基因芯片PGDPGS技术的临床应用课件.pptx

1、将已知序列的基因探针集成在 平面基质载体上，形成密集、有序的DNA分子微阵列样本DNA荧光标记与芯片上所含的基因序列 进行杂交通过扫描荧光信号的强度，检测样本基因表达情况。荧光 信号强度与基因拷贝数正相关G BandFISHaCGH、aSNPFishel et al.Fertil Steril 20101 Make amplified DNAGENOMIC DNA(200-400 ng)2 Incubate amplified DNA3 Fragment amplified DNA4 Precipitate&resuspend5 Prepare BeadChip6 Hybridize samp

2、les on BeadChipGenotype and LOH/CN analysis98 Image BeadChipDAY 1DAY 2DAY 3The first three babies born in the world from the SNP technology were from La Jolla IVF.CA.USA遗传病筛查和诊断23 对染色体非整倍体 拷贝数变异(CNVs)aCGH是 是aSNP是 是FISH最多10-12 对 否染色体结构不平衡0.5Mb0.05Mb有限的单基因病否否否线粒体突变否是否单亲二倍体否是否PCR聚合酶链式反应FISH荧光原位杂交aCGH 微

3、阵列比较基因组杂交aSNP微阵列单核苷酸多态性NGS二代测序Joyce C.et al.Hum Genet(2012)131:175186PGDPGS目的诊断出遗传学正常的胚 胎，从而使遗传学正常 的胎儿妊娠/出生染色体正常的胎儿 妊娠/出生适应症单基因病、X连锁遗传、亲代染色体异常高龄、反复妊娠失败、复发性流产、严重男性 不育生育力可生育不育、低生育力常用诊断技术FISH、PCR、arrayFISH、array未确诊结果的处理不移植可移植产前诊断建议与自然妊娠标准相同单基因疾病：常显、常隐、X连锁 染色体异常：结构、数目异常女方年龄35 岁、复发性流产 反复胚胎植入失败、不良孕产史 严重男性

4、不育症单基因疾病：常显、常隐、X连锁 染色体异常：结构、数目异常K.Haapaniemi Kouru et al.Human Reproduction,Vol.27,No.9 pp.28432849,2012瑞典斯德哥尔摩PGD中心1996年2009年256对夫妇569个促排卵周期我国普通人群染色体异常：0.47%不孕不育患者染色体异常：3%10%，显著增高时间检查 总例数数目异常结构异常 异常占比参考值2009.12-2014.028026对186 对313 对6.22%3%10%备注：数目异常包括：Turner、Klinefelter、XYY、XXX、及其嵌合体结构异常包括：易位，倒位，缺

5、失、重复，其中易位占结构异常的44.4%Turner 45,XOKlinefelter 47,XXY平衡易位46,XY t(1,6)(qter;q12)罗氏易位45,XY t(13,14)(q10;q10)染色体结构正常配子携带者异常配子异常类型平衡易位1/181/1816/18罗氏易位1/61/64/6倒位1/41/42/4缺失、重复1/201/2夫妇一方或双方染色体异常复发性流产不明原因复发性流产Helen Flynn et al。J.Obstet.Gynaecol.Res.Vol.40,No.1:109116,January 20141992.01-2011.03Helen Flynn

6、et al。J.Obstet.Gynaecol.Res.Vol.40,No.1:109116,January 2014总流产率55.6%F.Fiorentino et al.Human Reproduction,Vol.26,No.7 pp.19251935,2011F.Fiorentino et al.Human Reproduction,Vol.26,No.7 pp.19251935,2011Nathan R.Treff,et al.Fertility and Sterility Vol.95,No.5,April 2011女方年龄35 岁、复发性流产 反复胚胎植入失败、不良孕产史 严重男

7、性不育症AMA：高龄RM：复发性流产RIF：反复种植失败SMF：严重男性因素Human Reproduction Update,Vol.18,No.3 pp.234-247,2012欧洲人类生殖与胚 胎学协会PGD联盟19972007年57家中心超过27000个周期Franco Causio et al,2002,Chromosome analysis of spontaneous abortions after in vitro fertilization(IVF)and intracytoplasmicsperm injection(ICSI),European Journal ofObs

8、tetrics&Gynecology and Reproductive Biology635 aborted products：normal karyotypes were 44.9%(n=285;euploid 280,translocation 5)abnormal karyotypes were 55.1%(n=350;trisomy 199,double trisomy 22,monosomy 25,polyploidy38,tetraploidy 9,derivative of translocation 34,others 23)Pere Mir et al.J Assist Re

9、prodGenet(2013)30:143149患者指征筛查周期芯片检测卵裂球数 正常胚胎异常胚胎 筛查失败 移植周期 临床妊娠 临床妊娠率 早期流产率复发性流产31 个202 个53个（26.24%）145 个（71.78%）4个（1.98%）24 个16 例66.67%（16/24）6.25%（1/16）截止2014.02.2823-chromosome single nucleotide polymorphism(SNP)microarraypreimplantationgenetic screening(PGS)for recurrent pregnancy loss(RPL)in 6

10、87 in vitro fertilization(IVF)cycles and 5871 embryosMATERIALS AND METHODS:Aretrospective review was conducted of all embryos that underwent PGS by 23chromosome SNP microarrays from January 2010 to April 2012.Patients underwent standard IVF and PGS primarilydue to a history of 2 spontaneous miscarri

11、ages.CONCLUSION:PGS using 23 chromosome SNP microarrays in women with RPL was superior to FISH PGS.Furthermore,performing the biopsy at the blastocyst stage was superior to cleavage stage biopsy.The applicationof dense microarray 23 chromosome PGS at the blastocyst stage may substantially improve pregnancy outcomes in couples suffering from RPL.E.Greco et al.Advances in Reproductive Sciences 1(2013)7-14