Post-thaw Embryo Research Articles

P-722 Impact of blastocyst quality and post-thaw embryo culture factors in non-invasive PGT-A informativity on vitrified blastocysts.

Abstract Study question What are the most important factor/s involved in the informativity of non-invasive PGT-A (niPGT-A) of vitrified blastocysts? Summary answer The day of vitrification and post-thaw time in culture of vitrified blastocysts are the most important factors for niPGT-A informativity, without correlation with blastocyst quality. What is known already niPGT-A is a technique that allows the non-invasive study of the chromosomal content of blastocysts. The informativity and concordance rates for fresh (Rubio, 2020) and frozen-thawed embryos (Garcia-Pascual, 2022) have been related with the time in culture and the day of media collection. However, there is a need to understand the optimal conditions needed to obtain optimal informativity for frozen-thawed vitrified blastocyst undergoing niPGT-A. Study design, size, duration Prospective study including 135 spent blastocyst media (SBM) and the corresponding blastocyst samples from thawed day-5 and day-6 blastocysts from PGT-A (73) and niPGT-A (62) cycles collected from January 2021 until March 2022. Blastocysts were discarded under IRB approval. The embryos were divided into 3 groups depending on the day of development and the time in culture after thawing: group-1, day-5 blastocysts cultured 8h; group-2, day-5 blastocysts cultured 24h and group-3, day-6 blastocysts cultured 8h. Participants/materials, setting, methods Media and blastocysts were obtained from IVF patients (29-42 years-old). After thawing, each blastocyst was washed to remove remaining cumulus cells and individually transferred to a 10 μL droplet of fresh media or to an EmbryoScope dish with 20 μL per well. SBM and blastocysts were collected, frozen and analysed by Next Generation Sequencing. Correlation between concordance rates (overall agreement between SBM and blastocyst samples), informativity, embryo quality and time in culture were evaluated. Main results and the role of chance All the embryos had a good embryo quality (BB or greater using Gardner Criteria) but different expansion degree. Informativity was significantly lower when day-5 embryos were cultured for only 8 hours (group 1), regardless of the degree of expansion (expansion degree 3, 28.57%; degree 4, 64.29% and degree 5-6, 57.14%, p < 0.0005). The concordance rates between SBM and whole blastocysts were very high and no significant different in relation to the expansion degree when comparing day-5 embryos cultured for 8 hours (group 1, 90.5%) with 24 hours (group 2, 93.6%) or with day-6 embryos cultured for 8 hours (group 3, 92.3%). The data suggest that the important factor impacting the informativity rate is the day of vitrification, and the time in culture after thawing, but not the expansion degree of the embryos. Regarding the concordance rates, there is no impact of the time in culture nor the expansion degree. Limitations, reasons for caution The main limitation of this study is that all the embryos analysed had fair or good quality but different expansion degrees. Wider implications of the findings The high concordance rates observed in frozen blastocysts opens the possibility of expanding the use of niPGT-A to frozen blastocyst without an impact of the expansion degree in the results. Trial registration number Not applicable

Vitrifying expanded equine embryos collapsed by blastocoel aspiration is less damaging than slow-freezing

The cryotolerance of equine blastocysts larger than 300 μm can be improved by aspirating blastocoele fluid prior to vitrification; however, it is not known whether blastocoele aspiration also enables successful slow-freezing. The aim of this study was therefore to determine whether slow-freezing of expanded equine embryos following blastocoele collapse was more or less damaging than vitrification. Grade 1 blastocysts recovered on day 7 or 8 after ovulation were measured (>300–550 μm, n = 14 and > 550 μm, n = 19) and blastocoele fluid was aspirated prior to slow-freezing in 10% glycerol (n = 14), or vitrification (n = 13) in 16.5% ethylene glycol/16.5% DMSO/0.5 M sucrose. Immediately after thawing or warming, embryos were cultured for 24 h at 38 °C and then graded and measured to assess re-expansion. Control embryos (n = 6) were cultured for 24 h following aspiration of blastocoel fluid, without cryopreservation or exposure to cryoprotectants. Subsequently, embryos were stained to assess live/dead cell proportion (DAPI/TOPRO-3), cytoskeleton quality (Phalloidin) and capsule integrity (WGA). For 300–550 μm embryos, quality grade and re-expansion were impaired after slow-freezing but not affected by vitrification. Slow-freezing embryos >550 μm induced additional cell damage as indicated by a significant increase in dead cell proportion and disruption of the cytoskeleton; neither of these changes were observed in vitrified embryos. Capsule loss was not a significant consequence of either freezing method. In conclusion, slow-freezing of expanded equine blastocysts collapsed by blastocoel aspiration compromises post-thaw embryo quality more than vitrification.

Impact of Post-Thaw Incubation Time of Frozen Embryos on Clinical Pregnancy Rate.

Vitrification of embryos following a single-controlled ovarian stimulation has been the strategy practised now in many in vitro fertilisation clinics to minimise the risk of early ovarian hyper stimulation syndrome, to reduce multiple pregnancy rates and to improve cumulative pregnancy rates. In recent years, advances in vitrification techniques and improved culture conditions have led to good post-thaw embryo survival rates, thereby increasing pregnancy rates of frozen embryo transfer (FET) cycles. The aim of this study was to analyse the effect of post-thaw incubation time of frozen embryos on the clinical pregnancy rates (CPRs) of frozen embryo transfer (FET) cycles. This was a retrospective, comparative study done at a teaching hospital in assisted reproductive treatment. Three hundred and ten FET cycles were analysed, of which 125 had day 2 freezing and 185 had day 3 freezing. Depending upon the day of thawing and day of transfer, FET cycles were divided into six groups: Group 1 (day 2 thawing and day 3 transfer), Group 2 (day 2 thawing and day 4 transfer), Group 3 (day 2 thawing and day 5 transfer), Group 4 (day 3 thawing and day 3 transfer), Group 5 (day 3 thawing and day 4 transfer) and Group 6 (day 3 thawing and day 5 transfer). Statistical analysis was performed using version 14 R software version 4.0.1 (2020-06-06) (R foundation for Statistical Computing, Vienna, Austria). A P < 0.05 is taken as significant. The CPR of Group 4 was 42.4% which was more than that of the other groups but it did not reach statistical significance. Short incubation time of 2-4 h is as effective as an extended incubation time in terms of CPRs of FET cycles.

Post-Thaw Day 5 Blastocyst Culture Time Prior to Transfer Does Not Affect Assisted Reproduction Technology (ART) Outcomes in Frozen-Thawed Embryo Transfer Cycles.

The frozen embryo transfer (FET) technique has been progressively used more worldwide due to improved culture conditions, as well as enhanced survival rates after vitrification. However, little is known about the effect of the post-thaw blastocyst culture duration prior to transfer on live birth rate in FET cycles. In this retrospective observational study, we evaluated the influence of two distinct post-thaw blastocyst culture spans (2-4 h versus 20-22 h) on clinical pregnancy and live birth rate. A total of n = 1927 frozen-warmed cycles were included in the analysis. Among those, n = 885 warmed blastocysts were cultured for 2-4 h, and n = 1029 were kept in culture for 20-22 h prior to transfer; the remaining blastocysts did not survive the warming protocol. We observed no significant differences in live birth and clinical pregnancy rates between the two groups. The blastocyst morphological evaluation at transfer improved following the longer culture time. No differences between the two groups were found also for gestational and neonatal outcomes. This work shows that different post-thaw embryo culture timings do not negatively impact pregnancy outcomes. Overall, these results are important in the context of the embryological laboratory in order to better organize the workflow and avoid unnecessary timing-related workload.

Post-thaw viability, developmental and molecular deviations in in vitro produced bovine embryos cultured with l-carnitine at different levels of fetal calf serum

l-carnitine is a well-known an antioxidant that enhanced lipid metabolism. Therefore, this study investigated the influence of supplementing l-carnitine (LC) to in vitro culture medium on preimplantation development, quality, cryotolerance and transcription profile of candidate genes. Following in vitro fertilization, embryos at zygote stage were cultured with medium supplemented with LC at 1.5 mM and fetal calf serum (FCS) at 0, 2.5, 5, 7.5 and 10% of the CR1-aa culture media. Intracellular quality of produced embryos was measured using different fluorescent stains that measured reactive oxygen species (ROS), lipid and mitochondria intensities. In addition, total cell number and total apoptotic cells were counted per embryo. Quantitative expression of candidate genes was conducted to find out molecular response of embryos after treatment. Moreover, vitrification was done at day 8 of preimplantation development to evaluate post-thaw embryo viability. The results indicated improved blastocyst formation rate at day 8 of preimplantation development (day zero = day of IVF) when embryos cultured with LC supplementation at low FCS at levels of 2.5% (35.3%) and 5% (34.7%) compared to control (25.9%), LC + FCS 7.5% (26.5%) and LC + FCS 10% (28.1%) groups. The total number of blastocyst cells that were cultured with LC + FCS 2.5% and LC + FCS 5% was increased and the number of dead cells (apoptotic) was decreased compared to control counterparts. Intracellular mitochondria activity was enhanced and resulted in reduction of cytoplasmic lipid in embryos treated with LC + FCS 2.5% and LC + FCS 5% compared with other experimental embryo groups. In addition, intracellular reactive oxygen species level was reduced in LC + FCS 2.5%, LC + FCS 5% and LC + FCS 7.5% compared to control and LC + FCS 10% groups. The expression profile of genes regulating embryo quality (BCL2), metabolic activity (GLUT1, CPT2 and TFAM), lipolysis (LIPE, AMPKa1 and ACCα), resistance to stress (SOD2) and ability to induce pregnancy (IFNt) was up-regulated under low FCS (2.5% and 5%) combined with LC supplementation. On the other hand, genes regulating lipogenesis were down-regulated (ACSL3 and S1PR). It can be concluded that LC is an efficient culture media supplement when added with FCS at 2.5 and 5% which improved blastocyst development rate and quality. These improvements are due to enhanced utilization of intracellular embryo lipid that subsequently increased cryotolerance through orchestrating genes involved in various activities of bovine embryos.

P-187 Post-thaw embryo quality is not predictive of live birth rates in frozen embryo transfer cycles: a retrospective cohort study

Abstract Study question To determine whether post-thaw change in embryo quality is associated with live birth outcomes. Summary answer Post-thaw embryo morphology is not predictive of live birth rates in single frozen embryo transfer cycles. What is known already Embryo quality is typically evaluated via morphological assessment of embryos based on the Gardner criteria. For medical or elective reasons, embryos are commonly frozen for future use through the vitrification process. Research has shown that embryo quality pre-vitrification correlates highly with freeze-thaw survival, implantation, pregnancy, and subsequent live birth rates. Good-quality embryos are therefore more likely to be selected for vitrification. However, it is not unusual to find a decline in the quality of these vitrified embryos upon thawing them for embryo transfer. Study design, size, duration We retrospectively identified a patient cohort based out of the Ottawa Fertility Centre in Ottawa, Ontario, Canada, between 2016 and 2020. Participants/materials, setting, methods Frozen single embryo transfer (FET) cycles involving good-quality expanded blastocysts deriving from autologous oocytes were selected for inclusion. We compared FET cycles involving good post-thaw embryo quality to those with worsened/poor post-thaw embryo quality. The primary outcome was live birth after FET. Secondary outcomes included rates of positive serum human chorionic gonadotropin, clinical intrauterine pregnancy, miscarriage, and ectopic pregnancy. We fit a multivariate logistic regression model, adjusting for patient and cycle characteristics. Main results and the role of chance A total of 962 single FET cycles were analyzed. There were 826 embryos that preserved their pre-vitrification quality post-thaw and 136 embryos that exhibited poorer quality on post-thaw assessment. Baseline characteristics were mostly comparable between the two groups under study. In the multivariate regression model, the adjusted odds of live birth was not significantly different in the group with good post-thaw embryo quality compared to that in the group with worse/poor post-thaw embryo quality (odds ratio [OR] 1.30, 95% confidence interval [CI] 0.86-1.96, p = 0.21). Similarly, no significant associations were found between post-thaw embryo quality and the secondary outcomes of positive BhCG (OR 1.19, 95% CI 0.81-1.75), clinical intrauterine pregnancy (OR 1.29, 95% CI 0.87-1.87), miscarriage (OR 0.97, 95% CI 0.53-1.77), and ectopic pregnancy rates (OR 0.41, 95% CI 0.14-1.24). Limitations, reasons for caution Although a multivariate regression model was used to adjust for clinically relevant confounders, there remains the possibility for residual confounding given the observational nature of our study. Wider implications of the findings The results of our study suggest that, after adjusting for patient and cycle characteristics, post-thaw embryo quality does not impact live birth outcomes. Trial registration number Not applicable

P-212 Impact of post-thaw blastocyst culture time prior to transfer on live birth rate in frozen-thawed embryo transfer cycles: a retrospective observational study

Abstract Study question Does post-thaw blastocyst culture time prior to transfer influence the live birth rate in frozen-thawed embryo transfer cycles? Summary answer Our study revealed that different culture times of frozen-thawed blastocysts prior to transfer does not influence live birth rate. What is known already Several studies demonstrated a significantly increased clinical pregnancy rate after a frozen-thawed embryo transfer (FET) compared to a fresh embryo transfer (ET). FET reduces the risk of ovarian hyperstimulation syndrome without facing adverse effects of supraphysiological hormonal levels over endometrial receptivity. However, little is known on the effect of post-thaw embryo culture duration prior to transfer on live birth rate in FET cycles. Recent findings described an improved blastocyst morphological grade after a 20-22h post-thaw culture. Of note, the overnight culture of non-ideal morphology thawed blastocysts prior to transfer resulted in an enhanced live birth rate. Study design, size, duration We compared the live birth rate of patients undergoing FET after a short blastocyst culture time (2-4h) prior to transfer to that of patients in whom thawed blastocysts were transferred after a long embryo culture time (20-22h). Frozen-thawed transfers of blastocysts vitrified at Day 5 and subsequently thawed were evaluated. Only transfers of single blastocyst were included. Participants/materials, setting, methods The study was conducted at the Infertility Center – Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan. We included couples with indications for IVF cycles who underwent frozen-thawed blastocyst transfer procedure on the following natural cycles for freeze-all strategy or to transfer supernumerary embryos from 2014-2021. Data were analyzed using SPSS Statistics software. We evaluated statistically significant differences using various tests including Fisher's test, chi-squared test and Wilcoxon's test. Main results and the role of chance A total of 2012 frozen-thawed cycles were included in the analysis; in 977 of them, blastocysts were transferred after a 2-4h post-thaw period and in 1035, blastocysts were transferred after a 20-22h post thaw culture period. The age of the women was comparable (35.4 ± 4.2 and 35.3 ± 4.1; p = 0.5, respectively in the short and long culture period). According to statistical analysis, no significant difference in pregnancy outcomes was found between the two groups. Live birth rate was respectively 40% and 42% ( p = 0.2) in the short and long post-thaw blastocyst culture period prior to transfer. Limitations, reasons for caution Our study was limited by the retrospective nature and was performed in a single fertility center. Moreover, only blastocysts vitrified on day 5 were evaluated and findings might not be generalized to day 6 and 7 blastocysts. Wider implications of the findings These findings might be useful in the context of the organization of the laboratory workflow. Indeed, the possibility to thaw blastocysts on the day before transfer may represent an advantage in order to avoid an overload of the laboratory procedures during very busy days. Trial registration number 28_2022

Effect of vitrification on the expression of genes in porcine blastocysts derived from in vitro matured oocytes

This study aimed to examine the effect of vitrification on the expression of genes that are crucial for porcine early embryo development; cathepsin B (CTSB), growth differentiation factor 9 (GDF9), caudal type homeobox 2 (CDX2), and OCT-4, which play an important role in the maintenance of embryonic cell pluripotency. Their gene expression was investigated in expanded blastocysts (day 6–7) derived from in vitro matured oocytes. The quantitative real-time PCR method was used to assess the amount of relative specific transcripts in 20 vitrified (treatment group) and 32 fresh non-vitrified (control group) blastocysts. Vitrification was performed using 7.5% dimethyl sulfoxide (DMSO) plus 7.5% ethylene glycol (EG), and in the final step, 15% DMSO plus 15% EG and a 0.5 M sucrose solution and cryotop as a vitrification device. The blastocysts were warmed in 1 M, 0.5 M, and 0.25 M sucrose solution and kept in a culture medium for six hours before their fixation and further qPCR analysis. A significant upregulation in the targeted genes CTSB (p<.006), GDF9 (p<.04), and CDX2 (p<.003) was observed in the vitrified embryos compared to the fresh control group. Interestingly, the OCT-4 mRNA expression level was not affected by vitrification and remained comparable to that of the fresh non-vitrified embryos. In summary, the results of this pilot study showed, that vitrification induced substantial alteration in the expression of CTSB, GDF9, and CDX2 genes but did not influence the expression of OCT-4 gene in porcine in vitro derived blastocysts. Our data on the expression of developmentally important genes in vitrified porcine blastocyst may facilitate: (1) future improvements in culture conditions and/or cryopreservation protocol and (2) understanding the mechanism(s) of cryoinjuries inducing compromised post-thaw embryo development followed by the poor pregnancy outcome after blastocyst transfer.

Slow-freezing is more damaging than vitrification to post-thaw quality of large equine embryos collapsed by blastocoele aspiration

Embryo flushing in mares is best performed on day 7 or 8 after ovulation because the likelihood of recovering an embryo(s) is higher. However, embryos larger than 300 µm are less tolerant of cryopreservation unless the blastocoele is first collapsed. While vitrification of collapsed blastocysts has yielded good results, there are no reports of the relative success of slow-freezing punctured blastocysts. The aim of this study was to compare the effects on post-thaw embryo quality of vitrifying and slow-freezing day 7-8 equine blastocysts after blastocoele puncture. Embryos were collected from donor mares on day 7 or 8 after ovulation, graded (1 to 4), measured (µm), and divided into two size categories (330-550 µm, n= 11 and >550 µm, n= 16). Blastocoele fluid was aspirated using a micromanipulator, and the embryos then either slow-frozen (n= 14; 2 steps, 10% glycerol) or vitrified (n= 13; 2 steps,16.5% ethylene glycol/16.5% DMSO/0.5M sucrose). After thawing or warming, embryos were cultured for 24h before being graded and measured to determine degree of (re)expansion (%). Next, the embryos were stained with DAPI to identify dead cells, before being fixed and further labeled with Phalloidin (cytoskeleton), WGA (capsule), and TOPRO-3 (DNA) to assess live/dead cell proportions, cytoskeleton quality, and capsule integrity. Control embryos (>300-550 µm, n=3 or >550 µm, n=3)were collapsed and cultured for 24h without cryopreservation. Data were analyzed using one-way ANOVA, Kruskal-Wallis, or Fisher's exact tests. For 300-550 µm embryos, quality grade was negatively affected by cryopreservation but did not differ between slow-frozen (3±0) and vitrified (1.7±1) embryos. On the other hand, re-expansion was significantly reduced in slow-frozen (73±34%) compared to vitrified (148±26%) and control (179±10%) embryos. Live/dead cell proportion, cytoskeleton quality and capsule integrity were not influenced significantly by cryopreservation technique. For larger (>550 µm) embryos, re-expansion was also significantly reduced in slow-frozen (52±24%) compared to vitrified (115±7%) and control (129±12%) embryos. In addition, embryo quality was compromised more after slow-freezing (3±0.5) than vitrification (1.9±0.4) or 24h culture alone (control: 2±0). Furthermore, the proportion of dead cells and degree of cytoskeleton disruption was higher after slow-freezing than vitrification, although the incidence of capsule loss was not influenced by freezing method. In conclusion, slow-freezing of micromanipulator-collapsed equine blastocysts compromises embryo quality more than vitrification; therefore, after aspiration of blastocoel fluid, it is preferable to vitrify rather than slow-freeze equine embryos >300 µm.

Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos.

This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P < 0.05) furthermore, T2 had similar expression of pro-apoptotic genes to CG (P < 0.05). ROS formation revealed that CG presented lower percentage of embryo surface area affected (3.80 ± 0.40%) (P < 0.05), in contrast, no differences were found among the other groups. T1 was most significantly (P < 0.05) damaged by DNA fragmentation. The vitrified groups with melatonin had similar damage levels of CG (P > 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

The Effects of Different Post-thawed Culture Periods on Clinical Outcomes in Frozen Embryo Transfer Cycle.

The purpose of this study is to evaluate the effects of different post-thawed culture periods on the clinical outcomes. 9381 frozen embryo transfer (FET) cycles were divided into three groups according to female age: < 35, 35-39, and > 39years, and two groups depending on post-thawed culture period before transfer: short culture (2-3h) group (S) and long culture (18-20h) group (L). According to the increment number of post-thawed embryos, the L group divided into three groups: ≤ 2, one ≤ 2 and the other > 2, and > 2 groups. Pregnancy outcomes included the implantation rate (IR), clinical pregnancy rate (CPR), multiple pregnancy rate (MPR), live birth rate (LBR), and neonatal characteristics. Long post-thawed culture caused a significant increase in the IR, CPR, MPR, and LBR (p = 0.000, 0.004, 0.037, and 0.001; CI = 1.06-1.194, 1.042-1.237, 1.008-1.254, and 1.054-1.245, respectively), and blastomere increment number also had a significant effect on IR, CPR, MPR, and LBR (p = 0.000, 0.000, 0.000, and 0.000, respectively). No significant differences were present in neonatal characteristics between the two post-thawed culture groups. Singleton group had a higher average gestational age and birthweight as well as a lower cesarean section rate, preterm labor rate, and low birthweight rate than twins group. Long post-thawed culture was associated with higher IR, CPR, MPR, and LBR, and transferring a well-developed embryo after long post-thawed culture might be a viable embryo transfer strategy to decrease MPR while maintaining CPR and LBR.

28 Evaluation invitro of two protocols of vitrification from alpaca (Vicugna pacos) embryos

The reproductive efficiency of South American camelids as the alpaca is low, with a few number of animals having a good genetic characteristic. The transfer of cryopreserved embryos has great potential to disseminate valuable genetic, but the suitable protocol for such cryopreservation still needs to be developed. In this study, two protocols of vitrification of alpaca embryos were tested. Day 6.5 post-mating, embryos (n=66) were recovered from 14 female alpacas through a non-surgical technique and classified according to the characteristics of old world camelids reported by Skidmore et al. 2004 (Reprod. Fertil. Dev. 16, 605–609). Only quality 1 and 2 embryos were used for the study. They were placed together in 50-µL drops of holding medium for 30min and transferred to a 100-µL drop of equilibration solution 1, consisting of 7.5% (v/v) ethylene glycol (EG) + 0.25M sucrose. After 1min, embryos were transferred to equilibration solution 2, consisting of 15% (v/v) EG + 0.5M sucrose. After 2min, embryos were transferred into 2 consecutive drops of vitrification solutions A [SA: 30% (v/v) EG + 1M sucrose] for 20s each, then in 2 other drops of vitrification solution B [SB: 30% (v/v) EG + 3% glycerol + 1M sucrose] for 20s each. Thereafter, embryos were quickly loaded into open pulled straws (OPS) in a volume of 10µL and then plunged into liquid nitrogen. For warming, the OPS were held in air for 5s and subsequently thawed at 37°C for 50s. Straws were emptied into 1mL of prewarmed holding medium solution (HMS1) containing 1M sucrose for wash and the thawed blastocysts were transferred into a second 1mL of prewarmed HMS1. After 5min incubation at 37°C, the blastocysts were transferred into 1mL of warmed Holding medium solution 2 (HMS2) containing 0.5M sucrose maintained at room temperature (∼24°C) for evaluation. Data were analysed by the Chi-squared test. Post-thaw embryo expansion results were 81.3% and 58.8% for SA and SB (P&amp;lt;0.05), respectively. Post-thaw embryo quality (1 and 2) were found at 62.5% and 29.1% with SA and SB, respectively (P&amp;lt;0.05). In conclusion, the vitrification of alpaca embryos with the ethylene glycol:sucrose solution results in better post-thaw outcomes than the ethylene glycol:sucrose:glycerol. Further experiments with embryo transfer are needed. This research was funded by FONDECYT project no. 149-2017.

Improvement of bovine in vitro embryo production by fetal calf serum and cysteamine supplementation and investigation of freezability

The aim of this study was to investigate the effects of Cysteamine (Cys) and Fetal Calf Serum (FCS) in Synthetic Oviduct Fluid (SOF) and Charles and Rosenkrans (CR1aa) on the bovine in vitro embryo production and its ability of freeze. The oocytes were divided into two groups and allowed to mature in TCM-199, with and without cysteamine. They were divided into 4 subgroups according to whether they contain Cys and FCS in 2 different culture media (SOF and CR1aa). Accordingly, 8 groups were formed as SOF+FCS+Cys, SOF+Cys, SOF+FCS, S Dulbeco's OF, CR1aa+FCS+Cys, CR1aa+Cys, CR1aa+FCS and CR1aa. Embryos were cultured for 7-9 days and were frozen using vitrification method. Development of embryos was observed during the first 24 hr post-thaw period. The highest rate of compact morula was 20% in SOF+FCS+Cys and 19.4% in CR1aa+FCS+Cys. The rate of blastocyst in SOF+FCS and CR1aa+FCS were found 17.9% and 15.4% respectively and the difference between groups was statistically significant (P&amp;lt;0.05). During the 12 hr vitality assessment, the highest rate was determined in SOF+FCS (47%). The results indicate that FCS has a positive effect in reaching compact morula and blastocyst regardless of the presence of the culture medium or antioxidant used. There was no statistically difference between the values of post-thaw embryos.

The impacts of the number of prefreeze and postthaw blastomeres on embryo implantation potential: A systematic analysis.

To systematically analyze the potential of embryo implantation through comparison between the number of surviving blastomeres, the growth, and implantation rate.Retrospective analysis on implantation rate and the growth of prefreeze-postthaw embryos with different blastomeres in 1487 frozen embryo transfer cycles.In groups of postthaw embryos without damage, implantation rate and the average number of blastomere growth increased significantly with increasing number of blastomeres. The implantation rate and the number of blastomeres of embryos with 8-8c (the number of blastomeres in prefreeze embryo-the number of blastomeres in postthaw embryo) continued to grow at a significantly higher rate than that of 5-5c and 6-6c (P < .05). In groups of embryos with the same number of blastomeres before freezing and with partial damage after resuscitation, the implantation rates were lower and the average numbers of blastomere growth reduced as the number of damaged blastomeres increased. For embryos with good quality before freezing, 1 to 3 damaged blastomeres in postthawed embryos did not affect the development and implantation rate. Both implantation rate and growth rate of embryos with 8-6c were significantly higher than those of embryos with 6-6c (P < .05).The number of surviving blastomeres and growth in frozen-thawed embryos could be important index to predict embryo development potential and clinical outcome of implantation. For embryos with good quality, a small amount of damaged blastomeres would not weaken embryo development potential and implantation rate after being thawed.

Influence of post-thaw embryo culture interval on assisted reproduction techniques pregnancy rates

Influence of post-thaw embryo culture interval on assisted reproduction techniques pregnancy rates

Prolonged post-thaw culture of embryos does not improve outcomes of frozen human embryo transfer cycles: A prospective randomized study

Objective: To evaluate the impact of prolonged post-thaw embryos culture on pregnancy outcome during frozen embryo transfer cycles. Methods: This prospective cohort study evaluated 324 thaw transfer cycles with 819 embryos from 269 patients at the Center for Reproductive Endocrinology and Infertility of Hue University Hospital in Vietnam. These frozen embryo transfer cycles were divided into two groups at the time of thawing: the short culture group (2-hour post-thaw culture) and the overnight culture group (overnight culture for 18 h) before the embryo was transferred into the uterus. The rates of embryo intact, grade A embryo at frozen and transfer time and continuing cleavage were recorded. The clinical outcomes including serum beta-human chorionic gonadotropin, clinical pregnancy and implantation rate were evaluated after 14 days, 4 weeks, 6 weeks, respectively, after embryo transfer. Results: Human chorionic gonadotropin positive occurred in 39.5% of patients in the short culture group compared to 25.9% in the overnight culture group with risk difference (RD)=13.6%, relative risk (RR)=1.343, 95% confidence interval (CI) 1.085-1.663, P 35 years) and women who received 3 embryos, pregnancy outcomes were found to be significantly (P Conclusions: The prolonged post-thaw culture period does not increase pregnancy outcome in comparison with the short culture.

28 Effect of deuterium oxide on bovine oocyte cryotolerance

It is known that cryopreservation triggers spindle disassembly, increased aneuploidy risk, decreased post-thaw survival, fertilization, and embryo development. We hypothesised that a treatment with D2O before vitrification would slow down oocyte metabolism and reduce ice crystal formation by replacing water inside the cells. The aim of the study was to evaluate the effect of a 4-h treatment with different D2O concentrations (0, 3, 15, and 30%) on cryotolerance of bovine in vitro-matured oocytes. Abattoir-derived bovine oocytes were matured in vitro for 20h in TCM-199 medium with 15% of bovine serum (BS), 0.5µg mL−1 of FSH, 5µg mL−1 of LH, 0.8mM l-glutamine, and 50µg mL−1 of gentamicin at 39°C with 5% of CO2 and randomly divided into 5 experimental groups. A group of non-vitrified oocytes was used as the fresh oocyte control group, whereas the remaining oocytes were incubated for 4h in in vitro maturation medium with 0% (vitrified control; n=205), 3% (n=205), 15% (n=205), and 30% D2O (n=205) before vitrification. The experiment was repeated 4 times. Oocytes were denuded in HEPES-buffered TCM-199 (H199)+5% BS and vitrified using a cryotop freezing straw. The oocytes were incubated in 200μL of H199+20% BS with 7.5% ethylene glycol and 7.5% dimethyl sulfoxide for 3min. After that, oocytes were collected in 50μL of H199+20% fetal bovine serum with 15% ethylene glycol+15% dimethyl sulfoxide and 0.5M sucrose for 20s and plunged into LN2. One month later, oocytes were warmed in thawing media with decreasing concentrations of sucrose (1.35M to 0.31M) and then placed into in vitro maturation medium for 2h before IVF. Matured oocytes were IVF and cultured according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). Cleavage and blastocyst rates were evaluated after 7 days of culture. Data were analysed using the GLM procedure of SPSS (SPSS Inc., Chicago, IL, USA). The least statistical difference post-hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. As expected, both cleavage [60.5±4.6 (fresh control); 36.9±2.6 (0% D2O); 46.3±3.7 (3% D2O); 31.6±2.4 (15% D2O); and 24.4±2.6 (30% D2O)] and blastocyst rates [25.7±0.8 (fresh control); 9.0±0.8 (0% D2O); 9.0±0.7 (3% D2O); 3.6±0.2 (15% D2O); and 4.3±0.8 (30% D2O)] decreased in all vitrified groups compared with the fresh control group. Within vitrified oocytes, cleavage rate increased (P&amp;lt;0.05) with 3% D2O treatment compared with the other groups. However, pretreatment with higher (15-30%) D2O concentrations decreased (P&amp;lt;0.05) blastocyst rates of vitrified-warmed oocytes. In conclusion, a pretreatment with low concentrations (3%) of D2O improved the cleavage rate of bovine vitrified-warmed oocytes, suggesting a potential beneficial effect, whereas deleterious effects were observed using the higher concentrations. Therefore, further studies are required to assess a potential use of D2O to improve oocyte cryotolerance, likely testing different incubation times.

Effect of vitrification on apoptotic changes in feline embryos

The aim of this study was to evaluate the influence of a vitrification-warming procedure on the viability of cat embryos and blastocysts and the incidence of apoptotic changes in blastocysts subjected to vitrification and blastocyst that developed from vitrified embryos. In the first part of the experiment, post-thaw embryo development and blastocyst viability were evaluated based on morphological appearance and the ability to develop (embryos) or re-expand (blastocyst) compared to control. In the second part, blastocysts that were viable after vitrification-warming and blastocysts that developed from vitrified-warmed embryos were stained with Annexin-V and TUNEL to evaluate apoptotic changes. Most of the vitrified-warmed embryos were viable after thawing, 36.3% developed to morula, and 14.7% to the blastocyst stage. The overall re-expansion rate of blastocysts that were vitrified on day 7 was 55.6%. Vitrification significantly increased apoptotic and necrotic changes in blastocysts, but did not influence late apoptotic and necrotic changes in blastocysts that developed from vitrified-warmed embryos. The total number of blastomeres in blastocysts was similar in blastocysts that developed from vitrified-warmed embryos (99.1 ± 23.1), but lower in blastocysts vitrified on day 7 (82.1 ± 16.8), if compared to the control group (107.9 ± 24.2). These results show that the vitrification-warming procedure returns embryos capable of further development to a good quality blastocyst in vitro. Whereas, vitrification of blastocysts caused the progression of apoptotic changes in some blastomeres, however it did not affect the overall blastocyst viability.

Effect of post-thaw embryo quality on clinical outcome of pregnancy

Effect of post-thaw embryo quality on clinical outcome of pregnancy

208 Cryotolerance of In Vitro-Produced Cattle Embryos Produced with Different Maturation and Culture Media

In vitro-produced (IVP) bovine embryos suffer from damage due to suboptimal culture conditions involving altered metabolism, reactive oxygen species generation and high lipid accumulation, which could be impacted by oocyte maturation × embryo culture interaction. We hypothesised that optimizing oocyte and embryo culture conditions would lead to a higher post-thaw embryo survival rate. The experiment was replicated 9 times in a factorial design with 3 oocyte maturation media: TCM-199 plus 10% serum and gonadotropins (control), BO-HEPES-IVM (BO, IVF Bioscience UK), and HMM (HEPES-buffered maturation medium) and 3 embryo culture media: BO-IVC (BOC), BBH7 (Cooley Biotech, Gainesville, FL, USA), and SCF1 (SOF for Conventional Freezing 1; Owen et al. 2017 Reprod Fertil Dev. 29, 129-130). Bovine oocytes (n = 2907) were aspirated from 2- to 8-mm follicles of abattoir ovaries, matured at 38.5°C for 22 to 24 h in 5% CO2 (control) or in BO and HMM in air, fertilized with semen from 1 of 3 bulls, and cultured in BOC, BBH7, and SCF1 at 38.5°C in 5.5% CO2, 5% O2, and 90% N2. Cleavage and blastocyst rates were evaluated at Day 3 and 7, respectively, after IVF. Selected stage 7 blastocysts were slow frozen using 1.5 M ethylene glycol supplemented with 1 mm l-ascorbic acid for 20 min (equilibration). The embryos were cooled to and seeded at –6°C, and then cooled to −32°C at 0.5°C/min. Embryos were thawed and re-expansion was assessed at 24 and 48 h. Data (Table 1) was arcsin transformed and analysed by ANOVA, and means separated by l.s.d. Results indicate that oocytes matured in BO and embryos cultured in SCF1 had a higher blastocyst rate than all oocytes matured in control medium and all embryos cultured in BBH7 (P &lt; 0.05). However, oocytes matured in control medium and embryos cultured in BOC had lower post-thaw re-expansion rates than other treatment groups (P &lt; 0.05). These results suggest that a combination of oocyte maturation and embryo culture media plays an important role in post-thaw embryo survival, al though interactions of present treatments need to be further evaluated. Table 1.Effect of maturation and culture media on blastocyst rate and cryotolerance ± SEM)