STUDY QUESTION Are there cell lineage-related differences in the apoptotic rates and differentiation capacity of human blastocysts diagnosed as euploid, mosaic, and aneuploid after preimplantation genetic testing for aneuploidy (PGT-A) based on concurrent copy number and genotyping analysis? SUMMARY ANSWER Trophectoderm (TE) cells of mosaic and aneuploid blastocysts exhibit significantly higher levels of apoptosis and significantly reduced differentiation capacity compared to those of euploid blastocysts. WHAT IS KNOWN ALREADY Embryos diagnosed as mosaic after PGT-A can develop into healthy infants, yet understanding the reasons behind their reproductive potential requires further research. One hypothesis suggests that mosaicism can be normalized through selective apoptosis and reduced proliferation of aneuploid cells, but direct evidence of these mechanisms in human embryos is lacking. Additionally, data interpretation from studies involving mosaic embryos has been hampered by retrospective analysis methods and the high incidence of false-positive mosaic diagnoses stemming from the use of poorly specific PGT-A platforms. STUDY DESIGN, SIZE, DURATION Prospective cohort study performing colocalization of cell-lineage and apoptotic markers by immunofluorescence (IF). We included a total of 64 human blastocysts donated to research on Day 5 or 6 post-fertilization (dpf) by 43 couples who underwent in vitro fertilization treatment with PGT-A at IVI-RMA Valencia between September 2019 and October 2022. A total of 27 mosaic blastocysts were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS The study consisted of two phases: Phase I (caspase-3, n = 53 blastocysts): n = 13 euploid, n = 22 mosaic, n = 18 aneuploid. Phase II (terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), n = 11 blastocysts): n = 2 euploid, n = 5 mosaic, n = 4 aneuploid. Following donation for research, vitrified blastocysts were warmed, cultured until re-expansion, fixed, processed for IF, and imaged using confocal microscopy. For each blastocyst, the following cell counts were conducted: total cells (DAPI+), TE cells (GATA3+), inner cell mass (ICM) cells (GATA3−/NANOG+), and apoptotic cells (caspase-3+ or TUNEL+). The incidence of apoptosis was calculated for each blastocyst by dividing the number of caspase-3+ cells (Phase I) or TUNEL+ cells (Phase II) by the number of TE or ICM cells. Statistical analysis was performed according to data type and distribution (P < 0.05 was considered statistically significant). MAIN RESULTS AND THE ROLE OF CHANCE Phase I: Mosaic blastocysts displayed a similar number of total cells (49.6 ± 15 cells at 5 dpf; 58.8 ± 16.9 cells at 6 dpf), TE cells (38.8 ± 13.7 cells at 5 dpf; 49.2 ± 16.2 cells at 6 dpf), and ICM cells (10.9 ± 4.2 cells at 5 dpf; 9.7 ± 7.1 cells at 6 dpf) compared to euploid and aneuploid blastocysts (P > 0.05). The proportion of TE cells retaining NANOG expression increased gradually from euploid blastocysts (9.7% = 63/651 cells at 5 dpf; 0% = 0/157 cells at 6 dpf) to mosaic blastocysts (13.1% = 104/794 cells at 5 dpf; 3.4% = 12/353 cells at 6 dpf) and aneuploid blastocysts (27.9% = 149/534 cells at 5 dpf; 4.6% = 19/417 cells at 6 dpf) (P < 0.05). At the TE level, caspase-3+ cells were frequently observed (39% = 901/2310 cells). The proportion of caspase-3+ TE cells was significantly higher in mosaic blastocysts (44.1% ± 19.6 at 5 dpf; 43% ± 16.8 at 6 dpf) and aneuploid blastocysts (45.9% ± 16.1 at 5 dpf; 49% ± 15.1 at 6 dpf) compared to euploid blastocysts (26.6% ± 16.6 at 5 dpf; 17.5% ± 14.8 at 6 dpf) (P < 0.05). In contrast, at the ICM level, caspase-3+ cells were rarely observed (1.9% = 11/596 cells), and only detected in mosaic blastocysts (2.6% = 6/232 cells) and aneuploid blastocysts (2.5% = 5/197 cells) (P > 0.05). Phase II: Consistently, TUNEL+ cells were only observed in TE cells (32.4% = 124/383 cells). An increasing trend was identified toward a higher proportion of TUNEL+ cells in the TE of mosaic blastocysts (37.2% ± 21.9) and aneuploid blastocysts (39% ± 41.7), compared to euploid blastocysts (23% ± 32.5), although these differences did not reach statistical significance (P > 0.05). LIMITATIONS, REASONS FOR CAUTION The observed effects on apoptosis and differentiation may not be exclusive to aneuploid cells. Additionally, variations in aneuploidies and unexplored factors related to blastocyst development and karyotype concordance may introduce potential biases and uncertainties in the results. WIDER IMPLICATIONS OF THE FINDINGS Our findings demonstrate a cell lineage-specific effect of aneuploidy on the apoptotic levels and differentiation capacity of human blastocysts. This contributes to unravelling the biological characteristics of mosaic blastocysts and supports the concept of clonal depletion of aneuploid cells in explaining their reproductive potential. STUDY FUNDING/COMPETING INTEREST(S) This work was funded by grants from Centro para el Desarrollo Tecnológico Industrial (CDTI) (20190022) and Generalitat Valenciana (APOTIP/2019/009). None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER N/A.

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人类嵌合胚胎的滋养外胚层细胞显示出凋亡水平增加和分化能力受损：关于其生殖命运的分子线索？

研究问题 基于并行拷贝数和基因分型分析，在植入前非整倍体基因检测 (PGT-A) 后诊断为整倍体、嵌合体和非整倍体的人类囊胚的凋亡率和分化能力是否存在与细胞谱系相关的差异？摘要答案 与整倍体囊胚相比，嵌合体和非整倍体囊胚的滋养外胚层 (TE) 细胞表现出显着更高的细胞凋亡水平和显着降低的分化能力。已知的情况 PGT-A 后诊断为嵌合体的胚胎可以发育成健康婴儿，但了解其生殖潜力背后的原因需要进一步研究。一种假设表明，嵌合现象可以通过选择性凋亡和减少非整倍体细胞的增殖来正常化，但人类胚胎中缺乏这些机制的直接证据。此外，涉及嵌合胚胎的研究的数据解释受到回顾性分析方法的阻碍，以及由于使用特异性差的 PGT-A 平台而导致假阳性嵌合诊断的高发生率。研究设计、规模、持续时间 通过免疫荧光 (IF) 进行细胞谱系和凋亡标记物共定位的前瞻性队列研究。我们纳入了 2019 年 9 月至 2022 年 10 月期间在巴伦西亚 IVI-RMA 接受 PGT-A 体外受精治疗的 43 对夫妇在受精后第 5 或 6 天 (dpf) 捐赠的总共 64 个人类囊胚。分析了 27 个嵌合囊胚。参与者/材料、背景、方法 该研究包括两个阶段： I 期（caspase-3，n = 53 个囊胚）：n = 13 个整倍体，n = 22 个嵌合体，n = 18 个非整倍体。 II 期（末端脱氧核苷酸转移酶 dUTP 缺口末端标记 (TUNEL)，n = 11 个囊胚）：n = 2 个整倍体，n = 5 个嵌合体，n = 4 个非整倍体。捐赠用于研究后，玻璃化囊胚被加热、培养直至重新膨胀、固定、IF 处理，并使用共聚焦显微镜成像。对于每个囊胚，进行以下细胞计数：总细胞（DAPI+）、TE细胞（GATA3+）、内细胞团（ICM）细胞（GATA3-/NANOG+）和凋亡细胞（caspase-3+或TUNEL+）。通过将 caspase-3+ 细胞（第一阶段）或 TUNEL+ 细胞（第二阶段）的数量除以 TE 或 ICM 细胞的数量来计算每个囊胚的细胞凋亡发生率。根据数据类型和分布进行统计分析（P＜0.05认为有统计学意义）。主要结果和机会的作用第一阶段：马赛克囊胚显示相似数量的总细胞（5 dpf 时为 49.6 ± 15 个细胞；6 dpf 时为 58.8 ± 16.9 个细胞）、TE 细胞（5 dpf 时为 38.8 ± 13.7 个细胞；49.2 ±与整倍体和非整倍体囊胚相比，6 dpf 时为 16.2 个细胞）和 ICM 细胞（5 dpf 时为 10.9 ± 4.2 个细胞；6 dpf 时为 9.7 ± 7.1 个细胞）（P > 0.05）。保留 NANOG 表达的 TE 细胞比例从整倍体囊胚（5 dpf 时 9.7% = 63/651 个细胞；6 dpf 0% = 0/157 个细胞）逐渐增加到嵌合囊胚（5 dpf 时 13.1% = 104/794 个细胞） ；3.4% = 6 dpf 时的 12/353 个细胞）和非整倍体囊胚（5 dpf 时的 27.9% = 149/534 个细胞；4.6% = 6 dpf 时的 19/417 个细胞）（P < 0.05）。在 TE 水平，经常观察到 caspase-3+ 细胞（39% = 901/2310 细胞）。嵌合囊胚中 caspase-3+ TE 细胞的比例显着较高（5 dpf 时为 44.1% ± 19.6；6 dpf 时为 43% ± 16.8）和非整倍体囊胚（5 dpf 时为 45.9% ± 16.1；6 dpf 时为 49% ± 15.1） dpf）与整倍体囊胚相比（5 dpf 时为 26.6% ± 16.6；6 dpf 时为 17.5% ± 14.8）（P < 0.05）。相反，在 ICM 水平上，很少观察到 caspase-3+ 细胞（1.9% = 11/596 个细胞），仅在嵌合囊胚（2.6% = 6/232 个细胞）和非整倍体囊胚（2.5% = 5/ 197个细胞）（P>0.05）。 II 期：一致地，仅在 TE 细胞中观察到 TUNEL+ 细胞（32.4% = 124/383 细胞）。与整倍体囊胚 (23% ± 32.5) 相比，嵌合囊胚 (37.2% ± 21.9) 和非整倍体囊胚 (39% ± 41.7) TE 中 TUNEL+ 细胞的比例呈增加趋势，尽管这些差异并没有增加达到统计学显着性（P>0.05）。局限性、注意原因 观察到的对细胞凋亡和分化的影响可能并非非整倍体细胞所独有。此外，非整倍体的变化以及与囊胚发育和核型一致性相关的未探索因素可能会在结果中引入潜在的偏差和不确定性。研究结果的更广泛意义我们的研究结果证明了非整倍性对人类囊胚的细胞凋亡水平和分化能力的细胞谱系特异性影响。这有助于揭示嵌合囊胚的生物学特性，并支持非整倍体细胞克隆耗竭的概念来解释其生殖潜力。研究经费/竞争利益 这项工作由 Centro para el Desarrollo Tecnológico Industrial (CDTI) (20190022) 和 Generalitat Valenciana (APOTIP/2019/009) 资助。所有作者都没有任何需要声明的利益冲突。试用注册号 不适用。caspase-3+细胞很少观察到（1.9% = 11/596 个细胞），仅在嵌合囊胚（2.6% = 6/232 个细胞）和非整倍体囊胚（2.5% = 5/197 个细胞）中检测到（P > 0.05） ）。 II 期：一致地，仅在 TE 细胞中观察到 TUNEL+ 细胞（32.4% = 124/383 细胞）。与整倍体囊胚 (23% ± 32.5) 相比，嵌合囊胚 (37.2% ± 21.9) 和非整倍体囊胚 (39% ± 41.7) TE 中 TUNEL+ 细胞的比例呈增加趋势，尽管这些差异并没有增加达到统计学显着性（P>0.05）。局限性、注意原因 观察到的对细胞凋亡和分化的影响可能并非非整倍体细胞所独有。此外，非整倍体的变化以及与囊胚发育和核型一致性相关的未探索因素可能会在结果中引入潜在的偏差和不确定性。研究结果的更广泛意义我们的研究结果证明了非整倍性对人类囊胚的细胞凋亡水平和分化能力的细胞谱系特异性影响。这有助于揭示嵌合囊胚的生物学特性，并支持非整倍体细胞克隆耗竭的概念来解释其生殖潜力。研究经费/竞争利益 这项工作由 Centro para el Desarrollo Tecnológico Industrial (CDTI) (20190022) 和 Generalitat Valenciana (APOTIP/2019/009) 资助。所有作者都没有任何需要声明的利益冲突。试用注册号 不适用。caspase-3+细胞很少观察到（1.9% = 11/596 个细胞），仅在嵌合囊胚（2.6% = 6/232 个细胞）和非整倍体囊胚（2.5% = 5/197 个细胞）中检测到（P > 0.05） ）。 II 期：一致地，仅在 TE 细胞中观察到 TUNEL+ 细胞（32.4% = 124/383 细胞）。与整倍体囊胚 (23% ± 32.5) 相比，嵌合囊胚 (37.2% ± 21.9) 和非整倍体囊胚 (39% ± 41.7) TE 中 TUNEL+ 细胞的比例呈增加趋势，尽管这些差异并没有增加达到统计学显着性（P>0.05）。局限性、注意原因 观察到的对细胞凋亡和分化的影响可能并非非整倍体细胞所独有。此外，非整倍体的变化以及与囊胚发育和核型一致性相关的未探索因素可能会在结果中引入潜在的偏差和不确定性。研究结果的更广泛意义我们的研究结果证明了非整倍性对人类囊胚的细胞凋亡水平和分化能力的细胞谱系特异性影响。这有助于揭示嵌合囊胚的生物学特性，并支持非整倍体细胞克隆耗竭的概念来解释其生殖潜力。研究经费/竞争利益 这项工作由 Centro para el Desarrollo Tecnológico Industrial (CDTI) (20190022) 和 Generalitat Valenciana (APOTIP/2019/009) 资助。所有作者都没有任何需要声明的利益冲突。试用注册号 不适用。所有作者都没有任何需要声明的利益冲突。试用注册号 不适用。所有作者都没有任何需要声明的利益冲突。试用注册号 不适用。