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。     

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。     

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。     

多囊卵巢综合征患者中未成熟卵母细胞体外培养成熟后体外受精的初步研究

目的:探讨未成熟卵母细胞体外培养成熟后体外受精、胚胎培养技术在多囊卵巢综合征患者中的初步应用及其影响因素。方法:用小剂量促性腺激素促使卵泡生长后,根据优势卵泡直径分为2组,直径6～8mm者为组1,10—12mm者为组2。采集未成熟卵母细胞,经体外培养成熟后,再进行体外授精和胚胎培养。结果:组1的GV期卵的成熟率和受精率低于组2者,但两组的MI期卵的成熟率、受精率没有明显差别。两组卵裂率没有明显差别,但形成胚胎的质量组2优于组1。总计成熟率69.3％,成熟卵中正常受精率61.5％。结论:可以用小荆量Gn促使卵泡生长后,采集未成熟卵,用体外成熟-体外授精(IVM/IVF)的方法使多囊卵巢患者在避免OHSS的情况下获得质量良好的胚胎。     

人成熟卵母细胞玻璃化冻存时长对体外受精-胚胎移植结局的影响

目的:探讨玻璃化冷冻人成熟卵母细胞的时长是否会影响解冻后卵细胞的复苏率、受精率和临床妊娠结局。方法:回顾性分析2013年5月至2018年6月就诊于北京大学第三医院生殖医学中心行成熟卵母细胞玻璃化冷冻,随后进行解冻受精形成胚胎培养移植的110例患者114个冷冻卵母细胞周期的临床资料。根据卵细胞冻存时间分为3组,A组(冻存时间≤3个月)42例,B组(3个月<冻存时间≤12个月)42例,C组(冻存时间>12个月)30例,比较3组的卵细胞解冻复苏率、受精率、可利用胚胎率、优质胚胎率、临床妊娠率和活产率等指标。结果:114个冷冻卵母细胞周期中最短冻存时间4天,最长32个月,共行胚胎移植周期114个,其中新鲜胚胎移植周期77个,解冻胚胎移植周期37个。共解冻成熟卵母细胞1578枚,复苏率为77.69%(1226/1578)、受精率为69.35%(774/1116)、卵裂率为93.41%(723/774)、可移植胚胎率为42.19%(305/723)、优质胚胎率为33.06%(239/723)、每冻卵活产率为2.47%(39/1578)。3组年龄、不孕年限、基础卵泡刺激素(FSH)水平、窦卵泡数、解冻卵细胞数和男方精液情况等基本信息差异无统计学意义(P>0.05),而A组体质量指数(BMI)明显大于B组(P=0.038);3组的解冻复苏率、受精率、卵裂率、可利用胚胎率、优质胚胎率、临床妊娠率、早期流产率和活产率的差异均无统计学意义(P>0.05)。随访25例活产新生儿均未发现先天畸形。结论:人成熟卵母细胞的发育潜能不会随着冷冻时间的延长而减低,玻璃化冷冻卵母细胞可获得比较满意的复苏效果。     

多囊卵巢患者行未成熟卵母细胞体外成熟、受精和胚胎发育观察

目的:探索在不用或小剂量使用促性腺激素条件下对多囊卵巢患者的未成熟卵母细胞体外培养至成熟、受精、卵裂和妊娠的可行性。方法:阴道B超引导下穿刺取卵后将卵子分别培养于3种不同培养基:基本培养基(f-HTF+75 mIU/mL rFSH+500 mIU/mL rhCG)+10%SPS(SPS组), 基本培养基+20% hFF(FF组)和基本培养基+20% hFF+2 ng/mL rEGF(FF+EGF组),共培养25-48 h,然后行体外受精和胚胎培养。结果:体外培养卵丘-卵母细胞复合体102个,成熟率65.69%,受精率83.58%,2PN率71.43%,卵裂率51.72%,优质胚胎率40.00%。FF组较SPS组有更高的成熟、受精、卵裂和优质胚胎率,再添加EGF后卵子的成熟、受精和优质胚胎率增加。刺激组和未刺激组在卵子成熟、受精、卵裂和优质胚胎率方面均无显著差异。PCO患者卵子的成熟、受精、卵裂和优质胚胎率均高于PCOS患者,ICSI组优质胚胎率增加,但均无统计学差异。随培养时间延长,卵子的成熟、受精、卵裂和优质胚胎率均升高,其中正常受精率和卵裂率有显著差异(P<0.05)。结论:PCO和PCOS患者行IVM可行,二者无明显差异。培养液中添加激素、FF、EGF以及延长培养时间至48 h,可促进卵子成熟。但有关临床用药、培养条件仍有待改善,进一步提高卵子和胚胎质量以获得妊娠。     

人类未成熟卵母细胞体外培养成熟的研究进展

本文综述了人类未成熟卵母细胞体外培养成熟的现状和未来的发展方向。未成熟卵母细胞体外培养成熟包括二种方法 :卵泡体外培养 ( IVC)和卵母细胞体外成熟 ( IVM)。从原始卵泡发育到成熟卵母细胞 ,受精形成胚胎 ,胚胎移植后妊娠 ,仅在小鼠成功 ,有关人类此方面的研究正在进行中。人类卵母细胞体外成熟已成功应用于治疗不孕症 ,但成功率低。卵母细胞体外成熟受自身大小以及所处月经周期的影响。培养系统添加卵泡液、表皮生长因子、激活素 /抑制素、以及共培养系统有助于卵母细胞体外成熟。根据卵母细胞所处发育阶段选择不同的培养系统 ,探索卵母细胞的生长和成熟是未来的重要研究方向     

人未成熟卵母细胞体外培养成熟、受精及胚胎移植

目的 应用卵母细胞体外成熟(IVM)技术帮助卵泡成熟障碍的不孕症患者获得妊娠及分娩。方法 接受未成熟卵IVM技术治疗者30例35个周期,其中多囊卵巢综合征14例,有卵巢过度刺激综合征病史6例,体外受精和胚胎移植(IVF-ET)周期中卵巢反应不良患者10例。设计卵巢刺激方案,采用含人成熟卵泡液的IVM培养液,建立未成熟卵母细胞的体外培养方法。结果 35个周期共取得未成熟卵母细胞203个,平均每个周期5．8个。培养后有156个卵母细胞排出第一极体,IVM率76．8％(156／203);在卵胞浆单精子显微注射(ICSI)12～18h后观察原核,正常受精率为76．9％(120／156);共有移植周期33个,获8例临床妊娠,妊娠率24％(8／33);有5例共7个婴儿出生。结论 IVM对一些卵泡发育和成熟障碍,特别是顽固性多囊卵巢综合征患者,是一种有效的辅助生育措施。人成熟卵泡液含有理想的自然促卵母细胞成熟的成分。     

人未成熟卵母细胞培养体系研究进展

未成熟卵母细胞体外培养成熟(in vitro maturation,IVM)是指将GV期或MI期的卵母细胞在体外培养发育到第二次减数分裂中期(MII期),能够正常发育、受精和着床。人未成熟卵母细胞体外培养成熟(in vitro maturation,IVM)技术已有几十年历史,作为治疗不孕症的新手段,为赠卵以及女性生育力保存提供了新途径。近年随着细胞分子生物科学的迅猛发展,IVM取得了很大进展,向IVM培养系统添加促性腺激素、甾体激素、抗氧化剂、减数分裂抑制剂、生长因子、抑制素/激活素等有助于卵母细胞体外成熟。但仍面临卵母细胞成熟率不高、成熟后体外受精率低、妊娠率低的问题,完善IVM培养系统及相关辅助治疗是IVM的关键,如何获得稳定、有较高成功率的培养体系是亟需解决的问题。现对近几年一些较新的研究发现进行总结,主要从基础培养液、添加成分、培养条件的选择等方面予以综述。     

不同浓度促性腺激素对促排卵周期中人类未成熟卵子体外成熟的影响

目的:研究体外成熟培养基中添加不同浓度促性腺激素(卵泡刺激激素和黄体生成激素)对卵子体外成熟培养以及其受精和后续的早期胚胎发育的影响。方法:收集生殖助孕治疗中废弃的人类未成熟卵子,将生发泡(GV)期未成熟卵子随机分为3组,添加终浓度分别为75 U/L的低浓度促性腺激素(低浓度组)和750 U/L的高浓度促性腺激素(高浓度组),以及不添加促性腺激素(空白对照组)进行体外成熟培养20小时,每组入组的卵子数分别为80、83和82枚。将成熟卵子进行卵胞质内单精子显微注射,研究卵子的体外成熟率、受精率以及卵裂率、可用胚胎率和优质胚胎率。结果:低浓度组的卵子体外成熟率最高(80.00%),空白对照组最低(76.83%),但差异无统计学意义(P0.05);低浓度组的受精率最高(67.19%),空白对照组最低(65.08%),但差异无统计学意义(P0.05);高浓度组的卵裂率最高(70.45%),空白对照组最低(63.41%),但差异无统计学意义(P0.05);低浓度组的可用胚胎率最高(28.57%),空白对照组最低(19.23%),但差异无统计学意义(P0.05);高浓度组的优质胚胎率最高(16.13%),空白对照组最低(3.85%),但差异无统计学意义(P0.05)。结论:不同浓度促性腺激素对促排卵周期中人类未成熟卵子的体外成熟和受精,及其后续的早期胚胎发育没有发现显著的影响。     

体外加入hCG不能改善人卵子体外成熟及发育潜能

目的:研究体外培养过程中hCG对人未成熟卵子体外成熟和发育潜能的影响。方法:62例PCOS不孕患者进行了89个未成熟卵子体外成熟培养(IVM)周期,根据体外成熟培养液中有无hCG,将其分为A组(29个周期)采用常规IVM培养液培养;B组(30个周期)先在去除hCG的常规IVM培养液中培养10h,然后改在常规IVM培养液中培养;C组(30个周期)在去除hCG的常规IVM培养液中培养。所有卵子体外培养24-48h,成熟后卵子分批行单精子注射授精(ICSI),培养2-3d后进行胚胎移植。结果:A、B、C组32h、48h卵子体外成熟率分别为46.02%,69.25%;43.72%,64.51%;51.87%,67.51%;组间无显著差异。受精率、卵裂率、临床妊娠率及种植率各组间也均无显著差异(P>0.05)。结论:对于PCOS患者,有无hCG对卵子体外成熟、胚胎发育及临床结局均没有显著影响。     

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.     

The live birth in a woman with resistant ovary syndrome after in vitro oocyte maturation and preimplantation genetic testing for aneuploidy

We report the pregnancy and live birth achieved after in vitro maturation (IVM) of oocytes and PGT-A in a 23-year-old patient suffering from ovarian gonadotropin resistance. A woman with resistant ovary syndrome (ROS) had secondary amenorrhea, high FSH levels (25.34 mIU/mL) and LH (29.6 mIU/mL), low estradiol levels (15.2 pg/mL), and high serum AMH levels (38.0 ng/mL), associated with an increased antral follicle count (AFC) of 45. Without gonadotropin priming and HCG trigger, ultrasound-guided transvaginal oocyte retrieval was performed. Aspiration of antral-stage follicles allowed the retrieval of 15 immature oocytes. After oocyte collection, immature oocytes were cultured in the IVM medium. Following IVM, six of them reached metaphase II stage. Resultant matured oocytes were fertilized by intracytoplasmic sperm injection (ICSI). Embryos obtained were cultured to the blastocyst stage. On day 5, three embryos reached blastocyst stage. Trophectoderm biopsy and PGT-A were performed on two better quality embryos on day 5 after fertilization. Two biopsied embryos were reported to be euploid. PGT-A was performed utilizing next-generation sequencing (NGS\MPS). One embryo was transferred in an artificial thaw cycle and resulted in a viable intrauterine pregnancy and live birth. Our experience indicates that there is no requirement for gonadotropin stimulation and use of b-hCG trigger prior to IVM in patients with ROS. The results suggest that oocytes obtained with IVM in patients with ROS are capable of meiotic and mitotic division, fertilization, and generation of euploid embryos. IVM appears to be a valuable approach in patients with ROS, allowing them to have genetically connected offspring.

     

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。     

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。     

Absence of corpus luteum rescue by chorionic gonadotropin in women immunized with a contraceptive vaccine

OBJECTIVE: To investigate possible differences between using recombinant FSH (rFSH) and hMG for ovarian stimulation in IVF/intracytoplasmic sperm injection (ICSI) cycles. DESIGN: Parallel group design. Prospective, randomized clinical study. SETTING: A tertiary care infertility clinic. PATIENT(S): A total of 578 patients of our IVF/ICSI routine were recruited. INTERVENTION(S): Treatment with hMG was used for 282 patients (282 cycles), whereas 296 patients (296 cycles) were treated with rFSH. The number of cycles leading to an embryo transfer were 248 and 259, respectively. MAIN OUTCOME MEASURES: Primary: clinical pregnancy rate. Secondary: treatment days, total dose of gonadotropin administered, number of oocytes retrieved, number of mature oocytes, and embryo quality. RESULT(S):Of the cycles with embryo transfer, the pregnancy rates were 30.1% and 32.3% in the rFSH and the hMG groups, respectively. This difference is not statistically significant (P=0.798). Treatment with rFSH resulted in a significantly higher number of recovered oocytes compared with the hMG group but was also associated with a higher number of ampoules needed to reach the criterion for hCG administration. No significant differences were found with regard to the number of mature oocytes, the number of treatment days, and the embryo quality. CONCLUSION(S): In terms of the clinical pregnancy rate, no significant differences between the two stimulation regimens can be stated.     

反义寡核苷酸对激素敏感型单纯性肾病患儿外周血单个核细胞病毒基因表达的抑制作用

目的通过设计反义寡核苷酸(ASONs)抑制激素敏感型单纯性肾病(SRSNS)呼吸道病毒基因的表达，提供激素敏感型单纯性肾病呼吸道病毒感染的证据。 方法将ASONs转染至外周血单个核细胞(PBMC)后，通过RT PCR的方法检测呼吸道病毒基因的表达。 结果SRSNS活动期PBMC中呼吸道病毒基因的表达能被其相应的ASONs所抑制。呼吸道合胞病毒基因表达受抑制率为93.9%(14/15)，流感病毒受抑制率为87.5%(7/8)，总抑制率为91.3%(21/23)。 结论呼吸道病毒确实存在于SRSNS活动期病人体内，呼吸道病毒感染作为主要触发因素，可能为SRSNS的重要发病机制。     

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.