Ortega-Jaén David, Mora-Martinez Carlos, Capalbo Antonio, Mifsud Amparo, Boluda-Navarro Mireia, Mercader Amparo, Martín Ángel, Pardiñas María Luisa, Gil Julia, de Los Santos María José
IVIRMA Global Research Alliance, IVI Foundation, Health Research Institute La Fe, Valencia, Spain.
Department of Research, Health in Code, Valencia, Spain.
Hum Reprod. 2025 Feb 1;40(2):244-260. doi: 10.1093/humrep/deae265.
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?
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.
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.
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.
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.
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能否从RNA测序（RNA-Seq）数据中预测整倍体染色体构成，并识别与延长胚胎发育兼容的转录组图谱？
除了可能提示着床能力提高的胚胎转录组特征外，还能够获得与非整倍体植入前基因检测（PGT-A）相当的核型。
基于形态学和形态动力学对胚胎能力进行的传统评估缺乏分子层面的认识，且在预测倍性状态方面存在争议。了解胚胎转录组至关重要，因为基因表达会影响发育和着床。PGT提高了妊娠率，但当高质量的整倍体胚胎无法足月出生时，问题依然存在。事实上，只有约50%-60%的胚胎着床，其中10%会导致流产。包括RNA-Seq在内的综合方法有发现生殖能力分子标志物的潜力，理论上可与长达第14天的延长胚胎培养平台相结合，该平台可作为研究植入后阶段胚胎发育的替代方法。
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。共分析了30个在发育第5天（D5）或第6天（D6）进行过PGT-A诊断的玻璃化人囊胚：15个整倍体和15个非整倍体。最终，21个胚胎样本纳入研究；其余9个因测序前数据质量差（7个）或数据高度不一致（2个）被排除。
参与者/材料、设置、方法：解冻并重新扩张后，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，研究差异表达基因（DEG），以比较未附着或附着于培养板的胚胎：未附着胚胎（12个）与附着胚胎（9个）。因此，基于第11天在培养板上的附着情况，我们还获得了具有“理论”持续着床能力的胚胎的特定转录组特征。
通过RNA-Seq获得的数字核型与早期PGT-A数据显示出良好的一致性，灵敏度为0.81，特异性为0.83，Cohen's Kappa为0.66，ROC曲线下面积为0.9。在基因水平上，未附着与附着胚胎的比较中发现了76个具有统计学意义的DEG（Padj<0.05；FC>1）。为了探讨这些差异的功能影响，根据GO和KEGG类别确定了显著失调的通路。未附着囊胚的壁滋养外胚层（TE）显示出63个显著失调的条目，自噬、凋亡、蛋白激酶和泛素样蛋白连接酶活性上调，核糖体、剪接体、动粒（着丝粒）、分离和染色体凝聚过程下调。第11天仍附着在培养板上（理论上着床能力更高）的胚胎的总体转录组特征由501个基因组成，包括：EMP2、AURKB、FOLR1、NOTCH3、LRP2、FZD5、MDH1、APOD、GPX8、COLEC12、HSPA1A、CMTM7、BEX3，这些基因与着床和胚胎发育相关（原始P值<0.05；收缩LFC>1.1）。这些发现表明，在排除存在染色体改变的胚胎后，有可能识别出具有更强着床和发育能力的整倍体胚胎。
局限性、谨慎原因：本研究样本量小，样本间差异显著，RNA扩增成功率低。此外，未包括染色体结构异常，无法诊断嵌合胚胎。TE活检不能保证整个胚胎的染色体状态。体外发育的最长天数为第11天，且当天在培养板上的附着情况并不能明确指示着床能力和生存能力，本研究未对此进行测试。
这项试点研究的短期目标是扩大样本量，纳入更复杂异常的胚胎，并进行前瞻性体外临床前验证。在更长远的未来且取得最佳结果时，这项技术可能具有临床应用价值，从而通过评估染色体组成和转录组图谱提高临床疗效。
研究资金/利益冲突：瓦伦西亚企业竞争力研究所（IVACE）（IMIDCA/2022/39）和瓦伦西亚大区政府（CIACIF/2021/11）支持了本研究。A.C.是JUNO Genetics的员工。他因IBSA讲座和默克讲座获得了酬金。他还是IVIRMA Global的少数股东。其他作者无利益冲突声明。
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