两种不同胚胎培养模式对体外胚胎发育和妊娠结局的影响

目的:比较2种胚胎培养模式对胚胎质量和妊娠结局的影响。方法:收集首次行IVF-ET治疗、年龄38岁、采用长方案促排卵、常规IVF授精、正常受精卵数3枚且第3日有新鲜胚胎移植的518个周期的临床资料,根据正常受精卵采用的培养模式分成单胚胎培养组(n=254)和胚胎群组培养组(n=264)。分析患者的基本资料、胚胎发育情况以及妊娠结局。结果:患者的年龄、不孕年限、基础卵泡刺激素(FSH)、Gn用量、h CG注射日内膜厚度以及各种不孕因素的比例等参数组间差异均无统计学意义(P0.05);平均获卵数、成熟率、2PN率等指标组间也均无统计学差异(P0.05),但胚胎群组培养组的卵裂率、第3日优质胚胎率、剩余胚胎可利用囊胚形成率均明显高于单胚胎培养组,差异有统计学意义(P0.05);胚胎群组培养组的种植率和临床妊娠率高于单胚胎培养组,但差异无统计学意义(P0.05)。结论:采用胚胎群组培养模式可以得到更多的优质胚胎和更高的囊胚形成率,推荐可用于日常IVF治疗周期中的胚胎培养。     

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。     

常规体外受精-胚胎移植周期中改行未成熟卵母细胞体外培养的临床研究

目的 探讨常规体外受精-胚胎移植(IVF-ET)周期中改行未成熟卵母细胞体外培养(IVM)的临床疗效果.方法 回顾性分析2008年1月至2009年6月在温州医学院附属第一医院生殖医学中心行常规IVF治疗,在促排卵5-7 d后,双侧卵泡数目≥20个,或用药8-13 d后,卵泡发育缓慢且两侧卵泡数日≥15个,平均最大卵泡直径≤12 mm的155个周期,根据患者意愿,即刻停药取卵、改行IVM治疗60个周期(A组),继续按照IVF常规治疗95个周期(B组).比较两组促性腺激素(Gn)的用量、周期取消率、平均获卵数、受精率、优质胚胎率、移植周期数、卵巢过度刺激综合征(OHSS)的发生率及妊娠结局.结果 周期移植率A组为92%(55/60),B组为63%(60/95),两组比较,差异有统计学意义(P<0.05).平均Gn用昔、平均获卵数、卵裂率、OHSS发生率A组分别为(1030±468)U、(10±6)个、82.2%(231/281)、0,B组分别为(1544±338)U、(14±4)个、94.0%(502/534)、35%(21/60),两组比较,筹异均有统计学意义(P<0.05);两组的受精率和优质胚胎率比较,差异无统计学意义(P>0.05).A组29例临床妊娠,每移植周期的临床妊娠率为53%(29/55),多胎妊娠发生率为14%(4/29);B组28例临床妊娠,每移植周期的临床妊娠率为47%(28/60),多胎妊娠发牛率为32%(9/28);两组比较,差异无统计学意义(P>0.05).结论 常规IVF周期巾改行IVM,不仅可以避免OHSS的发生和减少促排卵药物的使用,还可获得较高的临床妊娠率.     

体外受精结合未成熟卵母细胞体外培养的临床应用研究

目的探讨常规体外受精(IVF)结合未成熟卵母细胞体外培养(IVM)的临床应用价值。方法对2004年6月至2006年3月在沈阳东方医疗集团菁华医院就诊的多囊卵巢综合征(PCOS)、既往和本次超促排卵用药反应不良的患者,在自然周期或者降调节超排用药周期10～14d超声监测,如有1～2个直径≥14mm的优势卵泡发育,其他卵泡均<10mm,则注射人绒毛膜促性腺激素(HCG)10000IU,36h后将优势卵泡和小卵泡内的卵子同时取出,成熟卵子当天、未成熟卵子体外培养28～30h后行体外受精,受精后观察48～72h行胚胎移植。结果行IVF IVM周期32个(共49个优势卵泡),移植30周期,26个周期获得31个优势卵子(当天成熟29个,成熟率93.5%,29/31),非优势卵泡共获得未成熟卵子359个,成熟194个(54.0%,194/359)。优势卵子及未成熟卵子的成熟率、受精率、卵裂率及优质胚胎率分别为93.5%、65.5%、100.0%、78.9%和54.0%、83.5%、95.7%、36.1%。18个有优势卵子参与移植的16个移植周期临床妊娠8例,没有优势卵泡参与移植的14个移植周期,临床妊娠2例。HCG日优势卵泡直径为14～17mm时,优势卵泡卵子成熟率、受精率,未成熟卵子成熟率、受精率,均无明显差别。结论IVF IVM可应用于PCOS和卵巢反应不良的患者,优势卵泡直径14～17mm可以作为注射HCG的时间。     

人类体外受精培养液内毒素水平的评价

目的:检测人类体外受精使用的商品性培养液的内毒素水平,评价人精子存活试验和小鼠2-细胞发育试验测试内毒素的差异。方法:36批次的商品性体外受精培养液,使用前(A组,n=36),及使用后的其中25份样品(B组,n=25)采用鲎试验法检测内毒素水平。另对比人精子存活试验和2-细胞胚胎发育试验的敏感性。结果:A组样品无内毒素阳性检出,B组有2份样品检出内毒素。A组和B组样品24h精子活动率改变与对照组比无显著性差异(P>0.05),但A组中有3份样品抑制小鼠2-细胞胚胎的发育。结论:体外培养环境和实验操作须防止内毒素污染。虽然商品性培养液未检出内毒素,仍须对其作严格的质量控制。人精子存活试验测试培养液质量和低内毒素水平的敏感性低于小鼠2-细胞发育试验。     

Use of a Medium Devoid of Any Human or Animal Compound (SMART2) for Embryo Culture in Intracytoplasmic Sperm Injection

Purpose: Our purpose wax to evaluate the efficiency of a medium, devoid of any human or animal compound and specially designed for early embryo development (from the zygote to the eight-cell stage), SMART2, in intracytoplasmic sperm injection (ICSI) and to compare it with a medium containing human serum albumin (EllioStep2). Methods: Oocytes from 50 ICSI attempts were randomly placed, after sperm injection, into either SMART2 or EllioStep2. After a 48-hr incubation, the embryos were examined for quality scoring before transfer or freezing. Results: The percentage of normally fertilized oocytes per intact oocytes was slightly higher using SMART2 (139/199 vs. 135/224, respectively, for SMART2 and EllioStep2: P < 0.05). The distribution of embryo scores and the percentage of embryos with a fair morphology (71/143 vs. 72/148, respectively, for SMART2 and EllioStep2; not significant) were identical in both media. Conclusions: These data show that SMART2 medium can be successfully used for early embryo growth and, because it is devoid of any human or animal compound, offers better safety for patients than conventional media.     

Synthetic serum substitute (SSS): A globulin-enriched protein supplement for human embryo culture

Objective The purpose of the present study was to evaluate whether an IVF protein supplement prepared from human serum albumin (HSA) and human globulins would retain performance characteristics equivalent to those reported for the commercial plasma expanders, Plasmatein (Alpha Therapeutics, Los Angeles, California) and Plasmanate (Cutter Biological, Miles Inc., Elkhart, Indiana).Methods Pronuclear-stage human embryos were randomly divided and cultured in human tubal fluid medium (HTF) supplemented with either HSA (5 mg/mL) or Plasmatein (10%, v/v; 5 mg/ml as a means of indirectly assessing the effect - and - globulins have on embryonic development. Those results coupled with the known composition characteristics of Plasmatein were used as the starting basis to formulate test lots of synthetic serum substitute (SSS).Results Significantly (P<0.05) more of the human embryos cultured in Plasmatein supplemented medium reached the four-cell or greater stage by 40 hr postinsemination than a comparable group cultured in HSA alone. Lot 1 of SSS, formulated with HSA (84% of total protein) and human globulins (16% of total protein) and an aqueous lipoprotein fraction derived from human plasma (Excyte IV; Miles Diagnostics, Kankakee, Illinois), produced accelerated early embryonic growth relative to control murine embryos grown in the presence of Plasmatein, however, the percentage of the embryos reaching the hatched blastocyst stage was decreased (45 vs 100%). Human embryos from seven patients, randomized to HTF medium supplemented with Plasmatein or lot 1 of SSS, showed equivalent growth at 36– 40 hr postinsemination. A microprecipitate developed in media supplemented with lot 1 after several days of culture. The Excyte IV concentration was reduced and, ultimately, eliminated from the subsequent and final prototype lots of SSS. Murine embryos grown in the presence of lipoprotein free SSS showed significantly accelerated (P<0.01) growth at 17 hr postthaw compared to Plasmatein and all embryos progressed to hatching by 41 hr. Human embryos, randomized to either Plasmatein or lot 3 of SSS, showed significantly accelerated growth (P<0.01) when scored at 38 hr following insemination.Conclusion Synthetic serum substitute provides a convient, standardized means of adding protein to media used in assisted reproductive technology (ART) procedures.Irvine Scientific, Santa Ana, California.Presented in part at the Conjoint Annual Meeting of the American Fertility Society and Canadian Fertility and Andrology Society, Montreal Convention Center, Montreal, Quebec, Canada, October 9–14, 1993.     

Sperm:Oocyte ratios in an in vitro fertilization (IVF) program

Purpose During the past few years, many oocyte insemination techniques, including microinjection, have evolved in the treatment of male-factor infertility. This preliminary study was designed to evaluate whether microdroplet insemination could be considered a reliable technique, especially for semen samples with male-factor defects. The first objective was to assess fertilization rates obtained by inseminating sibling oocytes using both the conventional IVF and the microdroplet method (Group 1). The second objective was to evaluate subsequent embryo development and pregnancy rates resulting from microdroplet insemination, in addition to formulating adequate sperm oocyte ratios for various semen categories (Group 2). Four semen categories were studied including fresh normal sperm, frozen/thawed normal sperm, and male-factor sperm with one defect and two or three defects.Results Group 1 consisted of 54 couples; no statistical significance was found in the fertilization rates between test tube and microdroplet insemination in all four semen categories. Based on these results, patients from Group 2 (48 couples) had their oocytes inseminated only in microdroplets with sperm:oocyte ratios ranging from 2000 to 10,000 motile sperm:1 oocyte. The average fertilization rate for male-factor sperm was 55%, with a 91% cleavage rate.Conclusion Higher fertilization rates were observed in the lowest range of spermoocyte ratios (2000–40001) for male-factor sperm with one defect and in the highest range (8000–10,0001) for male-factor sperm with two or three defects. Polyspermy occurred in only 0.4% of the oocytes inseminated. Microdroplet insemination is an alternative treatment for moderate to moderately severe male-factor infertility, establishing a bridge between conventional IVF and microinjection. With adequate spermoocyte ratios, this technique allows the natural selection process of fertilization in vitro to take place, without the high incidence of polyspermy or mechanical damage frequently observed in assisted fertilization techniques.     

滋养层细胞培养方法研究进展

滋养层细胞在胚胎的植入过程中扮演重要角色 ,其增殖、功能调节失常与多种妊娠相关疾病的发生发展有密切关系。滋养层细胞的培养是进行这些相关实验的基础。然而 ,目前正被应用的滋养层细胞分离、培养、纯化方法均存在一定程度的缺陷。原代培养的滋养层细胞存在产率低、杂细胞污染等方面的缺憾 ,而滋养层细胞又在细胞特性上发生了变化。此外 ,目前在鉴定分离培养的细胞是否就是滋养层细胞这一关键问题上尚无统一标准     

A mobile oocyte incubation unit (MOIU): A device for improvement of the gamete intrafallopian transfer (GIFT) program

Forty couples with infertility due to various causes were selected for the gamete intrafallopian transfer (GIFT) program at our hospital. When the first 21 couples (Group A) had been treated in the program, the rate of pregnancy achieved was 23.8%, which did not seem satisfactory. This might have been caused by the distance between the embryo laboratory and the operating room. To eliminate this defect, a Mobile Oocyte Incubation Unit (MOIU) was designed. This is actually a compact laboratory that can be placed in the operating room. After the MOIU was utilized, the rate of pregnancy for the following 19 couples (Group B) increased to 42.1%. The MOIU has helped improved the performance of the GIFT program by increasing the stability of the pH value of the culture medium (Chetkowski R,et al.: J Vitro Fert Embryo Transfer 1985;2:207), lessening the exposure of the gametes to air and room temperature, and most importantly, shortening the time required for a GIFT procedure from 45–100 to 15–30 min. We expect that the MOIU will eventually become an integral part of the standard equipment for the GIFT program and make the program more successful and reliable in the treatment of infertility.     

Increasing Synthetic Serum Substitute (SSS) Concentrations in P1 Glucose/Phosphate-Free Medium Improves Implantation Rate: A Comparative Study

Purpose: To assess the comparative efficacy of IVF medium (MediCult, with 5.2 mM glucose) and a glucose/phosphate-free medium, P1 (Irvine Scientific), and to investigate the influence of increasing the serum supplementation (synthetic serum substitute; SSS; Irvine Scientific) to P1 on embryo development and implantation.Methods: Patients were randomly assigned to IVF medium (Group 1, cycles n = 172) or P1 supplemented with 10% SSS (Group 2, cycles n = 229) according to the medium scheduled for use on the day of oocyte retrieval. Another 555 IVF consequent cycles (Group 3) were performed using increased SSS concentrations (20%) in P1 medium.Results and Conclusion: In this large series of IVF cycles, we herein demonstrate that significantly higher pregnancy and implantation rates were found when embryos were cultured in glucose/phosphate-free medium P1 supplemented with 20% SSS compared to supplementation with the lower SSS concentration and with IVF medium.     

Enhancement of the developmental potential of mouse oocytes matured in vitro by gonadotropins and ethylenediaminetetraacetic acid (EDTA)

We attempted to improve the developmental potential of mouse oocytes matured in vitro. First, the effect of gonadotropin supplementation of the oocyte maturation medium was tested. The addition of follicle-stimulating hormone (FSH) or luteinizing hormone (LH) alone significantly increased the rate of development of inseminated oocytes to two-cell embryos, resulting in a twofold increase in blastocyst development. There was no significant difference between FSH and LH supplementation. However, the beneficial effect of FSH or LH was abolished when both were added together. Next, we tested the effect of ethylenediaminetetraacetic acid (EDTA) supplementation of the embryo culture medium. The addition of 10 M EDTA significantly enhanced the development of embryos derived from oocytes matured in vitro, both to two-cell embryos and to blastocysts. These data suggest that the inadequate development of embryos from oocytes matured in vitro results from a defect similar to that inherent in outbred mouse embryos showing the two-cell block in vitro.