Cryopreservation Of Day Research Articles

P–254 The outcome of artificial reproductive technologies (ART) cycles with transfer of frozen-thawed blastocysts depending on whether expanded on day 5 or day 6

Abstract Study question Is there a difference in ART cycle results after frozen embryo transfer (FET), depending on whether blastocysts were cryopreserved on day 5 or 6? Summary answer There’s no statistical difference in the clinical pregnancy rate (CPR), life birth rate (LBR), miscarriage rate (MR) between embryos frozen on day 5 and 6. What is known already Currently, opinions differ regarding this topic. Previous studies demonstrated no difference in ongoing pregnancy rates between embryos frozen on day 5 (group A) or day 6 (group B) after FET. However, metanalysis (2019) suggested higher CPR and LBR after transferring embryos from group A rather than group B. It has also been established that ovarian stimulation leads to endometrial changes that result in deleterious effects on the implantation window and endometrial receptivity. Consequently, fresh transfers were excluded. Due to hormonal priming of endometrial receptivity, the same pregnancy outcomes should be expected with frozen-thawed blastocysts (day 5 vs 6). Study design, size, duration Retrospective cohort study was conducted between January 2015 and December 2018 with selected group of patients under 40 years of age. Group A consisted of 2275 cryotransfers of blastocyst expanded on day 5; group B included 170 cryotransfers of blastocyst on day 6. Both groups had an average of 1,52 embryos transferred per patient. Participants/materials, setting, methods Embryos were vitrified and warmed with Cryotop method (Kitazato, BioPharma). Blastocysts were scored according to Gardner and Schoolcraft grading system. Only expanded on day or 6 blastocysts of excellent and good (AA, AB, BA, BB) quality were selected. The embryos were cultured in CSC medium (Irvine Scientific) for 2–4 hours prior intrauterine transfer. The cycles with donor gametes, surrogacy and preimplantation genetic testing (PGT) were excluded. Statistical validity was assessed by Pearson’s chi-squared test. Main results and the role of chance The rates of the CPR, the ongoing pregnancy rate (OPR) and the LBR between group A and B were 50,8% (1157/2275) vs 46,5% (79/170) (p = 0,26), 37,4% (852/2275) vs 37,0% (63/170) (p = 0,91), 36,5% (832/2275) vs 35,2% (60/170) (p = 0,73) respectively and no significant differences were found in each category. Moreover, similarly there were no significant differences in the miscarriage rate 26,0% (301/1157) and 21,5% (17/79) (p = 0,37) as well Limitations, reasons for caution The study is limited due to uneven distribution of patients in both groups and by a low number of participants. The grading of blastocysts’ quality is also subjected to a human factor. Wider implications of the findings: This study confirms that frozen-thawed blastocysts do not seem to exhibit a difference in the CPR, OPR, LBR and MR whether they were expanded on day 5 or day 6. The cryopreservation of day 6 blastocyst can increase the chances of the patient for the positive outcome. Trial registration number Not applicable

Effect of different penetrating and non-penetrating cryoprotectants and media temperature on the cryosurvival of vitrified invitro produced porcine blastocysts.

The aim of this study was to determine the most efficient vitrification protocol for the cryopreservation of day 7 invitro produced (IVP) porcine blastocysts. The post-warm survival rate of blastocysts vitrified in control (17% dimethyl sulfoxide+17% ethylene glycol [EG]+0.4mol/L sucrose) and commercial media did not differ, nor did the post-warm survival rate of blastocysts vitrified in medium containing 1,2-propandiol in place of EG. However, vitrifying embryos in EG alone decreased the cryosurvival rate (55.6% and 33.6%, respectively, p<.05). Furthermore, the post-warm survival rates of blastocysts vitrified with either trehalose or sucrose as the non-penetrating cryoprotectant did not differ. There was also no significant difference in post-warm survival of blastocysts vitrified in control (38°C) media and room temperature (22°C) media with extended equilibration times, although when blastocysts were vitrified using control media at room temperature, the post-warm survival rate increased (56.8%, 57.3%, 72.5%, respectively, p<.05). The findings show that most cryoprotectant combinations examined proved equally effective at supporting the post-warm survival of IVP porcine blastocysts. The improved post-warm survival rate of blastocysts vitrified using media held at room temperature suggests that the cryoprotectant toxicity exerted in 22°C media was reduced.

Early steps of cryopreservation of day one honeybee (Apis mellifera) embryos treated with low-frequency sonophoresis

Honeybees, major providers of pollination, are endangered in many areas. Embryo cryopreservation may be a very useful tool to maintain their genetic diversity. However, it is complex in insects, because embryos are chill sensitive and are surrounded by two protectant membranes, the chorion and vitelline. These membranes prevent penetration of cryoprotectant in the embryos. This study aimed to test different conditions of embryo preparation before cryopreservation, including low-frequency sonophoresis, a physical method of permeabilization, and passages through cryoprotectant solutions. Apis mellifera ligustica embryos were collected in artificial cell plugs 7.5 h after queens had been caged, in two different seasons (winter, spring) and were then incubated in vitro overnight (16.5 h). Embryos were individually sonicated and then incubated in three cryoprotectant baths (B1 = 10%, B2 = 20% and B3 = 40% of cryoprotectant) and quenched in liquid nitrogen. Artificial cell plugs and in vitro incubation device were efficient in producing future embryos hatching. Embryos stained ruby red with rhodamine B after sonophoresis treatment indicated that low-frequency ultrasound had permeabilized embryos. According to the treatment, different significant hatching rates were obtained after sonophoresis (up to 25%). After three cryoprotectant incubations, best hatching rates were obtained after 10 min in B1 and B2, and 40 s in B3. These results show that sonophoresis is an efficient tool to permeabilize the chorion and vitelline membrane of the day one honeybee embryo allowing a hatching rate of more than 20%. They also show that the season is an important variability factor.

Cryopreservation of Day 8 equine embryos after blastocyst micromanipulation and vitrification

Pregnancy rates after cryopreservation of large equine blastocyst stage embryos have remained lower than other domesticated livestock species. It is generally accepted that the embryonic capsule is the primary barrier to cryoprotectant entry into the embryo proper and techniques need to be developed to circumvent this obstacle. Therefore, the objective of this study was to develop an efficient Day 8 equine embryo cryopreservation protocol through blastocyst micromanipulation and vitrification. Grade 1 and 2 embryos recovered from mares (n = 15) 8 days after ovulation were used in these experiments. In experiment 1, the effect of either one- or two-puncture treatments before aspiration of blastocoel fluid and exposure to vitrification solutions was evaluated. No difference was detected in mean embryo volume across treatment groups after exposure to vitrification solutions or after 1, 24, 48, and 72 hours of culture. Percent of embryos re-expanding at 24 hours and percent of embryos showing diameter increase at 48 and 72 hours during in vitro culture were 100%, 83%, and 75% compared with 93%, 67%, and 50% for one- and two-puncture treatment groups, respectively. Capsule loss was 25% for one-puncture and 50% for two-puncture treatment groups. In experiment 2, no difference was detected in mean embryo volume for indirect introduction (aspiration of blastocoel fluid + equilibration) and direct introduction (injection of cryoprotectant into blastocoel cavity) treatment groups, after exposure to dilution solution or to culture medium. There was no difference in mean embryo volume for the indirect and direct introduction treatment groups after 1, 24, 48, and 72 hours of culture. Percent of embryos re-expanding at 24 hours and percent of embryos showing diameter increases at 48 and 72 hours during in vitro culture were 100%, 76.9%, and 69.2%, respectively, for both treatment groups. Those embryos subjected to the direct introduction treatment had a higher (P = 0.05) percent capsule loss (70%) compared with the indirect introduction treatment group (31%). The pregnancy rate after transfer of vitrified expanded Grade 1 blastocysts using the indirect introduction method was 83% (5/6). Three pregnancies were allowed to continue to term and resulted in the birth of three healthy foals. The vitrification protocol used in this study has the potential to become a key tool for the successful cryopreservation of equine expanded blastocysts.

Cryopreservation of day 2–3 embryos by vitrification yields better outcome than slow freezing

Objective: To compare the outcome of vitrification versus slow freezing cryopreservation for cleavage stage day 2–3 embryos.Design: A retrospective observational study.Setting: All thawed embryos assisted reproduction cycles between January 2010 and December 2012 at a single IVF laboratory of a Tertiary Medical Center.Patients: Five hundred and thirty-nine cycles of day 2–3 thawed embryos.Interventions: In 327 of the thawed cycles, the embryos were vitrified and in 212 of the cycles the embryos were derived from slow freezing embryos.Main outcomes measure: Embryo survival rate, blastomere surviving rate and pregnancy rate.Results: Embryo survival rate was significantly higher after vitrification compared with slow freezing (81.6%, 647/793 versus 70.0%, 393/562 embryos, p < 0.0001). The clinical pregnancy rate per ET was significantly higher following vitrification compared to slow freezing, 20.0%, 63/314 versus 11.9%, 23/193, respectively (p = 0.02).Conclusions: Vitrification of day 2–3 cleavage stage embryos yields better cycle outcome in all the parameters compared to slow freezing.

Clinical-pregnancy outcome after vitrification of blastocysts produced from in vitro maturation cycles

This study was conducted to report the clinical-pregnancy outcome after vitrification of the blastocysts produced from in vitro maturation cycles. The survival rate after thawing was 92.0% (92/100). The clinical-pregnancy and implantation rates were 43.8% and 23.6%, respectively. These results suggest that the blastocyst-stage embryos produced from in vitro maturation cycles can be safely cryopreserved through vitrification.

Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application

To evaluate implantation of day 5 and day 6 vitrified and slow-frozen blastocysts. Retrospective analysis comparing two cryopreservation techniques. Private IVF clinic. Five hundred eight cryopreserved embryo transfer candidates. Supernumerary day 5 and day 6 blastocysts were vitrified or slow-frozen and transfered after warming or thawing. Comparison of two cryopreservation techniques with respect to survival rate, implantation, and pregnancy rates of day 5 and day 6 blastocysts. In 254 vitrified transfer cycles, survival, embryonic implantation, and clinical pregnancy rates for day 5 blastocysts were 95.9%, 33.4%, 48.7%, respectively, and for day 6 blastocysts 97.5%, 25.9%, 42.8%. In 254 slow-frozen transfer cycles, survival, embryonic implantation, and clinical pregnancy rates for day 5 blastocysts were 91.4%, 29.6%, 42.8%, respectively, and for day 6 blastocysts 94.8%, 28.2%, 43.1%. Overall there was a slightly, but not significantly, higher outcome regarding implantation and clinical pregnancy with the use of day 5 blastocysts (31.3% and 45.4%, respectively) versus day 6 blastocysts (26.7, and 42.9%, respectively). Vitrification technique yields the same implantation and pregnancy rate as slow-frozen blastocyst transfers. Slow growing embryos can be cryopreserved on day 6, because they yield a satisfactory survival, implantation, and pregnancy rate.

Vitrification demonstrates significant improvement versus slow freezing of human blastocysts

Blastocyst culture has reduced the number of embryos transferred per cycle, whilst simultaneously creating new quandaries regarding supernumerary blastocyst cryopreservation. This retrospective study was undertaken to compare a slow freezing protocol to a vitrification protocol for cryopreservation of day 5 and day 6 human blastocysts. To demonstrate this, the survival, implantation rate and pregnancy rates were compared after thawing, assessment and embryo transfer of 86 consecutive day 5 and day 6 thawed blastocyst transfer cycles from January 1, 2002 to December 31, 2003. Seventy-one day 5 slow-frozen (SF) blastocysts were thawed and 59 embryos survived the thawing (83.1%). An average of 2.5 SF blastocysts was replaced per embryo transfer, resulting in a pregnancy rate of 16.7% (4/24). Concurrently, 41 vitrified (VIT) blastocysts were thawed and all 41 survived the thawing process (100%). An average of 2.0 VIT blastocysts was replaced per embryo transfer, resulting in a pregnancy rate of 50% (10/20). Survival, pregnancy and implantation rates of day 5 VIT blastocysts have significantly increased ( P < 0.01, P < 0.02 and P < 0.01 respectively) over day 5 SF blastocysts. A similar trend was observed with day 6 blastocysts.

Can vitrifaction on cryoloops be used effectively for cryopreservation of day 3 cleavage stage embryos?

Objective: Cryopreservation of cleavage stage embryos has traditionally been performed using slow controlled rate freezing with a programmable freezer. An alternative to this methodology is vitrifaction. There are few reports of application of this technique to human embryos at the 6–8 cell stage. The purpose of this study was to examine post-thaw survival and development of embryos following vitrifaction on cryoloops and to assess the benefits of using this technique for cryopreservation of human embryos at the 6–8 cell stage.

Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification.

In 1996, with the introduction of sequential media, we set up a programme of cryopreservation of supernumerary morulae (day 4) and blastocysts (day 5) using a vitrification procedure. Our results showed that the efficiency of the vitrification method was dependent on the stage of embryo development and was negatively correlated with the expansion of the blastocoele. We postulated that a large blastocoele might disturb cryopreservative potential due to ice crystal formation during the cooling step. We analysed therefore the effectiveness of reducing before vitrification the volume of the blastocoelic cavity. Day 4 and day 5 embryos were vitrified in 40% ethylene glycol-18% Ficoll and 0.3 mol/l sucrose before plunging the straws directly into liquid nitrogen. Artificial shrinkage of the blastocyst was achieved after pushing a needle into the blastocoele cavity until it contracted. The survival rate post-thawing of day 4 and intact day 5 embryos was correlated with the volume of the blastocoele. In the control group only 20.3% blastocysts or expanded blastocysts survived as compared with 54.5 and 58.5% with morulae and early blastocyst respectively. After puncturing the blastocoelic cavity, an increase in the survival rate of up to 70.6% was noted. The pregnancy rates were improved after the artificial shrinkage procedure (20.5%) compared with the control intact blastocyst group (4.5%) (not significant). After reduction of the blastocoelic cavity, a significant increase in the implantation rate per vitrified blastocyst was observed (12.0 versus 1.4% P < 0.01). Our results showed that survival rates in cryopreserved expanded blastocysts could be improved by reducing the fluid content. This was presumably because mechanical damage caused by ice crystal formation was avoided. These observations should be considered when establishing a strategy and a protocol for cryopreservation of day 5 embryos.

Cryopreservation of day 6 and 7 bovine blastocysts derived from in vitro fertilized and DNA-injected zygotes

Cryopreservation of day 6 and 7 bovine blastocysts derived from in vitro fertilized and DNA-injected zygotes