氟化物转化细胞的超微结构观察

研究氟化物的致突变性和潜在致癌性,探讨其诱变机理。方法应用NaF诱导转化金黄地鼠胚胎细胞(SHE),以扫描和透射电镜观察其超微结构。结果该细胞群中少数细胞凋亡,大部分细胞则已逃脱凋亡转化成恶性细胞。结论氟化物对哺乳动物细胞可能具有诱变致癌性。     

Studies on Optimal Conditions of Two Freezing Methods for Preservation of Rabbit Embryos

Since the first report on the successful deep cryopreservation of mam-malian embryos in 1972,slow progressing cooling rate has been employ-ed in conventional embryos-freezing techniques;while more recent studieson freezing preimplantation embryos have focussed on the simplificationof cooling and thawing procedures and improvement of viability of embr-yos.Dimethylsulfoxide (DMSO)has been used as an effective cryoprotec-tant for freezing human and other mammalian embryos.It has been foundthat glycerol and other alcohols are effective to protect embryos fromcryoinjury during     

PGD周期中机械法和激光法活检对胚胎发育及妊娠结局的影响

目的:比较机械法和激光法进行卵裂球和极体活检对胚胎发育及着床前遗传学诊断(preimplantation genetic diagnosis,PGD)周期妊娠结局的影响。方法:本研究包括20对夫妇的21个PGD周期,其中18个周期分别用机械法或激光法于受精后第3天进行卵裂球活检,并用荧光原位杂交(fluorescence in situ hybridization,FISH)分析检出的卵裂球,于受精后第5或第6天移植信号正常的胚胎;2个周期分别用机械法和激光法在取卵后行极体活检,后行胞浆内单精子注射(introcytoplasmic sperm injection,ICSI)。同时将活检取出的极体进行FISH分析,于取卵后第3天移植经FISH检查正常的卵发育而来的胚胎。另外一个周期先用激光法实行了极体活检,由于FISH检查均无信号,后又用激光法对胚胎行卵裂球活检。结果:共活检胚胎145枚,其中109枚用机械法,36枚用激光法。活检后胚胎的继续发育率分别为72.48%和83.33%,囊胚形成率为33.94%和44.44%,临床妊娠率为38.46%和16.67%,着床率为21.43%和8.33%,两种方法无显著差异。对27枚卵行极体活检,其中12枚用机械法,15枚用激光法。活检后2PN受精率分别为58.33%和46.66%,继续发育率为66.67%和60.00%,亦无显著差异。对活检出的极体进行FISH分析,用机械法活检的极体信号阳性率为90.00%,显著高于激光法的28.57%。结论:用机械法和激光法行极体或卵裂球活检对胚胎发育的影响差异无统计学意义。但使用机械法活检卵裂球能获得较高的临床妊娠率和着床率。极体活检时能获得较高的受精率和继续发育率,故推荐使用机械法进行活检。     

4-细胞胚胎卵裂球全能性的体外实验研究

目的 对体外培养的4－细胞人类胚胎卵裂球发育及分化情况进行观察和研究。方法 采用控制性促超排卵获取人类卵子,体外受精获得早期胚胎,待发育到4－细胞阶段时将透明带去除,分散成为单个卵裂球进行培养,观察卵裂球发育及分化的情况。结果 20个4－细胞、2个3－细胞人类早期胚胎分散后获得单个卵裂球83个,经体外培养后有35个发育成为扩张囊胚。结论 4－细胞人类胚胎卵裂球具有发育成为完整囊胚的能力。     

不同扩增方法和探针长度对植入前单个卵裂球比较基因组杂交检测结果的影响

目的 探讨不同扩增方法和探针长度对植入前单个卵裂球比较基因组杂交(comparative genomic hybridization,CGH)检测结果的影响,为植入前产前诊断奠定基础.方法 随机将20枚植入前6～8细胞期胚胎卵裂球分为A和B组(各10枚),取1名正常男性外周血20枚淋巴细胞,分为C和D组(各10枚)作为对照.A和C组经简并寡聚核苷酸聚合酶链反应(degenerate oligonucleotide primed polymerase chain reaction,DOP-PCR)、B和D组经多重置换扩增(multiple displacement amplification,MDA)分别进行全基因组扩增(whole genome amplification,WGA),用上述全基因组扩增产物进行PCR扩增TBX1基因2号外显子,验证其特异性.将获得的WGA产物制备成250～750 bp和750～2000 bp 2种不同长度的探针与正常男性中期染色体核型进行杂交,Y染色体性别决定区(sex-determining region of Y,SRY)基因验证CGH检测结果.结果 (1)用DOP-PCR扩增时,A组10枚卵裂球中有9枚WGA成功,C组10枚淋巴细胞WGA均成功;但其扩增产物进一步扩增TBX1基因2号外显子时,出现较多非特异性条带.CGH检测中.5例卵裂球用长度250～750 bp的探针检测时,1例失败,1例CGH软件核型分析结果与SRY结果不一致.(2)经MDA的B和D组,均WGA成功;其产物进一步扩增TBX1基因2号外显子时,非特异性条带少.CGH检测中,探针长度为250～750 bp的5例卵裂球均检测成功,且与SRY验证结果一致.(3)用长度为750～2000 bp探针检测时,杂交效果不好.结论 单卵裂球全基因组扩增,MDA较DOP-PCR方法稳定,特异性强,CGH检测中,扩增产物酶切至250～750 bp制备的探针杂交图像均匀、清晰,软件分析核型结果稳定、准确.

Abstract：

Objective To investigate the effect of different amplification methods and probes with various length on the results of comparative genomic hybridization (CGH) analysis of pre-implanted single blastomere and to establish the basis for preimplantation genetic diagnosis.Methods Twenty blastomeres of embryo at 6-8 cells stage were randomly divided into A and B group with 10 in each.Twenty peripheral blood lymphocytes from a healthy man were similarly divided into C and D group with 10 in each.Degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) was used to amplify whole genomic DNA in group A and C,and multiple displacement amplification (MDA) was used in group B and D for whole genome amplification (WGA).The specificity of resultant products was confirmed by amplification of TBX1 gene exon 2.CGH was performed respectively with 250-750 bp and 750-2000 bp probes prepared from the amplified whole genomic DNA.The result of CGH was verified by sex-determining region of Y (SRY).Results (1) Nine of the 10 samples in group A and all in group C were amplifiable by DOP-PCR,but there were multiple non-specific bands in the amplification of TBX1 exon 2 when WGA products were used as templates.When 250～750 bp probe was used in CGH,1 of the 5 blastomeres was failed and another one had different karyotype from that analyzed by SRY.(2) All samples in group B and D were successfully amplified by MDA,and the non-specific bands were significantly less in the amplification of TBX1 exon 2.All 5 blastomeres were successful in CGH with the 250～750 bp probe.Moreover,the karyotype was in agreement with that of SRY.(3) When 750 ～ 2000 bp probe was used,the CGH results were suboptimal.Conclusions In WGA of single blastomere,MDA is superior to DOP-PCR in the stability and specificity.The karyotype image detected by CGH with the 250～750 bp probe is clear and homogenous.     

部分卵裂球损伤对冻融后胚胎发育潜能的影响

目的了解部分卵裂球损伤对冻融后优质胚胎发育能力的影响，为解冻周期移植胚胎的挑选提供依据。方法对502例解冻移植周期进行回顾性分析。所有移植胚胎分为两类，胚胎冻融后所有卵裂球均无损伤，卵裂球数〉6，碎片〈10％（A型）；冻融后胚胎有1～2个卵裂球损伤，但仍有至少6个完整无损伤卵裂球，碎片〈10％（B型）。无损伤组移植的所有胚胎均为A型（n=294周期），部分损伤组移植胚胎中至少包含1个B型胚胎（n=208周期）。比较2组临床妊娠率和着床率。结果无损伤组临床妊娠率为36．73％（108／294），着床率19．42％（153／788），部分损伤组为38．46％（80／208）和19．43％（110／566），2组临床妊娠率和着床率比较，差异无统计学意义（P〉0．05）。而且含1枚B型胚胎组（163周期）和2枚B型胚胎组（42周期）的临床妊娠率和着床率比较，差异无统计学意义（P〉0．05）。结论少量卵裂球损伤的冻融后优质胚胎，其发育潜能与无损伤优质胚胎相同。     

Clinical analysis of the patients with single fair cleavage-stage embryo on day 3

Objective: This study aimed to explore an appropriate selection for the patients with single fair cleavage-stage embryo on day 3.

Methods: This study included 469 fresh transfers and 220 frozen-thawed transfers from January 2014 to June 2016. Furthermore, in 72 patients who have only 4–6 fair embryos (4–5 blastomeres) on day 3, the blastocysts were cultured to day 5 for transfer.

Results: In the fresh transfers, the clinical pregnancy rate of 4–5 blastomeres group was significantly lower than 6–7 and 8–10 blastomeres group (5.88 vs. 30.13%, p<.001and 5.88 vs. 26.09%, p?p?=?.040 and 10.00 vs. 33.33%, p?=?.005). For the blastocyst transfers derived from fair embryos with 4–5 blastomeres, the clinical pregnancy rate was significantly higher than single and double fair embryo transfers of similar quality (44.44 vs. 7.04%, p?p?=?.013).

Conclusions: For the patients with single fair embryo (6–7 blastomeres or 8–10 blastomeres), transfer at the cleavage stage is feasible. For the patients with single fair embryo (4–5 blastomeres), transfer of single fair embryo at the blastocyst stage or accumulating two fair embryos might be worthy of consideration.     

植入前遗传学诊断四例临床分析

目的 探讨对遗传病高危夫妇采用单细胞荧光原位杂交（FISH）进行胚胎植入前遗传学诊断（PGD）的临床价值。方法 对曾生育过遗传病患儿的4对夫妇通过超排卵获得卵子,体外受精,体外培养至6-8细胞胚胎,每个胚胎取1-2个细胞,采用FISH进行遗传学分析。筛选无遗传病发病风险的胚胎移植入子宫。结果 4例患者共进行4个治疗周期,获得可供活检的胚胎12个,活检细胞20个,固定后有核细胞17个,FISH后除2个细胞无杂交信号外,其余杂交信号清楚,结果明确,活检后的12个胚胎继续发育,结合遗传学诊断,8个胚胎可供移植,其中1例妊娠,于2001年9月14日足月剖宫产分娩一女婴,发育正常,体重4270g,出生后染色体检查为正常女性核型。结论 对遗传病高危夫妇采用FISH技术进行PGD具有临床应用价值。     

X、Y、18三色探针同时检测植入前人胚的实验研究

目的:探讨X、Y、18 三色探针同时检测植入前人胚,以鉴定其性别及倍体的可行性 .方法:对体外受精(in vitro fertilizatio n, IVF)所获18个剩余胚104个卵裂球,采用X、Y、18三色着丝粒探针进行FISH分析. 结果:FISH分析成功率97.5%(77/79), 9例(9/11,81.9%)单精受精胚胎为二倍体, 性染色体嵌合未影响胚胎性别的诊断, 7例双精受精胚胎呈多种倍体异常. 结论: 采用X 、Y、18三色探针FISH法同时检测植入前人胚准确、有效,每胚分析2 个卵裂球具有代表性.