The effect of coenzyme Q10 on cryotolerance of in vivo-derived mouse embryos.

OBJECTIVE: Cryopreservation has some adverse effects on embryos including cell metabolism reduction, mitochondria and plasma membrane damage, excess production of 'Reactive Oxygen Species' and damage to DNA. In the present study. In this study we assessed the effect of coenzyme Q10 as an exogenous antioxidant on mouse embryos following cryopreservation. METHODS: We collected mice embryos at the morula stage from uterine horns on the third day of gestation. The morulae were divided into 9 groups (1 control, 2 vehicles and 6 experimental), then vitrified. The culture and/or vitrification media of the experimental groups were supplemented by 10 or 30 µM of CoQ10. After one week, the embryos were warmed and then cultured. After 48 hours of embryo culture, the blastocyst rate, total cell number, viability; and after 72 hours of embryo culture, we assessed the hatching rate. RESULTS: Blastocyst rate and hatching rate were significantly reduced in the groups containing 30 µM CoQ10 supplemented culture media compared to other groups (p<0.05). The hatching rate in the groups containing 10 µM CoQ10 supplemented in both culture and vitrification media was significantly higher than in the other groups (p<0.05). In groups containing 10 µM CoQ10 supplemented culture media, the viability was higher than that in the other groups (p<0.05). CONCLUSIONS: It seems that CoQ10 in a dose-dependent manner is able to improve hatching rate and viability following cryopreservation through its antioxidant and anti-apoptotic properties, and through the production of ATP.

Maternal and obstetric outcomes following the transfer of embryos warmed with fatty acid-supplemented solutions.

BACKGROUND: Vitrification procedures decrease intracytoplasmic lipid content and impair developmental competence. Adding fatty acids (FAs) to the warming solution has been shown to recover the lipid content of the cytoplasm and improve developmental competence and pregnancy outcomes. However, the influence of the FA supplementation on live birth rates after embryo transfers and perinatal outcomes remains unknown. In the present study, we examined the influence of FA-supplemented warming solutions on live birth rates, pregnancy complications, and neonatal outcomes after single vitrified-warmed cleavage-stage embryo transfers (SVCTs). METHODS: The clinical records of 701 treatment cycles in 701 women who underwent SVCTs were retrospectively analyzed. Vitrified embryos were warmed using solutions (from April 2022 to June 2022, control group) or FA-supplemented solutions (from July 2022 to September 2022, FA group). The live birth rate, pregnancy complications, and perinatal outcomes were compared between the control and FA groups. RESULTS: The live birth rate per transfer was significantly higher in the FA group than in the control group. Multivariate logistic regression analysis further demonstrated a higher probability of live births in the FA group than in the control group. Miscarriage rates, the incidence and types of pregnancy complications, the cesarean section rate, gestational age, incidence of preterm delivery, birth length and weight, incidence of low birth weight, infant sex, and incidence of birth defects were all comparable between the control and FA groups. Multivariate logistic regression analysis further demonstrated no adverse effects of FA-supplemented warming solutions. CONCLUSIONS: FA-supplemented warming solutions improved live birth rates after SVCTs without exerting any adverse effects on maternal and obstetric outcomes. Therefore, FA-supplemented solutions can be considered safe and effective for improving clinical outcomes and reducing patient burden.

Preservation of Bacillus subtilis' cellular liquid state at deep sub-zero temperatures in perchlorate brines.

Although a low temperature limit for life has not been established, it is thought that there exists a physical limit imposed by the onset of intracellular vitrification, typically occurring at ~-20 °C for unicellular organisms. Here, we show, through differential scanning calorimetry, that molar concentrations of magnesium perchlorate can depress the intracellular vitrification point of Bacillus subtilis cells to temperatures much lower than those previously reported. At 2.5 M Mg(ClO4)2, the peak vitrification temperature was lowered to -83 °C. Our results show that inorganic eutectic salts can in principle maintain liquid water in cells at much lower temperatures than those previously claimed as a lower limit to life, raising the prospects of active biochemical processes in low temperature natural settings. Our results may have implications for the habitability of Mars, where perchlorate salts are pervasive and potentially other terrestrial and extraterrestrial, cryosphere environments.

Effect of post-vitrification cryopreservation duration on singleton birth-weight in frozen-thawed blastocysts transfer cycles.

Introduction: This study aimed to explore the effect of cryopreservation duration after blastocyst vitrification on the singleton birth-weight of newborns to assess the safety of long-term preservation of frozen-thawed blastocyst transfer (FBT) cycles. Methods: This was a retrospective observational study conducted at the Gynecological Endocrinology and Assisted Reproduction Center of the Peking Union Medical College Hospital. Patients who gave birth to singletons between January 2006 and December 2021 after undergoing FBT cycles were included. Five groups were formed according to the duration of cryopreservation of embryos at FBT: Group I included 274 patients with a storage time < 3 months. Group II included 607 patients with a storage time of 3-6 months. Group III included 322 patients with a storage time of 6-12 months. Group IV included 190 patients with a storage time of 12-24 months. Group V included 118 patients with a storage time of > 24 months. Neonatal outcomes were compared among the groups. Multivariate linear regression analysis was performed to evaluate birth-weights and other birth-related outcomes. Results: A total of 1,511 patients were included in the analysis. The longest cryopreservation period was 12 years. The birth-weights of neonates in the five groups were 3344.1 ± 529.3, 3326.1 ± 565.7, 3260.3 ± 584.1, 3349.9 ± 582.7, and 3296.7 ± 491.9 g, respectively (P > 0.05). The incidences of preterm birth, very preterm birth, low birth-weight, and very low birth-weight were similar in all groups (P > 0.05). The large-for-gestational-age and small-for-gestational-age rates did not differ significantly among the groups (P > 0.05). After adjusting for confounding factors that may affect neonatal outcomes, a trend for an increased risk of low birth-weight with prolonged cryopreservation was observed. However, cryopreservation duration and neonatal birth-weight were not significantly correlated (P > 0.05). Conclusion: The duration of cryopreservation after blastocyst vitrification with an open device for more than 2 years had no significant effect on the birth-weight of FBT singletons; however, attention should be paid to a possible increase in the risk of low birth-weight.

Cryopreservation and assessment of genetic fidelity Acorus calamus Linn., an endangered medicinal plant.

BACKGROUND: Acorus calamus Linn. is a medicinally valuable monocot plant belonging to the family Acoraceae. Over-exploitation and unscientific approach towards harvesting to fulfill an ever-increasing demand have placed it in the endangered list of species. OBJECTIVE: To develop vitrification-based cryopreservation protocols for A. calamus shoot tips, using conventional vitrification and V cryo-plate. MATERIALS AND METHODS: Shoot tips (2 mm in size) were cryopreserved with the above techniques by optimizing various parameters such as preculture duration, sucrose concentration in the preculture medium, and PVS2 dehydration time. Regenerated plantlets obtained post-cryopreservation were evaluated by random amplified polymorphic DNA (RAPD) to test their genetic fidelity. RESULTS: The highest regrowth of 88.3% after PVS2 exposure of 60 min was achieved with V cryo-plate as compared to 75% after 90 min of PVS2 exposure using conventional vitrification. After cryopreservation, shoot tips developed into complete plantlets in 28 days on regrowth medium (0.5 mg/L BAP, 0.3 mg/L GA3, and 0.3 mg/L ascorbic acid). RAPD analysis revealed 100% monomorphism in all cryo-storage derived regenerants and in vitro donor (120-days-old) plants. CONCLUSION: Shoot tips of A. calamus that were cryopreserved had 88.3% regrowth using V cryo-plate technique and the regerants retained genetic fidelity. https://doi.org/10.54680/fr24210110412.

C-phycocyanin improves the developmental potential of cryopreserved human oocytes by minimizing ROS production and cell apoptosis.

PURPOSE: The cryopreservation process damages oocytes and impairs development potential. As a potent antioxidant, C-phycocyanin (PC) regulates reproductive performance. However, its beneficial effects on vitrified human oocytes remain unknown. METHODS: In this study, human GV-stage oocytes obtained from controlled ovarian hyperstimulation (COH) cycles were randomly allocated to three groups: fresh oocyte without freezing (F group), vitrification in medium supplemented with PC (P group), and vitrification in medium without PC as control group (C group). After warming, viable oocytes underwent in vitro maturation. RESULTS: Our results showed that 3 µg/mL PC treatment increased the oocyte maturation rate after cryopreservation. We also found that PC treatment maintains the regular morphological features of oocytes. After PC treatment, confocal fluorescence staining showed a significant increase in the mitochondrial membrane potential of the vitrified oocytes, along with a notable decrease in intracellular reactive oxygen species and the early apoptosis rate. Finally, after in vitro maturation and parthenogenetic activation, vitrified oocytes had a higher potential for cleavage and blastocyst formation after PC treatment. CONCLUSION: Our results suggest that PC improves the developmental potential of cryopreserved human GV-stage oocytes by attenuating oxidative stress and early apoptosis and increasing the mitochondrial membrane potential.

The effect of an extended culture period on birth weight among singletons born after single or double vitrified embryo transfer.

Aim: To evaluate the effect of an extended culture period on birth weight among singletons born after vitrified-warmed embryo transfer. Methods: A retrospective cohort study was performed among 12400 women who gave birth to 1015, 1027, 687, and 9671 singletons after single blastocyst transfer, single cleavage-stage embryo transfer, double blastocyst transfer, and double cleavage-stage embryo transfer, respectively. Results: The unadjusted birth weight of singletons born after vitrified blastocyst transfer were heavier than those born after cleavage-stage transfer (ß=30.28, SE=13.17, P=0.022), as were the adjusted birth weights (ß=0.09, SE=0.03, P=0.007). In addition, there was a 37% increased odd of having an infant with high birth weight after vitrified blastocyst transfer compared with vitrified cleavage stage transfer (OR=1.37, 95% CI:1.07-1.77). Conclusion: The unadjusted and adjusted birth weight and odds of having an infant with high birth weight significantly increased after blastocyst transfer compared with cleavage-stage embryo transfer in vitrified-warmed cycles.

C-Phycocyanin improves the quality of goat oocytes after in vitro maturation and vitrification.

In vitro maturation (IVM) and cryopreservation of goat oocytes are important for establishing a valuable genetic bank for domesticated female animals and improving livestock reproductive efficiency. C-Phycocyanin (PC) is a Spirulina extract with antioxidant, antiinflammatory, and radical scavenging properties. However, whether PC has positive effect on goat oocytes IVM or developmental competence after vitrification is still unknown. In this study, we found that first polar body extrusion (n = 293), cumulus expansion index (n = 269), and parthenogenetic blastocyst formation (n = 281) were facilitated by adding 30 µg/mL PC to the oocyte maturation medium when compared with the control groups and that supplemented with 3, 10, 100 or 300 µg/mL PC (P < 0.05). Although PC supplementation did not affect spindle formation or chromosome alignment (n = 115), it facilitated or improved cortical granules migration (n = 46, P < 0.05), mitochondria distribution (n = 39, P < 0.05), and mitochondrial membrane potential (n = 46, P < 10-4). Meanwhile, supplementation with 30 µg/mL PC in the maturation medium could significantly inhibit the reactive oxygen species accumulation (n = 65, P < 10-4), and cell apoptosis (n = 42, P < 0.05). In addition, PC increased the oocyte mRNA levels of GPX4 (P < 0.01), and decreased the mRNA and protein levels of BAX (P < 0.01). Next, we investigated the effect of PC supplementation in the vitrification solution on oocyte cryopreservation. When compared with the those equilibrate in the vitrification solution without PC, recovered oocytes in the 30 µg/mL PC group showed higher ratios of normal morphology (n = 85, P < 0.05), survival (n = 85, P < 0.05), first polar body extrusion (n = 62, P < 0.05), and parthenogenetic blastocyst formation (n = 107, P < 0.05). Meanwhile, PC supplementation of the vitrification solution increased oocyte mitochondrial membrane potential (n = 53, P < 0.05), decreased the reactive oxygen species accumulation (n = 73, P < 0.05), promoted mitochondria distribution (n = 58, P < 0.05), and inhibited apoptosis (n = 46, P < 10-3). Collectively, our findings suggest that PC improves goat oocyte IVM and vitrification by reducing oxidative stress and early apoptosis, which providing a novel strategy for livestock gamete preservation and utilization.

Vitrification of immature oocytes in pigs.

Cryopreservation of oocytes is an important technology for the in vitro gene banking of female germplasm. Although slow freezing is not feasible, porcine oocytes survive vitrification at high rates. Cryopreservation at the germinal vesicle stage appears to be more advantageous than that at the metaphase-II stage. Several factors are considered to affect the success of vitrification and subsequent utilization of immature porcine oocytes such as the device, the protocols for cryoprotectant application, warming, and the post-warming culture. Although live piglets could be obtained from vitrified immature oocytes, their competence to develop to the blastocyst stage is still reduced compared to their non-vitrified counterparts, indicating that there is room for further improvement. Vitrified oocytes suffer various types of damage and alteration which may reduce their developmental ability. Some of these can recover to some extent during subsequent culture, such as the damage of the cytoskeleton and mitochondria. Others such as premature nuclear progression, DNA damage and epigenetic alterations will require further research to be clarified and addressed. To date, the practical application of oocyte vitrification in pigs has been confined to the gene banking of a few native breeds.

Effect of melatonin on developmental competence, mitochondrial distribution, and intensity of fresh and vitrified/thawed in vitro matured buffalo oocytes.

BACKGROUND: In livestock breeding, oocyte cryopreservation is crucial for preserving and transferring superior genetic traits. This study was conducted to examine the additional effect of melatonin to maturation and vitrification media on the in vitro developmental capacity, mitochondrial distribution, and intensity of buffalo oocytes. The study involved obtaining ovaries from a slaughterhouse and conducting two phases. In the first phase, high-quality oocytes were incubated in a maturation medium with or without 10-9M melatonin for 22 h (at 38.5°C in 5% CO2). Matured oocytes were fertilized in vitro and cultured in SOF media for seven days. In the second phase, vitrified in vitro matured oocytes were stored in vitrified media (basic media (BM) containing a combination of cryoprotectants (20% Ethyl Glycol and 20% Dimethyl sulfoxide), with or without melatonin, and then stored in liquid nitrogen. Normal vitrified/thawed oocytes were fertilized in vitro and cultured as described. Finally, the matured oocytes from the fresh and vitrified/thawed groups, both with and without melatonin, were stained using DAPI and Mitotracker red to detect their viability (nuclear maturation), mitochondrial intensity, and distribution using a confocal microscope. The study found that adding 10-9M melatonin to the maturation media significantly increased maturation (85.47%), fertilization rate (84.21%)cleavage (89.58%), and transferable embryo (48.83%) rates compared to the group without melatonin (69.85%,79.88%, 75.55%, and 37.25% respectively). Besides that, the addition of melatonin to the vitrification media improved the recovery rate of normal oocytes (83.75%), as well as the cleavage (61.80%) and transferable embryo (27.00%) rates when compared to the vitrified TCM group (67.46%, 51.40%, and 17.00%, respectively). The diffuse mitochondrial distribution was higher in fresh with melatonin (TCM + Mel) (80%) and vitrified with melatonin (VS2 + Mel groups) (76.70%), Furthermore, within the same group, while the mitochondrial intensity was higher in the TCM + Mel group (1698.60) than other group. In conclusion, Melatonin supplementation improves the developmental competence and mitochondrial distribution in buffalo oocytes in both cases(in vitro maturation and vitrification).

Effects of Slow Freezing and Vitrification of Human Semen on Post-Thaw Semen Quality and miRNA Expression.

Semen cryopreservation has played an important role in medically assisted reproduction for decades. In addition to preserving male fertility, it is sometimes used for overcoming logistical issues. Despite its proven clinical usability and safety, there is a lack of knowledge of how it affects spermatozoa at the molecular level, especially in terms of non-coding RNAs. Therefore, we conducted this study, where we compared slow freezing and vitrification of good- and poor-quality human semen samples by analyzing conventional sperm quality parameters, performing functional tests and analyzing the expression of miRNAs. The results revealed that cryopreservation of normozoospermic samples does not alter the maturity of spermatozoa (protamine staining, hyaluronan binding), although cryopreservation can increase sperm DNA fragmentation and lower motility. On a molecular level, we revealed that in both types of cryopreservation, miRNAs from spermatozoa are significantly overexpressed compared to those in the native semen of normozoospermic patients, but in oligozoospermic samples, this effect is observed only after vitrification. Moreover, we show that expression of selected miRNAs is mostly overexpressed in native oligozoospermic samples compared to normozoospermic samples. Conversely, when vitrified normozoospermic and oligozoospermic samples were compared, we determined that only miR-99b-5p was significantly overexpressed in oligozoospermic sperm samples, and when comparing slow freezing, only miR-15b-5p and miR-34b-3p were significantly under-expressed in oligozoospermic sperm samples. Therefore, our results imply that cryopreservation of normozoospermic sperm samples can modulate miRNA expression profiles in spermatozoa to become comparable to those in oligozoospermic samples.

Should we should consider day 3 blastomere number during single vitrified-warmed blastocyst transfer cycle? A retrospective study.

The present study investigated whether day 3 blastomere number has an effect on the clinical outcomes during single vitrified-warmed blastocyst transfer cycles. A total of 3294 vitrified-warmed single day 5 blastocyst transferred cycles were analyzed in this retrospective study from January 2018 to December 2021. The cycles were divided into ≥ 7 and < 7 blastomere groups depending on the day 3 embryo blastomere number. The clinical outcomes were compared between the two groups, moreover multivariate logistic regression analysis was conducted to investigate the correlation between the number of day 3 blastomeres and clinical outcomes. The chi-square test demonstrated that the rates of clinical pregnancy and live birth were significantly higher in the ≥ 7 blastomere group compared to the < 7 blastomere group with respect to single high-quality blastocyst transfer cycles. Conversely, these rates were similar in the two groups with respect to single low-quality blastocyst transfer cycles. These results were confirmed by multivariate logistic regression analysis. However, the miscarriage rate was higher in the < 7 blastomere group than in ≥ 7 group during low-quality blastocyst transfer cycles. These results suggested that day 3 blastomere number should be considered during single vitrified-warmed blastocyst transfer cycles. Thus, blastocsyts derived from ≥ 7 blastomere embryos are preferred when choosing the same quality blastocysts.

Fatty acid supplementation during warming improves pregnancy outcomes after frozen blastocyst transfers: a propensity score-matched study.

This study aimed to examine the viability of human blastocysts after warming with fatty acids (FAs) using an in vitro outgrowth model and to assess pregnancy outcomes after a single vitrified-warmed blastocyst transfer (SVBT). For the experimental study, we used 446 discarded vitrified human blastocysts donated for research purposes by consenting couples. The blastocysts were warmed using FA-supplemented (FA group) or non-FA-supplemented (control group) solutions. The outgrowth area was significantly larger in the FA group (P = 0.0428), despite comparable blastocyst adhesion rates between the groups. Furthermore, the incidence of outgrowth degeneration was significantly lower in the FA group than in the control group (P = 0.0158). For the clinical study, we retrospectively analyzed the treatment records of women who underwent SVBT in natural cycles between January and August 2022. Multiple covariates that affected the outcomes were used for propensity score matching as follows: 1342 patients in the FA group were matched to 2316 patients in the control group. Pregnancy outcomes were compared between the groups. The rates of implantation, clinical pregnancy, and ongoing pregnancy significantly increased in the FA group after SVBTs (P = 0.0091-0.0266). These results indicate that warming solutions supplemented with FAs improve blastocyst outgrowth and pregnancy outcomes after SVBTs.

Low oxygen tension during in vitro embryo production improves the yield, quality, and cryotolerance of bovine blastocysts.

Mammalian oocytes undergo maturation and fertilization in the low-oxygen (O2) environment of the oviduct. To evaluate the effect of O2 tension during in vitro maturation and fertilization on embryo yield, quality, cryotolerance, and gene expression, we matured and fertilized bovine cumulus-oocyte complexes under low (5%) or high (20%) O2 tension. Presumptive zygotes from both groups were cultured at 5% O2 for 8 days. Blastocysts were vitrified, and then warmed, and cultured for further 24 h to assess their cryotolerance. Our findings indicate that low O2 during maturation and fertilization enhances embryo development and cell count in both fresh and vitrified/warmed blastocysts. In this study, the interaction of O2 tension and status (fresh or vitrified/warmed) affected the transcript abundance of SOD2, AQP3, and BAX in blastocysts. These results highlight the role of low O2 tension during bovine maturation and fertilization and provide support to using 5% O2 throughout all stages of bovine in vitro embryo production.

Long-term embryo vitrification is associated with reduced success rates in women undergoing frozen embryo transfer following a failed fresh cycle.

OBJECTIVE: To investigate the association of long-term embryo vitrification with the success rates and neonatal outcomes in frozen cycles. STUDY DESIGN: A single-center, retrospective cohort study was performed in Peking University Third Hospital. We included women who had undergone their first vitrified-warmed cycles following an unsuccessful fresh embryo transfer cycle between January 2013 and December 2019. Restricted cubic splines with 4 knots (at min-3.0 months, 3.1-6.0 months, 6.1-12.0 months, 12.1-max months) were used to map the non-linear relationship between live birth and embryo storage time as a continuous variable after adjustment for covariates. Multiple logistic regression was used to calculate crude odds ratios (OR) and adjusted OR (aOR) with 95 % confidence intervals (CI). RESULTS: A total of 10,167 women undergoing their first frozen cycle following an unsuccessful fresh embryo transfer cycle were included, among whom 3,708 resulted in a live birth (3,254 singleton live births). Restricted cubic splines, both before and after adjusting for covariates, showed that the predicted live birth rate (LBR) progressively decreased with an increase in the duration of embryo cryopreservation. This trend was also evident when women were categorized into four groups based on the length of cryopreservation. The live birth rate (LBR) was highest in the 0.8-3.0 months group (38 %) compared to the other groups. Multivariable logistic regression with the 0.8-3.0 months group as the reference, demonstrated that the 6.1-12.0 months group and >12.0 months group experienced lower live birth rates (aOR = 0.82 (0.72, 0.94) and aOR = 0.71 (0.57, 0.88), respectively). The LBR for the 3.1-6.0 months group was comparable to that of the 0.8-3.0 months group, with an aOR of 0.98 (0.90, 1.07). Sensitivity analyses in women who underwent single blastocyst transfer, in women with at least one good-quality embryo for transfer, and in women with age less than 36 at embryo transfer demonstrated a similar association between LBR and embryo frozen time. The neonatal outcomes were not significantly different among the four groups. CONCLUSIONS: Embryo vitrification greater than six months is associated with a reduction in success rate but does not appear to alter neonatal outcome.

External bending of cryodevice during vitrification leads to cryoprotectant cracks and damage to embryo blastomeres.

RESEARCH QUESTION: Embryo blastomeres and the zona pellucida are occasionally damaged during vitrification; is this a result of crack-induced mechanical damage in the glass state, caused by external bending of the device? DESIGN: A stereomicroscope was used to observe external bending-induced cracks in a cryoprotectant. Thereafter, 309 human cleavage-stage embryos derived from abnormally fertilized eggs were used to assess embryo damage under two external bending conditions: forward bending and backward bending, with three bending degrees applied. Three distinct embryo positions were used to examine the correlation between bending and embryo damage. Damage was assessed by looking at blastomere lysis rates, and overall rates of damaged and surviving embryos. RESULTS: A series of parallel cracks were identified in the cryoprotectant used for external bending, which led to damage to the embryo blastomeres. Compared with forward bending and control, the embryos were found to be more easily damaged by backward bending, indicated by significantly higher blastomere lysis and embryo damage rates, and lower embryo survival rate of backward bending than forward bending (P < 0.001). The degree of embryo damage also increased as the degree of external forces increased. Embryo position correlated with degree of embryo damage. CONCLUSIONS: Cryoprotectant crack-induced damage was identified as the cause of embryo damage. Mechanical damage to the glass state occurs because of improper external bending of the cryodevice strip in liquid nitrogen during vitrification. To prevent damage, bending of the strip should be avoided and the embryos should be placed near the tip of the strip.

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

RESEARCH QUESTION: What is the efficiency and efficacy of the novel Biorocks semi-automated vitrification system, which is based on a hydrogel? DESIGN: This comparative experimental laboratory study used mouse model and human day 6 blastocysts. Mouse oocytes and embryos were quality assessed post-vitrification. RESULTS: The 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. CONCLUSIONS: The 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.

In silico-designed vitrification protocols: an approach to improve survival of in vitro produced-bovine embryos.

In brief: In silico predictions validated in this study demonstrate the potential for designing shorter equilibration protocols that improve post-warming re-expansion and hatching rates of D7 and D8 in vitro-produced bovine embryos. Our results benefit the livestock industry by providing a refined and reproducible approach to cryopreserving bovine embryos, which, in addition, could be useful for other mammalian species. Abstract: The cryopreservation of in vitro-produced (IVP) embryos is vital in the cattle industry for genetic selection and crossbreeding programs. Despite its importance, there is no standardized protocol yielding pregnancy rates comparable to fresh embryos. Current approaches often neglect the osmotic tolerance responses to cryoprotectants based on temperature and time. Hereby, we propose improved vitrification methods using shorter dehydration-based protocols. Blastocysts cultured for 7 (D7) or 8 days (D8) were exposed to standard equilibration solution (ES) at 25ºC and 38.5ºC. Optimized exposure times for each temperature and their impact on post-warming re-expansion, hatching rates, cell counts, and apoptosis rate were determined. In silico predictions aligned with in vitro observations, showing original volume recovery within 8 min 30 s at 25ºC or 3 min 40 s at 38.5ºC (D7 blastocysts) and 4 min 25 s at 25ºC and 3 min 15 s at 38.5ºC (D8 blastocysts) after exposure to ES. Vitrification at 38.5ºC resulted in D7 blastocysts re-expansion and hatching rates (93.1% and 38.1%, respectively) comparable to fresh embryos (100.0% and 32.4%, respectively), outperforming the 25ºC protocol (86.2% and 24.4%, respectively; P < 0.05). No differences were observed between D7 and D8 blastocysts using the 38.5ºC protocol. Total cell number was maintained for D7 and D8 blastocysts vitrified at 38.5ºC but decreased at 25ºC (P < 0.05). Apoptosis rates increased post-warming (P < 0.05), except for D8 blastocysts vitrified at 38.5ºC, resembling fresh controls. In conclusion, based on biophysical permeability data, new ES incubation times of 3 min 40 s for D7 blastocysts and 3 min 15 s for D8 blastocysts at 38.5ºC were validated for optimizing vitrification/warming methods for bovine IVP blastocysts.

Effects of stearic acid on the embryo cryopreservation in mouse.

BACKGROUND: Intracellular lipids are sensitive to freezing. Lipidome modification is an important tool for studying the role of intracellular lipids in cryotolerance of mammalian oocytes and preimplantation embryos. OBJECTIVE: To study the effects of in vitro exposure of murine embryos to saturated stearic acid (SA) on the lipid content, embryo development and cryotolerance. MATERIALS AND METHODS: In vivo derived mouse embryos were cultured with 100 uM SA for 48 h up to the morula/blastocyst stage. Some of the SA-treated embryos were chosen for the evaluation of their development competence and the change in the lipidome, and other embryos were either slowly frozen or rapidly vitrified. RESULTS: Nile red staining combined with confocal laser scanning microscopy revealed a decrease in the total amount of lipids in the SA-treated embryos. Raman measurements showed that the lipid unsaturation was lower in embryos after in vitro SA culture. The addition of SA did not affect the embryo development before cryopreservation, but negatively affected the results of slow freezing cryopreservation and vitrification. CONCLUSION: In vitro SA exposure lowered the total amount of intracellular lipids and unsaturation in mouse embryos. The changes were accompanied with a significantly lower efficacy of embryo cryopreservation. https://doi.org/10.54680/fr24110110512.

Prioritized single vitrified blastocyst to be warmed between grades 3 or 4 blastocyst on day 5 transfer cycles.

PURPOSE: Selecting the optimal blastocyst to implant during cryopreservation and warming is critial for in vitro fertilization success. Therefore, the aim of this study was to explore which blastocyst should be prioritized to be thawed when facing a single vitrified blastocyst on day 5 transfer. METHODS: A retrospective study including 1,976 single vitrified-warmed blastocyst transfer cycles was conducted from January 2016 to December 2020. RESULTS: We found that grade 4 vitrified blastocyst had a higher clinical pregnancy (60.64% vs. 49.48%, P < 0.001) and live birth rates (50.12% vs 39.59%, P < 0.001) than the grade 3 vitrified blastocyst. However, no statistical difference was found between groups in miscarriage rate, birth weight, or gestational age. Besides, the grade 4 vitrified-thawed blastocyst had significant potential to develop into grade 6 blastocyst after further culturing for 16 h (73.68% vs. 48.60%, P < 0.001). The grade 6 transferred blastocyst was markedly higher in both clinical pregnancy rate (61.88% vs. 51.53%, P < 0.001) and live birth rate (50.91% vs. 40.46%, P < 0.001) compared to grade 5 transferred blastocyst. CONCLUSIONS: Grade 4 vitrified blastocyst is recommended when facing single vitrified blastocyst on day 5 transfer. More importantly, the "embryonic escape hypothesis" was firstly proposed to reveal the findings.