背景:对于体外受精(IVF),活检胚泡的滋养外胚层(TE)细胞中的线粒体DNA(mtDNA)水平已被认为与细胞的发育潜力有关。然而,对于使用mtDNA水平作为预测IVF结局的可靠生物标志物,学者们达成了不同的意见.因此,本研究旨在评估线粒体拷贝数与胚胎发育特征和倍性的相关性。
方法:这项回顾性研究分析了胚胎的发育特征和活检的滋养外胚层细胞中的mtDNA水平。从2021年9月至2022年9月,使用延时监测和下一代测序进行分析。对符合纳入标准的88例接受IVF的患者进行了515个囊胚活检。使用所有记录的图像在授精后118小时评估胚胎形态动力学和形态。在第5天或第6天具有适当形态的胚泡接受TE活检和非整倍性植入前遗传学测试(PGT-A)。统计分析涉及广义估计方程,皮尔森的卡方检验,费希尔的精确检验,和Kruskal-Wallis测试,显著性水平设置为P<0.05。
结果:为了检查低和高核分裂的胚泡之间胚胎特征的差异,胚泡根据其线粒体分为四分位数。关于形态动力学特征,发现大多数发育动力学和观察到的卵裂畸形没有显着差异。然而,丝裂核第1组的囊胚在tPNf后达到3细胞期的时间(t3;中位数:14.4h)长于丝裂核第2组(中位数:13.8h),第二个细胞周期(CC2;中位数:11.7h)长于丝裂核第2组(中位数:11.3h)和第4组(中位数:11.4h;P<0.05)的囊胚。此外,4组的囊胚的整倍体率(22.6%)和非整倍体率(59.1%)低于其他组(39.6-49.3%和30.3-43.2%;P<0.05)。丝裂核组4的全染色体改变率(63.4%)高于丝裂核组1(47.3%)和2(40.1%;P<0.05)。使用多变量逻辑回归模型来分析选择性囊胚的核分裂和整倍体之间的关联。在考虑了可能影响结果的因素后,mitoscore仍与整倍体可能性呈负相关(校正OR=0.581,95%CI:0.396-0.854;P=0.006).
结论:囊胚具有不同水平的线粒体DNA,通过活检确定,通过延时成像观察到的早期植入前发育特征相似。然而,线粒体DNA水平可作为整倍体的独立预测因子.
BACKGROUND: For in vitro fertilization (IVF), mitochondrial DNA (mtDNA) levels in the trophectodermal (TE) cells of biopsied blastocysts have been suggested to be associated with the cells\' developmental potential. However, scholars have reached differing opinions regarding the use of mtDNA levels as a reliable biomarker for predicting IVF outcomes. Therefore, this study aims to assess the association of mitochondrial copy number measured by mitoscore associated with embryonic developmental characteristics and ploidy.
METHODS: This retrospective study analyzed the developmental characteristics of embryos and mtDNA levels in biopsied trophectodermal cells. The analysis was carried out using time-lapse monitoring and next-generation sequencing from September 2021 to September 2022. Five hundred and fifteen blastocysts were biopsied from 88 patients undergoing IVF who met the inclusion criteria. Embryonic morphokinetics and morphology were evaluated at 118 h after insemination using all recorded images. Blastocysts with appropriate morphology on day 5 or 6 underwent TE biopsy and preimplantation genetic testing for aneuploidy (PGT-A). Statistical analysis involved generalized estimating equations, Pearson\'s chi-squared test, Fisher\'s exact test, and Kruskal-Wallis test, with a significance level set at P < 0.05.
RESULTS: To examine differences in embryonic characteristics between blastocysts with low versus high mitoscores, the blastocysts were divided into quartiles based on their mitoscore. Regarding morphokinetic characteristics, no significant differences in most developmental kinetics and observed cleavage dysmorphisms were discovered. However, blastocysts in mitoscore group 1 had a longer time for reaching 3-cell stage after tPNf (t3; median: 14.4 h) than did those in mitoscore group 2 (median: 13.8 h) and a longer second cell cycle (CC2; median: 11.7 h) than did blastocysts in mitoscore groups 2 (median: 11.3 h) and 4 (median: 11.4 h; P < 0.05). Moreover, blastocysts in mitoscore group 4 had a lower euploid rate (22.6%) and a higher aneuploid rate (59.1%) than did those in the other mitoscore groups (39.6-49.3% and 30.3-43.2%; P < 0.05). The rate of whole-chromosomal alterations in mitoscore group 4 (63.4%) was higher than that in mitoscore groups 1 (47.3%) and 2 (40.1%; P < 0.05). A multivariate logistic regression model was used to analyze associations between the mitoscore and euploidy of elective blastocysts. After accounting for factors that could potentially affect the outcome, the mitoscore still exhibited a negative association with the likelihood of euploidy (adjusted OR = 0.581, 95% CI: 0.396-0.854; P = 0.006).
CONCLUSIONS: Blastocysts with varying levels of mitochondrial DNA, identified through biopsies, displayed similar characteristics in their early preimplantation development as observed through time-lapse imaging. However, the mitochondrial DNA level determined by the mitoscore can be used as a standalone predictor of euploidy.