Survival Rate Of Blastocysts Research Articles

Ultra-Fast Warming Procedure of Vitrified Blastocysts Results in Maintained Embryology and Clinical Outcomes.

Vitrification has revolutionized embryo cryopreservation, but represents a significant workload in the IVF lab. We evaluated here an ultrafast blastocyst warming procedure in order to improve workflow while maintaining clinical outcome. We first evaluated the expression of main markers of lineage specification in a subset of blastocysts donated to research warmed with ultrafast protocol. We then performed a prospective pseudo-randomized pilot study comparing blastocyst survival, reexpansion and live birth rates between standard (3 steps, 15min), and ultrafast warming protocol (1 step, 2min). Finally, survival, reexpansion and live birth rates (LBR) obtained with ultrafast warming protocol were prospectively collected during 3months and compared with previous indicators. Immunofluorescence experiments showed that staining and spatial organization of cell fate markers were conserved with ultrafast protocol. Survival, reexpansion and LBR were strictly comparable between standard (n = 47 cycles) and ultrafast (n = 39 cycles) groups in the pilot study (100 vs 100%, 80 vs 76% and 29.8 vs 30.7% in standard and simplified groups respectively). Survival, expansion and LBR obtained with the ultrafast warming protocol over the next 3-month period (321 cycles, 336 embryos) were comparable with those obtained with the standard protocol throughout the 6months (547 FBT cycles, 578 embryos) preceding shifting protocol (97.6 and 29.6% vs 97.8 and 28.3% respectively, p > 0.05 for both). In conclusion, using an ultrafast blastocyst warming procedure results in similar embryology and clinical outcomes compared with standard protocol, but significantly shortens the technical procedure, ultimately improving the overall lab's workflow.

Influence of the shortened warming protocol on human blastocyst viability: an in vitro experimental study

Research QuestionDoes the shortened warming protocol impact the cell viability and outgrowth competence of human vitrified blastocysts warmed with or without fatty acids (FAs)? DesignIn this study, 326 discarded vitrified human blastocysts donated for research by consenting couples were used. The blastocysts were randomly allocated to five groups depending on the warming solutions, protocols, and recovery culture media: the control-conventional, control-shortened, FA-conventional, FA-shortened, and FA-shortened/recovery culture with FA (RF) groups. The blastocysts were warmed with or without FAs following the manufacturer's instruction (conventional method) or using the shortened method, in which blastocysts were immersed in a thawing solution for 1 min and then cultured in the recovery medium for 2 h. The embryo volume recovery, cell viability, intracytoplasmic lipid droplets, and outgrowth competence were evaluated. ResultsThe degree of blastocyst volume recovery was significantly higher after shortened warming than after conventional warming. Cell membrane collapse was observed during the shortened warming. The blastocyst survival rate and expansion status after the recovery culture were comparable among the five groups. However, the proportion of necrotic cells was increased in the control-shortened, FA-shortened, and FA-shortened/RF groups. The adhesion rates were comparable among all the groups; however, the outgrowth area was significantly higher in the FA-conventional group than in the other groups. ConclusionsThe efficacy of the shortened warming protocol depended on the warming solutions used. The shortened protocol effectively improves the daily workflow; however, each laboratory should examine its impact thoroughly before introducing it to prioritise clinical outcomes.

O-160 Do day of vitrification, grade of expansion and blastocyst quality affect embryo survival rate? An observational study in oocyte recipient patients

Abstract Study question Which parameters are the most predictive of embryo survival rate in oocyte recipient cryotransfer cycles? Summary answer We have identified that only day of vitrification and grade of expansion have an impact on embryo survival rate What is known already Embryo vitrification is a well stablished technique that has led to survival rates around ±96%. Recent studies, based on day of vitrification, grade of expansion and blastocyst quality, are trying to identify which vitrified blastocyst has the highest potential to survive and to implant. However, controversial results are present in the literature, improvements in laboratory quality parameters and embryo culture could unleash into no significant differences regardless of morphological parameters. Nevertheless, having an acceptable survival rate does not mean to have a good implantation rate, biomarkers to identify the best potential blastocyst to survive and to implant are needed Study design, size, duration An unicenter-based retrospective observational clinical study was performed at IVI Madrid, approved by the local institutional review board (IRB). The study included all the warmings at blastocyst stage from oocyte recipient patients from January 2019 to April 2022. A total of 4.196 of warmed blastocysts were assessed, out of which 4.006 blastocysts were transferred Participants/materials, setting, methods All warmed blastocysts were allocated in groups according to the day of vitrification, grade of expansion and quality. The primary outcome analysed was embryo survival rate that was calculated as the number of viable embryos divided by the total number of embryos warmed. Vitrification and warming were performed following Kitazato method (Cryotop, Kitazato, Biopharma). Embryo classification was assessed previous vitrification according to IVIRMA criteria. Statistical analyses were conducted using univariate and multivariate regression model Main results and the role of chance The mean maternal oocyte recipient age was 42.27±3.89 years old and body mass index 23±3.74 kg/m2). In a multivariate logistic regression analysis, a statistically significant association between embryo survival rate and the day of vitrification and grade of expansion was found. Chance of survival was significantly reduced for blastocyst vitrified on day 6 (n = 737) compared to day 5 (n = 3459) (93,2 % versus 96,0% [OR = 0.976 (0.960-0.993); p=0.05]). Regarding grade of expansion, cavitated blastocyst (n = 840) survival rate was 97,7% [OR = 1;p<0.001], expanded blastocyst (n = 1866) was 96,2% [OR = 0.095 (0.978-1.013)], hatching blastocysts (n = 1465) was 94% [OR = 0.978 (0.960-0.997); p=0.02) and fully hatched blastocysts (n = 25) was 60% [OR = 0.704 (0.648-0.765);p<0.001)]. However, no association was found neither between lower inner cell mass quality [OR = 0.964 (0.842-1.104); p=0.596) nor trophoectoderm [OR = 1.004 (0.981-1.028); p=0.746)] and survival rate. Analysing the combination of these three variables will give us the possibility to choose the best embryo to warm. The impact of day of vitrification, grade of expansion and blastocyst quality on implantation, clinical pregnancy, miscarriage, and live birth rates will be included in further studies Limitations, reasons for caution A weakness of this study is that it is retrospective in nature. The single-group design employed in retrospective studies limits the researchers’ ability to determine cause and effect. Further studies are needed to determine the impact of these variables on the survival rate Wider implications of the findings Based on our findings, blastocyst survival rate is influenced mostly by the day of vitrification and degree of expansion. To be able to select the best blastocyst to warm, a comparison between different day of vitrification when we have a similar morphology and embryo quality cohort is needed Trial registration number Not applicable

P-138 Protein concentration of 5 mg/mL in recovery medium for thawed blastocysts is equivalent to 10 mg/mL in post-thaw survival and pregnancy rates

Abstract Study question Are comparable survival and pregnancy rates obtained when recovery media are supplemented with 5mg/mL protein versus customary 10mg/mL protein in Frozen Embryo Transfer cycles? Summary answer Recovery medium containing 5mg/mL protein compared to 10mg/mL yielded similar outcomes in blastocyst survival and pregnancy rates. What is known already Accepted practice has been to culture thawed blastocysts in medium with the same protein concentration as the vitrification media (20% protein) to minimize osmotic stress and optimize survival and implantation rates. The potential to acquire similarly positive outcomes with supplementation of only 5 mg protein/ml has been explored and demonstrated, challenging the requirement for high protein levels in recovery media. Study design, size, duration From July through August 2022, a prospective study was conducted in a university laboratory to compare the survival and implantation rates in Frozen Embryo Transfer cycles using vitrified blastocysts recovered in Continuous Single Culture–NX Complete (CSCM-NXC) (5mg/mL Human Serum Albumin (HSA)) or global HP[LW1] (8.8mg/mL HSA and 1.2mg/mL α-and ß-globulins). 50 blastocysts were warmed per condition. Comparative analyses included survival and implantation rates, beta HCG levels, and clinical pregnancy rates by fetal cardiac activity. Participants/materials, setting, methods To evaluate the effect of protein level in recovery medium for vitrified blastocysts, CSCM-NXC and global HP, with 5mg/mL and 10mg/mL total protein, respectively, were compared. Blastocysts were vitrified in Irvine Scientific Vit Kit – Freeze and were warmed in Irvine Scientific Vit Kit – Warm. Minimum recovery time was the same for all blastocysts (30 minutes), and is defined as the time from completion of the warming procedure to the time of embryo transfer. Main results and the role of chance In both study arms, one blastocyst was removed from the data analysis due to embryo transfer cancellation that was not related to embryo survival or suitability for transfer, resulting in N = 49 for each group. Chi-square analysis was performed to assess statistical significance of all compared parameters, and a p value < 0.05 was considered significant. Average patient age (years) for CSCM-NXC was 36.1 and for global HP, 35.0. There was no difference between blastocysts warmed in CSCM-NXC and global HP in average recovery time, i.e., 3.8 and 3.9 hours, respectively, or survival rates, 98.4% and 98.1% respectively. Comparison of patients with blastocysts warmed in CSCM-NXC reflected a trend towards higher outcomes compared to those in global HP with initial positive beta HCG rates of 64.6% vs 56.3% and ongoing clinical pregnancy rates of 52.1% vs 45.8%, respectively. Implantation rates were 56.1% and 57.7% in CSCM-NXC and global HP, respectively. Incidence of monozygotic twinning of 0% in CSM-NXC and 3.3% in Global HP was observed. Limitations, reasons for caution As this is a challenge to accepted formulation of recovery media for vitrified blastocysts, specifically with respect to protein supplementation - a strategy for minimizing osmotic shock and potential damage to warming embryos - more expansive and robust studies must be undertaken to increase the statistical power of this study. Wider implications of the findings As frozen embryo transfers are utilized more routinely in assisted reproductive treatment, the clinical finding that a commercial recovery medium containing of 5mg/mL protein, the standard for routine embryo culture, can support the recovery of vitrified embryos and lead to successful pregnancy could increase the efficiency in the laboratory. Trial registration number na

P-296 Supernumerary blastocysts resulting from abnormal cleavage patterns: freeze or not to freeze? A practical question!

Abstract Study question After eSET, the supernumerary blastocysts that showed cleavage anomalies can be vitrified as second choice embryos. Do they display enough implantation chance to be frozen? Summary answer No pregnancy was obtained after frozen transfer of blastocysts with direct uneven cleavage and chaotic cleavage. It is not recommended to freeze those spare embryos. What is known already The use of time-lapse incubation offers stable culture conditions and provides comprehensive information on dynamic embryo development. Studies found that abnormal cleavage affects the genome of human embryos and can reduce its developmental potential with embryonic arrest. Aneuploidy contributes also to failed implantation. It was also reported that cleavage anomalies such as direct uneven cleavage and chaotic cleavage affect negatively implantation and live birth. Even if the good survival rate of blastocysts after vitrification has dramatically improved clinical outcome after frozen embryo transfer, implantation and live birth are correlated to embryonic ploidy and healthy cellular machinery. Study design, size, duration Retrospective qualitative assessment of time-lapse images for 186 vitrified blastocysts resulting from extended culture of 871 embryos showing morphokinetic abnormalities such as direct uneven cleavage first mitosis (DC1), direct cleavage second mitosis (DC2), fast cleavage (FC), complete chaotic cleavage (CC) and multiple anomalies (MA), during 2021-2022 in a single private hospital, and analysis of the clinical outcome after warming and SET. Participants/materials, setting, methods From 871 embryos in Embryoscope + (Vitrolife), 445 total blastocysts and 251 top quality were obtained according to Gardner’s grading. 186 of those were vitrified for further transfer. In 5 subgroups of anomalies (DC1, DC2, FC, CC, MA) pregnancy (PR) and ongoing pregnancy rate (OPR) per warming cycle and per transfer were calculated. The results were compared by SPSS software to overall 2021 center’s results. Main results and the role of chance The mean female age was 35.4 ± 0.5 years. From the 186 top quality blastocysts that were vitrified, 96 were warmed in 94 frozen cycles with a survival rate of 98.3%, resulting in 93 SET transfers. A total of 32 pregnancies were obtained from which 21 were ongoing pregnancies. There was no statistical difference in PR per warming cycle or per transfer, and in OPR per warming cycle or per transfer between the overall study group compared to the center’s annual results 2021: 34.0% vs 40.2%, 34.4% vs 40.1%, 22.3% vs 30.1%, and 22.6% vs 30.7%. Reasonable PR and OPR per transfer were obtained with blastocysts from DC2, FC and MA patterns, with caution to small sample size: 32.0% and 20.0%, 44.1% and 32.3%, 40.0% and 25.0%. The results were statistically comparable to the center’s annual result. All the transfers from DC1 and CC pattern failed to achieve a pregnancy. Limitations, reasons for caution One limitation of the study was its retrospective nature and the other was the small sample size in each subgroup of anomaly. The results should be confirmed prospectively and should compel the live birth rate after delivery of the ongoing pregnancies obtained during the second semester of 2022. Wider implications of the findings Abnormal cleavage patterns such as DC and CC are known to affect negatively implantation and live birth. Deselection of embryos showing these specific patterns could significantly increase successful implantation for the accordingly selected embryos. It is therefore non optimal to freeze the spare blastocysts resulting from these peculiar abnormal patterns. Trial registration number NA

A meta-analysis: Effect of androgens on reproduction in sows.

The mechanisms by which male hormones affect the development of ovaries and follicles has been studied by injecting exogenous androgens into sows. This may provide a reference for human polycystic ovary syndrome (PCOS), and can also provide guidance for improving the litter size of sows. We present a meta-analysis of studies published in the past 30 years on the effect of androgens on the ovulation rate of sows. A total of 517 papers were analyzed. The results showed that both testosterone (T) and dihydrotestosterone (DHT) injected into sows were positively related to the ovulation rate. T did not have a relevant effect on swine in vivo blastocyst survival rate. DHT had a negative phase with respect to blastocyst survival rate. Pig T-androgen receiver affinity was higher than the analogous affinity for DHT; this is different in humans. This suggests that sows are not suitable as human PCOS experimental animal models. To improve the litter size of sows, future research should focus on the mixed use of T and DHT, and the timing of use should be consistent with the periodic changes in androgen levels in sows. In addition, the welfare of experimental sows should be considered with reference to the clinical symptoms of PCOS.

EXTERNAL VALIDATION STUDY OF THE ULTRAFAST BLASTOCYST WARMING TECHNIQUE –OPTIMIZING EFFICIENCY WITHOUT COMPROMISING OUTCOMES

To evaluate blastocyst survival and re-expansion rates for biopsied and non-biopsied blastocyst stage embryos following warming with ultrafast warming (UFW) protocol compared to standard warming (SW) protocol.

O-082 Patients are requesting to ship their surplus frozen embryos to other countries so that they can be donated to other patients

Abstract Study question Can all patients decide on the destination of their frozen embryos? Summary answer Patients that don’t have the legal right to donate their surplus embryos, still have the opportunity to ship them to centres in Spain What is known already In most European countries the laws do not allow patients to decide on the destination of their surplus vitrified embryos. In Spain, however, all possible options are allowed but certain requirements must be met for each of them: donation, research and destruction. Even though, most couples do not decide on any of them and do not reply to the documents they receive from the Centre. The reasons for this behaviour are not due to an irresponsible attitude but to the fact that it presents them with a difficult situation, which triggers emotional conflicts. Study design, size, duration The goal of this study is to communicate that since 2018 we have received embryo shipment requests from people who have stored their surplus embryos in other countries, where they cannot donate them. We have evaluated 47 cases and accepted 17 from Italy, Belgium, Ireland and Greece. We evaluated the survival rate after thawing and pregnancy rates, and compared them with the results of surplus embryos that have been donated in our Centre. Participants/materials, setting, methods We received applications from patients from different countries. Given the legal difficulties, we only evaluated those from European Community countries. In order to accept the transfer, we require that they meet all the requirements to be “donation eligible”: clinical history and phenotypic features of parents and siblings born from that cycle, serology results prior to treatment, age under 35 years, good embryo morphology score, and all the information from the embryology laboratory of the Centre. Main results and the role of chance According to our Centre's data, only 3% of patients choose to donate their surplus vitrified embryos to others. In a retrospective review of the informed consents of a total of 5,605 IVF cases in which the patients had frozen supernumerary embryos we found that there are no significant differences in this percentage according to the biological origin of the embryos, neither if a couple or a single woman carried out the treatment. Differences are observed between patients from European countries that are close to each other and which are evidence of the idiosyncrasies of each culture. Donation is chosen by 3% of patients from Spain and the UK, 4% from France, 5.5% from Italy, 6% from Germany, 7% from Ireland. We surveyed patients about their reasons for choosing the destination of their embryos: the main reason why they rejected the possibility of donating embryos was the fear that in the future, their children might meet their biological siblings. In these 17 cases that have been shipped from other countries, blastocyst survival rate after thawing was 90% (compared to 97% in donated embryos in our Centre) and pregancy rate was 56% (compared to 59% in our Centre). Limitations, reasons for caution We don’t know the percentage of patients that would agree to donate their cross-border embryos, and most of them are not even aware of this possibility. They concur in a high socio-economic-cultural level that allows them to be informed and if they are accepted, they must pay the shipment fees. Wider implications of the findings We wonder whether all european patients that are interested in donating their embryos and that won’t resign to abandon them should be informed of this possibility. So far, we have had exquisite cooperation from the centres where they remain vitrified. May globalisation allow them to exercise their generosity! Trial registration number Not applicable

Time from trophectoderm biopsy to vitrification affects the developmental competence of biopsied blastocysts.

PurposeThe present study aimed to examine the correlations of the time interval from trophectoderm (TE) biopsy to vitrification with the blastocyst survival rate and blastocyst outgrowth ability.MethodsA total of 1,202 mouse blastocysts were randomly divided into control (non‐biopsy) and TE biopsy groups. The biopsied blastocysts were vitrified at various time points. The survival rate after warming, blastocyst adhesion rate, and outgrowth area was investigated. Several biopsied blastocysts were cultured in a time‐lapse incubator, and the time required for re‐expansion was measured.ResultsBlastocyst survival rates after warming and blastocyst adhesion rates were comparable between the control and biopsy groups. The area of trophoblast outgrowth in the 1‐h biopsy group was significantly smaller than that in the control, 0‐h biopsy, and 4‐h biopsy groups (p = 0.0304, p = 0.0058, and p = 0.0029, respectively). Re‐expansion of blastocysts was observed at a high incidence 1–2 h after TE biopsy.ConclusionsThe vitrification of biopsied blastocysts in the process of re‐expansion impairs outgrowth competence; therefore, blastocyst vitrification should be performed immediately after TE biopsy and before initiation of re‐expansion.

Does longer storage of blastocysts with equal grades in a cryopreserved state affect the perinatal outcomes?

AimAlthough mammalian embryos could be preserved in liquid nitrogen for thousands of years in theoretical models, the viability of cryopreserved blastocyst with varying grades remains to be speculated. In this study, we aimed to determine whether the longer storage time of blastocysts with equal grades could negatively affect the perinatal outcomes. Materials and methodsSingle vitrified-warmed blastocyst was divided into four grades (AA, AB/BA, BB, BC/CB) according to the blastocyst score when freezing, and each grade of blastocyst was categorized into four storage duration categories: 28 days-1 year, 1–3 years, 3–5 years, and ≥5 years. Then the perinatal outcomes with different storage time were analyzed. ResultsOur results revealed that for blastocysts with the same grade, the length of storage time had no statistical effect on blastocyst survival rate, clinical pregnancy/implantation rate, live birth rate, and abortion rate. In addition, more advanced developmental blastocyst could obtain better pregnancy outcomes regardless of the cryopreservation length. Similar neonatal outcomes were obtained over time. ConclusionsCryopreservation time could not negatively affect the perinatal outcomes of blastocysts with equal grades. Efficient blastocyst cryopreservation technology by vitrification can help older women obtain high-quality embryos at a young age.

P–251 To collapse or not to collapse blastocysts before vitrification? A matched case-control study on single vitrified-warmed blastocyst transfers

Abstract Study question Does laser-induced artificial blastocoel collapse result in better blastocyst cryopreservation survival and higher live birth rate (LBR) in comparison with intact counterparts? Summary answer Compared to vitrification of intact blastocysts, collapsed blastocysts resulted in higher survival and for 5% higher LBR. Neonatal outcomes were comparable in both groups. What is known already Blastocysts have long been considered a stage that is suboptimal for freezing-thawing procedures due to their high fluid content and different cell types. The development of a modified vitrification technique has enabled blastocysts to better survive cryopreservation compared to a slow freezing procedure. Many studies on the optimization of cryopreservation of blastocysts have mentioned the need for artificial collapsing of the blastocoel prior to cryopreservation, thereby reducing the risk of intracellular ice-crystals formation. However, the effectiveness of artificial collapsing on blastocyst survival rate, single vitrified-warmed blastocyst transfer (SVBT) outcome and on safety of such intervention remains to be confirmed. Study design, size, duration A retrospective matched case-control study of transfers of single blastocysts being artificially collapsed (case) or intact (control) before vitrification. A sample size of 306 cycles in both arms was needed to achieve 80% power to detect a difference between the groups of 10% with P < 0.05. Controls were matched to cases on a 1:1 ratio by female age, parity, fresh and frozen cycle protocol, blastocyst age and quality, getting 309 pairs of cases and controls. Participants/materials, setting, methods Artificial collapsing was introduced into clinical practice gradually. In fresh IVF cycles (performed in university clinic from 2012 until 2014) with supernumerary blastocysts, half of the blastocysts were randomly selected before vitrification for laser-induced artificial collapsing. The other half was vitrified in intact form. Only the first transfers of a single vitrified-warmed blastocyst (n = 818) were included in the study. By matching, 309 pairs of collapsed (study) and intact (control) SVBTs were identified. Main results and the role of chance Both groups were comparable by their characteristics in indications, female age, type and length of ovarian hyperstimulation, insemination method in fresh cycle, protocol for warmed blastocyst transfer, blastocyst quality and day of blastocyst vitrification. Survival rates in case and control group ((309/316) 97.8% and (309/323) 95.7%; P = 0.13) were comparable, but optimal survival rates (100% survival and re-expansion after warming) was significantly higher in artificial collapse group ((247/316) 78.2% and (225/323) 69.7%; P = 0.01). Clinical pregnancy rates ((120/309) 38.8% and (110/309) 35.6%; P = 0.4), miscarriage rates ((15/120) 12.5% and (24/110) 21.8%; P = 0.06) and LBR per transfer ((100/309) 32.4% and (85/309) 27.5%; P = 0.19) or LBR per warmed blastocyst ((100/316) 31.6% and (85/323) 26.3%; P = 0.14) were not statistically different between case and control groups. Since the study was powered to detect a 10% difference, the possibility of type 2 error cannot be excluded. Perinatal outcomes were available for 175 live births. There were 10.5% (10/95) preterm births in the study group vs. 16.3% (13/80) in control group (P > 0.05). Birth weights (3,308 g (SD 592 g) vs 3,308 g (SD 738 g) and sex ratio (50.7% vs 49.2% boys) were also comparable between both groups (P > 0.05). There were no major malformations detected in the study population. Limitations, reasons for caution The research is retrospective, but the cycles from both groups were performed in the same time period. The groups were balanced according to all possible confounders. Blastocysts for vitrification were first categorized by quality groups and embryos from each category were randomized for collapsing or for remaining intact. Wider implications of the findings: No significant difference was found in live births by this sample size. Nevertheless, increasing the success by 5% with the introduction of artificial collapsing can be an important step towards optimizing of blastocyst cryopreservation. To confirm a 5% improvement in results, a sample size of > 2500 cases would be needed. Trial registration number The study has been approved by the National Ethics Committee of the Republic of Slovenia (0120–204/2016–2).

P–586 Clinical outcome in frozen cycles using cryopreserved blastocysts derived from ovarian stimulation with follitropin delta

Abstract Study question To compare the live birth rate using frozen-thawed blastocysts obtained from ovarian stimulation with individualised follitropin delta dosing to conventional follitropin alfa dosing. Summary answer The live birth rate in cryo cycles conducted within 1 year after ovarian stimulation was comparable for individualised follitropin delta and conventional follitropin alfa treatment. What is known already It has been demonstrated that the follitropin delta (Rekovelle, Ferring Pharmaceuticals) in an individualised dosing regimen based on anti-Müllerian hormone (AMH) level and body weight is non-inferior to conventional follitropin alfa (Gonal-f, Merck Serono) dosing with respect to ongoing pregnancy and ongoing implantation rates in fresh cycles. The individualised approach also reduced the risk of ovarian hyperstimulation syndrome (OHSS) versus the conventional approach. Furthermore, treatment with follitropin delta and follitropin alfa gave comparable pregnancy rates in repeated fresh cycles. Study design, size, duration Analysis of frozen cycles using blastocysts obtained from a randomised trial comparing follitropin delta versus follitropin alfa in 1,326 IVF/ICSI patients (18–40 years) and a subsequent trial of up to two additional ovarian stimulation cycles. The clinical outcome includes women with cryopreserved blastocysts following ovarian stimulation and who underwent frozen cycles within 1 year after starting stimulation in their last cycle. Participants/materials, setting, methods A total of 917 women had at least one Day 5 blastocyst which was vitrified and stored following up to three ovarian stimulation cycles. A started cryo cycle was defined as warming of a blastocyst. After warming, 1–2 blastocysts were transferred in cryo cycles, using natural cycle or programmed regimens. Treatment differences and 95% confidence intervals (CI) were calculated with adjustment for age strata and accounting for repeated cycles within patient. Main results and the role of chance The proportion of women with frozen blastocysts was similar in the two treatment groups, with 69.5% in the follitropin delta group and 68.8% in the follitropin alfa group. Similar postwarming blastocyst survival rates were observed for the two groups, with 87.4% of the warmed blastocysts proceeding to transfer in the follitropin delta group and 88.8% in the follitropin alfa group. About half of the women (48.1% in each treatment group) with frozen blastocysts underwent at least one frozen cycle with transfer within the 1-year period, with an average of 1.5 cycles per woman in the follitropin delta group and 1.6 cycles per woman in the follitropin alfa group. The ongoing implantation rate was 27.6% in the follitropin delta group and 27.8% in the follitropin alfa group (adjusted difference 0.5% [95% CI: –7.1%; 8.2%]). The live birth rate per started cryo cycle was 32.0% in the follitropin delta group and 31.3% in the follitropin alfa group (adjusted difference 1.2% [95% CI: –6.8%; 9.3%]), while the live birth rate per cryo cycle with transfer was 33.2% and 31.9% (adjusted difference 1.9% [95% CI: –6.2%; 10.0%]), respectively. Limitations, reasons for caution The number of blastocysts to be transferred in the frozen cycles as well as the protocol for endometrial preparation was based on local centre practices. Wider implications of the findings: These findings suggest that the follitropin delta and follitropin alfa dosing regimens are equally effective in terms of live birth rate in frozen replacement cycles and add reassuring information to the clinical performance of cryopreserved blastocysts derived from ovarian stimulation with follitropin delta. Trial registration number NCT01956110, NCT01956123.

Prolonged interval time between blastocyst biopsy and vitrification compromised the outcomes in preimplantation genetic testing

This study aimed to evaluate to what extent the different interval times between trophectoderm (TE) biopsy and vitrification influence the clinical outcomes in preimplantation genetic testing (PGT) cycles. Patients who underwent frozen embryo transfer (FET) after PGT between 2015 and 2019 were recruited. In total, 297 cycles with single day 5 euploid blastocyst transfer were included. These cycles were divided into three groups according to the interval times: <1 h group, 1-2 h group, and ≥2 h group. Blastocyst survival, clinical pregnancy, miscarriage, and ongoing pregnancy rates were compared. The results showed that, in PGT-SR cycles, survival rate in the ≥2 h group (96.72%) was significantly lower than in the <1 h group (100%, P = 0.047). The clinical pregnancy rate in the ≥2 h group was 55.93%, significantly lower than in the <1 h group (74.26%, P = 0.017). The ongoing pregnancy rates in the 1-2 h group and the ≥2 h group were 48.28% and 47.46%, respectively, significantly lower than that in the <1 h group (67.33%, P < 0.05). The miscarriage rate in the 1-2 h group was 18.42%, significantly higher than that in the <1 h group (5.33%, P = 0.027). In PGT-A cycles, the clinical pregnancy and ongoing pregnancy rates in the <1 h group were 67.44% and 53.49%, respectively, higher than that in the 1-2 h group (52.94%, 47.06%, P > 0.05) and the ≥2 h group (52.63%, 36.84%, P > 0.05). In conclusion, vitrification of blastocysts beyond 1 h after biopsy significantly influences embryo survival and clinical outcomes and is therefore not recommended.

A comparative study of post-warming survival rates and clinical outcomes of human blastocysts vitrified/warmed by CryoTouch and Cryotop methods.

Advances in embryo culture conditions and the development of vitrification as a revolutionary cryopreservation method have allowed for routine use of blastocyst transfer in assisted reproduction technology (ART) cycles. Several vitrification/warming media and devices have been introduced for commercial use so far. The aim of this retrospective study was to compare post-warming survival rates and clinical outcomes of human blastocysts vitrified/warmed by two different commercial methods (CryoTouch and Cryotop) during ART cycles. This retrospective study assessed a total of 50 frozen embryo transfer (FET) cycles conducted on 56 warmed blastocysts between January 2018 and December 2020. Post-warming blastocyst survival rates and clinical outcomes including clinical pregnancy and live birth rates were calculated after single blastocyst transfer cycles. The results revealed no significant differences between two groups in post-warming survival rate (p-value=0.8381), clinical pregnancy rate (p-value=0.8157) and live birth rate (p-value=0.7041). Post-warming survival rates and clinical outcomes were comparable with no significant difference in blastocysts vitrified/warmed by CryoTouch and Cryotop commercial methods.

Does the number of embryos loaded on a single cryo-carrier affect post-vitrification survival rate?

We aimed to assess whether the survival rate of embryos is influenced by the number of embryos/oocytes loaded on a single cryo-carrier during vitrification. This was a retrospective study that included 974 patients who underwent thawing of 1896 embryo-warming cycles between September 2016 and January 2020. A distinct analysis was made for cleavage stage embryos (2-10-cell stage) and blastocysts. For vitrification, embryos were placed in a Cryotop™ open device using a SAGE vitrification kit following the manufacturer's instructions. Warming was carried using a SAGE warming vitrification kit according the manufacturer's instructions. Total post-vitrification survival rates of embryos at the cleavage stage or blastocyst stage was 94.8%. At the cleavage stage, cryo-preserving three embryos per single cryo-carrier gave the highest full intact embryo survival rate (91.5%) compared with one or two embryo(s) per single cryo-carrier (85.7%, P < 0.0002 and 87.3%, P < 0.004). Conversely, post warmed full intact blastocyst survival rate for two blastocysts was significantly lower compared with one blastocyst (76.7% vs. 87.9%, P < 0.0193) per single cryo-carrier. Post-thawing survival rate following vitrification is affected by the number of embryos per single cryo-carrier undergoing the vitrification equilibration phase, with the optimum number of three cleaved embryos or one blastocyst per single cryo-carrier. Further studies are required to determine the optimum number of cleaved embryos or blastocysts that should be loaded onto a single cryo-carrier vitrification device.

Inhibiting diacylglycerol acyltransferase-1 reduces lipid biosynthesis in bovine blastocysts produced in vitro

Diacylglycerol acyltransferase-1 (DGAT1) is one of the DGAT enzymes that catalyzes the final step in the synthesis of triacylglycerol, which is a major component of the lipid droplets in embryos. Intracellular lipids accumulated in embryos produced in vitro have been associated with reduced cryotolerance and quality. The objective of the present study was to investigate the influence of DGAT1 inhibition on embryo development, quality, and post-vitrification survival, in addition to expression profiles of selected lipid metabolism-regulating and oxidative stress genes. Bovine cumulus-oocyte complexes were matured and fertilized in vitro and were cultured in synthetic oviduct fluid (SOF) supplemented with 5% fetal calf serum (FCS) alone (Control) or with 1, 5, 10 or 50 μM DGAT1 inhibitor (A922500®; D1, D5, D10, and D50, respectively) or 0.1% dimethyl sulfoxide (CDMSO: vehicle for DGAT1 inhibitor dilution) from 54 h post-insemination until Day 8 post insemination. No differences were found in blastocyst yield on days 7 and 8 in Control, CDMSO, D10, and D50 groups. Embryos cultured with 10 or 50 μM DGAT1 inhibitor had greater mitochondrial activity (P < 0.01), and increased number of cells (P < 0.05), while the cytoplasmic lipid content was reduced (P < 0.01), the latter associated with altered expression profiles of selected genes regulating lipid metabolism or genes related with oxidative stress (transcript abundance increased for SLC2A1 and SLC2A5 and decreased for DGAT1 and GPX1). Importantly, the survival rate of blastocysts produced with 10 μM DGAT1 was higher than that of Control, CDMSO and D50 groups at 72 h after vitrification and warming (73.8 vs 57.1, 55.9 and 56.1%, respectively, P < 0.001). In conclusion, inhibition of DGAT1 synthesis in bovine embryos produced in vitro abrogates the negative effect of FCS by decreasing their lipid content, increasing mitochondria activity and improving embryo cryotolerance, as well as favoring the expression of lipid metabolism regulating and oxidative stress-related transcripts.

Autophagy and multivesicular body formation in blastocysts during the experimental diapause in mice

In experimentally induced diapause model in mice, blastocysts remain dormant for an extended period but resume implantation competency upon estrogen injection. The underlying mechanism by which extended longevity of dormant blastocysts is maintained is unclear. We have previously shown that dormant blastocysts, during experimentally induced diapause, exhibit heightened autophagic activation. Activation of autophagy appears to be a crucial adaptive response for survival in the unfavorable uterine environment, as inhibiting autophagy reduces the survival rate of dormant blastocysts. As a unique cell biological change occurring following estrogen supplementation to activate dormant blastocysts, multivesicular bodies (MVBs) accumulate in the trophectoderm. In various cellular contexts, autophagy and MVB formation are linked cell biological phenomena. Herein, we discuss the implications of these cell biological changes in dormant and activated blastocysts.

Cryopreservation of donkey embryos: Comparison of embryo survival rate after in vitro culture between conventional freezing and vitrification

Embryo cryopreservation ensures that genetic biodiversity is preserved over time. This study evaluates the survival of donkey embryos subjected to slow freezing and vitrification after thawing and in vitro culture. Seven-day-old in vivo produced donkey embryos were subjected to slow freezing (SF, N = 14) or vitrification (VIT, N = 22). After one year of cryopreservation, embryos were warmed, washed and placed in incubation for in vitro culture (IVC). In order to assess the embryo viability, the quality grade and developmental stage were recorded after thawing and after 24 and 48 h of IVC. Eleven embryos (SF = 4 and VIT = 7) were incubated under a time-lapse camera, for up to 68 h, in order to determine the area and growth. The survival rate was not influenced by the procedure but by the developmental stage: after 48 h of IVC blastocyst survival rate (1/8, 12.5%) was significantly lower compared to both morulas (8/12, 66.7%) and early blastocysts (11/16, 68.7%) (P < 0.05). Embryo diameter class at recovery did not significantly influence the survival rate. In terms of the embryos that were judged to be alive after 48 h of IVC, quality grade 1 was observed in 7/8 (88%) and 4/12 (33%) of the SF and VIT embryos, respectively (P < 0.05). After time-lapse analysis, the IVC embryo area as well as growth percentage were statistically higher in the SF than the VIT embryos (P < 0.05). In conclusion, no difference in survival rates was found between the two cryopreservation procedures, although embryo quality was more negatively affected by vitrification.

Effect of equilibration time on clinical and neonatal outcomes in human blastocysts vitrification.

PurposeProlonged exposure to equilibration solutions may be detrimental to an embryo's developmental potential, whereas a shorter exposure may affect the penetration of cryoprotectants into blastomeres. The purpose of this study was to evaluate the effects of different equilibration times on the clinical and neonatal outcomes of human blastocyst vitrification.MethodsThis is a retrospective study based on data collected between November 2008 and November 2015. A total of 192 blastocysts (80 non‐expanded and 112 expanded) obtained from 167 patients were analyzed. The blastocysts were divided into two groups according to their equilibration time: 8‐11 minutes or 12‐15 minutes. The clinical and neonatal outcomes of warmed blastocysts were evaluated.ResultsThe survival, implantation, and live birth rates of non‐expanded blastocysts were not different between the two groups, but they significantly improved for the expanded blastocysts in the 12‐15 minutes group compared to the 8‐11 minutes group. The results were similar for the neonatal outcomes after vitrified embryo transfer, when partitioned by equilibration time and blastocyst stage at vitrification.ConclusionsFor the non‐expanded blastocysts, a shortened equilibration time (8‐11 minutes) is sufficient for effective vitrification.

Should artificial shrinkage be performed prior to blastocyst vitrification? A systematic review of the literature and meta-analysis

Embryo vitrification is increasingly used in IVF. Artificial shrinkage (collapse) before vitrification has been proposed to maximise blastocyst survival after warming. However, its effectiveness on blastocyst survival rate and vitrified-warmed blastocyst transfer cycle outcome remains to be confirmed. Therefore, we performed a systematic MEDLINE search according to PRISMA guidelines on all articles published up to April 2018 and related to human blastocyst collapse before vitrification using the following keywords: (i) blastocyst; (ii) collapse; (iii) artificial shrinkage; and (iv) vitrification. The following outcomes were analysed and included in the meta-analysis: (i) blastocyst survival rate after warming; (ii) implantation rate; (iii) clinical pregnancy rate; and (iv) live birth rate after vitrified-warmed blastocyst transfer (commonly named frozen-thawed blastocyst transfer). Eight articles were included. Briefly, blastocyst survival (OR 5.04, 95% CI 2.43–10.46) and clinical pregnancy rate (OR 1.87, 95% CI 1.26–2.77) were significantly higher in collapse than in control group. However, implantation rate (OR 2.50, 95% CI 0.67–9.28) and live birth rate (OR 1.35, 95% CI 0.88–2.09) were comparable in both groups. In conclusion, this systematic review and meta-analysis suggests that artificial shrinkage before blastocyst vitrification improves survival and clinical pregnancy rate, but not implantation or live birth rate. Further randomised studies are warranted to improve the level of evidence and confirm these findings.