两种不同的氧气浓度对人类未成熟卵子体外成熟培养、受精及早期胚胎发育的影响

目的探讨两种不同的氧气浓度对人类未成熟卵子体外成熟培养(IVM)及受精和早期胚胎发育的影响,为临床辅助生殖技术治疗提供参考依据。方法将收集的未成熟卵子随机分为低浓度氧气(低氧组)和高浓度氧气(高氧组)条件进行IVM,将成熟卵子进行卵胞质内单精子显微注射(ICSI)并行体外胚胎培养,分析两种不同的氧气浓度培养对人类未成熟卵子的成熟率、受精率、卵裂率以及优质胚胎率的影响。结果对于有生发泡(GV)期卵子,低氧组的成熟率高于高氧组(84. 40%vs. 69. 81%),差异有统计学意义(P<0. 05);低氧组的受精率和优质胚胎率都高于高氧组(65. 22%vs. 64. 86%;20. 51%vs. 12. 50%),低氧组的卵裂率低于高氧组(65. 00%vs. 66. 67%),但差异无统计学意义(均P>0. 05)。对于减数分裂I (MI)期卵子,低氧组的成熟率高于高氧组(90. 84%vs. 78. 29%),差异有统计学意义(P<0. 05);低氧组的卵裂率低于高氧组(62. 34%vs. 65. 08%),但差异无统计学意义(P>0. 05);低氧组的受精率和优胚率都高于高氧组(64. 70%vs. 62. 38%;18. 75%vs. 17. 07%),但差异无统计学意义(均P>0. 05)。结论低浓度氧气培养相对高浓度氧气可以提高卵子的体外成熟率,但不能改善其受精和早期胚胎发育。     

Stereomicroscopic and histological examination of bovine embryos following extended in vitro culture

Attempts to support survival of mammalian embryos after hatching have met with limited success, although some mouse studies have reported growth at the post-implantation stage. The aim of the present research was to establish and characterise an in vitro culture system that could support extended growth and differentiation of bovine embryos. Abattoir-derived oocytes were matured and fertilised in vitro. Presumptive zygotes were cultured in modified synthetic oviduct fluid (SOFaaci) medium supplemented with 5% cow serum (CS). On Day 9, single hatched blastocysts (n = 160) were randomly allocated to SOFaaci supplemented with either 5% bovine serum albumin, 5% CS, 5% fetal calf serum (FCS) or SOF only and cultured on a collagen gel substrate for up to 45 days. Embryos were evaluated at various time-points until complete disaggregation or the total disappearance of embryonic cells. Blastocyst viability post hatching was severely compromised in protein-free SOFaaci medium. Addition of FCS generated increased embryonic growth for the longest time period (Day 45) when compared to the other groups. Long-term survival of embryonic cells was observed stereomicroscopically by the proliferation and development of three-dimensional tubular structures to 85% confluence in culture. Haematoxylin and eosin staining of morphological structures obtained from all treatment groups revealed embryos displaying trophoblast, inner cell mass and hypoblast development to varying degrees. Regardless of treatment, extended in vitro culture did not result in development comparable with that described for in vivo embryos. In the present work, however, there was evidence of extended culture of bovine embryos beyond that achieved previously. However, further research is required to identify the exact requirements for extended in vitro culture for bovine embryos.     

两种培养基的沙门菌检出率比较

沙门菌属是肠杆菌中的一个重要菌属,自1880年Eberth发现以来,截止2007年底,全世界报道并被国际权威机构认定的沙门菌血清型达到近3000个,我国共发现322个不同的血清型。沙门菌在环境中容易受到不利因素的影响而处于损伤状态,而且混杂在其他肠道菌中,其数量又相对较少,故检测之前必须采取复苏和抗干扰措施,如用一般的微生物检测方法则难以检出,因此,     

辅助生殖技术中低氧浓度胚胎培养的meta分析

目的:用meta分析方法探讨低氧浓度胚胎培养对体外受精/卵胞浆内单精子注射临床结局的影响。方法:计算机检索PubMed、Ebase、Cochrane Database、中国知网(CNKI)、中国生物医学文献服务系统(CBM)、万方数据库,收集国内外有关低氧浓度胚胎培养的随机对照试验(RCTs)。对纳入的研究进行质量评价,采用RevMan5.1软件对种植率、妊娠率等进行meta分析。结果:排除不符合纳入标准、失访人数不明确、重复发表、文献质量过低的研究后,共纳入10项RCTs,累计调查对象5520例,其中低氧组2645例,常氧组2875例。meta分析显示:与常氧培养相比,低氧培养可提高着床率(OR=1.16,95%CI:1.05～1.29,P=0.01)、继续妊娠率(OR=1.32,95%CI:1.12～1.55,P<0.001)及临床妊娠率(OR=1.28,95%CI:1.07～1.53,P=0.008),且不增加多胎妊娠率(OR=1.19,95%CI:0.67～2.14,P=0.32)及流产率(OR=0.98,95%CI:0.63～1.53,P=0.93)。结论:低氧浓度胚胎培养可以一定程度地提高种植率及妊娠率,且不增加多胎及流产的风险,但更确切的效果仍需更多观察指标的大规模长期随机对照研究加以确认。     

The duration of immunity in cattle following inoculation of rinderpest cell culture vaccine

The duration of immunity following a single administration of rinderpest cell culture vaccine, of 90 or more monolayer passages, was studied in E. African zebu (Boran) and grade (cross-bred European) cattle. All animals were kept for periods of 6-11 years in rinderpest-free environments; groups of them (in all 23 Borans and 10 grades) were then challenged by parenteral or intranasal inoculation of virulent virus or by contact exposure to reacting cattle. Nasal excretion of virus was studied daily over the 10-to 14-day period following challenge, and simultaneous attempts were made to detect viraemia. The neutralizing antibody response was followed at 6-month intervals over the whole post-vaccination period and then daily for 10 days and at longer intervals to 3 weeks after challenge. All 33 animals which were exposed by various routes failed to react clinically and a rinderpest viraemia was never detected. No transmission of virus from the vaccinates to susceptible in-contact controls occurred within 14 or more days, from the 20 animals which could be so tested. Clearcut serological responses to challenge were seen in six cattle (four Borans and two grades) which were challenged after 7 years or more; these reactions were all delayed to the 9th or 10th days, i.e. they were not typically 'anamnestic'. These results are discussed in relation to mass vaccination campaigns for the control of rinderpest and from the comparative viewpoint of measles vaccination in man.     

Effect of 2,4-dinitrophenol on the energy metabolism of cattle embryos produced by in vitro fertilization and culture

In cattle embryos, the proportion of ATP produced by glycolysis increases following the major activation of the embryonic genome, and development to the blastocyst stage is improved in the presence of 10 microM 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, from Day 5 to Day 7 of culture. In Experiment 1 of the present study, culture of cattle embryos in the presence of 10 microM DNP from Day 5 to Day 7 stimulated development to the blastocyst stage, but had no significant effects on oxygen, pyruvate or glucose uptake, or on lactate production. In Experiment 2, culture of cattle embryos in the presence of 10 microM DNP from Day 5 to Day 7, stimulated the metabolism of [2-14C]pyruvate (a measure of Krebs cycle activity) on all of Days 5, 6 and 7, and stimulated metabolism of [5-3H]glucose (a measure of glycolysis) on Day 7 only. The results show that 10 microM DNP stimulates oxidative and glycolytic metabolism in Day-5 to Day-7 cattle embryos, but this does not fully explain the observed increase in developmental competence. We propose that partial inhibition or uncoupling of oxidative phosphorylation may reduce the level of intracellular reactive oxygen species production, thereby facilitating development.     

Dissection of culture media for embryos: the most important and less important components and characteristics

Improvements in culture media formulations have led to an increase in the ability to maintain the mammalian embryo in culture throughout the preimplantation and pre-attachment period. Amino acids and specific macromolecules have been identified as being key medium components, whereas temporal dynamics have been recognised as important media characteristics. Furthermore, other laboratory factors that directly impact embryo development and viability have been identified. Such factors include the use of a reduced oxygen tension, an appropriate incubation system and an adequate prescreening of all contact supplies. With rigourous quality systems in place, it is possible to obtain in vivo rates of embryo development in vitro using new media formulations while maintaining high levels of embryo viability. The future of embryo culture will likely be based on novel culture chips capable of providing temporal dynamics while facilitating real-time analysis of embryo physiology.     

LIF and TNF alpha concentrations in embryo culture media are predictive for embryo implantation in IVF

ObjectiveThere is strong evidence that the cytokines leucemia inhibitory factor (LIF) and tumor necrosis factor (TNF) alpha are related to embryo development and implantation. The aim of this study was to determine the levels of LIF and TNF alpha in embryo culture media and to assess its relationship to the outcome of in-vitro fertilization and embryo transfer.MethodsA total of 99 patients were included in this prospective trial and underwent either IVF or ICSI procedure. A total of 865 oocytes were collected. Embryos were cultured in sequential media until day 5. A standardized morphology evaluation of all embryos, including a detailed pronuclear scoring, was performed daily during this period followed by the replacement of one or two selected embryos. Collected embryo culture fluids of days 3 and 5 were analysed for LIF and TNF alpha on days 3 and 5.ResultsMean TNF alpha concentration in culture media on day 3 was 0.54 and 0.37 pg/mL on day 5 and was significantly lower in women conceiving than in not conceiving (0.43 pg/mL versus 0.59 pg/mL on day 3). Mean LIF concentration on day 3 was 31.5 pg/mL and 35.5 pg/mL on day 5 and was significantly higher in women conceiving (56.2 pg/mL versus 22.2 pg/mL on day 3).ConclusionsThe results indicate that LIF could have a function in early embryogenesis and as a factor required for embryo implantation. High TNF alpha concentrations seem to be predictive of implantation failure.     

The duration of immunity in cattle following inoculation of rinderpest cell culture vaccine.

The duration of immunity following a single administration of rinderpest cell culture vaccine, of 90 or more monolayer passages, was studied in E. African zebu (Boran) and grade (cross-bred European) cattle. All animals were kept for periods of 6-11 years in rinderpest-free environments; groups of them (in all 23 Borans and 10 grades) were then challenged by parenteral or intranasal inoculation of virulent virus or by contact exposure to reacting cattle. Nasal excretion of virus was studied daily over the 10-to 14-day period following challenge, and simultaneous attempts were made to detect viraemia. The neutralizing antibody response was followed at 6-month intervals over the whole post-vaccination period and then daily for 10 days and at longer intervals to 3 weeks after challenge. All 33 animals which were exposed by various routes failed to react clinically and a rinderpest viraemia was never detected. No transmission of virus from the vaccinates to susceptible in-contact controls occurred within 14 or more days, from the 20 animals which could be so tested. Clearcut serological responses to challenge were seen in six cattle (four Borans and two grades) which were challenged after 7 years or more; these reactions were all delayed to the 9th or 10th days, i.e. they were not typically ''anamnestic''. These results are discussed in relation to mass vaccination campaigns for the control of rinderpest and from the comparative viewpoint of measles vaccination in man.     

Fertilization and larval development in sea urchins following exposure of gametes and embryos to cadmium

The gametes and embryos of three sea urchin species were exposed to cadmium chloride at concentrations ranging from 10^–8 M to 10^–3 M. When zygotes were reared in the presence of Cd²⁺, skeletal differentiation displayed some severe abnormalities or was suppressed, as a function of Cd²⁺ level. The embryotoxic action of Cd²⁺ was inversely related to salinity and to Ca²⁺ concentration. Cadmium-exposed larvae displayed similar abnormalities if Cd²⁺ was present throughout development or only after hatching, while pre-hatching exposure produced no developmental defects. No aberrations in mitotic figures were observed in cleaving eggs following acute exposure to Cd²⁺. The pretreatment of sperm or eggs did not affect the ensuing development of embryos, both for acutely toxic Cd²⁺ levels (up to 10^–2M), and for prolonged exposures in relatively low Cd²⁺ levels. The fertilization rate was differently affected depending on whether sperm or eggs were pretreated;i.e., the exposure of eggs to Cd²⁺ promoted fertilization at relatively high Cd²⁺ levels. If sperm was exposed to Cd²⁺, a depression of fertilizing capacity was observed at high Cd²⁺ levels, while lower Cd²⁺ levels, displayed an opposite action, resulting in an increase in fertilization rate after prolonged sperm exposure.     

Cysteamine supplementation of in vitro maturation medium, in vitro culture medium or both media promotes in vitro development of buffalo (Bubalus bubalis) embryos

The effects of supplementation of in vitro maturation (IVM) or in vitro culture (IVC) or both IVM and IVC media with cysteamine on the yield, hatching rate (HR) and total cell number (TCN) of buffalo blastocysts were examined. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF. The IVM or IVC media were supplemented with 0, 50, 100 or 200 microm cysteamine. Supplementation of IVM medium with 50 microm cysteamine increased (P < 0.01) the cleavage rate and blastocyst yield without affecting the HR and TCN whereas a higher concentration of 200 microm significantly (P < 0.05) reduced the blastocyst yield but not TCN. Similar increases in blastocyst yield, without any effect on HR and TCN were observed after supplementation of the IVC medium with 100 (P < 0.01) or 50 microm (P < 0.05) cysteamine, whereas 200 microm cysteamine was ineffective. Supplementation of both IVM medium with 50 microm cysteamine and of IVC medium with 100 microm cysteamine increased the yield of blastocysts and hatched blastocyst by over 100% (P < 0.01) compared with the controls without any adverse effects on HR or TCN. The results of the present study suggest that supplementation of both IVM and IVC media improves the yield of blastocysts without compromising their health.     

昆明鼠自体卵丘细胞与早期胚胎序贯共培养对胚胎体外发育潜能的影响

目的:评价昆明鼠胚胎与自体卵丘细胞序贯共培养对提高早期胚胎体外发育潜能的影响。方法:分两组进行实验,序贯共培养组使用分离培养的卵丘细胞与序贯培养液构建培养体系,与昆明鼠早期胚胎进行共培养;序贯培养组使用序贯培养液对早期胚胎进行序贯培养,观察两组胚胎各阶段囊胚形成率,检测囊胚总细胞数、囊胚细胞凋亡数,囊胚进一步培养获得的干细胞系数。结果:序贯共培养3d后总的囊胚形成率为68.13%,序贯培养组囊胚形成率为45.31%(P<0.05);囊胚总细胞数分别为76.52和38.57(P<0.05),细胞凋亡率分别为7.44%和16.28%(P<0.05)。将囊胚进一步培养,共培养组干细胞建系率为12.50%,序贯培养组未能建系。结论:昆明鼠胚胎与自体卵丘细胞序贯共培养与序贯培养比较,前者能更好地模拟体内环境,提高早期胚胎体外发育潜能。     

Fetal growth and development following temporary exposure of day 3 ovine embryos to an advanced uterine environment

The effect of exposing Day 3 ovine embryos to an advanced uterine environment for a period of 3 days on subsequent fetal growth and development between Day 35 and Day 135 of gestation was studied. Day 3 embryos were recovered from superovulated donor ewes and transferred to synchronous final or asynchronous temporary recipients for 3 days. Embryos were recovered from these temporary recipients and transferred to Day 6 final recipients. Gravid uteri were recovered, weighed and dissected on Days 35, 45, 60, 90, 110, 125 and 135 of gestation. Fetal weight and length data were analysed by fitting non-linear Gompertz models of the form log(e) y = a - be(-ct), where y is fetal size and t is time from conception. Various terms including treatment, gestational age, embryo stage at transfer and fetal sex were fitted to this model. Fetal development was assessed by relating organ weight to fetal bodyweight using the linear allometric equation log(e) y = log(e) a + b log(e) x, where y is organ weight and x is fetal weight. Temporary exposure of Day 3 embryos to an advanced uterine environment did not increase the rate of embryo development and had no effect on fetal growth and development between Days 35 and 135 of gestation in this study. A single Gompertz model (log(e) y = 10.134 - 17.047e(-0.1733t)) explained 99.8% of the variation in fetal weight. Of terms fitted to this model only gestational age and fetal sex influenced fetal weight, with male fetuses being 5% heavier (P<0.05) than female fetuses. Fetal development was also unaffected by experimental treatment in this study. Allometric coefficients established for various fetal components agreed well with those from previously published studies.     

The effect of low and ultra-low oxygen tensions on mammalian embryo culture and development in experimental and clinical IVF

ABSTRACT

Over the last forty years, many trials have been performed using mammalian embryo cultures with reduced oxygen tension (O₂) to encourage proper embryo development and increase the success rate for in vitro fertilization (IVF) outcome. Even if the use of atmospheric O₂ (20%) affects in vitro embryo development and intracellular redox balance, the use of low (5% O₂, physiologic) and ultra-low (close or less to 5% O₂) O₂ applied to in vitro embryo culture is still under debate. Numerous studies in various mammalian species have shown that embryo development improves when culturing embryos under low O₂, although culture conditions are not the only factors involved in the success of IVF. This article reviews the literature data of the last four decades and discusses the current evidence on the use of low and ultra-low O₂ in embryo culture, and examines the impact of multiple factors on IVF outcomes.     

Duration of clinical symptoms in children under two years of age with acute otitis media

Objective: To describe the course of symptoms during the first ten days of episodes of AOM in children under two years of age.

Methods: Within the framework of a placebo-controlled, double-blind, randomised trial studying the effect of amoxicillin on acute otitis media in children under two years of age in the Netherlands, symptoms were recorded in diaries by the parents. Durations of symptoms were plotted by means of Kaplan-Meier curves. Possible factors influencing the duration were analysed in a Cox regression.

Results: Data from 230 children were used in the analyses. The median duration of fever was two days and the median duration of the combination of earache and/or crying was eight days. The duration of earache and/or crying was not influenced by any factor analysed (including treatment allocation).

Conclusion: Fifty per cent of the children under two years with an episode of AOM had symptoms for more than eight days (95% CI: 6.9-9.1). Since antibiotic treatment did not influence this period, persistence of symptoms should not be a reason for changing antibiotic therapy.     

两种支原体培养基的应用比较及临床意义

由于解脲脲原体(Ureaplasma urealyticum,Uu)和人型支原体(Mycoplasma hominis,Mh)的检出与所用培养基关系密切,因此可直接导致各种文献报道中相似人群感染率不同的结果。为了解南京地区性病门诊及妇科门诊就诊者中Uu和Mh的临床感染情况,科学地评价支原体的致病作用,并