胚胎移植与囊胚移植的临床结局分析*

背景：国内关于卵裂期胚胎移植与囊胚移植的临床比较与分析大样本报道较少见。 目的：比较第2,3天卵裂期胚胎移植与第5天囊胚移植的临床结局。 方法：选择2008-01/2009-12北京大学深圳医院生殖医学科行体外受精/卵胞浆内单精子显微注射共1612周期,比较第2,3天卵裂期胚胎移植与第5天囊胚移植的临床妊娠率、胚胎种植率、流产率、多胎率、出生婴儿性别比例等差异。 结果与结论：第2,3,5天移植组分别195,1 162,255周期,临床妊娠率分别为33.33%,38.04%,44.71%,种植率分别为21.37%,24.70%,31.96%;第5天移植组临床妊娠率和种植率均高于第2,3天移植组(P < 0.05);第2天移植组宫外孕发生率高于第3天移植组(P < 0.05)。3组流产、单卵双胎、多胎、早产及出生婴儿性别比例无差异。说明囊胚移植提高了临床妊娠率和种植率的同时,未增加流产、多胎、早产等风险,对男女性别比例的平衡无负面影响。     

囊胚玻璃化冻存前激光打孔对冻融后移植效果的影响

背景：一系列研究均表明人工皱缩囊胚腔能明显提高囊胚的玻璃化冻融效果,但在皱缩过程中采用的物理方法和使用材料的不同可能会造成囊胚细胞的机械性损伤。 目的：探讨囊胚玻璃化冻存前激光打孔对冻融后移植效果的影响。 方法：3例不孕患者,平均年龄30岁,于囊胚玻璃化冻存前行激光打孔使囊胚皱缩,平均冻存时间为1年,确定移植前给予解冻。 结果与结论：3例患者中有2例取得了满意的妊娠结局,1例未妊娠。说明采用激光辅助打孔的方法使囊胚皱缩,结合玻璃化冻融后行囊胚移植安全有效。     

小鼠囊胚细胞凋亡和半胱氨酸天冬氨酸蛋白酶3表达与维甲酸的诱导作用★

背景：细胞凋亡是维甲酸导致早期胚胎异常发育的原因之一，而半胱氨酸天冬氨酸蛋白酶3介导的细胞凋亡是细胞凋亡的主要途径。 目的：观察维甲酸对体外培养小鼠囊胚细胞凋亡的影响，以及对囊胚细胞中半胱氨酸天冬氨酸蛋白酶3表达的改变。 方法：获取妊娠3.5 d小鼠囊胚，分别在含0，1和10 μmol/L维甲酸的M199培养基中培养24 h，用带有荧光标记的原位末端标记法观察维甲酸对小鼠囊胚细胞凋亡的效应；并用免疫组织化学S-P法检测维甲酸对小鼠囊胚半胱氨酸天冬氨酸蛋白酶3的表达。 结果与结论：所有组均观察到囊胚细胞凋亡和半胱氨酸天冬氨酸蛋白酶3表达，高剂量维甲酸组中囊胚细胞凋亡荧光值，半胱氨酸天冬氨酸蛋白酶3表达的平均吸光度值和阳性面积率均显著升高(P < 0.01)。结果提示维甲酸可促进体外培养小鼠囊胚细胞凋亡和及半胱氨酸天冬氨酸蛋白酶3表达，证实了维甲酸对小鼠胚胎的发育的细胞毒性作用。     

昆明小鼠3.5 d和4 d囊胚不同分离方法的比较*

背景：远交系的昆明小鼠作为中国自然科学研究主要实验动物,其胚胎干细胞建系的成功率一直很低。 目的：探讨昆明小鼠胚胎干细胞体外分离培养的最佳方法和最适合的采胚时间。 方法：采集孕3.5 d和4 d的囊胚分别以免疫外科法和全胚培养法在小鼠胚胎成纤维细胞饲养层上分离和克隆昆明小鼠胚胎干细胞集落。 结果与结论：免疫外科法和全胚法培养3.5 d囊胚的内细胞团贴壁率和原代克隆形成率差异均不显著(P > 0.05);全胚法培养4 d囊胚的内细胞团贴壁率显著高于免疫外科法(P < 0.05),但原代克隆形成率显著低于免疫外科法(P < 0.05);全胚法培养4 d囊胚的原代克隆形成率显著高于3.5 d囊胚(P < 0.05);免疫外科法分离4 d囊胚内细胞团的贴壁率和原代克隆形成率显著高于以同样方法分离培养的3.5 d囊胚(P < 0.05)。结果显示用免疫外科法分离4 d囊胚更适合于昆明小鼠胚胎干细胞的体外分离培养。     

小鼠囊胚在胚胎成纤维细胞饲养层上向胚胎干细胞的生长发育

背景：到目前为止已建立了数百个小鼠胚胎干细胞系,一般情况下实验室培养的胚胎干细胞有3个细胞来源,即胚泡内的内细胞群、胚胎生殖嵴的原始生殖细胞和应用克隆技术制造人体胚胎并提取干细胞。 目的：尝试用自制的小鼠胚胎成纤维细胞饲养层分离培养小鼠胚胎干细胞。 设计、时间及地点：细胞学体外观察,于2005-12/2007-09在辽宁医学院完成。 材料：妊娠14.5 d的孕鼠40只,由辽宁医学院实验动物中心提供。 方法：无菌条件下剪开孕鼠子宫壁,取出胚胎,去除头、尾、内脏和四肢后采用胰蛋白酶消化法分离培养小鼠胚胎成纤维细胞,经丝裂霉素C处理3.5 h后接种于6孔板中,细胞贴壁后即为成纤维细胞饲养层。收集3.5 d的小鼠囊胚,在显微镜下将囊胚置于上述6孔板的中央,五六天后取隆起生长的内细胞团块,分离后再培养。 主要观察指标：成纤维细胞饲养层的制备情况,胚胎干细胞的生长状态、碱性磷酸酶染色结果及分化趋势。 结果：光镜下小鼠胚胎成纤维细胞呈不规则形,生长较快,传代比例为1∶4,经丝裂霉素C处理后细胞贴壁较慢,失去分裂增殖能力。小鼠囊胚孵出透明带的时间为24~72 h,从胞膜中孵出约为1 d,3~5 d后可形成胚胎干细胞集落,7 d后集落呈“鸟巢”状。组织化学染色后可见细胞内有大量的蓝紫色颗粒沉积,连续培养20 d,期间不换培养液,可见胚胎干细胞团分化为亮度较小、界限清楚的单核细胞。 结论：小鼠囊胚在胚胎成纤维细胞饲养层上可发育成胚胎干细胞,并能进行传代。     

昆明小鼠胚胎干细胞最佳分离方法及培养液选择

背景：到目前为止已经建立数百个小鼠胚胎干细胞系,昆明小鼠是中国应用最多的实验小鼠,其建系成功报道极少。 目的：建立一简单有效的昆明小鼠胚胎干细胞分离培养体系,以提高昆明鼠胚胎干细胞建系成功率。 方法：用添加血清替代物或胎牛血清培养液的小鼠胚胎成纤维细胞制备饲养层。昆明小鼠经孕马血清促性腺激素和人绒毛膜促性腺激素促排卵,交配后3.5 d冲洗子宫取胚胎,将胚胎接种在饲养层中培养,观察胚胎贴壁、内细胞团集落形成率。4~ 6 d后在0.05%trypsin-0.02%EDTA消化液中用切割法处理增殖的内细胞团块,再接种到新的饲养层上。倒置显微镜下观察形成的胚胎干细胞样集落形态。 结果与结论：妊娠3.5 d孕鼠适合胚胎干细胞的分离培养;在0.05%trypsin-0.02%EDTA消化液中采用切割法进行内细胞团集落的分离,集落增殖快、分化低;在含胎牛血清培养液中进行干细胞培养较血清替代物培养液中传代次数多,此培养液更适宜进行分离培养昆明鼠胚胎干细胞。     

受精第3天胚胎的融合程度与植入潜能

背景：虽然辅助生殖技术发展迅速,但是胚胎植入率仍不理想,关于胚胎融合程度与植入潜能的关系尚无定论。 目的：探讨受精第3天胚胎早期融合与植入率的相关性。 方法：回顾性分析420例(共移植胚胎1 016个)患者在超促排卵、体外受精及胚胎移植后,植入成功胚胎早期融合情况与未植入成功胚胎早期融合情况的差别。 结果及结论：受精第3天移植早期融合胚胎共200个,其中植入成功48个;受精第3天移植未早期融合胚胎共816个,其中植入成功133个,两组比较差异显著(P < 0.05)。植入胚胎中有3个早期融合胚胎妊娠率达71.43%,有2个为31.91%,有1个为51.06%,0个为39.81%。可见,在目前的胚胎选择标准下,胚胎早期融合可以显著提高胚胎的植入率,但是其具体机制,还有待进一步研究。     

影响玻璃化冻融胚胎移植结局的相关因素分析

【摘要】目的 探讨辅助生殖技术中影响玻璃化冻融胚胎移植结局的相关因素。方法 对142例共154个周期的相关资料进行回顾性统计学分析,根据患者年龄、胚胎发育时期、受精方式、子宫内膜准备方案、移植日子宫内膜厚度、移植过程是否顺利和胚胎解冻复苏后成活细胞比例进行分组，比较各类分组的胚胎种植率和临床妊娠率。结果 不同年龄、受精方式、子宫内膜准备方案、子宫内膜厚度和移植过程是否顺利组间胚胎种植率及临床妊娠率无显著性差异(P>O.05)；受精第二天的胚胎冻融（D2）组和受精第三天的胚胎冻融（D3）组的临床妊娠率没有显著性差异，但胚胎种植率D3组明显高于D2组。胚胎复苏后成活细胞比例对胚胎种植率和临床妊娠率有显著影响(P<0.05)。结论 在冻融胚胎移植周期, 解冻后胚胎质量对妊娠的成功率起主要作用,准备适宜的子宫内膜厚度能提高临床妊娠率。     

人骨髓基质细胞核移植重构胚的建立

目的建立人体细胞核移植技术，为治疗性克隆的实验研究及临床应用奠定基础。方法应用显微操作技术将单个人骨髓基质细胞注入去核后的兔卵母细胞内，经电融合、离子霉素和6．甲氨基嘌呤激活后．培养于含10％胎牛血清的TCM-199中，使重构胚进一步发育至囊胚。结果245枚卵母细胞进行去注核显微操作，核移植后有72．6％（178／245）卵母细胞仍保持完整；经电融合后细胞融合率为62．8％（83／132）：体外培养后约65％（54／83）的重构胚可进入分裂期，3．7％（2／54）发育至桑囊期，囊胚孵化率为100％（2／2）。结论兔卵母细胞能使人骨髓基质细胞核重新编程形成核移植重构胚并可继续发育至囊胚，使得从核移植重构囊胚分离胚胎干细胞用于实验和临床治疗研究成为可能。     

利用冷冻胚胎建立人类胚胎干细胞系

背景：目前人胚胎干细胞建系的技术路线主要有囊胚法、核移植法和胚胎单卵裂球法,但由于破坏胚胎,涉及诸多伦理问题。 目的：探讨利用冷冻胚胎建立人类胚胎干细胞系的可行性。 设计、时间及地点：细胞学体内外实验,于2005-01/2006-08在沈阳市妇婴医院生殖中心完成。 材料：妊娠13.5 d的ICR鼠20只用于制备小鼠胚胎成纤维细胞饲养层,10周龄SICD鼠2只用于人胚胎干细胞体内分化实验。行IVF/ICSI助孕患者自愿捐献的冷冻胚胎,由沈阳市妇婴医院生殖中心提供。 方法：复苏冷冻的胚胎,采用序贯培养法进行囊胚培养,用免疫外科方法去除滋养细胞,将得到的胚胎内细胞团接种于丝裂霉素C灭活的小鼠胚胎成纤维细胞上培养并传代。 主要观察指标：取不同代次的培养细胞,分别进行生长特性、碱性磷酸酶染色、转录因子OCT-4、阶段特异性胚胎抗原SSEA-4、SSEA-1、肿瘤排斥抗原TRA-1-60、TAR-1-81、核型及体内分化全能性鉴定。 结果：20个解冻卵裂期胚胎,获得9个囊胚,分离完整的内细胞团共7个,从7个内细胞团培养出2个人胚胎干细胞系,其中一个细胞系连续培养76代(20个月)。所培养的细胞具有人胚胎干细胞的共同生物学特性,即细胞呈扁平或圆形,可见1~3个核仁;碱性磷酸酶以及SSEA-3,SSEA-4,TRA-1-81,TRA-1-60,OCT-4均呈阳性表达,而SSEA-1呈阴性;核型正常,为46,XX核型;将细胞注射入SCID鼠皮下形成肿瘤,病理切片显示为成熟畸胎瘤;短串联重复分型结果显示所形成的畸胎瘤与人胚胎干细胞为相同的遗传背景。 结论：实施体外受精-胚胎移植的夫妇怀孕后,多余的冷冻保存胚胎所分离培养的细胞具备人胚胎干细胞的所有特性,是建立人胚胎干细胞系很好的材料来源。     

Ipsilateral connections of the gyrus proreus in the cat

The effects of interruption of anterior inputs to the medial basal hypothalamus (MBH) from or through the preoptic area (POA) on blastocyst implantation were studied. Hypothalamic deafferentation (HDAF) was performed on different days of early pregnancy. When the HDAF was made on day 2 or 3 of pregnancy, implantation was effectively inhibited. No implantation site and no unimplanted blastocyst was seen. In rats receiving the HDAF on day 4, small uterine swellings developed, but in most cases, degeneration of decidua and embryos was noted. When the HDAF was carried out on day 5, all rats had implantation sites. Replacement therapy with prolactin was successful in inducing implantation in rats whose implantation had been blocked by the HDAF. However, treatment with HCG or estrogen was not effective in reversing the inhibitory effect of the HDAF on implantation. These results suggest that the failure to implant in HDAF rats can be attributed to deficiency in prolactin required for progesterone secretion. Anterior inputs to the MBH seem to be necessary for supporting pituitary prolactin release in early pregnancy.     

Endocrine basis for the initiation, maintenance and termination of pregnancy in humans

Channels of hormonal communication between the embryo and the mother are established early in the conceptual process. The preimplantation embryo exerts local uterine and systemic effects on the maternal organism to provide a suitable environment for embryonic development, to enable the development of a receptive endometrium for implantation, and to maintain corpus luteum function. In primates, implantation can be induced with progesterone alone if the proliferative effects of estrogens on the endometrium have already been achieved; estrogens may have a priming effect for progesterone on the endometrium, but it is not obligatory for implantation. Progesterone secretion from the corpus luteum is essential for the maintenance of early pregnancy until the placenta takes over the major role of steroidogenesis. This luteo-placental shift is completed at about the 50th day of human gestation. Withdrawal of progesterone effects by the administration of the competitive progesterone antagonist, RU 486, within 24 days after conception will terminate 85% of human pregnancies. Other nonsteroidal functions of the corpus luteum have been recently suggested. Relaxin and prorenin are peptides secreted by corpus luteum of pregnancy but their role remains putative at the present time.     

IL-10 inhibits nitric oxide synthesis in murine uterus

OBJECTIVES: Recent reports point to a role for the nitric oxide/nitric oxide synthase (NO/NOS) system in implantation. It has been suggested that inducible NOS expressed at peri-implantation would lead to enhanced NO production, which could promote the attachment of the blastocyst. Short-term administration of NO donors during the pre-implantation period reduced the pregnancy rate in a dose-dependent manner. Thus, it is thought that optimal levels of NO are critical for embryo implantation, so regulation of NOS must be crucial. Taking this into consideration, interleukin-10 (IL-10), synthesized and secreted by the embryo, could be modulating NOS during implantation. In this study we have investigated the in vitro effect of IL-10 on NOS in the uterus. METHODS: To determine the effect of IL-10, slices of uterus from estrogenized mice were pre-incubated for 60 min with different concentrations of IL-10 and NOS activity was measured. RESULTS: IL-10 (50 and 100 ng/ml in vitro) diminished NOS activity. The in vivo administration of lipopolysaccharide (LPS; 8 mg/kg) significantly increased the conversion of arginine into citrulline. This effect was abolished after 60 min of preincubation with IL-10 (100 ng/ml). The stimulatory effect of LPS and estrogen on NOS activity is exerted on the Ca-independent isoform and IL-10 in vitro abolished this increase. We observed that the uterus of pregnant mice on day 5 of gestation synthesized NO. This production was significantly inhibited by preincubation with IL-10 (100 ng/ml). CONCLUSIONS: This report demonstrates that IL-10 is capable of inhibiting NO synthesis in estrogenized, LPS-treated and pregnant rat uterus.     

Mother-offspring dialogue in early pregnancy: Impact of adverse environment on pregnancy maintenance and neurobiology

The mother-offspring dialogue begins even before implantation and is essential to signal pregnancy, establish robust contact, and maintain embryo growth and development. Any circumstance that disrupts the dialogue risks pregnancy problems. A new look at how stress impacts on pregnancy involves its adverse effects on the key pregnancy hormones of progesterone and prolactin. These effects have far-reaching consequences on pregnancy maintenance, maternal anxiety and embryo programming. This review focuses on early pregnancy and how stress might compromise the multi-layer, two-way communication between mother and embryo.     

Effect of thrombophylaxis on uterine and fetal circulation in pregnant women with a history of pregnancy complications

PURPOSE: The aim of this study was to investigate the effect of thromboprophylactic therapy on fetal and maternal Doppler flow parameters in pregnant women with severe complications in previous pregnancies and evidence of acquired or congenital thrombophilia in the current pregnancy. METHODS: Sixty-five patients with a history of recurrent abortions, intrauterine fetal death, intrauterine growth restriction (IUGR), and severe early-onset preeclampsia were tested for the presence of acquired or congenital thrombophilia. Those with positive findings were prescribed low-dose aspirin plus low-molecular-weight heparin (LMWH) (enoxaparin); the remainder received low-dose aspirin only. A Doppler flow study was performed before and after treatment and in the third trimester of pregnancy. RESULTS: Of the 65 pregnancies, four ended in spontaneous abortion and were excluded from the analysis. Of the 61 women with completed pregnancies, 37 (61%) had evidence of acquired or congenital thrombophilia: 22 (36%) protein S deficiency; 1 (2%) protein C deficiency; 2 (3%) activated protein C resistance (APC-R); 2 (3%) IgG for antiphospholipid antibodies; 1 (2%) circulating anticoagulant; and 9 (15%) a combined defect. This group showed a significant decrease in mean uterine artery pulsatility index (PI) before and after treatment (1.32+/-0.36 vs. 1.04+/-0.23, P=.006), whereas the remaining 24 patients treated with low-dose aspirin only had nonsignificant changes. Pearson's correlation test yielded no correlations of the pregnancy outcome parameters with Doppler flow values in the umbilical or uterine arteries. CONCLUSIONS: Thromboprophylactic therapy transiently improves maternal circulation parameters in patients with thrombophilia at risk of fetal loss and other severe complications of pregnancy, but not in correlation with their pregnancy outcome. Therefore, Doppler examination of maternofetal circulation in the second trimester is not predictive of pregnancy outcome.     

Late gestational maternal serum cortisol is inversely associated with fetal brain growth

To analyze the association between fetal brain growth and late gestational blood serum cortisol in normal pregnancy.Blood total cortisol was quantified at delivery in 432 Chinese mother/child pairs. Key inclusion criteria of the cohort were: no structural anomalies of the newborn, singleton pregnancy, no alcohol abuse, no drug abuse or history of smoking no hypertensive disorders and no impairment of glucose tolerance and no use of steroid medication during pregnancy. Differential ultrasound examination of the fetal body was done in early (gestational day 89.95 ± 7.31), middle (gestational day 160.17 ± 16.12) and late pregnancy (gestational day 268.89 ± 12.42). Newborn's cortisol was not correlated with any of the ultrasound measurements during pregnancy nor with birth weight. Multivariable regression analysis, considering timing of the ultrasound examination, the child's sex, maternal BMI, maternal age, maternal body weight at delivery, the timing of cortisol measurement and maternal uterine contraction states, revealed that maternal serum total cortisol was significantly negative correlated with ultrasound parameters describing the fetal brain: late biparietal diameter (R2=0.512, p=0.009), late head circumference (R2=0.498, p=0.001), middle biparietal diameter (R2=0.819, p=0.013), middle cerebellum transverse diameter R2=0.76, p=0.014) and early biparietal diameter(R2=0.819, p=0.013). The same analysis revealed that birth weight as well as ultrasound parameters such as abdominal circumference and femur length were not correlated to maternal cortisol levels. In conclusion, our study demonstrates that maternal cortisol secretion within physiological ranges may be inversely correlated to fetal brain growth but not to birth weight. It remains to be demonstrated whether maternal cortisol secretion negatively influencing fetal brain growth translates to adverse neurological outcomes in later life.     

Myasthenia gravis in pregnancy: Experience of a portuguese center

Introduction: We evaluated the clinical course during pregnancy and neonatal outcomes of a cohort of Portuguese women with myasthenia gravis (MG). Methods: Retrospective study. Results: Twenty‐five patients with 30 pregnancies were included. Mean maternal age was 32.4 ± 4.1 years. Miscarriage rate was 6.7%, with delivery of 28 newborns. Deterioration in MG during pregnancy occurred in 43.3%, and 46.4% occurred at postpartum. Eighty percent were medicated with pyridostigmine, 43.3% with corticosteroids, and 40% with intravenous immunoglobulin. There were no maternal or neonatal deaths. Mean gestational time at delivery was 38.2 weeks. No cases of fetal growth restriction, preeclampsia, preterm delivery, or fetal demise were observed. Global cesarean rate was 64.3%. Two newborns developed transient neonatal myasthenia. Conclusions: A high rate of clinical worsening of MG in the mother was observed in this retrospective study, which highlights the importance of a multidisciplinary approach for avoiding maternal adverse outcomes. Muscle Nerve 54 : 715–720, 2016