Embryo transfer and related techniques in domestic animals, and their implications for human medicine

This report was commissioned by the Canadian government on therelevance of new techniques in animal embryology to the social,ethical and clinical applications of assisted human reproduction.It briefly describes the history of animal breeding, and theregulation of the female reproductive tract, ovulation and fertilizationin laboratory and veterinary species. Embryo transfer is describedin detail, including the synchronization of reproductive cycles,superovulation and embryo growth in vitro. Methods of experimentalembryology, including bisection, sexing of spermatozoa and embryos,cloning and gene therapy are described. The relevance of thesestudies to human IVF are considered briefly.     

The influence of in-vitro culture versus stimulated and untreated oviductal environment on mouse embryo development and implantation.

A prospective randomised study was performed to evaluate stimulated versus natural oviductal environment in comparison with in-vitro culture for the developmental capacity of mouse embryos. Therefore, embryos of superovulated F1 hybrid CBAxC57Bl females were collected at 17, 22, 41 and 46 h after human chorionic gonadotrophin treatment and randomly divided into five groups. They were either transferred immediately to untreated pseudopregnant females, cultured in vitro for 5, 24 or 29 h before transfer, or cultured in vitro for 96 h to blastocysts. The transfers resulted in an impaired implantation (P < 0.001) and a lower numbers of living fetuses (P < 0.001) when embryos had been exposed longer to the stimulated oviductal environment. Similar results were obtained after a longer period of in-vitro culture (P < 0.05). However when embryos were flushed earlier from the superovulated mice and cultured longer in-vitro until the transfer was performed, the implantation rate was improved (P < 0.01). Blastocyst development, however, was better (P < 0.001) when embryos were flushed later. In conclusion, the stimulated oviductal environment impairs the developmental capacity of embryos in comparison with untreated pseudopregnant females. In-vitro culture is also suboptimal but better than the stimulated oviductal environment.     

不同时间注射PMSG和HCG对小鼠超排卵效果的影响

目的探讨不同时间注射孕马血清激素（PMSG）和人绒毛膜促性腺激素（HCG）对小鼠超排卵效果的影响。方法选用40只6周龄F1昆明小鼠,随机分成4组,分别在同一天的11：00,13：00,15：00,17：00注射PMSG5IU,48h后注射HCG5IU,然后与公鼠合笼;次日晨检查阴栓,阳性者于当日10：00处死取卵,计数受精卵的数量。结果11：00,13：00,15：00和17：00组平均每只获取可用受精卵9．8枚、24．8枚、11．5枚、4．7枚。结论在13：00注射PMSG,48h后注射HCG对小鼠超排效果最好。     

卵巢过度刺激综合征与血管内皮生长因子关系研究

[目的]探讨在超促排卵周期中体外受精患者血管内皮生长因子（VEGF）浓度与卵巢过度刺激综合征（OHSS）发生的相关性及预测价值。[方法]选择60例接受体外受精及胚胎移植者，采用酶联免疫吸附测定法分别测定HCG注射前和注射后取卵日血清及卵泡液中的VEGF浓度。[结果]取卵日OHSS组患者血清和卵泡液中的VEGF浓度均高于高危组及对照组。[结论]VEGF与OHSS的发生密切相关，测定取卵日血清和卵泡液中VEGF的浓度对OHSS的发生有预测价值。     

两种修正达必佳长方案在IVF-ET中的应用

目的:比较两种修正的达必佳长方案在体外受精-胚胎移植(IVF-ET)和卵母细胞单精子显微注射(ICSI)中的作用。方法:将行IVF-ET和ICSI的512个治疗周期按其停用达必佳的时间分为两组,比较其在诱发排卵中的效果:A组(早停组):上次月经第21天开始隔日一次皮下注射达必佳0.1mg至Gn第4天;B组(改良组):上次月经第21天开始隔日一次皮下注射达必佳0.1mg至注射HCG日。结果:两组Gn天数、ET评分、生化妊娠率、临床妊娠率、早期流产率、OHSS发生率、LH峰发生率比较无显著性差异(P>0.05);A组达必佳支数明显少于B组(P<0.01),且受精率、卵裂率、M2卵母细胞率高于B组(P<0.01)。结论:Gn第4天停用达必佳即可有效抑制内源性LH峰,且能减少达必佳用量,获得与改良长方案相同的临床效果。     

多剂量促性腺激素释放激素拮抗剂在体外受精-胚胎移植促超排卵中的应用效果

[目的]探讨促性腺激素释放激素拮抗剂(GnRH-Ant)改良多剂量应用方案在体外受精-胚胎移植(IVF-ET)促超排卵中的使用效果.[方法]回顾性比较分析本中心2006年1～4月接受IVF-ET助孕治疗的患者中采用GnRH-Ant改良多剂量方案应用于26例年龄偏大的患者和采用促性腺激素释放激素激动剂(GnRH-a)长方案降调节方案应用于患者41例,观察其临床效果.[结果]两组促性腺激素的使用天数、受精率、优胚数、HCG日内膜厚度和E2值、流产率、妊娠率分别为(8.58±1.84)d和(7.71±2.06)d、88.17±17.96%和88.91±14.55%、(3.23±1.48) 个和(3.78±2.31)个、(9.81±1.79)mm和(10.37±1.55)mm、(7060.27±4602.02)pmol/L和(7481.85±3852.84)pmol/L、11.54%和14.63%、50%和39.02%,两组之间比较无显著性差异;两组患者年龄、不孕年限、促性腺激素使用量、HCG日LH值、获卵数、MII卵子数分别为(34.73±15.46)和(28.76±3.08)岁、(6.54±4.11)和(4.2±2.08)年、(34.73 ±15.46 ) 支和(19.32±10.45)支、(3.14±3.2 6)IU/L 和(1.21±1.21)IU/L、(6.77 ±3.09) 个和(9.66±3.58)个、6.23±2.79和8.88±3.60个,两组之间比较有显著性差异.[结论]GnRH-Ant联合促性腺激素促超排卵方案较GnRH-a长方案同样有效,尤其对于年龄较大患者比较适用,并可减轻患者的费用.     

比利时蓝白花肉牛超数排卵及胚胎移植试验

用FSH +Ctoprostenol药物组合 ,超排处理 1 7头次比利时蓝白花肉牛 ,共采卵 70枚 (头均 4.1 2± 2 .74) ,可用胚 47枚 (头均 2 .76± 2 .30 ) ;以非手术方法移植 34头受体 ,妊娠 1 3头 ,妊娠率 38.2 % ,获 8头犊牛     

Optimization of superovulation in the reproductively mature mouse

Optimum gonadotropin doses and chronology were established for the induction of superovulation in sexually mature hybrid mice (BALB/cBy×C57BL/6By). A regime of 12 IU pregnant mares' serum gonadotropin (PMSG), followed 48 hr later by 20 IU human chorionic gonadotropin (hCG) administered 1 hr before the midpoint of the light cycle (1200), gave the maximum ovulatory response. There was no evidence that endogenous luteinizing hormone influenced the superovulation response to exogenous gonadotropins. Fewer than 50% of zygotes reached the blastocyst stage (90–93 hr post hCG), with the greatest rate of loss at the two-to four-cell stage. Litter size following superovulation was 19.6±0.9. There was no significant difference between the number of blastocysts observed and litter size. Similarly, counts of mature follicles in ovaries prior to hCG stimulation were not significantly greater than the number of secondary oocytes that subsequently ovulated. These data indicate that standard superovulation protocols may require finetuning to maximize productivity and confirm that embryo loss is greatest between the first cleavage division and blastocyst formation.     

Impact of gonadotrophin dose on pre-embryo recovery and development in superovulated mice

A dose—response analysis was performed by stimulatingmice with Humegon, which contains approximately equal amountsof follicle stimulating hormone (FSH) and luteinizing hormone(LH). The effect of increasing stimulation was assessed by monitoringthe number and developmental stages of preembryos flushed fromthe tubes, and the developmental potency of these pre-embryosafter culturing for 72 h. Increasing the stimulation doses resultedin an increased recovery of 2-cell pre-embryos. A maximal plateauof 40–50 2-cell pre-embryos was reached after stimulationwith 15–30 IU FSH/LH. Higher stimulation doses up to 50IU FSH/LH showed no further increase in pre-embryo recovery.Increased stimulation doses did not affect the number of 1-cellpre-embryos or the number of pre-embryos which developed furtherthan the 2-cell stage. An average of 88 ± 1.7% of theflushed pre-embryos were 2-cell pre-embryos. The fraction offlushed pre-embryos which developed into blastocysts after 72h of culturing was constant at all stimulation doses, with anaverage of 81 ± 1.9%. A maximal plateau of 35–40blastocysts was reached after stimulating with 15–30 IUFSH/LH. The group of pre-embryos at less advanced developmentalstages and the group of degenerated pre-embryos showed a smallbut dose dependent increase in number. In conclusion, we foundthat increasing doses of Humegon resulted in an increased recoveryof pre-embryos capable of preimplantation development. Thisincreased recovery occurred despite the high level of LH containedin this FSH/LH stimulation.     

Superovulation of mice with human menopausal gonadotropin or pure follicle-stimulating hormone in combination with human chorionic gonadotropin and the effects of oocyte aging on in vitro fertilization

The response of female mice of F₁ hybrids (CBA×C57/BL) to superovulatory doses of human menopausal gonadotropin (hMG) or pure follicle-stimulating hormone (FSH) in combination with human chorionic gonadotropin (hCG) was studied. Furthermore, the effect of oocyte aging in vivo on the subsequent rate of fertilization in vitro was also investigated. The oocytes were collected at 12, 18, and 24 hr after hCG injection and in vitro fertilization (IVF) was carried out in T6 medium. A higher proportion of animals responded to hMG stimulation (32/70) compared to pure FSH (15/66). Furthermore, hMG gave a higher oocyte recovery (454/32) than pure FSH (77/15). Fertilization rates of 57.8, 51.5, and 53.5% were obtained for the 12-, 18-, and 24-hr groups, respectively, after correction for parthenogenetic division of oocytes in the controls. No significant differences in fertilization rates were observed among the three time intervals used in recovering oocytes. However, as the degeneration and parthenogenetic division increased with the delay in collection of oocytes, 12 hr post-hCG injection was the best time to collect oocytes to obtain optimum results in in vitro fertilization.