兔抗小鼠胚胎干细胞免疫血清对8-细胞胚胎发育的影响

目的 制备兔抗小鼠胚胎干细胞免疫血清,观察其对小鼠胚胎发育的影响。方法 用小鼠胚胎培养基稀释兔抗血清,分别稀释至50倍、100倍、200倍,用其对小鼠8-细胞胚胎进行培养,通过胚胎致密化、囊胚率、胚胎体外贴壁生长、细胞计数、胚胎移植和免疫荧光染色观察胚胎发育状况。 结果 形态学上50倍稀释的抗血清能够抑制8 细胞胚胎的发育甚至使其死亡;100倍稀释的抗血清能够延迟8 细胞胚胎的致密化,并使得胚胎在囊胚化过程中出现空泡化的现象;200倍稀释的抗血清作用不明显。未作免疫处理的兔血清与空白对照组差异不显著。100倍稀释的抗血清能够显著抑制囊胚内细胞团（ICM）的发育、囊胚细胞分布紊乱、胚胎细胞数目减少以及囊胚畸形率增加。虽然囊胚的碱性磷酸酶和Oct4均呈阳性,但经胚胎移植后都不能正常发育。 结论 兔抗小鼠胚胎干细胞免疫血清能够抑制小鼠胚胎致密化过程和囊胚内细胞团的发育,使囊胚出现空泡化,细胞分布紊乱,抑制胚胎着床。     

囊胚培养液对小鼠胚胎的毒性:体外生殖辅助医疗器械安全性评价

背景:囊胚培养液是可以支持胚胎从受精卵发育到囊胚阶段的一种重要培养液,其安全性直接影响囊胚的质量。目的:观察囊胚培养液对小鼠胚胎发育的影响。方法:选用B6D2F1品系小鼠,将雌性小鼠进行超数排卵处理,与同品系雄鼠合笼过夜,次日收集1细胞期受精卵,在M16培养液中培养至4细胞期后,检查胚胎发育情况并转移到受试囊胚培养液中继续培养(实验组),5 d后检查囊胚发育情况并计算囊胚形成率。同时,用含有内毒素的囊胚培养液M16培养小鼠受精卵,作为阳性对照组;用已被证实没有胚胎毒性的囊胚培养液M16培养液培养1细胞期受精卵,作为阴性对照组。结果与结论:阳性对照组囊胚形成率为0,阴性对照组囊胚形成率为87.1%,实验组囊胚形成率为87.3%,实验组囊胚形成率大于80%,说明受试囊胚培养液可以支持小鼠受精卵正常发育到囊胚阶段,对胚胎没有毒性效应。     

MATER蛋白在人类卵母细胞及着床前胚胎中的表达

目的探讨Mater在人卵发生和胚胎早期发育中的表达情况。方法收集体外受精助孕过程中未成熟的卵母细胞及不适合移植及冷冻保存的胚胎，采用免疫荧光联合激光共聚焦显微镜技术检测人类卵母细胞及着床前胚胎MATER蛋白的表达。结果16个未成熟卵母细胞15个MATER蛋白表达阳性；58个2—8细胞期人类早期胚胎中40个MATER蛋白检测阳性；5个囊胚和4个孵化出囊胚内细胞团均为阴性，滋养层细胞为弱阳性。结论MATER蛋白在人类卵母细胞中存在，并随着早期胚胎的发育其表达阳性率逐渐降低，至囊胚期只有滋养层细胞少量表达，这符合母性效应基因在哺乳动物中的表达模式。     

输卵管积水对小鼠胚胎体外发育能力的影响

目的探讨输卵管积水对小鼠胚胎体外发育能力的影响。方法收集人输卵管积水，小鼠促超排卵，收集2细胞胚胎和囊胚，随机分配到含有不同浓度输卵管积水的培养液中，观察胚胎的发育，计算成囊率，囊胚孵出率。结果输卵管积水组的胚胎成囊率和囊胚孵出率低于无积水组，并呈剂量依赖性。结论输卵管积水能显著影响胚胎的早期发育潜能。     

人类胚胎早期发育潜能的研究

目的　本研究采用卵裂球分离技术获取不同发育阶段人类早期胚胎单个卵裂球 ,对分散的人类早期胚胎单个卵裂球进行体外培养。对其进一步发育和分化进行研究 ,发现其影响因素。结果　 2 0个 4-细胞、2个 3-细胞人类早期胚胎分散后获得单个卵裂球 83个 ,经体外培养后有 35个发育成为扩张囊胚。结论　 4-细胞人类胚胎卵裂球具有发育成为完整囊胚的能力     

菟丝子含药血清对大鼠胚胎肢芽形态发育的影响

目的探讨不同剂量菟丝子含药血清对SD大鼠胚胎肢芽形态发育的影响。方法采用中药血清药理学方法制备菟丝子含药血清。取孕第15天胚胎前肢芽,随机分为3个实验组、阴性对照组和阳性对照组。实验组和阴性对照组分别加入10%培养体积的高、中、低剂量菟丝子含药血清和正常血清,阳性对照组加入全反式维甲酸溶液(10 mg/mL)。采用体外肢芽悬浮培养法培养72h后收获肢芽,石蜡包埋、切片、HE染色观察肢芽细胞和软骨形态。每组取30个肢芽,测量肢芽长度。结果经HE染色后,菟丝子含药血清低、中剂量组和阴性对照组肢芽结构完整,细胞排列整齐,软骨轮廓清晰,指突明显。高剂量组和阳性对照组肢芽软骨出现,轮廓较清晰,但指突不明显。分段测量肢芽长度后,与阴性对照组比较,低和中剂量组前肢芽近端a段长度和远端b段长度无显著性差异(P>0.05)。而阳性对照组和高剂量组的肢芽长度显著小于阴性对照组,与之比较差异显著(P<0.05)。结论高剂量菟丝子含药血清对SD大鼠胚胎前肢芽生长有抑制作用,可显著抑制肢芽指突的生长,存在潜在的胚胎发育毒性。     

人废弃胚胎体外培养成囊胚的实验室结局

背景：有报道称可以利用废弃胚胎建立人类胚胎干细胞系,对形态发育滞后的胚胎冻融后移植仍有再利用的医学价值。 目的：观察人废弃胚胎在体外继续培养形成囊胚的潜能。 方法：收集接受体外受精-胚胎移植或卵胞浆内单精子注射患者治疗后第3天废弃的胚胎404枚,进行囊胚序贯培养。 结果与结论：①废弃胚胎404枚,形成囊胚 65枚,总囊胚形成率为16.1%。②囊胚形成与患者的病因分类、年龄、受精方式无相关性(P > 0.05)。③胚胎来源于≥三原核(3PN)的(6~8)细胞组的囊胚形成率最高(P < 0.05)。卵裂球数随细胞数的增加,囊胚率也增高,但>8细胞囊胚率反而下降、与<3细胞组的结果相近。④Ⅰ,Ⅱ胚胎形成囊胚速度快,形成率显著高于Ⅲ,Ⅳ胚胎(P < 0.05)。⑤有囊胚形成患者的临床妊娠率显著高于无囊胚形成患者(P < 0.05)。提示囊胚形成与患者病因分类、年龄、受精方式无相关性。优质胚胎与(6~8)细胞的胚胎囊胚形成率高,多原核胚胎形成囊胚的价值应引起关注。废弃胚胎再利用能预测临床结局,为人类胚胎干细胞提供有利资源。 关键词：人废弃胚胎;胚胎培养;囊胚形成;结局分析;胚胎干细胞 doi:10.3969/j.issn.1673-8225.2012.05.038     

姐妹胚胎单囊胚结局的影响因素分析

目的 探讨姐妹胚胎继续单囊胚培养后影响其囊胚形成的相关因素.方法 回顾性分析行D3优质胚胎移植、 剩余姐妹胚胎单囊胚培养的7662个胚胎的相关信息,根据D3胚胎质量分为优质胚胎组及非优质胚胎组;根据形成囊胚的情况分为无囊胚形成组及囊胚形成组,其中囊胚形成组又按时间分为D5组和D6组,按囊胚形成的质量分为优质囊胚组和非优质囊胚组.结果 D3优质胚胎组的囊胚形成率、 优质囊胚形成率、D5/D6囊胚形成率、D5/D6优质囊胚形成率均显著高于非优质胚胎组;0PN、2PN胚胎的囊胚形成率、 优质囊胚形成率均显著高于1PN胚胎;囊胚形成组与无囊胚形成组间D3优质胚胎、1PN、2PN胚胎比例有显著差异;优质囊胚形成组与非优质囊胚形成组之间0PN、1PN、D3优质胚胎比例有显著差异;D5与D6囊胚形成组组间0PN、1PN、2PN、D3优质胚胎比例均存在显著差异.Logistic回归结果表明,原核数、 胚胎质量均对单囊胚培养结局有显著影响.结论 原核数、D3胚胎质量与姐妹胚胎单囊胚培养的结局密切相关.     

IGF-Ⅱ对小鼠2-细胞胚胎体外发育的影响

目的研究胰岛素生长因子-Ⅱ对小鼠2-细胞胚胎体外发育的影响。方法在mKSOM培养液中添加不同浓度的IGF—Ⅱ（insulin—like growth factor—Ⅱ）对小鼠2-细胞胚胎进行体外培养,观察囊胚发育率、孵化率和囊胚细胞数的变化。结果（1）培养到72h,实验组囊胚率显著高于对照组。（2）培养到96h,0．1、1、10ng／ml IGF-Ⅱ添加组的孵化率显著高于对照组和100ng／ml IGF—Ⅱ添加组。（3）进行囊胚细胞计数,实验组与对照组比较,差异无显著性;四个实验组之间比较,差异亦无显著性。结论IGF-Ⅱ浓度为0．1、1、10ng／ml时,能促进小鼠2-细胞胚胎的体外发育。     

恶性肿瘤细胞对小鼠2-细胞胚胎体外发育的影响

目的： 探讨生命早期形式与恶性肿瘤细胞在相同微环境下的早期胚胎发育情况和肿瘤细胞的生物学行为。方法： 建立小鼠2-细胞胚胎-恶性肿瘤细胞(HepG2,SKOV3,HNE1,Hepa1-6,B16,CHO)体外共培养模型，观察小鼠胚胎的发育状况和恶性肿瘤细胞的生物学行为。结果： 处于不同种属源性和胚层来源恶性肿瘤微环境的小鼠胚胎4-细胞形成率，桑葚胚形成率和囊胚形成率及其形成的囊胚细胞数目与对照组比较无显著差异（P>0.05）。小鼠的2-细胞胚胎能在人源性和鼠源性不同胚层来源的恶性肿瘤细胞体外培养环境中按一定的时间顺序发生卵裂、卵裂球紧密化、分化、囊胚腔形成，而肿瘤细胞的形态、增殖及核分裂相与对照组比较没有明显差异。结论： 在共培养环境中，恶性肿瘤细胞对小鼠2-细胞胚胎的发育时程没有阻滞和破坏作用，且与恶性肿瘤细胞的种属和胚层来源无关。小鼠早期胚胎能在不同种属和胚层的恶性肿瘤环境中按正常体外培养发育时程发育，这可能与早期胚胎发育的自我组织、高适应性和肿瘤细胞分泌的某些因子有关。     

Embryo evaluation by analysing blastomere nuclei

BACKGROUND: To create a more effective selection standard for early embryos, we developed a new grading system consisting of conventional morphological evaluation in combination with analysis of blastomere nuclei. METHODS: A total of 744 embryos used during 459 cycles of embryo transfer on day 2 and blastocyst transfer were subjected to retrospective analysis. The overall implantation rate was 15.5% (115/744). Morphological evaluation of the embryos was performed on day 2 by referring to both the size of blastomere and fragmentation (conventional method) and the nucleic features of the blastomeres--either multinucleated or anucleic (nuclei counting method). The implantation rate for every transferred embryo and blastocyst was examined. RESULTS: Although a high implantation rate was observed with the highest quality embryos as judged by either the conventional method (24.1%; 57/237) or the nuclei counting method (26.1%; 104/399), the nuclei counting method predicted implantation rate better than the conventional method. The embryos that were considered to be high quality according to the conventional method, but low quality according to the nuclei counting method, had a limited implantation success rate of 6.3% (4/66). Also, after blastocyst transfer, implantation occurred most often when high quality embryos evaluated by the nuclei counting method were used (25.5%; 25/98), while the blastocysts from low quality embryos seldom implanted (3.2%; 2/63). CONCLUSIONS: When choosing which embryo to transfer, the normality of blastomere nuclei may be a more important index of quality than standard fragmentation features and/or blastomere uniformity analysis. When choosing among embryos, if nucleic status is identical, then embryos with the least fragmentation should be chosen. Moreover, in blastocyst transfer, a blastocyst whose nuclei were judged normal on day 2 should be selected on day 5 over any other blastocysts.     

Prevalence of Embryotoxic Factor in Sera From Women With Unexplained Recurrent Abortion

PROBLEM : The presence of embryotoxic factors in sera from women with recurrent spontaneous abortion (RSA) has been proposed as a basis for classification of unexplained RSA. To determine the prevalence of circulating embryotoxins among women with idiopathic RSA, sera from 160 women were studied using the mouse blastocyst assay. METHODS : Two-cell embryos were collected from superovulated mated CB6F1/J mice and cultured in media supplemented with fetal bovine serum (FBS) or 10% serum at 37°C with 5% CO₂ and high humidity. Each assay was run in triplicate using three mice with at least five embryos from each mouse. Results were determined by calculating the average percentage atresia for each mouse. FBS, known to support embryo proliferation, was used to control in each assay. RESULTS : The prevalence of embryotoxic factors among women experiencing RSA was 24.4% (39/160). There is no correlation found between the presence of embryotoxicity and phospholipid antibodies, lupus anticoagulant, and thyroglobulin/microsomal antibodies. CONCLUSION : The embryotoxicity assay can serve as a basis for a new approach for classification of unexplained recurrent spontaneous abortion.     

高值血清CA19-9稀释测定的影响因素研究

探讨高值血清CA19-9稀释后测定的结果与临床影响因素.对高值血清CA19-9用不同的稀释液(去离子水,Dil稀释液,健康人混合低值血清),不同稀释倍数(1∶2;1∶5;1∶10;1∶20)经手工稀释测定和仪器自动稀释检测.结果表明:用Dil稀释液进行稀释,手工法和仪器自动稀释法呈明显正相关;3种稀释液稀释高值血清CA...     

Preliminary clinical experience with human blastocyst development in vitro without co-culture

This preliminary analysis was designed to quantify blastocyst development of supernumerary embryos without the use of feeder cells, conditioned medium or whole serum. Embryos derived from in-vitro fertilization (IVF) that were not transferred or cryopreserved were included in this study. Ova were harvested for IVF after a standard ovarian stimulation with gonadotrophin-releasing hormone agonist/ human menopausal gonadotrophin (GnRHa/HMG) or follicle-stimulating hormone (FSH). Ova were collected and culture in 150 microliters droplets of P1 medium under mineral oil, in groups at 37 degrees C under 5% CO2, 5% O2, 90% N2 (group A) or under 5% CO2 in air (group B) environment. Embryo transfer was performed 72 h post-harvest. Viable embryos not transferred or cryopreserved were placed in blastocyst medium and cultured for an additional 48 h in 5% CO2 in air. Embryos that exhibited an expanded blastocoelic cavity and well-defined inner cell mass at 120 h were counted. Of 838 supernumerary embryos cultured, 448 (53.5%) reached the expanded blastocyst stage by 120 h of culture. Patients were given the option of cryopreservation at that time. The embryos were cryopreserved using a standard protocol with serial addition of glycerol. Embryos reaching the blastocyst stage after more than 120 h of culture were not included. There was no difference in the proportions of blastocyst development between group A, 217/410 (53.5%) and group B, 231/428 (54%). To date, 16 patients have each had up to three thawed blastocysts transferred, out of whom seven became pregnant. This report demonstrates that a simple system of sequential culture generated acceptable, viable blastocyst development (54%) with supernumerary embryos, without the use of feeder cells, conditioned medium or whole serum. Recognizing the differential metabolic requirements of early and late cleavage stage embryos has enabled the application of a glucose/phosphate-free simple culture medium (P1) for up to 72 h of culture and a complex, glucose-containing medium (blastocyst medium) for subsequent blastocyst development.     

归芪地黄汤干预对耳蜗干细胞治疗感音性耳聋的影响

BACKGROUND:Combining Chinese medicine with stem cell transplantation opens up a new avenue for the use of traditional Chinese medicine in the stem cell transplantation. It is expected to improve survival and differentiation of cochlear stem cells into hair cells through Chinese medicine interventions. OBJECTIVE:To observe the influence of Guiqi Dihuang Tang on the treatment of sensorineural deafness with cochlear stem cells. METHODS:Guinea pigs with sensorineural deafness were randomly assigned into three groups: combined group intervened with cochlear stem cell suspension with medicated serum, cell transplantation group with cochlear stem cell suspension, and blank control group with normal saline injection. At 7, 28 and 56 days after treatment, all guinea pigs were detected for auditory brainstem response and immunofluorescent observation. RESULTS AND CONCLUSION:Nestin positive cells and MyosinVIIA positive cells were observed in the combined and cell transplantation groups, and the number of positive cells was higher in the combined group than the cell transplantation group. Auditory brainstem response threshold of guinea pigs was decreased in the combined and cell transplantation groups, and the recovery of hearing was better in the former group. These findings indicate that the intervention of Guiqi Dihuang Tang can remarkably improve the survival rate of transplanted stem cells and the differentiation ratio of transplanted cells into hair cells.     

A retrospective analysis of the in-vitro development of 'spare' human in-vitro fertilization preimplantation embryos using 'in-house' prepared medium and 'Medi-Cult' commercial medium

In-house prepared medium was used routinely in our in-vitrofertilization (IVF) facility prior to the introduction of thecommercial ‘Medi-Cult’ products. A comparative studyof the in-vitro development of embryos cultured in two [T6 andEarle's balanced salt solution (EBSS)] humaninactivated serum(HlS)-supplemented media from days 0 to 5 showed that 44.7%(46/103) of the embryos developed to the blastocyst stage inthe T6 medium compared with 22.3% (23/103) in EBSS. Followingthe introduction of the commercial Medi-Cult IVF M2 medium,which is used routinely to culture fertilized eggs from days0 to 2, new baseline data were required for the in-vitro developmentof ‘spare’ embryos from days 2 to 5. When Medi-CultM3 medium was used, 35.6% (37/104) of the ‘spare’day 2 embryos achieved the blastocyst stage. However, if morphologicallysimilar (four normal nucleated blastomeres with no fragmentation)day 2 embryos were selected, an increase in the blastocyst rateto 50.0% (33/66) was achieved. This compared favourably withthe 45.0% blastocyst rate (published in the Medi-Cult literature)for M2/M3 medium cultured human embryos. A small series of experimentswith T6 $ HIS medium and human serum albumin (HSA)- supplementedHam's F-10, MCDB 302 and M3 media was undertaken to identifya suitable medium which could be used for the culture of M2medium day 2 embryos. Results show that M2 medium cultured embryosplaced in Ham's F-10 medium supplemented with 10 mg/ml HSA gavean acceptable 37.8% (14/45) blastocyst rate. Therefore, thismedium could be substituted for M3 medium in an emergency. Atotal of 483 IVF embryos donated by patients, which were surplusto the therapeutic IVF programme, were used for these studiesover a period of 30 months. Late day 2 IVF spare embryos wereassigned an embryo score based on a high-power phase-contrastmicroscopic examination prior to being placed in culture. Theembryo score provides an effective in-vitro parameter with whichembryos from different patients can be compared. The cleavageand development of individual embryos were monitored on days2 to 5. In some cases, the continuing normal development andviability of the day 5 cultured embryo were assessed by monitoringthe hatching, attachment and outgrowth of the cavitated blastocyst.     

Blastocyst formation--good indicator of clinical results after ICSI with testicular spermatozoa

BACKGROUND: The aim of this study was to evaluate the role of blastocyst culture in patients with azoospermia. METHODS: In 98 cycles embryos were cultured for 2 days and in 128 cycles for 5 days to reach the blastocyst stage; a maximum of two of the most developed embryos were transferred in each group. RESULTS: There was a negative correlation between a high (>/=20 IU/l) male serum FSH and embryo development, manifested as embryos not reaching the morula stage on day 5 (r = 0.387; P < 0.05). After prolonged culture, 23% of embryos reached the blastocyst stage. The pregnancy rates per transfer, and the abortion rates were approximately the same in the day 2 group and the day 5 group (20 versus 20% and 19 versus 18% respectively). After blastocyst transfer, a high clinical pregnancy rate (55%) and a low abortion rate (6%) were achieved, whereas the transfer of arrested embryos provided a low pregnancy rate (2%) and a high abortion rate (100%). If only blastocysts had been transferred on day 5, the clinical pregnancy rate per started cycle would have been approximately the same in both groups (13 versus 16%). CONCLUSIONS: Blastocyst formation is a good indicator of clinical results after ICSI with testicular sperm.     

Evaluation of serum-associated embryotoxicity/mutagenicity in females with reproductive dysfunctions using preimplantation mouse embryos in vitro

Reproductive failures may be caused by chromosomal anomalies,complex interactions of maternal/fetal genotype and environmentalfactors. Some of them have also been attributed to undefinedmaternal serum factor(s). We have developed an assay to evaluatematernal-serum associated embryotoxicity/mutagenicity usingthe preimplantation mouse embryo. It is based on in vitro cultureof 8-cell mouse embryos for 48 h in the presence of 10% serafrom females with reproductive dysfunction along with suitablecontrols. The human sera were obtained from three groups offemales: (i) with normal reproductive histories (negative controls),(ii) undergoing cancer chemotherapy (positive controls), and(iii) with a history of failing to achieve or maintain pregnancy(test group). The preimplantation mouse embryos were culturedin the three types of human sera and female rat serum (as aninternal control for each experiment) and evaluated for toxicity(embryo survival) and mutagenicity (as measured by SCE). Embryoscultured in serum from women with normal reproductive historiesand female rats had survival rates of 70.8 ± 4.3 and78.9 ± 0.9%, respectively. The SCE frequencies for thesetwo sera types were similar: 15.7 ± 0.2 SCEs/cell (rat)and 16.3 ± 0.3 SCEs/cell (human). Mouse embryos culturedin medium supplemented with serum from females undergoing chemotherapyhad significantly (P < 0.01) reduced survival (33.7 ±5.2%) and significantly (P < 0.01) increased SCEs/cell (26.5± 0.6). The effect of sera from women with historiesof reproductive failures on preimplantation mouse embryos wasvariable and individual specific. Approximately 40% of the testgroup female sera yielded survival and SCEs/cell comparableto the sera from patients undergoing chemotherapy. The resultssuggest that at least some of the females with reproductivefailures may possess serum substance(s) that is/are toxic topreimplantation mouse embryos in culture and is/are mutagenic.It is logical to hypothesize that at least in some cases thesesubstance(s) may contribute to or represent the cause of reproductiveproblems. Furthermore, the preimplantation mouse embryos maybe used to evaluate human serum embryotoxicity and mutagenicitybefore the onset of pregnancy which may provide strategies forappropriate preventative measures.     

Progression to the blastocyst stage of embryos derived from testicular round spermatids

Progression to the blastocyst stage of embryos derived from testicular round spermatids in men with non-obstructive azoospermia was studied. A total of 56 men were studied in whom partial spermatogenesis failure had occurred where only very few spermatozoa (fewer than the number of oocytes retrieved) were extracted from multiple testicular biopsy specimens. Oocytes remaining after intracytoplasmic injection of testicular spermatozoa (group 1) were injected with round spermatids (ROSI, group 2). Only embryos derived from group 1 were transferred. Remaining embryos were observed under culture for 8 days and their progression to the blastocyst stage was recorded. Of the 546 oocytes injected with testicular spermatozoa, 404 (73.9%) showed evidence of 2-pronuclear (2PN) fertilization. Injection of testicular round spermatids resulted in 2PN fertilization rate of 50% (P < 0.05). Using a four-point grading system, 53% of the good quality embryos (grade 1 or 2) in group 1 reached the blastocyst stage compared with 25% in group 2 (P < 0.05). The rate of progression to the blastocyst stage of grade 3 and grade 4 embryos was 46 and 8.5% in the two groups respectively (P < 0.05). Using a different three-point grading system for the blastocysts, 75.3% of the blastocysts in group 1 were either grade 1 or grade 2 and 24.7% were grade 3. However, in group 2 all blastocysts were grade 3. All embryos observed in group 1 reached the blastocyst stage by day 5 or 6 compared with 25% of the embryos reaching the blastocyst stage by this time in group 2. While 31.2% of the blastocysts in group 1 showed evidence of spontaneous hatching in vitro, none of the blastocysts in group 2 hatched. In conclusion, progression to the blastocyst stage occurred at a much lower and slower rate in embryos derived from testicular round spermatids. Furthermore, all blastocysts resulting from ROSI were of poor quality and none showed spontaneous hatching. These results may explain the dismal outcome associated with ROSI.