Metabolic imaging with the use of fluorescence lifetime imaging microscopy (FLIM) accurately detects mitochondrial dysfunction in mouse oocytes

Abstract

OBJECTIVE: To determine whether metabolic imaging using fluorescence lifetime imaging microscopy (FLIM) identifies metabolic differences between normal oocytes and those with metabolic dysfunction. DESIGN: Experimental study. SETTING: Academic research laboratories. PATIENT(S): None. MAIN INTERVENTIONS: Oocytes from mice with global knockout of Clpp (caseinolytic peptidase P; n=52) were compared to wild type (WT) oocytes (n=55) as a model of severe oocyte dysfunction. Oocytes from old mice (1-year old; n=29) were compared to oocytes from young mice (12-week-old; n=35) as a model of mild oocyte dysfunction. MAIN OUTCOME MEASURE(S): FLIM was used to measure the naturally occurring NADH and FAD autofluorescence in individual oocytes. Eight metabolic parameters were obtained from each measurement (4 per fluorophore): short (τ1) and long (τ2) fluorescence lifetime, fluorescence intensity (I), and fraction of the molecule engaged with enzyme (F). ROS levels and blastocyst development rates were measured to assess illumination safety. RESULTS: In Clpp-knockout oocytes compared to WT, FAD τ1 and τ2 were longer (p<0.0001) and I was higher (p<0.01), NADH τ2 was longer (p<0.0001), and F was lower (p<0.0001). In older oocytes compared to young ones, FAD τ1 was longer (p<0.001) and I was lower (p<0.01), while NADH τ1and τ2were shorter (p<0.0001 for both), I and F were lower (p<0.0001 and p<0.05, respectively). FLIM did not affect ROS levels or blastocyst development rates. CONCLUSIONS: FLIM parameters exhibit strong differentiation between Clpp-knockout vs WT, and old vs young oocytes. FLIM could potentially be used as a non-invasive tool to assess mitochondrial function in oocytes.

Metabolic imaging via fluorescence lifetime imaging microscopy for egg and embryo assessment

“…These parameters are highly sensitive to differences and deficiencies in the metabolic state of oocytes and embryos. In a recent proof-of-concept study, mouse oocytes with significant metabolic dysfunction due to a mutation in a mitochondrial stress response gene exhibited highly significantly different FLIM parameter values compared with wild-type (normal) oocytes (40). Within the same experimental system, mtDNA copy number was only marginally different between the groups.…”

“…Since the metabolism of embryos and oocytes is central to their viability, and since FLIM provides a means of noninvasively and quantitatively measuring metabolism, FLIM is a promising technique for assessing oocyte and embryo viability. We have recently carried out a number of studies on mouse oocytes and embryos to evaluate the safety and potential utility of FLIM within this context (40).…”

“…Within the same experimental system, mtDNA copy number was only marginally different between the groups. FLIM was also used to compare oocytes from old (1-year-old) versus young mice as a model for mild metabolic dysfunction and showed highly significant differences; mtDNA copy number was not significantly different between the groups (40). Furthermore, FLIM metabolic parameters change over the course of preimplantation embryo development, as the embryo's metabolism reconfigures.…”

“…Preliminary animal studies on oocytes and embryos indicate sensitivity to metabolic differences that are relevant in fertility. Recently, Sanchez et al (40) showed that FLIM measurements were able to sensitively distinguish between metabolic states that are known to be different: [1] old versus young mice (40) and [2] oocytes from wildtype and knockout mice for the gene, Clpp, a mutation affecting metabolic function and fertility (41,42).…”

Mitochondria as a biomarker for IVF outcome

“…Besides direct measurement of cellular DNA content, it is worth mentioning that there have also been forays into assessment of mitochondrial function through other methods. One such study utilized Fluorescence Lifetime Imaging Microscopy (FLIM) as a noninvasive means of detecting mitochondrial dysfunction in murine oocytes (Sanchez et al 2018). This technique measures inherent NADH and FAD autofluorescence in cells.…”