目的:荧光寿命成像显微镜(FLIM)可以检测卵丘细胞(CC)样品的代谢状态与相应胚胎的临床结局之间的关联吗?
结论:FLIM可以检测与导致临床妊娠的相应胚胎相关的CC代谢的显着变化。
背景:已知CC和卵母细胞代谢协同作用是获得发育能力所必需的。然而,反映卵母细胞活力和胚胎发育能力的可靠的CC生物标志物尚未建立。CC代谢的定量测量可用于辅助评估ART中的卵母细胞和胚胎质量。
方法:进行了一项前瞻性观察性研究。总的来说,纳入了从2018年2月至2020年5月在三级护理中心接受常规授精或ICSIIVF的223例患者。没有应用排除标准。
方法:该队列的平均产妇年龄为36.5±4.4岁,平均卵母细胞产量为16.9(范围1-50)。在取卵并玻璃化后,从每位患者收集一到四个CC簇。使用FLIM评估CC代谢状态以测量分子NAD(P)H和FAD+的自发荧光,对多种代谢途径至关重要。用其相应的卵母细胞和相关胚胎跟踪CC簇。患者年龄,第3天和第5/6天胚胎形态等级,并记录具有可追溯命运的胚胎的临床结局.为每个CC簇获得9个FLIM定量参数。我们调查了FLIM参数与患者产妇年龄之间的关联,胚胎形态等级,倍性,和临床结果,其中错误发现率P值<0.05被认为具有统计学意义。
结果:共收集了来自223例患者的851个卵丘-卵母细胞复合体的851个CC簇。在这些CC集群中,623使用FLIM成像。测得的CCFLIM参数均与第3天形态等级或相应胚胎的倍性相关,但FAD+FLIM参数与囊胚形态等级显著相关。与导致临床妊娠的胚胎相关的CC簇的FAD+FLIM参数(FAD+参与分数和短寿命)与没有的差异显著,以及与导致活产的胚胎相关的CC簇,与未导致活产的胚胎相比。
结论:我们的数据基于年龄较大的患者群体中相对较少数量的可追溯胚胎。此外,我们仅评估每位患者1~4个卵母细胞的CCs.未来的工作在更年轻的患者人群与更多的可追溯的胚胎,以及测量来自每个患者的所有卵母细胞的CC的代谢状态,将更好地了解该技术在卵母细胞/胚胎选择中的潜在用途。
结论:通过FLIM的代谢成像能够检测CC代谢与母亲年龄的关联,并检测与卵母细胞相关的CC代谢的变化,从而导致导致临床妊娠和活产的胚胎。我们的发现表明,CCs的FLIM可以作为一种新的方法来帮助评估临床ART中的卵母细胞和胚胎发育能力。
背景:美国国立卫生研究院授予NIHR01HD092550-03(至C.R.,和D.J.N.)。Becker和HicklGmbH以及波士顿电子公司赞助了FLIM设备贷款的研究。D.J.N.和C.R.是专利US20170039415A1的发明人。
背景:不适用。
OBJECTIVE: Can fluorescence lifetime imaging microscopy (FLIM) detect associations between the metabolic state of cumulus cell (CC) samples and the clinical outcome of the corresponding embryos?
CONCLUSIONS: FLIM can detect significant variations in the metabolism of CC associated with the corresponding embryos that resulted in a clinical pregnancy versus those that did not.
BACKGROUND: CC and oocyte metabolic cooperativity are known to be necessary for the acquisition of developmental competence. However, reliable CC biomarkers that reflect oocyte viability and embryo developmental competency have yet to be established. Quantitative measures of CC metabolism could be used to aid in the evaluation of oocyte and embryo quality in ART.
METHODS: A prospective observational study was carried out. In total, 223 patients undergoing IVF with either conventional insemination or ICSI at a tertiary care center from February 2018 to May 2020 were included, with no exclusion criteria applied.
METHODS: This cohort had a mean maternal age of 36.5 ± 4.4 years and an average oocyte yield of 16.9 (range 1-50). One to four CC clusters from each patient were collected after oocyte retrieval and vitrified. CC metabolic state was assessed using FLIM to measure the autofluorescence of the molecules NAD(P)H and FAD+, which are essential for multiple metabolic pathways. CC clusters were tracked with their corresponding oocytes and associated embryos. Patient age, Day 3 and Day 5/6 embryo morphological grades, and clinical outcomes of embryos with traceable fate were recorded. Nine FLIM quantitative parameters were obtained for each CC cluster. We investigated associations between the FLIM parameters and patient maternal age, embryo morphological rank, ploidy, and clinical outcome, where false discovery rate P-values of <0.05 were considered statistically significant.
RESULTS: A total of 851 CC clusters from 851 cumulus-oocyte complexes from 223 patients were collected. Of these CC clusters, 623 were imaged using FLIM. None of the measured CC FLIM parameters were correlated with Day 3 morphological rank or ploidy of the corresponding embryos, but FAD+ FLIM parameters were significantly associated with morphological rank of blastocysts. There were significant differences for FAD+ FLIM parameters (FAD+ fraction engaged and short lifetime) from CC clusters linked with embryos resulting in a clinical pregnancy compared with those that did not, as well as for CC clusters associated with embryos that resulted in a live birth compared those that did not.
CONCLUSIONS: Our data are based on a relatively low number of traceable embryos from an older patient population. Additionally, we only assessed CCs from 1 to 4 oocytes from each patient. Future work in a younger patient population with a larger number of traceable embryos, as well as measuring the metabolic state of CCs from all oocytes from each patient, would provide a better understanding of the potential utility of this technology for oocyte/embryo selection.
CONCLUSIONS: Metabolic imaging via FLIM is able to detect CC metabolic associations with maternal age and detects variations in the metabolism of CCs associated with oocytes leading to embryos that result in a clinical pregnancy and a live birth versus those that do not. Our findings suggest that FLIM of CCs may be used as a new approach to aid in the assessment of oocyte and embryo developmental competence in clinical ART.
BACKGROUND: National Institutes of Health grant NIH R01HD092550-03 (to C.R., and D.J.N.). Becker and Hickl GmbH and Boston Electronics sponsored research with the loaning of equipment for FLIM. D.J.N. and C.R. are inventors on patent US20170039415A1.
BACKGROUND: N/A.