Voros Charalampos, Darlas Menelaos, Athanasiou Diamantis, Athanasiou Antonia, Athanasiou Aikaterini, Bananis Kyriakos, Papadimas Georgios, Tsimpoukelis Charalampos, Gkirgkinoudis Athanasios, Sapantzoglou Ioakeim, Papapanagiotou Ioannis, Vaitsis Dimitrios, Koulakmanidis Aristotelis-Marios, Topalis Vasileios, Thomakos Nikolaos, Theodora Marianna, Antsaklis Panagiotis, Chatzinikolaou Fotios, Dahl Hans Atli, Daskalakis Georgios, Loutradis Dimitrios
1st Department of Obstetrics and Gynecology, 'Alexandra' General Hospital, National and Kapodistrian University of Athens, 80 Vasilissis Sofias Avenue, 11528 Athens, Greece.
IVF Athens Reproduction Center, 15123 Maroussi, Greece.
Biomedicines. 2025 Aug 18;13(8):2010. doi: 10.3390/biomedicines13082010.
Preimplantation genetic testing for aneuploidy (PGT-A) is a popular approach in assisted reproductive technology that improves embryo selection and implantation rates. Traditional approaches rely on trophectoderm (TE) biopsy, which is an invasive procedure that might jeopardize embryo integrity and create technical constraints such as mosaicism-related misclassification. Non-invasive preimplantation genetic testing (niPGT) has emerged as a possible alternative, using embryonic cell-free DNA (cfDNA) extracted from wasted culture media or blastocoel fluid to assess chromosomal status without requiring direct embryo manipulation. This systematic study investigates the molecular mechanisms behind cfDNA release, its biological properties, and the technological concerns that influence its utilization in niPGT. We look at recent advances in next-generation sequencing (NGS), whole-genome amplification (WGA), and bioinformatic techniques that improve cfDNA-based aneuploidy detection. In addition, we compare the sensitivity, specificity, and concordance rates of niPGT to conventional TE biopsy, highlighting the major aspects impacting its diagnostic performance. The release of cfDNA from embryos is influenced by apoptotic and necrotic processes, active DNA shedding, and extracellular vesicle secretion, which results in fragmented chromosomal material of different qualities and quantities. While niPGT has shown promise as a noninvasive screening approach, significant variability in cfDNA yield, maternal DNA contamination, and sequencing biases all have an impact on test accuracy. Studies show that niPGT and TE biopsies have moderate-to-high concordance, although there are still issues in detecting mosaicism, segmental aneuploidies, and DNA degradation artifacts. NiPGT is a safer and less intrusive alternative to TE biopsy, with potential clinical benefits. However, technical advancements are required to improve cfDNA collecting procedures, reduce contamination, and improve sequencing accuracy. Additional large-scale validation studies are needed to create standardized methodologies and ensure that niPGT achieves the diagnostic reliability requirements required for widespread clinical deployment in IVF programs.
植入前非整倍体基因检测（PGT-A）是辅助生殖技术中一种常用的方法，可提高胚胎选择和着床率。传统方法依赖于滋养外胚层（TE）活检，这是一种侵入性操作，可能会损害胚胎完整性，并产生技术限制，如与嵌合体相关的错误分类。非侵入性植入前基因检测（niPGT）作为一种可能的替代方法出现了，它使用从废弃培养基或囊胚腔液中提取的胚胎游离DNA（cfDNA）来评估染色体状态，而无需直接对胚胎进行操作。这项系统研究调查了cfDNA释放背后的分子机制、其生物学特性以及影响其在niPGT中应用的技术问题。我们关注下一代测序（NGS）、全基因组扩增（WGA）和生物信息学技术的最新进展，这些进展提高了基于cfDNA的非整倍体检测。此外，我们比较了niPGT与传统TE活检的敏感性、特异性和一致性率，突出了影响其诊断性能的主要方面。胚胎中cfDNA的释放受凋亡和坏死过程、活性DNA脱落以及细胞外囊泡分泌的影响，这导致了不同质量和数量的染色体片段化物质。虽然niPGT作为一种非侵入性筛查方法显示出了前景，但cfDNA产量的显著变异性、母体DNA污染和测序偏差都对检测准确性有影响。研究表明，niPGT和TE活检具有中度到高度的一致性，尽管在检测嵌合体、节段性非整倍体和DNA降解伪像方面仍然存在问题。NiPGT是TE活检的一种更安全、侵入性更小的替代方法，具有潜在的临床益处。然而，需要技术进步来改进cfDNA采集程序、减少污染并提高测序准确性。还需要额外的大规模验证研究来创建标准化方法，并确保niPGT达到体外受精项目广泛临床应用所需的诊断可靠性要求。
