家兔胚胎冻存和胚胎移植初探：第一批胚胎移植兔仔出生

成年杂种家兔，分自然排卵和药物超排卵两组，交配后６３ｈ收集桑椹胚，以４Ｍ甘油和０．２５Ｍ蔗糖为冻存保护剂，置于塑料麦管或安瓿，快速降温并存入液氮，冻存期为５－１６０天，冻存完好率在５０－８０％，两组之间没有显著性差异。受体雌兔自然发情经无效交配及ｈＣＧ注射后，于左侧子宫角移植入４个同步冻存的纯白兔胚，受体兔成功妊娠并出生两只纯白健康仔兔。     

PCOS患者不同子宫内膜准备方案冻胚移植结局分析

目的:探讨冻胚移植失败PCOS患者再次冻胚移植内膜准备的最佳方案。方法:回顾分析2011年8月~2013年7月在青岛市妇女儿童医院生殖中心行人工周期准备内膜冻融胚胎移植的PCOS患者的临床资料,对移植失败或因突破性出血取消周期尚有冻存胚胎的162例PCOS患者实施再次冻胚移植。将患者随机分为GnRHa+人工周期(降调节+人工周期组)、人工周期和诱导排卵方案3组,观察3组患者的年龄、体重指数(BMI)、不孕年限、胚胎冻存时间、突破性出血率、周期取消率、优质胚胎率、移植胚胎数、移植日子宫内膜厚度及类型、内膜增殖时间、内膜转化日血清雌激素浓度、种植率、妊娠率、流产率、异位妊娠率。结果:降调节+人工周期组和诱导排卵组无一例发生突破性出血。诱导排卵组内膜转化日E2平均水平为(2827.33±1148.49)pg/ml,高于其他2组,差异有统计学意义(P0.05);平均子宫内膜厚度为(9.14±0.90)mm,较其他2组增高,但差异无统计学意义(P0.05)。诱导排卵组的B级以下内膜所占比例最低,为9.76%,但差异无统计学意义(P0.05)。降调节+人工周期组的临床妊娠率和着床率分别为50.94%和22.54%,诱导排卵组分别为53.66%和22.77%,与人工周期组(31.03%,13.07%)比较,差异均有统计学意义(P0.05)。结论:对于PCOS患者,初次冻胚移植失败后再次冻胚移植时采用降调节+人工周期方案或诱导排卵方案,可降低突破性出血率,减少周期取消,提高妊娠率和着床率。     

GnRH类似物－Buserelin在体外受精与胚胎移植超排卵（长方案）治疗中的作用

从１９９４年４月至１９９５年７月在体外受精与胚胎移植超排卵中，应用ＧｎＲＨ类似物－Ｂｕｓｅｒｅｌｉｎ及ＦＳＨ／ｈＭＧ长方案治疗９例（Ⅰ组）与单独用ＦＳＨ／ｈＭＧ１２例（Ⅱ组）进行比较。结果：Ⅰ组的ＦＳＨ／ｈＭＧ用量大，用药时间长，其受精率、卵裂率及妊娠数均高于Ⅱ组。卵巢过度刺激综合征（ＯＨＳＳ）发生率明显低于Ⅱ组。本文研究显示了Ｂｕｓｅｒｅｌｉｎ长方案治疗能明显提高体外受精与胚胎移植的效果。     

GnRH类似物—Buserelin在体外受精与胚胎移植超排卵（长方案）治疗…

从１９９４年４月至１９９５年７月在体外受精与胚胎移植超排卵中，应用ＧｎＲＨ类似物－Ｂｕｓｅｒｅｌｉｎ及ＦＳＨ／ｈＭＧ长方案治疗９例与单独用ＦＳＨ／ｈＭＧ１２例进行比较。     

过夜培养对冻融胚胎移植周期妊娠结局的影响

目的:探讨冻融胚胎移植周期中胚胎复苏时间对妊娠结局的影响。方法:回顾分析行冻胚移植(FET)的206对夫妇共248个周期,根据解冻时间的不同分为A组(对照组):排卵后2～3d或注射黄体酮3～4d后解冻,培养2h移植;B组(实验组):提前1d解冻,培养20h移植。比较各组间患者一般资料、复苏胚胎存活率、完整率、临床妊娠率、胚胎着床率和流产率。结果:A、B组间患者年龄、不孕年限、不孕原因、继发不孕所占比例、第3日冷冻胚胎比例、冷冻胚胎数、移植时内膜厚度、优胚数、胚胎复苏存活率、完整率、平均移植胚胎数均未见差异,而B组临床妊娠率和胚胎着床率显著高于A组(46.36%、25.29%vs30.88%、13.54%,P<0.05),组间流产率未见差异。过夜培养后有胚胎生长组临床妊娠率高于无生长组(49.47%vs26.67%,P>0.05)。结论:冻胚移植周期中提前解冻可以改善妊娠结局。     

注射醋酸甲孕酮抑制家兔生育力

本文报告醋酸甲孕酮(Depo-Provera)对家兔生育力抑制的情况及其机理.研究采用19只成年雌性新西兰大白兔,其中4只皮下注射Depo-Provera 20mg,7只皮下注射Depo-Provera 50mg,前者于注射后40～65天,后者于注后15～83天与3只雄兔交配或以人工阴道取得的3只雄兔的精子人工授精,50mg组并给hCG,于交配或授精后一天处死,计数排卵点并取出输卵管,以2ml培养液冲洗,在解剖显微镜下检查冲洗液内卵子是否受精.结果表明皮下注射Depo-Provera 20mg后40～65天家兔排卵仍被抑制;而     

hMG对子宫内膜、卵泡发育及白细胞雌激素受体作用的探讨

目的：探讨ｈＭＧ对子宫内膜、卵泡发育及白血胞雌激素受体含量的变化及其临床意义。方法：应用阴道超声、放免法和放射配体结合法对１８例自然周期和１６例ｈＭＧ促排卵周期的不孕症患者子宫内膜厚度、分型、成熟卵泡数、血浆雌二醇及孕酮浓度和外周血白细胞雌激素受体含量进行测定比较。结果：①ｈＭＧ组围排卵期子宫内膜厚度大于自然周期组（Ｐ＜０．０５）;成熟卵泡数多于自然周期组（Ｐ＜０．０５）;子宫内膜Ａ、Ｂ型回声比例高于自然周期组（Ｐ＜０．０５）;②ｈＭＧ组围排卵期血浆雌二醇、孕酮浓度明显高于自然周期组（Ｐ＜０．０５）;③ｈＭＧ组围排卵期白细胞雌激素受体含量较自然周期组明显升高（Ｐ＜０．０１）。结论：①ｈＭＧ可使促排卵周期成熟卵泡数增加,子宫内膜分型改善,血浆Ｅ２浓度及白细胞雌激素受体含量升高,有改善子宫内膜容受性的作用;②ｈＭＧ可能有致卵泡过早黄素化的作用;③测定白细胞雌激素受体可间接反映子宫内膜雌激素受体的变化,具有无创、可靠、重复性好的优点     

国产hMG诱发排卵的临床研究

以国产ｈＭＧ治疗１０３例无排卵不孕症病人，共治疗１２５个周期，１１０个周期排卵，排卵率８８．０％，２１例获妊娠，妊娠率２０．４％，其中多胎妊娠４例，占３．９％，２５个周期发生卵巢地过度刺激征，发生率２０．０％，结果显示：国产ｈＭＧ的促排卵疗效与国外同类产品相近似，并发症发生率低，不失为临床医生治疗用药选择之一。     

自然周期冻融胚胎移植使用绒促性素诱发排卵后不同胚胎移植时间临床结局比较

目的:比较自然周期冻融胚胎移植(FET)使用绒促性素(HCG)诱发排卵后不同胚胎移植时间的临床结局,探讨自然周期FET时机。方法:回顾性分析2014年1月至2018年7月于河南省人民医院生殖中心行自然周期使用HCG诱导排卵的FET周期,根据移植胚胎的不同分为卵裂期胚胎(n=522)和囊胚期胚胎(n=251)共773个周期。根据移植日距HCG注射日天数进行分组,以HCG注射日为D0,HCG注射后第3天移植卵裂期胚胎为D3-3组;HCG注射后第4天移植卵裂期胚胎为D4-3组;HCG注射后第5天移植卵裂期胚胎为D5-3组。HCG注射后第5天移植囊胚为D5-5组;HCG注射后第6天移植囊胚为D6-5组;HCG注射后第7天移植囊胚为D7-5组。比较各组一般情况和妊娠结局相关指标,并对妊娠结局指标进行多元Logistic回归分析。结果:移植卵裂期胚胎的D4-3组移植日子宫内膜厚度高于D3-3组与D5-3组,差异有统计学意义(P0.05);移植囊胚期胚胎的D7-5组优胚移植周期率显著低于D5-5组与D6-5组,差异有统计学意义(P0.05);将患者体质量指数(BMI)、年龄、移植胚胎数、优胚移植周期率、移植日子宫内膜厚度作为可能对妊娠结局产生影响的混杂因素进行调整后,移植卵裂期胚胎的D3-3组、D4-3组、D5-3组相比较,移植囊胚期胚胎的D5-5组、D6-5组、D7-5组相比较,胚胎种植率、临床妊娠率、异位妊娠率、持续妊娠率、早期流产率差异无统计学意义(P0.05)。结论:在HCG诱导排卵的自然周期FET中,HCG注射后3～5天移植卵裂期胚胎,5～7天移植囊胚期胚胎均可取得相似的临床结局。     

用睾丸酮主动免疫后孕兔血浆促性腺激素和孕激素的浓度

有证据提示在卵泡的正常发育和成熟中雄激素具有重要作用。如用睾丸酮主动免疫兔引致排卵增加。离体条件下雄激素促进颗粒细胞产生孕酮。本文进一步研究了妊娠期用睾丸酮主动免疫后的各种内分泌指标。将16只雌兔任意分为两组,一组用牛血清白蛋白免疫,作为对照;另一组用偶联牛血清白蛋白的睾丸酮免疫(实验组)。免疫四个月后血清睾丸酮抗体滴度高于1∶1,000。在连续四天中与有生育力的雄兔交配,经血清孕酮测定证明5只对照兔及3只实验兔妊娠。在交配前24小时、1小时和交配后     

Electrical activation of rabbit oocytes increases fertilization and embryo development by intracytoplasmic sperm injection using sperm from deceased male

Purpose

We investigated the effect of electrical stimulation on rabbit oocyte activation using intracytoplasmic sperm injection (ICSI) to determine whether viable offspring can be produced from deceased rabbit sperm using ICSI.

Methods

Sperm were collected from a heterozygote GFP male rabbit 5 h after sacrifice and cryopreserved in liquid nitrogen. Mature oocytes were fertilized using ICSI. A series of electrical pulse procedures were used to activate oocytes before and/or after ICSI. Following ICSI, zygotes were cultured in B₂ medium for 4 days or transferred into the oviducts of recipient rabbits at the 2- or 4-cell stage.

Results

The blastocyst formation rate was significantly greater in oocytes that received one or two pulses prior to ICSI compared to controls and other electrically stimulated groups. In the single pulse before ICSI group, 23 % of the blastocysts expressed GFP, which was significantly greater than all other groups. However, those that received treatment before and after, or just following ICSI, showed a significant decrease in embryo survival. Finally, embryos from the single pulse before ICSI group were transferred into recipient female rabbits and a full-term kit was successfully delivered.

Conclusions

One pulse of electrical stimulation prior to sperm injection was an effective method to activate rabbit oocytes for fertilization. Sperm collected from a deceased rabbit is able to produce viable embryos through ISCI that are capable of normal fetal and kit development.     

输卵管积液对小鼠胚胎体外发育的影响

目的 :了解输卵管积液是否具有胚治毒性。方法 :收集兔输卵管液和机械诱导的输卵管积液 ;小鼠超数排卵 ,收集 2 -细胞胚胎 ,随机分配到含有不同浓度输卵管积液、输卵管液 (正常对照 )和培养液 (空白对照 )中培养 ,观察并记录胚胎形态 ,同时测定新形成囊胚的细胞数和有丝分裂指数。结果 :1 0 0 %输卵管积液组囊胚形成率和胚胎孵化率均明显低于两对照组 (P<0 .0 0 1 ) ;而低浓度 (2 0 %和 1 0 % )积液组囊胚形成率和胚胎孵化率与两对照组相比 ,均无明显差异。各实验组囊胚的细胞数和有丝分裂指数均明显低于两对照组(P<0 .0 5)。结论 :机械诱导的输卵管积液 ,会影响早期种植前胚胎质量     

Precoital single doses of delta9-tetrahydrocannabinol block ovulation in the rabbit.

delta9-Tetrahydrocannabinol (delta9-THC) inhibits pituitary gonadotropin secretion in castrated rhesus monkeys. Also, delta9-THC blocks the ovulatory reflux in rabbits. We report now the dose-response relationship of precoital single doses of delta9-THC on luteinizing hormone (LH) release and ovulation in the rabbit. Forty-five female rabbits in estrous were divided into nine groups of five animals. Groups 1 to 5 received a single intramuscular dose of delta9-THC (5,2.5, 1.25, 0.612, and 0.306 mg/kg, respectively) 2 hours before mating; animals of group 6 received vehicle only. In animals of groups 7 and 8 ovulation was induced with 100 IU of human chorionic gonadotropic (hCG), given intravenously 2 hours after the administration of delta9-THC (5 mg/kg) or vehicle. Rabbit luteinizing hormone (rLH) was measured in plasma 90 to 120 minutes after coitus or hCG administration. After the injection of 5 mg of delta9-THC, luteinizing hormone-releasing factor (LH-RF) (20 microgram intravenously) was administered to the animals of group 9. All animals of groups 6, 7, and 8 ovulated. A dose-response curve was observed in the animals treated with delta9-THC and natural mating. Whereas none of the animals treated with 5 or 2.5 mg/kg ovulated, one of the group treated with 1.25 mg/kg, two of the group treated with 0.612 mg/kg, and all treated with 0.312 mg/kg ovulated. Ovulations correlated with postcoital levels of rLH. All animals of group 9 ovulated, indicating that the site of action of delta9-THC is suprapituitary, probably hypothalamic.     

In vitro fertilization and embryo transfer of pre-ovulatory rabbit oocytes

The objective of this study was to develop an effective and successful technique of in vitro fertilization in rabbits that could lead us to start treatment of infertile couples by using extracorporal fertilization. Pre-ovulatory oocytes were harvested from follicles of virgin does 9 h after induction of ovulation by hCG injection, and incubated with in vivo capacitated sperm for 5 h. An 82% in vitro fertilization rate was achieved. The fertilized ova were cultivated for an additional 20, 44 and 68 h, and until they reached the hatched blastocyst stage. Most of these embryos (439) were transferred to 42 pseudopregnant recipients, asynchronous in the sense that they had been given hCG injections 0, 6, 12, 18, 24, 36 and 42 h later than the rabbits from which the ova had been obtained. With this type of synchronicity implantation rates of 31, 78, 52, 33, 52, 33, 51 and 10% were achieved, respectively. One hundred and twenty-eight embryos were not transferred to recipients but cultured in Ham's F-10 medium supplemented with 20% FCS. 77% of these embryos reached the stage of hatched blastocyst. A total of 143 young were born after 28-32 days of gestation and all are fertile till the third generation. Our results were obtained in a consecutive series of experiments, and demonstrated that in vitro fertilization can be made to work quite well in the rabbit, which in the early stages of embryogenesis has sufficient similarity to the human to make it a useful model for those wishing to acquire techniques applicable to human fertilization in vitro and embryo transfer.     

Role of progesterone in the ovulatory process of PMSG/hCG treated immature female rats

To determine exactly when progesterone (P) acts as a most important mediator in the ovulatory process, a dose of 10mg/kg of RU486, an antiprogesterone, was administered to PMSG/hCG treated immature female rats (22 days old) at 0 (RU0 groups), 2 (RU2 group), 4,6,8 or 10 hours after an hCG injection, respectively. In the RU0 groups, the ovulatory effect of P was investigated at 0.2 (P2 group), 4 (P4 group), 6 or 8 hours after the hCG injection. Ovulation rates were calculated 24 hours after the hCG injections. Serum P and estradiol (E2) concentrations in the RU0 groups, the P2 group and the control rats (C group), were determined. The results were as follows. 1) The numbers of ova in the RU0, RU2 and RU4 group rats were significantly (p less than 0.01, p less than 0.01 and p less than 0.01) lower than that in the C group rats. 2) In the P2 and P4 groups, the numbers of ova were significantly (p less than 0.01 and p less than 0.01) increased compared with the RU0 group and returned to the control range. 3) In the RU0 group, the serum P and E2 concentrations within 8 hours after an hCG injection did not show any significant differences compared with the C group. In contrast, in the P2 group, the P concentrations at 4 and 6 hours after hCG increased significantly (p less than 0.01 and p less than 0.01) compared to the corresponding RU0 group. These results clarified an indispensable role of P in the ovulatory process within 6 hours after an hCG injection.     

18-甲基炔诺酮抗家兔排卵作用机理的初步分析

本实验使用家兔诱导排卵动物模型,分别观察了消旋18-甲基炔诺酮和左旋18-甲基炔诺酮对于用铜盐、由PMSG和hCG以及由LH-RH诱导的家兔排卵的影响。实验分三组:一组于实验第1～3天皮下注射雌二醇(每只100μg/天),第4天经耳缘静脉注射1%醋酸酮(0.4ml/kg),24小时后处死并检查排卵点。二组于实验第1～2天皮下注射雌二醇,第4天皮下注射一次LH-RH(12.5μg/只),48小时后处死并观察和记数排卵点。三组于实验第2天皮下注射PMSG(12.5u),第4天经耳缘静脉注射50iuhCG,48小时后处死并检查排卵点。各组中的实验组于第1～4天肌肉注射消旋18-甲基炔诺酮或左旋18-甲基炔诺酮(每只8mg/天)对照组给予等量溶媒。结果表明,两种18-甲基炔诺酮皆明显抑制由铜盐诱导的家兔排卵,抗排卵率分别为83.3%和66.7%。然而,两者对于由LH-RH和由PMSG与hCG诱导的家兔排卵皆无明显影响。以上结果提示,消旋18-甲基炔诺酮和左旋18-甲基炔诺酮的抗排卵作用可能是通过干扰下丘脑LH-RH的释放而实现的,并非由于在垂体水平拮抗LH-RH的作用或在性腺水平上拮抗促性腺激素作用的结果。     

Controlled ovulation in the marmoset monkey (Callithrix jacchus) with human chorionic gonadotropin following prostaglandin-induced luteal regression

Controlled induction of ovulation in the marmoset monkey was attempted with a single injection of human chorionic gonadotropin (hCG; 50 IU) given on day 7 after prostaglandin-induced luteal regression. Animals given hCG (n = 12) ovulated within a 2-day period (days 9 and 10 after prostaglandin) compared with a 4-day period (days 9 to 12) in the control group (n = 12). The mean interval to ovulation was similar in both groups. There was no difference in the timing of the preovulatory estradiol (E2) peak between groups, although E2 levels on the day of hCG injection were lower than in controls on the day of the onset of the luteinizing hormone surge. All animals given hCG ovulated and 11 of 12 became pregnant. Ten of 11 embryos recovered surgically from six of these animals were normal blastocysts; 5 of the remaining 6 animals carried pregnancies to term. The results are of practical importance for experiments involving follicular and oocyte maturation and the collection and transfer of embryos.     

促性腺激素释放激素激动剂长、短方案在体外受精-胚胎移植中的比较性研究

目的　探讨促性腺激素释放激素激动剂（GnRH-a）长、短方案控制性超排卵在体外受精-胚胎移植（IVF-ET）中的疗效。方法　将2000年1～5月进行IVF和单精子卵胞浆注射（ICSI）助孕的不孕患者，按病历奇、偶数编号分为GnRH-a长方案组（55例）和短方案组（54例）。长方案组从使用促性腺激素（Gn）治疗周期前的黄体中期开始使用GnRH-a0.9mg/d，至垂体完全降调节后，加用Gn；短方案组从月经第2天开始使用GnRH-a0.45mg/d，同时加用Gn。两组均在优势卵泡达18mm时，肌内注射人绒毛膜促性腺激素（hCG），36h后取卵行IVF及ICSI。结果　长方案组较短方案组，使用Gn前血清促卵泡激素（FSH）和黄体生成素（LH）水平降低［（4.4±1.2）IU/L比（6.3±1.7）IU/L,（2.7±1.5）IU/L比（4.4±2.8）IU/L,P<0.01］；注射hCG前血清雌二醇（E2）和LH水平降低［（7119±3584）pmol/L比（9523±3587）pmol/L，（1.0±1.0）IU/L比（4.0±3.4）IU/L,P<0.01］；每个卵子E2水平降低［（610±315）pmol/L比（935±450）pmol/L,P<0.01］；Gn用量增多［（28.0±8.6）支比（23.4±8.7）支,P<0.01］，用药时间增长［（11.1±1.2）d比（10.1±1.5）d,P<0.01］；两组平均获卵数、第2次成熟分裂中期卵子数、受精卵数、卵裂数、优质胚胎数及妊娠率无显著差异。结论　在IVF-ET，GnRH-a长、短方案能获得相同的控制性超排卵效果，且GnRH-a短方案能减少Gn用量和缩短治疗时间。     

Effect of RU486 on collagenolytic enzyme activities in immature rat ovary

To investigate the role of progesterone (P) in the ovulatory process, RU486 (RU) (10mg/kg), an antiprogesterone, was administered to PMS-hCG-treated immature female rats (22 days old). As for the collagenolytic enzymes (CE), the activities of BANA hydrolase and DNP peptidase were measured by using synthetic substrates alpha-N-benzoyl-DL-arginine-2-naphthylamide HCl and DNP Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH, respectively. Serial assays were performed at 0, 2, 4, 6, 7, 8, 9, 10 and 12 hours after the hCG injection. In the control, the CE activities were significantly increased after the hCG injection at 8-9 hours in BANA hydrolase and 7-10 hours in DNP peptidase, respectively. By administering RU concomitant with hCG, the preovulatory increase in the CE activities was totally suppressed. Following the P(10mg/kg) injection at 4 hours after hCG and RU injections, the CE activities showed the greatest recovery, approaching the control range. In the ovulatory process of the rat ovary, P plays an important role through its regulatory effects on the collagenolytic enzymes, and this P effect seems to be most prominent around 4 hours after a LH surge.     

Blastomere multinucleation: Contributing factors and effects on embryo development and clinical outcome

Introduction.?The aim of the study was to discover which intracytoplasmic sperm injection (ICSI) cycle parameters could influence the presence of multinucleated blastomeres (MNBs) and how ICSI outcomes are influenced by this event.

Material and methods.?Embryos derived from normally fertilised oocytes were divided into two groups: embryos that had only mononucleated blastomeres (NBs group n = 2818) and embryos that had at least one multinucleated blastomere (MNB group, n = 404). The effects of ICSI cycle factors on multinucleation were investigated and embryo development was compared between the groups. The cycles were also split into those in which only NB embryos were present (NB cycles, n = 298) and cycles in which MNB embryos were present (MNB cycles, n = 203). ICSI outcomes were compared between the groups.

Results.?A higher incidence of MNB embryos arose in pituitary blockage with gonadotropin-releasing hormone agonists, male factor infertility and in cycles with higher number of retrieved oocytes. Embryos that had only one affected blastomere showed greater development than embryos with more than one affected blastomere. Finally, the implantation rate decreased when MNB embryos were transferred.

Conclusion.?Multinucleation events may be affected by aspects of the ICSI cycle and compromise embryo quality and implantation rate.