辅助生育技术与自然受孕双胎妊娠结局的临床分析

目的探讨辅助生育技术与自然受孕两种不同方式双胎妊娠的临床结局。方法回顾性分析我院2005年-2008年7月分娩的143例辅助生育技术受孕双胎孕妇（ART组）和108例自然受孕双胎孕妇（对照组）的孕期合并症、分娩方式及围产儿儿结局。结果（1）ART组孕妇平均年龄（33．1±4．0）岁,对照组为（28．2±4．0）岁,两组比较差异有极显著性（P〈0．001）。（2）不良孕产史发生率ART组高于对照组（13．7％vs3．7％）,差异有极显著性（P〈0．01）;初次产检孕周,ART组为（13．1±5．4）周,对照组为（17．4±6．9）周,ART组的孕期产检次数为（8．2±2．8）次,对照组为（6．7±3．1）,均有极显著性差异（P≤0．001）。（3）ART组分娩孕周为（35．1±2．1）周,对照组为（34．4±2．4）周,ART组34周及以上分娩率高于对照组（P〈0．05）;妊娠期高血压疾病的发生率ART组低于对照组。（4）ART组新生儿平均出生体重（2394．3±38．04）g,对照组为（2184．9±53．20）g,差异有极显著性（P=0．001）。ART组极低出生体重儿发生率低于对照组。新生儿窒息、围产儿死亡率、一胎胎死宫内、先天性畸形的发生率,两组均无显著差异。结论ART助孕双胎孕妇更加重视孕期保健,分娩孕周延长,妊娠期高血压疾病的发生率较低,ART助孕组单卵双胎的比例较低．围产儿结局与自然爱孕双胎相似．     

卵丘细胞凋亡对体外培养卵母细胞发育潜能的影响

目的 研究体外培养中卵丘细胞凋亡对卵母细胞结构的影响,探讨体外受精中可用卵丘细胞凋亡率预测卵母细胞发育潜能的原因.方法 对GV期人卵丘-卵母细胞复合体进行体外成熟培养,用HE染色、DAPI染色和原位末端标记(TUNEL)法标记3种方法对单卵卵丘细胞凋亡率进行检测.分为凋亡率高和低的2组,用光镜和透射电镜观察卵母细胞的结构.对MⅡ期卵母细胞进行体外受精,培养2d后在光镜下评价胚胎质量.结果 卵丘细胞凋亡率低的卵母细胞结构和发育潜能良好;卵丘细胞凋亡率高的卵母细胞发育潜能差,存在各种结构异常,包括围卵周隙不均,对应不同部位的细胞质发育不同步;细胞质中出现堆积不均的细胞器团、次级溶酶体、及可能由溶酶体降解造成的大量空隙;线粒体外膜和嵴模糊、膨大,呈现凋亡迹象;第一极体碎裂;透明带异常增厚或变薄.相应的卵丘细胞微绒毛和细胞连接减少.结论 揭示了卵丘细胞凋亡对卵母细胞结构的影响,揭示了卵丘细胞凋亡率影响卵母细胞发育潜能的原因.     

小鼠卵丘透明质酸结合蛋白的分布

目的：观察小鼠卵丘细胞外基质透明质酸结合蛋白（HABPs）的分布,探讨HABPs对小鼠卵丘的结构和功能的影响。方法：应用免疫荧光双重染色检测HABPs在野生型和bikunin基因敲除小鼠卵丘细胞外基质中的表达。结果：HABPs在两型小鼠卵丘细胞外基质呈阳性表达,HABPs的缺失损害小鼠卵丘细胞外基质完整。结论：HABPs对卵丘细胞外基质的完整是必需的。     

卵丘细胞在卵母细胞成熟和受精中的作用

卵泡是人类生殖的基本功能单位，卵丘细胞在卵母细胞发育、成熟和受精过程中有极其重要的作用，主要表现在参与卵母细胞减数分裂的调节、支持卵母细胞细胞质的成熟、促进精卵的结合，并且其形态和扩展程度影响卵母细胞成熟与质量。本文对卵丘细胞在卵母细胞成熟和受精中的作用进行系统的阐述，有助于进一步揭示卵母细胞成熟的机制。     

TSG-6和连接蛋白在小鼠卵丘-卵母细胞复合体的共位性分析

目的：探讨小鼠卵丘-卵母细胞复合体(COC)中 TSG-6和连接蛋白(LP)与透明质酸(HA)的相关性。方法：应用激光扫描共聚焦显微镜和免疫荧光双重染色技术观察小鼠 COC 中 TSG-6和 LP 的表达和分布。结果： TSG-6和 LP 在 COC 中呈阳性表达。结论：TSG-6和 LP 的阳性表达可能和 HA 在 COC 成熟中起重要作用。     

人卵巢壁层颗粒细胞和卵丘细胞体外分离及原代培养的比较

目的:为建立合适卵巢功能研究的细胞模型,本研究比较了人卵巢壁层颗粒细胞(mural granulosa cells,MGCs)与卵丘细胞(cumulus cells,CCs)体外分离及原代培养的效果。方法:收集16例行辅助生殖技术治疗患者取卵日的卵泡液及其卵丘-卵母细胞复合物(223个),采用密度梯度离心法分离MGCs,机械切割法+酶解法分离CCs。应用台盼蓝染色法比较CCs与MGCs的细胞总数和存活率,通过流式细胞术检测卵泡刺激素受体(follicle stimulating hormone receptor,FSHR)的表达来鉴定CCs与MGCs的细胞纯度,qPCR和Western blot方法检测微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)、P62和Bax mRNA和蛋白的表达水平。结果:与MGCs相比,CCs的分离耗时少,细胞存活率高(P<0.05),体外培养的细胞延展性好;流式细胞术显示分离后的CCs与MGCs的FSHR表达量分别为(92.23±2.66)%和(81.33±6.57)%,差异...     

辅助受体CCR5、CCR2及细胞趋化因子SDF1基因多态性与HIV-1异性传播的关系

目的 进一步阐述CCR5、CCR2和SDFl基因多态性与HIV-1异性传播的关系。方法 通过PCR／RFLP技术分析CCR5、CCR2和SDF1基因的多态性，继而分析基因多态性与HIV-1异性传播的关系。结果 在收集到的70对异性配对病例中，未能在汉族人群发现CCR5基因缺失突变，维吾尔族人CCR5Δ32基因频率为6．5％(6／92)，未发现纯合突变。有暴露史而未感染HIV-1者CCR2-64I基因频率明显高于受暴露后感染病毒者，说明CCR2-64I对异性传播可能具有一定保护作用。对SDFl基因的多态性研究发现，将病毒传播给配偶的指标病例(先感染：HIV-1一方)SDF-3′A的基因频率高于未发生传播者(较接近于统计学检验水准)，SDFl-3′A似乎是危险因素。结论 CCR5Δ32对汉族人群的HIV-1异性传播无明显意义，CCR2-64I对HIV-1异性传播可能具有一定的保护作用，而SDFl-3′A则有可能是危险因素，但有必要扩大样本量对后二者作进一步的深入研究。     

人卵丘颗粒细胞和壁层颗粒细胞体外培养方法比较

目的:建立有效分离、提纯及培养人卵丘颗粒细胞的方法,并与人壁层颗粒细胞的体外培养相比较。方法:收集卵胞浆内单精子显微注射取卵时的卵泡液和直接机械分离卵母细胞所得的卵丘颗粒细胞复合物,直接将卵丘颗粒细胞复合物接种于培养皿中培养。用密度梯度离心法分离卵泡液中的人壁层颗粒细胞。用免疫荧光法检测卵泡刺激素受体(follicle stimulating hormone receptor,FSHR)的表达;用CCK-8法检测细胞生长曲线;用ELISA检测这2种颗粒细胞分泌雌激素的能力。结果:直接法所得人卵丘颗粒细胞培养24 h后可贴壁,体外培养生长状态与人壁层颗粒细胞相似;免疫荧光检测显示两者均表达FSHR;CCK-8实验结果表明,两者体外培养生长曲线相似;ELISA结果显示两者分泌雌激素能力相当。结论:利用机械切割获得人卵子周围卵丘颗粒细胞复合物直接培养的方法操作简单,获得的人卵丘颗粒细胞具有与人壁层颗粒细胞相似的生长状态、生长曲线以及雌激素分泌能力,可作为人颗粒细胞亚群体外培养方法的补充。     

腹腔镜手术对输卵管妊娠患者术后生育结局的影响

目的探讨腹腔镜下不同术式对输卵管妊娠患者术后生育结局的影响。方法回顾分析了86例经腹腔镜手术及70例开腹手术的输卵管妊娠患者术后不孕、异位妊娠及宫内妊娠的发生率。结果经腹腔镜手术的患者术后宫内妊娠率50％高于行开腹手术者35．7％（P〈0.05）；在不同的腹腔镜术式中，行输卵管保守性手术的患者术后宫内妊娠率52．8％高于行患侧输卵管切除术者35．7％（P〈0.05）；在行保守性手术中，壶腹部妊娠及伞端妊娠患者术后宫内妊娠率分别为60％及61．1％，均高于峡部妊娠者31.6％（P〈0．05）。结论腹腔镜手术有利于改善输卵管妊娠患者术后生育结局，壶腹部妊娠及伞端妊娠患者宜行输卵管保守性手术。     

猪卵透明带纯化抗原主动免疫家兔后长期抗生育效应的观察

以猪卵透明带的纯化抗原主动免疫家兔,和对照组一起观察22个月,发现免疫家兔始终不孕.而对照组的生育不受影响.以放射免疫测定血清中抗体滴度的动态变化,发现在初次免疫后3个月,其峰值范围是1:116 000～360 000,维持7个月后,其滴度逐渐下降.在抗体高峰期.免疫动物动情周期延长.两组动物的组织学与免疫酶组织化学的观察结果基本一致.     

Inhibition of respiratory syncytial virus infection with the CC chemokine RANTES (CCL5)

Respiratory syncytial virus (RSV) is a major cause of respiratory tract disease in infants, aged adults, and immunosuppressed patients. The only approved medicines for RSV disease are administration of prophylatic antibodies or treatment with a synthetic nucleoside. Both approaches are expensive and the latter is not without risk and of controversial benefit. The present investigation studied whether pharmaceutical or biologic compounds based upon chemokines might be useful in preventing RSV disease. Of interest was RANTES/CCL5, which inhibits infection by HIV strains that use chemokine receptor (CCR)-5 as co-receptor. Herein, we report that prior or simultaneous treatment of HEp-2 cells with recombinant human CCL5 provides dose-dependent inhibition of infection with RSV. Other recombinant chemokines (MIP-1alpha/CCL3, MIP-1beta/CCL4, MCP-2/CCL8, eotaxin/CCL11, MIP-1delta/CCL15, stromal cell derived factor (SDF)-1alpha/CXCL12) were not inhibitory. The data suggested that CCL5 might inhibit infection by blocking fusion (F) protein-epithelial cell interactions. Infections by mutant RSV strains deleted of small hydrophobic and/or attachment proteins and only expressing F protein in the envelope were inhibited by prior treatment with CCL5 or a biologically inactive N-terminally modified met-CCL5. Inhibition was also observed when virus adsorption and treatment with CCL5 were performed at 4 degrees C. Flow cytometry further revealed that epithelial cells were positive for CCR3, but not CCR1 or CCR5. Thus, novel mimetics of CCL5 may be useful prophylatic agents to prevent respiratory tract disease caused by RSV.     

Relationship between antithyroid antibody and pregnancy outcome following in vitro fertilization and embryo transfer

Objective: To investigate the impact of antithyroid antibody on pregnancy outcome following the in vitro fertilization and embryo transfer (IVF-ET).Methods: A total of 90 patients (156 cycles) positive for antithyroid antibody (ATA+ group) and 676 infertile women (1062 cycles) negative for antithyroid antibody (ATA- group) undergoing IVF/ICSI from August 2009 to August 2010 were retrospectively analyzed.Results: There was no significant difference in the days of ovarian stimulation, total gonadotropin dose, serum E2 level of HCG day and number of oocytes retrieved between the two groups. The fertilization rate, implantation rate and pregnancy rate following IVF-ET were significantly lower in women with antithyroid antibody than in control group (64.3% vs 74.6%, 17.8% vs 27.1% and 33.3% vs 46.7%, respectively), but the abortion rate was significantly higher in patients with antithyroid antibody (26.9% vs 11.8%).Conclusion: Patients with antithyroid antibody showed significantly lower fertilization rate, implantation rate and pregnancy rate and higher risk for abortion following IVF-ET when compared with those without antithyroid antibody. Thus, the presence of antithyroid antibody is detrimental for the pregnancy outcome following IVF-ET.     

短时受精不同脱颗粒细胞方式对体外受精结局的影响

目的探讨体外受精-胚胎移植（IVF-ET）治疗中几种短时受精脱颗粒细胞方式对胚胎质量及临床结局的影响。方法将261个IVF-ET周期根据脱颗粒细胞时间分为：短时受精后18小时脱颗粒细胞组（A组）94个周期、短时受精后5h脱颗粒细胞组（B组）95个周期和短时受精5h后部分（1/3-1/2）脱颗粒细胞组（C组）72个周期。分别比较三组的受精率、多精受精率、卵裂率、优胚率、胚胎利用率、临床妊娠率、种植率和流产率。结果三组的受精率、正常受精率、多精受精率、卵裂率、临床妊娠率和流产率差异无统计学意义（P〉0.05）,A组的优胚率、胚胎利用率和胚胎种植率显著高于B组（P〈0.05）。结论短时受精后即刻剥除部分卵子颗粒细胞观察第二极体,在确保受精的情况下剩余卵母细胞保留自体卵周颗粒细胞共培养,可以提高常规IVF的胚胎质量,从而提高常规IVF总的临床妊娠率和胚胎种植率。     

A simple method for in vitro maturation,in vitro fertilization,and co-culture of bovine oocytes

Summary An efficient procedure has been developed and subsequently simplified for in vitro maturation and in vitro fertilization (IVF) of bovine follicular oocytes obtained from abattoir ovaries. After in vitro fertilization, successful cleavage and in vitro development were obtained using a simple and efficient cumulus cell co-culture method, which consistently produced 45 to 50% morulae during the treatment and culture of over 6000 bovine oocytes. Embryonic cell number for IVF-derived embryos was monitored at different stages of in vitro development during cumulus cell co-culture to evaluate embryo growth and assess embryo quality.     

CD52 is synthesized in cumulus cells and secreted into the cumulus matrix during ovulation

Problem Early studies have shown that an antibody to male reproductive tissue CD52 is a pathogenic factor of infertility. The molecule contains a unique carbohydrate antigen that induces antibodies interfering with sperm function. However, the characteristic properties of CD52 in female reproductive tissues are not known. We examined the expression and localization of CD52 in mature expanded cumulus masses. Method of study Mouse cumulus oocyte complexes were collected from [C57B1/6; DBA/2] F1 female mice having a superovulation treatment. Human cumulus cells were obtained from infertile patients taking in vitro fertilization-embryo transfer treatment under informed consent. CD52 messenger RNA (mRNA) and protein were detected using RT-PCR, quantitative PCR, western blotting and immunohistochemical methods. Results CD52 mRNA was found both in the human and mouse cumulus cells. Mouse CD52 mRNA was detected in cumulus cells but not oocytes and significantly increased after ovulation. The expression of the molecule was also confirmed at the protein level. Immunostaining with anti CD52 peptide antibody revealed that CD52 is present in cumulus cells and the extracellular matrix. Conclusion We first showed the expression of CD52 in human cumulus cells. CD52 has some functional roles around fertilization in females as well as in males.     

Human B cells express the orphan chemokine receptor CRAM-A/B in a maturation-stage-dependent and CCL5-modulated manner

Chemokines orchestrate the organization of leucocyte recruitment during inflammation and homeostasis. Despite growing knowledge of chemokine receptors, some orphan chemokine receptors are still not characterized. The gene CCRL2 encodes such a receptor that exists in two splice variants, CRAM-A and CRAM-B. Here, we report that CRAM is expressed by human peripheral blood and bone marrow B cells, and by different B-cell lines dependent on the B-cell maturation stage. Intriguingly, CRAM surface expression on the pre-B-cell lines Nalm6 and G2 is specifically upregulated in response to the inflammatory chemokine CCL5 (RANTES), a chemokine that is well known to play an important role in modulating immune responses. Although Nalm6 cells do not express any of the known CCL5 binding receptors, extracellular signal-regulated kinases 1 and 2 (ERK1/2) are phosphorylated upon CCL5 stimulation, suggesting a direct effect of CCL5 through the CRAM receptor. However, no calcium mobilization or migratory responses upon CCL5 stimulation are induced in B-cell lines or in transfected cells. Also, ERK1/2 phosphorylation cannot be inhibited by pertussis toxin, suggesting that CRAM does not couple to Gi proteins. Our results describe the expression of a novel, non-classical chemokine receptor on B cells that is potentially involved in immunomodulatory functions together with CCL5.     

精子DNA碎片率与男性年龄、精子活动力和体外受精结局关系的研究

目的 探讨精子DNA碎片率(DNA fragmentation index,DFI)与男性年龄、精液检查指标、体外受精(in vitro fertilization,IVF)的受精率、优质胚胎率、周期妊娠率和胚胎着床率等关系.方法 随机选取本院生殖中心111例IVF患者,使用流式细胞仪行精子DFI测定,DFI值按不同界...     

Functional and Molecular Alterations in T Cells Induced by CCL5

To delineate whether, and the extent to which, CCL5 could impact T cell function we examined cytokine production and proliferative ability following CCL5 treatment in vitro. We report a decreased ability of splenic T cells to produce IFN-γ and TNF-α as well as proliferate in response to crosslinking with antibody to CD3 after 72, but not 24 hours of CCL5 exposure. To identify a mechanism by which CCL5 modulated T cell function, we examined T cell receptor translocation and lipid raft clustering. After exposure to CCL5, T cells were less efficient at translocating the TCR and clustering lipid rafts. Since TCR translocation and lipid raft clustering are required for creation of an immunological synapse, these data suggest that extended exposure to CCL5 may impact T cell effector function by modulating the ability to create a functional immunological synapse.     

Ovary and ovulation: Application of image analysis cytometry in fofficular fluid cells obtained from in-vitro fertilization cycles: relationships to patient's age, oocyte maturity, fertilizability and in-vitro fertilization outcome

In an in-vitro fertilization (LVF)/embryo transfer pro grammegranulosa cells obtained from 59 individual preovulatory follicleswere analysed using multiparameter image analysis cytometry,in an attempt to determine whether their morphometric and DNA-cytometricparameters could prove useful in assessing follicle and oncytematurity and in predicting fertilizabifity and outcome of theseIVF cycles. Almost all morphometric and DNA- cytometric parameterswere not correlated with either the patient's age or oocytematurity, and did not predict oocyte fertilization or occurrenceof a clinical pregnancy. The only possible relevant parameterwhich, despite its inverse correlation to total luteinizinghormone administration, also proved to be inversely correlatedto pregnancy outcome (in the seven cases in which a pregnancyoccurred), was the percentage of granulosa cell nuclei withincreased DNA content (>5c). Finally, if granulosa cellsdo not reveal euploid polyploidization in spontaneous or inducedovulatory cycles, the detected cells with increased DNA contentshould be interpreted as aneuploid, i.e. with chromosomal aberrations,and so their presence could also be discussed in connectionwith the hypothetical risk of prospective neoplastic transformationof the tissue.     

精子染色质结构异常对体外受精-胚胎移植结局的影响

目的 探讨精子染色质结构异常对体外受精-胚胎移植(in vitro fertilization and embryo transfer,IVF-ET)的影响.方法 对IVF-ET治疗的不育夫妇136对,分别检测精子DNA碎片化和染色质包装缺陷,并分析精子染色质结构参数与受精及临床妊娠之间的关系.结果 精子DNA碎片化阳性率和染色质包装缺陷阳性率均与受精率呈负相关(r值分别为-0.198,P＜0.05,和-0.389,P＜0.01);在未获得临床妊娠的患者,精子DNA碎片化阳性率和染色质包装缺陷阳性率均高于获得临床妊娠的夫妇(分别为10.74%vs.5.40%,P＜0.01和23.58%vs.11.83%,P＜0.01).结论 IVF-ET治疗失败与精子染色质结构异常有关,精子染色质结构检测有助于评估IVF-ET失败的风险,以制订最佳治疗方案.

Abstract：

Objective To explore the effects of sperm chromatin structure abnormalities on the outcome of in vitro fertilization and embryo transfer (IVF-ET). Methods Sperm DNA fragmentation and chromatin packaging defects were assessed in 136 couples undergoing IVF-ET because of infertility. The relationship between sperm DNA fragmentation, chromatin packaging defects and fertilization rate and clinical pregnancy was evaluated. Results Both sperm DNA fragmentation and chromatin packaging defect had a negative correlation with fertilization rate (r= -0. 198, P＜0. 05, and r= -0. 389, P＜0. 01,respectively). Both parameters were higher in couples who failed to achieve pregnancy than those who achieved clinical pregnancy (10. 74% vs. 5. 40%, P＜0. 01 and 23. 58% vs. 11. 83%, P＜0. 01,respectively). Conclusion Abnormality of sperm chromatin structure is one of the reasons for IVF-ET failure. Examination of sperm chromatin structure is helpful in predicting he risk of IVF-ET failure and optimizing treatment of infertility.