目的:使用植入前遗传学检测(PGT)平台是否可以检测到拷贝数非整倍体(即倍性水平和微缺失(MDs))以外的染色体异常?
结论:拟议的综合方法可以准确评估倍性水平和引起基因组疾病的最常见致病性微缺失,扩大PGT的临床应用。
背景:非整倍性植入前遗传测试(PGT-A)中采用的标准方法可鉴定染色体非整倍性,但无法确定倍性水平或引起基因组疾病的复发性致病性MD的存在。携带这些异常的胚胎移植会导致流产,磨牙怀孕,以及后代的智力障碍和发育迟缓。开发一种整合其评估的测试策略可以解决当前的局限性,并增加有关胚胎遗传构成的宝贵信息,未在PGT中进行评估,为进一步了解植入失败和早期妊娠丢失的基因组原因提供了新的临床效用和有价值的知识。据我们所知,迄今为止,从未在植入前人类胚胎中研究过MD。
方法:这是一项回顾性队列分析,包括2018年2月至2021年11月在多个合作试管婴儿诊所从45岁以下的欧洲血统的准父母那里收集的囊胚活检,使用自体配子并对所有卵母细胞进行ICSI。使用164个已知倍性状态的胚胎样本(147个二倍体,9个三倍体,和8个单倍体)。检测九种常见的MD综合征(-4p=Wolf-Hirschhorn,-8q=Langer-Giedion,-1p=1p36删除,-22q=DiGeorge,-5p=Cri-du-Chat,-15q=Prader-Willi/Angelman,-11q=Jacobsen,-17p=Smith-Magenis)开发并使用28个阳性对照和97个阴性对照进行测试。稍后,该方法被盲目地应用于以下分析:(i)100个2个原核(2PN)来源的囊胚,这些囊胚先前被标准PGT-A定义为均匀整倍体;(ii)99个整倍体胚胎,其移植导致妊娠丢失。
方法:该方法基于对整个基因组中选定的多态性的靶向下一代测序,并在包括MD综合征的关键区域内富集。通过概率模型分析测序数据(即等位基因频率),该模型估计了倍性水平和MD存在的可能性,考虑测序噪声和群体遗传学模式(即连锁不平衡,LD,相关性)在1000基因组计划数据库的2504个全基因组测序数据中观察到。对通过单核苷酸多态性(SNP)阵列基因分型获得的分阶段亲本单倍型进行分析,以确认MD的存在。
结果:在分析验证阶段,该策略在倍性分类中都显示出极高的准确性(100%,CI:98.1-100%),并且在八个MD中的六个(99.2%,CI:98.5-99.8%)。为了改进基于杂合性丢失(LOH)的MD检测,根据参考人群中的单倍型频率和LOH发生率分析常见单倍型,从而发展了两个进一步的数学模型。因此,chr1p36和chr4p16.3区域由于可靠性差而被排除在MD鉴定之外,同时开发了纳入亲本DNA信息的临床工作流程以增强MD的识别。在临床应用阶段,在2PN来源的胚泡中检测到1例三倍体(i),在导致流产的移植胚胎活检标本中回顾性发现1例致病性MD(-22q11.21)(ii).对于后一种情况,基于家庭的分析显示,来自非携带者父母的不同兄弟姐妹胚胎(n=2/5)具有相同的MD,表明女性伴侣中存在生殖系镶嵌。当根据胚胎的遗传构成选择移植时,这种策略可以识别出倍性异常和/或非整倍体以外的MD的胚胎,在整倍体胚胎中,估计发生率为1.5%(n=3/202,95%CI:0.5-4.5%)。
结论:需要进行流行病学研究,以准确评估植入前胚胎,特别是整倍体流产中倍性改变和MD的发生率。尽管开发的检测方法具有很高的准确性,使用亲本DNA来支持诊断调用可以进一步提高测定的精确度。
结论:通过整合负责基因组疾病的最常见的致病性MD(从头和遗传的),这种新的检测方法显着扩展了PGT-A的临床应用,通常在后期通过侵入性产前检测进行评估。从基础研究的角度来看,这种方法将有助于阐明与整倍体胚胎植入失败和妊娠丢失的遗传学相关的基本生物学和临床问题.
背景:本研究未使用外部资金。S.C.,M.F.,F.C.,P.Z.,I.P.,L.G.,C.P.,M.P.,D.B.,J.J.-A.,D.B.-J.,J.M.-V.,和C.R.是Igenomix的员工,C.S.是Igenomix科学委员会的负责人。A.C.和L.P.是JUNOGENETICS的雇员。Igenomix和JUNOGENETICS是提供生殖遗传服务的公司。
背景:不适用。
Can chromosomal abnormalities beyond copy-number aneuploidies (i.e. ploidy level and
microdeletions (MDs)) be detected using a preimplantation genetic testing (PGT) platform?
The proposed integrated approach accurately assesses ploidy level and the most common pathogenic
microdeletions causative of genomic disorders, expanding the clinical utility of PGT.
Standard methodologies employed in preimplantation genetic testing for aneuploidy (PGT-A) identify chromosomal aneuploidies but cannot determine ploidy level nor the presence of recurrent pathogenic MDs responsible for genomic disorders. Transferring embryos carrying these abnormalities can result in miscarriage, molar pregnancy, and intellectual disabilities and developmental delay in offspring. The development of a testing strategy that integrates their assessment can resolve current limitations and add valuable information regarding the genetic constitution of embryos, which is not evaluated in PGT providing new level of clinical utility and valuable knowledge for further understanding of the genomic causes of implantation failure and early pregnancy loss. To the best of our knowledge, MDs have never been studied in preimplantation human embryos up to date.
This is a retrospective cohort analysis including blastocyst biopsies collected between February 2018 and November 2021 at multiple collaborating IVF clinics from prospective parents of European ancestry below the age of 45, using autologous gametes and undergoing ICSI for all oocytes. Ploidy level determination was validated using 164 embryonic samples of known ploidy status (147 diploids, 9 triploids, and 8 haploids). Detection of nine common MD syndromes (-4p=Wolf-Hirschhorn, -8q=Langer-Giedion, -1p=1p36 deletion, -22q=DiGeorge, -5p=Cri-du-Chat, -15q=Prader-Willi/Angelman, -11q=Jacobsen, -17p=Smith-Magenis) was developed and tested using 28 positive controls and 97 negative controls. Later, the methodology was blindly applied in the analysis of: (i) 100 two pronuclei (2PN)-derived blastocysts that were previously defined as uniformly euploid by standard PGT-A; (ii) 99 euploid embryos whose transfer resulted in pregnancy loss.
The methodology is based on targeted next-generation sequencing of selected polymorphisms across the genome and enriched within critical regions of included MD syndromes. Sequencing data (i.e. allelic frequencies) were analyzed by a probabilistic model which estimated the likelihood of ploidy level and MD presence, accounting for both sequencing noise and population genetics patterns (i.e. linkage disequilibrium, LD, correlations) observed in 2504 whole-genome sequencing data from the 1000 Genome Project database. Analysis of phased parental haplotypes obtained by single-nucleotide polymorphism (SNP)-array genotyping was performed to confirm the presence of MD.
In the analytical validation phase, this strategy showed extremely high accuracy both in ploidy classification (100%, CI: 98.1-100%) and in the identification of six out of eight MDs (99.2%, CI: 98.5-99.8%). To improve MD detection based on loss of heterozygosity (LOH), common haploblocks were analyzed based on haplotype frequency and LOH occurrence in a reference population, thus developing two further mathematical models. As a result, chr1p36 and chr4p16.3 regions were excluded from MD identification due to their poor reliability, whilst a clinical workflow which incorporated parental DNA information was developed to enhance the identification of MDs. During the clinical application phase, one case of triploidy was detected among 2PN-derived blastocysts (i) and one pathogenic MD (-22q11.21) was retrospectively identified among the biopsy specimens of transferred embryos that resulted in miscarriage (ii). For the latter case, family-based analysis revealed the same MD in different sibling embryos (n = 2/5) from non-carrier parents, suggesting the presence of germline mosaicism in the female partner. When embryos are selected for transfer based on their genetic constitution, this strategy can identify embryos with ploidy abnormalities and/or MDs beyond aneuploidies, with an estimated incidence of 1.5% (n = 3/202, 95% CI: 0.5-4.5%) among euploid embryos.
Epidemiological studies will be required to accurately assess the incidence of ploidy alterations and MDs in preimplantation embryos and particularly in euploid miscarriages. Despite the high accuracy of the assay developed, the use of parental DNA to support diagnostic calling can further increase the precision of the assay.
This novel assay significantly expands the clinical utility of PGT-A by integrating the most common pathogenic MDs (both de novo and inherited ones) responsible for genomic disorders, which are usually evaluated at a later stage through invasive prenatal testing. From a basic research standpoint, this approach will help to elucidate fundamental biological and clinical questions related to the genetics of implantation failure and pregnancy loss of otherwise euploid embryos.
No external funding was used for this study. S.C., M.F., F.C., P.Z., I.P., L.G., C.P., M.P., D.B., J.J.-A., D.B.-J., J.M.-V., and C.R. are employees of Igenomix and C.S. is the head of the scientific board of Igenomix. A.C. and L.P. are employees of JUNO GENETICS. Igenomix and JUNO GENETICS are companies providing reproductive genetic services.
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