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.

Should embryo rebiopsy be considered a regular strategy to increase the number of embryos available for transfer?

PURPOSE: To investigate whether embryo rebiopsy increases the yield of in vitro fertilization (IVF) cycles. METHODS: Retrospective study including 18,028 blastocysts submitted for trophectoderm biopsy and preimplantation genetic testing for aneuploidy (PGT-A) between January 2016 and December 2021 in a private IVF center. Out of the 517 embryos categorized as inconclusive, 400 survived intact to the warming procedure, re-expanded, and were suitable for rebiopsy. Of them, 71 rebiopsied blastocysts were transferred. Factors affecting the probability of obtaining an undiagnosed blastocyst and clinical outcomes from blastocysts biopsied once and twice were investigated. RESULTS: The overall diagnostic rate was 97.1%, with 517 blastocysts receiving inconclusive reports. Several blastocyst and laboratory features, such as the day of the biopsy, the stage of development, and the biopsy methodology, were related to the risk of obtaining an inconclusive diagnosis after PGT-A. A successful diagnosis was obtained in 384 of the rebiopsied blastocysts, 238 of which were chromosomally transferable. A total of 71 rebiopsied blastocysts were transferred, resulting in 32 clinical pregnancies [(clinical pregnancy rate (CPR)=45.1%], 16 miscarriages [(miscarriage rate (MR)=41%], and, until September 2020, 12 live births [(live birth rate (LBR)=23.1%]. A significantly lower LBR and higher MR were obtained after transferring rebiopsied blastocysts compared to those biopsied once. CONCLUSION: Although an extra round of biopsy and vitrification may cause a detrimental effect on embryo viability, re-analyzing the test-failure blastocysts contributes to increasing the number of euploid blastocysts available for transfer and the LBR.

Prediction of embryo survival and live birth rates after cryotransfers of vitrified blastocysts.

RESEARCH QUESTION: Which pre-vitrification parameters are the most predictive of survival and live birth in vitrified-warmed blastocyst transfer cycles? DESIGN: A retrospective study including 11,936 warmed blastocysts. Pre-vitrification morphological parameters analysed for blastocysts included day of vitrification; blastocyst expansion degree; trophoectoderm grade (A, B and C); and inner cell mass grade (A, B and C). Univariate and multivariate generalized estimating equations models were used to analyse survival, clinical pregnancy and live birth rate. A stepwise regression analysis was conducted to select and classify by order which outcomes were the most predictive. RESULTS: The odds of survival increased almost twice for blastocysts with lower expansion degree (OR 1.92; 95% CI 1.37 to 2.69; P < 0.001) and by about 50% for blastocysts vitrified on day 5 (OR 1.56; 95% CI 1.27 to 1.89; P < 0.001). Multivariate generalized estimating equations model showed that trophectoderm grade followed by the day of vitrification were the most significant predictors of live birth. The odds of live birth increased nearly three times for blastocysts with trophectoderm graded as A compared with those with trophectoderm graded as C (OR 2.85; 95% CI 2.48 to 3.27; P < 0.001), and double for blastocysts vitrified on day 5 compared with those vitrified on day 6 (OR 2.22; 95% CI 1.97 to 2.49; P < 0.001). The odds of live birth also increased in higher expansion degree blastocysts. CONCLUSIONS: Blastocysts vitrified on day 5 and those with higher trophoectoderm grade should be given priority when warming.

Number needed to freeze: cumulative live birth rate after fertility preservation in women with endometriosis.

RESEARCH QUESTION: How does the number of oocytes used affect the cumulative live birth rate (CLBR) in endometriosis patients who had their oocytes vitrified for fertility preservation? DESIGN: Retrospective observational study including data from 485 women with endometriosis who underwent fertility preservation from January 2007 to July 2018. Survival curves and Kaplan-Meier plots were used to analyse the CLBR according to the number of vitrified oocytes used. Endometriosis curves were compared with plots developed using elective fertility preservation (EFP) patients as control group. Log-rank, Breslow and Tarone-Ware tests were used to compare the survival curves. RESULTS: The CLBR increased as the number of oocytes used per patient rose, reaching 89.5% (95% confidence interval [CI] 80.0-99.1%) using 22 oocytes. Higher outcomes were observed in young women (≤35 years old versus >35 years old). In the younger group, the CLBR was 95.4% (95% CI 87.2-103.6%) using approximately 20 oocytes versus 79.6% (95% CI 58.1-101.1%) in older women (log-rank [Mantel-Cox] P = 0.002). The mean age was higher in EFP patients (37.2 ± 4.9 versus 35.7 ± 3.7; P < 0.001). The outcome was better in the endometriosis group as compared with EFP: a CLBR of 89.5% (95% CI 80.0-99.1%) versus 59.9% (95% CI 51.4-68.6%) when 22 oocytes were used (log-rank [Mantel-Cox] P < 0.00001). CONCLUSION: The probability of live birth increases as the number of oocytes used increases in patients with endometriosis, but better outcomes were observed among young women. The information provided here may be of interest to both patients and treating physicians for counselling purposes.

Effect of oocyte morphology on post-warming survival and embryo development in vitrified autologous oocytes.

RESEARCH QUESTION: Does the presence of dysmorphisms affect post-warming survival and embryo development in vitrified autologous oocytes? DESIGN: A retrospective study comparing post-warming survival, fertilization and embryo development between morphologically normal (n = 269) and dysmorphic oocytes (n = 147). RESULTS: The survival rate was 81.4% in the morphologically normal oocytes and 87.1% in the dysmorphic oocyte group (OR 1.53; 95% CI 0.86 to 2.72). The fertilization rate was 69.9 versus 66.4% (OR 0.85; 95% CI 0.53 to 1.36), the proportion of good-quality embryos on day 3 was 30.3% versus 32.0% (OR 1.08; 95% CI 0.59 to 1.97) and the blastocyst formation rate was 54.5% versus 60.5% (OR 1.27; 95% CI 0.60 to 2.72) for the morphologically normal and the dysmorphic oocytes group, respectively. No statistical differences were found when the number and type of dysmorphism were analysed. CONCLUSION: Oocyte dysmorphisms did not seem to affect survival, fertilization and embryo development in vitrified autologous oocytes, and yielded comparable results to the morphologically normal oocytes.

Vitrification of human oocytes.

In recent years, growing evidence for the safety and efficiency of oocyte vitrification has made this technique be increasingly proposed for fertility preservation (FP). The populations who could benefit from FP include oncological patients who need the option to preserve their gametes before undergoing potential sterilizing treatment, patients with non-oncologic conditions requiring gonadotoxic chemotherapy and women who wish to delay their motherhood for a variety of reasons. By vitrifying oocytes, women have the chance to conceive in the future, have their own genetic offspring and maintain their reproductive autonomy. This review focuses on describing current knowledge on oocyte vitrification as a means to preserve female fertility. We present the general experience of our group and others in FP for both oncological and non-oncological reasons.

Analysis of the morphological dynamics of blastocysts after vitrification/warming: defining new predictive variables of implantation.

OBJECTIVE: To describe the morphological dynamics of vitrified/warmed blastocysts and to identify quantitative morphological variables related to implantation. Subsequently, by using the most predictive parameters, to develop a hierarchical model by subdividing vitrified/warmed blastocysts into categories with different implantation potentials. DESIGN: Observational, retrospective, cohort study. SETTING: University-affiliated private IVF center. PATIENT(S): The study included 429 vitrified/warmed blastocysts with known implantation data, which were evaluated by time-lapse imaging. Blastocysts were routinely placed in EmbryoScope (Vitrolife) immediately after warming until transfer. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Embryos were vitrified and warmed by the Cryotop method (KitazatoBiopharma). The studied variables included the initial and minimum thicknesses of zona pellucida (µm), the initial and maximum areas (µm2), the area of inner cell mass (µm2), expansion (whether the embryo reexpands or not after warming), and collapsing or contraction after warming. After defining the optimal ranges according to the consecutive quartiles with the highest probability of implantation, a logistic regression analysis was performed by combining the former variables and the blastocyst morphological classification criteria defined by the Spanish Association of Embryologists into A, B, C, or D categories. RESULT(S): Reexpansion of vitrified/warmed blastocysts correlated strongly with implantation (44.6% for reexpanded vs. 6.5% for the blastocysts that did not reexpand after warming). Throughout the logistic regression analysis, the model identified the maximum blastocyst area, odds ratio (OR) = 0.41 (95% confidence interval [CI], 0.22-0.77), followed by the initial area, OR = 0.62 (95% CI, 0.35-1.08) as the most predictive variables related to implanting embryos. Blastocyst morphology was not considered relevant in our model. The hierarchical tree model subdivided embryos into four categories, A-D, with lowering expected implantation potentials (from 47.3% for A to 14.2% for D). CONCLUSION(S): The analysis of warmed blastocysts by time-lapse imaging may provide objective quantitative markers for the blastocyst implantation potential. We propose a hierarchical model to classify vitrified/warmed blastocysts according to their implantation probability. The observed correlations and the proposed algorithm should be validated in a prospective trial to evaluate its efficacy.

Effect of oocyte vitrification on embryo quality: time-lapse analysis and morphokinetic evaluation.

OBJECTIVE: To analyze whether oocyte vitrification may affect subsequent embryo development from a morphokinetic standpoint by means of time-lapse imaging. DESIGN: Observational cohort study. SETTING: University-affiliated private IVF center. PATIENT(S): Ovum donation cycles conducted with the use of vitrified (n = 631 cycles; n = 3,794 embryos) or fresh oocytes (n = 1,359 cycles; n = 9,935 embryos) over 2 years. INTERVENTIONS(S): None. MAIN OUTCOME MEASURE(S): Embryo development was analyzed in a time-lapse imaging incubator. The studied variables included time to 2 cells (t2), 3 cells (t3), 4 cells (t4), 5 cells (t5), morula (tM), and cavitated, early, and hatching blastocyst (tB, tEB, tHB) as well as 2nd cell cycle duration (cc2 = t3 - t2). All of the embryos were classified according to the hierarchic tree model currently used for embryo selection. The analyzed variables were compared with the use of analysis of variance or chi-square and included 95% confidence intervals (CIs). RESULT(S): The embryos that originated from vitrified oocytes showed a delay of ∼1 hour from the first division to 2 cells (t2) to the time of blastulation (tB). The embryos that originated from vitrified oocytes showed a delay of ∼1 hour from the 1st division to 2 cells (t2) to the time of blastulation (tB) (P<.05). The proportions of embryos allocated to categories A-E in the hierarchical tree were similar between groups. No differences in implantation rates between the fresh (51.3% [95% CI 47.1%-55.7%]) and vitrified (46.4% [95% CI 38.4%-54.4%]) groups were found. CONCLUSION(S): The embryo quality of vitrified oocytes was not impaired: cc2, quality according to our hierarchic morphokinetic model, and implantation rates were similar between fresh and vitrified oocytes. However, morphokinetic differences were observed from t2 to tB. Our main study limitation was the retrospective nature of the analysis, although a large database was studied.

A combination of hydroxypropyl cellulose and trehalose as supplementation for vitrification of human oocytes: a retrospective cohort study.

PURPOSE: This study aimed to determine whether the new formulation of vitrification solutions containing a combination of hydroxypropyl cellulose (HPC) and trehalose does not affect outcomes in comparison with using conventional solutions made of serum substitute supplement (SSS) and sucrose. METHODS: Ovum donation cycles were retrospectively compared regarding the solution used for vitrification and warming of human oocytes. The analysis included 218 cycles (N = 2532 oocytes) in the study group (HPC + trehalose) and 214 cycles (N = 2353 oocytes) in the control group (SSS + sucrose). RESULTS: No statistical differences were found in ovarian stimulation parameters and baseline characteristics of donors and recipients. The survival rate was 91.3% (95% confidence interval (CI) = 89.8-92.9) in the HPC + trehalose group vs. 92.1% (95% CI = 90.4-93.7) in the SSS + sucrose group (NS). The implantation rate (42.8%, 95% CI = 37.7-47.9 vs. 41.2%, 95% CI = 36.0-46.4), clinical pregnancy rate (CPR) per transfer (60.7%, 95% CI = 53.9-67.5 vs. 56.4%, 95% CI = 49.3-63.5), and ongoing pregnancy rate (OPR) per transfer (48.5%, 95% CI = 41.5-55.5 vs. 46.3%, 95% CI = 39.2-53.4) were similar for patients who received either HPC + trehalose-vitrified oocytes or SSS + sucrose-vitrified oocytes. Statistical differences were found when analyzing blastocyst rate both per injected oocyte (30.2%, 95% CI = 28.3-32.1 vs. 24.1%, 95% CI = 22.3-25.9) and per fertilized oocyte (40.8%, 95%CI = 38.5-43.1 vs. 33.2%, 95% CI = 30.8-35.5) (P < 0.0001). Delivery rate was comparable between groups (37.2%, 95% CI = 30.8-46.6 vs. 36.9%, 95% CI = 30.4-43.4; NS). CONCLUSIONS: Our data demonstrate that HPC and trehalose are suitable and safe substitutes for serum and sucrose. Therefore, the new commercial media can be used efficiently in the vitrification of human oocytes avoiding viral and endotoxin contamination risk.

Oocyte vitrification as an efficient option for elective fertility preservation.

OBJECTIVE: To provide a detailed description of the current oocyte vitrification status as a means of elective fertility preservation (EFP). DESIGN: Retrospective observational multicenter study. SETTING: Private university-affiliated center. PATIENT(S): A total of 1,468 women who underwent EFP because of age or having associated a medical condition other than cancer (January 2007 to April 2015). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Survival and cumulative live birth rate (CLBR) per consumed oocyte. RESULT(S): Mean age was higher with EFP due to age versus having an associated medical reason (37.7 y [95% confidence interval (CI) 36.5-37.9] vs. 35.7 y [95% CI 34.9-36.3]). In total, 137 patients (9.3%) returned to use their oocytes. Overall survival rate was 85.2% (95% CI 83.2-87.2). Live birth rate per patient was higher in women ≤35 years old than ≥36 years old (50% [95% CI 32.7-67.3] vs. 22.9% [95% CI 14.9-30.9]). CLBR was higher and increased faster in younger women. The gain in CLBR was sharp from 5 (15.4%, 95% CI -4.2 to 35.0) to 8 oocytes (40.8%, 95% CI 13.2-68.4), with an 8.4% gain per additional oocyte, in the ≤35-year-old group. The increase was slower with 10-15 oocytes, reaching a plateau CLBR of 85.2%. A milder increase (4.9% gain) was observed in the ≥36-year-old group (from 5.1% [95% CI -0.6 to 10.7] to 19.9% [95% CI 8.7-31.1] when 5-8 oocytes were consumed), reaching the plateau with 11 oocytes (CLBR 35.6%). Forty babies were born. CONCLUSION(S): At least 8-10 metaphase II oocytes are necessary to achieve reasonable success. Numbers should be individualized in women >36 years old. We suggest encouraging women who are motivated exclusively by a desire to postpone childbearing because of age, to come at younger ages to increase success possibilities.