人类囊胚活检和细胞化
Summary
需要进行胚泡活检和保并口授,以便有效地进行植入前基因检测。一种方法,导致在授精后的第5-7天连续打开区域细胞和检索7-8个营养细胞,限制了所需的操作次数和胚胎在次优环境条件下的暴露。
Abstract
在IVF周期内进行胚泡活检,通过植入前基因测试获得可靠的基因诊断。然后,理想的工作流程需要一个安全有效的牙化协议,由于诊断技术的周转时间,并在下面的自然周期中在生理子宫内膜上移植选定的胚胎。活检方法包括连续打开区域细胞和检索5-10个营养细胞(理想情况下为7-8)限制所需的操作数量和胚胎在次优环境条件下的暴露。经过适当的训练后,该技术在不同的操作者之间可重复活检的时间(±8分钟,根据每盘的胚胎数量进行活检3-22分钟),获得结论性诊断(+97.5%)和玻璃化加热的花体胚泡转移后活产率(>40%)。活检、硝化和变暖后的存活率高达99.8%。气候变暖后1.5小时的再膨胀率高达97%,主要取决于活检和抗化(理想情况下为±30分钟)、胚泡形态质量和活检日之间的时间。一般来说,最好对坍塌的胚泡进行压化;因此,在非PGT周期中,可以进行激光辅助人工收缩,以诱导胚胎在冷冻保存前崩溃。最有希望的未来观点是,在胚泡培养后对IVF培养基进行非侵入性分析,作为胚胎DNA的假定来源。然而,这种潜在的前卫仍在调查之中,还需要定义和验证一个可靠的协议。
Introduction
现代人类胚胎学的主要目标是使每个刺激周期的活产数量最大化,并降低成本、时间和实现怀孕的努力。为了实现这一目标,应采用经验证的胚胎选择方法,在IVF周期中获得的一组胚胎中确定具有生殖能力的胚胎。根据最新证据,胚泡培养1结合全面的染色体检测和玻璃化加热的幼倍体胚胎移植(ET)是提高IVF效率的最有效框架2。显然,非倍体试验需要胚胎标本,目前主要代表从营养素(TE)中检索到的细胞,即囊胚中给予妊娠期间胚胎附件(例如胎盘)来源的部分.除了核类型分析,还可以从TE活检中评估单个基因突变,作为称为植入前基因测试的临床策略的一部分(PGT;-A用于非倍体,-SR用于结构染色体重组,-M用于单源性疾病)。其他卵母细胞/胚胎活检方法在过去几十年中已被进行解剖和临床采用,即极性身体活检和卵波粒活检。然而,现在减少了它们的使用,因为它们的程序性缺陷(例如,更高的工作量和生殖影响的风险)和诊断限制(例如单细胞分析问题)隐含地阻碍了成本、风险和好处(有关审查,请参阅3)。
在本文中,对TE活检的主要方案之一以及随后所需的保质化、加热和转移程序进行了详尽描述。此处概述的工作流非常适合繁忙的 PGT 单元。
如我们组4,5前面所述,该过程涉及完全扩张的胚泡的分子的连续打开和去除少数TE细胞(平均7-8)。与第3天激光辅助孵化为基础的胚泡活检方法6相比,这个程序可以简化IVF单元的日常时间表,其中必须及时执行诸如胚泡活检和葡萄光化等精细程序。一旦胚泡达到其完全膨胀,活检可以通过选择TE细胞进行去除,从而防止内细胞质量(ICM)的增生风险,否则会使手术具有挑战性。在文献中,也描述了胚泡活检的第三个方案,即一旦胚胎到达胚泡阶段,在手术5、7前几个小时进行激光辅助孵化。然而,这种方法更耗时,主要适合在经验有限的操作员手中实施TE活检的IVF单元,并且考虑到日工作量适中。
如果旨在进行IVF中的遗传分析,则细胞内体精子注射(ICSI)8应该是一种综合技术。同样,一个适当的培养系统,安全地将胚胎收获到胚泡阶段,对于实施TE活检策略也至关重要。足够数量的培养箱,以及使用低氧张力是这个目的的关键先决条件,不损害胚泡率9。同时,需要一个有效的冷冻保存程序来安全地管理PGT周期。在过去的十年中,牙化的实施提高了胚胎的存活率,甚至高达>99%10,11。这提供了足够的时间进行基因测试,并将胚胎移植推迟到下面的月经周期,在非刺激和可能更容易接受的子宫内膜12。
TE 活检和 Vitaita 化都是要求严格技能的任务,其有效性可能因经验不足的操作员而异。因此,在允许每个操作员临床执行这些程序之前,提倡一个特定的培训期;此外,应定期通过监测冷冻保存和活检程序的关键绩效指标(KPI)来评估操作人员技能的维持情况。每个IVF诊所应为此设置内部KPI,其中必须近似于国际财团公布的KPI和/或参考实验室公布的结果。
TE 活检、保质化加热和见证程序是我们部门验证的技术,这些技术已在之前三份出版物 11、13、14 中报告的所有操作员中标准化.
Protocol
Representative Results
Discussion
只有经验丰富的熟练胚胎学家谁完成了他们的训练期应进行TE活检和胚泡性牙菌。此外,需要证人监测程序,并保证在i期间有效追踪活检胚泡从活检盘(补充图1)到活检后盘的移动(补充图1)(补充图1)),然后到化板(补充图1),最后到硝化支持(补充图1);ii) 活检TE细胞从活检盘转移到PCR管(补充图1);iii) 诊断后的加热和转移步骤。有关所有见证步骤…
Disclosures
The authors have nothing to disclose.
Acknowledgements
AG 和 RM 收集了数据并起草了手稿。DC 分析数据,起草代表性结果,进行统计并修订稿件。FMU 和 LR 对结果和整个手稿进行了批判性讨论。
Materials
| Equipment | |||
| Cold tube rack | Biocision | XTPCR96 | |
| Electronic pipette controller | Fisher Scientific | 710931 | |
| Flexipet adjustable handle set | Cook | G18674 | Stripper holder |
| Gilson Pipetman | Gilson | 66003 | p20 |
| IVF Electronic Witness System | CooperSurgical Fertility & Genomic Solutions | RI Witness ART Management System | |
| Inverted microscope | Nikon | Eclipse TE2000-U | |
| Laminar Flow Hood | IVF TECH | Grade A air flow | |
| Laser objective | RI | Saturn 5 | |
| Microinjectors | Nikon Narishige | NT-88-V3 | |
| Mini centrifuge for PCR tubes | Eppendorf | CSLQSPIN | for 0.2ml PCR tubes |
| Stereomicroscope | Leica | Leica M80 | |
| Thermostat | Panasonic | MCO-5AC-PE | |
| Tri-gas incubator | Panasonic | MCO-5M-PE | 02/CO2 |
| Consumables | |||
| Biopsy pipette | RI | 7-71-30FB35720 | 30µm ID, flat 35°C |
| Cryolock | Cryolock | CL-R-CT | |
| CSCM complete | Irvine Scientific | 90165 | IVF culture medium supplemented with HSA |
| Embryo Transfer Catheter | Cook | G17934 | |
| Flexipet pipette | Cook | G26712 | 140µm stripping pipette tip |
| Flexipet pipette | Cook | G46020 | 300µm stripping pipette tips |
| Holding pipette | RI | 7-71-IH35/20 | 30µm ID, flat 35°C |
| Human Serum Albumin | Irvine Scientific | 9988 | |
| IVF One well dish | Falcon | 353653 | |
| Mineral Oil for embryo culture | Irvine Scientific | 9305 | |
| Modified HTF Medium | Irvine Scientific | 90126 | Hepes-Buffered medium |
| Nuclon Delta Surface | Thermofisher scientific | 176740 | IVF dish 4-well plate with sliding lid |
| Primaria Cell culture dish | Corning | 353802 | 60x15mm |
| Reproplate | Kitazato | 83016 | |
| Serological pipette | Falcon | 357551 | 10ml |
| Sterile disposable Gilson tips | Eppendorf | 0030 075.021 | 200µl |
| Tubing Kit | Provided by the genetic lab | PCR tubes (0.2mL), loading solution, biopsy washing solution | |
| Vitrification media | Kitazato | VT801 | Equilibration and vitrification solutions |
| Warming media | Kitazato | VT802 | Thawing and dilution solutions |
References
- Glujovsky, D., Farquhar, C., Quinteiro Retamar, A. M., Alvarez Sedo, C. R., Blake, D. Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology. Cochrane Database of Systematic Reviews. (6), CD002118 (2016).
- Dahdouh, E. M., Balayla, J., Garcia-Velasco, J. A. Comprehensive chromosome screening improves embryo selection: a meta-analysis. Fertility and Sterility. 104 (6), 1503-1512 (2015).
- Cimadomo, D., et al. The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis. Biomedical Research International. 2016, 7193075 (2016).
- Capalbo, A., et al. Correlation between standard blastocyst morphology, euploidy and implantation: an observational study in two centers involving 956 screened blastocysts. Human Reproduction. 29 (6), 1173-1181 (2014).
- Capalbo, A., et al. Implementing PGD/PGD-A in IVF clinics: considerations for the best laboratory approach and management. Journal of Assisted Reproduction and Genetics. , (2016).
- McArthur, S. J., Leigh, D., Marshall, J. T., de Boer, K. A., Jansen, R. P. Pregnancies and live births after trophectoderm biopsy and preimplantation genetic testing of human blastocysts. Fertility and Sterility. 84 (6), 1628-1636 (2005).
- Kokkali, G., et al. Birth of a healthy infant following trophectoderm biopsy from blastocysts for PGD of beta-thalassaemia major. Human Reproduction. 20 (7), 1855-1859 (2005).
- Rienzi, L., Ubaldi, F., Anniballo, R., Cerulo, G., Greco, E. Preincubation of human oocytes may improve fertilization and embryo quality after intracytoplasmic sperm injection. Human Reproduction. 13 (4), 1014-1019 (1998).
- Wale, P. L., Gardner, D. K. The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction. Human Reproduction Update. 22 (1), 2-22 (2016).
- Rienzi, L., et al. Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance. Human Reproduction Update. 23 (2), 139-155 (2017).
- Cimadomo, D., et al. Associations of blastocyst features, trophectoderm biopsy and other laboratory practice with post-warming behavior and implantation. Human Reproduction. , (2018).
- Evans, J., et al. Fresh versus frozen embryo transfer: backing clinical decisions with scientific and clinical evidence. Human Reproduction Update. 20 (6), 808-821 (2014).
- Capalbo, A., et al. Consistent and reproducible outcomes of blastocyst biopsy and aneuploidy screening across different biopsy practitioners: a multicentre study involving 2586 embryo biopsies. Human Reproduction. 31 (1), 199-208 (2016).
- Cimadomo, D., et al. Failure mode and effects analysis of witnessing protocols for ensuring traceability during PGD/PGS cycles. Reproductive Biomedicine Online. 33 (3), 360-369 (2016).
- Gardner, D. K., Schoolcraft, B., Jansen, R., Mortimer, D. . Towards Reproductive Certainty: Infertility and Genetics Beyond 1999. , 377-388 (1999).
- Cimadomo, D., et al. Inconclusive chromosomal assessment after blastocyst biopsy: prevalence, causative factors and outcomes after re-biopsy and re-vitrification. A multicenter experience. Human Reproduction. , (2018).
- de Boer, K. A., Catt, J. W., Jansen, R. P., Leigh, D., McArthur, S. Moving to blastocyst biopsy for preimplantation genetic diagnosis and single embryo transfer at Sydney IVF. Fertility and Sterility. 82 (2), 295-298 (2004).
- Capalbo, A., Rienzi, L. Mosaicism between trophectoderm and inner cell mass. Fertility and Sterility. 107 (5), 1098-1106 (2017).
- McCoy, R. C., et al. Evidence of Selection against Complex Mitotic-Origin Aneuploidy during Preimplantation Development. PLoS Genetics. 11 (10), e1005601 (2015).
- 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).
- Lee, H., et al. Live births after transfer of rebiopsy and revitrification of blastocyst that had “no diagnosis” following trophectoderm biopsy. Fertility and Sterility. 106 (3), e164 (2016).
- Capalbo, A., et al. 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).
- Hammond, E. R., Shelling, A. N., Cree, L. M. Nuclear and mitochondrial DNA in blastocoele fluid and embryo culture medium: evidence and potential clinical use. Human Reproduction. 31 (8), 1653-1661 (2016).
- Vera-Rodriguez, M., et al. Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development. Human Reproduction. 33 (4), 745-756 (2018).
