Cryotop Method Research Articles

P-150 Predicting fetal heartbeat in vitrified blastocysts through artificial intelligence analysis of post-warming images and videos

Abstract Study question Is it possible to predict the fetal heartbeat of frozen blastocysts from post-warming time-lapse images and videos captured with EmbryoScope? Summary answer A significant positive correlation was found between the score given by the artificial intelligence algorithm and the fetal heartbeat rate of the vitrified blastocysts analyzed. What is known already Vitrification is the safest and most successful technique for cryopreserving human embryos, although some of the best morphologically graded blastocysts before freezing do not recover properly when thawed, collapsing or failing to re-expand. Artificial intelligence (AI) has been applied on videos and images of fresh embryos at different stages, and also on images of vitrified-warmed embryos. However, a single image does not give information about events such as collapses or re-expansion rate. This study evaluated the ability of an AI software to predict the fetal heartbeat of vitrified blastocysts from images and videos taken in the post-warming period. Study design, size, duration This retrospective single-center study included a video of the period between thawing and transfer of 537 vitrified blastocysts, and the last image of that video of 518 blastocysts (Feb 2022-May 2023). All blastocysts were vitrified and warmed using the Cryotop method (Kitazato, Biopharma, Japan), and placed in EmbryoScope (Vitrolife, Denmark) time-lapse incubators for 2-5 hours to let them re-expand until embryo transfer. Warmed embryos were assessed by experienced embryologists according to the ASEBIR morphological criteria. Participants/materials, setting, methods Images and videos were provided to the algorithm (EMBRYOAID v1.0, MIM Fertility), which returned scores from 0 to 10. Multivariate logistic regression analysis included embryo score and confounders: patient age and body mass index, oocyte age, oocyte origin (autologous vs. donated) and handling (fresh vs. vitrified), day of blastulation (5 vs. 6), preimplantation genetic testing (PGT-A), and post-thaw incubation time. Model performance was calculated using the area under the receiver operating characteristic (ROC) curve (AUC). Main results and the role of chance The mean score was different between embryos with negative (5.9±1.2) and positive (6.3±1.2)* fetal heartbeat outcome. The contribution of each increased unit of algorithm score to the fetal heartbeat was significant in both imaging (OR = 1.36 [95%CI: 1.16-1.60]**) and video (OR = 1.29 [95%CI: 1.12-1.50]*). The score predicted fetal heartbeat with AUCs of 0.61 in images and 0.60 in videos, and it performed better than the ASEBIR conventional morphology, which achieved an AUC of 0.58. The algorithm performance was higher in embryos not subjected to PGT-A (AUC=0.62) in images (n = 418) and videos (n = 435). Fetal heartbeat prediction was optimal in images of non-PGT-A embryos taken between 3.3-4h of post-warming incubation (n = 107) (OR = 1.76 [1.22-2.55]*), reaching an AUC of 0.74. In videos, the optimal prediction in non-PGT-A embryos was at 3.3-4.1h (n = 126) (OR = 1.93 [1.36-2.74]*), with an AUC of 0.69. 9.9% of the embryos collapsed and/or did not re-expand after thawing. These damaged embryos had an implantation rate of 15.2%, lower than the rest (46.8%**). Mean score differed between poor prognostic and normal embryos, both in images (4.8±1.1 vs. 6.1±1.1**) and videos (4.9±1.1 vs. 6.2±1.2**). Embryo score was related to the odds of collapse/non-re-expansion in images (OR = 3.07 [2.20-4.29]**) and videos (OR = 2.94 [2.13-4.05]**), with an AUC=0.81 *P<0.01, **P<0.001. Limitations, reasons for caution The algorithm tested in this study was developed externally. It was designed to evaluate videos of fresh embryo development and images at 120 h post-ICSI, but not post-thaw videos and images at days 5 and 6. The developers did not have access to any data or labels from our clinic. Wider implications of the findings Algorithm scoring on videos and images was related to fetal heartbeat rate and to collapse/non-re-expansion of vitrified blastocysts. Fetal heartbeat prediction performance reached 74% in images of non-PGT-A embryos taken at 3.3-4h post-warming. The application of predictive models in vitrified cycles may facilitate the choice of the best frozen embryo. Trial registration number Not applicable

P-472 Oocyte cryopreservation for social reasons in women ≥ 40 years who decline oocyte donation: anticipated outcomes, myth dispelling, and addressing concerns

Abstract Study question Is oocyte vitrification a reproductive viable alternative for women aged over 40 who decline oocyte donation? Under what specific conditions? Summary answer Oocyte vitrification could be considered as an acceptable alternative in this patient cohort, with patients being adequately informed about the realistic possibilities. What is known already In Argentina, legislation on Assisted Reproduction Technologies endorses assisted reproductive treatments employing women’s own oocytes up to the age of forty-four. This legislative framework has resulted in an increased number of such procedures for women over 40 who decline oocyte donation, as well as a rise in those women in this age group who wish to postpone maternity for social reasons. There are scarce published studies presenting reproductive outcomes using vitrified and thawed autologous oocytes in this specific demographic. Study design, size, duration This retrospective observational study, conducted between January 2008 and December 2023, involved 299 patients aged 40 and above who underwent 356 cycles of autologous oocyte cryopreservation (1.2 cycles per patient). Statistical analysis utilized the InStat GraphPad statistical package, including one-way ANOVA and two-tailed Student’s t-test as required. Participants/materials, setting, methods Eighty-seven patients thawed their oocytes and performed 95 ICSI procedures followed by 75 embryo transfers. The primary outcome was the live birth rate, with secondary outcomes including oocyte survival rate, fertilization rate, implantation rate, clinical pregnancy rate, and miscarriage rate. Vitrification and thawing of oocytes were performed using the Cryotop method. Main results and the role of chance Average age: 41.4± 1.8 years. Average vitrified oocytes: 4.5 ± 3.9 [1-24]. 29.1%(87/299) patients thawed their eggs. Average age at thawing:44.0± 2.7 years [41-50]. Average storage time until use was 2.6 ± 1.9 years. 465 oocytes were thawed (4.9 ± 3.0 oocytes per patient). 31.0% used a donor semen sample and 69% used their partne’s semen sample. Survival rate was 87.1%(405/465). Fertilization rate was 74.3% (301/405). Cleavage rate was 98.0 % (295/301). 78.9% (75/95) of ICSI procedures had embryo transfer (1.9 ± 0.6 embryos per patient). Implantation rate was 8.9% (13/146). Clinical pregnancy rate per transfer was 17.3% (13/75). Miscarriage rate was 23.1% (3/13). Miscarriage rate per transfer was 4.0% (3/75). Live birth rate per transfer was 13.3% (10/75), 11.5% (10/87) per patient who thawed their oocytes and 10.5% (10/95) per ICSI procedure performed. Live birth rate per cryopreserved oocyte was 2.1% (10/465) in the general population and 9.1% (10/110) in those women who achieved at least clinical pregnancy. Women who achieved a healthy live birth had a greater number of vitrified oocytes (7.1 ± 3.1) than those who did not achieve pregnancy (4.9 ± 3.0) (p = 0.02). Limitations, reasons for caution This retrospective study’s conclusions are pertinent to a well-defined group of patients, specifically women aged over 40 who decline oocyte donation and have undergone oocyte vitrification. Wider implications of the findings Despite the absence of an assurance for a live birth through oocyte vitrification in this cohort, offering clear counsel grounded in variables such as the number of retrieved oocytes and age at the moment of cryopreservation allows for informed communication of realistic possibilities for those patients who decline oocyte donation. Trial registration number na

P-391 Association between meteorological season at the time of embryo transfer and pregnancy outcomes in freeze-all cycles: lessons from seasonal reproductive mammals

Abstract Study question Is there any effect of meteorological season at the time of embryo transfer on clinical outcomes of freeze-all cycles? Summary answer Optimal conditions for implantation appear to be associated with spring and summer. Embryo transfers performed in autumn or winter are associated with reduced ongoing pregnancy. What is known already Extensive research has consistently shown that most mammals are seasonal breeders, and as day length increases, gonadotropin secretion increases, to anticipate reproduction in the spring and summer months. There is intriguing evidence that there is a seasonal variance in human reproduction, but the data are inconclusive. Therefore, the goal for the present study was to evaluate the effect the meteorological season at the time of embryo transfer on clinical outcomes of freeze-all cycles. Study design, size, duration This historical cohort study was performed in a private university-affiliated IVF center between February 2018 and December 2022. A total of 4952 frozen-thawed embryos transferred to 3725 cycles in a single clinic were analyzed. Participants/materials, setting, methods Both vitrification and the warming procedures were performed using the Cryotop method. Embryos survival and clinical outcomes were analyzed by the season, at the time of embryo transfer, using generalized linear models adjusted for potential confounders. Main results and the role of chance The mean maternal age was 37.0 ± 4.7 years. Embryos were cryopreserved for a mean of 8.1 ± 17.3 months. Autumn was designated as the reference season for all analyses. No significant differences were observed in embryo survival rates when the seasons were compared, however, lowest implantation and pregnancy rates were observed when embryo transfers occurred in autumn (44.6%), followed by winter (45.8%, OR: 1.049, CI: 0.876 – 1.257, p = 0.603) and summer (49.0%, OR: 1.196, CI: 1.010 – 1.415, p = 0.038), while spring embryo transfers had a 30% increased odds of pregnancy (51.2%, OR: 1.302, CI: 1.100 – 1.541, p = 0.002). An increased miscarriage rate was observed when embryo transfers occurred in autumn (13.9%), followed by winter (11.4%, OR: 0.510, CI: 0.323 – 0.805, p = 0.004) and summer (10.4%, OR: 0.461, CI: 0.292 – 0.727, p = 0.001), while spring embryo transfers had a 63% decreased odds of miscarriage (5.6%, OR: 0.369, CI: 0.230 – 0.592, p < 0.001). Limitations, reasons for caution The use of historical cohort groups is a drawback. Despite the eligibility criteria for inclusion in the analysis, potential differences in the baseline characteristics cannot be ruled out. Wider implications of the findings In seasonal breeding mammals, melatonin increases the gonadotropins’ release during the transition period. Melatonin rise favors embryo implantation and protects offspring by increasing progesterone levels. Our evidence suggests that human reproductive potential may be influenced by climatic variations. Trial registration number N/A

P-114 The timing from post-insemination to blastocyst vitrification correlates with ploidy and gender

Abstract Study question Which morphology parameter of blastocysts has the strongest correlation with the euploid rate? Summary answer The vitrification times post-insemination and trophectoderm grade (TE) are more predictive parameters for euploid blastocysts, and female blastocysts require less time for vitrification. What is known already In current ART, the use of preimplantation genetic tests for aneuploidy (PGT-A) has increased by 2 to 25 percent in different countries. PGT-A analyses have helped patients achieve an increased pregnancy rate, choose the gender of the embryo and prevent miscarriages. Moreover, the potential for blastocyst euploid is strongly correlated with the developmental speed and morphology grades. However, for patients without PGT-A, the criteria for selecting transferred blastocysts are mostly based on morphology and the day of the blastocyst. Therefore, it is essential to clarify which parameter is more predictive of euploid embryos during elective FET cycles. Study design, size, duration This retrospective analysis included 713 blastocysts from 417 patients treated at the Ojinmed IVF Center, Mongolia, between May 2019 and June 2023. The morphology parameters (morphology grade, blastocyst formation time, vitrification time and inner diameter) were compared between the PGT-A results and the control results. Blastocyst quality was evaluated with the Gardner and Schoolcraft grading system, and the inner diameter of the blastocyst was measured. Participants/materials, setting, methods All patients underwent a clomiphene-based minimal ovarian stimulation protocol or drug-free natural cycle of IVF treatment. When the blastocyst diameter exceeded 160 μm, a trophectoderm (TE) biopsy was taken, and the blastocyst was immediately vitrified using the Cryotop method. The time of vitrification and formation of the blastocyst were recorded from the insemination. The patients were analyzed using high-resolution NGS PGT-A (10 Mb resolution for copy number variations and 30%-70% for mosaicism). Main results and the role of chance The PGT-A analysis reported euploid, aneuploid, and mosaic rates. Euploid, aneuploid, and mosaic rates were significantly correlated with blastocyst formation time (114.78 ± 4.49 h, 116.12 ± 6.42 h and 116.33 ± 6.87 h, p = 0.003) and vitrification blastocyst time (126.65 ± 9.8 h, 131.57 ± 10.8 h and 130.44 ± 11.19 h, p < 0.001), along with women’s age (34.9 ± 4.32, 38.14 ± 3.77, and 35.84 ± 4.5, p < 0.001). In logistic regression analysis, aneuploid rates were significantly lower in blastocysts with A and B grade of ICM (OR 0.36, CI 95% 0.24-0.32, p < 0.01 and OR 0.45, CI 95% 0.32-0.63, p < 0.01) and A and B grade of TE (OR 0.20, CI 95% 0.11-0.37, p < 0.01 and OR 0.20, CI 95% 0.25-0.48, p < 0.01) compared to C grades, respectively. The AUCs for prediction of euploid were maternal age 0.66 (95% CI 0.62-0.70), TE grade 0.64 (95% CI 0.57-0.64), blastocyst vitrification time 0.62 (95% CI 0.58-0.66), ICM grade 0.60 (95% CI 0.61-0.68), blastocyst formation time 0.55 (95% CI 0.51-0.59) and inner diameter 0.46 (95% CI 0.42-0.5). Although blastocyst vitrification time is correlated with blastocyst gender (female blastocyst 127.9 ± 10.25h vs male blastocyst 129.68 ± 10.84h, p = 0.02). Limitations, reasons for caution The results of the retrospective study are limited to data from a single IVF center and conventional incubators were used in this study. Wider implications of the findings These findings might help non PGT-A patients in deciding on embryo selection for transfer. The vitrification time and TE grade of blastocysts are strongly correlated with euploid rate than the ICM, inner diameter and formation time. Also, female blastocysts take less time to vitrification time than male blastocysts. Trial registration number *

Vitrification of pig embryos dysregulates the microRNA transcriptome profile

This study examined how the vitrification of pig blastocysts using either the superfine open pulled straw (SOPS) or Cryotop method affects the expression profile of embryonic microRNA (miRNA) transcriptomes, as well as its relation to changes in the expression of target genes (TGs). Surgically collected pig blastocysts were vitrified using either the SOPS method (n = 60; 4–6 embryos/device) or the Cryotop system (n = 60; 20 embryos/device). Embryos were cultured in vitro for 24 h after warming. Fresh blastocysts (n = 60) cultured for 24 h served as controls. After in vitro culture, five pools of eight viable blastocysts from each group were prepared for miRNA expression analysis based on a microarray approach. Then, biological interpretation of miRNAs profiles and integrative analysis of miRNA and mRNA transcriptome data were performed. Survival after 24 h of in vitro culture was similar (>96 %) for both the vitrification systems and the control group (100 %). Compared with the controls, the SOPS-vitrified blastocysts had 94 (one upregulated and 93 downregulated) differentially expressed (DE) miRNAs, and the Cryotop-vitrified blastocysts had 174 DE miRNAs (one upregulated and 173 downregulated). One DE miRNA (miR-503) in the SOPS group and three DE miRNAs (miR-7139–3p, miR-214 and miR-885–3p) in the Cryotop group were annotated for Sus scrofa. The integrative analysis showed that 27 and 61 DE TGs were regulated by the DE miRNAs in blastocysts vitrified with the SOPS and Cryotop systems, respectively. The TGs enriched one pathway (the TGF-β signaling pathway) for the SOPS system and four pathways (HIF-1, Notch, ascorbate and aldarate metabolism and glycosphingolipid biosynthesis-ganglio series) for the Cryotop system. In summary, vitrification via the SOPS and Cryotop systems dysregulates miRNAs, with slight differences between methods. The altered miRNAs identified in this study were related mainly to cell proliferation, apoptosis, and the response to cell stress. Further studies are needed to clarify the consequences of dysregulation of miRNAs involved in the TGF-β (SOPS-vitrified blastocyst) and Notch (Cryotop-vitrified blastocyst) signaling pathways, particularly if they can affect embryonic development.

Blastocoel fluid aspiration improves vitrification outcomes and produces similar sexing results of in vitro-produced cattle embryos compared to microblade biopsy

The potential applications of in vitro-produced (IVP) cattle embryos are significantly enhanced when combined with genotype selection and cryopreservation techniques. While trophectoderm (TE) biopsies are frequently used for genotyping, cell-free DNA (cfDNA) found in blastocoele fluid (BF) arises as a less-invasive method. Moreover, the blastocoel collapse produced by BF aspiration could be beneficial for embryo cryotolerance. This study was conducted to test the BF as a source of cell free-DNA (cfDNA) and to compare the BF to the TE biopsy in terms of sexing efficiency/accuracy, embryo survival and gene expression after vitrification/warming. IVP day 7 expanded blastocysts were artificially collapsed by aspiration of BF (VIT-Collapsed) or biopsied (VIT-Biopsied). After sample collection, embryos were vitrified/warmed by the Cryotop method and individually cultured in vitro. Intact fresh non-vitrified and vitrified/warmed blastocysts served as Fresh Control and VIT-Control, respectively. After sex identification of BF or TE biopsies and the corresponding surviving embryos, amplification efficiency and sexing accuracy were assessed. There were no differences between the BF and TE biopsy samples in terms of sexing accuracy or efficiency. Although all vitrified groups showed lower post-warming re-expansion rates (p < 0.05), the blastocyst re-expansion rates in the VIT-Collapsed group were comparable to those in the Fresh Control group whereas biopsied blastocysts showed the lowest (p < 0.05) re-expansion rates. VIT-Collapsed blastocysts had hatching rates that were comparable to those of Fresh Control blastocysts but significantly higher than those of the other vitrification treatments. Proapoptotic gene BAX was overexpressed in VIT-Biopsied embryos, whereas BCL2 transcripts were more abundant in the VIT-Collapsed group. On the other hand, VIT-Biopsied embryos showed altered ATP1B1- and AQP3-mRNA levels. The analysis of the cfDNA present in the BF is an efficient, minimally invasive approach to sex IVP cattle embryos. Besides, the artificial collapse of blastocoel prior to vitrification resulted in higher re-expansion and hatching ability than when embryos were vitrified after being biopsied.

Oocyte Vitrification Reduces its Capability to Repair Sperm DNA Fragmentation and Impairs Embryonic Development.

Oocytes play a crucial role in repairing sperm DNA damage, which can affect the next generation; however, certain factors can impair this ability. This study examined whether oocyte vitrification, a widely used method for fertility preservation, negatively affects repair ability. Male DBA/2 mice (n = 28) were injected with 101.60µmol/100g body weight of tert-Butyl hydroperoxide (tBHP) for 14days to induce sperm DNA damage. Histological changes, sperm functions, and DNA fragmentation were assessed using the TUNEL assay. Cumulus-oocyte-complexes (COCs) of superovulated female DBA/2 mice (n = 28) were vitrified using the Cryotop method. Fresh and vitrified oocytes were then fertilized by tBHP-treated and untreated sperms, and subsequent embryonic development was monitored. Additionally, the expression of Mre11a, Rad51, Brca1, and Xrcc4 was assessed in resulting zygotes and blastocysts using real-time PCR. The sperm tBHP treatment reduced differentiated spermatogenic cells in the testicular tissue, sperm concentration, and motility, while increasing DNA fragmentation (P < 0.05). The fertilization rate was decreased in the tBHP-treated sperm-vitrified oocyte group (P < 0.05), and the two-cell rate diminished in tBHP-treated sperm-fresh and vitrified oocyte groups (P < 0.05). The four-cell to blastocyst rate decreased in the untreated sperm-vitrified oocyte and the tBHP-treated sperm-fresh and vitrified oocyte groups (P < 0.05), and the tBHP-treated sperm-vitrified oocyte groups had the lowest blastocyst rate. In zygotes, Brca1 was upregulated in the tBHP-treated sperm-vitrified oocyte group (P < 0.05). Also, in blastocysts, Rad51, Brca1, and Xrcc4 were significantly upregulated in the untreated sperm-vitrified oocytes group (P < 0.05). Damages to the oocyte due to vitrification can disrupt the repair of sperm DNA fragmentation and consequently impair the embryo development.

A compact, high-throughput semi-automated embryo vitrification system based on hydrogel

Research questionWhat is the efficiency and efficacy of the novel Biorocks semi-automated vitrification system, which is based on a hydrogel? DesignThis comparative experimental laboratory study used mouse model and human day 6 blastocysts. Mouse oocytes and embryos were quality assessed post-vitrification. ResultsThe Biorocks system successfully automated the solution exchanges during the vitrification process, achieving a significantly improved throughput of up to 36 embryos/oocytes per hour. Using hydrogel for cryoprotective agent delivery, 12 vessels could be processed simultaneously, fitting comfortably within an assisted reproductive technology (ART) workstation. In tests involving the cryopreservation of oocytes and embryos, the system yielded outcomes equivalent to the manual Cryotop method. For example, the survival rate for mouse oocytes was 98% with the Biorocks vitrification system (n = 46) and 95% for the manual Cryotop method (n = 39), of which 46% and 41%, respectively, progressed to blastocysts on day 5 after IVF. CC-grade day 6 human blastocysts processed with the Biorocks system (n = 39) were associated with a 92% 2 h re-expansion rate, equivalent to the 90% with Cryotop (n = 30). The cooling/warming rates achieved by the Biorocks system were 31,900°C/minute and 24,700°C/minute, respectively. Oocyte quality was comparable or better post-vitrification for Biorocks than Cryotop. ConclusionsThe Biorocks semi-automated vitrification system offers enhanced throughput without compromising the survival and developmental potential of oocytes and embryos. This innovative system may help to increase the efficiency and standardization of vitrification in ART clinics. Further investigations are needed to confirm its efficacy in a broader clinical context.

Effect of IL-10 and TNF-α on the competence and cryosurvival of in vitro produced Bos indicus embryos

In vitro-produced embryos are constantly exposed to stressful conditions that can lead to the activation of the apoptotic pathway. The nuclear Kappa B factor (NF-κB) is an inflammatory mediator that induces the expression of tumor necrosis factor (TNF-α), a pro-inflammatory cytokine, while interleukin-10 (IL-10), an anti-inflammatory cytokine, inhibits NF-κB activity. This study aimed to investigate the effects of IL-10 and TNF-α on the competence and cryosurvival of in vitro-produced bovine embryos. Embryos were produced in vitro using standard protocols, and Grade I blastocysts were vitrified using the Cryotop method. Non-vitrified and vitrified blastocysts were subjected to the TUNEL assay. In Experiment I, on day 6.5 (156 h post-insemination), the embryos were treated with PBS (control), 50 ng/mL of IL-10, or a combination of 25 ng/mL of TNF-α and 50 ng/mL of IL-10. Embryonic development and apoptotic rates were monitored. In Experiment II, the same groups were set up, with the addition of a group treated with 25 ng/mL of TNF-α alone. Grade I blastocysts were vitrified 5 h after treatment, and cryosurvival was monitored at until 48 h post-warming. The apoptosis rate and total cell number were investigated in the vitrified-hatched blastocysts. IL-10 alone did not affect developmental competence or cryosurvival (P > 0.05). The IL-10-treated embryos, when exposed in combination with TNF-α, presented a detrimental effect (P < 0.05) in the embryonic development of non-vitrified embryos. However, vitrified blastocysts had no negative effect (P > 0.05). The TNF-α treatment reduced (P < 0.05) the re-expansion rate at 6 h post-warming and increased (P < 0.05) the apoptosis rate in vitrified hatched blastocysts, whereas no effect (P > 0.05) of the treatments was detected in the hatching rate and total cell number post-warming. In conclusion, TNF-α has a detrimental effect on embryonic developmental competence and cryosurvival by compromising the development of non-vitrified embryos and apoptotic-related events of vitrified blastocysts, whereas IL-10, when in combination with TNF-α, appears to attenuate the detrimental effects of TNF-α.

Cryopreservation of Bovine Oocyte using Vitrification Solution and Cryotop Techniques

Cryopreservation is used to preserve biological samples over an extended period at ultra-low temperatures. This process evolved into vitrification, a more advanced and superior technology in which fluids or water molecules form a glass-like structure without forming ice crystals. Unlike fresh cells, cryopreservation is reported to reduce oocyte viability and developmental competency. This study employed two vitrification techniques, vitrification solution (VS) and Cryotop, to investigate the meiotic resumption in bovine. Oocytes were extracted from cow ovaries collected from slaughterhouses in Banting and Shah Alam, Selangor, Malaysia. The oocytes were grouped (A, B, and B’) based on cumulus morphology and matured in vitro in a culture dish (humidified 5% carbon dioxide incubator at 38.5°C) for 20 to 24 hr. Oocytes were vitrified after maturation using straws or aids of Cryotop sheets, then submerged in liquid nitrogen and stored for five days before defrosting for cryoprotectant elimination. By using Giemsa staining, the maturation state of fresh and vitrified bovine oocytes was evaluated through five parameters: zygotene, pachytene, diakinesis, metaphase I, and metaphase II. The maturation rate demonstrated only slight differences in the three groups of oocytes treated with VS (A: 44.79%; B: 30.97%; B’: 20.70%) and Cryotop (A: 39.42%; B: 37.27%; B’: 28.97%), which were significantly lower than fresh oocytes (A: 55.83%; B: 44.82%; B’: 56.17%). Both VS and Cryotop methods were viable options for cryopreserving oocytes, but the Cryotop technique was more effective in increasing the meiotic competence of poor-quality oocytes.

O-158 Prediction of implantation of vitrified-warmed blastocysts using a deep learning algorithm on a single post-warming image of each embryo

Abstract Study question Can a deep learning algorithm determine the implantation likelihood of vitrified blastocysts from single time-point post-warming time-lapse images obtained from EmbryoScope? Summary answer A significant positive correlation was found between the score given by the deep learning algorithm and the implantation rate of the frozen blastocysts analyzed. What is known already Despite the safety and success of the vitrification technique, many of the best morphologically classified embryos prior to vitrification do not maintain their characteristics when returned to room temperature. Since artificial intelligence (AI) has been shown to be useful in assessing the quality of fresh embryos at different stages, it could also be used to predict the outcome of frozen embryo transfers. This is the first attempt to distinguish vitrified blastocysts that will implant from those that will not by applying deep learning on a single image taken in the period between warming and transfer. Study design, size, duration This retrospective single-centre study included 689 blastocysts with known implantation data. All of them were vitrified and warmed using the Cryotop method (Kitazato, Biopharma, Japan). Warmed embryos were assessed by experienced embryologists according to the ASEBIR morphological scoring system and the degree of expansion. Immediately after warming, blastocysts were placed in EmbryoScope (Vitrolife, Denmark) time-lapse incubators for 2-5 hours to let them re-expand until embryo transfer. Participants/materials, setting, methods A pre-transfer embryo image was provided to the algorithm (Life Whisperer Viability), which returned scores from 0 to 10. Data were analyzed by chi-square test or one-way ANOVA. Multivariate logistic regression analysis was performed including embryo score, oocyte origin (donated vs. autologous), oocyte age, patient age, oocyte handling (fresh vs. vitrified eggs), and day of vitrification (5 vs. 6). The area under the receiver operating characteristic (ROC) curve (AUC) was used to calculate evaluation performance. Main results and the role of chance There was a significant difference between the scores given to vitrified implanted and non-implanted embryos. The mean value was 4.98 [95% CI 4.70-5.26]* for non-implanted embryos and 6.16 [95% CI 5.84-6.48]* for implanted embryos. When dividing the embryo score by quartiles, the implantation rate for each quartile was 27.7% for Q1, 33.7% for Q2 (Odds ratio (OR)=1.31 [0.82–2.09]), 44.8% for Q3 (OR = 2.05 [1.30–3.22]*) and 48.3% for Q4 (OR = 2.46 [1.56–3.87]*). The contribution of each increased unit of the algorithm score to the implantation (OR = 1.16 [95% CI: 1.09–1.23]*) was statistically significant. The deep learning algorithm score successfully predicted implantation with an AUC of 0.65 [95% CI: 0.60–0.69]*. Moreover, the algorithm performed better than the ASEBIR morphological classification, which achieved an AUC of 0,62 [95% CI: 0.58–0.66] in predicting implantation. *P &amp;lt; 0.001. Limitations, reasons for caution Our clinic was not involved in the development of the algorithm. Furthermore, it was designed to assess fresh embryos prior to vitrification but not thawed ones, so this study should be considered an external trial. A post-warming full video analysis by AI could possibly provide additional information. Wider implications of the findings The use of predictive models on vitrified cycles may help to select the best frozen embryo for transfer and predict which embryos will result in implantation failure early enough to thaw another one with a better chance of success. It also indicates that AI can generalize to thawed embryo assessment. Trial registration number -

Refinements in embryo manipulation applied to CRISPR technology in livestock

The implementation of CRISPR technology in large animals requires further improvements in embryo manipulation and transfer to be applied with commercial purposes. In this study we report (a) developmental competence of CRISPR/Cas microinjected zygotes subjected to in vitro culture in large scale programs in sheep; (b) pregnancy outcomes after early-stage (2–8-cell) embryo transfer into the oviduct or the uterine horn; and (c) embryo survival and birth rate after vitrification/warming of CRISPR/Cas microinjected zygotes. Experiment 1 consisted of a retrospective analysis to evaluate embryo developmental rate of in vitro produced zygotes subjected to CRISPR/Cas microinjection (n = 7,819) compared with a subset of non-microinjected zygotes (n = 701). Development rates to blastocyst on Day 6 were 20.0% for microinjected zygotes and 44.9% for non-injected zygotes (P < 0.05). In Experiment 2, CRISPR/Cas microinjected zygotes were transferred on Day 2 after in vitro fertilization (2-8 cell embryos) into the oviductal ampulla (n = 262) or into the uterine horn (n = 276) in synchronized recipient ewes at prefixed time (i.e., approximately two days after ovulation). Pregnant/transferred recipients (24.0% vs. 25.0%), embryo survival/transferred embryos (6.9% vs. 6.2%), and born lambs/pregnant embryos (72.2% vs. 100.0%) did not differ significantly in the two groups. In Experiment 3, CRISPR/Cas microinjected zygotes were maintained under in vitro culture until blastocyst stage (Day 6), and subjected to vitrification/warming via the Cryotop method (n = 474), while a subset of embryos were left fresh as control group (n = 75). Embryos were transferred into the uterine horn of recipient females at prefixed time 8.5 days after the estrous synchronization treatment (i.e., approximately six days after ovulation). Pregnancy rate (30.8% vs. 48.0%), embryo survival rate (14.8% vs. 21.3%), and birth rate (85.7% vs. 75.0%) were not different (PNS) between vitrified and fresh embryos, respectively. In conclusion, the current study in sheep embryos reports (a) suitable developmental rate after CRISPR/Cas microinjection (i.e., 20%), even though it was lower than non-microinjected zygotes; (b) similar outcomes when Day 2-embryos were placed into the uterine horn instead of the oviduct, avoiding both time-consuming and invasive oviduct manipulation, and extended in vitro culture during one week; (c) promising pregnancy and birth rates obtained with vitrification of CRISPR/Cas microinjected embryos. This knowledge on in vitro embryo development, timing of embryo transfer, and cryopreservation of CRISPR/Cas microinjected zygotes have practical implications for the implementation of genome editing technology in large animals.

Detrimental effects of vitrification on integrin genes (α9 and β1) and in vitro fertilization in mouse oocytes.

Integrins are known as key molecules that importantly involve in fertilization. This study aimed to evaluate effects of vitrification on fertilization rate and expression of integrin genes, α9 and β1, on mice oocytes in GV and MІІ stages. From the ovarian tissue and fallopian tube of NMRI mice, germinal vesicle (GV, n = 200) and metaphase II (MII, n = 200) oocytes were obtained. Then, oocytes were distributed into 4 groups including non-vitrified GV, non-vitrified MII, vitrified GV, and vitrified MII. Cryotop method was used for vitrification and oocytes (for 4 weeks) were kept in liquid nitrogen. After that, by using an inverted microscope, the rate of survived oocytes was assessed. Also, in vitro fertilization (IVF) for oocytes, obtained from in vitro maturated MII and mice ovaries (ovulated MII), was done to assess embryos at differenced stages (2-cells, morula, and hatched). Finally, RT-qPCR was performed to investigate the mRNA expression of integrin genes (α9 and β1). After vitrification, the rate of survived oocytes, 68.65%for GV and 65.07% % for MII, did not show a remarkable difference related to non-vitrified groups, while the fertilization rate in vitrified groups remarkably decrease compared to non-vitrified groups (p < 0.05). Also, the expression of α9 and β1 genes was significantly altered in vitrified groups when compared to non-vitrified groups (p < 0.05). There was no significant difference in embryo developmental rates for non-vitrified and vitrified groups. Cryotop method for vitrification caused an alternation in oocyte quality by reducing fertilization rate and integrin gene expression.

Development of an Open Microfluidic Platform for Oocyte One-Stop Vitrification with Cryotop Method.

Oocyte vitrification technology is widely used for assisted reproduction and fertility preservation, which requires precise washing sequences and timings of cryoprotectant agents (CPAs) treatment to relieve the osmotic shock to cells. The gold standard Cryotop method is extensively used in oocyte vitrification and is currently the most commonly used method in reproductive centers. However, the Cryotop method requires precise and complex manual manipulation by an embryologist, whose proficiency directly determines the effect of vitrification. Therefore, in this study, an automatic microfluidic system consisting of a novel open microfluidic chip and a set of automatic devices was established as a standardized operating protocol to facilitate the conventional manual Cryotop method and minimize the osmotic shock applied to the oocyte. The proposed open microfluidic system could smoothly change the CPA concentration around the oocyte during vitrification pretreatment, and transferred the treated oocyte to the Cryotop with a tiny droplet. The system better conformed to the operating habits of embryologists, whereas the integration of commercialized Cryotop facilitates the subsequent freezing and thawing processes. With standardized operating procedures, our system provides consistent treatment effects for each operation, leading to comparable survival rate, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) level of oocytes to the manual Cryotop operations. The vitrification platform is the first reported microfluidic system integrating the function of cells transfer from the processing chip, which avoids the risk of cell loss or damage in a manual operation and ensures the sufficient cooling rate during liquid nitrogen () freezing. Our study demonstrates significant potential of the automatic microfluidic approach to serve as a facile and universal solution for the vitrification of various precious cells.

Cryoprotectant role of exopolysaccharide ID1 in the vitrification/in-straw warming of in vitro-produced bovine embryos.

The cold-adapted bacterium Pseudomonas sp. ID1 produces the extracellular exopolysaccharide ID1 (EPS ID1) with cryoprotective activity. This study was designed to optimize the vitrification/in-straw warming protocol of in vitro-produced (IVP) blastocysts by adding EPS ID1 to the vitrification media. Day 7-expanded blastocysts were vitrified/warmed using the VitTrans device after the addition of 0 or 100 μg/mL EPS ID1 to the vitrification media. Blastocysts vitrified by the Cryotop method and fresh non-vitrified blastocysts served as controls. Outcomes were assessed in the warmed embryos in terms of survival rates and mRNA relative abundances of BAX, BCL2, GPX1, and CDX2 genes. No differences in survival rates were observed at 3h post-warming between vitrification treatments. At 24 h post-warming, the addition of EPS prior to vitrification with the VitTrans device produced similar survival rates to Cryotop-vitrified embryos and similar hatching rates to fresh non-vitrified or Cryotop-vitrified embryos. No differences emerged in BCL2 gene expression. Lower BAX (p < .05) and higher GPX1 (p < .05) and CDX2 (p < .1) gene expression were observed in expanded and/or hatched blastocysts derived from VitTrans-EPS-vitrified embryos when compared to those from the non-supplemented group. In conclusion, addition of EPS not only promoted blastocyst survival and hatching after VitTrans vitrification/warming but also modified the expression of genes associated with better embryo quality.

P-377 Does C-Section history affect the live birth outcomes after IVF in case of frozen embryo transfers?

Abstract Study question Do previous Caesarean section deliveries (CD) affect reproductive outcomes in case of frozen embryo transfers (FET), including live births (LB), after IVF or ICSI? Summary answer Previous CD does not impair LBR after IVF when cycle segmentation is performed and intracavitary fluid (ICF) is eliminated prior frozen embryo transfer. What is known already Successful implantation requires not only a receptive endometrium, but also the process of ‘apposition’ and ‘attachment’ of the embryo to the endometrial surface. This process could be severely disturbed by previous CD and data are contradictory regarding its impact on ART outcome. Former CD might not only increase the difficulty of the ET-procedure, but might also lead to the presence of ICF. Therefore, special attention has to be paid to exclude fluid accumulation during preparation for FET. When ICF is excluded, the reproductive outcomes of FET cycles appear to be uncompromised, regardless of the previous delivery mode. Study design, size, duration This single center retrospective cohort study included a total of 412 single/double euploid FET cycles between March 2017 and October 2019. Trophectoderm biopsy samples were subjected to Next Generation Sequencing to diagnose the ploidy state. Vitrification and warming were performed using the Cryotop method (Kitazato, Biopharma). No embryo transfer was performed when ICF was visible during the endometrial preparation for FET. Participants/materials, setting, methods Patients with secondary infertility, having at least one euploid embryo after a previous IVF/ICSI-cycle with embryo vitrification, undergoing FET, were included. Clinical pregnancy rate (CPR), early pregnancy loss (EPL) and LBR were evaluated in patients after CD and after vaginal delivery (VD). Main results and the role of chance Patients in the CD group were significantly older than in the VD group (35.02±4.62 vs 34.11±5.03 years, respectively) (p = 0.028). FET was performed in a Natural cycle (NC) (n = 82, 46.86%) or in a Hormonal replacement therapy (HRT) cycle (n = 93, 53.14%) in CD group versus NC (n = 77, 32.49%) or HRT cycle (n = 160, 67.51%) in VD group (p = 0.003). Patient characteristics (AMH, BMI, duration of infertility, presence of intrauterine abnormalities, endometrial thickness, embryo quality, presence of blood on the transfer catheter and number of SETs/DETs) were similar between groups. Embryo transfer difficulty and the presence of mucus on the transfer catheter were significantly higher in CD group versus VD group (p = 0.040, p=0.004, respectively). Pregnancy outcomes CPR (61.71% vs 63.71%), EPL (12% vs 12%) and LBR (60% vs 60.34%) did not differ statistically between the CD and VD groups. In the multivariate regression model with embryo-quality, cycle-regimen, ET-difficulty, presence of blood/mucus, age, AMH, number of transferred embryos, only embryo quality remained significantly associated with LBR in CD-group (p = 0.001). In VD-group, cycle regimen was also significant parameter besides embryo quality (p = 0.001, p = 0.001 respectively). When CD and VD groups are categorized in terms of cycle regimen, CP and LBR were similar (p = 0.828/p=0.618 in HRT; p = 0.826/p=0.150 in NC). Limitations, reasons for caution This study is limited to its retrospective design and the fact that no distinction existed between patients with one or several previous CDs. Furthermore, this analysis is limited to patients, for whom ICF was excluded prior to ET. Wider implications of the findings This study confirmed that in patients after CD, the chance for CP and LBR is not compromised, when ICF is excluded during the endometrial preparation for FET. The existence of C-section scar may increase the ET-difficulty and the presence of the mucus on the transfer catheter. Trial registration number NA

P-137 ICM and TE quality significantly impact the live birth in euploid frozen blastocyst transfer cycles

Abstract Study question Is the live birth rate (LBR) in euploid frozen blastocyst transfer (FET) affected by the quality of ICM (Inner cell mass) and TE (Trophectoderm)? Summary answer ICM and TE significantly impacts the LBR with a decline of LB from 57.3% (ICM-A) to 48.5% (ICM-B) to 22.1% (ICM-C) (p &amp;lt; 0.001) What is known already The morphological blastocyst grading system proposed by Gardner-Schoolcraft remains the most accepted system to identify blastocysts with higher implantation potential. It relies on morphological features within the blastocyst, including ICM and TE. Several studies tried to identify the individual contribution of each. However, the conclusions remain contradictory and no clear consensus has yet been achieved. Due to heterogeneity of parameters evaluated between different publications, where embryos with unknown ploidy status were transferred in conjunction with a variability of stimulation protocols and in the number of transferred embryos, the real effect of the ICM and TE is difficult to infer. Study design, size, duration This two-center retrospective observational study includes a total of 977 euploid single FET cycles between March 2017 and March 2020 at ART Fertility Clinics Muscat, Oman and Abu Dhabi, UAE. Participants/materials, setting, methods Trophectoderm biopsies were analyzed with Next Generation Sequencing (NGS). All blastocysts available on D5 or D6 with a quality ≥ BL3CC were subjected to TE biopsy for PGT-A analysis and LBR was recorded. Vitrification/warming of blastocysts was performed using Cryotop method (Kitazato). Bivariate and multivariate analysis were performed between LB outcomes and ICM and TE grade while controlling for confounding factors. Main results and the role of chance A total of 977 single FET cycles were analyzed: 651 in hormone replacement therapy (HRT) and 326 in natural cycle regimen (NC) resulting in a 46.88% LBR. The mean patients’ age was 33.80 years with a mean Body Mass Index (BMI) of 26.80 kg/m2. Though all qualities of ICM and TE were associated with LB, blastocyst ICM-A LBR was statistically significantly higher (57.3%) than ICM-B (48.4%) and ICM-C (22.1%) (p &amp;lt; 0.001). Similarly, blastocyst TE-A LBR was statistically significantly higher (59.2%) than TE- B (48.6%) and TE- C (30.3%) (p &amp;lt; 0.001). Miscarriage rate was similar in all groups. The grade of ICM and TE were significantly associated with Anti-Mullerian-Hormone (AMH) and day of blastocyst biopsy. Mean AMH (ng/ml) was higher in ICM groups (A: 3.78, B: 3.24, p &amp;lt; 0.001) and TE group (A: 3.63, B: 3.38, p &amp;lt; 0.05) compared to lower grade (ICM-C: 2.86, TE-C: 2.82). In multivariate analysis, endometrial preparation for FET, BMI and AMH were the parameters influencing LBR: OR:1.45, [CI:1.07-1.96], p &amp;lt; 0.015) for NC; OR 0.96 [CI:0.93-0.99], p = 0.004 for BMI; OR 0.95 [CI:0.90-1.00], p = 0.033 for AMH; Both, ICM-C and TE-C, resulted in a significantly lower chance of LB [ICM: OR 0.32, CI:0.17-0.61, p &amp;lt; 0.001; TE: OR 0.44, CI:0.27-0.73, p = 0.002), compared to grade A. Limitations, reasons for caution The retrospective nature of the study and inter-observer variability in blastocyst scoring is a limitation. The physician/embryologist performing the embryo transfer could not been standardized due to the multicenter design. Randomized controlled studies are needed to determine whether ICM or TE should be prioritized in the selection of the blastocyst. Wider implications of the findings The ICM and TE scoring in FET may influence the LBR and should be considered as an important factor for the success of embryo transfer cycles. Whether these results can be extrapolated to fresh embryo transfer and to blastocysts with unknown ploidy status, needs further investigation. Trial registration number not applicable

P-757 Clinical and neonatal outcome of single vitrified-warmed blastocyst transfer with complete zona pellucida removal using laser-assisted hatching

Abstract Study question Complete zona pellucida (ZP) removal using laser assisted hatching can improve clinical and neonatal outcome in single vitrified-warmed blastocyst transfer (SVBT) cycle? Summary answer Implantation rate, clinical pregnancy rate and live birth rate can be improved by complete removal of zona pellucida, whereas no impact on neonatal outcomes. What is known already Assisted hatching (AH) technique widely used in assisted reproductive technology for improving clinical outcome in frozen embryo transfer (FET), which could impair successful embryo hatching out of the zona pellucida (ZP) due to ZP hardening during vitrification-warming. The zona thinning and drilling methods of AH doesn’t affect clinical, live births outcomes and it could increase monozygotic twin birth, risk of placenta associated diseases. But recent studies showed that the complete removal of ZP may increase the chance of blastocyst adhesion and outgrowth afterwards, which required to be proven. Study design, size, duration Retrospective study data collected from July 2015 to November 2019 included SVBT in the Ojinmed IVF center. We divided into 2 groups as follows: case group 160 (randomly selected complete removal of ZP) and control group 160. The clinical outcomes were implantation rate (IR), clinical pregnancy rate (CPR), live birth rate (LBR), miscarriage and neonatal outcomes including monozygotic twin, birth baby sex, birth weight, height and gestational weeks. All parameters were compared between two groups. Participants/materials, setting, methods 320 vitrified blastocyst transfer cycles in 215 patients underwent a clomiphene-based minimal ovarian stimulation protocol or drug-free natural cycles. All embryos were vitrified-warmed by Cryotop method (Kitazato, Japan). Complete removal of ZP selected an survived blastocysts after warming and used an immediate laser drilling (Laser: 1.8-2.2 ms, Octax NaviLaseTM) in perivitelline space. And gently glass pipetting done for complete removal of ZP. Statistical analysis performed by Chi-square test and logistic regression analysis using STATA 12. Main results and the role of chance A total 320 SVBT cycles, including ZP completely removed 160 cycles and control 160 cycles. In analysis of the patient's age, fertility causes and fertilization methods were not significant (p &amp;lt; 0.05) between two groups. The IR (55.2 % and 66.2 %, p &amp;lt; 0.01), CPR (39.3 % and 52.5 %, p = 0.01) and LBR (29.3 % and 43.7 %, p &amp;lt; 0.01) are highly correlated between the control and the completely removal of ZP group, respectively. In multivariable logistic regression analysis showed the completely removal of ZP group had significance for IR (aOR 0.54, CI 95 % 0.31-0.93, p = 0.02), CPR (aOR 0.56, CI 95 % 0.32-0.96, p = 0.03) and LBR (aOR 0.56, CI 95 % 0.32-0.99, p = 0.048), which adjusted by patient age, blastocyst grade and vitrification day. Miscarriage rate didn’t show significance (23.8% and 21.4%, p = 0.73) between groups. The neonatal outcomes including monozygotic twin, sex of the births, average gestation week and birthweight were non significant (p &amp;lt; 0.05) between two groups (49 for control group and 66 for case group) children. Limitations, reasons for caution The result of the current retrospective study is limited to data from a single IVF center. Wider implications of the findings Our study suggests that complete removal of ZP using laser assisted can improve clinical outcomes including implantation, clinical pregnancy and live birth after SVBT. And it may doesn’t affect to miscarriage rate and neonatal outcome. Trial registration number not applicable

P-276 Embryo development of fresh versus vitrified metaphase II oocytes in low prognosis patients: a retrospective sibling-oocyte monocentric study

Abstract Study question Do fresh and vitrified/warmed oocytes have the same developmental potential in low prognosis patients defined by POSEIDON criteria? Summary answer Embryos derived from vitrified/warmed oocytes show lower competence for embryo development when compared to those obtained from fresh oocytes in low prognosis patients. What is known already The strategy of oocyte accumulation has been proposed for the management of low-prognosis patients with the aim to increase the number of oocytes available for fertilization and further the number of embryos. The effect of oocyte vitrification/warming on the embryo developmental potential has mainly been studied in oocyte donation programs. Investigations on sibling-oocyte failed to show difference in the morphological embryo development, but the evaluation stopped after 2-3 days of embryo culture. In addition, morphokinetic evaluation revealed delayed development of embryos derived from vitrified oocytes, from the first cleavage to the time of blastulation. Study design, size, duration Single-center sibling-oocyte retrospective monocentric study including low-prognosis patients, defined by POSEIDON criteria, who had undergone an accumulation of oocyte strategy for managing their infertility, between November 2013 and January 2021. Only patients presenting fresh and vitrified/warmed oocytes at the time of insemination were analyzed for the sibling-oocyte comparison. Main study outcomes were fertilization, cleavage, blastulation rates, day-2 and day-5 embryo quality, and the rate of embryos available for transfer/cryopreservation. Participants/materials, setting, methods Forty-five patients were included, with a mean age of 34.8 ± 3.4 years. Oocyte vitrification/warming was performed using the Cryotop method. Top-quality and good-quality embryos were defined at day-2 respectively as 4 and 3-5 adequate-sized blastomeres, without multinucleation and containing &amp;lt;20% of cytoplasmic fragmentation, and at day-5 according to Gardner and Schoolcraft’s classification, top ≥B4 (AB/BA/AA) and good ≥ B3BB. Statistical analyses used mixed effects logistic regression to take into account the sibling-oocyte design. Main results and the role of chance A total of 656 oocytes were inseminated, 225 fresh and 431 vitrified/warmed oocytes. The oocyte survival rate after warming was 82%, 95%CI [76-87%]. There was no difference between fresh and vitrified/warmed oocytes groups regarding the degeneration rate following ICSI (5.0% vs 4.8% respectively, p = 0.95), the fertilization rate (74.6% vs 71.4% respectively, p = 0.42) or the cleavage rate on day-2 (98.3% vs 95.4% respectively, p = 0.15). However, the embryo development was significantly different from day 2, with higher day 2 top-quality and good-quality embryos rates in the fresh oocytes group when compared to the vitrified/warmed oocytes group (top-quality: 38.3% vs 24.8% respectively, p = 0.01; good-quality 58.0% vs 45.2% respectively, p = 0.02). The same results were observed on blastocysts when extended culture was decided, with a higher blastulation rate in the fresh oocyte group (56.7% vs 31.7% respectively, p &amp;lt; 0.001) and higher top-quality and good-quality day 5 blastocyst rate (top-quality: 23.4% vs 5.6% respectively, p = 0.01; good-quality 23.9% vs 7.9%, p = 0.01).The rate of embryos available for transfer/cryostorage was similar in both groups (40,7 % vs 38,8 %, p = 0,71). The cumulative live-birth rate was 33,3% (4/45 from vitrified oocytes group, 5/45 from fresh oocytes group, 6/45 from mixed oocytes group). Limitations, reasons for caution The retrospective nature of the study, on a limited number of limited number of patients represents an inherent limitation. Embryo assessment was subjective, the evaluator knowing the oocyte’s source group. The impact of the morphological alterations observed in embryos derived from vitrified/warmed oocytes need to be confirmed with clinical outcomes. Wider implications of the findings The majority of studies comparing fresh and vitrified/warmed oocytes were performed in egg-donation programs. This study focuses on low-prognosis patients, who reflect perhaps more the patients who consult in ART centers. Further prospective studies are needed to confirm our findings. Trial registration number Not applicable

Post-warming survival rates and clinical outcomes of human cleavage stage embryos vitrified/warmed using CryoTouch and Cryotop methods

BackgroundVitrification has become the method of choice for cryopreservation of human embryos and gametes. There are multiple commercial media, containing different combinations and concentrations of cryoprotectants, available for vitrification and warming procedures. The aim of this retrospective study was to compare post-warming survival rate and clinical outcomes of cleavage stage embryos vitrified/warmed using two different commercial methods (CryoTouch and Cryotop) during intracytoplasmic sperm injection/frozen embryo transfer (ICSI/FET) cycles. This retrospective study evaluated a total of 173 FET cycles performed on 446 warmed cleavage stage embryos between January 2018 and December 2020. Post-warming embryo survival rate and clinical outcomes including clinical pregnancy, implantation, and live birth rates were calculated.ResultsThe results showed no significant differences between two groups in terms of post-warming survival rate (p value = 0.5020), clinical pregnancy rate (p value = 0.7411), implantation rate (p value = 0.4694), and live birth rate (p value = 0.5737).ConclusionsCollectively, high successful rates were observed in outcomes of vitrified/warmed cleavage stage embryos using both CryoTouch and Cryotop commercial methods.