慢速程序化冷冻与玻璃化冷冻对第3天分裂期胚胎发育潜能的影响

目的比较慢速程序化冷冻法（慢速法）和玻璃化冷冻法（玻璃化法）对第3天分裂期胚胎发育潜能的影响。方法选择因输卵管阻塞或男性少弱精因素行体外受精（IVF）或卵母细胞胞质内单精子注射（ICSI）治疗的患者,挑选在行IVF或ICSI后第3天剩余优质胚胎数目超过4个者80例进入本研究。随机将每例患者的2个胚胎用玻璃化法冷冻,另外2个用慢速法冷冻;冷冻复苏后随机抽取40例患者移植慢速法冷冻的胚胎,另40例患者移植玻璃化法冷冻的胚胎,比较2种冷冻方法对胚胎发育潜能的影响。结果冷冻复苏后待移植的胚胎共160个,其中慢速法冷冻80个,复苏后存活73个（91％,73／80）,移植后40例患者中15例（38％,15／40）获得临床妊娠,其中3例（20％,3／15）为双胎妊娠,余为单胎妊娠,胚胎着床率为25％（18／73）;玻璃化法冷冻胚胎80个,复苏后存活71个（89％,71／80）。移植后40例患者中19例（48％,19／40）获得临床妊娠,其中9例（47％,9／19）为双胎妊娠,余为单胎妊娠,胚胎着床率为39％（28／71）,与慢速法相比,玻璃化法的临床妊娠率和双胎妊娠率均有提高,但差异无统计学意义（P〉0．05）,而胚胎着床率则显著提高,差异有统计学意义（P〈0．05）。结论玻璃化法能够更好地保存胚胎复苏后的发育潜能,更适合第3天分裂期胚胎的冷冻保存。     

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。     

培养液中添加不同促性腺激素对卵母细胞体外成熟的影响

目的:比较卵母细胞体外成熟培养液中添加不同促性腺激素对未成熟卵母细胞体外成熟结局的影响。方法:将行卵母细胞体外成熟(IVM)的35例患者共42个新鲜取卵周期,随机分成A组:22个取卵周期将重组人促卵泡激素(果纳芬,rFSH)和重组人绒毛膜促性腺激素(艾泽,hCG)按1∶1的比例混合添加,终浓度为75 mIU/ml;B组:20个取卵周期添加终浓度为75 mIU/ml的尿源性促性腺激素(hMG),进行未成熟卵母细胞体外成熟培养。35例患者中新鲜取卵周期未移植或移植后未孕者行解冻胚胎移植。比较组间患者的卵母细胞成熟率、受精率、卵裂率、优质胚胎率、累计临床妊娠率及胚胎着床率。结果:取卵均于月经周期第12日或最大卵泡发育至10￣12 mm时进行,故所获卵均为未成熟卵。A组获卵181枚,经培养后成熟84枚,行卵胞浆内单精子注射(ICSI)84枚,受精60枚,卵裂55枚,优质胚胎20枚;新鲜胚胎移植9例,获1例临床妊娠,解冻胚胎移植5例,获1例临床妊娠,累计临床妊娠率为14.29%,胚胎着床率为7.14%。B组获卵176枚,经培养后成熟120枚,行ICSI 120枚,受精97枚,卵裂90枚,优质胚胎41枚,新鲜胚胎移植6例,获4例临床妊娠,解冻胚胎移植9例,获3例临床妊娠,累计临床妊娠率为46.67%,胚胎着床率为33.33%。结论:卵母细胞体外成熟培养液中添加尿源性促性腺激素可获得较添加重组人促卵泡激素和重组人绒毛膜促性腺激素高的卵母细胞成熟率、临床妊娠率及胚胎着床率。     

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。     

玻璃化冷冻保存技术对体外成熟卵母细胞发育潜能和转录组的影响

目的探索玻璃化冷冻保存技术对体外成熟(in vitro maturation,IVM)卵母细胞发育潜能和转录组的影响。方法选择2014年9月—2016年3月期间在北京大学第三医院生殖医学中心接受腹腔镜手术联合经阴道穿刺取卵行卵母细胞IVM治疗的不孕症患者为研究对象进行前瞻性队列研究,将研究对象分为IVM组(A组,n=13)和IVM-冻卵组(B组,n=24)。分析卵母细胞的早期胚胎发育状况和单细胞水平的转录组学情况。结果 IVM后玻璃化冷冻-解冻对存活卵母细胞的早期胚胎发育没有明显影响,受精率、卵裂率、优质胚胎率和可移植胚胎率组间差异均无统计学意义。但IVM后行玻璃化冷冻-解冻对转录组存在一定影响,与A组相比,B组共有1 913个基因呈现差异表达,其中570个基因表达量升高,主要富集到59个生物学过程;1 343个基因表达量降低,主要富集到140个生物学过程,而且一些差异基因参与了卵母细胞质量、受精和胚胎发育潜能的调控。结论 IVM后行玻璃化冷冻-解冻对早期胚胎发育没有明显影响,但对转录组具有一定影响,且发生表达量变化的基因与卵母细胞质量、受精和胚胎发育潜能有关,提示玻璃化冷冻-解冻有可能影响子代安全性。     

冻融胚胎移植周期中胚胎因素的分析研究

目的:分析冻融胚胎移植周期中胚胎卵裂球损伤程度与有丝分裂的恢复对于临床结局的影响。方法:171例患者共进行冻融胚胎移植206个周期,采用慢速冷冻法冻存d3胚胎,快速解冻法复苏后继续培养4h后移植,根据复苏后卵裂球的损伤程度分为完整组、混合组和损伤组,另根据复苏后4h胚胎进行分裂的情况分为分裂组,混合组和未分裂组,比较各组间的临床妊娠率和单胚着床率。结果:206个周期共复苏胚胎632枚,复苏存活率85.44%(540/632),移植胚胎508个,周期妊娠率36.41%(75/206),单胚着床率19.88%(101/508),完整组与损伤组间、分裂组与未分裂组间的临床妊娠率和单胚着床率都有显著性差异(P<0.05)。结论:冻融胚胎移植周期中胚胎卵裂球的损伤程度与有丝分裂的恢复是影响临床结局的重要因素。     

多囊卵巢综合征患者卵丘形态与未成熟卵母细胞发育潜能关系

目的 研究多囊卵巢综合征患者无刺激周期取出的不同形态未成熟卵母细胞的发育潜能。方法 43例PCOS不孕患者进行了47个未成熟卵母细胞体外成熟培养（IVM）周期。所有患者均未经促卵泡素刺激,予以HCG36h后取卵。根据取出的卵-冠-丘复合物形态将其分为3组：卵丘紧密组、卵丘松散组、无卵丘组。比较3组的体外成熟率、受精率和优质胚胎率。结果 47个IVM周期共收集未成熟卵母细胞874枚,体外成熟率61.19%,受精率71.07%,着床率13.13%。卵丘松散组的体外成熟率明显高于卵丘紧密组（72.26%vs49.54%,P〈0.05）,受精率、优质胚胎率三组间无差异。结论 PCOS患者无刺激周期取出的未成熟卵母细胞中,卵丘松散、扩张的卵母细胞具有更好的体外成熟潜力。     

影响冻融胚胎发育潜能及妊娠结局因素的探讨

目的:探讨胚胎冷冻复苏后,卵裂球存活状态对胚胎发育的影响以及体外授精(IVF)和卵胞质内单精子注射(ICSI)2种授精方式对冷冻胚胎复苏后体外发育能力和妊娠结局的影响。方法:回顾性分析142例患者,150个复苏周期,共769个冷冻胚胎复苏后培养至囊胚期进行移植的结果。结果:共复苏胚胎769个,存活胚胎702个,复苏率91.3%。220个胚胎(31%)到达囊胚阶段。在卵裂球全部存活胚胎中,囊胚形成率35%,部分存活胚胎中囊胚形成率24%,两者有统计学差异(P<0.01)。在卵裂球全部存活胚胎中,来源于IVF的胚胎囊胚形成率为40%,来源于ICSI的为26%,两者有统计学差异(P<0.01);部分卵裂球存活胚胎中来源于IVF的胚胎囊胚形成率为26%,来源于ICSI的为19%,差异无统计学意义(P>0.05)。在全部220个囊胚中,IVF组的优质囊胚率为38.6%,ICSI组的优质囊胚率为21.0%,差异有统计学意义(P<0.05)。临床妊娠率IVF组和ICSI组分别为61.05%与61.11%;胚胎种植率分别为37.50%与36.67%,活产率分别为81.03%与78.79%,差异均无统计学意义(P>0.05)。结论:胚胎冷冻复苏后卵裂球的损伤削弱了囊胚形成能力,影响卵裂期胚胎进一步发育,这与ICSI和冷冻胚胎复苏后发育潜能的降低有关,但其对临床结果和妊娠结局的影响不大。     

PCOS患者未成熟卵体外成熟培养后妊娠结局分析

目的:探讨PCOS患者未成熟卵母细胞体外成熟(in vitro maturation,IVM)治疗的有效性和安全性。方法:分析比较PCOS合并不孕症自愿要求行IVM或IVF治疗的患者临床结局。结果:IVF组的平均优质胚胎数明显高于IVM组(P<0.05),两者的临床妊娠率(39.22%vs 40.32%)和着床率(24.37%vs 23.89%)无差异(P>0.05)。IVM组的妊娠丢失率为38.0%,高于IVF患者(21.1%,P<0.05),出生婴儿(单胎)的平均体质量组间无统计学差异(P>0.05)。结论:对于PCOS患者进行IVM治疗可以获得与IVF周期相类似的临床妊娠率和着床率;但是IVM治疗后流产率明显增加。     

人未成熟卵母细胞体外成熟培养影响因素的研究

未成熟卵母细胞体外成熟培养(IVM)技术的研究已成为世界生殖医学研究的热点之一,目前,IVM技术仍面临着卵子成熟率不高,成熟后体外受精率低及妊娠率较低等困难,所以IVM过程中各种影响因素的调控对卵母细胞的成熟及体外受精(IVF)后胚胎的发育甚为重要。     

Successful birth following transfer of frozen–thawed embryos produced from in-vitro matured oocytes

It is well established that ovarian hyperstimulation syndrome (OHSS) is more frequent in patients with polycystic ovarian syndrome. In-vitro maturation (IVM) of immature oocytes presents a potential alternative for the fertility treatment and prevention of OHSS for these patients. This report describes the case of a 26-year old woman with a successful pregnancy and delivery following the transfer of frozen–thawed embryos derived from in-vitro matured oocytes. She had three failed cycles of ovarian stimulation (using low-dose step-up gonadotrophin protocol) with or without intrauterine insemination cycles, an ovulation-induction cycle with luteal long protocol, two fresh IVM cycle and one frozen–thawed IVM cycle. During the IVF cycle, she developed moderate OHSS and required hospitalization for 3 weeks. Following four unsuccessful IVF or IVM cycles, 15 months after the last cryopreservation, six fertilized oocytes were thawed for a scheduled embryo transfer. Following thawing, four fertilized oocytes survived and cleaved. Four frozen–thawed embryos were transferred. Six weeks after embryo transfer an ongoing intrauterine single pregnancy with fetal heartbeat was confirmed by transvaginal ultrasound. An uneventful pregnancy and delivery via Caesarean section at 39 weeks resulted in the birth of a normal healthy infant.     

IVF versus ICSI for the fertilization of in-vitro matured human oocytes

Traditional dogma suggests that intracytoplasmic sperm injection (ICSI) should be performed to ensure successful oocyte fertilization in an in-vitro maturation (IVM) cycle. This study postulated that there would be no difference in the fertilization rate when ICSI was compared with IVF. This hypothesis was tested in a randomized trial of IVF versus ICSI in IVM. A total of 150 immature oocytes were collected in eight cycles of IVM for patients diagnosed with polycystic ovarian syndrome (PCOS). Patients were primed with minimal FSH before transvaginal oocyte aspiration. Sibling oocytes were inseminated by 50% IVF and 50% ICSI. There was no significant difference in fertilization, useable or total blastocyst development between the two insemination technique groups. Clinical pregnancy results for combined fresh and cryopreserved transfers were identical between the two insemination techniques with a total of two fresh and five cryopreserved IVF-inseminated embryos resulting in three clinical pregnancies (42.9%) and five fresh and two cryopreserved ICSI-derived embryos resulting in three clinical pregnancies (42.9%). This research has shown IVF to be a legitimate fertilization technique for IVM oocytes in PCOS patients and provides a greater awareness of the use of a fertilization method previously not utilized with IVM.In-vitro maturation (IVM) is an alternative treatment method to traditional IVF. Due to the minimal use of stimulating hormones in this treatment, IVM has a lower risk of ovarian hyperstimulation syndrome, it can be used for fertility preservation in cancer patients and it is more cost conservative. Early research into the effects of IVM showed a hardening effect on the membrane surrounding the egg (the zona pellucida). It was initially believed that, to overcome this hardening in order to allow the egg to be fertilized, spermatozoa would need to be injected into the egg using intracytoplasmic sperm injection. Due to recent advances in hormonal stimulation protocols (FSH priming) and culture conditions, we postulated that, for patients suffering from polycystic ovarian syndrome (PCOS), fertilization, embryo development and clinical pregnancy would not be superior in the injected oocytes compared with those inseminated by IVF. We found that by using the two insemination techniques on sibling oocytes from eight PCOS patients, there was no significant difference in fertilization, useable or total blastocyst development (day 5 or 6 embryos) and that clinical pregnancy results were identical. This research provides a greater awareness of a fertilization technique which is not normally utilized for IVM treatment, providing a less invasive, more cost-effective approach for the patient.     

冻融胚胎移植术用于补救卵胞浆内单精子注射治疗周期的临床价值

目的：探讨冻融胚胎移植在补救卵胞浆内单精子注射（ICSI）治疗周期中的临床价值。方法：回顾分析常规体外受精完全失败的306个周期,根据补救ICSI的时间不同分为6～8h补救ICSI组（A组）和18～20h补救ICSI组（B组）,其中行新鲜胚胎移植且有胚胎冷冻的分别记为AI组（n=108）和BI组（n=51）,随后行冻融胚胎移植的分别记为AⅡ组（n：74）和BⅡ组（n=46）,观察各组的妊娠结局。结果：A组正常受精率、卵裂率、优质胚胎率和临床妊娠率均明显高于B组（P〈0．05）;BⅡ组临床妊娠率和胚胎种植率明显高于BI组（32．61％VS11．76％,15．08％VS5．04％,P〈0．05）;AII组临床妊娠率和胚胎种植率较AI组高（44．59％VS34．26％,21．86％VS19．40％）,但无明显的统计学差异（P〉0．05）。结论：合理的补救时间是影响补救ICSI结局的关键因素,进一步的冻融胚胎移植可以改善补救ICSI治疗周期的妊娠结局。     

A comparison of in vitro maturation and in vitro fertilization for women with polycystic ovaries

OBJECTIVE: To establish the relative success of treatment by unstimulated in vitro maturation (IVM) of oocytes or stimulated in vitro fertilization (IVF) in women with polycystic ovaries undergoing assisted conception treatment. METHODS: The case-control study included 107 IVM and 107 IVF cycles matched for age and cause of infertility. In vitro maturation patients underwent transvaginal recovery of immature oocytes during an unstimulated cycle, in vitro oocyte maturation, and fertilization. Those in the IVF group underwent ovarian stimulation after pituitary suppression. Embryos were transferred in the same cycle in both groups. Main outcome measures included numbers of mature oocytes and embryos produced, and rates of implantation, pregnancy, live birth, and complications. RESULTS: In the IVM group after in vitro culture, 7.8 mature oocytes and 6.1 embryos were obtained per retrieval. With IVF, 12.0 mature oocytes (P <.01) and 9.3 embryos (P <.01) were obtained. The IVM pregnancy and live birth rates per retrieval were 26.2% and 15.9% compared with 38.3% and 26.2% for IVF (nonsignificant). The implantation rate of IVF-derived embryos was higher (17.1% versus 9.5%) than that for IVM (P <.01). There were 12 cases (11.2%) of moderate or severe ovarian hyperstimulation syndrome in IVF patients, compared with none in the IVM group (P <.01). CONCLUSION: Our results suggest that for women with polycystic ovaries who require assisted conception, IVM is a promising alternative to conventional IVF treatment.     

Pronuclear stage cryopreservation after intracytoplasmic sperm injection and conventional IVF: implications for timing of the freeze

Objective: To compare clinical outcomes of frozen embryo transfers using cryopreserved pronuclear stage oocytes that had undergone either intracytoplasmic sperm injection (ICSI) or conventional IVF.

Design: Observational.

Setting: A tertiary referral reproductive medicine unit.

Patient(s): Couples undergoing either ICSI or conventional IVF from January 1, 1995 to December 31, 1997.

Intervention(s): Patients underwent a standard controlled ovarian hyperstimulation protocol and transvaginal ultrasound-guided oocyte retrieval. All normally fertilized (2PN) oocytes exceeding a specified embryo number designated for fresh transfer were immediately cryopreserved at the pronuclear stage. Our cryopreservation method included timing of the freeze according to pronuclear morphology. Subsequent frozen embryo thaw-transfer cycles were usually performed by thawing only the intended number of embryos for transfer.

Main Outcome Measure(s): Thaw survival rate, implantation rate, clinical pregnancy rate, delivery rate.

Result(s): Ninety-six thaw-transfer cycles (n = 72) and 93 thaw-transfer cycles (n = 67) were undertaken in patients who had previously undergone conventional IVF or ICSI, respectively. Embryo thaw survival rates (IVF, 90.4%; ICSI, 91.1%) were similar. Clinical pregnancy (IVF, 40.6%; ICSI, 44.1%) and delivery (IVF, 36.4%; ICSI, 39.8%) rates per transfer, as well as implantation (IVF, 19.1%; ICSI, 19.9%) rates, were also similar. There were only four clinical pregnancy losses in both groups.

Conclusion(s): Embryo thaw survival is similar for cryopreserved pronuclear stage oocytes derived from ICSI and conventional IVF. Clinical pregnancy, implantation and delivery rates were also similar for the two groups. In addition, there was no increase in the rate of pregnancy loss in ICSI patients after frozen embryo transfers.     

Fertilization, embryo development, and clinical outcome of immature oocytes obtained from natural cycle in vitro fertilization

Purpose

To analyze the fertilization, embryo development, and clinical outcome of immature oocytes obtained from natural cycle IVF in women with regular cycles.

Methods

Natural cycle IVF was performed in 28 patients who had normal ovaries, > 6 antral follicle counts and were less than 40 years old (n = 28 cycles). An hCG trigger of 10,000 IU was administered 36 h before oocyte collection when the diameter of the dominant follicle (DF) was over 12 mm. Oocytes were retrieved from DF as well as from the cohort of smaller follicles. Embryological aspects of the mature and immature oocytes retrieved from these cycles as well as the implantation and clinical pregnancy rates depending on the origin of the embryos transferred were evaluated.

Result(s)

Overall clinical pregnancy and implantation rates were 20.8 % and 6.7 %, respectively. There were no differences in in vitro maturation (IVM), fertilization and embryo development between immature oocytes retrieved with and without in vivo matured oocytes. However, the clinical and implantation rates in cycles with embryos produced from in vivo matured oocytes transferred were better than the cycles where only IVM embryos were transferred (30.8 %, 9.1 % vs. 9.1 %, 3.2 %).

Conclusion(s)

Although our results show that immature oocytes from natural cycle IVF can fertilize normally and can be used to increase the number of embryos available for transfer, the embryos derived from the immature oocytes in natural cycles IVF have a poorer reproductive potential.     

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。     

In-vitro maturation versus IVF with GnRH antagonist for women with polycystic ovary syndrome: treatment outcome and rates of ovarian hyperstimulation syndrome

In-vitro maturation (IVM) treatment has gained popularity for decreasing the incidence of ovarian hyperstimulation syndrome (OHSS) by eliminating or minimizing the use of gonadotrophins in women with polycystic ovary syndrome (PCOS). Studies have shown that IVF with GnRH-antagonist protocol is associated with a lower incidence of OHSS. Data comparing the relative success of these two treatments is, however, lacking. Treatment outcome and rates of OHSS were compared in patients with PCOS who underwent assisted conception with either IVM or IVF with GnRH-antagonist protocol between 2006 and 2011. The number of oocytes retrieved was higher in the IVM group, whereas the number of mature oocytes, fertilization rate and number of embryos cleaved were comparable. The implantation rate was higher in the IVF group. The clinical pregnancy rates per embryo transfer were not statistically different (IVF: 45.8% versus IVM: 32.4%). The live-birth rate was higher in the IVF group (IVF: 40.7% versus IVM: 23.5%; P = 0.04). Five women developed moderate or severe OHSS in the IVF group, whereas none did in the IVM group. Both IVM and IVF with GnRH-antagonist protocol seem to be effective treatment regimens in women with PCOS, although IVM is associated with a lower risk of OHSS.