不同操作温度对小鼠卵母细胞体外受精的影响

目的：观察不同操作温度对小鼠体外受精以及后续胚胎发育的影响,探讨较合适的实验室受精及操作温度,为提高人类体外受精-胚胎移植（IVF-ET）技术提供实验依据。方法对IVF实验室温度共设置19℃、21℃、23℃、25℃、27℃、29℃及31℃温度点,选择10~12周龄（20~25g）的ICR雌性小鼠及雄性小鼠168只。每个温度点设自然受精组和体外受精组,随机分配雌鼠8只、雄鼠4只。在上述实验室温度下对小鼠自然受精卵团与体外受精卵团进行操作、观察,并统计受精率、卵裂率、囊胚形成率及囊胚孵出率。结果体外受精组中25℃组与19℃、21℃、29℃、31℃四组间比较受精率（94.83%、52.69%、78.98%、82.76%、66.67%）、囊胚形成率（87.12%、54.17%、68.75%、70%、56.56%）、囊胚孵出率（86.62%、46.15%、66.23%、67.857%、51.78%）有统计学差异（P＜0.05）,尤以19℃、31℃差异明显;体外受精组中25℃组与19℃、21℃、31℃三组间比较卵裂率（98.79%、81.82%、90.32%、89.83%）有统计学差异（P＜0.05）,尤以19℃、31℃差异明显;顺次两组间比较,19℃与21℃、29℃与31℃组受精率、囊胚形成率、囊胚孵出率有统计学差异（P＜0.05）;组内比较19℃、21℃、29℃、31℃组各自的受精率、卵裂率、囊胚形成率、囊胚孵出率有统计学差异（P＜0.05）,尤以19℃、31℃组差异明显。结论实验室温度在23℃~27℃之间时,小鼠卵母细胞体外受精组与自然受精组在受精率、卵裂率、囊胚形成率和囊胚孵出率等方面较其他温度具有显著优势,提示23℃~27℃为较合适的实验室受精及胚胎操作温度范围。     

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。     

注核方法对大鼠-小鼠种间体细胞核移植重组胚发育的影响

目的 :探讨胞质内直接注核法和电融合法对大鼠 -小鼠种间体细胞核移植重组胚发育的影响。方法 :采用血清饥饿法同步化处理的 SD大鼠成纤维细胞作为供体细胞 ,常规超排 KM小鼠获得卵母细胞 ,采用盲吸法去核 ,分别采用直接注核法和电融合法构建重组胚胎 ,6- DMAP+ CCB+乙醇激活重组胚 ,CZB培养液中培养。结果 :电场强度 ( V/cm)和脉冲 ( ms)为 :1 5 0 0 /30 ;1 5 0 0 /40 ;1 80 0 /30 ;1 80 0 /40时 ,融合率为 :81 .5 % ;83.3% ;77.6% ;82 .0 %。直接注核法和电融合法构建重组胚的卵裂率分别为 33.1 %和 2 8.9% ,桑椹胚发育率为 1 7.8%和 1 5 .5 %。结论 :电场强度为 1 5 0 0～ 1 80 0 V/cm,脉冲为 30 ms或 40 ms时 ,都能获得较高的融合率 ;直接注核法构建的核移植重组胚胎的卵裂率要比电融合法略高 ,但统计学分析差异不显著     

咽后壁滴注柴油机尾气颗粒物对雌性小鼠生殖功能的影响

目的 观察柴油机尾气颗粒物(DEP)亚急性暴露对雌性小鼠生殖功能的影响.方法 将168只ICR( Institute of Cancer Research)雌性小鼠按数字表法随机分为低(B)、中(C)、高(D)剂量DEP暴露组和对照组(A),每组42只,分别向B、C、D组小鼠咽后壁滴注0.8、3.0、12.0 μg/μl DEP混悬液及PBS各30μl,每3天重复滴注1次,共4次.测量小鼠体重及卵巢重量,计算卵巢重量/体重,检测卵母细胞的存活率、胚泡破裂百分率、第一极体释放百分率、受精率、线粒体DNA含量和超微结构的变化.结果 A、B、C、D组卵巢重量/体重分别为(15.4±7.3) ×10-5、(14.1±6.8) ×10-5、(8.2±0.7) ×10-5和(7.2±2.5)×10-5 (F=3.841,P＜0.05).A、B、C、D组加入精子后第48小时,卵母细胞存活率分别为64.3％、56.8％、39.5％、32.9％(x2=21.575,P＜0.05),第一极体释放率分别为75.5％、65.3％、37.0％、27.1％ (x2 =52.772,P＜0.05),2细胞胚胎率分别为27.9％、39.1％、17.6％、12.5％(x2=20.148,P＜0.05),2细胞以上胚胎率分别为45.3％、32.2％、12.5％、13.9％ (x2 =32.135,P＜0.05).A、B、C、D组卵母细胞线粒体DNA的拷贝数对数值分别为6.54±0.13、6.48±0.09、5.57±0.15和5.41±0.07(F =89.241,P＜0.05).C、D组卵母细胞许多线粒体肿胀、空泡化,A、B组卵母细胞结构无明显改变.结论 DEP具有雌性生殖毒性.亚急性DEP暴露可对卵巢及卵母细胞造成不同程度损伤,降低卵巢功能和卵母细胞受精能力.     

Oogonia and oocytes degeneration and the nutritive macrophages in the process of the development of the ovary in embryos of the domestic pig (Sus scrofa dom. L.)

Summary In the development the embryonic ovary numerous oogonia and oocytes degenerate. A histochemical study was carried out on the degenerated oogonia and oocytes in different stages of differentiation and development of the ovary in the pig embryos. In the oogonia the degenerative changes include chiefly the chromatin in interphasic and mitotic nuclei. The most numerous degenerations of the nuclei are in meiotic prophase. The degenerative process involves the changes in nuclear volume. Both, the large swollen nuclei, with chromosomes displaced and clustered at one pole, and also the small pycnotic nuclei are observed. Different kinds of oocytes degenerations are discussed.Special attention was paid to the gonadal cord cells and their role in nutrition of gonocytes and in the follicle formation. Some of the cord cells are transformed into macrophages, which ingeste degenerated oocytes. The supposed nutritive functions of these macrophages (nutritive macrophages) are discussed.     

未成熟卵母细胞在TCM199与P1培养体系体外成熟的比较

目的 探讨人未成熟卵母细胞适宜的培养体系及培养时间.方法 将卵母细胞-卵丘复合体(0CC)332枚和裸卵(DO)393枚随机置入TCM199和P1两种培养体系体外成熟.OCC体外培养48 h,观察其成熟情况.DO分别在24、30、48 h观察第1极体排出情况,计算体外成熟率.OCC和DO成熟后称为成熟的卵母细胞,成熟的卵母细胞行精子卵浆内注射技术(ICSI)受精,比较其在两种培养体系的受精率、卵裂率及囊胚形成率.结果 在TCM199和P1培养体系中,OCC的成熟率、成熟后成为成熟的卵母细胞的受精率和卵裂率的差异无统计学意义(P均>0.05),但成熟的卵母细胞的囊胚形成率为53.7%比37.8%(P<0.05).DO在TCM199和P1培养体系中体外培养24、30、48 h时成熟率的差异无统计学意义;由DO成熟后形成的成熟的卵母细胞在P1培养体系中的受精率、卵裂率和囊胚形成率高于其在TCM199培养体系中,但只有受精率差异具有统计学意义(73.5%比62.9%,P<0.05);DO在两种培养体系中体外培养48 h和30 h的成熟率均明显高于24 h(P<0.05),但48 h与30 h的成熟率相比无差异.结论 OCC适宜在TCM199培养体系的培养;DO适官在P1培养体系的培养.DO体外成熟形成成熟的卵母细胞的时间可以缩短至30 h.     

Controlled ovarian hyperstimulation (COH) parameters associated with euploidy rates in donor oocytes

Although oocyte donors are young and are expected to provide a high rate of euploid oocytes, significant differences of euploidy rates for donor embryos exist between different IVF centers (1). Laboratory conditions can lead to differences of euploidy (2,3,4,5,6,7); but, the role of COH has not been investigated. In this study, we investigated whether euploidy rates in the embryos created from donor oocytes are influenced by controlled ovarian hyperstimulation parameters used during assisted reproduction. Euploidy rates in egg donor cycles undergoing PGT-A (N = 423) were examined retrospectively for associations with donor age, gonadotropin doses (dose per day), the fraction of gonadotropin provided by hMG (F(hMG)), days of stimulation, estradiol per mature oocyte on day of trigger, number of mature oocytes retrieved, number of embryos biopsied, incidence of euploidy and physician of record. Differences in euploidy rates between physicians were examined using analysis of variance. The proportion of euploid embryos per donor cycle was examined for associations with COH parameters using pairwise post-hoc comparisons, adjusting for multiple testing. The set of variables from this analysis was then submitted to a principal component analysis. Linear regression analysis was used to assess the relationships between stimulation parameters and the incidence of euploidy (the dependent variable). Euploidy rates and cycle parameters varied significantly among treating physicians. Euploidy rates (expressed as a fraction of biopsied embryos) were associated (p = 0.01) only with the F(hMG) but not with the number of MII retrieved or other variables. On the other hand, the number of euploid embryos (in contrast to the euploidy rate) was associated with the number of MII produced. Donor euploidy rates are significantly associated with the fraction of total gonadotropin comprising human menopausal gonadotropin (or F(hMG)) during controlled ovarian hyperstimulation but are not associated with other cycle parameters. The study provides the first suggestion that patient stimulation parameters can affect the incidence of euploidy in embryos generated through the use of standard assisted reproductive techniques. The study is limited by its retrospective approach and because the aCGH analysis used is less sensitive than more recent NGS technology. Further, it provides a suggestion that the use of hMG is beneficial for obtaining euploid embryos.     

卵母细胞单精子显微注射治疗男性因素及不明原因不育156个周期临床分析

目的阶段性回顾应用卵母细胞单精子显微注射（ＩＣＳＩ）技术治疗严重男性因素及不明原因不育的临床效果。方法收集１９９５年１２月２０日至１９９８年１月１６日,在我院生殖医学研究中心接受ＩＣＳＩ技术治疗的严重男性因素及不明原因不育患者１４０对（１５６个周期）为研究对象,以１９９７年５月２８日为分界点,将其前的７４个周期划为第一阶段,其后的８２个周期为第二阶段,分析比较两阶段的临床结果。采用常规超排卵方案治疗,成熟卵母细胞行单精子显微注射。没有无核碎片及无核碎片＜２０％为Ａ类胚胎。结果两个阶段的成熟卵母细胞数和卵裂率差异无显著性,但注射后存活卵子数、正常受精数、卵裂球的质量和临床妊娠率,第二阶段分别为７０３个（８９５％）、４８７个（６９３％）、Ａ类胚胎占７４２％和３６６％,较第一阶段的５９２个（８５１％）、３８８个（６５５％）、Ａ类胚胎占６６０％和２７０％,有显著性提高。结论ＩＣＳＩ的操作直接影响胚胎质量,随着ＩＣＳＩ技术的不断成熟和完善,其在治疗男性因素及不明原因不育中的作用是肯定的     

对常规IVF授精后4小时未排出第二极体的卵母细胞行补救ICSI

目的比较常规IVF授精后4h和20h补行卵胞浆内单精子注射（rescue ICSI）的效果。方法回顾分析2006年1月-2007年12月在我院生殖医学中心接受常规IVF治疗的21例非男性因素不育患者,授精后4h行补救ICSI,并与另组实施的18个常规IVF授精后20h行补救ICSI周期进行比较。结果 4h补救ICSI组的受精率为76.3%,高于20h补救ICSI组（53.0%）,有统计学意义（P〈0.05）。4h补救ICSI组中11例临床妊娠（52.3%）,其中2个双胎,8个单胎和一个流产。而20h补救ICSI组无一例妊娠。结论和授精后20h行补救ICSI相比,授精后4h行补救ICSI可以获得较好的受精率、妊娠率和种植率。