新建体外受精实验室中挥发性有机化合物对鼠胚体外发育的影响

目的:探讨新建成的体外受精(IVF)胚胎实验室挥发性有机化合物(VOC)的浓度变化及不同浓度VOC环境下对小鼠IVF和胚胎发育的影响和去除实验室内VOC的方法。方法:按照胚胎在培养环境中是否接触培养室内空气分为实验组和对照组,在实验室装修后每个月测定两组中VOC水平,比较两组不同浓度的VOC对小鼠体外受精率、2-细胞率、卵裂率和囊胚率的影响,比较排风扇抽风、Coda空气净化器和单纯活性炭吸附去除室内VOC的效果。结果:1装修后第1个月,实验组VOC浓度高于对照组(384.00±5.16 ppb vs 199.00±2.58 ppb,P0.05);装修后第2个月,实验组VOC浓度高于对照组(279.50±3.70 ppb vs 201.75±3.50 ppb,P0.05);装修后第3个月两组VOC浓度比较,差异无统计学意义(P0.05)。2装修后第1、2个月时实验组受精率、2-细胞率、卵裂率和囊胚率低于对照组,差异均有统计学意义(P0.05);装修后第3个月时两组以上4项指标差异无统计学意义(P0.05)。3空气净化器的VOC去除率[(60.15±2.18)%]明显高于排风扇抽风[(51.88±2.23)%]和单纯活性炭吸附[(28.95±1.35)%],差异有统计学意义(P0.05)。结论:实验室内高浓度的VOC会对小鼠IVF胚胎造成严重的胚胎毒性,导致胚胎质量下降和发育受阻;空气净化器是一种有效地去除实验室内VOC的方法。     

氧浓度对体外培养胚胎发育的影响

目的:探讨体外受精/卵胞质内单精子注射(IVF/ICSI)后移植前胚胎在不同氧浓度培养条件下的发育情况。方法:将接受IVF或ICSI的患者随机分组成常氧组(体积分数20%的氧浓度,包括IVF卵裂期胚胎患者150例,IVF囊胚期胚胎患者51例,ICSI卵裂期胚胎患者81例,ICSI囊胚期胚胎患者39例)和低氧组(体积分数5%的氧浓度,包括IVF卵裂期胚胎患者108例,IVF囊胚期胚胎患者56例,ICSI卵裂期胚胎患者79例,ICSI囊胚期胚胎患者45例),观察各组胚胎在受精后第3日的卵裂胚和第5日的囊胚发育情况以及临床妊娠结局。结果:在患者年龄、不孕年限、基础FSH值、Gn用量、获卵数和MⅡ卵率组间均无统计学差异的基础上,1与常氧组相比,低氧组的胚胎IVF或ICSI后的受精率、卵裂率以及第3日形成的优质胚胎率无统计学差异(P0.05),临床妊娠率和着床率组间也无统计学差异(P0.05);2低氧组胚胎IVF或ICSI后形成的Ⅴ级囊胚多于常氧组(P0.05),总囊胚形成率组间有显著的统计学差异(P0.01),临床妊娠率和着床率组间无统计学差异(P0.05)。结论:与常氧培养条件相比,低氧培养条件并不能显著促进IVF/ICSI后卵裂期胚胎的发育,但能够促进囊胚的形成,特别是囊胚的孵出,因此低氧培养可以作为囊胚培养的首选条件,但不是卵裂期胚胎培养的必要条件。     

鼠胚实验在新建体外受精胚胎培养室质量控制中的作用

目的:检测新建体外受精实验室培养体系的可靠与否,是否符合人类胚胎体外培养的要求。方法:对昆明白小鼠进行超促排卵,手术获取昆明白小鼠雌雄配子进行体外受精和体内受精的胚胎;并进行体外培养观察两组鼠胚的受精率、2-细胞率、囊胚率及采用密度梯度离心法和上游法两种精液处理方法对小鼠体外受精胚胎发育情况的影响。结果:进行了30个周期的体内受精体外培养实验,共获卵1136枚;进行了35个周期的体外受精实验,共获卵1486枚。体内受精组获得的受精率(93.13±1.46)%和2-细胞率(94.52±1.67)%高于体外受精组的受精率(84.39±2.87)%和2-细胞率(87.80±3.21)%,两组间差异有统计学意义(P0.05)。密度梯度离心法处理精液后,其受精率(90.32±3.14)%和2-细胞率(88.54±2.28)%高于上游法的受精率(76.46±2.35)%和2-细胞率(82.67±2.62)%,两者差异有统计学意义(P0.05)。结论:小鼠卵母细胞体内受精和体外受精后均有高的成胚率,体内受精后进行体外培养优于体外受精后培养,采用密度梯度离心处理精液实施体外受精优于上游法,鼠胚实验可对新建胚胎培养室的培养体系进行评估。     

胚胎碎片对早期胚胎发育影响的研究

目的:探讨胚胎碎片对早期胚胎发育的影响。方法:回顾分析3PN胚胎的囊胚形成率,根据胚胎碎片含量分为A组(10%)、B组(25%碎片≥10%)、C组(50%碎片≥25%)、D组(≥50%),检测各组活性氧(ROS)的含量。选取碎片含量10%～50%的胚胎,根据是否行显微碎片清除分为实验组(行显微去碎片)和对照组(未行显微去碎片),观察囊胚形成率。结果:D组的囊胚形成率显著低于A、B、C组,差异有统计学意义(P0.05)。随着碎片增多,胚胎的ROS逐渐增多,B组和C组间无显著差异,其余各组间有显著差异(P0.05)。去除碎片后,囊胚形成率为27.70%,显著高于对照组,差异有统计学意义(P0.05)。结论:ROS增加可能是胚胎碎片形成的主要因素之一,去除碎片可提高囊胚形成率,但去除碎片对胚胎发育潜能的远期影响,有待进一步研究。     

IVF-ET周期中1原核(PN)及0PN(2Pb)胚胎的染色体组成分析

目的:初步探寻体外受精-胚胎移植(IVF-ET)周期中高发育潜能的1原核(PN)及0PN(2Pb)胚胎的方法。方法:观察787个IVF周期患者的PN及卵裂情况,培养至第6日,观察其囊胚形成情况。取囊胚滋养层细胞活检,采用胚胎植入前遗传学筛查(PGS)技术检测0PN(2Pb)、1PN及2PN囊胚的染色体组成。结果:787个IVF周期共获卵8 352枚,2PN、0PN(2Pb)、1PN胚胎的形成率分别为64.35%、4.93%、3.65%,囊胚形成率分别为45.71%、30.03%、18.18%。PGS结果显示,1PN及0PN(2Pb)囊胚染色体正常的比例明显低于2PN囊胚,差异有统计学意义(P0.05)。0PN(2Pb)囊胚的染色体正常比例较1PN囊胚的略高,差异无统计学意义(P0.05)。此外,发育至第6日的0PN(2Pb)囊胚及1PN囊胚较第5日胚胎的正常染色体比例高。结论:对于本周期无可利用胚胎的患者,建议移植1PN及0PN(2Pb)来源的第6日评分较好的囊胚。     

添加抗氧化物维生素E对人类胚胎体外发育的影响

目的:探讨抗氧化物维生素E添加与人类胚胎体外发育潜力之间的关系.方法:选择2009年10月至2010年5月在我院进行常规体外受精-胚胎移植和卵胞浆内单精子注射的不孕患者106例,116个周期,共432个正常受精2个原核(2PN)来源的废弃胚胎,随机分为对照组和维生素E组,在维生素E组受精后第3天(D3)废弃胚胎的培养液中添加100 μmol维生素E,对照组不添加.观察两组囊胚的形成情况,并通过荧光染料Hoechst33342对胚泡进行染色,检测囊胚的总细胞数.结果:维生素E组的早期囊胚、扩张囊胚、孵化囊胚发育率均高于对照组,两组比较差异有统计学意义(P＜0.05);维生素E组孵化囊胚细胞总数(80.42±0.60)%高于对照组(76.11±0.82)%,差异有统计学意义(t=-7.365,P＜0.05).结论:添加维生素E能提高人类胚胎的体外发育率和胚胎质量.     

干细胞条件培养液对小鼠体外成熟卵母细胞发育潜能的影响

目的:探讨骨髓间充质干细胞(MSCs)条件培养液对小鼠体外成熟卵母细胞发育潜能的影响。方法:分离、培养小鼠MSCs,获得MSCs条件培养液;收集4~6周小鼠生发泡期(GV)卵母细胞,在MSCs条件培养液中体外成熟(IVM);同时部分小鼠促排处理,收集体内成熟卵母细胞;体外受精(IVF)后,比较受精率、4-细胞形成率及囊胚形成率差异;Hoechst33342染色后观察内细胞团(ICM)和滋养层细胞形成情况,计数内细胞团数。结果:IVM卵母细胞IVF后的受精率、4-细胞形成率和囊胚形成率及囊胚内细胞总数与体内成熟组比较,无显著差异(P0.05);IVM组各时期的胚胎形态正常,囊胚内细胞染色后的荧光图像与体内成熟组基本一致。结论:MSCs条件培养液能保持小鼠IVM卵母细胞良好的质量和发育潜能,是一种较理想的IVM培养体系。     

不同精子来源及授精方式对剩余胚胎继续囊胚培养结局的影响

目的:探讨不同精子来源及不同授精方式对胚胎继续发育能力的影响。方法:分析499例患者499个取卵周期剩余胚胎继续培养形成囊胚的情况,按精子来源不同分为供精IVF(D-IVF)组和夫精IVF(H-IVF)组,按授精方式不同分为IVF组和ICSI组,ICSI组按精子来源分为新鲜精液组、附睾精子和睾丸精子组,比较不同精子来源及授精方式获得剩余胚胎的囊胚形成率、胚胎利用率和无囊胚移植率。结果:① D-IVF组和H-IVF组受精率、卵裂率、优质胚胎率、第3日和第5日胚胎种植率、临床妊娠率和流产率均无统计学差异(P0.05),组间剩余胚胎囊胚形成率、胚胎利用率和无囊胚移植率亦无统计学差异(P0.05);②ICSI组与IVF组比较,其受精率较高(P0.05),但优质胚胎率显著下降,有统计学差异(55.11%vs 61.30%,P0.05),组间第3日卵裂期胚胎和剩余胚胎囊胚种植率、临床妊娠率无统计学差异(P0.05),但ICSI组与IVF组比较,其剩余胚胎囊胚形成率、胚胎利用率稍低,无囊胚移植率较IVF组稍高,差异有统计学意义(56.13%vs 65.32%,48.18%vs 55.39%,21.68%vs 13.20%,P0.05)。③新鲜精液组的优质胚胎率、胚胎利用率显著低于附睾精子和睾丸精子组(P0.05),各组囊胚移植的种植率和临床妊娠率无统计学差异(P0.05)。结论:D-IVF可获得H-IVF相似的结局,其剩余胚胎都有较高的发育潜能,ICSI获得的剩余胚胎发育潜能低于IVF组。附睾精子和睾丸精子ICSI后获得的胚胎比新鲜精液精子ICSI后胚胎发育潜能高。针对不同的授精方式可能需要制定相应的剩余胚胎囊胚培养标准。     

废弃胚胎继续囊胚培养研究

目的:探讨体外受精治疗周期中废弃胚胎的体外发育潜能。方法:通过囊胚序贯培养法将无原核(0PN)、单个原核(1PN)、多个原核(≥3PN)和卵裂期发育延缓的2原核(2PN)废弃胚胎培养至囊胚期。比较不同来源胚胎囊胚形成情况和d3胚胎的卵裂球数、质量分级等;并利用废弃胚胎囊胚形成情况对体外受精妊娠结局进行预测。结果:共收集801个废弃胚胎,经序贯培养,形成209个囊胚(26.09%),其中58个为优质囊胚(27.75%)。1PN胚胎、0PN胚胎、d3卵裂球数为7-9-细胞胚胎、d3评分为Ⅰ-Ⅱ级胚胎以及卵裂球数为偶数的胚胎囊胚形成率较高(P均<0.05)。废弃胚胎中有囊胚形成者的临床妊娠率明显高于无囊胚形成者(P<0.05)。结论:废弃胚胎有不同程度的发育潜能,部分可发育为囊胚;特别是:①0PN和1PN胚胎;②d3的4-9-细胞胚胎,偶数卵裂球者更佳;③I级和Ⅱ级胚胎。     

体外受精胚胎细胞数量和碎片比例对囊胚形成的影响

目的:探讨体外受精(IVF)周期中胚胎细胞数量和碎片比例对囊胚形成的影响。方法:回顾性分析2 404个IVF周期患者的13 037个进行囊胚培养的胚胎。根据第3日细胞数量将胚胎分为2~3-细胞组(2C~3C)、4-细胞组(4C)、5-细胞组(5C)、6-细胞组(6C)、7-细胞组(7C)、8-细胞组(8C)、9-细胞组(9C)、10-细胞组(10C)、11-细胞组(11C)、12-细胞组(12C)、≥13-细胞组(≥13C),分析囊胚培养细胞数量和碎片比例与囊胚形成的关系。结果:碎片为1~2级的胚胎,8C组和≥13C组囊胚形成率明显高于其他各组(P0.05);7C、9C~12C组之间囊胚形成率无统计学差异(P0.05)。碎片为3级的胚胎,8C组的囊胚形成率高于其他各组(P0.05),且与碎片1~2级7C、9C~12C组的胚胎相比无统计学差异(P0.05);9C组囊胚形成率高于7C组,且差异显著(P0.05)。结论:第3日8C胚胎的发育潜能是最优的,可适当放宽对8C碎片的评估;随着碎片比例增大可减弱胚胎的发育潜能。     

激光辅助孵化在冻融胚胎移植中的临床研究

目的:探讨激光辅助孵化对冻融胚胎移植周期中胚胎种植率的影响。方法:分析192个冻融胚胎移植周期,其中行激光辅助孵化后移植86个周期(激光组),常规冻融胚胎移植106个周期(常规组),比较二组患者临床妊娠率和胚胎种植率。结果:激光组的临床妊娠率为31.4%,与常规组的23.6%比较,无统计学差异(P>0.05);胚胎种植率为19.2%,显著高于常规组的12.0%(P<0.05);特别当透明带厚度≥17mm时,激光组胚胎种植率显著高于常规组(P<0.05)。结论:在冻融胚胎移植前对透明带厚度≥17mm的胚胎行激光辅助孵化有助于提高胚胎种植率。     

Embryo-derived platelet activating factor,a marker of embryo quality and viability following ovarian stimulation for in vitro fertilization

Embryo-derived platelet-activating factor (PAF) may be an important mediator of early maternal recognition of pregnancy. PAF was higher in media associated with clinical pregnancies when compared to preclinical pregnancies but not higher in pregnant vs nonpregnant groups. The production of PAF by the preimplantation embryo was not related to follicle size or embryo morphology. However, differences in PAF concentrations in the culture media were related to the age of the embryo culture medium and the developmental stage of the embryo.     

Assessing efficacy of day 3 embryo time-lapse algorithms retrospectively: impacts of dataset type and confounding factors

This study investigated the efficacy of four published day 3 embryo time-lapse algorithms based on different types of datasets (known implantation data [KID] and single embryo transfer [SET]), and the confounding effect of female age and conventional embryo morphology. Four algorithms were retrospectively applied to three types of datasets generated at Fertility North between February 2013 and December 2014: (a) KID dataset (n?=?270), (b) a subset of SET (n?=?144, end-point?=?implantation), and (c) SET (n?=?144, end-point?=?live birth), respectively. All four algorithms showed progressively reduced predictive power (expressed as area under the receiver operating characteristics curve and 95% confidence interval [CI]) after application to the three datasets (a–c): Liu (0.762 [0.701–0.824] vs. 0.724 [0.641–0.807] vs. 0.707 [0.620–0.793]), KIDScore (0.614 [0.539–0.688] vs. 0.548 [0.451–0.645] vs. 0.536 [0.434–0.637]), Meseguer (0.585 [0.508–0.663] vs. 0.56 [0.462–0.658] vs. 0.549 [0.445–0.652]), and Basile (0.582 [0.505–0.659] vs. 0.519 [0.421–0.618] vs. 0.509 [0.406–0.612]). Furthermore, using KID dataset, the association (expressed as odds ratio and 95% CI) between time-lapse algorithms and implantation outcomes lost statistical significance after adjusting for conventional embryo morphology and female age in 3 of the 4 algorithms (KIDScore 1.832 [1.118–3.004] vs. 1.063 [0.659–1.715], Meseguer 1.150 [1.021–1.295] vs. 1.122 [0.981–1.284] and Basile 1.122 [1.008–1.249] vs. 1.038 [0.919–1.172]). In conclusion, SET is a preferred dataset to KID when developing or validating time-lapse algorithms, and day 3 conventional embryo morphology and female age should be considered as confounding factors.     

PCOS患者移植冻融胚胎后妊娠结局

目的:探讨多囊卵巢综合征(PCOS)患者在优质冻融胚胎移植(FET)后是否有更高的妊娠丢失率。方法:回顾性分析诊断为PCOS并初次接受体外受精-胚胎移植(IVF-ET)或卵胞质内单精子注射(ICSI)FET的患者,选择移植冻融胚胎中含有卵裂期优质胚胎且生化妊娠阳性的157个周期的临床资料为PCOS组,同期年龄、体质量指数(BMI)、不孕年限与PCOS组匹配的、因单纯输卵管因素接受IVF-ET助孕治疗并生化妊娠阳性的389个FET周期的临床资料为对照组,比较组间的生化妊娠丢失率、早期流产率、晚期流产率、活产率。结果:在接受优质卵裂期FET的PCOS患者妊娠后,生化妊娠丢失率、早期流产率、晚期流产率、活产率与对照组相比(8.28%vs 5.14%、7.64%vs 8.13%、6.94%vs 5.15%、79.86%vs 84.28%),差异无统计学意义(P0.05)。结论:在固定胚胎质量这个重要的影响妊娠早期丢失的影响因子的基础上,排除年龄、BMI、自然流产次数、是否多胎妊娠、移植日内膜厚度等影响因素的干扰,患PCOS并不增加IVF/ICSI的生化妊娠丢失率和临床妊娠流产率。     

全胚冷冻再行解冻胚胎移植周期的临床结局分析

目的:探讨新鲜移植周期与冻融胚胎移植(FET)周期妊娠结局的差异。方法:回顾性分析本中心刺激周期行新鲜胚移植(190例)和全部胚胎冷冻后再行FET(97例)周期的临床妊娠率、种植率以及流产率。结果:190例刺激周期新鲜胚胎种植后的妊娠率、种植率、流产率分别为47.4%(90/190)、30.2%(103/341)、10.0%(9/90),97例全部胚胎冷冻后行FET后的妊娠率、种植率、流产率分别为60.8%(59/97)、47.0%(86/183)、10.2%(6/59),组间妊娠率与种植率均有统计学差异(P<0.05),流产率无统计学差异(P>0.05)。结论:对于有OHSS风险等不适宜进行新鲜胚胎移植的患者,选择全部胚胎冷冻并择期进行FET,并不降低胚胎种植率和临床妊娠率,从而预防迟发型OHSS的发生,可获得更为理想的妊娠结局。     

Assessment of hamster blastocysts derived from eight-cell embryos cultured in hamster embryo culture medium-2 (HECM-2): Cell numbers and viability following embryo transfer

The viability of hamster blastocysts, cultured from the eight-cell stage using hamster embryo culture medium-2, was examined by embryo transfer. Approximately 15–20 cultured hamster blastocysts were surgically transferred unilaterally to uterine horns of pseudopregnant recipient hamsters that had been mated to vasectomized males 3 days previously. Control recipients received in vivo developed, freshly recovered eight-cell embryos or blastocysts on day 2 or 3 of pseudopregnancy, respectively. Of the successful embryo transfers, the experimental group (receiving cultured blastocysts;n=10) gave 51.8% implantations and 28.2% live pups. These values were closely similar to those of the controls; the percentages of control implantations and offspring were 51.1 and 34.0%, respectively, for eight-cell embryo transfer (n=7,P>0.69) and 48.5 and 28.9% for blastocyst transfer (n=6,P>0.52). To evaluate the quality of cultured hamster blastocysts, the following two parameters were examined. (1) The mean number of cells per blastocysts was 24.4±0.7 for cultured blastocysts. This value was similar to that (range, 14–24) obtained in this laboratory for in vivo developed freshly recovered blastocysts. (2) Oxygen consumption analysis revealed that cultured blastocysts actively respired at a level close to that observed with freshly recovered eight-cell embryos (slopes of oxygrams: 0.25 and 0.26, respectively). From these results, it is concluded that hamster blastocysts, cultured from the eight-cell stage, are (a) qualitatively similar to freshly recovered in vivo developed blastocysts and (b) biologically viable as revealed by the production of live offspring upon embryo transfer. Therefore, hamster embryo culture medium-2 could be useful for studies relating to embryo-derived proteins involved in the process of implantation and for the production of transgenic hamster embryos, for example, using embryonic stem cells.     

冻融胚胎移植周期无创性内膜刺激胚胎移植策略的临床疗效探究

目的探讨无创性内膜刺激胚胎移植(stimulation of endometrium embryo transfer,SEET)技术在冻融胚胎移植(frozen-thawed embryo transfer,FET)周期中对妊娠结局的影响。方法回顾性分析2016年3月—2017年2月在本中心进行体外受精-胚胎移植(IVF-ET)并首次行单囊胚FET的不孕症患者的临床资料,按移植方式分组:A组(实验组)57个周期,FET时采用SEET技术;B组(对照组)56个周期,FET时采用传统囊胚移植技术。结果 A组采用SEET技术后的胚胎种植率(64.9%)和临床妊娠率(64.9%)显著高于B组(44.6%,44.6%)。结论 SEET技术可以显著提高临床妊娠率,为改善IVF结局提供了一种新的移植策略。     

Use of follicle-stimulating hormone (FSH) to increase the in vitro fertilization (IVF) efficiency of mice

Purpose The possibility of increasing the efficiency of an in vitro fertilization system (IVF) for Swiss OF1 mice was studied. The experimental protocol proposed analyzed the use of FSH as a superovulatory inducing hormone in comparison to traditional PMSG treatment. At the same time, the quality of IVF-derived embryos was evaluated both in vitro, with culture in CZB medium and fixation in advanced stages of development, and in vivo, by transfer to female recipients.Results Treatment with FSH induced a much higher ovulation number compared to PMSG (64.26 vs 33.85; P<0.01). With this gonadotropin, IVF provided a positive tendency to normal fecundation (67.76 vs 64.72; P<0.1) and a much lower index of abnormal division in embryos (10.57 vs 15.11; P<0.05). The viability of embryos obtained from donors treated with hormones was similar, although differences did exist regarding embryo origin: those obtained following natural fertilization showed a higher developmental capacity both in vitro (P<0.05) and in vivo (P<0.05).Conclusions We conclude that FSH is an improved superovulation alternative treatment in comparison to PMSG for IVF. It provides a higher number of embryos with the same in vitro and in vivo viability as those obtained from PMSG.     

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。     

原核期胚胎的原核模式与胚胎发育及妊娠关系的研究

目的:探讨原核期胚胎的原核模式与胚胎发育及妊娠能力的关系。方法:在IVF/ICSI后16～18 h,根据原核期胚胎的核仁前体数目和分布,将其分为模式0～5;并进行随后的胚胎形态学评估和计算妊娠率。结果:模式0的优良胚胎率(83.13％)高于其它各模式之和(76.11％,P<0.05);单纯移植模式0、含模式0和不含模式0组的妊娠率分别为48.08％、32.14％和21.28％,三组间差异有显著性(P<0.05)。结论:原核期胚胎的原核模式对预测胚胎质量和种植能力有一定参考价值。