胚胎活檢時(shí)機(jī)及部位在植入前遺傳學(xué)診斷中的新進(jìn)展

1、胚胎活檢時(shí)機(jī)及部位在植入前遺傳學(xué)診斷中的新進(jìn)展北京大學(xué)深圳醫(yī)院生殖醫(yī)學(xué)中心王佳睿，錢衛(wèi)平【摘要】胚胎活檢是胚胎植入前遺傳學(xué)診斷的重要步驟，胚胎活檢既需要獲取生物樣本以滿足遺傳學(xué)檢測(cè)的需求，又要盡可能的降低對(duì)胚胎的損傷。它包括極體活檢、卵裂球活檢和囊胚期滋養(yǎng)外胚層活檢；近期發(fā)現(xiàn)囊胚腔液及培養(yǎng)基中存在游離DNA可反映胚胎遺傳特性，本文將對(duì)不同胚胎活檢時(shí)機(jī)及部位的應(yīng)用及新進(jìn)展進(jìn)行詳述。關(guān)鍵詞：胚胎植入前遺傳學(xué)診斷、胚胎活檢、無(wú)創(chuàng)性活檢胚胎植入前遺傳學(xué)診斷（PGD）指在體外受精過(guò)程中，對(duì)具有遺傳風(fēng)險(xiǎn)患者的胚胎進(jìn)行種植前活檢和遺傳學(xué)分析，以選擇無(wú)遺傳學(xué)疾病的胚胎植入宮腔，從而獲得正常胎兒的診斷方法。廣泛

2、應(yīng)用于攜帶單基因遺傳病、染色體病、性連鎖遺傳病等疾病的夫婦中。而胚胎活檢是PGD中至關(guān)重要的步驟，它既需要獲取生物樣本以滿足遺傳學(xué)檢測(cè)的需求，又要盡可能的降低對(duì)胚胎的損傷。故選擇一個(gè)合適時(shí)機(jī)及部位進(jìn)行胚胎活檢在PGD技術(shù)中顯得尤為重要的。1 極體活檢當(dāng)卵母細(xì)胞成熟時(shí)，完成第一次減數(shù)分裂，排出第一極體；受精后排出第二極體；通過(guò)對(duì)極體的染色體或基因狀態(tài)進(jìn)行分析，可以間接的推測(cè)卵子的染色體結(jié)構(gòu)、數(shù)目是否異?；蛘呤欠駭y帶有致病基因。由于極體不是胚胎發(fā)育所必需的，不影響卵子的受精功能或胚胎的正常發(fā)育，不會(huì)引起胚胎遺傳物質(zhì)的減少，所以這項(xiàng)技術(shù)廣泛應(yīng)用于立法禁止胚胎活檢的國(guó)家，如德國(guó)、奧地利、瑞士和意大利

3、Montag M, Kster M, Strowitzki T, Toth B. Polar body biopsy. Fertil Steril 2013;100:6037.。分析極體的染色體整倍體性/非整倍體性，可以間接推測(cè)胚胎的整倍體/非整倍體性；若母親為攜帶遺傳病基因的雜合子狀態(tài)，極體中測(cè)出攜帶有致病基因，則表示卵細(xì)胞攜帶的基因異常。另外，極體活檢可以提供的遺傳學(xué)診斷時(shí)間相對(duì)較長(zhǎng)，不會(huì)錯(cuò)過(guò)胚胎移植時(shí)間，有利于胚胎在新鮮周期移植。 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，利用極體活檢只能間接反映來(lái)自于母親的遺傳信息，不能分析父親的遺傳信息。而且從胚胎發(fā)育的角度看，并不是每一枚卵母細(xì)胞都會(huì)發(fā)育至可利用胚胎，如果進(jìn)行極體PGD/PGS，有可能會(huì)增加無(wú)效的工作及診斷花費(fèi)，造成資源浪費(fèi)。因此一定程度上限制了極體活檢的應(yīng)用。在對(duì)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檢采用卵裂期胚胎的1-2個(gè)卵裂球活檢進(jìn)行胚胎遺傳學(xué)分析，可以同時(shí)分析來(lái)自父母雙方的遺傳信息，世界首例PGD嬰兒即采用的卵裂期胚胎活檢 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對(duì)顯微操作十分敏感，活檢時(shí)容易受損傷；桑葚期胚胎細(xì)胞之間連接緊密，操作困難，故卵裂球胚胎活檢即在胚胎發(fā)育至6-8個(gè)細(xì)胞時(shí)取1-2個(gè)卵裂球進(jìn)行活檢。一般認(rèn)為，這個(gè)時(shí)期胚胎的卵裂球是具有全能性的，因此活檢1-2個(gè)卵裂球不會(huì)嚴(yán)重影響胚胎的發(fā)育潛能。但是，卵裂期胚胎嵌合比例較高，40-60%的卵裂期胚胎為嵌合體 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.雖然一般認(rèn)為卵裂球是具有全能性的，但近年來(lái)，越來(lái)越多的證據(jù)表明，卵裂期活檢對(duì)胚胎發(fā)育有負(fù)面影響。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年設(shè)計(jì)的一項(xiàng)隨機(jī)對(duì)照研究，比較卵裂球活檢與囊胚期滋養(yǎng)外胚層活檢后胚胎著床率的分析，所有病例均移植2枚胚胎，1枚活檢胚胎及1枚未活檢胚胎，妊娠后通過(guò)母親血中胚胎DNA指紋驗(yàn)證妊娠胚胎來(lái)源，發(fā)現(xiàn)卵裂球活檢后胚胎著床率顯著低于囊胚期活檢胚胎，卵裂球活檢胚胎著床率較未活檢胚胎下降39%，而囊胚期活檢胚胎與未活檢胚胎著床率無(wú)統(tǒng)計(jì)學(xué)差異。在對(duì)子代安全性的研究中，Desmyttere等對(duì)102名卵裂球活檢的PGD/PGS子代進(jìn)行隨訪，發(fā)現(xiàn)該子代在孕期至產(chǎn)后2年內(nèi)的生長(zhǎng)發(fā)育指標(biāo)與常規(guī)卵胞漿內(nèi)單精子注射（Intracytoplasmic sperm injection, ICSI

20、）的子代無(wú)差異。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.綜上所述，由于卵裂期胚胎嵌合現(xiàn)象發(fā)生率高，且處于對(duì)活檢胚胎發(fā)育影響及子代安全性考慮，目前卵裂球活檢所占比例在逐漸下降。3 囊胚時(shí)期的滋養(yǎng)滋養(yǎng)外胚層活檢體外培養(yǎng)的胚胎通常在第5-6天發(fā)育至囊胚，此時(shí)胚胎的細(xì)胞數(shù)目明顯增多，可以達(dá)到100個(gè)以上，囊胚滋養(yǎng)外胚層將來(lái)會(huì)發(fā)育成胎盤或胎膜，不參與形成胎兒部分，對(duì)滋養(yǎng)外胚層細(xì)胞進(jìn)行活檢，不損傷決定胚胎發(fā)育的內(nèi)細(xì)胞團(tuán)細(xì)胞。因此，囊胚期活檢不僅能夠提供較多的細(xì)胞進(jìn)行遺傳學(xué)分析，增加遺傳檢測(cè)的可靠性，而且也能夠避免對(duì)胎兒部分的損傷。當(dāng)胚胎

24、發(fā)育至囊胚期時(shí)，染色體嵌合比例較卵裂期胚胎明顯降低，能提高遺傳學(xué)診斷的準(zhǔn)確性，降低誤診率。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，發(fā)育至囊胚期的胚胎基因表達(dá)更完整，更有利于選擇好的胚胎移植，減少移植胚胎的數(shù)目，可以有效的減少多胎妊娠。但是，目前的胚胎培養(yǎng)技術(shù)條件下，并不是所有的胚胎都能發(fā)育成囊胚，囊胚期活檢可能會(huì)出現(xiàn)沒(méi)有囊胚形成，無(wú)胚可檢的情況。另外，雖然囊胚期胚胎染色體嵌合體比例顯著低于卵裂期胚胎，但仍存在內(nèi)細(xì)胞群與滋養(yǎng)外層細(xì)胞的核型不一致的嵌合體現(xiàn)象，有可能

26、導(dǎo)致誤診。囊胚期活檢提供給遺傳學(xué)分析的時(shí)間較短，通常需要凍存胚胎。4 其他活檢方法4.1囊胚腔液活檢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，囊胚腔液活檢仍處?kù)秾?shí)驗(yàn)階段，其游離DNA的來(lái)源尚未明確，是否能代表內(nèi)細(xì)胞團(tuán)的DNA仍需更多的實(shí)驗(yàn)驗(yàn)證。另外，在目前的胚胎培養(yǎng)技術(shù)條件下，并不是所有的胚胎都能發(fā)育成囊胚，且由于囊胚腔液體積較少，部分樣本有擴(kuò)增失敗可能。此項(xiàng)技術(shù)目前仍處于實(shí)驗(yàn)階段，有較好的發(fā)展和應(yīng)用前景。4.2培養(yǎng)基活檢培養(yǎng)基活檢是利用培養(yǎng)液中游離DNA來(lái)推測(cè)胚胎的遺傳學(xué)特征，其游離DNA來(lái)源主要是細(xì)胞壞死、細(xì)胞凋亡以及細(xì)胞分泌而來(lái)。培養(yǎng)基活檢的

35、優(yōu)勢(shì)在于完全不損傷胚胎細(xì)胞，達(dá)到真正的無(wú)創(chuàng)性檢測(cè)，同時(shí)樣本獲取方便，收集方法簡(jiǎn)單。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