Cryotop Method Research Articles

33 Assessment of spindle morphology and reactive oxygen species production after vitrification of bovine oocytes following invitro maturation in the presence of glutathione ethyl ester

Oocyte cryopreservation by vitrification affects spindle integrity and causes oxidative stress induced by reactive oxygen species (ROS). Glutathione (GSH) ensures the correct assembly of microtubules, prevents ROS from attacking tubulin assembly, and ensures normal spindle function during meiosis. We hypothesised that addition of GSH ethyl ester (GSH-OEt), a cell-permeable GSH donor, to IVM medium before vitrification may improve oocyte cryotolerance by maintaining spindle morphology and preventing ROS production. For the IVM, viable cumulus-oocyte complexes were randomly distributed into two groups: 1) control: conventional IVM media with tissue culture medium-199 + 20% fetal calf serum + epidermal growth factor and 2) GSH-OEt: IVM medium supplemented with 5mM GSH-OEt at 38.5°C in a 5% CO2 humidified air atmosphere. After 22h of IVM, half of the oocytes from each group were vitrified/warmed using the Cryotop method (Kitazato Corp.). After 24h of IVM, oocytes were denuded, fixed, and microtubule and chromosome distribution were analysed by immunofluorescence. To measure ROS, oocytes were denuded and incubated in 5μM 2′,7′-dichlorodihydrofluorescein diacetate. Fluorescence was acquired, and resulting images were quantified with ImageJ software. A linear mixed effect followed by a pairwise comparison test (Tukey adjustment) were performed to analyse differences in meiotic spindle configuration and ROS production (P<0.05). No significant differences in percentages of MII or normal spindle configuration were observed among treatments. Treatment with GSH-OEt before vitrification resulted in similar percentages of abnormal spindle configuration to control oocytes, whereas vitrified oocytes showed significantly higher percentages of abnormal spindle configuration relative with control oocytes (Table 1). When the content of ROS was measured, vitrification resulted in significantly higher levels (15.1E+06±1.2E+06; P<0.05) when compared with other treatments. However, oocytes vitrified after IVM with GSH-OEt (11.8E+06±0.6E+06) showed similar ROS levels to control (10.7E+06±0.2E+06) and GSH-OEt (10.0E+06±0.1E+06) groups. In conclusion, addition of GSH-OEt to IVM before vitrification did not have detrimental effects on spindle morphology and was able to reduce ROS content. Further experiments are warranted to assess whether the addition of GSH-OEt to IVM medium can improve oocyte development competence after vitrification. Table 1.Assessment of spindle morphology after oocyte vitrification (VIT) following IVM in the presence of glutathione ethyl ester (GSH-OEt) Item n MII (%) Spindle configuration (%) Chromosome distribution (%) Normal Abnormal Absent Dispersed Decondensed Absent Control 127 79.4±92 74.1±4.4 16.2±5.6a 9.7±1.1 16.2±3.8 9.7±1.1 0 GSH-OEt 113 76.5±6.5 65.6±5.7 26.2±9.0ab 8.1±4.3 19.5±2.6 13.7±3.0 1.1±1.1 VIT 71 60.0±5.6 54.8±7.8 32.7±10.8b 12.4±1.4 34.4±8.7 10.7±6.4 0 GSH-OEt VIT 53 57.4±10.7 58.5±8.8 31.1±9.3ab 10.4±5.8 34.8±5.6 6.7±6.7 0 a,bDifferent superscripts indicate significant differences (P<0.05). *Percentage referred to the total number of oocytes at MII. This work was supported by MCIU, Spain (AGL2016-79802-P) and Generalitat de Catalunya (García-Martínez, 2017_FI_00451).

Reprint of: Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method

Reprint of: Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method

Effect of Knockout Serum Replacement During Postwarming Recovery Culture on the Development and Quality of Vitrified Parthenogenetic Porcine Blastocysts.

The postwarming recovery culture, as one of the steps in cryopreservation process, is directly correlated with the survival and quality of embryos. Generally, recovery medium includes undefined serum or serum components that may cause the instability of results and other problems. The objective of this study was to evaluate the effect of knockout serum replacement (KSR) as a substitute for serum during recovery culture on the development and quality of vitrified parthenogenetic porcine blastocysts. Fetal bovine serum (FBS) was used as a positive control. The expanded blastocysts on day 5 were vitrified by the Cryotop method, and recovered with 10% (v/v) KSR or 10% (v/v) FBS for 48 hours after warming. Survival and hatching rates of vitrified blastocysts were significantly increased by KSR or FBS supplementation. The vitrified blastocysts recovered in KSR or FBS exhibited significantly decreased percentages of membrane damage and apoptosis, and increased total cells. Addition of KSR or FBS during recovery culture significantly reduced reactive oxygen species levels, and improved mitochondrial activity and adenosine triphosphates content in the vitrified blastocysts. Vitrification did not affect the gene expression of PCNA, CDX2, and CPT1, but significantly increased mRNA levels of POU5F1 and uPA. KSR added to the recovery medium significantly upregulated mRNA levels of PCNA and CPT1, and downregulated POU5F1 mRNA levels. The expression levels of PCNA, CDX2, CPT1, and uPA in vitrified blastocysts were significantly upregulated by addition of FBS to recovery medium. Moreover, the BAX: BCL2L1 ratio was similar between fresh and vitrified blastocysts, and KSR or FBS supplementation had no effect on the value. In conclusion, our data showed that KSR supplementation during recovery culture can improve the development and quality of vitrified parthenogenetic porcine blastocysts. These findings provide a useful reference that KSR could be used to replace FBS as a defined serum supplement for recovery culture of vitrified blastocysts.

78 Evaluation of the genomic estimated breeding value of carcass traits in blastocyst-stage embryos derived from Japanese Black cattle

Genomic selection based on a high-throughput microarray for genotyping single nucleotide polymorphism (SNP) is expected to accelerate genetic improvement in cattle. Recently, a genomic evaluation system for carcass traits, such as carcass weight and marbling score, is being established in Japanese Black cattle. To further increase genetic improvement efficiency in this breed, establishing a genomic evaluation system for pre-implantation embryos before embryo transfer (ET) is required. Here, we examined the correlation between genomic estimated breeding value (GEBV) of carcass traits calculated from embryonic (blastocyst) biopsy cells and from a corresponding calf produced by ET (Experiment 1); we also evaluated the pregnancy rate following ET of GEBV-evaluated blastocysts (GEBV blastocysts) preserved by vitrification (Experiment 2). In total, 16 Japanese Black dams and cryopreserved semen from 6 Japanese Black sires were used for producing in vivo blastocysts (Day 7-8). In Experiment 1, four blastocysts (IETS code 1) were divided into biopsy cells (15-20 cells) and biopsied embryos using a micromanipulator equipped with a micro blade. Biopsy cells were processed for DNA extraction and whole-genome amplification. Freshly biopsied embryos were transferred to recipient cows, and DNA was extracted from the blood or ear cells of the resulting 4 calves. Then SNP genotyping was performed using Illumina bovine LD BeadChip (Illumina, San Diego, CA, USA). The GEBV of 6 carcass traits (carcass weight, ribeye area, rib thickness, subcutaneous fat thickness, estimated yield percent, and marbling score) were calculated using phenotypic and genotypic data from 4,311 Japanese Black steers, and these were compared between biopsy cells and the corresponding calf. In Experiment 2, 134 blastocysts (IETS code 1 and 2) in total were biopsied (10-20 cells), and the biopsied embryos were vitrified by the cryotop method. Biopsy cells were processed for SNP genotyping as in Experiment 1, and the samples in which the call rate was more than 85% were used for GEBV calculation. Based on GEBV records, 24 vitrified GEBV blastocysts were warmed, cultured for 3 to 5h, and 22 GEBV blastocysts that survived (re-expanded) post-culture were transferred to recipient cows. Pregnancy in these cows was diagnosed using ultrasonography during Day 55 to 60 of gestation. In Experiment 1, the SNP call rates of the biopsy cells and corresponding calf were 98.5 to 99.3% and 99.7 to 99.8%, respectively. The GEBV of 6 carcass traits from biopsy cells and from the corresponding calf had almost the same values. In Experiment 2, the SNP call rates of the biopsy cells were ranged from 26.1 to 99.3%. The GEBV of 6 carcass traits varied among full-sib embryos. The pregnancy rate following ET of vitrified GEBV blastocysts was 40.9% (9/22). These results suggest the possible application of a genomic evaluation system for carcass traits at the blastocyst stage in Japanese Black cattle. Further large-scale assessment of pregnancy rates following ET of cryopreserved GEBV blastocysts is required for practical application of the evaluation system.

Cryopreservation of donkey embryos by the cryotop method: Effect of developmental stage, embryo quality, diameter and age of embryos

Cryopreservation of embryos has the potential to become a valuable tool for the conservation of endangered donkey breeds. However, there are several factors that can affect cryosurvival of embryos. This study evaluates the effectiveness of the Cryotop method to vitrify donkey embryos and factors affecting the survival of vitrified-warmed embryos. Day 6–8 embryos were measured and morphologically evaluated. Embryos were then vitrified-warmed using the Cryotop technique. After 24 h post-warming, the embryos were measured and evaluated for their morphology, development and viability (Propidium Iodide-Hoechst 33342 dyes). A total of 25 embryos were used, of which 17 were classified as Grade 1 (excellent), 5 as Grade 2 (good) and 3 as Grade 3 (fair). Based on their diameter, embryos were grouped as follows: ≤220 μm (n = 10), >220–300 μm (n = 8), and >300 μm (n = 7). Post-warming survival of vitrified embryos was similar (P > 0.05) to the control fresh embryos, regardless of embryonic diameter, developmental stage, and age of the embryos before vitrification. However, the proportion of embryos that survived vitrification procedures was numerically higher but not significantly different (P > 0.05) for Day 7 embryos (84.6%). The ability of Grade 1 (70.6%) and 2 (80%) embryos to survive vitrification procedures was higher (P = 0.0214) than those of Grade 3 (0%). The proportion of dead cells in Grade 3 embryos (56.5%) was higher (P < 0.01) than that of Grade 1 (3.2%) and 2 (1.5%) embryos. In conclusion, the Cryotop technique seems to be useful for Grade 1 and 2 donkey embryos. It is likely that donkey embryos show similar survival rates after vitrification in Cryotops irrespective of age, diameter and development stage.

Intent to treat analysis reveals significantly improved clinical outcomes with aneuploidy testing in an age-matched population

Preimplantation genetic testing for aneuploidy (PGT-A) has been proposed as an improved embryo selection strategy to decrease pregnancy loss and increase live birth rates for infertility patients undergoing in vitro fertilization (IVF). In an intent to treat analysis, we investigated clinical outcomes with PGT-A, compared to patients who have freeze-all blastocyst cycles with embryo selection based on morphology alone. Prospective cohort age-matched study. Infertility couples were identified during their IVF consult as intent to treat with either a freeze-all blastocyst cycle or IVF with PGT-A. Female patients were maternally age-matched between the two treatment groups (mean 32.5 ±3.7 years; n=46 per group). For the ‘Freeze All’ group, blastocysts were vitrified using the Cryotop method on either day 5 (D5) or day 6 (D6) of development. In contrast, for the ‘PGT-A’ group, prior to vitrification, D5 or D6 blastocysts were biopsied and analyzed for chromosome numeration using the VeriSeq™ platform (Illumina). Standard protocols for a hormone replacement frozen embryo transfer (FET) were utilized, with either blastocyst morphology alone (Freeze All) or euploid (PGT-A) embryo selection. Primary outcomes measured included implantation (fetal heart tone), ongoing clinical pregnancy, miscarriage, and live birth rates. Statistical analysis included Student’s t-test and Fisher’s exact test where appropriate, significance at P<0.05. In each treatment group, 46 retrievals were performed with no significant differences in number of oocytes retrieved, fertilized or number of usable blastocysts between the two treatment groups (P≥0.05; ns). Two retrievals (4.4%) resulted in no fertilization and four (8.7%) with no blastocyst development in the Freeze All group, while one (2.2%) resulted in no fertilization and four (8.7%) with no blastocyst development in the PGT-A group (P≥0.05; ns). Additionally, following PGT-A, two (4.4%) cycles ended with all aneuploid blastocysts. Remaining patients underwent FET with significant clinical improvements for primary outcomes measured in the PGT-A group (P<0.05; Table 1). From the prospective of intent to treat analysis, at the time a clinical decision is made to proceed with IVF therapy, the inclusion of PGT-A resulted in significantly improved clinical outcomes in a younger maternally age-matched population. This data reflects that embryo selection incorporating PGT-A should be considered for infertility patients undergoing IVF.Table 1FET OutcomesFreeze All (n=40)PGT-A (n=39)Implantation Rate (fetal heart tone)48.2%78.9%*Ongoing Clinical Pregnancy Rate60%84.6%*Miscarriage Rate16.7%6.1%Live Birth Rate per FET50%79.5%*Live Birth Rate per Intent to Treat43.5%65.2%**P<0.05 Open table in a new tab

Efficiency of elective oocyte vitrification for reproductive aging women seeking fertility preservation to delay motherhood

There is an increasing demand for fertility preservation for women who wish to delay motherhood for a variety of non-medical reasons. The efficiency and safety of oocyte vitrification allows women the opportunity to conceive in the future with their own genetic offspring. The aim of this study was to analyze the efficacy of oocyte vitrification techniques in preserving female fertility for reproductive aging women. Retrospective matched cohort study. Female patients (n=98) that underwent an elective autologous oocyte vitrification cycle for fertility preservation (mean maternal age = 37.4±3.9 years) and returned to undergo in vitro fertilization (IVF) at a later date, were directly compared to maternally aged-matched female patients that underwent IVF using fresh autologous oocytes. Mature oocytes were vitrified using the Cryotop method following standard controlled ovarian stimulation. All normally fertilized embryos were cultured to the blastocyst stage prior to embryo vitrification. Statistical analysis included Students t-test and Fishers exact test where appropriate, significance at P<0.05. Fertility preservation patients (n=98) returned for an oocyte warming cycle at a mean maternal age of 40.7±4.6 years, representing on average, three years and three months after oocyte vitrification. IVF outcomes with vitrified oocytes were significantly reduced compared to maternally age-matched infertility patients utilizing fresh autologous oocytes (Table 1). Nevertheless, 84% of oocytes survived vitrification resulting in 3.8 usable blastocysts per elective fertility preservation patient (Table 1). To date, there have been 72 frozen embryo transfers from these vitrified oocytes that have resulted in a respectable 51.4% live birth rate. Even though this clinical outcome is reduced compared to FETs with fresh oocytes, only 76.4% of the fertility preservation cycles underwent preimplantation genetic testing for aneuploidy (PGT-A) (Table 1). In conclusion, women in their mid-late thirties seeking elective oocyte vitrification for fertility preservation can expect good IVF outcomes when they return in their early forties. Although these outcomes are lower than if they had utilized their own fresh oocytes at the time of oocyte vitrification, they can be presumed to be higher than if they had initiated treatment in their early forties without fertility preservation.Table 1Fresh OocytesVitrified OocytesMean # oocytes retrieved or warmed19.7±10.015.2±8.7*Mean # oocytes survived post warm (%)N/A12.4±7.7 (84%)Mean # oocytes fertilized11.7±6.18.9±5.8*Mean # usable blastocysts (%)6.2 (57%)3.8 (39%*)Live birth rate (%)67.7%51.4%*PGT-A cycles (%)100%76.4%*P<0.05 Open table in a new tab

Effect of vitrification procedures on the subsequent development of invitro matured swamp buffalo oocytes following invitro fertilization.

Nowadays, the efficiency of buffalo oocytes cryopreservation is still low. The purpose of this study was to evaluate effects of two combinations of cryoprotectant agents (CPAs) and two vitrification devices for vitrification of swamp buffalo oocytes on their survival after vitrification warming, and subsequent developmental ability after invitro fertilization. In vitro matured (IVM) oocytes were vitrified by either Cryotop (CT) or solid surface vitrification (SSV) interacting with vitrification solution A (VA) or B (VB). In the VA or VB solution exposed test, the oocytes showed similar survival rates, but decreased blastocyst rates after invitro fertilization compared with that of untreated oocytes. After vitrification, the CT method combined with VA solution yielded a higher survival rate (91.3±5.84%) of vitrified oocytes than that combined with VB solution (69.8±4.19%-75.8±4.55%); however, all the vitrification treatments showed lower blastocyst rates (1.1±0.07%-5.2±0.24%) compared with that of untreated oocytes (18.0±1.09%). Our results indicated that combined vitrification treatments in this study did not improve the decreased ability of vitrified oocytes developing to the blastocyst stage.

Sucrose ‘Versus’ Trehalose Cryoprotectant Modification in Oocyte Vitrification : A Study of Embryo Development

Numerous studies reported that vitrification, an ultra-rapid cooling technique, seems to be highly effective and could increase oocyte survival rate rather than slow freezing. The successful of oocyte vitrification depends on the proper combination of type and concentration of cryoprotectant. This study was addressed to determine the effects of the combination of type and concentration of cryoprotectants of vitrification media, notably in the embryo development. This experimental research was conducted by using oocyte obtained from thirty-two adult female Deutschland, Denken and Yoken (DDY) mice (7-8 weeks old). The MII mice oocytes were vitrified within 24 h after retrieval using the Cryotop method with cryoprotectants as follow : sucrose (16.5% EG, 16.5% DMSO, 0.5 mol/l sucrose), trehalose (16.5% EG, 16.5% DMSO, 0.5 mol/l trehalose) and Kitazato. The embryo development and morphological grading was observed at 2-cell and 8-cells under reverse phase light microscope and inverted microscope. This study demonstrated a good embryo development and morphological grading in sucrose and trehalose vitrification media. In embryo development, trehalose medium seems more superior compared to sucrose medium, even though Kitazato was the most superior compared to both. In the morphological grading, in 2-cells embryo, there were no significant differences between the three cryoprotectants, While, in 8-cells embryo, trehalose medium appeared to be superior compared to sucrose medium, even though seemed more inferior compared to Kitazato. The appropriate type and concentration of sugar as extracellular cryoprotectant was trehalose in oocyte vitrification based on embryo development, compared to sucrose.

Cryostorage duration does not affect pregnancy and neonatal outcomes: a retrospective single-centre cohort study of vitrified–warmed blastocysts

A retrospective cohort study of 8736 autologous single vitrified-warmed blastocyst transfer cycles was conducted in a single centre to investigate the effect of cryostorage on clinical and neonatal outcomes. Cryostorage duration was classified into three groups: (A) 0-2 months (n = 4702); (B) 2-13 months (n = 2853) and (C) 13-97 months (n = 1181). Blastocysts were vitrified using the Cryotop method. No significant differences were observed in live birth rates: (A) 37.3%; (B) 34.9%; (C) (35.2%). Gestational period was significantly shorter in group C: (A) 38.7 ± 1.8; (B) 38.6 ± 1.6; (C) 38.1 ± 1.7; P < 0.05. This was clinically unimportant as the average gestational age was more than 38 weeks. No significant differences between groups were observed in birth weight: (A) 3060 ± 455 g; (B) 3052 ± 449 g; (C) 2992 ± 445 g, or congenital malformation rates: (A) 2.2%; (B) 1.9%; (C) 1.8%. The limitation of this study was that maximum storage duration was 8 years; most blastocysts were in cryostorage for much shorter periods. Long-term storage of blastocysts that are vitrified using an open device vitrification system has no negative effect on pregnancy and neonatal outcomes.

Freeze-all embryo strategy in poor ovarian responders undergoing ovarian stimulation for in vitro fertilization

This retrospective study determined the efficacy of the ‘freeze-all’ embryo strategy in poor ovarian responders undergoing ovarian stimulation for in vitro fertilization (IVF). A total of 559 poor responders who met Bologna criteria between January 2012 and December 2014 were included in this study: 256 in the fresh embryo transfer group and 303 in the freeze-all group. Vitrification and warming of day 3 embryos were performed using the Cryotop method. The poor responders treated with fresh embryo transfer and those treated with freeze-all strategy showed similar live birth rates per cycle (12.1% vs. 16.2%, p = .172) and per transfer (15.9% vs. 20.9%, p = .182). Multivariate logistic regression analysis showed that maternal age at retrieval (odds ratio, 0.919; 95% confidence interval, 0.865–0.977; p = .006) and number of good-quality embryos transferred (odds ratio, 1.953; 95% confidence interval, 1.346–2.835; p < .001) were significantly associated with the live birth rate. Freeze-all cycle is an acceptable treatment in poor ovarian responders, and it should be suggested by physicians as an alternative to cycle cancelation in case in which a fresh transfer would not be advantageous.

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

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

Embryo survival and birth rate after minimum volume vitrification or slow freezing of in vivo and in vitro produced ovine embryos

The objective was to evaluate pregnancy outcomes and birth rate of in vivo derived vs. in vitro produced ovine embryos submitted to different cryopreservation methods. A total of 197 in vivo and 240 in vitro produced embryos were cryopreserved either by conventional freezing, or by vitrification with Cryotop or Spatula MVD methods on Day 6 after insemination/fertilization. After thawing/warming and transfer, embryo survival rate on Day 30 of gestation was affected by the source of the embryos (in vivo 53.3%, in vitro 20.8%; P < 0.05) and by the method of cryopreservation (conventional freezing 26.5%, Cryotop 52.0%, Spatula MVD 22.2%; P < 0.05). For in vivo derived embryos, survival rate after embryo transfer was 45.6% for conventional freezing, 67.1% for Cryotop, and 40.4% for Spatula MVD. For in vitro produced embryos, survival rate was 7.3% for conventional freezing, 38.7% for Cryotop, and 11.4% for Spatula MVD. Fetal loss from Day 30 to birth showed a tendency to be greater for in vitro (15.0%) rather than for in vivo produced embryos (5.7%), and was not affected by the cryopreservation method. Gestation length, weight at birth and lamb survival rate after birth were not affected by the source of the embryo, the cryopreservation method or stage of development (average: 150.5 ± 1.8 days; 4232.8 ± 102.8 g; 85.4%; respectively). This study demonstrates that embryo survival and birth rate of both in vivo and in vitro produced ovine embryos are improved by vitrification with the minimum volume Cryotop method.

Effect of different cryo-devices on in vitro maturation and development of vitrified-warmed immature buffalo oocytes

The aim of the study was to identify a cryo-device that would be best suited for the vitrification of buffalo immature cumulus-oocyte complexes (COCs) as judged by viability and meiotic competence of the vitrified-warmed oocytes and their development ability following in vitro fertilization (IVF). The expression of oocyte secreting factors and their receptors (GDF9, BMP15, BMPR2, TGFBR1) and apoptosis related genes (BCL2, BAX, P53, C-MYC) were compared in vitrified-warmed oocytes after in vitro maturation. COCs from the ovaries of slaughtered buffaloes were vitrified in a combination of dimethyl sulfoxide, ethylene glycol, and sucrose using either a conventional straw (CS), open pulled straw (OPS), cryoloop (CL), hemistraw (HS) or cryotop (CT). The fresh COCs were exposed to vitrification and warming solutions as in other vitrification methods without plunging in to liquid nitrogen (EC). The viability of vitrified-warmed COCs, 2 h post warming in HS and CT was similar to fresh and EC groups but significantly higher than CS and OPS methods. The proportions of oocytes with first polar body after 24 h in vitro maturation were significantly higher in HS and CT methods than in CS, OPS and CL methods. The development ability of these vitrified-warmed oocytes to blastocyst stage following IVF in all vitrified groups was significantly lower than control and EC groups. Among the vitrified groups, the blastocyst rate in HS, CT and CL groups was significantly higher than in OPS and CS groups. It was also observed that the expression levels of GDF9, BMP15, BMPR2, TGFBR1, BCL2, BAX, P53 and C-MYC genes in vitrified-warmed COCs in CT, HS and CL groups were similar to control. The results indicated that HS, CT and CL are more suitable cryo-devices for vitrification of buffalo immature oocytes.

Reduced competence of immature and mature oocytes vitrified by Cryotop method: assessment by in vitro fertilization and parthenogenetic activation in a bovine model.

This study aimed to evaluate the embryo development competence, the nuclear maturation and the viability of germinal vesicle (GV) and metaphase II (MII) oocytes vitrified by the Cryotop method. Cumulus-oocyte complexes were derived from bovine ovaries and three experiments were conducted. In Experiment 1, GV oocytes were vitrified and underwent in vitro maturation (IVM) or not and their nuclear maturation was assessed by orcein staining. In Experiment 2, GV oocytes and MII oocytes were vitrified or not and the viability was assessed by calcein/ethidium homodimer-1 staining. In Experiment 3, MII oocytes matured before or after vitrification were submitted to in vitro fertilization (IVF) and parthenogenetic activation (PA) in order to evaluate embryo development. No difference was found for the nuclear maturation rate in the GV group (50%) and the GV control group (67%; P = 0.23) and for viability rate (56%; 77%; P = 0.055, respectively). However, in the MII group (27%) viability was significantly lower than that of the MII control group (84%; P < 0.0001). The cleavage rate by IVF and PA was similar in the GV group and the MII group. In contrast, vitrified MII oocytes showed no capacity for blastocyst development after IVF or PA and vitrified GV oocytes were able to develop to blastocysts only after PA, but not after IVF. In conclusion, oocyte vitrification by the Cryotop method reduced the capacity for embryo development. Vitrification of GV oocytes, however, did not influence the capacity of meiotic nuclear maturation and they exhibited higher viability following vitrification at the MII stage.

Optimal embryo transfer strategy in poor response may include freeze-all.

In this retrospective cohort study, we investigated the best embryo transfer strategy in ICSI cycles with ≤4 oocytes collected at oocyte retrieval. Women who underwent antagonist co-treatment COS for ICSI treatment between January 2010 and December 2015 at a private ART clinic (N = 2263). Eight hundred seventy-nine women (group 1) had ≤4 oocytes collected at oocyte retrieval, of whom 645 (group A) had cleavage stage embryo transfer (ET), and 234 (group B) had blastocyst ET. One thousand three hundred eighty-four women (group 2) had 10-15 oocytes collected at oocyte retrieval, of whom 676 (group C) had cleavage stage ET, and 708 women (group D) had blastocyst ET. Blastocyst vitrification was performed using the Cryotop method and FET using artificial cycles. In group 1, the cancellation rate was significantly lower in group A (25.2 vs 38%). The pregnancy rate (PR), clinical PR, implantation rate (IR), and live birth rate (LBR) per ET and per oocyte retrieval were all lower in group A. The clinical PR, IR, and LBR per ET of vitrified-warmed blastocyst ET were significantly the highest. In group 2, the cycle cancellation rate was significantly lower in group C (3.5 vs 13.4%). The PR, clinical PR, and IR per ET and per oocyte retrieval were all lower in group C. The LBR per ET was significantly lower, but the LBR per oocyte retrieval was not significantly lower in group C. Again, the PR, clinical PR, and IR per ET of vitrified-warmed blastocyst ET were significantly the highest. Day 5 ET strategy has been reserved for normal or high responders. The improved pregnancy outcomes from blastocyst culture and cryopreservation may challenge ART to extend this benefit to poor responders.

Generation of rats from vitrified oocytes with surrounding cumulus cells via in vitro fertilization with cryopreserved sperm.

The objective of this study was to evaluate fertility and full-term development of rat vitrified oocytes after in vitro fertilization (IVF) with cryopreserved sperm. Oocytes with or without surrounding cumulus cells were vitrified with 30% ethylene glycol + 0.5 mol/L sucrose + 20% fetal calf serum by using the Cryotop method. The warmed oocytes were co-cultured with sperm. Although the denuded/vitrified oocytes were not fertilized, some of the oocytes vitrified with cumulus cells were fertilized (32.7%) after IVF with fresh sperm. When IVF was performed with cryopreserved sperm, vitrified or fresh oocytes with cumulus cells were fertilized (62.9% or 41.1%, respectively). In addition, to confirm the full-term development of the vitrified oocytes with surrounding cumulus cells after IVF with cryopreserved sperm, 108 vitrified oocytes with two pronuclei (2PN) were transferred into eight pseudopregnant females, and eight pups were obtained from three recipients. The present work demonstrates that vitrified rat oocytes surrounded by cumulus cells can be fertilized in vitro with cryopreserved sperm, and that 2PN embryos derived from cryopreserved gametes can develop to term. To our knowledge, this is the first report of successful generation of rat offspring derived from vitrified oocytes that were fertilized in vitro with cryopreserved sperm.

Crocetin improves the quality of in vitro–produced bovine embryos: Implications for blastocyst development, cryotolerance, and apoptosis

The aim of this work was to assess the effect of supplementation of bovine culture medium with the natural antioxidant crocetin on in vitro blastocyst development and quality. This was evaluated as cryotolerance, apoptosis index, and total cells number and allocation. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid medium, supplemented with 0, 1, 2.5, and 5 μM crocetin (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed and the blastocysts were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO, and 0.5 M sucrose. Finally, blastocysts produced on Day 8 in the absence (control) and presence of 1 μM crocetin were used for terminal deoxynucleotidyl transferase–mediated dUTP nick end labelling and differential staining to evaluate, respectively, the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm (TE) lineages (experiment 2). Embryo development was higher in the 1 μM crocetin group compared to the control, both in terms of total embryo output (37.7 ± 4.2%, 52.9 ± 6.3%, 40.9 ± 7.6%, and 42.4 ± 8.7%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.01) and grade 1 and 2 blastocysts (33.6 ± 4.9%, 46.1 ± 7.3%, 37.8 ± 7.9%, and 39.4 ± 7.9%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.05). Moreover, the percentage of fast-developing embryos increased in 1 μM crocetin group compared to the control (23.4 ± 4.7%, 32.7 ± 6.6%, 27.2 ± 6.6%, and 30.1 ± 7.2%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.05). In addition, the enrichment of culture medium with 1 μM crocetin improved embryo cryotolerance compared to the control, as indicated by higher hatching rates recorded after 48 hours postwarming culture (46.5% vs. 60.4%; P < 0.05). Furthermore, 1 μM crocetin decreased both the average number (9.9 ± 0.4 vs. 7.1 ± 0.3) and the percentage of apoptotic cells (7.1 ± 0.4 vs. 4.2 ± 0.2) in blastocysts compared to the control (P < 0.01). However, no differences were recorded in the average number of ICM, TE, and total cells between 1 μM crocetin and control groups. In conclusion, the enrichment of bovine culture medium with 1 μM crocetin increased both blastocyst yield and quality, as indicated by the improved chronology of embryo development, increased resistance to cryopreservation, and reduced incidence of apoptosis.

Stability of the cytoskeleton of matured buffalo oocytes pretreated with cytochalasin B prior to vitrification

Stabilizing the cytoskeleton system during vitrification can improve the post-thaw survival and development of vitrified oocytes. The cytoskeleton stabilizer cytochalasin B (CB) has been used in cryopreservation to improve the developmental competence of vitrified oocytes. To assess the effect of pretreating matured buffalo oocytes with CB before vitrification, we applied 0, 4, 8, or 12 μg/mL CB for 30 min. The optimum concentration of CB treatment (8 μg/mL for 30 min) was then used to evaluate the distribution of microtubules and microfilaments, the expression of the cytoskeleton proteins actin and tubulin, and the developmental potential of matured oocytes that were vitrified-warmed by the Cryotop method. Western blotting demonstrated that vitrification significantly decreased tubulin expression, but that the decrease was attenuated for oocytes pretreated with 8 μg/mL CB before vitrification. After warming and intracytoplasmic sperm injection, oocytes that were pretreated with 8 μg/mL CB before vitrification yielded significantly higher 8-cell and blastocyst rates than those that were vitrified without CB pretreatment. The values for the vitrified groups in all experiments were significantly lower (P < 0.01) than those of the control groups. In conclusion, pretreatment with 8 μg/mL CB for 30 min significantly improves the cytoskeletal structure, expression of tubulin, and development capacity of vitrified matured buffalo oocytes.

Intrauterine embryo transfer with canine embryos cryopreserved by the slow freezing and the Cryotop method.

Canine embryos (8-cell to blastocyst stages) frozen-thawed using the slow-freezing method with glycerol (four recipients) or dimethyl sulfoxide (three recipients) as a cryoprotectant and vitrified-warmed using the Cryotop method (five recipients) were surgically transferred into the unilateral uterine horn of recipient bitches. As a result, the morphology of embryos frozen-thawed using the slow-freezing method was judged to be normal, but no conception occurred in any of the recipient bitches. Two of the five bitches that received transferred embryos (morula to early blastocyst stages) vitrified-warmed using the Cryotop method became pregnant and produced normal pups (1/9 embryos, 11.1% and 1/6 embryos, 17.0%). It was concluded that the Cryotop method was more appropriate for canine embryo cryopreservation than the slow-freezing method, which is used for the cryopreservation of embryos of other mammalian species.