Effects of Fluoride on Lipid Peroxidation, DNA Damage and Apoptosis in Human Embryo Hepatocytes

Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage, apoptosis, and cell cycle analysis were measured after in vitro cultured L-02 cells were exposed to sodium fluoride at different doses (40 μg/mL, 80 μg/mL, and 160 μg/mL) for 24 hours. Results Fluoride caused an increase of LPO levels and a decrease of GSH content in L-02 cells. There appeared to be an obvious dose-effect relationship between the fluoride concentration and the observed changes. Fluoride also caused DNA damage and apoptosis and increased the cell number in S phase of cell cycle in the cells tested. There was a statistically significant difference in DNA damage and apoptosis when comparing the high dose of fluoride treated cells with the low dose of fluoride treated cells. Conclusion Fluoride can cause lipid peroxidation, DNA damage, and apoptosis in the L-02 cell experimental model and there is a significant positive correlation between fluoride concentration and these pathological changes.

Total saponins of Panax ginseng effects on proliferation and differentiation of human embryonic neural stem cells and in a Parkinson's disease mouse model

BACKGROUND： Total saponins of Panax ginseng （TSPG） exhibits neuroprotection against Parkinson＇s disease in the substantia nigra. OBJECTIVE： To investigate the effects of TSPG on human embryonic neural stem cells （NSCs） proliferation and differentiation into dopaminergic neurons using in vitro studies, and to observe NSC differentiation in a mouse model of Parkinson＇s disease, as well as behavioral changes before and after transplantation. DESIGN, TIME AND SETTING： In vitro neural cell biology trial and in vivo randomized, controlled animal trial were performed at the Institute of Basic Medical Sciences, Chongqing Medical University between September 2004 and December 2007. MATERIALS： TSPG （purity 〉 95%） was isolated, extracted, and identified by Chongqing Academy of Chinese Materia Medica. Recombinant human basic fibroblast growth factor （bFGF） and recombinant human epidermal growth factor （EGF） were purchased from PeproTech, USA. A total of 25 C57/BL6J mice, aged 18-20 weeks were included. Twenty were used to establish a Parkinson＇s disease model with i.p. injection of MPTP （1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine） and TSPG alone or combined with interleukin-1 （IL-1）-treated NSCs prior to transplantation into the corpus striatum. The remaining five mice were pretreated for 3 days with TSPG prior to MPTP injection, serving as the TSPG prevention group. METHODS： Primary NSCs were isolated, cultured and purified from embryonic cerebral cortex. Immunocytochemistry was employed to detect specific antigen expression in the NSCs. In vitro experiment： （1） to induce proliferation, NSCs were treated with TSPG, EGF＋bFGF, or TSPG＋EGF＋bFGF, respectively; （2） to induce dopaminergic neuronal differentiation, NSCs were treated with TSPG, IL-1, or TSPG＋IL-1, respectively. MAIN OUTCOME MEASURES： In vitro experiment： the effects of TSPG on NSCs proliferation were evaluated with flow cytometry and MTT assay. Tyrosine hydroxylase expression was determined by immunocytochemistry assay to observe effects of TSPG on dopaminergic neuronal differentiation. In vivo experiment： differentiation of grafted NSCs in the mouse brain was determined by immunohistochemical staining. Behavioral changes were evaluated by spontaneous activity frequency, memory function, and score of paralysis agitans. RESULTS： （1） NSCs were cultured and passaged for more than three passages. Immunocytochemistry revealed positive nestin staining, as well as neurofilament protein and glial fibrillary acidic protein. （2） TSPG significantly increased NSC proliferation, in particular when combined with EGF and bFGF, which was twice as effective as FGF or bFGF alone. TSPG also induced dopaminergic differentiation in NSCs, in particular when TSPG was added together with IL-1, resulting in an effect five times greater than that of IL-1 alone. （3） At day 30 following transplantation, most NSCs in the TSPG prevention group differentiated into dopaminergic neurons, and the scores of paralysis agitans, spontaneous activity, and memory function were significantly increased compared with TSPG alone or TSPG＋IL-1 groups （P 〈 0.05）. CONCLUSION： TSPG stimulated NSC proliferation, in particular when combined with FGF and bFGF. TSPG significantly induced dopaminergic neuronal differentiation of NSCs, and the effect was greater when combined with IL-1. In addition, TSPG greatly improved behavior in the Parkinson＇s disease mouse model following NSC transplantation. Following NSC transplantation, TSPG pretreatment exhibited superior efficacy over either TSPG alone or TSPG in combination with IL-1, in terms of behavioral improvements in the Parkinson＇s disease mouse model.

Neuroprotective effects of human telomerase reverse transcriptase on beta-amyloid fragment 25-35-treated human embryonic cortical neurons

BACKGROUND： Numerous current studies have suggested that human telomerase reverse transcriptase （hTERT） gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses; however, the mechanism of action remains unknown. OBJECTIVE： To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 （AI325-35）. DESIGN, TIME AND SETTING： The randomized, controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research, Zhongshan School of Medicine, Sun Yat-sen University, China, from September 2005 to June 2008. MATERIALS： AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University, China. Human cortical neurons were derived from 12-20 week old aborted fetuses, obtained from the Guangzhou Maternal and Child Health Hospital, China. Mouse anti-Odk5 and mouse anti-p16 monoclonal antibodies （Lab Vision, USA）, and mouse anti-hTERT monoclonal antibody （Epitomics, USA）, were used in this study. METHODS： （1） Recombinant adenovirus vectors, encoding hTERT （Ad-hTERT） and green fluorescent protein （Ad-GFP）, were constructed using the AdEasy-1 Expression System. Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days. Likewise, human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days. Human embryonic cortical neurons in the control group were cultured as normal. （2） Human embryonic cortical neurons in the Ad-hTERT group were treated with 10 pmol/L Aβ25-35 for 24 hours. Normal human embryonic cortical neurons treated with 10 pmol/Lβ25.35 for 24 hours served as a model group. Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with Aβ25-35. MAIN OUTCOME MEASURES： Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay. Telomerase activity was measured using a PCR-based telomeric repeat amplification protocol （TRAP） ELISA kit. Neural activity in human embryonic cortical neurons was examined by MTT assay; apoptosis was measured using TUNEL assay; and Cdk5 and p16 protein expressions were measured by Western blot. RESULTS： Expression of hTERT protein was significantly increased and peaked at day 3 post-transfection in the Ad-hTERT group. No hTERT expression was detected in the Ad-GFP and control groups. Telomerase activity was significantly greater in the Ad-hTERT group compared with the Ad-GFP and control groups （P 〈 0.01）. Compared with the control group, cell activity was significantly decreased （P 〈 0.05）, and cell apoptotic rate, Cdk5 and p16 expression were significantly increased （P 〈 0.01） in the model group. Compared with the model group, cell activity was increased in the Ad-hTERT group, and peaked at day 3 post-transfection （P 〈 0.05）. Neuroprotective effects also peaked at day 3 post-transfection; and the apoptotic rate, Cdk5 and p16 expression significantly decreased （P 〈 0.01）. CONCLUSION： Expression of hTERT in human embryonic cortical neurons can relieve Aβ25-35-induced neuronal apoptosis. The possible mechanism by which hTERT produces these neuroprotective effects may be associated with inhibition of Cdk5 and p16 expression.

Human cytomegalovirus induces alteration of β-actin mRNA and microfilaments in human embryo fibroblast cells

Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV.RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

Human Embryo Neuronal Culture <i>in Vitro</i>: A Model to Study Cellular Physiology, Receptors, Power and Toxicity of Cytostatic Drugs for Human Use

Neural cells cultures from human embryo brain of 9° - 11°W gestational age have been used to study ERα (Estrogens Receptor α) and to perform toxicity test for Mitomycin C and Methotrexate. Histochemical confirmation of cellular neuronal phenotype was based on histochemical evidence of NSE (Neuron Specific Enolase).The detection of ERα in neuronal cells was performed with a rabbit Monoclonal Antibody. ERα was absent both on neurons grown in vitro and on tissue brain specimens. This finding is apparently in contrast with the positive immunoreactivity of ERα and ERβ reported by other Authors on foetal and adult CNS (Central Nervous System). The absence of nuclear ERα on neurons in culture and in brain tissue specimens in our experiment is not in contrast with the relevant physiologic role of estrogens on nervous central system, but it could be correlated to the embryonic period of life and could represent a protection of male brain from an undue estrogens imprinting. The mitomycin C, alkylation agent, has shown in our experiment a major neurotoxic and cytostatic power in comparison with methotrexate. Our conclusion is that human embryo neuronal culture in vitro is a powerful instrument for physiology and human therapy for cancer and neurodegenerative diseases.

Co-Culture of Early Embryo with Human Decidual Stromal Cells in vitro by Improvement of Early Embryo Development

Summary: An early embryo co-culture system with human decidual stromal cells was established to study its effect on early embryonic cleavage and growth in vitro. Three hundred and eight 2-cell mouse embryos were co-cultured with human decidual stromal cell monolayer in MEM+0. 4 % bovine serum albumin (BSA) and 163 embryos cultured in MEM+15 % FCS alone as control. Among the mouse 2-cell embryos co-cultured with human decidual stromal cells, 72.73 % developed to the morula stage and 67.21 % cavitated to blastocysts with 59. 74 % hatching, as compared with 61. 34 % to morula stage, 48. 47 % to blastocysts and none hatching in the controls, respectively. Co-cultured embryos cleaved slightly faster than controls and showed no or less fragmentation than those in the control. These results suggested that human decidual stromal cells can support early embryonic development and yield a reasonable number of embryos with good quality up to blastocyst stage.

Location and expression of neurotrophin-3 and its receptor in the brain of human embryos during early development

BACKGROUND： Cell culture in vitro trials have demonstrated that neurotrophin-3 （NT-3） can enhance the survival of sensory neurons and sympathetic neurons, and can also support embryo-derived motor neurons. This effect is dependent on nerve growth factor on the surface of cells. Understanding the role of NT-3 and its receptor in the early development of human embryonic brains will help to investigate the correlation between early survival of nerve cells and the microenvironment of neural regeneration. OBJECTIVE： To observe the proliferation of cerebral neurons in the development of human embryonic brain, and to investigate the location, expression and distribution of NT-3 and its receptor TrkC during human brain development. DESIGN, TIME AND SETTING： An observation study on cells was performed in the Department of ttuman Anatomy, Histology and Embryology, Chengdu Medical College in September 2007. MATERIALS： Fifteen specimens of flesh human embryo, aged 6 weeks, were used in this study. METHODS： The proliferation of cerebral neurons was detected using proliferating cell nuclear antigen, and the immunocytochemistry ABC technique was applied to observe the location, expression and distribution of NT-3 and its receptor TrkC in the brain of the human embryo. MAIN OUTCOME MEASURES： Location, expression and distribution of NT-3 and its receptor in the brain of the human embryo. RESULTS： In the early period （aged 6 weeks） of human embryonic development, proliferating cell nuclear antigen-positive reactive substances were mainly observed in the nucleus of the forebrain ventricular zone and subventricular zone, and the intensity was stronger in the subventricular zone than the forebrain ventricle. NT-3 positive reactive substance was mainly distributed in the cytoblastema of the forebrain neuroepithelial layer and nerve cell process, while TrkC was mainly distributed in the cell membrane of the forebrain ventricular zone and subventricular zone. During embryonic development, NT-3 and TrkC showed a positive immune reaction to a greater or lesser extent in ependymal epithelium. CONCLUSION： During early human embryonic development, cerebral nerve cells proliferate in the ventricular zone and subventricular zone, and NT-3 is expressed in the neural axon. The results show that the highly expressed NT-3 could promote the proliferation of neural axons and maintain the neuron body＇s survival.

Apoptotic gene expression in the neural tube during early human embryonic development

Neural tube development comprises neural induction, neural epithelial cell proliferation, and apoptosis, as well as migration of nerve cells. Too much or too little apoptosis leads to abnormal nervous system development. The present study analyzed expression and distribution of apoptotic-related factors, including Fas, FasL, and caspase-3, during human embryonic neural tube development. Experimental results showed that increased caspase-3 expression promoted neural apoptosis via a mitochondrial-mediated intrinsic pathway at 4 weeks during early human embryonic neural tube development. Subsequently, Fas and FasL expression increased during embryonic development. The results suggest that neural cells influence neural apoptosis through synergistic effects of extrinsic pathways. Therefore, neural apoptosis during the early period of neural tube development in the human embryo might be regulated by the death receptor induced apoptotic extrinsic pathways.

A Critique on the Application of the Principle of Subsidiarity Concerning Human Embryonic Stem Cell Research in South Africa

Researchers from all around the world emphasize on the enormous possible benefits that stem cells may have for the treatment of diseases. However, this technology is considered morally problematic when the source of the stem cell is from a human embryo. Nonetheless, there is a consensus that of all the types of stem cells, hESC （human embryonic stem ceils） are the most promising for particular and important research and therapies. Yet, there are controversial issues regarding the ＂killing＂ of the human embryo for stem cell derivation. There are two general ethical conditions that should govern the instrumental use of embryo. One of them, the principle of subsidiarity, which is defined as ＂a state we have that we have to choose the less contentious means of achieving the intended goal＂. Based on this principle, we ought only to use hESC when there are no other alternatives, which are less morally controversially. Subsidiarity is based on the assumption that there is something ethically unsound about the use ofhESC. However, this principle only makes sense if it is based on consistently upheld views of the moral status of embryo, moreover, the law should also not limit or prohibit hESC research based on this principle. In this paper, I argue---using the South African law for hESC technology--that criterion for deciding which type of stem cells to use should be based on their potential and suitability for advancing scientific knowledge and development of new therapies which will be greatly beneficial in alleviating human suffering.

论非法植入基因编辑、克隆胚胎罪保护法益

随着世界范围内基因编辑技术的发展,人类有了攻克遗传疾病的希望。但是“基因编辑婴儿”案的出现,使我们必须思考如何通过法律来防范基因编辑技术对人类生活的冲击。在刑法规制层面,准确把握非法植入基因编辑、克隆胚胎罪的保护法益是进一步思考的前提。既往的研究将该罪法益限定为人性尊严、医疗秩序或人类遗传安全,但这并非真正的适格法益,无助于法益机能的发挥。合理界定该罪法益的方向应当是综合分析规范目的、法益性质、价值判断以及行为对象。基于以上方法,将非法植入基因编辑、克隆胚胎罪的保护法益确定为人类遗传基因具有合理性。该法益界定一方面明确了“情节严重”的类型,另一方面可以作为出罪事由的判定标准,划定了犯罪行为与科研行为的界限。

人类胚胎干细胞法律规制的路径研究

人类胚胎干细胞具有很高的研究和临床应用价值,而目前我国关于人类胚胎干细胞研究的法规还在探索阶段,故尚存在临床研究备案制不足以防范伦理风险、对科研机构及其工作人员法律责任的认定不够细化、有关捐赠者与受赠者的知情同意规定不完善等问题;由剖析英、美、德等对人类胚胎干细胞研究的法律规范,对我国人体胚胎干细胞研究之科学立法原则、机构准入、科研机构及其工作人员法律责任和相关人员知情同意书等提出有针对性的完善性建议。

人类体外胚胎利用活动的私法回应——以《民法典》第一千零九条为中心

面对人类体外胚胎利用活动,《民法典》不仅选择积极介入而且决定有限开放。为避免潜在风险,《民法典》不仅直接规定了多种风险预防机制,如提高合法标准、设置三大底线和强调人格尊严,而且还间接引致了多种风险预防机制,如划定行为禁区、限制利用对象和实施伦理审查。即便是在《民法典》的框架下,对人类体外胚胎的利用活动所引发的损害进行救济依然是可能的。但是,《民法典》所能供给的侵权责任方案存在很多局限。因此,后续立法应同时引入其他损害救济方案。从我国现行立法来看,潜在选择有强制责任保险方案、商业保险方案、社会救助基金方案和国家补偿方案。后续立法甚至可以建立同时包含四者的综合救济体系。为确保受害人能获得救济,应由国家兜底。

IVF-ET胚胎培养液中sHLA-G、MMP-9与胚胎质量及临床妊娠关系

目的:分析体外受精-胚胎移植(IVF-ET)胚胎培养液中可溶性人类白细胞抗原G(sHLA-G)、基质金属蛋白酶(MMP-9)与胚胎质量、临床妊娠的关系。方法:收集2017年1月-2023年1月在本院生殖中心接受IVF-ET治疗的女性患者107例临床资料及标本,新鲜胚胎移植后收集胚胎培养液,分别取未移植的正常受精胚胎中优质胚胎、中等胚胎、劣质胚胎对应胚胎培养液,酶联免疫吸附实验检测胚胎培养液中sHLA-G、MMP-9水平,比较不同胚胎质量对应的胚胎培养液sHLA-G、MMP-9水平及其相关性;记录IVF-ET患者临床妊娠情况并分为临床妊娠组、未妊娠组,比较两组胚胎培养液中的sHLA-G、MMP-9水平及其他临床资料,logistic回归分析胚胎培养液中sHLA-G、MMP-9水平与临床妊娠关系。结果:107例未移植的正常受精胚胎中共收集127份胚胎培养液标本,其中优质胚胎培养液44份、中等胚胎培养液52份、劣质胚胎培养液30份,其胚胎培养液sHLA-G、MMP-9水平逐渐降低,胚胎培养液sHLA-G、MMP-9水平与胚胎质量正相关(均P<0.01)。107例IVF-ET获得临床妊娠69例,临床妊娠率64.6%,未妊娠组胚胎培养液sHLA-G(8.11±1.63 ng/ml)、MMP-9水平(0.45±0.17 ng/ml)均低于临床妊娠组(10.63±2.41 ng/ml、0.61±0.12 ng/ml),第三日卵裂球数(6.0±1.8个)、优质胚胎率(45.6±12.1)%低于临床妊娠组(8.9±1.8个、64.8%±15.9%)(均P<0.05);logistic回归分析,sHLA-G、MMP-9、第三日卵裂球数、优质胚胎率等均是影响IVF-ET患者临床妊娠因素。结论:IVF-ET患者胚胎培养液中sHLA-G、MMP-9水平与胚胎质量正相关,sHLA-G、MMP-9、第三日卵裂球数、优质胚胎率等降低均是IVF-ET患者临床妊娠影响因素。

人类早期胚胎发育体外模型研究进展

人类早期胚胎发育阶段对于胎儿的健康出生至关重要。然而,由于伦理和技术的限制,人类早期胚胎发育的具体调控机制仍未完全解密。除了人类胚胎体外培养技术以外,以干细胞为基础模拟人类真实胚胎结构的体外模型被构建出来,被称为“类胚胎/胚胎模型”。通常人类胚胎模型可大致分为两类:非整合型和整合型胚胎模型。整合型胚胎模型通常包含胚内和胚外细胞类型并具有发育成完整胎儿的潜力,而非整合型胚胎模型则不包含任何相关的胚外组织。本文系统总结了人类体外非整合型和整合型胚胎模型的最新研究进展,探讨了有关国际干细胞研究的伦理政策,并简要阐述了人类胚胎模型潜在的应用前景和未来机遇。以期为研究人类早期胚胎发育过程中不同细胞谱系的特化轨迹,以及早期胚胎发育缺陷等重大疾病的临床药物筛选和再生医学提供新的研究思路。

冷冻胚胎处置医疗纠纷的裁判困境与破解之道

在人类辅助生殖实施过程中,若夫妻一方或双方在冷冻胚胎移植之前死亡,死者亲属与医疗机构往往因能否继续移植或取回冷冻胚胎引发民事纠纷。其中,由人类辅助生殖机构出具、夫妻双方签字的知情同意书仅为机构履行告知说明义务的证明,不具备合同法上的效力。生育权作为我国民法典未列明的自由性人格权,是当下冷冻胚胎私法秩序建构中的重要理论工具,可借助人格权理论协调特殊情形下冷冻胚胎处置过程中的权利冲突。在夫妻一方死亡的情形下,另一方有权基于生育权保存、植入或废弃冷冻胚胎;在夫妻双方死亡的情形下,涉及冷冻胚胎的生育权主体不复存在,考虑到儿童利益最佳原则,该冷冻胚胎原则上不应被继续孕育成人。

孕早期血清孕酮、雌二醇、人绒毛膜促性腺激素水平与胚胎停育关系的研究进展

近年来,在胚胎停育发生率不断升高的趋势下,通过何种方法进行预测并给予提前干预,从而降低其发生风险成为临床急需解决的一大难题。胚胎停育的诱因较多且复杂,其中内分泌因素属于临床研究的重要内容。现阶段,临床上在孕早期进行妊娠结局预测的主要指标涉及孕酮(P)、雌二醇(E_(2))及人绒毛膜促性腺激素(HCG)等,临床发现有胚胎停育史者上述指标水平存在异常表达的现象。通过查阅诸多相关文献发现,不同孕龄者上述指标水平存在一定规律,即孕4周P、E_(2)通常处于最佳临界值状态,孕6周前HCG上升最快,而进入孕7周后P、E_(2)处于黄体-胎盘转换期,到孕8~10周上升至最高水平,此阶段也是决定妊娠能否顺利进行的关键阶段。因此,对不同孕龄者进行P、E_(2)、HCG检测,了解其与妊娠结局存在的相关性,可有效反映胚胎发育状态,为早期治疗及干预提供一定参考。

拮抗剂方案中HCG扳机日直径16~18 mm卵泡比例对PCOS患者IVF-ET结局的影响

目的探讨拮抗剂方案中人绒毛膜促性腺激素(HCG)扳机日直径16~18 mm卵泡比例对接受拮抗剂方案的多囊卵巢综合征(PCOS)患者体外受精-胚胎移植(IVF-ET)结局的影响。方法回顾性分析2016年1月至2021年12月于南京医科大学附属妇产医院行IVF-ET助孕的PCOS患者的临床资料。根据HCG扳机日双侧卵巢直径16~18 mm卵泡比例以三分位法将患者分为三组,该比例<0.43为A组(262例),0.43~0.56为B组(267例),≥0.56为C组(247例)。比较三组一般临床资料、超促排卵周期情况、胚胎发育情况、体外受精周期后首次冻融胚胎移植(FET)情况以及妊娠、围产期结局。结果B组基础促黄体生成素(LH)、基础LH与基础促卵泡激素(FSH)比值显著高于A组和C组,差异有统计学意义(P<0.05)。A组促性腺激素(Gn)使用总量高于B组和C组,HCG扳机日雌二醇(E_(2))水平低于B组、C组,差异有统计学意义(P<0.05)。B组和C组获卵数、2原核(PN)数、卵裂数、囊胚形成数以及可移植胚胎数高于A组,差异有统计学意义(P<0.05)。C组囊胚形成率高于A组和B组,高评分囊胚率高于B组,差异有统计学意义(P<0.05)。三组移植胚胎数、移植胚胎天数、内膜准备方案及内膜厚度比较差异无统计学意义(P>0.05)。三组胚胎着床率、生化妊娠率、临床妊娠率、流产率、活产率、妊娠期糖尿病发生率及妊娠期高血压发生率等比较差异无统计学意义(P>0.05)。结论在拮抗剂方案中,HCG扳机日直径16~18 mm卵泡比例可反映卵母细胞发育潜能,当该比例≥0.56时可获得较好的获卵数、2PN数、卵裂数、囊胚形成数以及可移植胚胎数,在一定程度上可作为PCOS患者HCG扳机时机的参考指标,但该比例并不影响后续首次FET的妊娠结局。

Estimating zona pellucida hardness under microinjection to assess oocyte/embryo quality: Analytical and experimental studies

The precise determination of zona pellucida (ZP) hardness is largely unknown due to the lack of appropriate measuring and modelling methods. In this study, we have used experimental and theoretical models to describe the mechanical behavior of a single oocyte cell to improve the assisted reproductive technology (ART) outcomes by assessing oocyte/embryo quality. This paper presents the development of: i) a microinjection model to estimate the force of ZP penetration, ii) a micropipette aspiration model to determine the corresponding hardness, and iii) an experimental procedure to generate the required data for these two models. Our results show that the estimated penetration force provides a performance target for the penetration process during intracytoplasmic sperm injection (ICSI), while the estimated corresponding hardness serves as an indicator of the extent of deformation sustained by the oocyte prior to penetration. Evaluation of these results shows that a routine assessment of ZP hardness under microinjection would allow for the identification of certain oocyte pools for which further manipulation is recommended in order to improve injection, hatching and finally ART outcomes.

Evaluation on Sensitivity of the Human Sperm Motility Assay for Detecting Endotoxin in Culture Medium

Objective To investigate the sensitivity of the human sperm motility assay for detecting endotoxin in culture mediumMaterials &. Methods Motile sperm were separated and exposed to different concentrations of endotoxin (0.5 ng/mL, 1ng/mL, 10ng/mL, 1000ng/mL, 10 000ng/ mL, and 50 000ng/mL), and sperm motility was determined after incubation. Effects of endotoxin on sperm motility in media without albumin were also examined. In addition, at the same concentrations of endotoxin (0. 5ng/mL, 1 ng/mL, and 10 ng/ mL ) , the sensitivity of the human sperm motility assay was compared to those of 1-cell and 2-cell mouse embryo bioassays.Results At levels of 0. 5ng/mL-1000ng/mL endotoxin in media with 2mg/mL albumin, sperm did not show significant change in motility during 24 h of incubation when compared with the control (P>0. 05). However, the sperm motility was significantly inhibited at endotoxin dosages of 10 000 and 50 000 ng/mL. In the absence of albumin supplementation, at endotoxin levels of 50 000ng/mL, and 1 000ng/mL, there was a marked decrease in sperm motility compared with the control after 2 h or 8 h of incubation, respectively (P<0. 01). In media containing 0. 5 ng/mL and 1 ng/ mL endotoxin, 1-cell and 2-cell mouse embryos had significantly reduced developmental rates in all developmental stages, and at the level of 10ng/mL, the development of the embryos was arrested. Conclusion The human sperm motility assay could detect high levels of endotoxin in culture medium but its sensitivity to endotoxin would be inferior to that of the 1-cell or 2-cell mouse embryo bioassay. In the absence of albumin supplementation, the sensitivity of the sperm motility assay could be improved.

Evaluation of Endotoxin in Culture Medium for Human in vitro Fertilization

To determine whether the presence of bacterial endotoxin in the commercial culture media utilized for human in vitro fertilization （IVF）, and evaluate the difference in detecting endotoxin in culture medium between the human sperm motility assay and the 2-cell mouse embryo assay. Methods Thirty-six batches of culture media commonly used in IVF laboratories from 3 manufacturers were determined for thepresence ofendotoxin before using the medium for the assisted reproductive programs （group A）. After being used, 25 specimens among above media were also tested （group B）. The chromogenic limulus amoebocyte lysate （LAL） test was used for quantification the content of endotoxin. In addition, the human sperm motility assay was compared with the 2-cell mouse embryo assay to evaluate the difference in detecting endotoxin in culture medium. Results Endotoxin was not detected in group A. However, 2 samples were positive in group B, Sperm did not show significant change in motility in group A during 24 h of incubation when compared with the control （P〉0. 05）. However, in group A the 2-cell embryo development to blastocyst was suppressed in 3 batches of media. Conclusions Regular screening of each batch of culture medium should be performed if possible although there was no evidence of endotoxin contamination in commercially prepared pre-tested media. Culture environment should be stringently controlled in case the medium is polluted. The sensitivity of the sperm motility assay was lower than that of the mouse embryo assay for detecting low levels of endotoxin or toxic compounds in the medium.