重组人钙调素对小鼠胚胎发育及凋亡的影响

目的探讨外源重组人钙调素(rhCAM)对小鼠早期胚胎体外发育及凋亡的影响。方法小鼠超促排卵,收集2-细胞胚胎置于含不同浓度rhCAM的IVF-30培养液中,观察并记录胚胎形态,TUNEL法检测囊胚细胞数及囊胚细胞凋亡情况。结果20μg/ml rhCAM组的胚胎体外培养48、72 h的囊胚形成率和孵化囊胚率均高于对照组,该组体外培养72 h囊胚细胞凋亡指数显著低于对照组。200μg/ml rhCAM组的胚胎囊胚形成率低于对照组,凋亡指数显著高于对照组。结论一定浓度的rhCAM可促进胚胎体外发育并可抑制胚胎细胞的凋亡。     

小鼠致密化前胚胎卵裂球体外培养体系的研究

目的探索改善小鼠致密化前胚胎卵裂球体外发育能力。方法用酶消化与机械法结合分离小鼠2-、4-、8-细胞胚胎卵裂球为单个卵裂球及2/4、4/8多卵裂球胚胎,并将其装入空透明带后于兽用人绒毛膜促性腺激素(hCG)注射后138 h,观察透明带包裹、胚胎密度及胰岛素(Ins)、葡萄糖(Glu)添加对各卵裂球、囊胚形成率及囊胚细胞数目的影响。结果透明带包裹提高了2-细胞胚胎卵裂球来源的单腔囊胚的发育率。25/50μl胚胎密度及培养基添加Ins、Glu能显著提高2-细胞胚胎卵裂球囊胚发育率和囊胚细胞数;且对4-和8-细胞胚胎卵裂球发育具有同样的促进作用。结论采用透明带包裹、卵裂球密集培养、培养基添加Ins、Glu的方法成功建立了一种维持致密化前胚胎卵裂球体外高效发育的培养体系。其中,透明带在卵裂球发育中具维持囊胚正常形态作用,避免了多腔囊胚的形成;卵裂球密集培养和培养基添加Ins、Glu均提高囊胚形成率和(或)囊胚细胞数目。该体系有效改善了致密化前胚胎卵裂球体外发育质量。     

体外受精与体外培养对小鼠囊胚发育潜能的影响

目的评估体外受精和体外培养对囊胚期胚胎细胞数及发育潜能的影响。方法 (1)雌性ICR小鼠,自然交配获取囊胚为In vivo组,体内受精-体外培养获取囊胚为IVC组,体外受精获取囊胚为IVF组;(2)对三组囊胚行免疫杀伤法检测并在荧光显微镜下计数总细胞数、内细胞团细胞数、内细胞团与总细胞数的比值;(3)提取三组小鼠囊胚mRNA,采用实时荧光定量聚合酶链反应(qRT-PCR)方法检测Pou5f1等细胞因子的mRNA表达水平;(4)三组囊胚移植入D3.5假孕鼠,取D10.5胚胎进行形态学检测,计算胚胎植入率、成活率、流产率。结果 (1)IVF组和IVC组囊胚与In vivo组相比,细胞总数、滋养外胚层(TE)细胞数、内细胞团(ICM)细胞数均显著降低(P0.01),TE/ICM细胞数的比值三组之间无显著差异(P0.05);(2)IVF组和IVC组囊胚与In vivo组相比,多潜能基因OCT4、Nanog、Sox2表达水平相对上调,而早期细胞命运分化相关基因Fgf4、Gata4、Sox17、Eomes、Elf5、Lif表达水平下调,差异有统计学意义(P0.05);(3)三组间胚胎植入率无显著性差异(P0.05),而IVC组、IVF组活产率显著低于In vivo组,流产率显著高于In vivo组,差异有统计学意义(P0.05)。结论体外受精会导致囊胚质量下降,并影响胚胎植入后的进一步发育,导致胚胎成活率降低。     

脑源性神经营养因子在着床前小鼠胚胎中的表达及其对胚胎发育的影响

目的检测脑源性神经营养因子(BDNF)及其受体TrkB在植入前胚胎中的表达情况,探讨BDNF对小鼠早期胚胎发育和着床过程中的作用。方法收集小鼠受精后1~4d的胚胎,利用免疫组化和逆转录聚合酶链反应(RT-PCR)检测BDNF及其受体TrkB在各阶段胚胎中的表达情况。将2细胞阶段的胚胎放入添加不同浓度的BDNF的培养基中,添加或不添加TrkB受体阻断剂k252a/k252b,观察胚胎发育情况,并对形成的囊胚进行细胞计数。在注射人绒毛膜促性腺激素(HCG)后72h、76h、84h和88h分别腹腔注射10μg K252a/k252b,收集胚胎,计算扩张期囊胚数和囊胚细胞数。结果 BDNF及TrkB均表达于小鼠植入前各阶段的胚胎中,在培养基中添加BDNF后能够促进小鼠胚胎的体外发育,能够提高囊胚形成率和囊胚孵化率,并能够提高滋养层细胞数,此作用能够通过TrkB受体阻断剂k252a而阻断。体内研究进一步证实,k252a处理后能够抑制囊胚细胞数而明显抑制早期胚胎发育。结论 BDNF/TrkB信号系统可能通过某种自分泌或旁分泌的作用方式促进植入前胚胎的发育,同时也可能参与胚胎的着床过程。     

罗比卡因和利多卡因对小鼠体外受精和早期胚胎发育的影响

目的 :应用昆明小鼠胚胎体外培养模式和小鼠体外受精模型探讨罗比卡因与利多卡因对体外受精和早期胚胎发育的影响。方法 :( 1 ) 2 -细胞期胚胎培养 :取 4～ 6周昆明雌鼠 ,下午 4～ 6时腹腔注射孕马血清 ( PMSG) 5IU,48h后腹腔注射人绒毛膜促性腺激素 ( h CG) 5IU超排卵 ,随即雌雄合笼。次日早上 9时检查阴栓 ,阳性者于注射 h CG42h后取 2 -细胞期胚胎 ,分别放入含 1、1 0及 1 0 0μg/ ml不同浓度的罗比卡因和利多卡因的M1 6培养液中培养 ,培养液中加入输卵管上皮 ,72 h后观察桑椹胚或囊胚形成率。 ( 2 )体外受精 :从 F1代 ( C57× CBA)杂交雄鼠附睾和输精管取精子 ,从昆明雌鼠取卵母细胞 ,卵母细胞体外受精前暴露于不同浓度的利多卡因和罗比卡因 3 0 min,统计体外受精率。结果 :罗比卡因只在高浓度 1 0 0 μg/ ml时对 2 -细胞至囊胚期的胚胎发育产生明显抑制影响。利多卡因则在 1μg/ ml的浓度即对 2 -细胞期胚胎发育产生明显抑制。而 1 0和 1 0 0μg/ ml的利多卡因和罗比卡因均未对体外受精率有明显影响。结论 :提示罗比卡因可能用于体外受精技术中有关配子和合子取出或植入时的麻醉。     

人输卵管积液对小鼠胚胎基质金属蛋白酶分泌的影响

目的探讨人输卵管积液对小鼠胚胎体外培养胚胎发育及着床能力的影响机制。方法收集人输卵管积液,小鼠促超排卵,自然受精后适时收集囊胚,并将其随机分配到含有不同浓度的输卵管积液的培养液中,进行体外培养,观察囊胚的发育,计算囊胚孵出率;采用逆转录聚合酶链反应及免疫印迹法测定囊胚和培养液中的基质金属蛋白酶9(MMP-9)和基质金属蛋白酶组织抑制因子1(TIMP-1)的含量。结果输卵管积液组的囊胚孵出率低于无积液组,囊胚及培养液中的MMP-9和TIMP-1低于无积液组,并呈剂量依赖性,MMP-9/TIMP-1随积液浓度增加而降低。结论输卵管积液通过降低胚胎MMP-9/TIMP-1的分泌和影响胚胎早期发育潜能,从而影响胚胎着床的能力。     

褪黑素对老化卵母细胞体外发育能力的改善效果评价

目的探讨体外培养阶段(IVC)添加褪黑素(MT)对卵母细胞老化引起发育受损的改善效果。方法采用不同浓度的过氧化氢(H_2O_2)处理小鼠MII期卵母细胞诱导老化,浓度分别为0(对照组)、10、50、100、150μmol/L,体外受精(IVF)后统计各组二细胞率、囊胚形成率,检测老化卵母细胞的线粒体活性及丰度(Mitotracker Red、JC-1)、活性氧(ROS)水平、线粒体拷贝数等指标;在100μmol/L H_2O_2处理条件下,老化卵母细胞在IVF后的培养阶段分别添加不同浓度褪黑素(10-5、10-7、10-9 mol/L),统计二细胞率、囊胚率,并且分别检测各组获得囊胚的细胞数及凋亡率。结果不同浓度H_2O_2诱导卵母细胞老化后,囊胚发育率随H_2O_2浓度的升高而降低,50μmol/L和100μmol/L H_2O_2组囊胚形成率分别为(26.27±0.06)%和(28.46±3.45)%,相比对照组的(34.90±1.77)%显著降低,差异有统计学意义(P0.05);在H_2O_2诱导老化卵母细胞中,100μmol/L、150μmol/L H_2O_2浓度时,ROS水平相比对照组显著升高,差异具有统计学意义(P0.05);而活性线粒体的丰度及拷贝数呈现下降趋势,膜电位呈上升趋势,但相比对照组无显著性差异(P0.05);100μmol/L H_2O_2处理诱导卵母细胞老化后,在培养液中添加10-9 mol/L褪黑素组与老化对照组相比,囊胚率[(29.42±2.39)%vs.(20.87±4.12)%]、囊胚细胞数(39.36±9.78vs.37.91±4.25)均显著升高,凋亡率显著下降(2.57%vs.3.18%),差异均有统计学意义(P0.05);并且这些指标均达到与未经老化处理组的相似发育水平。结论老化卵母细胞体外受精后发育率和发育质量偏低的现象,可以通过在受精后体外培养阶段添加褪黑素得到改善。     

固定时间原核评估中0PN来源胚胎的发育潜能及其染色体整倍体率研究

目的 分析常规IVF后固定时间原核评估中零原核(0PN)来源胚胎的发育潜能及其染色体整倍体率。方法 回顾性分析2019年10月至2020年9月于本中心行常规IVF治疗的20对不孕不育夫妇的临床资料。共纳入183枚卵母细胞,剔除14枚0PN卵母细胞(处于未受精状态)后,根据原核评估以及细胞分裂情况分为两组：2个原核(2PN)来源胚胎为2PN胚胎组(n=116),0PN来源胚胎为0PN胚胎组(n=53),比较两组患者的胚胎发育情况;同时,为了分析0PN来源胚胎的染色体是否正常,选取0PN来源囊胚为0PN囊胚组(n=42),选取同一时期的植入前遗传学检测-单基因病(PGT-M)周期中2PN来源囊胚为2PN囊胚组(n=251),比较两组患者的染色体整倍体率。结果 0PN胚胎组Day 2(D2)卵裂球数[(5.74±1.40)个vs.(4.32±1.01)个]、D3卵裂球数[(10.15±2.29)个vs.(7.95±1.97)个]、囊胚形成率(86.8%vs. 60.3%)、可利用囊胚率(91.3%vs. 66.0%)均显著高于2PN胚胎组(P<0.05);0PN胚胎组的优质囊胚率(47...     

激光削薄透明带对小鼠囊胚形成和孵出的影响

目的探索激光削薄透明带对体外培养昆明小鼠囊胚形成和孵出的影响。方法激光孵化组为应用1．48μm二极管激光对186枚2细胞期昆明小白鼠胚胎实施透明带削薄处理;对照组不削薄透明带,在离胚胎一个直径处发射激光。比较两组囊胚形成率、孵出率、完全孵出率、透明带厚度和胚胎直径。结果激光组和对照组的囊胚形成率分别为73．7％（137／186）和71．6％（53／74）,两组比较差异无统计学意义。激光组的孵出率（86．9％,119／137）明显高于对照组（71．7％,38／53,P〈0．05）;囊胚嵌顿率（74．8％,89／119）明显高于对照组（23．1％,9／38,P〈0．001）。结论激光辅助孵化对体外培养昆明小鼠囊胚形成无不良影响且可以提高囊胚孵出率,但囊胚嵌顿率显著升高。     

植入前小鼠胚胎活检的实验研究

目的　以小鼠胚胎进行植入前胚胎活检的研究。　方法　通过输卵管冲洗 ,获得 4～ 8细胞期胚胎 ,用“一”字机械法和化学法分别对 4、8细胞的胚胎进行活检后继续培养 ,比较其囊胚形成及孵出情况。　结果　“一”字机械法胚胎活检后胚胎的囊胚形成率 (4细胞期 78 8% ,n =6 6 ;8细胞 99 0 % ,n =10 0 )、囊胚孵出率 (4细胞期 84 6 % ,n =5 2 ;8细胞期 94 9% ,n =99) ;化学法胚胎活检后胚胎的囊胚形成率 (4细胞期 74 1% ,n =5 8;8细胞期 92 9% ,n =12 7)、囊胚孵出率 (4细胞期88 4 % ,n =4 3;8细胞期 95 8% ,n =118)。两法均与未活检对照组比较无显著性差异 (P >0 .0 5 ) ,且两者的囊胚形成率、囊胚孵出率均无显著性差异 (P >0 .0 5 )。Logistic回归分析表明 ,细胞数越多越有利于胚胎进一步发育 ;而是否活检与活检方法对胚胎发育潜能无显著影响。　结论　用“一”字机械法在小鼠胚胎 4或 8细胞期进行单个卵裂球活检 ,不影响胚胎的发育 ,是一种较好的胚胎活检方法。     

Stimulatory and inhibitory effects of glucose and insulin on rat blastocyst development in vitro

The effect of glucose and insulin on the in vitro development of the rat preimplantation embryo was studied by incubating rat blastocysts recovered on days 5 or 6 of pregnancy in the absence or presence of increasing levels of glucose and/or insulin for 24 or 48 h. A differential cell-staining method allowed the separate counting of inner cell mass (ICM) and trophectoderm (TE) cells at the end of the incubation period. In a high-glucose medium (17 mM), ICM and, to a lesser extent, TE developments were significantly and irreversibly inhibited. Low insulin concentrations (3 pM) stimulated ICM and TE development in the presence of 1.1 or 6 mM glucose. Higher insulin levels (30-600 pM) in a 6-mM glucose medium, resulted in a dose-dependent inhibition of ICM and, to a lesser extent, TE development after both 24 and 48 h. This insulin-induced inhibition was reversible if insulin was removed from the medium after 24 h. In the absence of glucose in the medium, insulin was neither stimulatory nor inhibitory on ICM growth. Dead-cell occurrence in ICM after a 48-h incubation increased with increasing glucose concentration in the medium. Insulin alone did not increase dead-cell number but enhanced the effect of glucose. These results show that, in the presence of glucose, insulin might be stimulatory (at low concentrations) or inhibitory (at higher concentrations) on ICM development. A high glucose level was also inhibitory and increased dead-cell occurrence. The data suggest that insulin and glucose might interact and modulate blastocyst development as a function of their respective concentrations.     

两步极体活检对小鼠卵母细胞体外受精和胚胎发育的影响

目的评价两步活检一、二极体对小鼠卵母细胞体外受精(IVF)和相应胚胎发育的影响。方法8～12周龄雌、雄昆明小鼠。雌鼠促排卵,取成熟卵母细胞行卵胞浆内单精子注射(ICSI),序贯培养胚胎至囊胚孵出。分别于ICSI后激光透明带打孔活检第一极体,两步活检一、二极体,在受精后打孔同时活检一、二极体。比较各处理组与对照组受精、卵裂、优质胚胎、囊胚形成、扩张和孵出情况。结果两步活检一、二极体和同时活检一、二极体受精率、卵裂率、优质胚胎率和囊胚形成率与对照组均无明显差异。启动孵出率明显高于对照组;完全孵出率虽高于对照组,但无统计学意义。结论一、二极体活检不影响小鼠卵母细胞IVF和相应胚胎发育,而且可能有助于胚胎孵出,是安全可行的植入前遗传学诊断(PGD)活检取材方法,以两步活检法为最佳极体活检方案。     

Autoradiographic evidence for insulin and insulin-like growth factor binding to early mouse embryos

Insulin binding to mouse oocytes and preimplantation embryos was assessed by light-microscopic autoradiography. Significant insulin binding was present on the cells of morulae and increased twofold at the blastocyst stage of development. Insulin binding was markedly decreased by native insulin and to a lesser extent by insulin-like growth factors (IGFs). No specific insulin binding was detected on oocytes or embryos throughout the eight-cell stage. Specific binding of IGF-I and IGF-II was also observed on the cells of blastocyst outgrowths. The findings demonstrate that specific binding of insulin and IGF is temporally expressed on the cells of pre- and peri-implantation mouse embryos. These results confirm and extend our previous immunofluorescence study.     

Maternal diabetes and retarded preimplantation development of mice

The streptozocin-induced diabetic (STZ-D) mouse was found to be a suitable model for studying the effects of maternal diabetes on the preimplantation embryo. This study looked at the effects of maternal diabetes on embryonic growth. Female Quakenbush mice were made diabetic (plasma glucose levels greater than 20 mM) by injection of 190 mg/kg i.p. STZ and were superovulated by standard methods. The blastocysts collected on day 4 from diabetic mothers had 8.5% fewer cells and a 35% lower protein synthetic rate than control embryos. Their cellular protein synthetic rate was 19% less than that in controls. Morulae from diabetic mothers also displayed a reduced protein synthetic rate, but this reduction was not seen in the two-cell embryo. Furthermore, blastocysts cultured in vitro from two-cell embryos from diabetic and control mothers displayed similar protein synthetic rates. This infers that the two-cell embryos from diabetic mothers are normal, and the retardation seen in later development in vivo occurs after the two-cell stage while the embryo is still free in the oviductal and uterine environment. Treatment of the diabetic mice with ultralente insulin every 12 h raised the protein synthetic rate of those blastocysts toward control levels, whereas treatment with lente insulin every 8 h recovered the embryo to the same rate as the control embryos. Because insulin has been shown to be mitogenic and stimulates protein synthesis of morulae and blastocysts in vitro, the absence of insulin in the diabetic mothers may be the cause of the retardation observed in their preimplantation embryos.     

Fertilization and mouse embryo development in the presence of midazolam.

Mouse embryo in vitro development elucidates the effect of a pharmacologic agent on cellular differentiation. Midazolam provides conscious sedation for patients undergoing egg retrieval for in vitro fertilization and is found in patient follicular fluid. Mouse preimplantation embryo formation and development were evaluated in the presence of midazolam. Midazolam was cocultured with two-cell mouse preimplantation embryos over 72 h and injected systemically just before ovulation and coitus. Concentrations to 12.5 micrograms/mL displayed no significant toxic effects on in vitro two-cell-to-blastocyst development. Doses to 35.0 mg/kg did not prevent or impair in vivo fertilization. Midazolam has no adverse effect on in vitro development of two-cell-to-blastocyst-stage embryos nor on in vivo fertilization and cell division at concentrations approximating and exceeding those that ova are exposed to during clinical anesthesia. Midazolam is recommended for use to induce sedation in human in vitro fertilization where association with gametes and zygotes is probable.     

胰岛素释放试验异常对PCOS患者IVF/ICSI结局的预测作用

目的探讨胰岛素释放试验异常对多囊卵巢综合征(PCOS)患者体外受精/卵胞浆内单精子注射-胚胎移植(IVF/ICSI-ET)妊娠结局的预测作用。方法选取就诊于青岛市妇女儿童医院生殖中心行助孕治疗的PCOS患者119例,所有入组的PCOS患者均行口服葡萄糖耐量试验(OGTT)及胰岛素释放试验。根据胰岛素释放试验将PCOS患者分为两组,胰岛素释放试验正常组50例(空腹胰岛素为3.0~25.0mU/L,2h胰岛素低于1h胰岛素,血胰岛素200mU/L),胰岛素释放试验异常组69例。比较2组患者临床结局。结果胰岛素释放试验正常组双原核(2PN)数、优质胚胎数及鲜胚移植时生化妊娠率、胚胎种植率、临床妊娠率明显高于胰岛素释放试验异常组,差异有统计学意义(P0.05)。胰岛素释放试验正常组患者获卵数、成熟卵数有高于胰岛素释放试验异常组的趋势,胰岛素释放试验正常组患者早期流产率有低于胰岛素释放试验异常组的趋势,但差异均无统计学意义(P0.05)。结论胰岛素释放试验可以作为预测PCOS患者IVF/ICSI-ET妊娠结局的良好指标。胰岛素释放试验异常对PCOS患者IVF/ICSI-ET妊娠结局造成不利影响,对于此类患者在进行助孕治疗前最好给予预处理以改善其妊娠结局。