Fluorescence lifetime imaging microscopy (FLIM) detects differences in metabolic signatures between euploid and aneuploid human blastocysts

Abstract

STUDY QUESTION Can non-invasive imaging with fluorescence lifetime imaging microscopy (FLIM) detect metabolic differences in euploid versus aneuploid human blastocysts? SUMMARY ANSWER FLIM has identified significant metabolic differences between euploid and aneuploid blastocysts. WHAT IS KNOWN ALREADY Prior studies have demonstrated that FLIM can detect metabolic differences in mouse oocytes and embryos and in discarded human blastocysts. STUDY DESIGN, SIZE, DURATION This was a prospective observational study from August 2019 to February 2020. Embryo metabolic state was assessed using FLIM to measure the autofluorescence metabolic factors nicotinamide adenine dinucleotide dehydrogenase together with nicotinamide adenine phosphate dinucleotide dehydrogenase (NAD(P)H) and flavin adenine dinucleotide (FAD). Eight metabolic FLIM parameters were obtained from each blastocyst (four for NAD(P)H and four for FAD): short (T1) and long (T2) fluorescence lifetime, fluorescence intensity (I) and fraction of the molecules engaged with enzymes (F). The redox ratio (NAD(P)H-I)/(FAD-I) was also calculated for each image. PARTICIPANTS/MATERIALS, SETTING, METHODS This study was performed at a single academically affiliated centre where there were 156 discarded frozen blastocysts (n = 17 euploids; 139 aneuploids) included. Ploidy status was determined by pre-implantation genetic testing for aneuploidy (PGT-A). Discarded human blastocysts were compared using single FLIM parameters. Additionally, inner cell mass (ICM) and trophectoderm (TE) were also evaluated. Multilevel models were used for analysis. A post-hoc correction used Benjamini–Hochberg’s false discovery rate, at a q-value of 0.05. MAIN RESULTS AND THE ROLE OF CHANCE Comparing euploid (n = 17) versus aneuploid (n = 139) embryos, a significant difference was seen in NAD(P)H-F (P < 0.04), FAD-I (P < 0.04) and redox ratio (P < 0.05). Euploid ICM (n = 15) versus aneuploid ICM (n = 119) also demonstrated significantly different signatures in NAD(P)H-F (P < 0.009), FAD-I (P < 0.03) and redox ratio (P < 0.03). Similarly, euploid TE (n = 15) versus aneuploid TE (n = 119) had significant differences in NAD(P)H-F (P < 0.0001) and FAD-I (P < 0.04). LIMITATIONS, REASONS FOR CAUTION This study utilized discarded human blastocysts, and these embryos may differ metabolically from non-discarded human embryos. The blastocysts analysed were vitrified after PGT-A biopsy and it is unclear how the vitrification process may affect the metabolic profile of blastocysts. Our study was also limited by the small number of rare donated euploid embryos available for analysis. Euploid embryos are very rarely discarded due to their value to patients trying to conceive, which limits their use for research purposes. However, we controlled for the imbalance with the bootstrap resampling analysis. WIDER IMPLICATIONS OF THE FINDINGS These findings provide preliminary evidence that FLIM may be a useful non-invasive clinical tool to assist in identifying the ploidy status of embryos. STUDY FUNDING/COMPETING INTEREST(S) The study was supported by the Blavatnik Biomedical Accelerator Grant at Harvard University. Becker and Hickl GmbH and Boston Electronics sponsored research with the loaning of equipment for FLIM. D.J.N. is an inventor on patent US20170039415A1. There are no other conflicts of interest to declare. TRIAL REGISTRATION NUMBER N/A

Vitrification within a nanoliter volume: oocyte and embryo cryopreservation within a 3D photopolymerized device

“…In these studies, metabolism was measured using metabolites present in spent media. The use of label-free autofluorescence to non-invasively assess oocyte and embryo metabolism is increasing in popularity [39,[49][50][51]. Compared to analysis of spent medium, recording cellular autofluorescence provides a point in time measurement that also enables spatial information on the metabolic activity of individual cells [39].…”

“…As with oocytes, the differences in metabolism between the vitrified groups and the non-vitrified control group observed may indicate an increased demand for energy required for post-warming recovery processes [ 47 ]. Hence, this study highlights the need to consider the impact of cryopreservation on metabolism when developing diagnostic tools that use cellular autofluorescence to predict developmental competency [ 39 , 51 , 56 , 57 ].…”

Status of maternal serum B vitamins and pregnancy outcomes: New insights from in vitro fertilization and embryo transfer (IVF-ET) treatment

“…Bioactive derivatives of riboflavin and niacin including flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide (NAD+) are universally implicated in the maintenance of mitochondrial function and energy homeostasis (33,34). Previous evidence demonstrates that the elimination of influx of riboflavin via immuno-blocking of riboflavin carrier protein in primate ovulated oocytes can cause early embryo degeneration and that lower intracellular flavins or FAD+ levels are associated with an age-related decline of oocyte quality or chromosome instability in human preimplantation embryos (35)(36)(37). This evidence suggests that riboflavin deficiency may impair oocyte quality and embryo development.…”

Investigation of refractive index dynamics during embryo development using digital holographic microscopy

“…Towards developing an objective and accurate diagnostic for embryo quality, there has been a recent surge in the use of optical approaches within the field. For example, identification of chromosomal aberrations, traditionally performed via embryo biopsy -may now be determined using hyperspectral imaging [4] or fluorescence lifetime imaging microscopy (FLIM) [5]. Further, optical coherence microscopy (OCM), a label-free and non-invasive imaging technique, has been shown to detect changes in subcellular architecture that were associated with embryo quality [6,7].…”