: group A (<90 μm²), group B (90‐120 μm²), and group C (>120 μm²).

Results

Overall, 389 (29.9%) oocytes were classified into group A, 662 (50.8%) into group B, and 251 (19.3%) into group C. The fertilization rate of the group B oocytes was significantly higher than for the A and C oocytes. The blastocyst formation rate in group B was significantly higher than in group A. In addition, the pregnancy rate in group B was significantly higher than in the other two groups.

Conclusion

The oocytes with a spindle size of 90‐120 μm² showed higher fertilization, blastocyst formation, and clinical pregnancy rates than those with larger or smaller spindles. The measurement of the meiotic spindle size thus has a positive predictive value for identifying human embryo developmental potential clinically.     

Abnormal chromosome behavior in human oocytes which remained unfertilized during human in vitro fertilization

Chromosomal abnormalities and abnormal embryonic development have previously been observed after human in vitro fertilization (IVF). Chromosomal abnormalities may arise not only after fertilization but even earlier during meiotic maturation of human oocytes in culture. Since chromosomal analysis is simple in oocytes during meiotic maturation, the chromosomal status was analyzed in oocytes which remained unfertilized in a human in vitro fertilization program. In 50 fertilization attempts the chromosomes of 62 unfertilized oocytes could be analyzed; 45 of them were in the process of meiotic maturation. In three oocytes two small polar bodies were observed 16–18 hr after insemination in the absence of fertilization. In one oocyte abnormal chromosome behavior was found during the first meiotic division, and in four oocytes during metaphase of the second meiotic division. These data suggest that chromosomal analysis of unfertilized oocytes in human IVF may improve the understanding of human oocyte maturation and fertilization.     

Do quantitative birefringence characteristics of meiotic spindle and zona pellucida have an impact on implantation in single embryo transfer cycles?

Objective

The aim of this study was to determine whether quantitative PolScope characteristics of meiotic spindle and zona pellucida could be used as a non-invasive marker to predict implantation success in elective single embryo transfer cycles.

Methods

Quantitative birefringence parameters; including mean retardance, area, length and polar body deviation angle of meiotic spindle and mean retardance and width of inner zona pellucida belonging to 53 transfer oocytes from elective single embryo transfer cycles were retrospectively analyzed. The relevant PolScope features were compared between 20 conception and 33 non-conception cycles.

Results

Meiotic spindle mean retardance, area, length and inner zona pellucida mean retardance and width did not reveal a statistically significant difference between transfer oocytes from conception and non-conception cycles. Deviation angle of the polar bodies was also comparable between the groups. Spindle and inner zona PolScope characteristics of transfer oocytes were not correlated with the maternal age.

Conclusion

Quantitative PolScope features of meiotic spindle and inner zona pellucida can not be used as a non-invasive marker to predict assisted reproductive technology success in elective single embryo transfer cycles.     

The relationship between meiotic spindle imaging and outcome of intracytoplasmic sperm injection: a retrospective study

Meiotic spindle analysis with a non-invasive technique, the PolScope, is used to protect the meiotic spindle from damage during microinjection. To evaluate the predictive feature of PolScope, we have designed a retrospective study to analyse the correlation between the meiotic spindle visualisation with regard to spindle location and outcomes of assisted reproductive technologies (ART), including patient age, previous cycles, the number of the collected oocytes, fertilisation rates (FR), pronuclear scoring (PNS) and embryo scoring of the days from two to five. All of the data belonging to 1496 oocytes from 190 patients were statistically analysed. We found that the oocytes having PolScope visualised spindle have higher FR, and also observed that when the spindle located at 0°–30° according to the first polar body, gave the highest FR. PNS gave higher scores in the spindle visualised group, but spindle angle did not affect PNS outcomes. Although a correlation was found between spindle visualisation and developed embryo qualities, particularly at day 2 and 3, spindle angles did not affect embryo quality. We conclude that PolScope microscopy has an efficiency to estimate FR, and cleavage stage embryo development.