IVF／ICSI受精失败的原因

全部卵母细胞受精失败（或低受精率）是体外受精（IVF）实验室经常遇到的困惑之一。尽管在熟练掌握和广泛使用卵胞浆内单精子注射（ICSI）技术后,全卵不受精现象已显著降低,但在IVF实验室内仍然存在着难以预测和不明原因的全卵不受精现象。事实上,无论是采取常规IVF或ICSI助受精方式,都避免不了某些病例出现全卵受精失败。     

冻融人卵子胞浆内单精子注射临床妊娠成功

目的 提供一种适合于临床应用的缓慢低温冷冻和快速解冻复苏的贮存人卵母细胞的方法。方法 接受试管婴儿(IVF)或卵子胞浆内单精子注射(ICSI)治疗的6例患者剩余的卵母细胞共33个，通过程序化缓慢低温冷冻方法贮存于含有蔗糖的1，2丙二醇中。6例因不同原因需要受卵者的子宫内膜用激素替代治疗(HRT)法调整或采用自然周期，当受卵者子宫内膜处于合适时机时，复苏冻存的卵母细胞6次共33个，共28个成熟卵子通过ICSI受精，记录卵母细胞复苏率、复苏卵ICSI后的受精率，及随后胚胎发育情况，选择质量好的胚胎进行移植，胚胎移植后2周检查受孕情况，孕4周B超检查。结果 当用高浓度的蔗糖冻存液保存时，慢冻快融法得到了很高的存活率。复苏后的卵子存活率97％(32／33)，复苏卵经ICSI后的受精率82％(23／28)，受精胚胎分裂率91％(21／23)，6个胚胎移植周期共移植胚胎21个。4例获临床妊娠，目前1例孕17周，1例孕12周，1例孕7周，均为单胎妊娠，状态良好；另外1例妊娠9周后自然流产。结论 人卵母细胞慢冻快融低温贮藏方法能够提高人卵母细胞冻存后的存活率。该法操作简便、费用低、复苏率高，较好地解决了现有人卵母细胞的技术冻存效果不理想、复苏率低的问题，为进一步人卵母细胞冷冻技术的常规应用提供了经验。     

早期补救卵胞浆内单精子注射在常规体外受精不受精或低受精周期中的应用

常规体外受精-胚胎移植(in vitro fertilization-embryo transfer,IVF-ET)周期中,卵母细胞受精低下或不受精的发生率约为10％～20％[1].补救性卵胞浆内单精子注射(rescue intracytoplasmic sperm injection,rescue ICSI)作为受精失败的一种补救措施已在许多生殖中心实施,早期多行晚期补救,一般在受精后16～20 h观察原核形成情况,如未见原核形成则通过ICSI再次受精.     

影响补救性卵胞浆内单精子注射周期妊娠的因素

常规体外受精（IVF）周期中不受精或者低受精（周期受精率≤25％）在临床上的发生率一般占常规IVF的10％～30％,补救性卵胞浆内单精子注射（rescue—ICSI）作为一种受精失败的补救措施在临床上的应用已有十余年,但rescue—ICSI的临床结局不如人意,对于这部分患者,目前国内外仍无有效的改善措施。本文通过对rescue～ICSI周期的回顾,分析影响周期妊娠的因素,为临床实践提供参考。     

常规体外受精失败卵母细胞行早期补救卵胞浆内显微注射的临床应用价值

目的探讨常规体外受精-胚胎移植（IVF—ET）中受精障碍患者行早期补救卵胞浆内单精子注射（ICSI）的可行性。方法回顾性分析我院生殖中心2007年1月至2009年7月常规IVF—ET治疗877周期,其中2008年3月至2009年7月期间开展短时受精并对受精障碍周期行早期补救ICSI的546周期作为研究组,2007年1月至2008年2月期间未开展短时受精、早期补救ICSI的331周期作为对照组。研究组通过IVF后6h观察卵母细胞是否排出第2极体评估受精,对完全未受精和低受精周期中未见第二极体排出的成熟卵母细胞立即行早期补救ICSI,比较两组临床及实验室指标。研究组中行早期补救ICSI的70周期实验室指标和临床指标与同期179个常规ICSI周期相比较。结果研究组通过早期补救ICSI,受精率、周期冷冻率、优质胚胎率均比对照组显著提高（P〈O．01）,因受精失败取消移植率显著降低（P〈O．01）。早期补救ICSI周期受精率、可移植胚胎率、临床妊娠率及胚胎种植率与常规ICSI相似,正常受精率、卵裂率和优质胚胎率显著低于常规ICSI,≥3原核（PN）异常受精的比率较常规ICSI略有升高,但无显著性差异。结论IVF后6h行早期补救ICSI能提高常规IVF卵的利用率,并获得与常规ICSI相似的临床妊娠结局。     

常规体外受精失败周期早补救卵胞浆内单精子注射与部分-卵胞浆内单精子注射受精方式的回顾性分析

目的比较早补救卵胞浆内单精子注射（ICSI）与部分（Half）-ICSI两种授精方式在体外受精（IVF）助孕中的应用。方法回顾性分析76个常规IVF发生受精失败的移植周期。其中30个周期为早补救ICSI授精方式（早补救ICSI组）,46个周期为Half-ICSI授精方式（Half-ICSI组）。分析受精情况、胚胎质量、种植率、妊娠率、抱婴回家率、出生缺陷和卵母细胞利用情况。结果早补救ICSI组与Half—ICSI组的双原核（2PN）受精率、多PN受精率、1PN受精率、优质胚胎率、胚胎种植率、妊娠率、抱婴回家率、出生缺陷无显著差异（P〉0．05）。但早补救ICSI组卵母细胞利用率高于Half-ICSI组,平均受精卵数分别为（6．30±2．96）和（4．20±1．75）（P〈0．001）,胚胎数分别为（6．20±2．89）和（4．11±1．68）（P〈0．001）,冷冻胚胎数分别为（3．20±2．76）和（1．56±1．68）（P〈0．01）、冷冻周期率分别为76．67％和50．00％（P〈0．05）。结论在常规IVF完全受精失败的周期,早补救ICSI授精方式与Half-ICSI授精方式的受精率、胚胎质量和妊娠结局类似,但前者有更好的卵母细胞利用率和更多可利用胚胎。     

人工卵母细胞激活在ICSI受精失败周期中的应用

在辅助生殖技术领域,人工卵母细胞激活(AOA)是解决单精子卵胞浆内显微注射(ICSI)低受精或完全未受精周期中精子缺陷所致卵母细胞激活失败的重要措施。文献报道AOA的方法包括机械刺激、电刺激和化学激活。但与传统ICSI相比,ICSI联合AOA(ICSI-AOA)对ICSI受精失败周期的有效性和安全性尚难以评估。本文就AOA在ICSI受精失败周期中的应用,包括卵母细胞激活的机制、ICSI受精失败原因、AOA的方法和AOA的有效性及安全性进行综述,并探讨AOA在临床应用中存在的问题,以期更好地服务于临床。     

ICSI受精失败的原因与对策

卵细胞胞质内单精子注射(ICSI)是一种新型的体外受精技术,有很高的单精受精率,但ICSI技术不能解决所有的受精失败,可能的原因包括卵母细胞质量、受精激活障碍、精子异常以及ICSI技术等问题.IVF超促排卵可能引起卵母细胞内凋亡增加,从而导致受精失败.卵母细胞激活障碍有多种人工辅助的激活方法.ICSI时精子引起的受精失...     

体外受精失败MII期人卵母细胞的免疫荧光研究

目的探讨体外受精中MII期人卵母细胞受精失败的原因。方法收集体外受精后24～48h仍未受精的MII期卵母细胞,进行免疫荧光染色和碘化丙啶(PI)复染,在荧光显微镜下对其失败原因进行分类。结果卵母细胞内未见精子的在常规体外受精(IVF)周期有55.8%,显著多于卵胞浆内单精子注射(ICSI)周期中的9.7%(P<0.01);卵母细胞活化失败两者分别为14.9%和58.1%,有显著性差异(P<0.01);原核形成和(或)迁移缺陷的在两者分别为25.3%和32.3%(P>0.05);其他异常两者分别为3.9%和0.0%。结论IVF中MII期卵母细胞的受精失败主要是缺乏精子的穿透,ICSI周期中的主要原因是卵母细胞活化不完全。     

常规体外受精失败后补救卵胞浆内单精子注射治疗39例分析

在常规体外受精-胚胎移植（IVFET）周期中完全不受精或低受精率（受精率≤25％）的发生率约为10％～20％。补救性卵胞浆内单精子注射（ICSI）作为受精失败的一种补救措施在临床应用已有十余年,虽然有获得妊娠的报道,但其临床应用价值目前还存在争议。本文回顾性分析了我院生殖医学中心2004年1月至2008年12月常规IVF周期完全不受精或低受精率的39个周期,     

Oocyte membrane maturation and oocyte-sperm relationship: transmission electron microscopy study

The success of in vitro maturation (IVM) depends greatly on the acquisition of immature oocytes. Immature oocytes in prophase I (PI) and metaphase I (MI), aspirated after controlled ovarian hyperstimulation, were incapable of fertilization, leading to a lower fertilization rate. Therefore, they must be evaluated on a fine structure level for their in vitro maturation (IVM) processes and their relationship with sperm. Oocyte membrane maturation and oocyte-sperm relationship were studied using transmission electron microscopy. A total of 55 human oocytes obtained from 20 patients at various times and 83 oocytes obtained from the dissected ovarians of female Wistar rats were used for transmission electron microscopy (TEM) evaluation. Despite being in either prophase I and metaphase I or in metaphase II, the oocytes were not fertilized after 48 h of incubation. At the various stages of maturation between PI and MII, the number and the size of microvilluses on the oocyte membrane increased as MII approached and decreased after full maturation. Oocyte activation was related to oocyte membrane maturation and has an effect on the oocyte sperm penetration.     

The human acrosome reaction

We developed tests of sperm-oocyte interaction: sperm-zona binding, zona-induced acrosome reaction, spermzona penetration and sperm-oolemma binding, using oocytes which failed to fertilise in clinical in vitro fertilization (IVF). Although oocyte defects contribute to failure of sperm oocyte interaction, rarely are all oocytes from one woman affected. Low or zero fertilization in standard IVFwas usually caused by sperm abnormalities. Poor sperm-zona pellucida binding was frequently associated with failure of standard IVF and obvious defects of sperm motility or morphology. The size and shape of the acrosome is particularly important for sperm binding to the oocyte. The proportion of acrosome intact sperm in the insemination medium was related to the IVF rate. Inducing the acrosome reaction with a calcium ionophore reduced sperm-zona binding. Blocking acrosome dispersal with an acrosin inhibitor prevented spermzona penetration. Sperm-zona penetration was even more highly related to IVF rates than was sperm-zona binding. Some patients had low or zero fertilization rates with standard IVF but normal sperm by conventional tests and normal sperm-zona binding. Few of their sperm underwent the acrosome reaction on the surface of the zona and none penetrated the zona. In contrast, fertilization and pregnancy rates were high with intracytoplasmic sperm injection. We call thiscondition defective zona pellucida induced acrosome reaction. Discovery of the nature of the abnormalities in the signal transduction and effector pathways of the human zona pellucida induced acrosome reaction should result in simpler tests and treatments for the patients and also provide new leads for contraceptive development.     

In vitro fertilization failure: identification of gamete defects by investigation of sperm-zona pellucida binding capacity of unfertilized oocytes

Summary. The objective of the study was to determine whether fertilization failure was due to spermatozoal or oocyte factors. Twenty-five unfertilized oocytes from 12 IVF/GIFT couples showing total or partial fertilization failure were evaluated for sperm zona binding potential under hemizona assay (HZA) conditions. Hemizonae were separately incubated with a sperm sample from the husband and that of a fertile control. Tight sperm binding to hemizonae was assessed. First, among the 12 patients, results showed a possible zona defect thought to be the cause of fertilization failure in five cases. Second, in two cases, fertilization failure was possibly caused by poor sperm binding potential of spermatozoa. Third, in two cases, fertilization failure was possibly caused by an oocyte defect, and fourth, three cases showed a mixture of possible causes. The results stress the need to develop a sequential analytic programme for those couples with repeated total or partial fertilization failure.     

Differential effects of polysulphates between mouse and hamster during in vitro fertilization

The interaction between zona pellucida polysulphates and sperm receptors appears to be a widespread mechanism used by mammals during gamete interaction. In this work, the effect of heparin on binding, penetration and fertilization of mouse and hamster oocytes was assessed. We found that heparin inhibited oocyte penetration and fertilization in both species. Heparin as well as fucoidan (a fucose-sulphate polymer) inhibited the proteolytic activity of acrosomal enzymes in both species. Our results suggest that zona pellucida penetration in both species may be modulated by polysulphates acting on either the proteolytic rate of degradation of the zona and/or its interaction with acrosome-reacted sperm (secondary binding).     

卵泡液中血管内皮生长子和甾体激素水平对卵母细胞成熟和受精的影响

目的 探讨卵胞浆内单精子注射（ICSI）周期取卵日卵泡液中血管内皮生长因子（VEGF）和甾体激素水平与卵母细胞成熟和受精的关系.方法 收集在本中心接受ICSI治疗的男方因素不育患者取卵日卵泡液105例,采用电化学发光法（ECLIA）检测卵泡液中雌二醇（E2）、孕酮（P）、睾酮（T）水平,酶联免疫吸附法（ELISA）检测VEGF水平.卵泡液按照相应卵母细胞成熟度及激素VEGF水平分别进行分组.结果 成熟组卵母细胞对应的卵泡液中VEGF水平高于未成熟组（P〈0.05）,而低于退化组（P〈0.05）;卵泡液VEGF水平在500～650 ng/L时卵母细胞成熟率、受精率最高（P〈0.05）;E2水平在退化组显著低于成熟组（P〈0.05）;P水平三组比较均无显著性差异（P〉0.05）;T水平在未成熟组和退化组均显著高于成熟组 （P〈0.05）;10≤P/E2≤30组卵子成熟率最高（91.11%）（P〈0.05）.结论 卵泡液中,VEGF水平在500～650 ng/L时卵母细胞成熟率、受精率较高.卵母细胞的成熟及退化与卵泡液中甾体激素环境关系密切,卵泡液P/E210～30范围时卵母细胞成熟率较高.     

Paternal DNA damage resulting from various sperm treatments persists after fertilization and is similar before and after DNA replication

In spite of its highly condensed state, sperm DNA is vulnerable to damage that can originate from oxidative stress, the activity of sperm-specific nucleases, or both. After fertilization, in the oocyte, paternal chromatin undergoes dramatic changes, and during this extensive remodeling, it can be both repaired and degraded, and these processes can be linked to DNA synthesis. Here, we analyzed sperm response to damage-inducing treatments both before and after fertilization and before or after zygotic DNA replication. Epididymal mouse spermatozoa were either frozen without cryoprotection (FT) or treated with detergent Triton X-100 coupled with dithiothreitol (TX+DTT) to induce DNA damage. Fresh, untreated sperm served as control. Immediately after preparation, spermatozoa from 3 groups were taken for comet assay, or for intracytoplasmic sperm injection into prometaphase I oocytes to visualize prematurely condensed single-chromatid chromosomes, or into mature metaphase II oocytes to visualize chromosomes after DNA replication. Comet assay revealed increased DNA fragmentation in treated sperm when compared with control, with FT sperm more severely affected. Chromosome analysis demonstrated paternal DNA damage in oocytes injected with treated, but not with fresh, sperm, with FT and TX+DTT groups now yielding similar damage. There were no differences in the incidence of abnormal paternal karyoplates before and after DNA synthesis in all examined groups. This study provides evidence that subjecting sperm to DNA damage-inducing treatments results in degradation of highly condensed sperm chromatin when it is still packed within the sperm head, and that this DNA damage persists after fertilization. The difference in DNA damage in sperm subjected to 2 treatments was ameliorated in the fertilized oocytes, suggesting that some chromatin repair might have occurred. This process, however, was independent of DNA synthesis and took place during oocyte maturation.     

Relationship between sperm apoptosis signalling and oocyte penetration capacity

Human sperm have been documented to display apoptosis-like features such as externalization of phosphatidylserine (EPS), disruption of the transmembrane mitochondrial potential (MMP) and activation of caspases. Our aim was to evaluate possible association between activation of the apoptosis cascade in human sperm and its oocyte penetration capacity using the zona free hamster oocyte penetration assay (SPA). Semen specimens from 76 unselected donors were subjected to double density gradient centrifugation followed by incubation under capacitating conditions for 3 h and SPA. Apoptosis signalling was monitored by assessment of EPS, disruption of MMP and activation of caspase-3 by flow cytometry. Semen samples with subnormal SPA values (<20% penetrated oocytes) contained significantly higher amounts of spermatozoa with EPS, disrupted MMP and activated caspase-3 compared with those samples with normal SPA values (>20% penetrated oocytes, p < 0.01). All three apoptosis markers showed a significantly negative correlation with the percentage of penetrated oocytes (p < 0.01). Apoptosis-related signalling appears to have a negative association with sperm-oocyte penetration. The exclusion of sperm presenting with those apoptosis-related features during assisted reproduction may improve success rates of procedures such as intrauterine insemination and in vitro fertilization.     

IVF过程受精失败的预测

在发明和常规应用卵胞浆内单精子注射（ICSI）之前,十多年的临床体外受精（IVF）治疗实践中,受精率低下很常见,大约有20％～35％的IVF患者受精率很低（〈35％的卵子受精）和受精完全失败（所有卵子都不受精）。虽然受精失败与精子或卵子的质量有关,但相当一部分患者受精失败与精液质量或精子功能低下有很密切相关性。最常见的是严重的少精,弱精和畸形精子症患者。