人未成熟卵玻璃化冷冻初步实验探讨

目的:对人未成熟卵母细胞玻璃化冷存保存技术进行初步实验探讨。方法:采用OPS玻璃化冷冻方法,对未成熟的卵子进行冷冻,解冻后体外成熟培养,ICSI受精,观察胚胎培养情况,并对某些影响因素进行探讨。结果:冷冻了GV期卵62个,M I期卵40个。两期卵细胞复苏后存活率、成熟率、受精率和卵裂率分别为:80.65%、77.50%,52.00%、41.94%,57.69%、61.54%,60.00%、62.50%,两组间比较无统计学差异(P>0.05);采用HTF或G-Mops两种缓冲液配制玻璃化冷冻液冷冻解冻未成熟卵效果无统计学差异(P>0.05);未成熟卵解冻后置于含性激素的TCM199与单纯IVF30中成熟培养效果无统计学差异(P>0.05)。结论:应用OPS玻璃化冷冻方法能够有效冻存未成熟卵母细胞。     

玻璃化冷冻法在未成熟卵子冷冻技术中的应用

目的:探讨玻璃化冷冻法在未成熟卵子冷冻方面的应用价值。方法:选取GV期卵母细胞744枚,随机分为5组,A组(128枚),GV期卵子直接进行未成熟卵母细胞体外培养(IVM);B组(220枚),GV期卵子直接行玻璃化冷冻;C组(155枚),GV期卵子直接行IVM,待培养至MⅡ期,再行玻璃化冷冻;D组(112枚),GV期卵子去除冠-丘细胞,行玻璃化冷冻;E组(129枚),GV期卵子保留除冠-丘细胞行玻璃化冷冻。比较各组卵母细胞的存活率、成熟率、受精率、卵裂率及囊胚形成率。结果:与A组相比,B组和C组的成熟率、受精率、卵裂率和囊胚形成率均降低(P<0.05);B组与C组间存活率、成熟率、受精率、卵裂率和囊胚形成率无差异(P>0.05)。E组的成熟卵子率较D组提高(P<0.05),但两组在解冻卵子存活率、受精率、2细胞数和>2细胞数方面无差异(P>0.05)。结论:未成熟卵子的玻璃化冷冻保留冠-丘细胞可显著提高冻融效果。     

不同方法冻融小鼠未成熟与成熟卵母细胞的研究

目的:探讨不同冷冻方法对小鼠生殖泡(GV)期与成熟期(MⅡ)卵母细胞体外成熟、体外受精及胚胎发育能力的影响。方法:收集ICR小鼠GV期与MⅡ期卵母细胞,分别行程序化冷冻(慢速冷冻-快速解冻)与玻璃化冷冻。解冻后比较各组成熟率、受精率、卵裂率及8细胞胚胎形成率。结果:采用玻璃化冷冻法冷冻GV期卵母细胞(Ⅰ组),解冻后存活率达84.2%,显著高于MⅡ期卵母细胞(Ⅱ组)60.5%及程序化冷冻组(Ⅲ组)56.9%、(Ⅳ组)55.7%(均P<0.01)。各组成熟率、受精率、卵裂率及8细胞胚胎形成率比较,差异无统计学意义(P>0.05)。结论:在进行卵母细胞冷冻保存时,宜选择玻璃化冷冻法,且GV期卵母细胞冷冻效果好于MⅡ期卵母细胞。     

人卵母细胞体外成熟培养研究进展

卵母细胞体外成熟(IVM)是指卵母细胞在体外从生殖泡(GV)期发育到第二次减数分裂中期(MⅡ).IVM后成熟的卵母细胞受精率较高,其卵裂率与常规的体外受精(IVF)、胞浆内单精子注射(ICSI)相近,可代替IVF前常规超促排卵过程,出生的新生儿未见异常.     

细胞松弛素B在人类体外成熟卵子冷冻中的作用

目的:探讨细胞松弛素B在人类体外成熟卵子玻璃化冷冻中的作用。方法:收集常规胞浆内单精子显微注射-胚胎移植(ICSI-ET)周期中未成熟卵母细胞234枚(包括生发泡期即GV期和第1次减数分裂中期即MⅠ期),在体外培养24～48 h,181枚卵母细胞成熟(排出第2极体),随机分成3组并行玻璃化冷冻。A组(43枚)用细胞松弛素B(CB)预处理30 min后行玻璃化冷冻;B组(66枚)用CB预处理20 min后行玻璃化冷冻,C组(72枚)未用CB预处理直接行玻璃化冷冻。D组为对照组30枚体内成熟卵子未用CB处理直接玻璃化冷冻。各组卵子冷冻3周后解冻,复苏的卵子行ICSI辅助授精,观察各组成活率、受精率、卵裂率及囊胚形成率。结果:冻融后的体外成熟卵子,其成活率、受精率、卵裂率及囊胚形成率均显著低于体内成熟卵子(P<0.05)。A组的成活率显著低于B、C两组(P<0.05),A、B、C 3组间的受精率、卵裂率差异无统计学意义(P>0.05),A、B两组未获囊胚,C组仅有1枚囊胚。结论:冻融体外成熟卵子,成活率、受精率、卵裂率均下降,囊胚形成率低,胚胎后期发育潜能显著降低。CB预处理未能提高卵子冷冻的成活率、受精率、卵裂率及胚胎的发育潜能。     

人卵母细胞体外成熟培养研究进展

卵母细胞体外成熟（IVM）是指卵母细胞在体外从生殖泡（GV）期发育到第二次减数分裂中期（MⅡ）。IVM后成熟的卵母细胞受精率较高，其卵裂率与常规的体外受精（IVF）、胞浆内单精子注射（ICSI）相近，可代替IVF前常规超促排卵过程。出生的新生儿未见异常。     

卵母细胞体外成熟调节的研究进展

虽然已有一定数量的未成熟人卵母细胞体外成熟(IVM)、体外受精-胚胎移植(IVF-ET)后得到的正常后代,但其发育潜能显然不如体内成熟的卵母细胞好,这主要是由于对灵长类动物卵母细胞胞质成熟及卵母细胞获得发育能力方面的分子机理和生理生化过程知之甚少,现有的IVM条件不足以维持卵母细胞的胞质成熟.所以,改良IVM培养基的有效成分,积极探索卵母细胞IVM的条件,成功建立IVM、IVF和体外胚胎培养(IVC)程序,对生殖生物学及生殖工程的发展具有重要意义.就未成熟卵母细胞的IVM调节因素的研究进展进行综述.     

卵丘细胞在未成熟卵母细胞玻璃化冷冻保存中的作用

卵母细胞冷冻技术是生殖医学的一大热点,未成熟卵母细胞冻存是保存女性生育力的重要方法,具有广阔的临床应用前景。卵丘细胞与卵母细胞共处同一微环境,二者通过缝隙连接进行复杂的双相调控和物质交换,目前已知卵丘细胞在人类未成熟卵母细胞体外成熟过程中具有重要作用,但在冷冻过程中的作用尚无定论。随着玻璃化冷冻技术的发展,卵丘细胞对未成熟卵母细胞冷冻的影响有了较多的研究,卵丘细胞可减缓冷冻剂渗透速率,从而避免细胞渗透压的突变对未成熟卵母细胞的损伤,提高细胞冻融后存活率和受精率并支持未成熟卵母细胞的体外成熟。就卵丘细胞在人类未成熟卵母细胞玻璃化冷冻保存中的作用进行综述,为癌症化疗、延迟生育年龄等需进行的未成熟卵母细胞冷冻提供重要的理论基础及可靠的实验依据。     

小鼠卵母细胞程序化和玻璃化冷冻效果的比较

目的本实验通过建立小鼠的实验模型,比较程序化和OPS玻璃化冷冻效果,探讨卵子冷冻的具体方法,为卵子冷冻技术应用于临床提供实验依据。方法通过促排卵获取小鼠MII期卵母细胞,随机分组,观察不同冷冻方法、高浓度的冷冻保护液,对卵子的成活及进一步发育潜能的影响;不同的IVF前培养时间对冻融卵子的受精及继续发育的影响。结果（1）OPS玻璃化冷冻组与程序化冷冻组的存活率、受精率、卵裂率及6—8cell胚形成率均无显著差异。（2）玻璃化冷冻组与暴露组的卵母细胞存活率分别为35．3％,100％,有显著性差异（P〈0．05）。暴露组与对照组比较的受精率、卵裂率,无显著性差异（P〉0．05）。而暴露组与对照组的6—8cell的胚形成率分别为41％,63％,两组比较,有显著差异（P〈0．05）。（3）无论OPS玻璃化冷冻法还是程序化冷冻法,冻融后的培养2h组和培养1h组的,受精率、卵裂率。两组比较,均有显著性差异（P〈0．05）。结论本实验证实,OPS玻璃化冷冻法至少可以达到程序化冷冻法同样的效果,而OPS玻璃化冷冻法有好于程序化冷冻法的趋势。加之,其简便、省时、经济等优点,因而,更有发展前景。     

卵子玻璃化冷冻在体外受精胚胎移植周期中的临床应用

目的:探讨卵子玻璃化冷冻保存的可行性,更好提高卵子复苏率、受精率,提高卵子冻存患者的妊娠率。方法:在河北医科大学第二医院生殖医学门诊行体外受精胚胎移植(IVF-ET)患者中,由于取卵日男方取精失败行卵子冻存。于2011年2月～2012年8月共行5例患者卵子冻存并复苏。结果:冷冻MII卵子55枚,复苏率94.5%(52/55),受精率92.3%(48/52),卵裂率95.8%(46/48),移植胚胎11枚,临床妊娠3例,冷冻保存胚胎22枚。结论:卵子玻璃化冷冻是可行的,可应用于取卵日取精困难的患者。     

添加两种不同培养成分对人未成熟卵体外成熟培养的影响

目的:研究添加两种不同培养成分对人未成熟卵母细胞体外成熟培养(IVM)的效果。方法:选择接受ICSI治疗的77名不孕患者共收集未成熟卵177枚,随机分A组(激素组)33例,添加0.075 IU/ml FSH及0.075 IU/ml LH至基础IVM培养液中;B组(成熟卵泡液组)44例,50%基础IVM培养液与50%人成熟卵泡液配成。将两组收集的未成熟卵母细胞放入不同培养液中培养48 h,每24 h倒置显微镜下观察卵母细胞的形态;对MII卵进行ICSI,ICSI操作后继续培养,ICSI后16～20 h进行受精观察,24及48 h分别进行卵裂观察及胚胎评分。结果:①B组未成熟卵母细胞48 h成熟率和MI期卵母细胞48 h成熟率显著高于A组,差异有统计学意义(P<0.05),两组培养液GV期卵母细胞24和48 h成熟率比较差异无统计学意义(P>0.05)。②B组2PN卵裂率显著高于A组,差异有统计学意义(P<0.05),两组2PN受精率及可利用胚胎率差异无统计学意义(P>0.05)。结论:人成熟卵泡液内可能含有一些未知的有利于卵母细胞成熟的因子,深入研究卵泡液的成分,进一步明确其促卵母细胞成熟机制对开发新的IVM培养液、改善IVM的临床结局有重要意义。     

体外培养成熟技术对未成熟卵母细胞冻融的影响

目的:探索影响未成熟卵母细胞冻融技术的关键因素。方法:A组:来源于体外受精-胚胎移植(IVF-ET)周期中自愿捐献的多余的成熟卵母细胞(MⅡ期),共56个;B组:来源于手术切除的卵巢组织中的未成熟卵母细胞(GV或MⅠ),共67个。不同成熟时期卵母细胞经慢冻快融后培养和体外受精,观察其体外成熟、受精及胚胎发育情况。结果:A组与B组相比,两组冻融后的存活率有显著性差异(60.71%vs 77.61%,P<0.05);A组受精率高于B组,但两组比较无显著性差异(61.76%vs 50.00%,P>0.05);A组与B组相比,两组冻融后的卵裂率和优质胚胎率有显著性差异(76.19%vs 37.50%,47.62%vs 12.50%,P<0.05)。A组获2枚囊胚,而B组没有囊胚培养成功。结论:体外成熟培养技术可能是影响冻融后未成熟卵母细胞发育的关键因素。     

BDNF对小鼠卵母细胞体外成熟及发育能力的影响

目的:观察脑源性神经营养因子(BDNF)对小鼠未成熟卵的体外成熟及发育能力的影响。方法:以α-MEM为基础培养液,添加不同浓度(0、1、5、10 ng/ml)的BDNF以及FSH、FBS培养小鼠未成熟卵,并进行体外受精,观察卵母细胞成熟率、受精率和胚胎发育至囊胚的能力,了解不同培养条件下BDNF对卵母细胞发育能力的影响。结果:当体外成熟培养液中含有FSH和10%的FBS时,与体外成熟对照组比较,BDNF虽然不影响卵母细胞的成熟率和受精率,但BDNF 5 ng/ml组的囊胚形成率(75.00%)显著高于体外成熟对照组(56.63%),而接近体内成熟组(76.92%);当培养液中仅含FBS时,各组间卵母细胞成熟率和受精率没有差异,但与对照组比较,BDNF显著提高囊胚形成率;当培养液中不含FBS、FSH时,虽然无囊胚形成,但BDNF显著提高了卵母细胞的受精率。结论:BDNF能促进小鼠未成熟卵胞质的发育,提高卵母细胞的发育能力。     

卵母细胞体外成熟治疗多囊卵巢综合征不孕症

[目的]应用卵母细胞体外成熟（IVM）技术治疗多囊卵巢综合征不孕症。[方法]23例患者中的152个未成熟卯母细胞进行体外培养,成熟后行单精子卵胞浆内注射。受精后取优质胚胎移植宫腔,同时对其影响因素进行探讨。[结果]共培养成熟105个卵子,成熟率69．1％。受精率92．0％,卵裂率79．4％。5例妊娠,周期妊娠率27．8％,1例已出生1个正常男婴。IVM结局与末成熟卵子的形态及体外培养系统有关。[结论]IVM对卵泡发育和成熟障碍,特别是难治性多囊卵巢综合征患者助孕有效。     

未成熟卵体外成熟技术在卵巢高反应患者IVF-ET中的应用

目的:探讨未成熟卵体外成熟(IVM)技术在卵巢高反应患者体外受精-胚胎移植(IVF-ET)中的应用价值。方法:在IVF-ET促排治疗中,对双卵巢卵泡数过多,有可能发生卵巢过度刺激综合征(OHSS)或继续治疗可能发生重度OHSS的患者,根据其意愿即刻停药,全部取卵改行IVM治疗12个周期(A组)或取部分小卵泡改行IVM治疗,同时保留部分卵泡继续行IVF-ET常规治疗18个周期(B组)。小卵泡体外培养成熟后,通过卵胞浆内单精子注射(ICSI)获得受精卵并行胚胎移植或冷冻。统计分析未成熟卵的成熟率、卵母细胞的受精率、胚胎的发育情况及临床结局。结果:两组30个取卵周期,共获未成熟卵240个,经IVM、ICSI和体外培养后,成熟率、受精率、正常卵裂率及优质胚胎率分别为61.25%(147/240),77.55%(114/147),92.98%(106/114)和29.25%(31/106)。A组8例行IVM新鲜胚胎移植(8周期)4例临床妊娠,A、B两组有8例行IVM解冻胚胎移植(9周期)3例临床妊娠,已有3例分娩。A组12例无OHSS发生,促性腺激素用量少于B组,B组18例中3例有OHSS风险而取消胚胎移植。结论:对常规IVF促排周期中卵巢高反应患者及时改行IVM,可以避免周期取消及OHSS的发生,减少促排卵药物的使用量,同时获得较好的临床妊娠率。     

Comparison of pregnancy outcomes in natural cycle IVF/M treatment with or without mature oocytes retrieved at time of egg collection

The objective of this study is to compare the pregnancy and live birth rates of a natural cycle in vitro fertilization (IVF) combined with in vitro maturation (IVM) treatment (natural cycle IVF/M) by the presence or absence of mature oocytes retrieved. Infertile women were divided into two groups: (A) patients with mature oocytes found at retrieval and (B) patients with only immature oocytes at retrieval. Patients of group A were further divided into three subgroups: (A1) mature oocytes retrieved from both the leading and the small follicles, (A2) mature oocytes retrieved from the leading follicles only, and (A3) mature oocytes retrieved from the small follicles only. Pregnancy and implantation rates were compared. The results indicate that the clinical pregnancy rates were 40.1% (126/314) and 34.5% (19/55) for groups A and B, respectively. There were no differences in pregnancy rates among the subgroups: A1=44.0% (66/150), A2=34.9% (30/86), and A3=38.5% (30/78). In addition there were no differences in implantation rates among the groups (16.2%?=139/859, 15.0%?=22/147, 16.8%?=69/410, 14.7%?=34/232, and 16.6%?=36/217, respectively). However, the live birth and miscarriage rates were significantly different between the group A and group B (29.6%?=93/314 vs. 16.4%?=9/55 and 26.2%?=32/126 vs. 52.6%?=10/19, respectively). In conclusion, for natural cycle IVF/M treatment, although the clinical pregnancy rates are not different regarding the retrieval of mature oocytes or the time of the egg retrieval, the live birth rate is higher (P < 0.05) when the mature oocytes are obtained at the time of the egg retrieval.     

Improvement of embryonic development and clinical outcomes of germinal vesicle stage oocytes using a microvibration culture system

ABSTRACT

In vitro maturation (IVM) has evolved as a clinical treatment option in assisted reproductive technology. However, the poor developmental potential of germinal vesicle (GV)-stage oocytes is still suboptimal. This study’s objective was to evaluate the effect of a microvibration culture system (MVC) during IVM and/or in vitro culture (IVC) on the clinical outcomes and the embryonic development potential of human GV-stage oocytes collected from human chorionic gonadotropin (HCG)-primed IVM and fertilization-embryo transfer (IVM/F-ET) cycles of patients with polycystic ovaries (PCO). A total of 206 HCG-primed IVM/F-ET cycles were divided into four groups according to the microvibration and static culture system applied during IVM and/or IVC: Group SS (static system during both IVM and IVC); Group SV (static system during IVM alternated with microvibration system during IVC); Group VS (microvibration system during IVM alternated with static system during IVC), and Group VV (microvibration system during both IVM and IVC). The results indicate that the rates of in vitro MII oocytes per cycle, fertilization, and cleavage were not significantly different between the groups. The rate of good-quality embryos in Group SV tended to be higher than the rate in Groups SS and VS, but there was no significant difference between Group SS and Group SV. Clinical pregnancy, implantation, and live birth rates of Groups SV and VS were slightly higher than those of Group SS. However, the rate of good-quality embryos with at least six cells on day 4, the clinical pregnancy, implantation, and live births in Group VV were significantly higher than those in Group SS. These results indicate that, compared with the static culture system, the MVC system applied for both IVM and IVC seems to improve the clinical outcomes and the quality of embryos of GV oocytes derived from HCG-primed IVM/F-ET cycles in PCO patients.

Abbreviations: PCO: polycystic ovaries; HCG: human chorionic gonadotropin; GV: germinal vesicle; MII: metaphase II; IVM: in vitro maturation; IVF: in vitro fertilization; IVC: in vitro culture: MVC: microvibration culture; SC: static culture; ICSI: intracytoplasmic sperm injection; IVM/F-ET: IVM and fertilization-embryo transfer; AMH: anti-Mullerian hormone; OHSS: ovarian hyperstimulation syndrome     

Reduced polyspermic fertilization of porcine oocytes utilizing elevated bicarbonate and reduced calcium concentrations in a single-medium system

The development of efficient systems for in vitro production of porcine embryos has been hampered by a high incidence of polyspermic fertilization. A recently developed single-medium system for porcine in vitro maturation (IVM), IVF and in vitro embryo culture (IVC) (Purdue Porcine Medium; PPM) was modified with elevated bicarbonate (44 mM) and reduced calcium concentrations (1.7 mM) for IVF (PPMfert.2). Oocyte penetration was evaluated after maturation in PPMmat (0.5 mg mL(-1) hyaluronan, 0.6 mM cysteine, 10 ng mL(-1) epidermal growth factor (EGF), 0.1 U mL(-1) porcine LH and FSH, and 1 x Minimal Essential Medium (MEM) vitamins) and fertilization (5 h with 5 x 10(5) sperm mL(-1)) in either PPMfert.2 or mTBM (20 mM Tris, 0.0 mM bicarbonate, 7.5 mM calcium). Embryonic development (cleavage and blastocyst stages) was assessed after culture in PPM1 and PPM2. Although penetration was lower in PPMfert.2 (69.9%) compared with mTBM (83.9%), 48.8% of penetrated oocytes were fertilized normally in PPMfert.2 compared with only 27.8% normal fertilization in mTBM. More oocytes cleaved in PPMfert.2 (77.9% v. 53.7%), but development to the blastocyst stage was not different between treatments (14.1% v. 14.3%). Further work is needed to improve embryonic development, but reduced polyspermic penetration is an important step in the optimization of the PPM system for in vitro porcine embryo production.