使用废培养基对人类植入前胚胎进行染色体筛选：样品采集和染色体倍性分析
1Centre for Reproductive Medicine, Department of Obstetrics and Gynecology,Peking University Third Hospital, 2National Clinical Research Center for Obstetrics and Gynecology(Peking University Third Hospital), 3Ministry of Education,Key Laboratory of Assisted Reproduction (Peking University), 4Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, 5Department of Clinical Research,Yikon Genomics Co. Ltd.
Summary
本研究报告了一种使用废培养基对人类胚胎进行染色体筛选的方案，该方案避免了胚胎活检并使用 NGS 进行染色体倍性鉴定。本文介绍了详细的过程，包括培养基的制备、全基因组扩增（WGA）、下一代测序（NGS）文库的制备和数据分析。
Abstract
在临床体外受精 （IVF） 中，PGT-A 的常用方法需要对滋养外胚层 （TE） 中的一些细胞进行活检。这是形成胎盘的谱系。然而，这种方法需要专业技能，具有侵入性，并且存在假阳性和假阴性，因为 TE 中的染色体数量和发育成胎儿的内细胞团 （ICM） 并不总是相同的。NICS是一种需要对从TE和ICM释放到培养基中的DNA进行测序的技术，可能为这些问题提供一条出路，但先前已被证明疗效有限。本研究报告了NICS的完整方案，包括培养基采样方法、全基因组扩增（WGA）和文库制备，以及通过分析软件进行NGS数据分析。考虑到不同胚胎实验室的冷冻保存时间不同，胚胎学家有两种收集胚胎培养基的方法，可以根据试管婴儿实验室的实际情况进行选择。
Introduction
辅助生殖技术（ART）越来越多地用于治疗不孕症。然而，ART如试管婴儿的成功率一直有限，流产率明显高于正常人群1。这些问题的主要原因是染色体异常，这通常存在于植入前人类胚胎中2。PGT-A是在植入前筛选胚胎染色体平衡的有效方法3,4。一些研究证明，PGT-A可以降低流产率，提高怀孕率5,6,7,8。然而，PGT-A需要复杂的技术专长，需要特定的培训和经验。侵入性胚胎活检程序也可能对胚胎造成损害9.研究表明，卵裂球活检会阻碍后续发育，活检TE的数量可能会影响植入率10。尽管胚胎活检的长期生物安全性问题尚未在人类中得到彻底评估，但动物研究表明其对胚胎发育的负面影响11,12,13。
先前的报道表明，在胚胎发育过程中，微量的DNA物质被分泌到培养基中，并且已经努力使用废胚胎培养基14,15,16,17,18进行全面的染色体筛选（CCS）。然而，检测的检出率和准确性尚未满足临床广泛使用的要求。本研究报告了 NICS 检测方法在提高检测率和 NICS 测试准确性方面的改进19.近年来，囊泡液 （BF） 已被研究为微创 PGT-A 的分析样本。然而，囊胚液样本中成功的全基因组扩增和可检测 DNA 的比例从 34.8% 到 82%20,21,22 不等。各种研究中报告的 BF 体积范围为 0.3 nL 至 1 μL。鉴于BF中DNA含量低，可以通过混合囊胚液和培养基来增加游离DNA的量，以提高检测的成功率和一致性。库兹涅佐夫等人。23和Li等24用激光处理透明带，向培养基中释放囊胚液，提高胚胎DNA总量，WGA后合并培养基/BF样品的扩增率分别为100%和97.5%。Jiao等25也用同样的方法获得了100%的扩增成功率。
本研究报告了一个详细的方案，包括废培养基样品制备、NGS 制备和数据分析。通过小心地从卵母细胞中去除卵丘细胞，本研究进行了卵胞浆内单精子注射（ICSI）和囊胚培养。收集第 4 天-第 5 天/第 6 天用过的培养基用于 WGA 和 NGS 文库制备。通过使用NICS技术，本研究在大约3 h内简化了WGA和NGS文库的制备步骤，并在大约9 h内获得了无创的CCS结果。
Protocol
获得北京大学第三医院伦理委员会的伦理许可。 1. 准备工作 注意： 所需的材料和设备列在 材料表中。 试剂在使用前，在37°C，5%CO2 和5%O2 中预热并平衡（平衡）20-30μL配子培养基/受精培养基和裂解/囊胚期培养基（覆盖有矿物油）和透明质酸酶（在密盖管中）过夜。 在通风橱的工作表面上将透明质酸酶?…
Representative Results
本研究将所提出的方法应用于患者。在应用NICS分析之前，已获得IRB的批准和知情同意。本研究从患者身上获取了 6 个囊胚，并在第 4 天至第 5 天培养基上对所有 6 个胚胎进行了 NICS检测。采用NICS法在5条染色体中检测到由父母平衡易位引起的染色体异常;因此，它们不能用于转移（图4A-E）。两个胚胎的NICS结果显示相同的核型45，XN和-18（×1）都是18号染色体缺失（…
Discussion
修改和故障排除
如果NICS结果被亲本遗传物质污染，请确保去除所有日冕状积云细胞，并确保进行ICSI受精。避免了不正确的培养基储存或模板制备过程，这可能会降解DNA。使用DNase和RNase去污试剂对工作空间进行了彻底净化。为了避免其他胚胎的污染，一个胚胎总是在单滴培养基中培养，以避免从第4天开始的交叉污染。当延迟胚胎在最终培养液滴中的放置时，污?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢 Shiping Bo 和 Shujie Ma 在 NGS 数据分析方面的帮助。基金资助：本工作由国家重点研发计划（批准号：2018YFC1003100）资助。
Materials
| 1.5 mL EP tube, 0.2 mL PCR tube | Axygen | MCT-150-C, PCR-02-C | DNase/RNase free, Low Binding PCR tubes and 1.5 mL micro-centrifuge tubes are recommended. |
| 10 µL, 200 µL, 1000 µL DNase /RNase Free Tips | Axygen | T-300-R-S, T-200-Y-R-S, T-1000-B-R-S | This can be replaced by other brand/For sample transfer |
| 100 % ethanol | Sinopharm Chemical | 10009218 | This can be replaced by other brand/For DNA library purification |
| Barcode Primer1-48 | Yikon Genomics | Reagent in NICSInst library preparation kit | For library amplificaton |
| BD Falcon Organ Culture Dish, Sterile | BD Bioscience | 363037 | This can be replaced by other brand/For embryo culture |
| BD Falcon Tissue culture Dishes (Easy Grip) , Sterile | BD Bioscience | 353001 | This can be replaced by other brand/For embryo culture |
| BD Falcon Tissue culture Dishes, Sterile | BD Bioscience | 353002 | This can be replaced by other brand/For embryo culture |
| Cell Lysis Buffer | Yikon Genomics | Reagent in NICSInst library preparation kit | For culture medium pre-treatment |
| Cell Lysis Enzyme | Yikon Genomics | Reagent in NICSInst library preparation kit | For culture medium pre-treatment |
| ChromGo software | Yikon Genomics | Data analysis | |
| CMPure Magbeads | Yikon Genomics | Reagent in NICSInst library preparation kit | For library purification |
| Cryotop open systerm | KITAZATO BioPharma | 81110 | This can be replaced by other brand/For embryo vitrification |
| Distill water | Yikon Genomics | Reagent in NICSInst library preparation kit | To dissolve DNA |
| ES (Vitrification kit) | KITAZATO BioPharma | Reagent inVitrification kit | This can be replaced by other brand/For embryo vitrification |
| HOLDNIG | ORIGIO | MPH-MED-35 | This can be replaced by other brand/For ICSI |
| Hyaluronidase solution, 80 U/mL | SAGE | ART4007-A | This can be replaced by other brand/Digest oocyte-corona-cumulus complex |
| ICSI | ORIGIO | MPH-35-35 | This can be replaced by other brand/For ICSI |
| Illumina MiSeq® System | Illumina | SY-410-1001 | For library sequencing |
| Incubator | Labotect | Inkubator C16 | This can be replaced by other brand/For embryo culture |
| Library buffer | Yikon Genomics | Reagent in NICSInst library preparation kit | For library amplificaton |
| Library Enzyme Mix | Yikon Genomics | Reagent in NICSInst library preparation kit | For library amplificaton |
| Magnetic Stand | DynaMagTM-2 | 12321D | For library purification |
| Microscope | OLYMPUS | 1X71 | This can be replaced by other brand/For embryo observation |
| Mini-centrifuge | ESSENSCIEN | ELF6 | For separation |
| MT Enzyme Mix | Yikon Genomics | Reagent in NICSInst library preparation kit | For culture medium pre-treatment |
| NICSInst library preparation kit | Yikon Genomics | KT1000800324 | Whole genome amplification and library construction |
| NICSInst Sample Prep Station | Yikon Genomics | ME1001003 | Amplificate DNA |
| Nunc IVF 4-Well Dish | Thermo Scientific | 144444 | This can be replaced by other brand/For embryo washing and blastocyst culture |
| Pasteur Pipette | Oirgio | MXL3-IND-135 | This can be replaced by other brand/For embryo tansfer |
| Pasteur pipettes | ORIGIO | PP-9-1000 | This can be replaced by other brand/For IVF laboratory |
| Pre-Lib Buffer | Yikon Genomics | Reagent in NICSInst library preparation kit | Pre-library preparation |
| Pre-Lib Enzyme | Yikon Genomics | Reagent in NICSInst library preparation kit | Pre-library preparation |
| Qubit® 3.0 Fluorometer | Thermo Scientific | Q33216 | For library quantification |
| Quinn's Advantage Blastocyst Medium | SAGE | ART-1029 | For embryo blastocyst stage culture |
| Quinn's Advantage Cleavage Medium | SAGE | ART-1026 | This can be replaced by other brand/For embryo cleavage stage culture |
| Quinn's Advantage Fertilization Medium | SAGE | ART-1020 | This can be replaced by other brand/For oocyte and sperm fertilization |
| Quinn's Advantage m-HTF Medium with HEPES | SAGE | ART-1023 | This can be replaced by other brand/For embryo clutrure |
| Quinn's Advantage SPS Serum protein Substitute Kit | SAGE | ART-3010 | This can be replaced by other brand/To denude the oocyte |
| Quinn's Advantage Tissue culture mineral oil | SAGE | ART-4008P | This can be replaced by other brand/To cover the culture medium |
| STRIPPER TIPS | ORIGIO | MXL3-IND-135 | This can be replaced by other brand/For denudating granulosa cells |
| Vitrification Cryotop Open systerm | KIZTAZATO | 81111 | This can be replaced by other brand/For embryo vitrification |
| Vitrification kit | KITAZATO BioPharma | VT101 | This can be replaced by other brand/For embryo vitrification |
| Vortexer | Qilinbeier | DNYS8 | Sample mix |
| VS (Vitrification kit) | KITAZATO BioPharma | Reagent inVitrification kit | This can be replaced by other brand/For embryo vitrification |
| ZILOS-tk Laser System | Hamilton Thorne | CLASS 1 laser | This can be replaced by other brand/For artificial blastocoele collapse |
References
- Barlow, P. Early pregnancy loss and obstetrical risk after in-vitro fertilization and embryo replacement. Human Reproduction. 3 (5), 671-675 (1988).
- Munne, S. Chromosome abnormalities and their relationship to morphology and development of human embryos. Reproductive BioMedicine Online. 12 (2), 234-253 (2006).
- Harton, G. L. Diminished effect of maternal age on implantation after preimplantation genetic diagnosis with array comparative genomic hybridization. Fertility and Sterility. 100 (6), 1695-1703 (2013).
- Hodes-Wertz, B. Idiopathic recurrent miscarriage is caused mostly by aneuploid embryos. Fertility and Sterility. 98 (3), 675-680 (2012).
- Keltz, M. D. Preimplantation genetic screening (PGS) with Comparative genomic hybridization (CGH) following day 3 single cell blastomere biopsy markedly improves IVF outcomes while lowering multiple pregnancies and miscarriages. Journal of Assisted Reproduction and Genetics. 30 (10), 1333-1339 (2013).
- Scott, R. T. Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial. Fertility and Sterility. 100 (3), 697-703 (2013).
- Forman, E. J. In vitro fertilization with single euploid blastocyst transfer: a randomized controlled trial. Fertility and Sterility. 100 (1), 100-107 (2013).
- Yang, Z. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study. Molecular Cytogenetics. 5 (1), 24 (2012).
- Cimadomo, D. The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis. BioMed Research International. 2016, 7193075 (2016).
- Scott, R. T., Upham, K. M., Forman, E. J., Zhao, T., Treff, N. R. Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy does not: a randomized and paired clinical trial. Fertility and Sterility. 100 (3), 624-630 (2013).
- Wu, Y. Blastomere biopsy influences epigenetic reprogramming during early embryo development, which impacts neural development and function in resulting mice. Cellular and Molecular Life Sciences. 71 (9), 1761-1774 (2014).
- Zhao, H. C. Aberrant epigenetic modification in murine brain tissues of offspring from preimplantation genetic diagnosis blastomere biopsies. Biology of Reproduction. 89 (5), 117 (2013).
- Zeng, Y. Preimplantation genetic diagnosis (PGD) influences adrenal development and response to cold stress in resulting mice. Cell and Tissue Research. 354 (3), 729-741 (2013).
- Palini, S. Genomic DNA in human blastocoele fluid. Reproductive BioMedicine Online. 26 (6), 603-610 (2013).
- Gianaroli, L. Blastocentesis: a source of DNA for preimplantation genetic testing. Results from a pilot study. Fertility and Sterility. 102 (6), 1692-1699 (2014).
- Stigliani, S., Anserini, P., Venturini, P. L., Scaruffi, P. Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation. Human Reproduction. 28 (10), 2652-2660 (2013).
- Stigliani, S. Mitochondrial DNA in Day 3 embryo culture medium is a novel, non-invasive biomarker of blastocyst potential and implantation outcome. Molecular Human Reproduction. 20 (12), 1238-1246 (2014).
- Wu, H. Medium-Based Noninvasive Preimplantation Genetic Diagnosis for Human α-Thalassemias-SEA. Medicine. 94 (12), e669 (2015).
- Xu, J. Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization. Proceedings of the National Academy of Sciences. 113 (42), 11907-11912 (2016).
- Capalbo, A. Diagnostic efficacy of blastocoel fluid and spent media as sources of DNA for preimplantation genetic testing in standard clinical conditions. Fertility and Sterility. 110 (5), 870-879 (2018).
- Tobler, K. J. Blastocoel fluid from differentiated blastocysts harbors embryonic genomic material capable of a whole-genome deoxyribonucleic acid amplification and comprehensive chromosome microarray analysis. Fertility and Sterility. 104 (2), 418-425 (2015).
- Magli, M. C. Preimplantation genetic testing: polar bodies, blastomeres, trophectoderm cells, or blastocoelic fluid?. Fertility and Sterility. 105 (3), 676-683 (2016).
- Kuznyetsov, V. Evaluation of a novel non-invasive preimplantation genetic screening approach. PLoS One. 13 (5), e0197262 (2018).
- Li, P. Preimplantation Genetic Screening with Spent Culture Medium/Blastocoel Fluid for in Vitro Fertilization. Scientific Reports. 8 (1), 9275 (2018).
- Jiao, J. Minimally invasive preimplantation genetic testing using blastocyst culture medium. Human Reproduction. 34 (7), 1369-1379 (2019).
- Palermo, G. D. Births after intracytoplasmic injection of sperm obtained by testicular extraction from men with nonmosaic Klinefelter’s syndrome. New England Journal of Medicine. 338 (9), 588-590 (1998).
- Alpha Scientists in Reproductive, M., & Embryology, E. S. I. G. o. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Human Reproduction. 26 (6), 1270-1283 (2011).
- . Qubit dsDNA HS Assay Kit Available from: https://www.thermofisher.com/order/catalog/product/Q32851?ICID=search-product (2015)
- . Miseq system use guide Available from: https://support.illumina.com/downloads/miseq_system (2016)
- Lane, M. Ability to detect aneuploidy from cell free DNA collected from media is dependent on the stage of development of the embryo. Fertility and Sterility. 108 (3), (2017).
- Rubio, C. Multicenter prospective study of concordance between embryonic cell-free DNA and trophectoderm biopsies from 1301 human blastocysts. American Journal of Obstetrics and Gynecology. 223 (5), 751-751 (2020).
- Rubio, C. Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: concordance rate and clinical implications. Fertility and Sterility. 112 (3), 510-519 (2019).
- Lledo, B. Consistent results of non-invasive PGT-A of human embryos using two different techniques for chromosomal analysis. Reproductive BioMedicine Online. 42 (3), 555-563 (2021).
- Kuznyetsov, V. Minimally Invasive Cell-Free Human Embryo Aneuploidy Testing (miPGT-A) Utilizing Combined Spent Embryo Culture Medium and Blastocoel Fluid -Towards Development of a Clinical Assay. Scientific Reports. 10 (1), 7244 (2020).
Cite This Article
Huang, J., Yao, Y., Jia, J., Zhu, X., Ma, J., Wang, J., Liu, P., Lu, S. Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis. J. Vis. Exp. (175), e62619, doi:10.3791/62619 (2021).