A propensity score-based, comparative study assessing humid and dry time-lapse incubation, with single-step medium, on embryo development and clinical outcomes.

STUDY QUESTION: Does culture in a high relative humidity atmosphere improve clinical outcomes when using a time-lapse integrated incubator and single-step culture medium? SUMMARY ANSWER: Using an integrated time-lapse system and single-step culture medium, culture in a high relative humidity atmosphere increases the likelihood of embryos, especially those subjected to preimplantation genetic testing for aneuploidies, to achieve a pregnancy compared to those cultured in dry conditions. WHAT IS KNOWN ALREADY: The use of a humid atmosphere inside incubators can reduce changes in culture media osmolality, which has been reported to have a significant effect on embryo quality and morphokinetics. Studies assessing the effect of humid culture (HC) in clinical outcomes are, however, scarce and inconclusive, mostly due to a high variability in culture conditions and reduced sample size. STUDY DESIGN, SIZE, DURATION: Retrospective cohort study performed over 1627 ICSI cycles performed during 3 consecutive years in which embryo cohorts were cultured in a time-lapse incubator with three dry and three humidified chambers, and using single-step culture medium. Clinical outcomes were compared between treatments in which embryo cohorts were cultured in either humid (n = 833) or dry (n = 794) conditions. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study includes autologous treatments, with (N = 492) and without (N = 372) preimplantation genetic testing for aneuploidies (PGT-A) and ovum donation treatments (N = 763), performed in three university-affiliated private IVF centres. Stimulation, oocyte pickup and fertilization were performed according to the standard procedures of the clinic. All embryo cohorts were cultured in the same model of time-lapse incubator, distributed to either a dry or humidified chamber, while the rest of the culture variables remained equal. The population was weighted by the inverse probability of treatment to control for all measured confounders. The association between HC and the main outcome was assessed by logistic regression over the weighted population. The E-value was reported as a way of considering for unmeasured confounders. Differences in embryo development and other secondary outcomes between the study groups were assessed by Pearson Chi-squared test, ANOVA test and Kaplan-Meier survival analysis. MAIN RESULTS AND THE ROLE OF CHANCE: An univariable logistic regression analysis, weighted by the inverse probability of treatment, determined that embryos cultured in humid conditions are more likely to achieve a clinical pregnancy than those cultured in dry conditions (odds ratio (OR) = 1.236 (95% CI 1.009-1.515), P = 0.041, E = 1.460). Through stratification, it was determined that said effect is dependent on the type of treatment: no improvement in clinical pregnancy was present in ovum donation or autologous treatments, but a statistically significant positive effect was present in treatments with preimplantation genetic testing (OR = 1.699 (95% CI 1.084-2.663), P = 0.021, E = 1.930). Said increase does not relate with an improvement in later outcomes. Differences were also found in variables related to embryo developmental morphokinetics. LIMITATIONS, REASONS FOR CAUTION: The retrospective nature of the study makes it susceptible to some bias linked to the characteristics of the treatments. To lessen the effect of possible biases, cases were weighted by the inverse probability of treatment prior to the evaluation of the outcome, as means to assess for measured confounders. In addition, the E-value of the weighted OR was calculated as a sensitivity analysis for unmeasured confounders. A randomized prospective study could be performed for further assessing the effect of humid conditions in clinical outcome. WIDER IMPLICATIONS OF THE FINDINGS: These results support that embryo culture under conditions of high relative humidity contributes to optimize clinical results in undisturbed culture in a time-lapse incubator with single-step medium. To our knowledge, this is the largest study on the matter and the first performing a propensity score-based analysis. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the ''Centro para el Desarrollo Tecnologico Industrial'' from the Spanish Ministry of Science, Innovation, and Universities (CDTI-20170310) and Generalitat Valenciana and European Social Fund (ACIF/2019/264). None of the authors have any competing interest to declare. TRIAL REGISTRATION NUMBER: N/A.

A time to look back: analysis of morphokinetic characteristics of human embryo development.

OBJECTIVE: To describe the times associated with the morphological changes that occur in the embryo during preimplantation development based on the largest sample size described with time lapse. DESIGN: Cohort study. SETTING: University-affiliated private center. PATIENT(S): A total of 9,530 embryos from 1,806 intracytoplasmic sperm injection (ICSI) cycles. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Using a time-lapse system, embryo images were acquired for at least 68 hours, in some cases reaching 120-130 hours. Embryo cleavage time points up to 8-cell-stage (t2-t8) as well as morulae (tM) and blastocyst formation (tB) were registered in hours after ICSI. Additionally, duration of the cell cycle (cc) and synchrony (s) of the second and third cell cycles were defined. Finally, four subgroups of embryos were considered: the "regular divisions" group excluded embryos with a direct cleavage from 1 to 3 or 2 to 5 cells, and the "viable 8-cell," the "viable blastocyst," and "implanted embryos" groups included only embryos viable to the 8-cell stage, blastocyst stage, or transferred and successfully implanted, respectively. RESULT(S): Averages of times in the general population were: t2 = 27.9 hours, t3 = 38.2 hours, t4 = 40.7 hours, t5 = 51.0 hours, t6 = 54.1 hours, t7 = 56.7 hours, t8 = 59.1 hours, tM = 86.6 hours, tB = 104.1 hours, cc2 = 10.3 hours, cc3 = 12.8 hours, s2 = 2.7 hours, and s3 = 9.9 hours. Comparison between groups showed significant differences between regular divisions and viable 8 cells for t2, t3, t5, cc2, cc3, s2, and s3; between 8 cells and blastocyst for t5, t8, tM, cc3, and s2; and between blastocyst and implanted embryos for t8, tM, tB, and s2. Differences in timing related to morphology of cleavage- and blastocyst-stage embryos were detected. CONCLUSION(S): A time-lapse monitoring system applied to embryology allows accuracy and objectivity when defining the basis of embryo development within a clinic. The sample size is the largest ever described that provides consistent information about the normal distribution of embryo developmental timings.

Reduced oxygen tension improves embryo quality but not clinical pregnancy rates: a randomized clinical study into ovum donation cycles.

OBJECTIVE: To investigate the effect of low O2 tension during in vitro culture in terms of ongoing pregnancy rates in ovum donation cycles. DESIGN: Randomized trial. SETTING: Private university-affiliated IVF center, university-based hospital. PATIENT(S): A total of 1,125 cycles of ovum donation. INTERVENTION(S): Embryo culture in an atmosphere of 5.5% CO2, 6% O2, and 88.5% N2 versus a dual-gas system of 5.5% CO2 in air. MAIN OUTCOME MEASURE(S): Ongoing clinical pregnancy rates per intention-to-treat (ITT) patients. RESULT(S): The use of low O2 tension achieved a 41.3% ongoing pregnancy rate per ITT compared with a 40.8% rate obtained for 5% CO2 in air. The mean number of blastomeres and the percentage of top-quality embryos were significantly higher after lower O2 concentration during in vitro culture (7.1 ± 3.6 and 28.6% vs. 7.3 ± 8.4 and 32.1%, respectively). CONCLUSION(S): In the ovum donation cycles undergoing day-3 embryo transfers, the use of low O2 tension did not improve ongoing pregnancy rates per cycle and per transfer. However, it benefited embryo quality, demonstrating the potential negative impact of high O2 tension on the in vitro embryo development.

Outcome of cryotransfer of embryos developed from vitrified oocytes: double vitrification has no impact on delivery rates.

OBJECTIVE: Evaluate the outcome of cryotransfer of embryos developed from vitrified oocytes. DESIGN: Retrospective cohort study. SETTING: Private university-affiliated IVF center. PATIENT(S): Women undergoing warming cycles in which vitrified embryos were developed from vitrified or fresh oocytes. INTERVENTION(S): Vitrification by the Cryotop open device. MAIN OUTCOME MEASURE(S): Delivery rate (DR) per warming cycle. RESULT(S): A total of 471 warming cycles of 796 vitrified embryos developed from vitrified oocytes (group 1) and 2,629 warming cycles of 4,394 vitrified embryos derived from fresh oocytes (group 2) were evaluated. Overall survival rates were 97.2% [95% confidence interval [CI] 95.9%-98.6%] vs. 95.7% [95% CI 94.9-96.4], respectively. DRs per warming cycle were 33.8% (group 1) and 30.9% (group 2). Double vitrification had no effect on DR (odds ratio [OR] 0.877, 95% CI 0.712-1.080). Confounding factors (ovum donation or autologous cycles; day-3 or blastocyst embryo transfer [ET]; natural or hormonal replacement therapy for ET; single or double ET; previous cycles, number of oocytes, doses of gonadotropins and E2 levels on the day of hCG) did not modify the effect of double vitrification on DR (OR 0.872, 95% CI 0.702-1.084). CONCLUSION(S): Vitrification at early cleavage or blastocyst stage of embryos obtained from previously vitrified oocytes has no effect on DR/warming cycle.

The singleton, term gestation, and live birth rate per cycle initiated: a 1-year experience in in vitro fertilization cycles with native and donated oocytes.

OBJECTIVE: To investigate the singleton, term gestation, and live birth rate per cycle initiated in our IVF program during a 1-year period. DESIGN: Retrospective study of all first IVF cycles performed in the year 2002, with or without intracytoplasmic sperm injection (ICSI), with day 2/3 embryo transfer and using native or donated oocytes. SETTING: Instituto Valenciano de Infetilidad (IVI), Valencia, Spain. PATIENT(S): Of 3,158 IVF cycles initially considered, 165 were excluded because of embryo freezing, follow-up loss, or embryo reduction. Of the remaining cycles, only 1,836 were first cycles with day 2/3 embryo transfer; of these, native oocytes were employed in 1,095 and donated oocytes in 741. INTERVENTION(S): No patient underwent any additional procedure or intervention. MAIN OUTCOME MEASURE(S): The singleton, term gestation, and live birth rate per cycle initiated was used as a primary outcome measure. Results were analyzed according to the origin of the oocytes (native vs. donated) and the woman's age (<37 and > or =37 years old). RESULT(S): The ectopic pregnancy rate was higher in the native oocyte group. The singleton, term gestation, and live birth rate per cycle initiated was similar in native and donated oocyte groups (15.3% vs. 13.4%). In the native oocyte group, patients <37 years old showed a significantly better outcome. The singleton, term gestation, and live birth rate per cycle initiated was 16.7% and 10.8% in younger and older women, respectively. CONCLUSION(S): The singleton, term gestation, and live birth rate per cycle initiated constitutes an essential parameter for determining the real possibility of a healthy baby for a specific assisted reproduction technology (ART).