目的:利用小鼠囊胚培养液探讨无创胚胎植入前非整倍体筛查(niPGT-A)的可行性,为优化和发展无创胚胎植入前遗传学筛查技术提供理论依据和数据支持。方法:本研究共收集11枚小鼠囊胚,同一囊胚分别取少量滋养外胚层细胞(TE)作为金标准、内细胞团(ICM)细胞作为阳性对照、胚胎培养液(BCM)作为待验证样本。所有胚胎样本基于全基因组测序数据进行染色体状态分析,并比较同一胚胎不同样本间染色体倍性一致性。结果:在11枚小鼠囊胚中,BCM、TE和ICM的PGT-A检出率分别为82.8%(9/11)、72.7%(8/11)和100%(11/11)。BCM与TE、BCM与ICM及TE与ICM的染色体一致性分别为85.7%(6/7),88.9%(8/9),87.5%(7/9),任意两组间差异无统计学意义(P＞0.05)。结论:在小鼠模型中,相比于同期活检的滋养外胚层细胞,利用囊胚培养液中的游离DNA行无创胚胎植入前非整倍体检测可获得更高的检出率和更为准确的染色体检测结果。
Objective: To explore the feasibility of non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) using mouse blastocyst culture medium, and to provide theoretical basis and data support for optimizing and developing non-invasive preimplantation genetic screening technology. Methods: A total of 11 mouse blastocysts were collected. A small amount of trophectoderm cells (TE) were taken as the gold standard, inner cell mass (ICM) cells as the positive control, and blastocyst culture medium (BCM) as the sample to be verified. All embryo samples were analyzed for chromosome status based on whole genome sequencing data, and the chromosome ploidy consistency between different samples of the same embryo was compared. Results: In 11 mouse blastocysts, the detection rates of PGT-A in BCM, TE and ICM were 82.8% (9/11), 72.7% (8/11) and 100% (11/11), respectively. The chromosome consistency between BCM and TE, BCM and ICM, TE and ICM was 85.7% (6/7), 88.9% (8/9), 87.5% (7/9), respectively. There was no significant difference between any two groups (P＞0.05). Conclusion: In mouse model, noninvasive preimplantation genetic testing for aneuploidy using cell free DNA in blastocyst culture medium can obtain higher detection rate and more accurate chromosome detection results than trophoblastic ectoderm cells biopsy at the same time.
[1] DuRQ, ZhaoDD,KangK, et al. A review of pre-implantation genetic testing technologies and applications[J]. Reprod Dev Med, 2022,7(1):20-31.
[2] Ji H, Zhang MQ, Zhou Q, et al. Trophectoderm biopsy is associated with adverse obstetric outcomes rather than neonatal outcomes[J]. BMC Pregnancy Childbirth, 2023,23(1):141.
[3] Li M, Kort J, Baker VL. Embryo biopsy and perinatal outcomes of singleton pregnancies: an analysis of 16, 246 frozen embryo transfer cycles reported in the Society for Assisted Reproductive Technology Clinical Outcomes Reporting System[J]. Am J Obstet Gynecol, 2021,224(5):500.e1-500.e18.
[4] Lledo B, Morales R, Ortiz JA, et al. Consistent results of non-invasive PGT-A of human embryos using two different techniques for chromosomal analysis[J]. Reprod Biomed Online, 2021,42(3):555-563.
[5] Gleicher N, Patrizio P, Mochizuki L, et al. Previously reported and here added cases demonstrate euploid pregnancies followed by PGT-a as “mosaic” as well as “aneuploid” designated embryos[J]. Reprod Biol Endocrinol, 2023,21(1):25.
[6] Cai L, Zeng Q, Gao C, et al. Majority of transferred mosaic embryos developed healthy live births revealed by a preclinical study using embryonic morphology assessment and noninvasive PGT-A on cell-free DNA in blastocoel fluid[J]. J Assist Reprod Genet, 2022,39(11):2483-2504.
[7] Popovic M, Dhaenens L, Boel A, et al. Chromosomal mosaicism in human blastocysts: the ultimate diagnostic dilemma[J]. Hum Reprod Update, 2020,26(3):313-334.
[8] Victor AR, Tyndall JC, Brake AJ, et al. One hundred mosaic embryos transferred prospectively in a single clinic: exploring when and why they result in healthy pregnancies[J]. Fertil Steril, 2019,111(2):280-293.
[9] Zore T, Kroener LL, Wang C, et al. Transfer of embryos with segmental mosaicism is associated with a significant reduction in live-birth rate[J]. Fertil Steril,2019,111(1):69-76.
[10] Hong B, Hao Y.The outcome of human mosaic aneuploid blastocysts after intrauterine transfer: a retrospective study[J]. Medicine (Baltimore), 2020,99(9):e18768.
[11] Stigliani S, Anserini P, Venturini P L, et al. Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation[J]. Hum Reprod, 2013,28(10):2652-2660.
[12] Palini S, Galluzzi L, De Stefani S, et al. Genomic DNA in human blastocoele fluid[J]. Reprod Biomed Online, 2013,26(6):603-610.
[13] Xu JJ, Fang R, Chen L, et al. Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization[J]. Proc Natl Acad Sci USA, 2016,113(42):11907-11912.
[14] Shitara A, Takahashi K, Goto M, et al. Cell-free DNA in spent culture medium effectively reflects the chromosomal status of embryos following culturing beyond implantation compared to trophectoderm biopsy[J]. PLoS One, 2021,16(2):e0246438.
[15] Hanson BM, Tao X, Hong KH, et al. Noninvasive preimplantation genetic testing for aneuploidy exhibits high rates of deoxyribonucleic acid amplification failure and poor correlation with results obtained using trophectoderm biopsy[J]. Fertil Steril, 2021,115(6):1461-1470.
[16] Capalbo A, Romanelli V, Patassini C, et al. Diagnostic efficacy of blastocoel fluid and spent media as sources of DNA for preimplantation genetic testing in standard clinical conditions[J]. Fertil Steril, 2018,110(5):870-879.e5.
[17] Huang L, Bogale B, Tang YQ, et al. Noninvasive preimplantation genetic testing for aneuploidy in spent medium may be more reliable than trophectoderm biopsy[J]. Proc Natl Acad Sci USA, 2019,116(28):14105-14112.
[18] Rubio C, Rienzi L, Navarro-Sánchez L, et al. Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing:concordance rate and clinical implications[J]. Fertil Steril, 2019,112(3):510-519.
[19] Rubio C, Navarro-Sánchez L, García-Pascual CM, et al. Multicenter prospective study of concordance between embryonic cell-free DNA and trophectoderm biopsies from 1301 human blastocysts[J]. Am J Obstet Gynecol, 2020,223(5):751.e1-751.e13.
[20] Wu YT, Dong ZH, Li C, et al. The effect of blastomere loss during frozen embryo transfer on the transcriptome of offspring’s umbilical cord blood[J]. Mol Biol Rep, 2020,47(11):8407-8417.
[21] Kotlarska M, Winiarczyk D, Florek W, et al. Blastomere removal affects homeostatic control leading to obesity in male mice[J]. Reproduction, 2021,161(1):61-72.
[22] Gardner DK, Lane M, Stevens J, et al. Reprint of: Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer[J]. Fertil Steril, 2019,112(4 Suppl1):e81-e84.
[23] Telugu BP, Pence L. Development of pre-implantation mammalian blastocyst[J]. Adv Anat Embryol Cell Biol, 2021,234:21-40.
[24] Leigh D, Cram DS, Rechitsky S, et al. PGDIS position statement on the transfer of mosaic embryos 2021[J]. Reprod Biomed Online, 2022,45(1):19-25.
[25] Leaver M, Wells D. Non-invasive preimplantation genetic testing (niPGT):the next revolution in reproductive genetics?[J]. Hum Reprod Update, 2020,26(1):16-42.
[26] Yeung QSY, Zhang YX, Chung JPW, et al. A prospective study of non-invasive preimplantation genetic testing for aneuploidies (NiPGT-A) using next-generation sequencing (NGS) on spent culture media (SCM)[J]. J Assist Reprod Genet, 2019,36(8):1609-1621.
[27] Deng CC, Liu JL, Liu S, et al. Maternal and fetal factors influencing fetal fraction: a retrospective analysis of 153, 306 pregnant women undergoing noninvasive prenatal screening[J]. Front Pediatr, 2023,11:1066178.
