Pre-clinical validation of a closed surface system (Cryotop SC) for the vitrification of oocytes and embryos in the mouse model.

Vitrification is currently a well-established technique for the cryopreservation of oocytes and embryos. It can be achieved either by direct (open systems) or indirect (closed systems) contact with liquid nitrogen. While there is not a direct evidence of disease transmission by transferred cryopreserved embryos, it was experimentally demonstrated that cross-contamination between liquid nitrogen and embryos may occur, and thus, the use of closed devices has been recommended to avoid the risk of contamination. Unfortunately, closed systems may result in lower cooling rates compared to open systems, due to the thermal insulation of the samples, which may cause ice crystal formation resulting in impaired results. In our study, we aimed to validate a newly developed vitrification device (Cryotop SC) that has been specifically designed for being used as a closed system. The cooling and warming rates calculated for the closed system were 5.254 °C/min and 43.522 °C/min, respectively. Results obtained with the closed system were equivalent to those with the classic Cryotop (open system), with survival rates in oocytes close to 100%. Similarly, the potential of the survived oocytes to develop up to good quality blastocysts after parthenogenetic activation between both groups was statistically equivalent. Assessment of the meiotic spindle and chromosome distribution by fluorescence microscopy in vitrified oocytes showed alike morphologies between the open and closed system. No differences were found either between the both systems in terms of survival rates of one-cell stage embryos or blastocysts, as well as, in the potential of the vitrified/warmed blastocysts to develop to full-term after transferred to surrogate females.

Combination of metabolism measurement and a time-lapse system provides an embryo selection method based on oxygen uptake and chronology of cytokinesis timing.

OBJECTIVE: To evaluate correlations between oxygen consumption (OC) measurements before and after embryo cytokinesis, observing OC during embryo cleavages and combining that information with morphokinetics to relate to implantation potential. DESIGN: Prospective cohort study. SETTING: University-affiliated private IVF unit. PATIENT(S): A total of 1,150 injected oocytes in 86 first oocyte donation cycles with embryo transfer on day 3. INTERVENTION(S): None. MAIN OUTCOME MEASUREMENT(S): We analyzed the embryo OC and combined this data with the cytokinesis event, exact timing (in hours) of blastomeric cleavages, with the use of an incubator equipped with time-lapse videography, gathering a total of 7,630 measurements during the cytokinesis (active phase) and consecutive measurements after this division (passive phase), correlating this data with embryo outcome. RESULT(S): OC was found to increase during embryo cleavage, showing high levels during first division with a strong correlation with implantation success. Moreover, those embryos with slow or fast development gave rise to lower OC levels, whereas higher levels were associated with optimal embryo division ranges linked to higher implantation potential. CONCLUSION(S): A detailed analysis of OC by time-lapse observations enhances the value that these measurements represented as markers of embryo quality, especially during the cytokinesis events produced during preimplantation development.

Day-3 embryo metabolomics in the spent culture media is altered in obese women undergoing in vitro fertilization.

OBJECTIVE: To determine whether the global metabolomic profile of the spent culture media (SCM) of day-3 embryos is different in obese and normoweight women undergoing in vitro fertilization (IVF). DESIGN: Prospective cohort analysis. SETTING: IVF clinic. PATIENT(S): Twenty-eight young, nonsmoking women with normoweight, nonsmoking male partners with mild/normal sperm factors undergoing a first IVF attempt for idiopathic infertility, tubal factor infertility, or failed ovulation induction: obese ovulatory women (n = 12); obese women with polycystic ovary syndrome (PCOS; n = 4); normoweight ovulatory women (n = 12). INTERVENTION(S): Fifty µl of SCM collected from two day-3 embryos of each cohort. MAIN OUTCOME MEASURE(S): Metabolomic profiling via ultrahigh performance liquid chromatography coupled to mass spectrometry of SCM from a total of 56 embryos. RESULT(S): The untargeted metabolomic profile was different in obese and normoweight women. Partial least squares discriminant analysis resulted in a clear separation of samples when a total of 551 differential metabolites were considered. A prediction model was generated using the most consistent metabolites. Most of the metabolites identified were saturated fatty acids, which were detected in lower concentrations in the SCM of embryos from obese women. The metabolomic profile was similar in obese women with or without PCOS. CONCLUSION(S): The metabolomic profile in the SCM of day-3 embryos is different in normoweight and obese women. Saturated fatty acids seem to be reduced when embryos from obese patients are present. CLINICAL TRIAL REGISTRATION NUMBER: NCT01448863.

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.

Effect of vitrification on human oocytes: a metabolic profiling study.

OBJECTIVE: To evaluate the effect of oocyte vitrification in the metabolomic profile of embryos developed from vitrified and fresh oocytes in our ovum donation program. DESIGN: Analysis of the metabolic profiles of spent culture medium samples corresponding to embryos developed from vitrified and fresh oocytes. SETTING: In vitro fertilization (IVF) unit/metabolomic facility. PATIENT(S): Oocyte donors between the ages of 18 and 35 years. INTERVENTION(S): Metabolomic profile liquid chromatography coupled with mass spectrometry (LC-MS) of spent media samples. MAIN OUTCOME MEASURE(S): Identification of spent media components and metabolites present and absent in vitrified and fresh day-3 embryos. RESULT(S): We obtained a total of 190 spent media samples: vitrification group (65), fresh group (59), and a matched control media group (66). Multivariate data analysis was performed after global metabolomic and amino acid profiles did not reveal any statistically significant differences in day-3 embryos derived from fresh and vitrified oocytes, indicating that other metabolic differences between the samples (e.g., patient-to-patient variability, analytical variation) are greater than those between the vitrified and fresh sample groups. Univariate statistical analysis revealed a series of possible biomarkers, such as tryptophan, phenylalanine, and 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), although only alpha-CEHC was statistically significant after correction for multiple testing. CONCLUSION(S): Multivariate data analysis did not reveal statistically significant differences between the analyzed groups, suggesting that oocyte vitrification does not disturb embryonic metabolomic profiles.

Outcomes of vitrified early cleavage-stage and blastocyst-stage embryos in a cryopreservation program: evaluation of 3,150 warming cycles.

OBJECTIVE: To assess the outcomes achieved after Cryotop vitrification of both early cleavage and blastocyst-stage embryos and to determine whether the embryo developmental stage and embryo quality as well as the origin of the embryos (ovum donation cycles, patients' own oocytes) and the endometrial preparation for the embryo transfer had any effect on the final outcome. DESIGN: Observational study. SETTING: Private university-affiliated IVF center. PATIENT(S): Women undergoing 3,150 warming cycles whose embryos were vitrified due to various reasons. INTERVENTION(S): Vitrification by the Cryotop open device. MAIN OUTCOME MEASURE(S): Delivery rate (DR) per warming cycle. RESULT(S): Survival rate was 95% (5,722 out of 6,019 embryos). The percentage of intact embryos at warming showing 100% blastomere survival was 93% (95% CI 90.1%-95.3%) for day 2 and 95% (95% CI 94.3%-95.7%) for day 3; 3,057 embryo transfers were performed (3% cancellation rate). The DR/warming cycle was 32.5% (95% CI 30.9%-34.2%). Slight differences in survival rate were found [94.9% (95% CI 93.0%-96.8%) for day 2, 94.2% (95% CI 93.4%-94.9%) for day 3, 95.7% (95% CI 94.5%-96.9%) for day 5, and 97.6% (95% CI 96.9%-98.6%) for day 6]. Overall implantation, clinical pregnancy, ongoing pregnancy, and live birth rates per warming cycle were 35.5% (95% CI 33.5%-38.5%), 41.7% (95% CI 39.9%-43.4%), 32.6% (95% CI 31.0%-34.2%), and 38.1% (95% CI 36.4%-39.8%) respectively. The linear regression model considering embryo developmental stage, ovum donation or patient's own oocytes, and hormonal replacement therapy or natural cycle for endometrial preparation (odds ratio 1.179; 95% CI 0.912-1.277) showed no impact on the DR. CONCLUSION(S): Highly successful cryopreservation of all embryo developmental stages is possible with the use of the Cryotop system. There are no variables clearly exerting a negative effect on the survival and delivery rates.