Developmental kinetics of cleavage stage mouse embryos are related to their subsequent carbohydrate and amino acid utilization at the blastocyst stage.

What is the relationship between cleavage stage embryo kinetics, blastocyst metabolism and subsequent embryo viability? Embryos cleaving faster at the first cleavage division resulted in blastocysts with a larger inner cell mass (ICM), higher glucose consumption, lower glycolytic rate, higher aspartate uptake, lower global amino acid turnover and higher percentage of developing fetuses on E13.5 when compared with blastocysts that developed from slower cleaving embryos. Previous research has shown that morphokinetics, blastocyst carbohydrate metabolism and cleavage stage amino acid metabolism of the preimplantation embryo can be used independently as markers of its developmental competence and subsequent viability. Morphokinetics of in vitro fertilized mouse zygotes were observed using a time-lapse imaging system and they were identified as 'fast' or 'slow' cleaving embryos. Spent culture media from resultant blastocysts were analysed for carbohydrate and amino acid utilization. Blastocysts either had their ICM and trophectoderm (TE) cell number determined, were cultured further in an outgrowth assay or transferred to a recipient female to assess implantation and fetal development. Morphokinetics of in vitro fertilized C57BL/6xCBA (F1) zygotes individually cultured in 2 µl drops of G1/G2 media with HSA under Ovoil in 5% O2, 6% CO2 and 89% N2 were analysed using a time-lapse incubator. At 72 h post-insemination, blastocysts were separated into quartiles derived from timing of the first cleavage division. Blastocysts were cultured for a further 24 h and spent media samples, including controls containing no embryos, were frozen and subsequently analysed for amino acid utilization using liquid chromatography-mass spectrometry. These blastocysts were then analysed over a further 1.5 h period for carbohydrate utilization and subsequently stained to determine ICM and TE cells. To analyse implantation potential, fetal quality and viability, additional 'fast' and 'slow' blastocysts were cultured further in an outgrowth model or transferred to recipient females. Embryos cleaving faster at the time of first cleavage (first quartile, designated 'fast') were on average 2.5 h ahead of slower embryos (fourth quartile, designated 'slow', 15.1 ± 0.1 versus 17.6 ± 0.1 h, P < 0.001). On Day 5 of culture, blastocysts developed from 'fast' embryos had a larger ICM number (17.4 ± 2.1 versus 7.4 ± 2.0, P < 0.01), a higher glucose consumption (21.2 ± 1.2 versus 14.3 ± 1.0 pmol/embryo/h, P < 0.001) and a lower glycolytic rate (expressed as the percentage of glucose converted to lactate) (49.6 ± 2.8 versus 59.7 ± 2.8%, P < 0.05) compared with 'slow' embryos. Further non-invasive metabolomic analysis revealed that 'fast' blastocysts consumed more aspartate (2.2 ± 0.1 versus 1.8 ± 0.1 pmol/embryo/h, P < 0.05) and produced little or no glutamate compared with 'slow' blastocysts (0.02 ± 0.07 consumed versus 0.32 ± 0.11 pmol/embryo/h produced, P < 0.05). Transfer of 'fast' blastocysts to pseudo-pregnant recipients resulted in higher fetal survival post-implantation compared with 'slow' blastocysts (69.6 versus 40.4%, P < 0.01). The timing of the first cleavage division was used to classify blastocysts as 'fast' or 'slow' embryos; however, a combination of several developmental kinetic markers (cleavage time of 3- to 8-cell, duration between cleavage division times) may be used to more accurately determine an embryo as 'fast' or 'slow'. Only the fastest and slowest quartiles (those embryos with the fastest and slowest first cleavage division) were analysed in this study. These findings show that kinetically different embryos develop into blastocysts with different metabolic profiles and viability. Work is now being undertaken to determine if using these viability markers in combination will increase embryo selection efficacy and further improve implantation and pregnancy rates. The study was funded by the University of Melbourne. The authors have no conflicts of interest to declare.

Lee YS ，Thouas GA ，Gardner DK 《-》

Antioxidants improve mouse preimplantation embryo development and viability.

What is the effect of three antioxidants (acetyl-L-carnitine, N-acetyl-L-cysteine and α-lipoic acid), when used individually and in combination, on mouse embryo development in culture, and subsequent fetal development post-transfer? A combination of antioxidants resulted in significant increases in blastocyst cell number, maintained intracellular glutathione (GSH) levels, supported earlier cleavage times from 5-cell stage to expanded blastocyst, and improved fetal developmental irrespective of incubator oxygen concentration. Acetyl-L-carnitine, N-acetyl-L-cysteine and α-lipoic acid have been shown to have beneficial effects individually in several tissues, and most recently on developing embryos, in the presence of oxidative stress. Morphokinetics of mouse embryos were quantitated using time-lapse imaging. GSH levels in pronucleate oocytes were measured. Blastocysts underwent differential nuclear staining for inner cell mass and trophectoderm cells or were transferred to recipient females to assess implantation and fetal development. Pronucleate oocytes from F1 mice were cultured in 5 or 20% oxygen either individually or in groups of 10, in media G1/G2, in the presence or absence of 10 µM acetyl-L-carnitine /10 µM N-acetyl-L-cysteine /5 µM α-lipoic acid, either individually or in combination. Controls were embryos cultured without antioxidants. Intracellular levels of reduced glutathione were quantitated in pronucleate oocytes. Embryo development and viability were analysed through time-lapse microscopy and embryo transfers. Antioxidants significantly increased mouse blastocyst cell numbers compared with control when used individually (P< 0.05) and to a greater effect when all three were used in combination (P< 0.01) in 20% oxygen. The combination of antioxidants resulted in faster development rates to 5-cell cleavage stage, which continued until the expanded blastocyst stage when cultured in 20% oxygen. The beneficial effects of combining the antioxidants were greater for embryos cultured individually as opposed to in groups of 10 and for those embryos cultured in 20% compared to 5% oxygen. Levels of GSH were significantly decreased in control embryos that were incubated in the absence of antioxidants in 20% oxygen (P< 0.01), compared with in vivo flushed embryos. However, when embryos were cultured with antioxidants the level of GSH was not different to that of in vivo developed embryos. Embryos cultured in the presence of antioxidants in 20% oxygen and transferred resulted in significantly longer crown-rump length (11.6 ± 0.1 mm versus 11.3 ± 0.1 mm; P< 0.01), heavier fetuses (209.8 ± 11.8 mg versus 183.9 ± 5.9 mg; P< 0.05) and heavier placentas (103.5 ± 3.1 mg versus 93.6 ± 2.7 mg; P< 0.01) compared with controls (all data are mean ± SEM). Further, a post-implantation benefit of the antioxidant combination was also evident after culture in 5% oxygen. Embryo development and implantation was only examined in the mouse. These findings show that a combination of antioxidants in the culture media has a highly beneficial effect on mouse preimplantation embryo development in vitro and on subsequent fetal development post-transfer. These data indicate a potential role for the inclusion of specific antioxidant combinations in human embryo culture media irrespective of oxygen concentration. However, before application to human embryos, a proper evaluation of this approach in prospective, preferably randomized, trials will be required. This work was funded by a research grant from Vitrolife AB (Sweden). The authors have no conflict of interest to declare.

Truong TT ，Soh YM ，Gardner DK 《-》

Early cleaving embryos result in blastocysts with increased aspartate and glucose consumption, which exhibit different metabolic gene expression that persists in placental and fetal tissues.

Using time-lapse microscopy, previous research has shown that IVF mouse embryos that cleave earlier at the first division ('fast') develop into blastocysts with increased glucose consumption and lower likelihood of post-implantation loss as compared to slower cleaving embryos ('slow'). Further, metabolomics analysis employing LC-MS conducted on groups of 'fast' blastocysts revealed that more aspartate was consumed. With the worldwide adoption of single blastocyst transfer as the standard of care, the need for quantifiable biomarkers of viability, such as metabolism of specific nutrients, would greatly assist in embryo selection for transfer. Here we describe the development of a targeted enzymatic assay to quantitate aspartate uptake of single blastocysts. Results demonstrate that the rates of aspartate and glucose consumption were significantly higher in individual 'fast' blastocysts. Blastocysts, together with placental and fetal liver tissue collected following transfer, were analysed for the expression of genes involved in aspartate and carbohydrate metabolism. In 'fast' blastocysts, expressions of B3gnt5, Slc2a1, Slc2a3, Got1 and Pkm2 were found to be significantly higher. In placental tissue derived from 'fast' blastocysts, expression of Slc2a1, Got1 and Pkm2 were significantly higher, while levels of Got1 and Pkm2 were lower in fetal liver tissue compared to tissue from 'slow' blastocysts. Importantly, this study shows that genes regulating aspartate and glucose metabolism were increased in blastocysts that have higher viability, with differences maintained in resultant placentae and fetuses. Consequently, the analysis of aspartate uptake in combination with glucose represents biomarkers of development and may improve embryo selection efficacy and pregnancy rates.

Lee YSL ，Gardner DK 《-》

Correlation between standard blastocyst morphology, euploidy and implantation: an observational study in two centers involving 956 screened blastocysts.

Capalbo A ，Rienzi L ，Cimadomo D ，Maggiulli R ，Elliott T ，Wright G ，Nagy ZP ，Ubaldi FM ... -  《-》

Timing of human preimplantation embryonic development is confounded by embryo origin.

To what extent do patient- and treatment-related factors explain the variation in morphokinetic parameters proposed as embryo viability markers? Up to 31% of the observed variation in timing of embryo development can be explained by embryo origin, but no single factor elicits a systematic influence. Several studies report that culture conditions, patient characteristics and treatment influence timing of embryo development, which have promoted the perception that each clinic must develop individual models. Most of the studies have, however, treated embryos from one patient as independent observations, and only very few studies that evaluate the influence from patient- and treatment-related factors on timing of development or time-lapse parameters as predictors of viability have controlled for confounding, which implies a high risk of overestimating the statistical significance of potential correlations. Infertile patients were prospectively recruited to a cohort study at a hospital fertility clinic from February 2011 to May 2013. Patients aged <38 years without endometriosis were eligible if ≥8 oocytes were retrieved. Patients were included only once. All embryos were monitored for 6 days in a time-lapse incubator. A total of 1507 embryos from 243 patients were included. The influence of fertilization method, BMI, maternal age, FSH dose and number of previous cycles on timing of t2-t5, duration of the 2- and 3-cell stage, and development of a blastocoel (tEB) and full blastocoel (tFB) was tested in multivariate, multilevel linear regression analysis. Predictive parameters for live birth were tested in a logistic regression analysis for 223 single transferred blastocysts, where time-lapse parameters were investigated along with patient and embryo characteristics. Moderate intra-class correlation coefficients (0.16-0.31) were observed for all parameters except duration of the 3-cell stage, which demonstrates that embryos from one patient elicit clustering at a patient level. No single patient- and treatment-related factor was found to systematically influence the timing from cleavage to blastocyst stage, which indicates that no individual patient-related factor can be identified that separately explains the clustering throughout the entire developmental stages. The blastocyst parameters were more affected by patient-related factors than cleavage stage parameters, as tEB occurred significantly later with older age (0.29 h/year (95% confidence interval: CI 0.03; 0.56)), while both tEB and tFB occurred significantly later with increasing dose of FSH (tEB: 0.12 h/100 IU FSH (95% CI 0.01;0.24); tFB 0.14 h/100 IU FSH (95% CI 0.03;0.27)) and with more previous attempts (tEB: 1.2 h/attempt (95% CI 0.01;2.5); tFB 1.4 h/attempt (0.10;2.7)). Fertilization method affected timing of the first division, with ICSI embryos cleaving significantly faster than IVF embryos (-3.6% (95% CI -6.4; -0.77)), whereas no difference was found in the subsequent divisions. The univariable regression analysis identified female age, cumulative FSH dose, degree of blastocyst expansion, score of the inner cell mass and timing of full blastocyst formation as predictors of live birth. The timing of full blastocyst formation (tFB) did not remain significant when adjusting for age, number of previous cycles and cumulative FSH dose, which were the parameters shown to influence tFB in the mixed regression model. Only good prognosis patients were enrolled, so these results may not be generalized to all infertile women. Not all patient-related factors were investigated. Our findings underline the importance of treating embryos as dependent observations and suggest a high risk of patient-based confounding in retrospective studies. The impact of confounders and the embryo origin needs to be addressed in order to apply appropriate statistical models in observational studies. Furthermore, this observation emphasizes the need for RCTs for evaluating use of time-lapse parameters for embryo selection. Funding for the cohort study was provided by the Lippert Foundation, the Toyota Foundation, the Aase og Einar Danielsen foundation and NordicInfu Care research grant. Research at the Fertility Clinic, Aarhus University Hospital is supported by an unrestricted grant from MSD and Ferring. K.K. is funded by a grant from the Danish Council for Independent Research Medical Sciences. The authors declare no competing interest.

Kirkegaard K ，Sundvall L ，Erlandsen M ，Hindkjær JJ ，Knudsen UB ，Ingerslev HJ ... -  《-》