STUDY QUESTION Is it possible to predict an euploid chromosomal constitution and identify a transcriptomic profile compatible with extended embryonic development from RNA sequencing (RNA-Seq) data? SUMMARY ANSWER It has been possible to obtain a karyotype comparable to preimplantation genetic testing for aneuploidy (PGT-A), in addition to a transcriptomic signature of embryos which might be suggestive of improved implantation capacity. WHAT IS KNOWN ALREADY Conventional assessment of embryo competence, based on morphology and morphokinetic, lacks knowledge of molecular aspects and faces controversy in predicting ploidy status. Understanding the embryonic transcriptome is crucial, as gene expression influences development and implantation. PGT has improved pregnancy rates, but problems persist when high-quality euploid embryos do not reach term. In fact, only around 50–60% implant, of which 10% result in miscarriage. Comprehensive approaches, including RNA-Seq, offer the potential to discover molecular markers of reproductive competence, and could theoretically be combined with extended-embryo culture platforms up to Day 14 that can be utilized as a proxy to study embryo development at post-implantation stages. STUDY DESIGN, SIZE, DURATION This prospective pilot cohort study was conducted from March 2023 to August 2023. A total of 30 vitrified human blastocysts with previous PGT-A diagnosis on Day 5 (D5) or Day 6 (D6) of development were analysed: n = 15 euploid and n = 15 aneuploid. Finally, 21 embryo samples were included in the study; the rest (n = 9) were excluded due to poor quality pre-sequencing data (n = 7) or highly discordant data (n = 2). PARTICIPANTS/MATERIALS, SETTING, METHODS Following warming and re-expansion, embryos underwent a second trophectoderm (TE) biopsy. The embryos were then cultured until day 11 to assess their development. Biopsy analysis by RNA-Seq, studied the differential expressed genes (DEG) to compare embryos which did not or did attach to the plate: unattached embryos (n = 12) versus attached embryos (n = 9). Thus, we also obtained a specific transcriptomic signature of embryos with a “theoretical” capacity for sustained implantation, based on plate attachment on day 11. MAIN RESULTS AND THE ROLE OF CHANCE The digital karyotype obtained by RNA-Seq showed good concordance with the earlier PGT-A data, with a sensitivity of 0.81, a specificity of 0.83, a Cohen’s Kappa of 0.66, and an area under the ROC of 0.9. At the gene level, 76 statistically significant DEGs were found in the comparison unattached versus attached embryos (Padj < 0.05; FC > 1). To address the functional implications of these differences, significantly deregulated pathways according to GO and KEGG categories were identified. The mural trophectoderm (TE) of the unattached blastocysts showed 63 significantly deregulated terms, displaying upregulation in autophagy, apoptosis, protein kinase and ubiquitin-like protein ligase activity, and downregulation of ribosome, spliceosome, kinetochore, segregation, and chromosome condensation processes. The overall transcriptomic signature specific to embryos still attached to the plate on day 11 (with a theoretically higher implantation capacity) consists of 501 genes, including: EMP2, AURKB, FOLR1, NOTCH3, LRP2, FZD5, MDH1, APOD, GPX8, COLEC12, HSPA1A, CMTM7, BEX3, which are related to implantation and embryonic development (raw P-value < 0.05; shrunk LFC > 1.1). These findings indicate that it might be possible to identify euploid embryos with a greater capacity for implantation and development, after excluding those embryos that present chromosomal alterations. LIMITATIONS, REASONS FOR CAUTION This study included a small sample size, remarkable variability between samples, and low success rate of RNA amplification. Also, structural chromosomal abnormalities were not included, and it was not possible to diagnose mosaic embryos. TE biopsy does not assure the chromosomal status of the whole embryo. The maximum day for in vitro development was Day 11, and attachment to the plate on this day does not provide a clear indication of implantation capacity and viability, which was not tested in this study. WIDER IMPLICATIONS OF THE FINDINGS The short-term goals following on from this pilot study is to expand the sample size with embryos of more complex abnormalities, and to perform a prospective in vitro preclinical validation. In a more distant future and with optimal results, this technique could have clinical application, thus increasing clinical outcomes by assessing both chromosomal content and transcriptomic profiling. STUDY FUNDING/COMPETING INTEREST(S) The Institut Valencià de Competitivitat Empresarial (IVACE) (IMIDCA/2022/39) and Generalitat Valenciana (CIACIF/2021/11) supported the present study. A.C. is an employee of JUNO Genetics. He has received honoraria for an IBSA lecture and a Merck lecture. He is also a minor shareholder of IVIRMA Global. The other authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER N/A.

中文翻译：

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转录组学植入前基因检测 （PGT-T） 的初步研究：迈向胚胎选择的新步骤？


研究问题 是否有可能从 RNA 测序 （RNA-Seq） 数据中预测整倍体染色体构成并确定与扩展胚胎发育相容的转录组学特征？总结答案 除了可能表明植入能力增强的胚胎转录组特征外，还可以获得与植入前非整倍体基因检测 （PGT-A） 相当的核型。已知的基于形态学和形态动力学的胚胎能力的传统评估缺乏分子方面的知识，并且在预测倍性状态方面面临争议。了解胚胎转录组至关重要，因为基因表达会影响发育和植入。PGT 提高了妊娠率，但当高质量的整倍体胚胎未足月时，问题仍然存在。事实上，只有大约 50-60% 的植入物，其中 10% 导致流产。包括 RNA-Seq 在内的综合方法为发现生殖能力的分子标志物提供了潜力，理论上可以与延长胚胎培养平台相结合，直到第 14 天，可以用作研究植入后胚胎发育的代理。研究设计、规模、持续时间 这项前瞻性试点队列研究于 2023 年 3 月至 2023 年 8 月进行。分析了总共 30 个在发育第 5 天 （D5） 或第 6 天 （D6） 诊断为 PGT-A 的玻璃化人囊胚：n = 15 整倍体和 n = 15 非整倍体。最后，研究包括 21 个胚胎样本;其余 （n = 9） 由于测序前数据质量差 （n = 7） 或数据高度不一致 （n = 2） 而被排除在外。 参与者/材料、设置、方法 在加热和再扩增后，胚胎进行第二次滋养外胚层 （TE） 活检。然后将胚胎培养至第 11 天以评估其发育情况。通过 RNA-Seq 进行活检分析，研究了差异表达基因 （DEG），以比较未附着或附着在板上的胚胎：未附着的胚胎 （n = 12） 与附着的胚胎 （n = 9）。因此，我们还获得了胚胎的特异性转录组特征，基于第 11 天的板附着，具有持续植入的“理论”能力。主要结果和机会的作用 RNA-Seq 获得的数字核型与早期的 PGT-A 数据具有良好的一致性，灵敏度为 0.81，特异性为 0.83，Cohen's Kappa 为 0.66，ROC 下面积为 0.9。在基因水平上，在未附着胚胎与附着胚胎的比较中发现了 76 个具有统计学意义的 DEGs（Padj < 0.05;FC > 1） 的 1 个。为了解决这些差异的功能意义，根据 GO 和 KEGG 类别确定了显着失调的途径。未附着的囊胚的壁滋养外胚层 （TE） 显示 63 个显著失调的项，显示自噬、细胞凋亡、蛋白激酶和泛素样蛋白连接酶活性的上调，以及核糖体、剪接体、着丝粒、分离和染色体浓缩过程的下调。第 11 天仍附着在板上的胚胎的总体转录组特征（理论上具有更高的植入能力）由 501 个基因组成，包括：EMP2、AURKB、FOLR1、NOTCH3、LRP2、FZD5、MDH1、APOD、GPX8、COLEC12、HSPA1A、CMTM7、BEX3，它们与植入和胚胎发育有关（原始 P 值 < 0.05;收缩 LFC > 1.1）。 这些发现表明，在排除那些存在染色体改变的胚胎后，有可能识别出具有更强植入和发育能力的整倍体胚胎。局限性，谨慎的原因 这项研究包括样本量小、样本间显著的变异性以及 RNA 扩增的成功率低。此外，结构染色体异常不包括在内，因此无法诊断嵌合胚胎。TE 活检不能确保整个胚胎的染色体状态。体外发育的最长天数是第 11 天，在这一天附着在板上并不能明确指示植入能力和活力，这在本研究中没有测试。研究结果的更广泛影响 这项试点研究之后的短期目标是用更复杂异常的胚胎扩大样本量，并进行前瞻性体外临床前验证。在更遥远的将来，该技术可能会有临床应用，从而通过评估染色体内容和转录组学分析来提高临床结果。研究资金/利益争夺 瓦伦西亚竞争研究所 （IVACE） （IMIDCA/2022/39） 和瓦伦西亚政府 （CIACIF/2021/11） 支持本研究。A.C. 是 JUNO Genetics 的员工。他曾获得 IBSA 讲座和 Merck 讲座的酬金。他也是 IVIRMA Global 的小股东。其他作者没有需要声明的利益冲突。试验注册号 N/A。