This study used noninvasive, fluorescence lifetime imaging microscopy (FLIM)-based imaging of NADH and FAD to characterize the metabolic response of mouse embryos to short-term oxygen deprivation. We investigated the response to hypoxia at various preimplantation stages. Mouse oocytes and embryos were exposed to transient hypoxia by dropping the oxygen concentration in media from 5–0% over the course of ~1.5 h, then 5% O2 was restored. During this time, FLIM-based metabolic imaging measurements of oocyte/embryo cohorts were taken every 3 minutes. Experiments were performed in triplicate for oocytes and embryos at the 1- to 8-cell, morula, and blastocyst stages. Maximum hypoxia response for each of eight measured quantitative FLIM parameters was taken from the time points immediately before oxygen restoration. Metabolic profiles showed significant changes in response to hypoxia for all stages of embryo development. The response of the eight measured FLIM parameters to hypoxia was highly stage-dependent. Of the eight FLIM parameters measured, NADH and FAD intensity showed the most dramatic metabolic responses in early developmental stages. At later stages, however, other parameters, such as NADH fraction engaged and FAD lifetimes, showed greater changes. Metabolic parameter values generally returned to baseline with the restoration of 5% oxygen. Quantitative FLIM-based metabolic imaging was highly sensitive to metabolic changes induced by hypoxia. Metabolic response profiles to oxygen deprivation were distinct at different stages, reflecting differences in metabolic plasticity as preimplantation embryos develop.

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Mitochondrial (Mt) metabolism is pivotal to embryo viability, and oxygen (O2) availability is a key control parameter for Mt activity. Recent data suggest that ultra-low (2%) vs. low (5%) O2 may be beneficial for human embryos cultured from cleavage to blastocyst (i.e. from day 3 to days 5/6). However, whether these two O2 exposures result in different Mt metabolic profiles, and how these profiles may change from cleavage through blastulation are unknown. The present study was designed to address these knowledge gaps using a powerful new non-invasive system of time-lapse 'Metabolic Imaging' - fluorescence lifetime imaging microscopy (FLIM) - which measures NADH and FAD+. Human embryos were imaged from days 3 to 6 in 2% versus 5% O2 to acquire metabolic time courses for assessment of Mt function and to detect any Mt metabolic shifts during development. Frozen day 3 sibling embryos donated for research purposes under informed consent by IVF patients were allocated, after matching by day 3 morphology within each patient, to 2% vs. 5% O2 exposure (n=15 and 12, respectively). Thawed embryos were imaged individually in microwells that shared 50μL of equilibrated Global Total under oil. Comprehensive metabolic measurements were acquired every 2 hours and on day 6, embryos were classified as arrested/morula, poor or good quality blastocysts. To reduce effects of embryo heterogeneity, analyses were restricted to good quality blastocysts (n=15: 7 in 2% and 8 in 5%). After synchronizing to compaction times, metabolic time courses, averaged among embryos within each O2 tension group, were analyzed to detect metabolic shifts during development; and to determine whether decreasing O2 to 2% significantly impacted Mt metabolic function. Student's t-test with p<0.05 was used to assess significance. No significant differences in Mt metabolic function were observed for 2% vs. 5% O2 culture conditions. However, several distinct changes in metabolic state occurred during embryo development, including a marked decrease in NADH brightness from 0.47±0.08 to 0.34±0.04 (∼photons/μm2; p<0.05) during blastocoel expansion, and a decrease at the morula stage in the percentage of bound NADH from 22.6±0.2% to 21.5±0.1% (p<0.05). Our results indicate that metabolic state is similar for embryos cultured in 2% vs. 5% O2. However, this study, using this powerful new non-invasive method of assessing Mt state, has revealed significant metabolic shifts during embryo development. This work provides a foundation for further elucidating these potentially important metabolic shifts, with possible implications for developing a new non-invasive platform for embryo selection.

Mitochondria are cellular organelles that are required for ATP production, metabolism, and calcium homeostasis, and play a key role in oocyte and embryo development. In this study we aimed to determine whether metabolic imaging using FLIM to detect autofluorescence of NADH and FAD+ (key components of oxidative phosphorylation) can identify metabolic differences between normal oocytes and those with metabolic dysfunction. Experimental study. Oocytes from old mice (1-year old) (n=21) were compared to oocytes from young (12-week old) (n=37) mice as a model of mild oocyte dysfunction. Oocytes obtained from mice with global knockout of Clpp (n=55) were compared to wild type (WT) oocytes (n=52) as a model of severe oocyte dysfunction. CLPP (caseinolytic peptidase P) mediates mitochondrial unfolded protein response (mt-UPR) and helps maintain homeostasis in response to metabolic and cellular stress. CLPP is required for oocyte and early embryo development, and ClpP-knockout results in female infertility and accelerated reproductive aging. Cumulus oophorus complexes (COCs) were collected 48h after injection with 5IU pregnant mare serum gonadotropin (PMSG, Sigma), and germinal vesicle (GV) oocytes were isolated. Fluorescence lifetime imaging microscopy (FLIM) was used to measure the naturally occurring autofluorescence of NADH and FAD+ in individual oocytes. A total of 8 metabolic parameters were obtained from each measurement (4 for each fluorophore) that are sensitive to differences in metabolic state: short (T1) and long (T2) fluorescence lifetime, fluorescence brightness (IRR), and fraction of the molecule bound to enzyme (FB). Matlab was used for statistical analysis. In older oocytes compared to young ones, FAD+ short fluorescence time (T1) was longer (p<0.001) and fluorescence brightness (IRR) was lower (p<0.01), while NADH short and long fluorescence times (T1 and T2) were shorter (p<0.0001 for both), and brightness (IRR) and fraction bound (FB) were lower (p<0.0001 and p<0.05, respectively). In ClpP-knockout oocytes compared to WT, FAD+ short and long fluorescence times (T1 and T2) were longer (p<0.0001) and brightness (IRR) was higher (p<0.01), while NADH long fluorescence time (T2) was longer (p<0.0001), and fraction bound (FB) was smaller (p<0.0001). In this study we used metabolic imaging to characterize the metabolic state of oocytes with mild (old) and severe (ClpP-knockout) mitochondrial dysfunction. We found that multiple parameters exhibit strong differentiation between old vs young, and ClpP-knockout vs WT oocyte groups. Our findings suggest that FLIM could potentially be used as a non-invasive tool to assess metabolic efficiency and mitochondrial function of human oocytes and embryos.

Oocyte and embryo mitochondrial metabolism is a key determinant of viability in ART procedures; however, there are currently no methods of accurately assessing mitochondrial function in oocytes and embryos. NADH and FAD are naturally fluorescent and integral components of cellular respiration. Our aim was to develop a non-invasive method of assessing oocyte/embryo metabolism by measuring the fluorescence of NADH and FAD with Fluorescence Lifetime Imaging Microscopy (FLIM). We perform metabolic assessment of mitochondrial function in oocytes and embryos using FLIM, optimized for NADH and FAD fluorescence. We collect data over the course of embryo development from 1-cell to blastocyst to determine correlations between metabolic signatures and successful blastocyst development. We performed appropriate controls to verify that FLIM measurements reflect real physiological changes in metabolism, varying oxygen availability and using metabolic chemical inhibitors of the electron transport chain (ETC). Mouse embryos were imaged in an on-stage incubation system to allow for continuous monitoring during development. Comprehensive metabolic measurements were taken of embryos every 2h for 4 days. We varied oxygen concentration by exchanging premixed gases into the sample chamber with different oxygen concentrations, measuring metabolic responses with FLIM. We also used potassium cyanide, an ETC inhibitor, and FCCP, an ETC uncoupler, to actively alter NADH and FAD levels. Our results demonstrate that FLIM-based metabolic imaging is highly sensitive to differences in mitochondrial function. In monitoring metabolism during development, we observed distinct changes in metabolic profile as embryos grow. These shifts were highly conserved for all embryos that successfully developed to blastocyst. We observed the expected changes in NADH and FAD in response to O2 and chemical perturbations. This fundamental work establishes a framework and methods for non-invasively monitoring embryo metabolism. This could facilitate numerous new research studies into the role of metabolism in embryo development. These results also indicate promise for the use of metabolic imaging as a potential embryo selection tool.

Mitochondrial function is essential for reproduction. We hypothesized that the decreased viability of aging oocytes may be partly due to impaired mitochondrial function. We investigated key mitochondrial stress parameters in association with aging in mouse oocytes, and assessed mtDNA quantity and NADH/FAD fluorescence as potential invasive and non-invasive biomarkers of age-related changes, respectively. Experimental study. Mature (metaphase II) ocytes from old (12 months) and young (9 weeks) C57BL/6J mice were compared. Metabolic stress was assessed by determining the levels of reactive oxygen species (ROS) under baseline conditions and following H2O2 treatment (using carboxy-H2DCFDA fluorescent staining); and by quantifying expression of mitochondrial unfolded protein response (mt-UPR) genes (Clpp, Dnaja3, Haspd1, Haspe1) via qRT-PCR. Absolute mtDNA levels were quantified via cloning of mitochondria specific gene (Cox3) as a standard, followed by qPCR analysis of individual oocytes (20 young and 20 old). Fluorescence lifetime imaging microscopy (FLIM) was used to obtain non-invasive measurements of intracellular NADH and FAD. FLIM measurements were performed on individual oocytes (19 young and 14 old) in an on-stage incubator system to avoid environmental effects. Oocytes assessed by FLIM also underwent mtDNA quantification as described above. ROS levels in aged MII oocytes were higher following pretreatment with H2O2 (p<0.05). The expression of mt-UPR gene Hspd1 was also elevated in aged MII oocytes (p<0.05). Oocytes from old mice had significantly less mtDNA compared to young ones (58,222 +/- 15,429 copies/oocyte vs. 162,106 +/- 19,302 copies/oocyte [mean +/- SEM; p<0.01]). FLIM analysis showed a weak but statistically significant correlation with age and mtDNA copy number (p<0.05). Aging is associated with a significant increase in ROS levels in oocytes under stressful conditions and elevated expression of mitochondrial stress response gene Hspd1. Importantly, aged mouse oocytes have lower mtDNA levels that correlate with NADH/FAD levels detected by FLIM. Further delineation of mitochondrial changes associated with ageing may help the development of diagnostic biomarkers and therapeutic tools in assisted reproduction.

OBJECTIVE: Mitochondrial function is essential for reproduction. We hypothesized that the decreased viability of aging oocytes may be partly due to impaired mitochondrial function. We investigated key mitochondrial stress parameters in association with aging in mouse oocytes, and assessed mtDNA quantity and NADH/FAD fluorescence as potential invasive and non-invasive biomarkers of age-related changes, respectively. DESIGN: Experimental study.MATERIALS AND METHODS: Mature (metaphase II) ocytes from old (12 months) and young (9 weeks) C57BL/6J mice were compared. Metabolic stress was assessed by determining the levels of reactive oxygen species (ROS) under baseline conditions and following H2O2 treatment (using carboxy-H2DCFDA fluorescent staining); and by quantifying expression of mitochondrial unfolded protein response (mt-UPR) genes (Clpp, Dnaja3, Haspd1, Haspe1) via qRT-PCR. Absolute mtDNA levels were quantified via cloning of mitochondria specific gene (Cox3) as a standard, followed by qPCR analysis of individual oocytes (20 young and 20 old). Fluorescence lifetime imaging microscopy (FLIM) was used to obtain non-invasive measurements of intracellular NADH and FAD. FLIM measurements were performed on individual oocytes (19 young and 14 old) in an on-stage incubator system to avoid environmental effects. Oocytes assessed by FLIM also underwent mtDNA quantification as described above. RESULTS: ROS levels in aged MII oocytes were higher following pretreatment with H2O2 (p< 0.05). The expression of mt-UPR gene Hspd1 was also elevated in aged MII oocytes (p< 0.05 …