Elective single day 3 embryo transfer halves the twinning rate without decrease in the ongoing pregnancy rate of an IVF/ICSI programme

BACKGROUND: Data on the effect of elective single embryo transfer (eSET) on the total and multiple pregnancy rates of an IVF/ICSI programme are reported. METHODS AND RESULTS: A retrospective cohort analysis of eSET was carried out over a 4 year period. A total of 1559 cycles resulted in 1464 transfers; 299 transfers of one top quality embryo (20.4%) and 86 of one non-top quality embryo (5.9%) yielded 149 conceptions (49.8%) with 105 ongoing pregnancies (35.1%) and 26 conceptions (30.2%) with 19 ongoing implantations (22.1%) respectively; 1079 transfers of two (n = 853; 58.3%) or more than two (n = 226; 15.4%) embryos yielded 366 ongoing pregnancies (33.9%). The ongoing pregnancy rates for the years between 1998 and 2001 were 35.9, 27.9, 31.9 and 31.0% per oocyte retrieval and 38.5, 29.4, 34.1 and 33.2% per transfer. There were no differences in pregnancy rates between any of the years. The average ongoing pregnancy rate (>12 weeks) over the 4 years was 31.5% per started cycle and 33.5% per transfer; the average number of embryos transferred decreased from 2.26 (1998) to 1.79 (2001); the multiple pregnancy and twinning rates dropped from 33.6 and 29.5% (1998) to 18.6 and 16.3% (2001) respectively. CONCLUSIONS: Judicious application of eSET can halve the twinning rate while maintaining the overall pregnancy rate.     

抗人大肠癌单链抗体的构建、表达及其体内外生物学活性的检测

目的 将抗人大肠癌单克隆抗体ND-1(mAb)的VR和VL基因进行重组，构建和表达ND－1scFv，并对其在体内外的生物学活性进行检测。采用RT－PCR技术，从能够分泌mAb ND-1的鼠杂交瘤细胞中扩增VH和RL基因，通过重叠延伸拼接PCR在VH和VL基因间引入连接短肽，体外构构建ND－1scFv基因，并在大肠杆菌中表达。采用间接免疫荧光（IFA）EY IF工IMPG-1scFv的免疫学活性。用^99Tc^m标记ND－1scFv后，将偶联物给予荷瘤裸鼠，观察其在动物体内的显像及生物学分布。结果 SDS－PAGEW显示，重组蛋白Mr为30000，同预期结果一致。IFA及ELISA检测表明，ND－1scFv保留了与亲本抗体相近的免疫学活性，对表达相应抗原的靶细胞具有行异结合活性。体内放射免疫实验显示，^99Tc^m-ND-1scFv在荷瘤小鼠体内的生物学分布，呈明显的肿瘤积聚趋向，注入体内1h血中T／NT即在2．61。结论 获得免疫学活性良好的ND－1scFv，对荷瘤动物体内肿瘤的定位快速，准确，可望成为有效的肿瘤诊断和治疗的导向载体。     

PPARY2基因Pro12Ala和C1431T多态性及其单倍型与2型糖尿病和肥胖的相关性研究

目的 研究过氧化物体增殖活化受体γ2(peroxisome proliferator activated receptorγ2,PPARγ2)基因Pro12Ala和C1431T多态性及其单倍型与汉族人2型糖尿病、肥胖的关系.方法 应用聚合酶链反应-限制性片段长度多态性的方法,对207例2型糖尿病患者和101名非糖尿病对照者进行PPARγ2基因Pro12Ala和C1431T多态性研究.结果 (1)在非糖尿病对照人群中Aal 12等位基因频率是0.064,T1431等位基因频率是0.252.单倍型分析显示Pro12Ala和C1431T两个位点连锁不平衡(D'=0.63,r2=0.074),组成了3种常见单倍型Pro-C、Pro-T和Ala-T.(2)Pro12Ala和C1431T多态性分布及其单倍型分布频率在2型糖尿病组与对照组组间差异均无统计学意义(P>0.05).(3)Pro12Ala变异与糖尿病患者的血压、血脂相关,地等位基因降低非肥胖糖尿病患者的舒张压(P<0.05),而对肥胖糖尿病患者的血脂水平无保护作用(P<0.05);C1431T多态性与糖尿病患者的超重和肥胖相关,超重和肥胖的糖尿病者T等位基因频率相对较高(P<0.05).结论 Pro12Ala和C1431T多态性可能在汉族人糖尿病发病中不是起主要作用;C1431T多态性与糖尿病患者的超重和肥胖相关.     

DNA fingerprinting of sister blastomeres from human IVF embryos

BACKGROUND: Previously published single cell DNA fingerprinting systems have been plagued by high rates of allele drop-out (ADO) and preferential amplification (PA) preventing clinical application in preimplantation genetic diagnosis. METHODS: Tetranucleotide microsatellite markers with high heterozygosity, known allelic size ranges and minimal PCR stutter artefacts were selected for chromosomes X, 13, 18 and 21 and optimized in a multiplex fluorescent (FL)-PCR format. FL-PCR products were analysed using the ABI Prism 377 DNA sequenator and Genescan software. Validation of the DNA fingerprinting system was performed on single diploid (n = 50) and aneuploid (n = 25) buccal cells and embryonic blastomeres (n = 21). RESULTS: The optimized pentaplex PCR DNA fingerprinting system displayed a high proportion of successful amplifications (>91%) and low ADO and PA (<6%) when assessed on 50 human buccal cells. DNA fingerprints of single cells from a subject with Down's syndrome detected the expected tri-allelic pattern for the chromosome 21 marker, confirming trisomy 21. In a blind study on 21 single blastomeres, all embryos were identifiable by their unique DNA fingerprints and shared parental alleles. CONCLUSIONS: A highly specific multiplex FL-PCR based on the amplification of five highly polymorphic microsatellite markers was developed for single cells. This finding paves the way for the development of a more complex PCR DNA fingerprinting system to assess aneuploidy and single gene mutations in IVF embryos from couples at genetic risk.     

Combined analysis of T cell receptor gamma and immunoglobulin heavy chain gene rearrangements at the single-cell level in lymphomas with dual genotype

By prospectively studying immunoglobulin heavy chain gene (IgH) and T cell receptor gamma (TCRgamma) gene rearrangements in 398 lymphoma cases, a dual genotype was observed in 13% of B cell and 11% of T cell lymphomas. According to histological subtype, the highest incidence was observed for mantle cell lymphomas (32%) and lymphoplasmacytic lymphoma (21%) among B cell lymphomas, and for angioimmunoblastic lymphoma (AILT) (46%) and Sézary syndrome (SS) (50%) among T cell lymphomas. To determine whether the dual genotype corresponds to the presence of two distinct monoclonal populations or to the presence of both rearrangements within the same lymphoma cells, single-cell microdissection was used after immunohistochemistry and a single-cell combined IgH and TCRgamma gene analysis was designed after a whole-genome amplification step. This protocol was applied to the study of two nodal B cell lymphomas (one diffuse large B cell lymphoma and one mantle cell lymphoma) and two cutaneous T cell lymphomas (one AILT and one SS). Two cases (SS and mantle cell lymphoma) were true bigenotypic lymphomas, as both IgH and TCRgamma monoclonal rearrangements were detected in the same cells. Conversely, in the diffuse large B cell lymphoma and AILT cases, large CD22+ single cells exhibited only the monoclonal IgH rearrangement but not the TCRgamma gene that was detected in CD3+ single cells. Such an approach allows the identification of true bigenotypic lymphoma among dual genotypic lymphoma. Specific genetic alterations may be further amplified from microdissected cryopreserved material, such as the t(11;14) breakpoint detected in bigenotypic B cells of the mantle cell lymphoma case.     

5′HOXD基因与单纯性马蹄内翻足的相关性分析

目的检测HOXD10、HOXD12、HOXD13基因内4个已知单核苷酸多态(single nucleotide poly-morphisms,SNP)rs2593778、rs847154、rs847151、rs13392701在单纯性马蹄内翻足核心家系中的分布情况,分析各个SNP位点及所构成单倍型与单纯性马蹄内翻足的相关性。方法应用限制性片段长度多态性技术结合测序,分析84个单纯性马蹄内翻足核心家系4个SNP位点基因型;应用ETDT软件统计分析各SNP位点基因型与单纯性马蹄内翻足的相关性;应用TRANSMIT软件构建单倍型并统计分析单倍型频率是否存在差异。结果位于HOXD12基因5′侧翼序列的SNP位点rs847154和位于HOXD13第1外显子的SNP位点rs13392701在单纯性马蹄内翻核心家系中存在传递不平衡(P<0.05);位于HOXD12基因第1外显子的SNP位点rs847151未检测到多态;位于HOXD10第1外显子的SNP位点rs2593778经ETDT分析无统计学意义。结论HOXD12基因5′侧翼序列的SNP位点rs847154和位于HOXD13第1外显子的SNP位点rs13392701与单纯性先天性马蹄内翻足有明显的相关性,提示HOXD12、HOXD13可能是单纯性马蹄内翻足重要的易感基因。     

Skewed sex ratios in familial holoprosencephaly and in people with isolated single maxillary central incisor

Autosomal dominant holoprosencephaly is a rare but well documented entity and it can be the result of mutations in the Sonic Hedgehog gene (SHH). The transmitting parent may be normal or have a single maxillary central incisor. We describe a skewed sex ratio among the transmitting parents with SHH mutations, with more mothers than fathers having the mutation (p=0.002). The mechanism underlying this skewed sex ratio is not clear; the SHH mutations do not involve triplet repeats, imprinting is plausible but untested, and there is no evidence that the risk of holoprosencephaly is greater among males carrying such a mutation (p=0.15). We considered the possibility that males with such a mutation are at greater risk of other malformations outside the central nervous system, which could reduce their reproductive fitness. To avoid ascertainment bias in identifying children with various malformations in kindreds with familial holoprosencephaly, we reviewed the reports of people with single maxillary central incisor and no other congenital malformations. Of the 16 cases identified, 13 were female (p=0.0085). We suggest that boys with mutations associated with autosomal dominant holoprosencephaly may be at greater risk of major malformations outside the central nervous system than girls.     

Reverse enzyme-linked immunospot assay (RELISPOT) for the detection of cells secreting immunoreactive substances

A reverse modification of the recently described enzyme-linked immunospot assay (ELISPOT), based on localized enzyme-substrate reactions in gel, is described for the enumeration of antigen-secreting cells using petri dishes coated with specific antibodies. As a model the detection of mouse and human immunoglobulin-secreting cells has been evaluated. Simple and sensitive, this new method, termed RELISPOT, can be adapted for the quantitation of secreted antigen thus providing additional information on the metabolic state of the population of cells tested.     

Detailed investigation of factors influencing amplification efficiency and allele drop-out in single cell PCR: implications for preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) of single gene disorders relies on PCR-based tests performed on single cells (polar bodies or blastomeres). Despite the use of increasingly robust protocols, allele drop-out (ADO; the failure to amplify one of the two alleles in a heterozygous cell) remains a significant problem for diagnosis using single cell PCR. In extreme cases ADO can affect >40% of amplifications and has already caused several PGD misdiagnoses. We suggest that an improved understanding of the origins of ADO will allow development of more reliable PCR assays. In this study we carefully varied reaction conditions in >3000 single cell amplifications, allowing factors influencing ADO rates to be identified. ADO was found to be affected by amplicon size, amount of DNA degradation, freezing and thawing, the PCR programme, and the number of cells simultaneously amplified. Factors found to have little or no affect on ADO were local DNA sequence, denaturing temperature (94 or 96 degrees C) and cell type. Consideration of the causal factors identified during this study should permit the design of PGD protocols that experience little ADO, thus improving the accuracy of PGD for single gene disorders.