Optical imaging detects metabolic signatures associated with oocyte quality†

Abstract

Oocyte developmental potential is intimately linked to metabolism. Existing approaches to measure metabolism in the cumulus oocyte complex (COC) do not provide information on the separate cumulus and oocyte compartments. Development of an assay that achieves this may lead to an accurate diagnostic for oocyte quality. Optical imaging of the autofluorescent cofactors reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD) provides a spatially resolved indicator of metabolism via the optical redox ratio (FAD/[NAD(P)H + FAD]). This may provide an assessment of oocyte quality. Here, we determined whether the optical redox ratio is a robust methodology for measuring metabolism in the cumulus and oocyte compartments compared with oxygen consumption in the whole COC. We also determined whether optical imaging could detect metabolic differences associated with poor oocyte quality (etomoxir-treated). We used confocal microscopy to measure NAD(P)H and FAD, and extracellular flux to measure oxygen consumption. The optical redox ratio accurately reflected metabolism in the oocyte compartment when compared with oxygen consumption (whole COC). Etomoxir-treated COCs showed significantly lower levels of NAD(P)H and FAD compared to control. We further validated this approach using hyperspectral imaging, which is clinically compatible due to its low energy dose. This confirmed lower NAD(P)H and FAD in etomoxir-treated COCs. When comparing hyperspectral imaged vs non-imaged COCs, subsequent preimplantation development and post-transfer viability were comparable. Collectively, these results demonstrate that label-free optical imaging of metabolic cofactors is a safe and sensitive assay for measuring metabolism and has potential to assess oocyte developmental competence.

Keywords: FAD; NAD(P)H; autofluorescence; cellular metabolism; non-invasive; oocyte assessment; optical imaging; optical redox ratio; oxygen consumption rate.

Figure 1

Optical imaging of autofluorescent metabolic cofactors in immature COCs reflects changes in oxygen consumption rate. Metabolism was measured in immature COCs in response to oligomycin (oligo: 2.0 μM), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP: 1 μM) and Rotenone/antimycin A (Rot/AA: 2.5 μM). The metabolic response to these inhibitors of the electron transport chain was measured by either laser scanning confocal microscopy (intracellular NAD(P)H and FAD) or extracellular flux analysis (oxygen consumption rate). Representative images of NAD(P)H and FAD autofluorescence are shown in (A). The intensity of NAD(P)H (B and E) and FAD (C and F) was quantified for the oocyte (B and C) and cumulus cells (E and F). The optical redox ratio (FAD / [NAD(P)H + FAD]) was calculated for the oocyte (D) and cumulus cells (G) as an indicator of overall metabolic activity. The optical redox ratio for the oocyte and cumulus cell compartments was compared with the oxygen consumption rate for whole COCs (H). Data are presented as mean ± SEM; optical redox ratio: n = 12 COCs per drug treatment, four independent experimental replicates; OCR: n = 16 wells (20 COCs/well; OCR was normalized using the number of COCs per well and presented as pmol/min/COC), four independent experimental replicates. Data were analyzed either by a Kruskal-Wallis with Dunn multiple comparison test (B and D) or a one-way ANOVA with Holm-Šídák multiple comparison test (C and E–G). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. Scale bar = 80 μm.

Figure 2

Optical imaging of metabolic cofactors in mature COCs is associated with oocyte quality. Metabolism was measured in mature COCs in response to oligomycin (oligo: 2.0 μM), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP: 1 μM) and Rotenone/antimycin A (Rot/AA: 2.5 μM). In a separate cohort, COCs were matured in vitro in the absence or presence of etomoxir, an inhibitor of fatty acid metabolism (β-oxidation). The metabolic response to inhibitors/uncoupler (A–F) or etomoxir (G–L) was measured by laser scanning confocal microscopy (intensity of NAD(P)H and FAD autofluorescence). The intensity of NAD(P)H (A, D, G, and J) and FAD (B, E, H, and K) was quantified in the oocyte (A, B, G, and H) and cumulus cells (D, E, J, and K). The optical redox ratio (FAD / [NAD(P)H + FAD]) was calculated as indicator of metabolic activity in oocytes (C and I) and cumulus cells (F and L). The effect of inhibiting fatty acid metabolism during IVM on oocyte developmental competence was assessed by subsequent development to the blastocyst stage (M; blastocyst-rate calculated from starting oocyte number). Data were analyzed by either a Kruskal-Wallis with Dunn multiple comparison test (B) or a one-way ANOVA with Holm-Šídák multiple comparison test (A and C–F) or two-tailed unpaired Student t-test (G and M) or Mann–Whitney test (H–L). Data presented as mean ± SEM, three independent experimental replicates; n = 12 COCs per treatment (A–F); n = 33 for control COCs, n = 32 for etomoxir-treated COCs (G–L); n = 174 and 138 embryos developed from control and etomoxir-treated COCs, respectively (M). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

Figure 3

Hyperspectral microscopy detects metabolic changes associated with oocyte quality. COCs were matured in vitro in the absence or presence of etomoxir. Etomoxir is a known inhibitor of fatty acid metabolism (β-oxidation) and negatively affects oocyte developmental competence (see Figure 2M). Matured COCs were imaged using hyperspectral microscopy. Autofluorescence intensity was quantified for the oocyte (A–D) and cumulus cells (E–H) in hyperspectral channels that match the spectral properties of NAD(P)H: Channel 1 (A and E) and Channel 2 (B and F); and FAD: Channel 24 (C and G) and Channel 25 (D and H). Data were analyzed by a two-tailed unpaired Student t-test (A–C and H) or Mann–Whitney test (D–G). Data presented as mean ± SEM, three independent experimental replicates; n = 12 for control COCs, n = 16 for etomoxir-treated COCs. ^*P < 0.05, ^**P < 0.01.

Figure 4

Altered metabolism during oocyte maturation persists in resultant blastocyst-stage embryos. COCs were matured in vitro in the absence or presence of etomoxir, an inhibitor of fatty acid metabolism (β-oxidation), which negatively affects oocyte developmental competence (see Figure 2M). Following in vitro maturation, COCs were fertilized in vitro and developed to the blastocyst stage in the absence of etomoxir. Embryos were imaged using hyperspectral microscopy. Autofluorescence intensity within the inner cell mass was quantified for hyperspectral channels that matched the spectral properties of NAD(P)H: Channel 1 (A) and Channel 2 (B); and FAD: Channel 24 (C) and Channel 25 (D). Data were analyzed by either a two-tailed unpaired Student t-test (C and D) or Mann–Whitney test (A and B). Data presented as mean ± SEM, three independent experimental replicates; n = 24 for control and n = 29 for blastocysts developed from control and etomoxir-treated COCs, respectively. ^**P < 0.01, ^***P < 0.001.

Figure 5

Hyperspectral imaging of the COC does not affect subsequent post-natal outcomes. Mature COCs were either imaged or not imaged using the hyperspectral microscope. COCs from both groups were fertilized in vitro and allowed to develop to the blastocyst-stage. Resultant blastocysts were transferred to pseudopregnant recipients with imaged COCs resulting in the birth of live pups (A). The weight of offspring at weaning was recorded for each group (B). Data are estimated marginal mean ± SEM and were analyzed by linear mixed model with litter size as a covariate. Each datum point represents the average weight per litter. n = 9 L per group. See Supplementary Figure SS3 for offspring data according to sex.