L26/P-287 Impact of PGT-A timing on the relationship between mitochondrial DNA content and blastocyst quality: a comparison of fresh and cryopreserved-warmed embryos

Abstract Study question Does the timing of trophectoderm biopsy—performed on fresh versus post-warming cryopreserved blastocysts—alter the observable biological association between mitochondrial DNA (mtDNA) content and morphological quality? Summary answer High-quality fresh embryos exhibited significantly lower mtDNA (p = 0.032), but this biological signal was notably attenuated and lost significance following cryopreservation and warming What is known already Elevated mitochondrial DNA (mtDNA) in blastocysts is increasingly interpreted as a compensatory response to metabolic stress or cellular aging rather than a direct predictor of implantation success. Despite its potential as a marker of embryo integrity, clinical findings remain remarkably inconsistent across the literature. We hypothesized that this heterogeneity stems from the timing of PGT-A. Specifically, the physiological impact of cryopreservation and warming may introduce biological noise, potentially masking the subtle endogenous signals that characterize an embryo’s fresh state Study design, size, duration This retrospective cohort study analyzed 753 blastocysts undergoing NGS-based PGT-A during a focused 2025 period. By restricting the analysis to a single year, we minimized potential technical variability in laboratory protocols. Embryos were categorized by biopsy timing: fresh (n = 607) versus cryopreserved-warmed (n = 146). This rigorous, single-center design ensures that observed mtDNA differences reflect biological reality rather than longitudinal shifts in culture conditions Participants/materials, setting, methods MtDNA content was quantified via NGS and log-transformed to ensure statistical normality. Blastocyst quality scores (BQS) were treated as a continuous variable. Linear regression models evaluated the association between BQS and log(mtDNA), adjusting for maternal age as a covariate. We specifically tested for interaction effects between embryo quality and clinical groups—fresh versus cryopreserved—to ensure findings remained robust. All analyses were performed on high-resolution NGS data from a single-center laboratory Main results and the role of chance In the fresh PGT-A group (n = 607), a significant inverse association was observed between embryo quality and mtDNA content (beta = -0.0022, p = 0.0319), suggesting that lower mtDNA levels characterize high-quality, undisturbed blastocysts. In contrast, this association was not statistically significant in the cryopreserved-warmed group (n = 146, p = 0.1884), despite showing a similar negative trend (beta = -0.0038). Marginal means plots confirmed that this inverse relationship remained directionally consistent across all maternal age groups, with no significant interaction between age and quality (p > 0.05). However, the overall explanatory power was low (R-squared < 0.02), indicating that mtDNA content reflects subtle metabolic variations rather than serving as a definitive standalone selection marker. These findings suggest that the freezing-warming process introduces technical or biological variability that may mask endogenous mitochondrial signals. Consequently, the timing of PGT-A is a critical factor for the clinical interpretation of mtDNA; its association with embryo morphology is most evident in the fresh state and appears attenuated following cryopreservation Limitations, reasons for caution The retrospective design and smaller cryo-warmed sample (n = 146) may have limited statistical power to detect subtle associations post-warming. Additionally, the low R-squared values indicate that mtDNA should be viewed as a supplementary metabolic marker rather than a primary selection tool, requiring further validation in larger, prospective cohorts Wider implications of the findings Our results suggest that mtDNA signals of embryo integrity are context-dependent, appearing most transparent in fresh states but obscured by cryopreservation. This provides a potential explanation for existing literature inconsistencies. Accounting for PGT-A timing could refine mitochondrial biomarkers, moving toward more nuanced, individualized embryo assessment. Trial registration number No