小鼠精母细胞在体外分化为精子细胞

目的:探讨未成熟小鼠生精细胞在体外培养条件下的分化情况。方法:用支持细胞(TM4)条件培养液培养出生后14d的BALB/c小鼠睾丸细胞,对照组采用添加1%胎牛血清的MEM培养液维持细胞存活,在不同时段收获细胞,实时定量RT-PCR分析精子细胞特异性基因Tnp2表达的变化。结果:实时定量RT-PCR显示,在体内,Tnp2的表达随小鼠日龄增长而升高,与精子细胞发生过程同步;在睾丸细胞体外培养过程中,Tnp2的表达在TM4条件培养液培养d5显著升高(P<0.05),而对照组始终无变化。结论:在TM4条件培养液的作用下,小鼠精母细胞在体外分化为精子细胞。     

新生大鼠卵巢体外培养体系的研究

目的:探讨最适宜于大鼠原始卵泡体外生长发育的培养体系。方法:取出生2d的SD大鼠卵巢,分别在Waymouth和DMEM中培养8d,石蜡制片,HE染色,统计分析各级正常卵泡数;于培养d4、d8观察各级卵泡形态;并用免疫组化方法检测在这两种培养体系的卵巢中增殖细胞核抗原(PCNA)的表达定位情况。结果:①在Waymouth中,由原始卵泡启动生长成为初级卵泡和次级卵泡的百分率分别是25.51%和6.93%,高于DMEM中的17.06%和3.56%(P<0.01)。②在Waymouth中培养的卵泡形态都较正常,而在DMEM中培养的异常卵泡较多。③PCNA作为原始卵泡生长的指标,也说明卵巢在Waymouth中能良好生长。结论:Waymouth培养体系是研究原始卵泡生长的一个理想的培养体系。     

妊高征与体外培养内皮细胞

国外学者近年来在妊娠高征女血液对体外培养内皮细胞的影响研究表明,如同体同内皮细胞损伤一样,妊高征妇女的血液对体外培养的皮内细胞会产生同样的影响,而另外一些研究却得出完全相反的结论,对于两种结论产生的原因作了相关解释。     

卵母细胞的体外培养和成熟

卵母细胞的体外培养和成熟付永伦，严敬明（上海医科大学附属妇产科医院研究所，上海，２０００１１）随着体外受精和胚胎移植（ＩＶＦ－ＥＴ）技术水平的不断提高，越来越多的不孕妇女将接受ＩＶＦ－ＥＴ的治疗。在治疗中，她们不得不忍受和经受“超排卵”所带来的精神和...     

体外培养胚胎发育潜能的非损伤性评价方法

目前人类体外受精获得的胚胎,大多是采用形态学评分法进行评价.方法虽然快捷、简单,但具有一定的局限性和主观性,所以研究者采用生物学指标评价胚胎发育潜能的相关研究.本文仅对其中部分研究的结果进行综述,其中包括氨基酸、血小板活性因子、活性氧与抗氧化物、糖类代谢产物以及胚胎的呼吸率.     

卵母细胞减数分裂转变过程的调控机制

卵母细胞的成熟包括第一次减数分裂恢复、第一次减数分裂向第二次减数分裂转变、第二次减数分裂中期阻滞等过程,其中第一次减数分裂向第二次减数分裂的转变是卵母细胞成熟的关键步骤之一,但其机制目前尚不清楚。丝裂原激活蛋白激酶、成熟促进因子、内源性减数分裂抑制因子、Separase/Securin蛋白、Aurora激酶等多种已知或未知的蛋白可通过泛素-蛋白酶体通路、磷脂酰肌醇通路、受体酪氨酸激酶-Ras蛋白通路、环磷酸腺苷通路等多个信号通路调控卵母细胞的成熟。通过研究这些通路中关键蛋白间的相互作用有助于进一步探索卵母细胞成熟的调控机制。     

人类卵子体外培养成熟后冻存研究

目的探讨体外培养成熟的人类卵子冻存的可行性。方法中山大学附属第一医院从2003年3月至2003年9月在ICSI治疗周期中共收集到101个GV期卵子和53个MI期卵子,根据卵子发育阶段不同将不成熟卵子分为GV期组和MI期组,体外培养成熟后应用慢速冷冻方法进行卵子冷冻,解冻后用ICSI受精并进行胚胎体外培养,分别比较GV期组和MI期组卵子的复苏率、受精率、优质胚胎率和囊胚发育情况。结果经体外培养后GV期组和MI期组分别有64个和44个卵子成熟,GV期组卵子体外成熟率为634%(64/101),显著低于MI期组的830%(44/53)(P<005),冷冻后复苏率分别为719%(46/64)和818%(36/44),受精率分别为609%(28/46)和639%(23/36),优质胚胎率分别为536%(15/28)和652%(15/23),在统计学上差异均无显著性意义。结论在ICSI治疗周期中的GV期和MI期的卵子经体外成熟后可以成功地进行冷冻保存。     

人未成熟卵母细胞体外成熟培养影响因素的研究

未成熟卵母细胞体外成熟培养(IVM)技术的研究已成为世界生殖医学研究的热点之一,目前,IVM技术仍面临着卵子成熟率不高,成熟后体外受精率低及妊娠率较低等困难,所以IVM过程中各种影响因素的调控对卵母细胞的成熟及体外受精(IVF)后胚胎的发育甚为重要。     

孕酮和表皮生长因子在体外培养新生大鼠卵巢原始卵泡生长启动中的作用

目的:探讨孕酮(P_4)和表皮生长因子(epidermal growth factor,EGF)在原始卵泡生长启动中的作用。方法:取出生2d的SD大鼠卵巢,分别在含不同浓度P_4、EGF和P_4+EGF的Waymouth培养体系中培养,以单纯Waymouth培养体系培养的大鼠卵巢为对照,于培养8 d后石蜡包埋切片, HE染色,观察各级卵泡形态,并统计分析各级正常卵泡数。结果:P_4各浓度组的原始卵泡数明显多于对照组(P<0.01),50 ng/ml EGF组的初级卵泡和次级卵泡数明显多于对照组(P<0.01),但1 000 ng/ml EGF组的初级卵泡数和次级卵泡数均明显少于对照组(P<0.01);50 ng/ml EGF+10~(-5) mol/L P_4与相同剂量单独作用的P_4和EGF比较各级卵泡数有统计学差异(P<0.05),与对照组比较无明显差异(P<0.05)。结论:在Waymouth培养体系中孕酮对原始卵泡生长有抑制作用,一定浓度的EGF能促进原始卵泡生长,而同时加入P_4和EGF,两者的作用有部分抵消。     

辅助生育体外培养系统的内毒素控制

内毒素是革兰氏阴性菌胞壁外膜表面的一种大分子物质 ,本文介绍了内毒素的结构特点 ,体外培养系统内毒素的可能来源 ,内毒素对配子和胚胎的毒性效应 ,内毒素检测及去除。辅助生育项目要尽可能减少内毒素污染 ,对内毒素进行控制有助于改善辅助生育的成功率。     

In vivo development of in vitro fertilized bovine oocytes matured in vivo versus in vitro

In vivo developmental potentials of in vivo and in vitro matured oocytes fertilized in vitro were assessed in cattle. One-cell stages produced from in vivo matured oocytes developed into a pregnancy when transferred to the ampulla part of oviducts of synchronized heifers. In vitro matured oocytes achieved high penetration and cleavage rates but did not develop into pregnancies when transferred to synchronized heifers.     

Assessment of uterine receptivity by the endometrial-subendometrial blood flow distribution pattern in women undergoing in vitro fertilization-embryo transfer

OBJECTIVE: To examine whether in vitro differentiation of germ cells from men with maturation arrest is improved by augmenting FSH and T concentrations above the values effective in samples from men with normal spermatogenesis. DESIGN: Prospective, controlled in vitro study. SETTING: Private assisted reproduction centers and a university department. PATIENT(S): Men with meiotic or postmeiotic maturation arrest. INTERVENTION(S): Testicular spermatid extraction, in vitro culture of testicular biopsy samples, intraoocyte injection of elongated spermatids, embryo culture and transfer. MAIN OUTCOME MEASURE(S): Progression of in vitro germ cell differentiation, fertilization, and pregnancy outcomes with in vitro cultured germ cells. RESULT(S): In some cases of meiotic and postmeiotic maturation arrest, more advanced germ cell stages were achieved by in vitro culture in the presence of 500 IU/L FSH as compared with 50 IU/L FSH. The beneficial effect of 500 IU/L FSH was further potentiated by a simultaneous increase of T concentration from 1 to 10 microM. Fertilizations with germ cells recovered after incubation with these pharmacological hormone concentrations gave rise to viable embryos and the births of five healthy babies. CONCLUSION(S): Pharmacological concentrations of FSH and T are beneficial for in vitro maturation of germ cells from some men with in vivo maturation arrest.     

Study of the In Vitro Maturation of Mouse Oocytes Induced by Microinjection of Maturation Promoting Factor (MPF)

Purpose: Maturation promoting factor (MPF) acts at theresumption of meiosis and nonspecifically throughout theanimal species. There exists a considerable body of literatureon MPF, but little work has been done to study the inductionof maturation of mammalian oocytes by microinjection ofextracted MPF. Methods: Immature (GV-stage) mouse oocytes weremicroinjected MPF extracted from matured Xenopus eggs in thepresense of dbcAMP. Results: The rate of germinal vesicle breakdown (GVBD)induced at 24 hr after MPF injection was significantly higher(90.5%) than that of the control (2.2%), which was injectedwith HTF medium containing dbcAMP (P < 0.0001). Therate of extrusion of the first polar body at 24 hr after MPFinjection was significantly higher (84.1%) than that of thesame control (1.1%) (P < 0.0001). Conclusions: From these results, it is concluded that thematuration of mammalian oocytes can be induced by themicroinjection of MPF extracted from other species.     

Are Cumulus Cells Necessary for the Spontaneous Maturation of Germinal Vesicle-Stage Oocytes to Metaphase II

Purpose: In this study we investigated the need of the support from cumulus cells for germinal-vesicle (GV) oocytes collected from stimulated ovaries to complete their maturation to metaphase II (MII). Methods: We compared the maturation rate of GV oocytes after coculture with cumulus cells (study group) with their spontaneous maturation in culture medium alone (control group). Results: Sixty-four and nine-tenths percent of the GV oocytes matured to metaphase II in the coculture group, and of these, 43.5% gave normal 2pn zygotes following intracytoplasmic sperm injection (ICSI), while 73.8% of the GV oocytes spontaneously matured to the MII stage and 30% of these reached the zygote stage after ICSI. Conclusions: It is probable that a follicular factor is responsible for this arrested maturation in the human and that maturation occurs spontaneously when the oocytes are separated from their follicular fluid environment after collection.     

Successful in vitro maturation for urgent fertility preservation despite hormonal contraception by continuous progestin application

We report a unique case of a rare utilization of IVM. This case shows the successful retrieval of immature oocytes followed by in vitro maturation (IVM) for fertility preservation in a patient undergoing chronic progestin contraception. A 24-year-old patient with anaplastic astrocytoma requiring chemotherapy with temozolomide for 12 cycles as soon as possible with wish for fertility preservation while using a long acting etonogestrel birth control implant presented in our unit for fertility preservation in May 2017. The currently used implant should be preserved for further contraception. As the ovaries presented with a high, pco-like, antral follicle count, IVM was offered; the patient agreed. A transvaginal follicular puncture in general anesthesia without any hormonal intervention and IVM of gained oocytes was performed. As the patient actually had no spouse, she decided to freeze unfertilized metaphase II stage oocytes (MII). Thirteen oocytes were obtained, eight of them could be matured and cryopreserved. IVM could be a possibility for fertility preservation in patients with polycystic ovaries when no time is available for stimulation for conventional in vitro fertilization. Even use of continuous progestin application for contraception is no obstacle.     

Prediction of chromosome misalignment among in vitro matured human oocytes by spindle imaging with the PolScope

OBJECTIVE: To examine whether spindle morphologic features imaged with the LC-PolScope (Cambridge Research and Instrumentation, Woburn, MA) in living human oocytes matured in vitro can be used to predict chromosome configuration and select oocytes with normal chromosomes. DESIGN: Morphological study. SETTING: Academic IVF clinic. PATIENT(S): Women undergoing oocyte retrieval for ICSI treatment. INTERVENTION(S): Oocytes were examined after in vitro maturation. MAIN OUTCOME MEASURE(S): The study examined meiotic spindle morphologic features and chromosome alignments. RESULT(S): After culture for 22 to 24 hours, 77.1% of oocytes reached metaphase II stage, with 51.9% of oocytes showing birefringent spindles. Confocal microscopy revealed that 71% of oocytes with the birefringent spindles had normal chromosome alignment, and 29% of oocytes with birefringent spindles and all oocytes without birefringent spindles had abnormal microtubule organization and abnormal chromosome alignment. CONCLUSION(S): The spindle images obtained with the PolScope in living human oocytes are coordinate with those in fixed oocytes as imaged by confocal microscopy. Spindle images with the PolScope can be applied to human in vitro fertilization to help predict chromosomally normal oocytes for insemination.     

Isolation,in vitro maturation,and fertilization of germinal vesicle oocytes obtained from the intact murine ovary

The purpose of this investigation was to attempt to develop a process, utilizing a murine model, which would allow more efficient harvesting from the intact ovary and maturation in vitro of germinal vesicle (GV) oocytes. The recovery process yielded 25.5±4.5 ( ±SE) cumulusfree GV oocytes per animal. Treatment groups included culture medium (CM) supplemented with either estradiol (E₂), follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG), or prolactin (PRL). Among the hormone-free controls 83.2±1.6% of oocytes underwent GV breakdown, whereas 25.3±2.6% developed to the first polar body stage (PB-1) following 18 hr of incubation (n=29 trials). Oocytes progressing to the PB-1 stage were inseminated in vitro. In vitro fertilization (IVF) of pooled in vitro matured (IVM) PB-1 oocytes (judged by two-cell formation) was 19.9%, which was significantly lower than in the group of in vivo matured oocytes (74.7%). E₂ significantly increased the percentage of GV breakdown (control, 76.8±2.5%; E₂ at 10 ng/ml, 92.9±2.5%,P<0.001; E₂ at 100 ng/ml, 93.7±2.1%,P<0.001; and E₂ at 1 g/ml, 86.7±3.3%,P<0.05) but not PB-1 formation. Neither FSH nor hCG significantly increased GV breakdown or PB-1 formation. Prolactin treatment resulted in an increased percentage of PB-1 formation (control, 25.3±2.5%; PRL at 2 g/ml, 35.0±2.9%; and PRL at 20 g/ml, 34.1±1.9%;P<0.01), fertilization (control, 15.3±5.1%; PRL, 33.6±8.5;P<0.01), and subsequent development (control, 3.5±2.3%; PRL, 18.8±5.6%;P<0.01). We conclude that recovery and IVM of GV oocytes is feasible, however, further work is necessary to define optimal conditions.     

人类未成熟卵母细胞体外培养成熟的研究进展

本文综述了人类未成熟卵母细胞体外培养成熟的现状和未来的发展方向。未成熟卵母细胞体外培养成熟包括二种方法 :卵泡体外培养 ( IVC)和卵母细胞体外成熟 ( IVM)。从原始卵泡发育到成熟卵母细胞 ,受精形成胚胎 ,胚胎移植后妊娠 ,仅在小鼠成功 ,有关人类此方面的研究正在进行中。人类卵母细胞体外成熟已成功应用于治疗不孕症 ,但成功率低。卵母细胞体外成熟受自身大小以及所处月经周期的影响。培养系统添加卵泡液、表皮生长因子、激活素 /抑制素、以及共培养系统有助于卵母细胞体外成熟。根据卵母细胞所处发育阶段选择不同的培养系统 ,探索卵母细胞的生长和成熟是未来的重要研究方向