STUDY QUESTION Does fluorescence lifetime imaging microscopy (FLIM)-based metabolic imaging assessment of human blastocysts prior to frozen transfer correlate with pregnancy outcomes? SUMMARY ANSWER FLIM failed to distinguish consistent patterns in mitochondrial metabolism between blastocysts leading to pregnancy compared to those that did not. WHAT IS KNOWN ALREADY FLIM measurements provide quantitative information on NAD(P)H and flavin adenine dinucleotide (FAD+) concentrations. The metabolism of embryos has long been linked to their viability, suggesting the potential utility of metabolic measurements to aid in selection. STUDY DESIGN, SIZE, DURATION This was a pilot trial enrolling 121 IVF couples who consented to have their frozen blastocyst measured using non-invasive metabolic imaging. After being warmed, 105 couples’ good-quality blastocysts underwent a 6-min scan in a controlled temperature and gas environment. FLIM-assessed blastocysts were then transferred without any intervention in management. PARTICIPANTS/MATERIALS, SETTING, METHODS Eight metabolic parameters were obtained from each blastocyst (4 for NAD(P)H and 4 for FAD): short and long fluorescence lifetime, fluorescence intensity, and fraction of the molecule engaged with enzyme. The redox ratio (intensity of NAD(P)H)/(intensity of FAD) was also calculated. FLIM data were combined with known metadata and analyzed to quantify the ability of metabolic imaging to differentiate embryos that resulted in pregnancy from embryos that did not. De-identified discarded aneuploid human embryos (n = 158) were also measured to quantify correlations with ploidy status and other factors. Statistical comparisons were performed using logistic regression and receiver operating characteristic (ROC) curves with 5-fold cross-validation averaged over 100 repeats with random sampling. AUC values were used to quantify the ability to distinguish between classes. MAIN RESULTS AND THE ROLE OF CHANCE No metabolic imaging parameters showed significant differences between good-quality blastocysts resulting in pregnancy versus those that did not. A logistic regression using metabolic data and metadata produced an ROC AUC of 0.58. In contrast, robust AUCs were obtained when classifying other factors such as comparison of Day 5 (n = 64) versus Day 6 (n = 41) blastocysts (AUC = 0.78), inner cell mass versus trophectoderm (n = 105: AUC = 0.88) and aneuploid (n = 158) versus euploid and positive pregnancy embryos (n = 108) (AUC = 0.82). LIMITATIONS, REASONS FOR CAUTION The study protocol did not select which embryo to transfer and the cohort of 105 included blastocysts were all high quality. The study was also limited in number of participants and study sites. Increased power and performing the trial in more sites may have provided a stronger conclusion regarding the merits of the use of FLIM clinically. WIDER IMPLICATIONS OF THE FINDINGS FLIM failed to distinguish consistent patterns in mitochondrial metabolism between good-quality blastocysts leading to pregnancy compared to those that did not. Blastocyst ploidy status was, however, highly distinguishable. In addition, embryo regions and embryo day were consistently revealed by FLIM. While metabolic imaging detects mitochondrial metabolic features in human blastocysts, this pilot trial indicates it does not have the potential to serve as an effective embryo viability detection tool. This may be because mitochondrial metabolism plays an alternative role post-implantation. STUDY FUNDING/COMPETING INTEREST(S) This study was sponsored by Optiva Fertility, Inc. Boston IVF contributed to the clinical site and services. Becker Hickl, GmbH, provided the FLIM system on loan. T.S. was the founder and held stock in Optiva Fertility, Inc., and D.S. and E.S. had options with Optiva Fertility, Inc., during this study. TRIAL REGISTRATION NUMBER The study was approved by WCG Connexus IRB (Study Number 1298156).

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人类胚胎的代谢成像可预测倍性状态，但与临床妊娠结果无关：一项试点试验

研究问题 冷冻移植前基于荧光寿命成像显微镜 (FLIM) 的人类囊胚代谢成像评估是否与妊娠结局相关？摘要答案 FLIM 未能区分导致妊娠的囊胚与未妊娠的囊胚之间线粒体代谢的一致模式。已知信息 FLIM 测量可提供 NAD(P)H 和黄素腺嘌呤二核苷酸 (FAD+) 浓度的定量信息。胚胎的新陈代谢长期以来一直与其活力相关，这表明代谢测量在帮助选择方面具有潜在的效用。研究设计、规模、持续时间 这是一项试点试验，招募了 121 对 IVF 夫妇，他们同意使用非侵入性代谢成像来测量冷冻囊胚。加热后，105 对夫妇的优质囊胚在受控温度和气体环境中接受了 6 分钟的扫描。然后将经过 FLIM 评估的囊胚进行移植，无需任何管理干预。参与者/材料、设置、方法 从每个囊胚中获得八个代谢参数（4 个用于 NAD(P)H，4 个用于 FAD）：短荧光寿命和长荧光寿命、荧光强度以及与酶结合的分子分数。还计算了氧化还原比（NAD(P)H 的强度）/（FAD 的强度）。将 FLIM 数据与已知的元数据相结合并进行分析，以量化代谢成像区分妊娠胚胎和未妊娠胚胎的能力。还测量了去鉴定的废弃非整倍体人类胚胎（n = 158），以量化与倍性状态和其他因素的相关性。使用逻辑回归和接受者操作特征 (ROC) 曲线进行统计比较，并使用随机抽样重复 100 次以上的 5 倍交叉验证进行平均。AUC 值用于量化区分类别的能力。主要结果和机会的作用 代谢成像参数没有显示出妊娠的优质囊胚与未妊娠的囊胚之间存在显着差异。使用代谢数据和元数据的逻辑回归得出的 ROC AUC 为 0.58。相比之下，在对其他因素进行分类时获得了稳健的 AUC，例如第 5 天 (n = 64) 与第 6 天 (n = 41) 囊胚 (AUC = 0.78)、内细胞团与滋养外胚层 (n = 105) 的比较：AUC = 0.88 ）和非整倍体（n = 158）与整倍体和阳性妊娠胚胎（n = 108）（AUC = 0.82）。局限性和注意事项 该研究方案没有选择移植哪个胚胎，并且 105 个胚胎队列中的囊胚都是高质量的。该研究的参与者数量和研究地点也受到限制。增加功率并在更多地点进行试验可能会为临床使用 FLIM 的优点提供更有力的结论。研究结果的更广泛意义 FLIM 未能区分导致妊娠的优质囊胚与未妊娠的囊胚之间线粒体代谢的一致模式。然而，囊胚倍性状态是高度可区分的。此外，FLIM 一致地揭示了胚胎区域和胚胎日。虽然代谢成像检测人类囊胚中的线粒体代谢特征，但该试点试验表明它不具备作为有效的胚胎活力检测工具的潜力。这可能是因为线粒体代谢在植入后发挥着替代作用。研究经费/竞争利益 本研究由 Optiva Fertility, Inc. 赞助。波士顿 IVF 为临床地点和服务做出了贡献。Becker Hickl, GmbH 借用了 FLIM 系统。TS 是 Optiva Fertility, Inc. 的创始人并持有其股票，在本研究期间，DS 和 ES 拥有 Optiva Fertility, Inc. 的期权。试验注册号 该研究得到了 WCG Connexus IRB 的批准（研究编号 1298156）。