人废弃胚胎单卵裂球体外发育潜能研究

目的:探讨体外受精治疗周期废弃胚胎来源的单卵裂球体外发育潜能。方法:收集21例患者授精培养后第3日不适于移植和冷冻的废弃胚胎共60枚,其中2-细胞期胚胎10枚,3-细胞期胚胎8枚,4-细胞期胚胎26枚,5-细胞期胚胎12枚,6-细胞期胚胎4枚。采用化学法去除透明带并分割胚胎获得单卵裂球,继续体外培养,每24h观察卵裂球发育情况。结果:60枚胚胎共获得229枚单卵裂球。以分割取得单卵裂球日作为第1日(d1),继续培养,其发育似乎遵循d2分裂、d3紧密化、d4出现囊胚腔、d5扩张,延续分割前的程序,但提前进入紧密化期,致使囊胚细胞数少,囊胚期体积亦比正常胚胎小。其中,4-细胞期废弃胚胎来源的卵裂球分裂率、紧密化率、囊胚形成率显著高于来自其它细胞数的单卵裂球(P<0.05),且其大部分内细胞团(ICM)细胞的碱性磷酸酶(AKP)表达呈阳性。结论:废弃胚胎来源的单卵裂球能在体外继续发育至囊胚,发育潜能与供体胚胎发育质量密切相关,其发育似乎延续原有供体卵裂球的节律,而形成囊胚所需时间明显缩短,细胞数亦明显减少,但囊胚ICM细胞显示AKP阳性,深信其利用价值会得到进一步证实。     

小鼠4-细胞期胚胎单卵裂球体外发育潜能的实验研究

目的:探讨小鼠4-细胞期胚胎的单卵裂球(4-cell stage mice embryo derived-blastomeres, 4c-MEDB)的全能性。方法:将小鼠4-细胞期胚胎分割产生单个卵裂球,在体外无透明带培养条件下,不加外源性细胞因子,观察其发育潜能及特性,并将得到的囊胚与正常囊胚进行形态学、免疫学鉴定比较,同时将4c-MEDB来源囊胚(individual blastomere derived-blastocysts,IBDB)移植到假孕母鼠子宫,观察其发育情况。结果:①体外无透明带培养条件下,4c-MEDB较正常胚胎发育滞后,囊胚获得率低;②4c-MEDB具有发育成为完整囊胚的潜能;③IBDB移植入假孕小鼠子宫后具有一定发育能力,能发育到原条期,但未获得足月妊娠。结论:在未添加外源性细胞因子的体外无透明带培养条件下,小鼠4-细胞期胚胎单卵裂球具有发育为完整囊胚的能力,所获囊胚移植入假孕小鼠子宫后可以发育到原条期。     

B超早期诊断胚胎停止发育的原因探讨

胚胎停止发育最终导致胚胎丢失 ,是流产前的病理过程 ,可导致稽留流产和不全流产。探讨早期诊断胚胎停止发育的方法 ,可以减少产妇并发症。我院应用B超检查对妊娠 5～ 9+ 6周胚胎发育形态结构进行监测 ,总结出胚胎停止发育的B超检查参数 ,提出早期诊断标准 ,现报道如下。1　材料与方法1 1　研究对象我院门诊接诊妊娠 5～ 9+ 6周孕妇 12 2 9例 ,其中 12 5例有流产征兆或重复、习惯性流产史。1 2　方法1 2 1　计算孕龄　选择月经周期正常妇女 ,适度膀胱充盈 ,用Alora6 30扇扫 ,进行胚胎发育监测 ,孕龄 >6周用腹部B超 ,≤ 6周用阴道B超检查…     

人卵泡液脂肪酸谱评估卵母细胞发育潜能的初步研究

目的:分析体外受精(IVF)患者单卵泡液脂肪酸谱表达差异与卵母细胞发育潜能的相关性。方法:利用气相色谱分析技术检测卵泡液脂肪酸谱,以第3日胚胎碎片评分(EFS)及胚胎细胞数目(ECN)为卵母细胞发育结局的评价指标,行相关性分析。结果:单卵泡液中普遍存在6种脂肪酸:十一烷酸、肉蔻豆酸、棕榈酸、硬脂酸、油酸和亚油酸。Spearman分析显示油酸与EFS呈正相关(r=0.357,P=0.014),各种脂肪酸与ECN无明显相关性。Logistic回归进一步分析显示油酸浓度为EFS阳性预测因子(OR=1.035,95%CI 1.007~1.064)。结论:人卵泡液油酸含量在一定程度上可预测卵母细胞发育潜能。     

慢速程序化冷冻与玻璃化冷冻对第3天分裂期胚胎发育潜能的影响

目的比较慢速程序化冷冻法（慢速法）和玻璃化冷冻法（玻璃化法）对第3天分裂期胚胎发育潜能的影响。方法选择因输卵管阻塞或男性少弱精因素行体外受精（IVF）或卵母细胞胞质内单精子注射（ICSI）治疗的患者,挑选在行IVF或ICSI后第3天剩余优质胚胎数目超过4个者80例进入本研究。随机将每例患者的2个胚胎用玻璃化法冷冻,另外2个用慢速法冷冻;冷冻复苏后随机抽取40例患者移植慢速法冷冻的胚胎,另40例患者移植玻璃化法冷冻的胚胎,比较2种冷冻方法对胚胎发育潜能的影响。结果冷冻复苏后待移植的胚胎共160个,其中慢速法冷冻80个,复苏后存活73个（91％,73／80）,移植后40例患者中15例（38％,15／40）获得临床妊娠,其中3例（20％,3／15）为双胎妊娠,余为单胎妊娠,胚胎着床率为25％（18／73）;玻璃化法冷冻胚胎80个,复苏后存活71个（89％,71／80）。移植后40例患者中19例（48％,19／40）获得临床妊娠,其中9例（47％,9／19）为双胎妊娠,余为单胎妊娠,胚胎着床率为39％（28／71）,与慢速法相比,玻璃化法的临床妊娠率和双胎妊娠率均有提高,但差异无统计学意义（P〉0．05）,而胚胎着床率则显著提高,差异有统计学意义（P〈0．05）。结论玻璃化法能够更好地保存胚胎复苏后的发育潜能,更适合第3天分裂期胚胎的冷冻保存。     

0～6岁农村儿童早期智力发育的调查分析

目的:调查分析0～6岁农村儿童智力发育的现状。方法:随机对1124名0～6岁农村儿童进行入户调查,采用丹佛发育筛查量表(DDST)对应人能、细动作应物能、言语能、运动能四个能区逐项测试,对结果异常或者可疑者询问母亲文化程度及孕期情况、儿童身体状况及受教育方式。结果:1124例接受调查儿童中,116例(10.32%)DDST测试异常,确证智力低下(MR)7例(0.623%)。其中,1～6月儿童1例(0.8%),-12月16例(13.79%),-2岁25例(21.55%),-3岁22例(18.97%),-4岁21例(18.10%),-5岁18例(15.79%),-6岁13例(11.21%)。曾患新生儿疾病42例(36.21%)、早产18例(15.52%)、低体重15例(12.93%)、营养性疾病13例(12.21%)、神经系统疾病9例(7.76%)、外伤17例(14.66%)、先天性疾病2例(1.72%)。母亲文化程度:高中32例(27.59%)、初中72例(62.07%)、小学12例(10.34%)。孕期情况:母亲妊娠高血压综合征(简称妊高征)或其他疾病23例(19.83%)、怀孕早期患病(感冒或其他情况)服药...     

授精第3日发育迟缓胚胎的冷冻价值及可行性方案探讨

目的:探讨授精第3日发育迟缓(5~6-细胞)的胚胎冷冻价值及如何提高其利用价值的可行性方案。方法:回顾性分析进行冷冻复苏移植第3日发育迟缓胚胎的124个周期的临床资料,根据不同的复苏移植方案和培养条件,分为第3日冷冻,复苏移植周期提前1 d解冻者(A组,32个周期)、解冻后培养至囊胚再移植组(B组,62个周期)、当日解冻组(C组,30个周期),再与同期新鲜周期第3日发育迟缓行囊胚培养再冷冻复苏囊胚(复苏囊胚组,D组)的58个周期比较。结果:1各组在女方年龄、不孕年限、排卵日子宫内膜厚度、移植日激素水平等方面无统计学差异(P0.05)。2D组的周期临床妊娠率(55.4%)和种植率(42.2%)都高于其他组(P0.05)。A组和B组的周期取消率(12.5%和35.5%)均高于其他组(P0.05),B组的临床妊娠率(40.0%)高于C组(33.7%)(P0.05),但与A组(39.3%)比较无统计学差异(P0.05)。结论:1授精第3日5~6-细胞胚胎可以通过新鲜周期延长体外培养时间至第6日,获得囊胚后再进行冷冻复苏,可以得到更好的临床结局。2对于胚胎少的患者,可以在患者知情同意下选择5~6-细胞胚胎第3日直接冷冻,复苏周期提前解冻来挑选具有发育潜能的胚胎进行移植。     

早期综合干预对高危儿行为发育影响的研究

目的:探讨早期干预对高危儿行为发育的效果。方法:将197例高危儿随机分为干预组、未干预组,并设正常婴儿为对照组,参照鲍氏早期教育资料制定干预计划,采用门诊康复训练和家庭干预模式相结合,用Gesell婴幼儿发育检查量表测出两组高危儿的行为发育商(DQ)并作比较,同时还与正常新生儿作比较。结果:1岁时Gesell智能检查结果显示:干预组各行为能区的DQ均大于未组,存在统计学差异(P<0.05),与对照组比较,各行为能区的DQ差异均无显著性。结论:门诊的康复训练和家庭干预模式相结合可以促进高危儿的行为发育,改善其预后。     

体外受精中3原核胚胎的发育及可利用价值的探讨

目的:探讨体外受精(IVF)中异常的3原核(PN)胚胎的发育及可利用价值。方法:收集IVF治疗周期中废弃的3PN受精卵204个进行体外培养,观察其发育能力,并与同周期的1 138个2PN受精卵进行比较;采用胚胎植入前遗传学筛查(PGS)技术对由3PN发育成的19枚囊胚进行非整倍体分析。结果:3PN组和2PN组的卵裂率无统计学差异(P0.05);但3PN组囊胚形成率显著低于2PN组[9.6%(19/97)vs 37.9%(204/342),P0.01]。整倍体分析显示,10.5%(2/19)的3PN来源的囊胚为正常二倍体核型。结论:3PN受精卵有继续发育能力;囊胚培养和高通量测序可作为有效筛选异常PN受精卵中正常核型胚胎的一种方法。     

表皮生长因子(EGF)和胰岛素样生长因子(IGF-Ⅱ)对小鼠1-细胞胚胎体外发育的影响

目的:研究表皮生长因子(epidermalgrowthfactor,EGF)在小鼠早期胚胎体外发育中的作用,以及EGF联合胰岛素样生长因子-Ⅱ(insulin-likegrowthfactor-II,IGF-Ⅱ)对小鼠早期胚胎体外发育是否有协同促进作用。方法:①在mKSOM培养液中添加0ng/ml(对照组)、0.1ng/ml、1ng/ml、10ng/ml、100ng/ml的EGF、10ng/mlIGF-Ⅱ、1ng/mlEGF+1ng/mlIGF-Ⅱ、1ng/mlEGF+10ng/mlIGF-Ⅱ、10ng/mlEGF+1ng/mlIGF-Ⅱ、10ng/mlEGF+10ng/mlIGF-Ⅱ培养小鼠1-细胞胚胎,每组胚胎在培养箱中连续培养120h,每24h观察胚胎发育情况,分别计算2-细胞率、4-细胞率、桑椹胚率、囊胚率和孵化率,并进行囊胚细胞计数。结果:添加1ng/ml、10ng/mlEGF组的囊胚率及孵化率显著增加;EGF+IGF-Ⅱ组较单一添加组、对照组囊胚率及孵化率显著增加(P<0.05),且EGF+IGF-Ⅱ组囊胚细胞计数显著高于其他组(P<0.05),其中10ng/mlEGF+10ng/mlIGF-Ⅱ组的孵化率及囊胚细胞数最高(P<0.05)。结论:EGF浓度在1￣10ng/ml范围内可以提高体外培养鼠胚的囊胚率及孵化率;EGF及IGF-Ⅱ对体外培养鼠胚的发育有协同促进作用;本实验范围内10ng/mlEGF+10ng/mlIGF-Ⅱ为最优组合。     

Effect of morphokinetics and morphological dynamics of cleavage stage on embryo developmental potential: A time-lapse study

Objective

Using a non-invasive method to select the most competent embryo is essential in in vitro fertilization (IVF). Since the beginning of clinical application of time-lapse technology, several studies have proposed models using the time-lapse imaging system for predicting the IVF outcome. This study used both morphokinetic and morphological dynamic parameters to select embryos with the highest developmental potential.

Blastomere cytoplasmic granularity is unrelated to developmental potential of day 3 human embryos

Purpose : To examine whether the increase in cytoplasmic granularity observed in some human embryos on day 3 of development is of any predictive value as to embryo developmental potential. Methods : Retrospective study comparing outcomes of treatment attempts in three groups of patients after day 3 embryo transfer. Attempts in which only embryos with clear cytoplasm were transferred form Group I, those in which only embryos with granulated cytoplasm were transferred constitute Group II, and Group III consists of cases with mixed transfer combining both types of embryos. Each group was further divided according to the female age. Results : Clinical pregnancy rates in Groups I (314 attempts), II (173 attempts), and III (323 attempts) were 33.8, 36.4, and 31.3%, respectively. Implantations rates for Groups I–III were 17.0, 17.3, and 14.8%, respectively. No significant differences between groups concerning these and other values, including the number of oocytes and of metaphase II oocytes recovered, fertilization and cleavage rates were found. The proportion of good-morphology embryos was also similar between the different groups (74.3, 72.7, and 70.2% respectively). The representation of women of advanced age (>36 years) was also similar in each group, and intergoup differences remained insignificant were only younger or only older women were taken into account. Conclusion(s) : These data show that the appearance of cytoplasmic granulation in blastomeres of day 3 human embryos is of no prognostic value as to embryo quality and appears to be unrelated to the female age.     

Influence of post-thaw culture on the developmental potential of human frozen embryos

Purpose

Apart from freezing/thawing related cryodamage, several additional factors have been identified as major players in the reduction of success rates after frozen embryo transfers. The post-thaw culture is particularly relevant as it may amplify environmental influences over a stressed embryo. In the present study the influence of the post-thaw culture duration on the implantation and developmental potential of cleavage stage embryos was evaluated.

Methods

In this retrospective evaluation, that spanned an 8-year period, 631 frozen-thawed embryos were allocated to one of two study groups, depending on their post-thaw culture period: 1) the long (18–24 h), or 2) the short (2–5 h) culture group. Groups were compared regarding implantation rate and live birth rate per embryo transferred. This comparison was corrected for the most common confounding factors such as maternal age at oocyte pick-up, number of transferred embryos, developmental day at freezing, blastomere survival after thawing, catheter used for transfer and year of procedure.

Results

Implantation and live birth rate per embryo transferred were inversely related to the duration of the post-thaw culture, as diminishing this period significantly increased both rates. Moreover, no advantage could be found for a long post-thaw culture period, even for embryos with observed mitotic activity.

Conclusion

This retrospective analysis indicates that a short post-thaw culture period is associated with higher implantation and live birth rates per embryo. This study supports selection of frozen-thawed embryos strictly based on blastomere cryosurvival and raises the hypothesis that environmental factors may have an important role on embryo implantation and developmental potential during post-thaw culture.     

Using the Eeva Test™ adjunctively to traditional day 3 morphology is informative for consistent embryo assessment within a panel of embryologists with diverse experience

PurposeSince many transferred, good morphology embryos fail to implant, technologies to identify embryos with high developmental potential would be beneficial. The Eeva™ (Early Embryo Viability Assessment) Test, a prognostic test based on automated detection and analysis of time-lapse imaging information, has been shown to benefit embryo selection specificity for a panel of three highly experienced embryologists (Conaghan et al., 2013). Here we examined if adjunctive use of Eeva Test results following morphological assessment would allow embryologists with diverse clinical backgrounds to consistently improve the selection of embryos with high developmental potential.MethodsProspective, double-blinded multi-center study with 54 patients undergoing blastocyst transfer cycles consented to have embryos imaged using the Eeva System, which automatically measures key cell division timings and categorizes embryos into groups based on developmental potential. Five embryologists of diverse clinical practices, laboratory training, and geographical areas predicted blastocyst formation using day 3 morphology alone and day 3 morphology followed by Eeva Test results. Odds ratio (OR) and diagnostic performance measures were calculated by comparing prediction results to true blastocyst outcomes.ResultsWhen Eeva Test results were used adjunctively to traditional morphology to help predict blastocyst formation among embryos graded good or fair on day 3, the OR was 2.57 (95 % CI=1.88–3.51). The OR using morphology alone was 1.68 (95 % CI=1.29–2.19). Adjunct use of the Eeva Test reduced the variability in prediction performance across all five embryologists: the variability was reduced from a range of 1.06 (OR=1.14 to 2.20) to a range of 0.45 (OR=2.33 to 2.78).ConclusionsThe Eeva Test, an automated, time-lapse enabled prognostic test, used adjunctively with morphology, is informative in helping embryologists with various levels of experience select embryos with high developmental potential.

Electronic supplementary material

The online version of this article (doi:10.1007/s10815-014-0366-1) contains supplementary material, which is available to authorized users.     

卵母细胞分泌因子在人卵丘颗粒细胞中的表达及其与卵母细胞发育潜能的关系

目的:探讨单卵母细胞所对应的卵丘颗粒细胞(cumulus cells,CCs)中生长分化因子-9(GDF-9)、骨形成蛋白-15(BMP-15)、转化生长因子β1(TGFβ1)、Smad3的表达与对应单卵母细胞质量及其发育潜能的关系以及在不同促排卵方案中的表达。方法:因男方因素或曾有过体外受精(IVF)不受精史接受卵胞质内单精子显微注射(ICSI)治疗的患者41例,单卵母细胞的CCs 332份(Gn RH-a组21例172份CCs,非垂体降调节组20例160份CCs)。拣卵后机械法剥离单卵子周围的CCs,将卵母细胞与收集的CCs一一对应进行分析。M_Ⅱ卵均采用ICSI授精,并观察其受精、卵裂及囊胚发育情况。采用Real-time PCR方法检测,比较各组CCs中GDF-9、BMP-15、TGFβ1和Smad3的相对含量。结果:1 CCs收集及PCR扩增产物可满足实验要求。2 Gn RH-a组及非垂体降调节组中,上述4种卵母细胞分泌因子,在正常受精组的相对含量均显著高于异常受精组;优质卵裂胚形成组的相对含量均显著高于非优质卵裂胚形成组;优质囊胚形成组中的相对含量显著高于非优质囊胚形成组。3 Gn RH-a组的CCs中BMP-15的表达量相对更高;而GDF-9、TGFβ1和Smad3在非降调节组的CCs中相对表达量较高。结论:1采用单卵母细胞的CCs的RNA进行研究,实际操作是可行的,RNA的质量和数量可达到实验要求。2 GDF-9、BMP-15、TGFβ1、Smad3可作为评价卵母细胞质量、预测卵母细胞发育潜能的客观指标。3降调节与否可能影响CCs中的卵母细胞分泌因子的表达水平,且与M_Ⅱ期卵母细胞发育潜能有关。     

From a circle to a sphere: the ultrasound imaging of ovarian follicle with 2D and 3D technology

Ultrasound follicular count (antral follicle count, AFC) is a necessary tool for measuring ovarian reserve, whereby the estimated number of follicles responsive to FSH can predict the number of oocytes retrieved in IVF cycles and may be the basis for individualized ovarian stimulation therapy. Advances in the ultrasound technology have recently lead to the improvement in resolution and quality of the image. Moreover the automatic measurements of follicular diameter by using some specific 3D software seems associated to several advantages when compared to the 2D technique. Examination time is reduced because the ultrasound scan data are stored and can be analyzed in detail at a later time. These data can be reconstructed in any plane, regardless of the original scan plane facilitating the detailed analysis. Another advantage is that this new technique reduces the operator’s influence on scan interpretation and objectivity; therefore, interobserver variability is reduced. Using follicular volume obtained with sono AVC as the measure of follicular growth combined with volume-based criteria for the hCG triggering may in the future improve the treatment outcome compared to that achieved with conventional monitoring with follicular diameter. Better knowledge in this area could be helpful to optimize IVF outcome, by refining ovarian stimulation protocols and obtain high quality oocytes.     

Monoclonal antibody-based immunotherapy of ovarian cancer: Targeting ovarian cancer cells with the B7-H3-specific mAb 376.96

Objective

The high rate of relapse in patients with advanced ovarian cancer likely reflects the chemoresistance of cancer initiating cells (CICs). We evaluated the anti-tumor activity of monoclonal antibody (mAb) 376.96, which recognizes a B7-H3 epitope expressed on ovarian carcinoma cells (OCCs), in combination with the tyrosine kinase inhibitor Sunitinib and chemotherapy on chemosensitive and chemoresistant cells and CICs.

Methods

Eight ovarian cancer cell lines including platinum- and taxane-resistant cell lines were analyzed by flow cytometry to establish expression of the mAb 376.96-defined-B7-H3-epitope on differentiated ovarian cancer cells and CICs. Samples from 10 ovarian cancer patients were analyzed via immunohistochemistry for mAb 376.96-defined-B7-H3-epitope expression. In vitro studies assessed mAb 376.96 alone and in combination with Sunitinib on the growth of chemosensitive and chemoresistant cell lines and on the content of CICs.

Results

The mAb-376.96-defined-B7-H3 epitope is expressed on both differentiated cells and CICs in chemosensitive and chemoresistant ovarian cancer cell lines and 10 patient derived ovarian cancer tumors. In vitro treatment of chemoresistant cell lines with mAb 376.96 resulted in decreased cell viability. mAb 376.96 enhanced the cytotoxicity of Sunitinib and reduced the content of CICs.

Conclusion

The mAb-376.96-defined-B7-H3-epitope was found to be expressed on both differentiated ovarian cancer cells and CICs in chemosensitive and chemoresistant ovarian cancer cell lines. mAb 376.96 inhibited the in vitro growth of chemosensitive and chemoresistant OCCs and reduced the content of CICs when used with Sunitinib. Further studies examining B7-H3 as a potential target of mAb-based immunotherapy for this type of malignancy are warranted.