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

STUDY QUESTION: To what extent do patient- and treatment-related factors explain the variation in morphokinetic parameters proposed as embryo viability markers? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LIMITATIONS, REASONS FOR CAUTION: Only good prognosis patients were enrolled, so these results may not be generalized to all infertile women. Not all patient-related factors were investigated. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTERESTS: 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.

Nuclear magnetic resonance metabolomic profiling of Day 3 and 5 embryo culture medium does not predict pregnancy outcome in good prognosis patients: a prospective cohort study on single transferred embryos.

STUDY QUESTION: Does the metabolomic profile, obtained with nuclear magnetic resonance (NMR), of spent culture media from human embryos correlate with reproductive potential in a cohort of good prognosis patients? SUMMARY ANSWER: In a large cohort of single transferred blastocysts from a homogeneous group of good prognosis patients, we find a high degree of individual variation in the metabolome that, however, has no relation to pregnancy outcome. WHAT IS KNOWN ALREADY: Differences among various specific metabolites have been linked to reproductive potential. Although results from retrospective near infrared (NIR) spectroscopy analyses of spent culture medias from transferred embryos were promising, randomized controlled trials were unable to demonstrate that NIR analysis improved pregnancy rates. Therefore, a more detailed investigation of the relation between embryo metabolism and reproductive potential is required. NMR is a powerful technique that provides detailed structural and dynamic information. STUDY DESIGN, SIZE, DURATION: A prospective cohort study was conducted at the Fertility Clinic, Aarhus University Hospital between February 2011 and July 2012. Infertile patients aged <38 years without endometriosis were offered participation and their embryos were included if greater than or equal to eight oocytes were retrieved. In total, 161 infertile patients were included in the cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS: Spent culture media was collected on Days 3 and 5 after oocyte retrieval from 148 single transferred embryos. NMR spectra were obtained from 12 µl of spent media. Data were quantitatively analysed using multivariate analysis with respect to pregnancy outcome, defined as a live fetus by ultrasound in gestational Week 8, along with patient and treatment related variables such as embryo score, age, BMI, fertilization method and cause of infertility. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 148 cycles were included in the analysis [embryo transfer cancelled (n = 12), no media collected (n = 1)]. Clinical pregnancy was confirmed in 47 patients (32%). We obtained high quality NMR spectra for 141 Day 3 and 137 Day 5 samples. Our spectra show a high degree of individual variation. Multivariate data analysis was performed on spectral data with several different pre-processing combinations, i.e. binning, alignment, normalization and scaling in the attempt to develop a valid prediction model. Different strategies of multivariate analysis showed, however, no correlation between the NMR profiles and pregnancy outcome, patient or treatment characteristics. No model could therefore be developed for prediction of pregnancy outcome. We conclude that within this group of good prognosis patients, large-scale metabolic variations between embryos detected with NMR have no apparent association with pregnancy outcome. LIMITATIONS, REASONS FOR CAUTION: Although this study is the largest we know of using NMR to investigate metabolomic profiles of single-transferred embryos, there may be differences that would be detected with a larger study. When analysing such a small sample volume, even small variations in the amount of media and dilution may introduce a large uncertainty in the results. WIDER IMPLICATIONS OF THE FINDINGS: Our study questions the usefulness of the entire metabolome for embryo selection, which should direct the search for viability markers in the culture media towards individual components. STUDY FUNDING/COMPETING INTERESTS: Funding was provided by Aarhus University, the Lippert Foundation, the Toyota Foundation, the Aase og Einar Danielsen foundation. Research at the Fertility Clinic, Aarhus Universtity Hospital is supported by an unrestricted grant from MSD and Ferring. The authors declare no competing interest. TRIAL REGISTRATION NUMBER: NCT01139268.

Time-lapse parameters as predictors of blastocyst development and pregnancy outcome in embryos from good prognosis patients: a prospective cohort study.

STUDY QUESTION: Do early time-lapse parameters predict which embryos will develop to high-quality blastocysts and does timing of development differ between embryos that implant and those that do not. SUMMARY ANSWER: Development to high-quality blastocysts could be predicted within the first 48 h of culture, whereas time-lapse parameters could not predict pregnancy. WHAT IS KNOWN ALREADY: Historical cohort studies on embryos from unselected groups of patients have suggested several putative kinetic markers of viability. Before well-designed randomized studies can be conducted, relevant selection models based on solid data must be developed. So far conclusions from the previous studies are ambiguous. STUDY DESIGN, SIZE, DURATION: A prospective cohort study conducted from February 2011 to June 2012. A total of 571 ICSI embryos from 92 patients were included in the blastocyst development analysis and 84 single embryo transfers were included in the pregnancy outcome analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryos from women aged <38 years, with no endometriosis and ≥ 8 oocytes retrieved. University affiliated clinic. Embryos were cultured in a time-lapse incubator till Day 6. Logistic regression analysis was performed with variables selected based on indication. MAIN RESULTS AND THE ROLE OF CHANCE: Duration of the first cytokinesis, duration of the 3-cell stage and direct cleavage to 3-cells predicted development to high-quality blastocyst. We found no difference in timing between implanted and non-implanted embryos. LIMITATIONS, REASONS FOR CAUTION: A larger study might detect differences in timing between implanted and non-implanted embryos. The cohort consisted of good prognosis patients only and may not be representative of the entire IVF population. WIDER IMPLICATIONS OF THE FINDINGS: Our results in context with the lack of consistency in previous studies and the presumed influences of different external factors indicate that a universal algorithm for optimal timing of development might not be feasible. The apparent negative significance of division patterns that differ from the expected may imply that time-lapse will facilitate de-selection of embryos. STUDY FUNDING/COMPETING INTEREST(S): Funding for the present study was provided by Aarhus University, the Lippert Foundation, the Toyota Foundation, the Aase og Einar Danielsen foundation and by an unrestricted grant from MSD and Ferring. The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: The study was registered at ClinicalTrial.gov with accession number NCT01139268.