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Multicolour detection of every chromosome as a means of detecting mosaicism and nuclear organisation in human embryonic nuclei

机译：对每条染色体进行多色检测，以检测人类胚胎细胞核中的镶嵌和核组织

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Fluorescence in situ Hybridisation (FISH) revolutionised cytogenetics using fluorescently labelled probes with high affinity with target (nuclear) DNA. By the early 1990s FISH was adopted as a means of PGD sexing for couples at risk of transmitting X-linked disorders and later for detection of unbalanced translocations. Following a rise in popularity of PGD by FISH for sexing and the availability of multicolour probes (5-8 colour), the use of FISH was expanded to the detection of aneuploidy and selective implantation of embryos more likely to be euploid, the rationale being to increase pregnancy rates (referral categories were typically advanced maternal age, repeated IVF failure, repeated miscarriage or severe male factor infertility). Despite initial reports of an increase in implantation rates, reduction in trisomic offspring and spontaneous abortions criticism centred around experimental design (including lack of randomisation), inadequate control groups and lack of report on live births. Eleven randomised control trials (RCTs) (2004-2010) showed that PGS with FISH did not increase delivery rates with some demonstrating adverse outcomes. These RCTs, parallel improvements in culturing and cryopreservation and a shift to blastocyst biopsy essentially outdated FISH as a tool for PGS and it has now been replaced by newer technologies (array CGH, SNP arrays, qRT-PCR and NGS). Cell-by-cell follow up analysis of individual blastomeres in non-transferred embryos is however usually prohibitively expensive by these new approaches and thus FISH remains an invaluable resource for the study of mosaicosm and nuclear organization. We thus developed the approach described herein for the FISH detection of chromosome copy number of all 24 human chromosomes. This approach involves 4 sequential layers of hybridization, each with 6 spectrally distinct fluorochromes and a bespoke capturing system. Here we report previously published studies and hitherto unreported data indicating that 24 chromosome FISH is a useful tool for studying chromosome mosaicism, one of the most hotly debated topics currently in preimplantation genetics. Our results suggest that mosaic embryo aneuploidy is not highly significantly correlated to maternal age, probably due, in part, to the large preponderance of post-zygotic (mitotic) errors. Chromosome loss (anaphase lag) appears to be the most common mechanism, followed by chromosome gain (endoreduplication), however 3:1 mitotic non- disjunction of chromosomes appears to be rare. Nuclear organisation (i.e. the spatial and temporal topology of chromosomes or sub-chromosomal compartments) studies indicate that human morula or blastocyst embryos (day 4-5) appear to adopt a "chromocentric" pattern (i.e. almost all centromeric signals reside in the innermost regions of the nuclear volume). By the blastocyst stage however, a more ordered organisation with spatial and temporal cues important for embryo development appears. We have however found no association between aneuploidy and nuclear organization using this approach despite our earlier studies. In conclusion, while FISH is mostly "dead and buried" for mainstream PGS, it still has a place for basic biology studies; the development of a 24 chromosome protocol extends the power of this analysis.
机译：荧光原位杂交（FISH）使用与目标（核）DNA具有高亲和力的荧光标记探针彻底改变了细胞遗传学。在1990年代初期，FISH被用作对有传播X连锁疾病风险的夫妇进行PGD性别鉴定的一种手段，后来又用于发现不平衡易位。随着FISH在性别鉴定中使用PGD的普及和多色探针（5-8色）的普及，FISH的用途已扩展到非整倍性的检测和选择性植入更有可能是整倍体的胚胎，其原理是提高怀孕率（推荐的类别通常是高龄产妇，反复IVF失败，反复流产或严重的男性不育）。尽管最初有报道植入率增加，但三体性后代减少和自然流产的批评主要集中在实验设计（包括缺乏随机性），对照组不足和活产报告方面。十一项随机对照试验（RCT）（2004-2010年）显示，含FISH的PGS不能提高分娩率，但有一些不良反应。这些RCT，在培养和冷冻保存方面的并行改进以及向囊胚活检的转变，基本上已经超过了FISH作为PGS的工具，现在已被更新的技术（CGH阵列，SNP阵列，qRT-PCR和NGS阵列）取代。然而，通过这些新方法对未转移的胚胎中的单个卵裂球进行逐个细胞的追踪分析通常是非常昂贵的，因此FISH仍然是研究镶嵌和核组织的宝贵资源。因此，我们开发了本文所述的用于FISH检测所有24条人类染色体的染色体拷贝数的方法。此方法涉及4个连续的杂交层，每个杂交层具有6种光谱不同的荧光染料和定制的捕获系统。在这里，我们报道以前发表的研究和迄今未报道的数据，表明24染色体FISH是研究染色体镶嵌术的有用工具，染色体镶嵌术是当前植入前遗传学中最热门的话题之一。我们的结果表明，镶嵌胚胎的非整倍性与产妇年龄没有高度相关性，这可能部分是由于合子后（有丝分裂）错误的大量发生。染色体丢失（后期滞后）似乎是最常见的机制，其次是染色体获得（内复制），但是，染色体的3：1有丝分裂不分离似乎很少见。核组织（即染色体或亚染色体区室的时空拓扑）研究表明，人类桑ula或胚泡胚胎（第4-5天）似乎采用“以色心为中心”的模式（即几乎所有着丝粒信号都位于最内层区域）核体积）。然而，到了胚泡期，出现了一个更加有序的组织，具有对胚胎发育很重要的时空线索。然而，尽管我们进行了较早的研究，但我们发现使用这种方法在非整倍性与核组织之间没有关联。总而言之，尽管FISH对于主流PGS大多是“死而复生”的，但它仍然有基础生物学研究的空间。 24染色体协议的发展扩展了这种分析的能力。

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