胚胎活检时机及部位在植入前遗传学诊断中的新进展

1、胚胎活检时机及部位在植入前遗传学诊断中的新进展北京大学深圳医院生殖医学中心王佳睿，钱卫平【摘要】胚胎活检是胚胎植入前遗传学诊断的重要步骤，胚胎活检既需要获取生物样本以满足遗传学检测的需求，又要尽可能的降低对胚胎的损伤。它包括极体活检、卵裂球活检和囊胚期滋养外胚层活检；近期发现囊胚腔液及培养基中存在游离DNA可反映胚胎遗传特性，本文将对不同胚胎活检时机及部位的应用及新进展进行详述。关键词：胚胎植入前遗传学诊断、胚胎活检、无创性活检胚胎植入前遗传学诊断（PGD）指在体外受精过程中，对具有遗传风险患者的胚胎进行种植前活检和遗传学分析，以选择无遗传学疾病的胚胎植入宫腔，从而获得正常胎儿的诊断方法。广泛

2、应用于携带单基因遗传病、染色体病、性连锁遗传病等疾病的夫妇中。而胚胎活检是PGD中至关重要的步骤，它既需要获取生物样本以满足遗传学检测的需求，又要尽可能的降低对胚胎的损伤。故选择一个合适时机及部位进行胚胎活检在PGD技术中显得尤为重要的。1 极体活检当卵母细胞成熟时，完成第一次减数分裂，排出第一极体；受精后排出第二极体；通过对极体的染色体或基因状态进行分析，可以间接的推测卵子的染色体结构、数目是否异常或者是否携带有致病基因。由于极体不是胚胎发育所必需的，不影响卵子的受精功能或胚胎的正常发育，不会引起胚胎遗传物质的减少，所以这项技术广泛应用于立法禁止胚胎活检的国家，如德国、奥地利、瑞士和意大利

3、Montag M, Kster M, Strowitzki T, Toth B. Polar body biopsy. Fertil Steril 2013;100:6037.。分析极体的染色体整倍体性/非整倍体性，可以间接推测胚胎的整倍体/非整倍体性；若母亲为携带遗传病基因的杂合子状态，极体中测出携带有致病基因，则表示卵细胞携带的基因异常。另外，极体活检可以提供的遗传学诊断时间相对较长，不会错过胚胎移植时间，有利于胚胎在新鲜周期移植。 HYPERLINK /pubmed/?term=Feichtinger MAuthor&cauthor=true&cauthor_uid=26024488 F

4、eichtinger HYPERLINK /pubmed/?term=Feichtinger MAuthor&cauthor=true&cauthor_uid=26024488 Feichtinger M, HYPERLINK /pubmed/?term=Stopp TAuthor&cauthor=true&cauthor_uid=26024488 Stopp T, et al. Increasing live birth rate by preimplantation genetic screening of pooledpolar bodiesusingarraycomparative g

5、enomic hybridization. HYPERLINK /pubmed/26024488 o PloS one. PLoS One.2015 May 29;10(5):e0128317 Montag M, Kster M, Strowitzki T, Toth B. Polar body biopsy. Fertil Steril 2013;100:6037. HYPERLINK /pubmed/?term=Feichtinger MAuthor&cauthor=true&cauthor_uid=26024488 Feichtinger M, HYPERLINK /pubmed/?te

6、rm=Stopp TAuthor&cauthor=true&cauthor_uid=26024488 Stopp T, et al. Increasing live birth rate by preimplantation genetic screening of pooledpolar bodiesusingarraycomparative genomic hybridization. HYPERLINK /pubmed/26024488 o PloS one. PLoS One.2015 May 29;10(5):e0128317. HYPERLINK /pubmed/?term=Chr

7、istopikou DAuthor&cauthor=true&cauthor_uid=23477909 Christopikou D, HYPERLINK /pubmed/?term=Tsorva EAuthor&cauthor=true&cauthor_uid=23477909 Tsorva E,et al. Polar bodyanalysis byarraycomparative genomic hybridization accurately predicts aneuploidies of maternal meiotic origin in cleavage stage embry

8、os of women of advanced maternal age. HYPERLINK /pubmed/23477909 o Human reproduction (Oxford, England). Hum Reprod.2013 May;28(5):1426-34.但是，利用极体活检只能间接反映来自于母亲的遗传信息，不能分析父亲的遗传信息。而且从胚胎发育的角度看，并不是每一枚卵母细胞都会发育至可利用胚胎，如果进行极体PGD/PGS，有可能会增加无效的工作及诊断花费，造成资源浪费。因此一定程度上限制了极体活检的应用。在对子代安全性的研究中， HYPERLINK /pubmed/?te

9、rm=Strom CMAuthor&cauthor=true&cauthor_uid=11015504 Strom HYPERLINK /pubmed/?term=Strom CMAuthor&cauthor=true&cauthor_uid=11015504 Strom CM, HYPERLINK /pubmed/?term=Strom CMAuthor&cauthor=true&cauthor_uid=11015504 Strom CM, HYPERLINK /pubmed/?term=Levin RAuthor&cauthor=true&cauthor_uid=11015504 Levi

10、n R,et al. Neonataloutcomeofpreimplantation genetic diagnosisbypolar bodyremoval: the first 109 infants. HYPERLINK /pubmed/11015504 o Pediatrics. Pediatrics.2000 Oct;106(4):650-3.2 卵裂期胚胎的卵裂球活检采用卵裂期胚胎的1-2个卵裂球活检进行胚胎遗传学分析，可以同时分析来自父母双方的遗传信息，世界首例PGD婴儿即采用的卵裂期胚胎活检 HYPERLINK /pubmed/?term=Handyside AHAuthor

11、&cauthor=true&cauthor_uid=2330030 Handyside AH, HYPERLINK /pubmed/?term=Kontogianni EHAuthor&cauthor=true&cauthor_uid=2330030 HYPERLINK /pubmed/?term=Handyside AHAuthor&cauthor=true&cauthor_uid=2330030 Handyside AH, HYPERLINK /pubmed/?term=Kontogianni EHAuthor&cauthor=true&cauthor_uid=2330030 Kontog

12、ianni EH, et al. Pregnanciesfrombiopsiedhumanpreimplantation embryossexedby Y-specific DNA amplification. HYPERLINK /pubmed/2330030 o Nature. Nature.1990 Apr 19;344(6268):768-70. HYPERLINK /pubmed/?term=De Rycke MAuthor&cauthor=true&cauthor_uid=26071418 De Rycke M, HYPERLINK /pubmed/?term=Belva FAut

13、hor&cauthor=true&cauthor_uid=26071418 Belva F, et al. ESHRE PGD Consortium data collection XIII: cycles from January to December 2010 with pregnancy follow-up to October 2011. HYPERLINK /pubmed/?term=De_Rycke_M+Author+Hum+Reprod.+2015+Aug;30(8):1763 o Human reproduction (Oxford, England). Hum Reprod

14、.2015 Aug;30(8):1763-89.由于卵裂早期的胚胎对显微操作十分敏感，活检时容易受损伤；桑葚期胚胎细胞之间连接紧密，操作困难，故卵裂球胚胎活检即在胚胎发育至6-8个细胞时取1-2个卵裂球进行活检。一般认为，这个时期胚胎的卵裂球是具有全能性的，因此活检1-2个卵裂球不会严重影响胚胎的发育潜能。但是，卵裂期胚胎嵌合比例较高，40-60%的卵裂期胚胎为嵌合体 HYPERLINK /pubmed/?term=European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium/Embryology S

15、pecial Interest GroupCorporate Author HYPERLINK /pubmed/?term=European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium/Embryology Special Interest GroupCorporate Author European Society for Human Reproduction andEmbryology(ESHRE)PGD Consortium / EmbryologySpecialInterestGroup. E

16、SHREPGDConsortium/EmbryologySpecialInterestGroup - bestpracticeguidelinesforpolarbody andembryobiopsy for preimplantation genetic diagnosis / screening(PGD/PGS). HYPERLINK /pubmed/?term=ESHRE+PGD+Consortium/Embryology+Special+Interest+Group+best+practice+guidelines+for+polar+body+and+embryo+biopsy+f

17、or+preimplantation+genetic+diagnosis/screening+(PGD/PGS) o Human reproduction (Oxford, England). Hum Reprod.2011 Jan; 26(1): 41-6.虽然一般认为卵裂球是具有全能性的，但近年来，越来越多的证据表明，卵裂期活检对胚胎发育有负面影响。Scott HYPERLINK /pubmed/?term=Scott RT JrAuthor&cauthor=true&cauthor_uid=23773313 Scott RT Jr, HYPERLINK /pubmed/?term=Uph

18、am KMAuthor&cauthor=true&cauthor_uid=23773313 Upham KM, et al. Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy does not: a randomized and paired clinical trial. HYPERLINK /pubmed/23773313 o Fertility and sterility. Fertil Steril.2013 Sep;100

19、(3):624-30.等2013年设计的一项随机对照研究，比较卵裂球活检与囊胚期滋养外胚层活检后胚胎着床率的分析，所有病例均移植2枚胚胎，1枚活检胚胎及1枚未活检胚胎，妊娠后通过母亲血中胚胎DNA指纹验证妊娠胚胎来源，发现卵裂球活检后胚胎着床率显著低于囊胚期活检胚胎，卵裂球活检胚胎着床率较未活检胚胎下降39%，而囊胚期活检胚胎与未活检胚胎着床率无统计学差异。在对子代安全性的研究中，Desmyttere等对102名卵裂球活检的PGD/PGS子代进行随访，发现该子代在孕期至产后2年内的生长发育指标与常规卵胞浆内单精子注射（Intracytoplasmic sperm injection, ICSI

20、）的子代无差异。YU Y HYPERLINK /pubmed/?term=Scott RT JrAuthor&cauthor=true&cauthor_uid=23773313 Scott RT Jr, HYPERLINK /pubmed/?term=Upham KMAuthor&cauthor=true&cauthor_uid=23773313 Upham KM, et al. Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy d

21、oes not: a randomized and paired clinical trial. HYPERLINK /pubmed/23773313 o Fertility and sterility. Fertil Steril.2013 Sep;100(3):624-30. Yu Y, et al. Evaluation of blastomere biopsy using a mouse model indicates the potential high risk of neurodegenerative disorders in the offspring. Mol Cell Pr

22、oteomics. 2009 Jul;8(7):1490-500. Yu Y, et al. Assessmentof therisk ofblastomerebiopsy during preimplantation genetic diagnosis in a mouse model: reducing female ovary function with an increase in age by proteomics method. HYPERLINK /pubmed/?term=Yu+Y,+assessment+of+the+risk+of+blastomere o Journal

23、of proteome research. J Proteome Res.2013 Dec 6;12(12):5475-86.综上所述，由于卵裂期胚胎嵌合现象发生率高，且处于对活检胚胎发育影响及子代安全性考虑，目前卵裂球活检所占比例在逐渐下降。3 囊胚时期的滋养滋养外胚层活检体外培养的胚胎通常在第5-6天发育至囊胚，此时胚胎的细胞数目明显增多，可以达到100个以上，囊胚滋养外胚层将来会发育成胎盘或胎膜，不参与形成胎儿部分，对滋养外胚层细胞进行活检，不损伤决定胚胎发育的内细胞团细胞。因此，囊胚期活检不仅能够提供较多的细胞进行遗传学分析，增加遗传检测的可靠性，而且也能够避免对胎儿部分的损伤。当胚胎

24、发育至囊胚期时，染色体嵌合比例较卵裂期胚胎明显降低，能提高遗传学诊断的准确性，降低误诊率。Scott HYPERLINK /pubmed/?term=Scott RT JrAuthor&cauthor=true&cauthor_uid=23731996 Scott RTJr, et al. Blastocystbiopsywith comprehensive chromosomescreeningand fresh embryo transfer significantly increases in vitro fertilization implantation and delivery r

25、ates: a randomized controlled trial. HYPERLINK /pubmed/23731996 o Fertility and sterility. Fertil Steril.2013 Sep;100(3):697-703.另外，发育至囊胚期的胚胎基因表达更完整，更有利于选择好的胚胎移植，减少移植胚胎的数目，可以有效的减少多胎妊娠。但是，目前的胚胎培养技术条件下，并不是所有的胚胎都能发育成囊胚，囊胚期活检可能会出现没有囊胚形成，无胚可检的情况。另外，虽然囊胚期胚胎染色体嵌合体比例显著低于卵裂期胚胎，但仍存在内细胞群与滋养外层细胞的核型不一致的嵌合体现象，有可能

26、导致误诊。囊胚期活检提供给遗传学分析的时间较短，通常需要冻存胚胎。4 其他活检方法4.1囊胚腔液活检Chen HYPERLINK /pubmed/?term=Chen SUAuthor&cauthor=true&cauthor_uid=16210007 Chen SU, HYPERLINK /pubmed/?term=Lee THAuthor&cauthor=true&cauthor_uid=16210007 Lee TH, et al. Microsuction of blastocoelic fluid before vitrification increased survival HYP

27、ERLINK /pubmed/?term=Chen SUAuthor&cauthor=true&cauthor_uid=16210007 Chen SU, HYPERLINK /pubmed/?term=Lee THAuthor&cauthor=true&cauthor_uid=16210007 Lee TH, et al. Microsuction of blastocoelic fluid before vitrification increased survival andpregnancy ofmouseexpandedblastocysts, butpretreatmentwith

28、thecytoskeletalstabilizerdid notincreaseblastocystsurvival. HYPERLINK /pubmed/?term=Microsuction+of+blastocoelic+fluid+before+vitrification+increased+survival+and+pregnancy+of+mouse+expanded+blastocysts,+but+pretreatment+with+the+cytoskeletal+stabilizer+did+not+increase+blastocyst+survival o Fertili

29、ty and sterility. Fertil Steril.2005 Oct;84 Suppl 2:1156-62. HYPERLINK /pubmed/?term=Palini SAuthor&cauthor=true&cauthor_uid=23557766 Palini S et al. GenomicDNAinhumanblastocoelefluid. HYPERLINK /pubmed/?term=Genomic+DNA+in+human+blastocoele+fluid o Reproductive biomedicine online. Reprod Biomed Onl

30、ine.2013 Jun;26(6):603-10. HYPERLINK /pubmed/?term=Zhang YAuthor&cauthor=true&cauthor_uid=26899834 Zhang Y, HYPERLINK /pubmed/?term=Li NAuthor&cauthor=true&cauthor_uid=26899834 Li N, et al. Molecular analysis of DNA in blastocoele fluid using next-generationsequencing. HYPERLINK /pubmed/?term=Molecu

31、lar+analysis+of+DNA+in+blastocoele+fluid+using+next-generation+sequencing o Journal of assisted reproduction and genetics. J Assist Reprod Genet.2016 May;33(5):637-45. HYPERLINK /pubmed/?term=Magli MCAuthor&cauthor=true&cauthor_uid=26658131 Magli MC, HYPERLINK /pubmed/?term=Pomante AAuthor&cauthor=t

32、rue&cauthor_uid=26658131 Pomante A, et al. Preimplantationgenetic testing:polar bodies, blastomeres, trophectoderm cells, or blastocoelic fluid? HYPERLINK /pubmed/26658131 o Fertility and sterility. Fertil Steril.2016 Mar;105(3):676-83. HYPERLINK /pubmed/?term=Tobler KJAuthor&cauthor=true&cauthor_ui

33、d=26006737 Tobler KJ, HYPERLINK /pubmed/?term=Zhao YAuthor&cauthor=true&cauthor_uid=26006737 Zhao Y, et al. Blastocoelfluidfromdifferentiatedblastocysts harbors embryonic genomic material capable of a whole-genome deoxyribonucleic acid amplification and comprehensive chromosome microarray analysis.

34、HYPERLINK /pubmed/26006737 o Fertility and sterility. Fertil Steril.2015 Aug;104(2):418-25.目前，囊胚腔液活检仍处於实验阶段，其游离DNA的来源尚未明确，是否能代表内细胞团的DNA仍需更多的实验验证。另外，在目前的胚胎培养技术条件下，并不是所有的胚胎都能发育成囊胚，且由于囊胚腔液体积较少，部分样本有扩增失败可能。此项技术目前仍处于实验阶段，有较好的发展和应用前景。4.2培养基活检培养基活检是利用培养液中游离DNA来推测胚胎的遗传学特征，其游离DNA来源主要是细胞坏死、细胞凋亡以及细胞分泌而来。培养基活检的

35、优势在于完全不损伤胚胎细胞，达到真正的无创性检测，同时样本获取方便，收集方法简单。Assou HYPERLINK /pubmed/?term=Assou SAuthor&cauthor=true&cauthor_uid=25182520 Assou S, HYPERLINK /pubmed/?term=A%C3%AFt-Ahmed OAuthor&cauthor=true&cauthor_uid=25182520 At-Ahmed O, et al. Non-invasive pre-implantation HYPERLINK /pubmed/?term=Assou SAuthor&caut

36、hor=true&cauthor_uid=25182520 Assou S, HYPERLINK /pubmed/?term=A%C3%AFt-Ahmed OAuthor&cauthor=true&cauthor_uid=25182520 At-Ahmed O, et al. Non-invasive pre-implantation genetic diagnosis ofX-linked disorders. HYPERLINK /pubmed/?term=Non-invasive+pre-implantation+genetic+diagnosis+of+X-linked+disorders o Medical hypotheses. Med Hypotheses.2014 Oct;83(4):506-8. HYPERLINK /pubmed/?term=Xu JAuthor&cauthor=true&cauthor_uid=27688762 Xu J, HYPERLINK /pubmed/?term=Fang RAuthor&cauthor=true&cauthor_uid=276