Embryo long-term storage does not affect assisted reproductive technologies outcome: analysis of 58,001 vitrified blastocysts over 11 years.

BACKGROUND: Recently, the potential detrimental effect that the duration of storage time may have on vitrified samples has raised some concerns, especially when some studies found an association between cryostorage length and decreased clinical results. OBJECTIVE: This study aimed to evaluate the effects of the storage time length of day-5 vitrified blastocysts in 2 study groups: freeze-all cycles and nonelective frozen embryo transfers. STUDY DESIGN: This was a retrospective study that included 58,001 vitrified/warmed day-5 blastocysts from 2 different populations, according to the reason for frozen embryo transfer. Elective frozen embryo transfer comprised freeze-all cycles (N=16,615 blastocysts and 16,615 patients) in which only single embryo transfers and only the first frozen embryo transfer were included. The nonelective frozen embryo transfer group included 41,386 embryos from 25,571 patients where frozen embryo transfer took place using supernumerary embryos after fresh embryo transfer. All the possible frozen embryo transfers were included. Both single embryo transfer and double embryo transfers were included. Donor and autologous oocytes were used. The period covered by this study was 11 years. The blastocyst sample was clustered into deciles, which provided specific storage duration categories. The main outcome was the live birth rate, and secondary outcomes were embryo survival, miscarriage, and clinical and ongoing pregnancy rates according to storage duration. The impact of storage time was assessed by univariable analyses in both groups. The comparison was made between each decile and the last one. A multivariable logistic regression analysis was conducted, including the variables with significant association found in the univariate analysis. Student t test and chi-square tests, or an analysis of variance, were used wherever appropriate. P<.05 was considered statistically significant. RESULTS: There were statistical differences in baseline characteristics of patients included in the study groups. Storage durations ranged from ≤0.67 to ≥4.34 and from ≤1.8 to ≥34.81 months in freeze-all and nonelective frozen embryo transfer, respectively. Embryo survival did not show statistical differences across the categories of storage time in freeze-all and nonelective frozen embryo transfer groups. Statistical differences were found for the live birth rate across some, but not all, the subgroups of storage duration. The multivariable analysis showed no association between storage time and the live birth rate in both groups (nonsignificant). Blastocyst quality, body mass index, number of retrieved oocytes, endometrial preparation, male factor, and uterine factor were related to the drop in the live birth rate in the freeze-all group (P<.05). In the nonelective frozen embryo transfer group, the variables that showed significant association with the live birth rate were age at retrieval and frozen embryo transfer, type of frozen embryo transfer (single embryo transfer or double embryo transfers), number of retrieved oocytes, body mass index, endometrial preparation, origin of sperm sample, and female factor. CONCLUSION: This large study demonstrated no association between storage time and clinical outcome. Other variables, such as the patient's age, embryo quality, body mass index, and etiology, are somewhat responsible for impacting the outcome. This provides evidence for the safety of embryo vitrification, even after long storage periods. This is reassuring for both in vitro fertilization practitioners and patients undergoing frozen embryo transfer of either elective or nonelective embryos.

Human embryo polarization requires PLC signaling to mediate trophectoderm specification.

Apico-basal polarization of cells within the embryo is critical for the segregation of distinct lineages during mammalian development. Polarized cells become the trophectoderm (TE), which forms the placenta, and apolar cells become the inner cell mass (ICM), the founding population of the fetus. The cellular and molecular mechanisms leading to polarization of the human embryo and its timing during embryogenesis have remained unknown. Here, we show that human embryo polarization occurs in two steps: it begins with the apical enrichment of F-actin and is followed by the apical accumulation of the PAR complex. This two-step polarization process leads to the formation of an apical domain at the 8-16 cell stage. Using RNA interference, we show that apical domain formation requires Phospholipase C (PLC) signaling, specifically the enzymes PLCB1 and PLCE1, from the eight-cell stage onwards. Finally, we show that although expression of the critical TE differentiation marker GATA3 can be initiated independently of embryo polarization, downregulation of PLCB1 and PLCE1 decreases GATA3 expression through a reduction in the number of polarized cells. Therefore, apical domain formation reinforces a TE fate. The results we present here demonstrate how polarization is triggered to regulate the first lineage segregation in human embryos.

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.

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.

Storage of human oocytes in the vapor phase of nitrogen.

OBJECTIVE: To evaluate the effectiveness of long-term vapor-phase nitrogen storage of vitrified human oocytes as a strategy for preventing the risk of cross-contamination due to direct contact with the liquid nitrogen (LN). DESIGN: Prospective randomized study. SETTING: Private infertility center, IVI, Valencia. PATIENT(S): Oocyte donors (n = 44) and recipients (n = 46). INTERVENTION(S): Vitrification by the Cryotop method. Storage of vitrified oocytes in a vapor-phase nitrogen storage freezer and a traditional LN storage tank. Donation of the surviving oocytes and evaluation of fertilization, embryo development, and clinical results. MAIN OUTCOME MEASURE(S): Survival, fertilization, and cleavage rates. Embryo quality and clinical outcome. RESULT(S): Survival was 95.3% (vapor-phase nitrogen) and 94.5% (LN). Fertilization rates (73.1% and 71.7%) or cleavage on day 2 (95.6% and 94.7%), day 3 (84.5% and 79.9%), and blastocyst formation (54.7% and 53.9%) were similar between vapor-phase nitrogen and LN. Implantation, clinical, and ongoing pregnancy rates were similar for vapor-phase nitrogen (40.5%, 58.1%, and 48.8%, respectively) and LN groups (33.7%, 53.3%, and 46.6%, respectively). CONCLUSION(S): The vapor-phase nitrogen system permits the storage of oocytes vitrified, maintaining their potential to develop into competent embryos in a similar manner as those stored in a traditional LN freezer. This approach represents a practical alternative that prevents cross-contamination during the storage of vitrified samples.

Effect of sperm glutathione peroxidases 1 and 4 on embryo asymmetry and blastocyst quality in oocyte donation cycles.

OBJECTIVE: To prospectively determine the impact of concrete components of the sperm oxidative glutathione stress system in terms of enzymatic activity and mitochondrial RNA (mRNA) expression on embryo quality and reproductive outcome. Human spermatozoa use the glutathione system to inactivate reactive oxygen metabolites, and there is a close correlation between some components of the glutathione system and male fertility. However, very few data are published regarding this system in sperm cells and its effect on fertilization ability and embryo development in human beings. DESIGN: An oocyte-donation model, used to homogenize the female factor. SETTING: University-affiliated private IVF setting. PATIENT(S): Semen samples from infertile males (n = 43) of couples undergoing oocyte-donation cycles (n = 43). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Gene expression and activity of glutathione peroxidases (GPXs) 1 and 4, glutathione reductase, and intracellular glutathione (GSH) by fluorescent quantitative polymerase chain reaction and spectrophotometry, respectively. RESULT(S): Fertilization rate, pronuclear number, asymmetry, and pronuclear body distribution were not correlated with any sperm glutathione parameters that were considered. When day 3 embryo parameters were evaluated, only GPX4 mRNA expression in sperm cells was statistically significantly lower when asymmetric embryos were observed. Also, worst embryo development and morphology on day 5 was statistically significantly correlated with lower sperm GPX1 activity (101.07 vs. 258.8 IU/mg protein). Glutathione system analysis in fresh sperm was not statistically significantly different in patients achieving pregnancy compared with those who not, and we did not find any correlation with implantation rate. CONCLUSION(S): We have been able to correlate embryo morphology on day 3 with the sperm expression of GPX family members. The results indicate that sperm-derived mRNA may condition human embryo quality and persist even to blastocyst stage. The correlation of the sperm GPX family mRNA expression with embryo health appears quite promising for discovery of molecular causes of male infertility.