Effect of time post warming to embryo transfer on human blastocyst metabolism and pregnancy outcome.

PURPOSE: This study is aiming to test whether variation in post warming culture time impacts blastocyst metabolism or pregnancy outcome. METHODS: In this single center retrospective cohort study, outcomes of 11,520 single frozen embryo transfer (FET) cycles were analyzed from January 2015 to December 2020. Patient treatments included both natural and programmed cycles. Time categories were determined using the time between blastocyst warming and embryo transfer: 0 (0- <1h), 1 (1-<2h), 2 (2-<3h), 3(3-<4h), 4 (4-<5), 5 (5-<6), 6 (6-<7) and 7 (7-8h). Non-invasive metabolic imaging of discarded human blastocysts for up to 10h was also performed using Fluorescence lifetime imaging microscopy (FLIM) to examine for metabolic perturbations during culture. RESULTS: The mean age of patients across all time categories were comparable (35.6 ± 3.9). Live birth rates (38-52%) and miscarriage rate (5-11%) were not statistically different across post-warming culture time. When assessing pregnancy outcomes based on the use of PGT-A, miscarriage and live birth rates were not statistically different across culture hours in both PGT-A and non-PGT cycles. Further metabolic analysis of blastocysts for the duration of 10h of culture post warming, revealed minimal metabolic changes of embryos in culture. CONCLUSION: Overall, our results show that differences in the time of post warming culture have no significant impact on miscarriage or live birth rate for frozen embryo transfers. This information can be beneficial for clinical practices with either minimal staffing or a high number of patient cases.

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

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.

Metabolic state of human blastocysts measured by fluorescence lifetime imaging microscopy.

STUDY QUESTION: Can non-invasive metabolic imaging via fluorescence lifetime imaging microscopy (FLIM) detect variations in metabolic profiles between discarded human blastocysts? SUMMARY ANSWER: FLIM revealed extensive variations in the metabolic state of discarded human blastocysts associated with blastocyst development over 36 h, the day after fertilization and blastocyst developmental stage, as well as metabolic heterogeneity within individual blastocysts. WHAT IS KNOWN ALREADY: Mammalian embryos undergo large changes in metabolism over the course of preimplantation development. Embryo metabolism has long been linked to embryo viability, suggesting its potential utility in ART to aid in selecting high quality embryos. However, the metabolism of human embryos remains poorly characterized due to a lack of non-invasive methods to measure their metabolic state. STUDY DESIGN, SIZE, DURATION: We conducted a prospective observational study. We used 215 morphologically normal human embryos from 137 patients that were discarded and donated for research under an approved institutional review board protocol. These embryos were imaged using metabolic imaging via FLIM to measure the autofluorescence of two central coenzymes, nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD+), which are essential for cellular respiration and glycolysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Here, we used non-invasive FLIM to measure the metabolic state of human blastocysts. We first studied spatial patterns in the metabolic state within human blastocysts and the association of the metabolic state of the whole blastocysts with stage of expansion, day of development since fertilization and morphology. We explored the sensitivity of this technique in detecting metabolic variations between blastocysts from the same patient and between patients. Next, we explored whether FLIM can quantitatively measure metabolic changes through human blastocyst expansion and hatching via time-lapse imaging. For all test conditions, the level of significance was set at P < 0.05 after correction for multiple comparisons using Benjamini-Hochberg's false discovery rate. MAIN RESULTS AND THE ROLE OF CHANCE: We found that FLIM is sensitive enough to detect significant metabolic differences between blastocysts. We found that metabolic variations between blastocyst are partially explained by both the time since fertilization and their developmental expansion stage (P < 0.05), but not their morphological grade. Substantial metabolic variations between blastocysts from the same patients remain, even after controlling for these factors. We also observe significant metabolic heterogeneity within individual blastocysts, including between the inner cell mass and the trophectoderm, and between the portions of hatching blastocysts within and without the zona pellucida (P < 0.05). And finally, we observed that the metabolic state of human blastocysts continuously varies over time. LIMITATIONS, REASONS FOR CAUTION: Although we observed significant variations in metabolic parameters, our data are taken from human blastocysts that were discarded and donated for research and we do not know their clinical outcome. Moreover, the embryos used in this study are a mixture of aneuploid, euploid and embryos of unknown ploidy. WIDER IMPLICATIONS OF THE FINDINGS: This work reveals novel aspects of the metabolism of human blastocysts and suggests that FLIM is a promising approach to assess embryo viability through non-invasive, quantitative measurements of their metabolism. These results further demonstrate that FLIM can provide biologically relevant information that may be valuable for the assessment of embryo quality. STUDY FUNDING/COMPETING INTEREST(S): 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. TRIAL REGISTRATION NUMBER: N/A.