BACKGROUND A normal chromosomal constitution defined through PGT-A assessing all chromosomes on trophectoderm (TE) biopsies represents the strongest predictor of embryo implantation. Yet, its positive predictive value is not higher than 50–60%. This gap of knowledge on the causes of euploid blastocysts’ reproductive failure is known as ‘the black box of implantation’. OBJECTIVE AND RATIONALE Several embryonic, maternal, paternal, clinical, and IVF laboratory features were scrutinized for their putative association with reproductive success or implantation failure of euploid blastocysts. SEARCH METHODS A systematic bibliographical search was conducted without temporal limits up to August 2021. The keywords were ‘(blastocyst OR day5 embryo OR day6 embryo OR day7 embryo) AND (euploid OR chromosomally normal OR preimplantation genetic testing) AND (implantation OR implantation failure OR miscarriage OR abortion OR live birth OR biochemical pregnancy OR recurrent implantation failure)’. Overall, 1608 items were identified and screened. We included all prospective or retrospective clinical studies and randomized-controlled-trials (RCTs) that assessed any feature associated with live-birth rates (LBR) and/or miscarriage rates (MR) among non-mosaic euploid blastocyst transfer after TE biopsy and PGT-A. In total, 41 reviews and 372 papers were selected, clustered according to a common focus, and thoroughly reviewed. The PRISMA guideline was followed, the PICO model was adopted, and ROBINS-I and ROB 2.0 scoring were used to assess putative bias. Bias across studies regarding the LBR was also assessed using visual inspection of funnel plots and the trim and fill method. Categorical data were combined with a pooled-OR. The random-effect model was used to conduct the meta-analysis. Between-study heterogeneity was addressed using I2. Whenever not suitable for the meta-analysis, the included studies were simply described for their results. The study protocol was registered at http://www.crd.york.ac.uk/PROSPERO/ (registration number CRD42021275329). OUTCOMES We included 372 original papers (335 retrospective studies, 30 prospective studies and 7 RCTs) and 41 reviews. However, most of the studies were retrospective, or characterized by small sample sizes, thus prone to bias, which reduces the quality of the evidence to low or very low. Reduced inner cell mass (7 studies, OR: 0.37, 95% CI: 0.27–0.52, I2 = 53%), or TE quality (9 studies, OR: 0.53, 95% CI: 0.43–0.67, I2 = 70%), overall blastocyst quality worse than Gardner’s BB-grade (8 studies, OR: 0.40, 95% CI: 0.24–0.67, I2 = 83%), developmental delay (18 studies, OR: 0.56, 95% CI: 0.49–0.63, I2 = 47%), and (by qualitative analysis) some morphodynamic abnormalities pinpointed through time-lapse microscopy (abnormal cleavage patterns, spontaneous blastocyst collapse, longer time of morula formation I, time of blastulation (tB), and duration of blastulation) were all associated with poorer reproductive outcomes. Slightly lower LBR, even in the context of PGT-A, was reported among women ≥38 years (7 studies, OR: 0.87, 95% CI: 0.75–1.00, I2 = 31%), while obesity was associated with both lower LBR (2 studies, OR: 0.66, 95% CI: 0.55–0.79, I2 = 0%) and higher MR (2 studies, OR: 1.8, 95% CI: 1.08–2.99, I2 = 52%). The experience of previous repeated implantation failures (RIF) was also associated with lower LBR (3 studies, OR: 0.72, 95% CI: 0.55–0.93, I2 = 0%). By qualitative analysis, among hormonal assessments, only abnormal progesterone levels prior to transfer were associated with LBR and MR after PGT-A. Among the clinical protocols used, vitrified-warmed embryo transfer was more effective than fresh transfer (2 studies, OR: 1.56, 95% CI: 1.05–2.33, I2 = 23%) after PGT-A. Lastly, multiple vitrification-warming cycles (2 studies, OR: 0.41, 95% CI: 0.22–0.77, I2 = 50%) or (by qualitative analysis) a high number of cells biopsied may slightly reduce the LBR, while simultaneous zona-pellucida opening and TE biopsy allowed better results than the Day 3 hatching-based protocol (3 studies, OR: 1.41, 95% CI: 1.18–1.69, I2 = 0%). WIDER IMPLICATIONS Embryo selection aims at shortening the time-to-pregnancy, while minimizing the reproductive risks. Knowing which features are associated with the reproductive competence of euploid blastocysts is therefore critical to define, implement, and validate safer and more efficient clinical workflows. Future research should be directed towards: (i) systematic investigations of the mechanisms involved in reproductive aging beyond de novo chromosomal abnormalities, and how lifestyle and nutrition may accelerate or exacerbate their consequences; (ii) improved evaluation of the uterine and blastocyst-endometrial dialogue, both of which represent black boxes themselves; (iii) standardization/automation of embryo assessment and IVF protocols; (iv) additional invasive or preferably non-invasive tools for embryo selection. Only by filling these gaps we may finally crack the riddle behind ‘the black box of implantation’.

中文翻译：

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打开黑匣子：为何整倍体囊胚无法着床？系统回顾和荟萃分析

背景 通过PGT-A评估滋养外胚层（TE）活组织检查上的所有染色体来定义正常的染色体构成，代表了胚胎植入的最强预测因子。然而，其阳性预测值不高于50-60%。这种关于整倍体囊胚生殖失败原因的知识空白被称为“植入黑匣子”。目的和基本原理 对一些胚胎、母体、父体、临床和 IVF 实验室特征进行了仔细检查，以确定它们与整倍体囊胚生殖成功或植入失败的假定关联。检索方法 系统性的文献检索截至 2021 年 8 月，不受时间限制。关键词为“（囊胚或第 5 天胚胎或第 6 天胚胎或第 7 天胚胎）AND（整倍体或染色体正常或植入前基因检测）AND（植入或植入失败或流产或堕胎或活产或生化妊娠或反复植入失败）'。总共识别和筛选了 1608 个项目。我们纳入了所有前瞻性或回顾性临床研究和随机对照试验 (RCT)，这些研究评估了 TE 活检和 PGT 后非镶嵌整倍体囊胚移植中与活产率 (LBR) 和/或流产率 (MR) 相关的任何特征-A。总共选择了 41 篇评论和 372 篇论文，根据共同焦点进行聚类，并进行了彻底的审查。遵循PRISMA指南，采用PICO模型，并使用ROBINS-I和ROB 2.0评分来评估推定偏倚。还使用漏斗图的目视检查和修剪和填充方法来评估有关 LBR 的研究偏差。分类数据与汇总 OR 相结合。使用随机效应模型进行荟萃分析。使用 I2 解决研究间的异质性。当不适合荟萃分析时，纳入的研究仅对其结果进行简单描述。该研究方案在 http://www.crd.york.ac.uk/PROSPERO/ 上注册（注册号 CRD42021275329）。结果 我们纳入了 372 篇原始论文（335 篇回顾性研究、30 篇前瞻性研究和 7 篇随机对照试验）和 41 篇综述。然而，大多数研究都是回顾性的，或者样本量较小，因此容易出现偏倚，从而将证据质量降低到低或极低。内细胞量减少（7 项研究，OR：0.37，95% CI：0.27–0.52，I2 = 53%）或 TE 质量减少（9 项研究，OR：0.53，95% CI：0.43–0.67，I2 = 70%） ，总体囊胚质量比 Gardner 的 BB 级差（8 项研究，OR：0.40，95% CI：0.24–0.67，I2 = 83%），发育迟缓（18 项研究，OR：0.56，95% CI：0.49–0.63， I2 = 47%），以及（通过定性分析）通过延时显微镜查明的一些形态动力学异常（异常卵裂模式、自发囊胚塌陷、桑葚胚形成时间较长 I、囊胚形成时间 (tB) 和囊胚形成持续时间）所有这些都与较差的生殖结果有关。据报道，≥38 岁的女性 LBR 略低，即使在 PGT-A 的情况下也是如此（7 项研究，OR：0.87，95% CI：0.75–1.00，I2 = 31%），而肥胖与较低的 LBR 相关（2 项研究，OR：0.66，95% CI：0.55–0.79，I2 = 0%）和更高的 MR（2 项研究，OR：1.8，95% CI：1.08–2.99，I2 = 52%）。先前反复植入失败 (RIF) 的经历也与较低的 LBR 相关（3 项研究，OR：0.72，95% CI：0.55–0.93，I2 = 0%）。通过定性分析，在激素评估中，只有移植前异常的孕酮水平与 PGT-A 后的 LBR 和 MR 相关。在使用的临床方案中，PGT-A 后玻璃化加热胚胎移植比新鲜移植更有效（2 项研究，OR：1.56，95% CI：1.05–2.33，I2 = 23%）。最后，多次玻璃化加温周期（2项研究，OR：0.41，95％CI：0.22-0.77，I2 = 50％）或（通过定性分析）大量细胞活检可能会略微降低LBR，而同时进行透明带透明体开口和 TE 活检比基于第 3 天孵化的方案获得更好的结果（3 项研究，OR：1.41，95% CI：1.18–1.69，I2 = 0%）。更广泛的影响 胚胎选择的目的是缩短怀孕时间，同时最大限度地降低生殖风险。因此，了解哪些特征与整倍体囊胚的生殖能力相关对于定义、实施和验证更安全、更高效的临床工作流程至关重要。未来的研究应面向：（i）系统研究除新生染色体异常之外的生殖衰老机制，以及生活方式和营养如何加速或加剧其后果；(ii) 改进对子宫和囊胚-子宫内膜对话的评估，这两者本身就是黑匣子；(iii) 胚胎评估和 IVF 方案的标准化/自动化；(iv)用于胚胎选择的额外侵入性或优选非侵入性工具。只有填补这些空白，我们才有可能最终破解“植入黑匣子”背后的谜团。I2 = 50%）或（通过定性分析）大量细胞活检可能会略微降低 LBR，而同时打开透明带和 TE 活检可得到比第 3 天基于孵化的方案更好的结果（3 项研究，OR：1.41 ，95% CI：1.18–1.69，I2 = 0%）。更广泛的影响 胚胎选择的目的是缩短怀孕时间，同时最大限度地降低生殖风险。因此，了解哪些特征与整倍体囊胚的生殖能力相关对于定义、实施和验证更安全、更高效的临床工作流程至关重要。未来的研究应面向：（i）系统研究除新生染色体异常之外的生殖衰老机制，以及生活方式和营养如何加速或加剧其后果；(ii) 改进对子宫和囊胚-子宫内膜对话的评估，这两者本身就是黑匣子；(iii) 胚胎评估和 IVF 方案的标准化/自动化；(iv)用于胚胎选择的额外侵入性或优选非侵入性工具。只有填补这些空白，我们才有可能最终破解“植入黑匣子”背后的谜团。I2 = 50%）或（通过定性分析）大量细胞活检可能会略微降低 LBR，而同时打开透明带和 TE 活检可得到比第 3 天基于孵化的方案更好的结果（3 项研究，OR：1.41 ，95% CI：1.18–1.69，I2 = 0%）。更广泛的影响 胚胎选择的目的是缩短怀孕时间，同时最大限度地降低生殖风险。因此，了解哪些特征与整倍体囊胚的生殖能力相关对于定义、实施和验证更安全、更高效的临床工作流程至关重要。未来的研究应面向：（i）系统研究除新生染色体异常之外的生殖衰老机制，以及生活方式和营养如何加速或加剧其后果；(ii) 改进对子宫和囊胚-子宫内膜对话的评估，这两者本身就是黑匣子；(iii) 胚胎评估和 IVF 方案的标准化/自动化；(iv)用于胚胎选择的额外侵入性或优选非侵入性工具。只有填补这些空白，我们才有可能最终破解“植入黑匣子”背后的谜团。