Post-thaw Survival Research Articles

Developmental potential of surplus morulas with delayed and/or incomplete compaction after freezing-thawing procedures

BackgroundMorulas with delayed growth sometimes coexist with blastocysts. There is still limited evidence regarding the optimal disposal of surplus morulas. With the advancement of vitrification, the freezing-thawing technique has been widely applied to zygotes with 2 pronuclei, as well as embryos at the cleavage and blastocyst stages. The freezing of morulas, however, has rarely been discussed. The purpose of this study was to investigate whether these poor-quality and slow-growing morulas are worthy of cryopreservation.MethodsThis is a retrospective, observational, proof-of-concept study. A total of 1033 day 5/6 surplus morulas were cryopreserved from January 2015 to December 2018. The study included 167 women undergoing 180 frozen embryo transfer cycles. After the morulas underwent freezing-thawing procedures, their development was monitored for an additional day. The primary outcome was the blastocyst formation rate. Secondary outcomes were clinical pregnancy rate, live birth rate and abortion rate.ResultsA total of 347 surplus morulas were thawed. All studied morulas showed delayed compaction (day 5, n = 329; day 6, n = 18) and were graded as having low (M1, n = 54), medium (M2, n = 138) or high (M3, n = 155) fragmentation. The post-thaw survival rate was 79.3%. After 1 day in extended culture, the blastocyst formation rate was 66.6%, and the top-quality blastocyst formation rate was 23.6%. The day 5 morulas graded as M1, M2, and M3 had blastocyst formation rates of 88.9, 74.0, and 52.8% (p < 0.001), respectively, and the top-quality blastocyst formation rates were 64.8, 25.2, and 9.0% (p < 0.001), respectively. The clinical pregnancy rate was 33.6%.ConclusionsThe post-thaw blastocyst formation rate was satisfactory, with approximately one-half of heavily fragmented morulas (M3) developing into blastocysts. Most of the poor-quality morulas were worth to freeze, with the reasonable goal of obtaining pregnancy and live birth. This alternative strategy may be a feasible approach for coping with poor-quality surplus morulas in non-PGS (preimplantation genetic screening) cycles.

Development of a programmable freezing technique on larval cryopreservation in Mytilus galloprovincialis

This study investigated the factors important to the development of a programmable larval cryopreservation technique in Mytilus galloprovinvialis, including (1) larval developmental stages; (2) cryoprotectant agents (CPAs); (3) thawing temperatures; (4) sucrose concentrations to remove CPA after thawing and (5) straw volumes, using D-larval rate as the post-thaw survival indicator. The results showed that larvae at 25 h post-fertilization (PF) had the highest resistance to cryopreservation. A post-thaw D-larval rate higher than 80% was achieved when the larvae were cryopreserved with 10% ethylene glycol +7.5% Ficoll +0.2% polyvinylpyrrolidone, thawed at 28 °C and used 9% sucrose solution as the medium to remove CPA after thawing. No significant difference in post-thaw D-larval rates (P > 0.05) was found between larvae cryopreserved in 0.25 mL and 0.5 mL straws. The performance comparison experiment showed that although a significantly lower survival rate (P < 0.05) was found in cryopreserved larvae than that in fresh larvae at day 2 PF (D larvae), no significant difference (P > 0.05) was shown on relative mortality rate from day 8 PF (early umbonal larvae) to day 32 PF (spat) and on shell length at day 8 PF and day 32 PF between fresh and cryopreserved larvae. Therefore, the larval cryopreservation technique developed in this study would enhance the breeding program and all year-round hatchery production in this species.

Strategies to improve the fertility of frozen-thawed boar semen for artificial insemination

Although cryopreservation of boar semen for artificial insemination (Al) was developed 35 years ago, cryopreservation conditions and Al strategies are still considered sub-optimal. Al with excessive numbers of frozenthawed sperm (5-6 x 109 cells), still does not achieve fertility levels similar to Al using liquid semen because of reduced sperm survival. Frozenthawed (FT) spermatozoa have therefore not been the preferred option for commercial breeding programmes. However, substantial progress has been made regarding boar sperm cryopreservation. Adjustment of cooling and re-warming rates to biophysical properties of boar spermatozoa, new sperm package systems and the achievement of accurately consistent freezing of large numbers of samples using programmable freezers have contributed to post-thaw survival rates above 50%, a threshold similar to that used for bull AI-semen. Moreover, these post-thaw sperm survival rates are consistent within a large population of boars selected for sperm freezability potential, as occurs with Al-bull sires. When such post-thaw boar semen is deposited intra-utero, acceptable fertility (in terms of farrowing rates and litter size) is obtained. Currently, the most effective application of FT-semen for Al is achieved using deep uterine-Al (DUI) which allows placement of a minimal semen dose (in volume 0.5 to 10 mL and sperm number 0.5 to 1 x109 total spermatozoa) into the anterior 1/3 of one uterine horn, with levels of fertility close to Al with liquid semen. However, owing to their shorter life span, FT-boar spermatozoa require an AI-to-ovulation interval not longer than 4-6 h, making peri-ovulatory Al a pre-requisite to obtain the highest possible fertility. Spontaneous ovulation most often occurs when two-thirds of oestrus has passed. Estimation of the duration of oestrus, taking into account the weaning-to-oestrus interval, is helpful when establishing appropriate AI-schedules. However, as the length of oestrus varies within and between farms, different Al strategies should be established a priori. The development of bio-sensors for spontaneous ovulation will widen the use of Al with frozen-thawed frozen semen.

Me2SO- and serum-free cryopreservation of human umbilical cord mesenchymal stem cells using electroporation-assisted delivery of sugars

Cryopreservation is the universal technology used to enable long-term storage and continuous availability of cell stocks and tissues for regenerative medicine demands. The main components of standard freezing media are dimethyl sulfoxide (hereinafter Me2SO) and fetal bovine serum (FBS). However, for manufacturing of cells and tissue-engineered products in accordance with the principles of Good Manufacturing Practice (GMP), current considerations in regenerative medicine suggest development of Me2SO- and serum-free biopreservation strategies due to safety concerns over Me2SO-induced side effects and immunogenicity of animal serum.In this work, the effect of electroporation-assisted pre-freeze delivery of sucrose, trehalose and raffinose into human umbilical cord mesenchymal stem cells (hUCMSCs) on their post-thaw survival was investigated. The optimal strength of electric field at 8 pulses with 100 μs duration and 1 Hz pulse repetition frequency was determined to be 1.5 kV/cm from permeabilization (propidium iodide uptake) vs. cell recovery data (resazurin reduction assay).Using sugars as sole cryoprotectants with electroporation, concentration-dependent increase in cell survival was observed. Irrespective of sugar type, the highest cell survival (up to 80%) was achieved at 400 mM extracellular concentration and electroporation. Cell freezing without electroporation yielded significantly lower survival rates. In the optimal scenario, cells were able to attach 24 h after thawing demonstrating characteristic shape and sugar-loaded vacuoles. Application of 10% Me2SO/90% FBS as a positive control provided cell survival exceeding 90%. Next, high glass transition temperatures determined for optimal concentrations of sugars by differential scanning calorimetry (DSC) suggest the possibility to store samples at −80 °C. In summary, using electroporation to incorporate cryoprotective sugars into cells is an effective strategy towards Me2SO- and serum-free cryopreservation and may pave the way for further progress in establishing clinically safe biopreservation strategies for efficient long-term biobanking of cells.

Dimethyl Sulfoxide-Free Cryopreservation of Chondrocytes Based on Zwitterionic Molecule and Polymers.

Cartilage tissue engineering highly relies on the ability to store and transport chondrocytes in order to be clinically successful. Cryopreservation is a most reliable technology for chondrocyte storage, but it suffers from the intrinsic toxicity of current state-of-the-art cryoprotectant, dimethyl sulfoxide (DMSO). In this work, we used the first fully zwitterionic compound-based approach for effective chondrocyte cryopreservation. A zwitterionic molecule combined with zwitterionic polymers could balance intra/extracellular osmotic stress and prevent ice formation, which were the keys of successful cryopreservation. Moreover, this zwitterionic combination showed noncytotoxicity due to its high biocompatibility, superior to cytotoxic DMSO. On the basis of these performances, chondrocytes could be well cryopreserved (∼90% post-thaw survival efficiency) for a long time without any addition of DMSO, and the recovered cells could maintain their normal functionalities. In view of the association between polymer molecular weight and cryopreservation efficacy, further mechanism of cryoprotection provided by zwitterionic molecule/polymer was proposed. This work opens a new window of opportunity for DMSO-free cryopreservation using biocompatible zwitterionic materials.

Prolonged semen cryopreservation decreases motile concentration

There is a paucity of data regarding the viability of cryopreserved sperm. Good laboratory practice involving cryopreservation include QA/QC, equipment maintenance and stable temperatures of -196°C. These ensure tissues remain viable for later use. However, cryopreservation has been shown to alter the structure and function of stored spermatozoa. This study aims to determine if sperm viability is affected by long-term storage. Before-After Study. Patients sperm samples that were abandoned over the years of 1995-2014 (average 14.5 years) were thawed and analyzed before discard. Samples were considered abandoned if patients couldn’t be contacted within the past 5 years to continue storage. Prior to initial freeze, semen analyses were performed. Semen samples were stored in a 1:1 mixture of test yolk buffer, aliquoted into a 1.5 ml cryovial and suspended in nitrogen vapor for 30 minutes prior to being plunged into liquid nitrogen. Vials were thawed by placing cryovials into 37°C heat blocks for 20 minutes. Post-thaw survival of sperm was determined by calculating sperm concentration, motility and progression. Pre and post analyses were analyzed using regression analyses with a cluster analysis to account for pairwise comparisons. 131 patient semen samples were grouped into four categories listed in Table 1. Age, initial sperm concentration and motility were not different between groups. Overall there is a significant decline in sperm motility with years of storage as vials stored for 20 years and longer show a 70% decrease compared to 10 years and less (p=0.0012). The patient age or initial sperm concentration at the time of freeze has no impact on sperm survival. Despite appropriate measures to maintain specimen in storage, it appears that prolonged storage in liquid nitrogen may impact sperm survival. This result warrants further study as viability of tissues may be affected over time and reduce the success of fertility outcomes.Table 1Summary of difference between initial and post-thaw motile concentrationsYears StorednPt AgeInitial Motile Concentration (M/ml)Post-Thaw Motile Concentration (M/ml)Δ<103038.8±4.937.6±33.519.6±29.80.26±0.2610-143637.7±7.528.2±29.316.6±220.28±0.1815-193638±7.768.8±3610.1±21.90.18±0.21>202934.7±8.745±58.211.7±24.10.09±0.13**p<0.05 Open table in a new tab

Effect of supplementation of vitrification solution on post-thaw survivability rate of porcine follicular oocytes

Oocytes (510) with two or more cumulus cell layers adhered to zona pellucida obtained by aspiration from follicles (2–8 mm dia) of 127 abattoir porcine ovaries were utilized to study the effect of supplementation of 0.5 M sucrose (Suc), 50 mg/ml polyvinyl pyrrolidone (PVP) and Suc + PVP into two different vitrification solutions, i.e. 35% concentration of both ethylene glycol (EG)+ propylene glycol (PG) and EG + dimethyl sulphoxide (DMSO). Vitrified post-thaw oocytes with intact zona pellucida and vitelline membrane, normal spherical shape and dark and evenly granulated cytoplasm under a stereo-zoom microscope were considered as viable. Out of the three supplements treatments, supplementation with sucrose + PVP combination and sucrose alone resulted in significantly higher mean post-thaw survivability rate of vitrified oocytes both for EG + PG and EG + DMSO as compared to the supplementation with PVP alone. It was concluded that addition of sucrose alone or in combination with PVP in vitrification solution, significantly improved survivability of vitrified porcine follicular oocytes.

Cryopreservation of incomplete compacted morulae and preliminary biopsy of excluded fragments.

The complexity of predicting embryo development potential at the cleavage stages and the emergence of epigenetic risks during prolonged in vitro culture of pre-implantation embryos made it more advantageous to transfer embryos at the morula stage to the uterine cavity. The criteria for estimating embryos at this stage that allow prediction of cryopreservation outcomes have been poorly described. All day 4 embryos (n = 224) were graded 1, 2, 3, 4 or 5 according to blastomere compaction degree (BCD = 100, 75, 50, 25 or 0%, respectively) and the survival and blastocyst formation rate of these morulae were studied after cryopreservation. An inverse dependence was found between survival rate and BCD. Excluded fragments were characterized by low osmotic reaction during exposure to cryoprotective medium and, after freeze-thawing, they were destroyed. As damaged necrotic areas of the embryo can affect their further development rate we proposed blastomeres and biopsy fragments of incomplete compacted morula be removed before embryo cryopreservation. This step led to significant increase in the post-thawing survival rate up to 93.1 ± 4.1%, 75 ± 8.8% and blastocyst formation rate up to 85.2 ± 10.4%, 59.4 ± 5.2% in grade 2 and grade 3 embryos, respectively. There was no significant difference in grade 4 embryos. Therefore the removal of blastomeres and biopsy fragments in incomplete compacted morulae can improve cryopreservation outcomes of grade 2 and grade 3 embryos with BCD.

Should we discard cryopreserved embryos with poor post-thaw survival? – Report of a successful pregnancy with &lt;50% blastomere survival

The objective of the present study is to report a case of successful ongoing pregnancy following transfer of two cleavage-stage day 3 embryos with one-two blastomeres survival. The study was conducted at a private clinical-assisted reproductive technology center. A 31-year-old female with a 3-year history of primary infertility underwent an in vitro fertilization cycle followed by a frozen embryo transfer cycle. The main outcome measure was clinical pregnancy. Ultrasonographic examination at 6 weeks revealed a single gestational sac with positive cardiac activity. Follow-up scans at 10, 12, and 14 weeks of pregnancy showed fetal age parameters corresponding to gestational age and normal value of nuchal translucency. Noninvasive prenatal screening test confirmed a euploid fetal development. Transfer of frozen-thawed embryos with only one–two blastomeres survival resulted in an ongoing pregnancy with euploid fetus development; therefore, we suggest that embryo with

Comparison of clinical outcome of frozen embryo transfer after embryo selection based on morphokinetic versus morphologic criteria for freezing

Background: Results from the latest meta-analysis, in fresh cycles, showed that the application of time-lapse monitoring (TLM) together with an embryo-evaluating algorithm was associated with a significantly higher rate of ongoing pregnancy and a lower rate of early pregnancy loss. The aim of this study was to compare the clinical outcomes of frozen embryos classified according to morphokinetic versus morphologic criteria.&#x0D; Methods: This was a retrospective cohort study, conducted at IVFAS, An Sinh Hospital, Vietnam, from July 2014 to July 2017. Patients undergoing in vitro fertilization (IVF) treatment with antagonist protocol and having freeze-only on day 5 were included. Exclusion criteria were patients (i) treated with in-vitro maturation, (ii) having obstructive azoospermia, or (iii) having uterine abnormalities. Embryos were cultured up to day 5 in TLM system (Primo Vision, Vitrolife, Sweden) or in benchtop (G185, K System, Denmark). The quality of frozen embryos was evaluated based on morphokinetic or morphologic criteria. In the subsequent cycle, endometrial preparation was done by using exogenous estradiol and progesterone. Embryos were thawed and up to 2 embryos were transferred to the uterus. The primary outcome was ongoing pregnancy. The rate of post-thaw survival, post-thawed good/moderate embryo, clinical pregnancy, implantation, miscarriage and ectopic pregnancy were used as secondary endpoints.&#x0D; Results: A total of 276 patients were recruited, with 138 patients in the morphokinetic group and 138 patients in the morphologic group. Baseline characteristics were comparable between the two groups. There was no significant difference in ongoing pregnancy rate in morphokinetic versus morphologic group (57.2% vs. 60.1%, p=0.71). All secondary outcomes were comparable between the two groups.&#x0D; Conclusion: In frozen day-5 embryo transfer, the clinical outcomes were similar when embryos were classified according to morphokinetic versus morphologic criteria for freezing. Using morphokinetic criteria to select embryos for freezing did not improve the ongoing pregnancy rate, as compared to morphologic criteria.&#x0D;

29 Development and survival of bovine vitrified sexed IVF-derived embryos in vitro matured with pituitary or human recombinant follicle-stimulating hormone

Improving in vitro production efficiency involves the development of effective oocyte in vitro maturation conditions. Although &amp;gt;80% of cumulus-oocyte complexes (COC) undergo nuclear maturation, only approximately 30 to 35% of immature bovine COC develop to the blastocyst stage. Also, animal-sourced FSH is typically used in IVF, so an effective alternative using recombinant DNA technology is desirable. The aim of this study was to compare the effect of porcine (p) and recombinant human (rh)FSH concentrations on in vitro performance and post-thaw survival. Ovaries were collected from an abattoir and oocytes were aspirated from 2- to 6-mm follicles. The COC with compact and complete cumulus cell layers were selected and matured in groups of 25 COC in 200µL of M199 medium supplemented with alanyl-glutamine (0.1mM), Na pyruvate (0.2mM), gentamicin (5µg mL−1), epidermal growth factor (50ng mL−1), pLH (5µg mL−1), cysteamine (0.1mM), and 10% FBS for 22 to 24h in humidified air and 5% CO2. Oocytes were divided into the following groups: 1× pFSH (2µg mL−1), 1× rhFSH (0.01 UI mL−1), 1× pFSH+1× rhFSH, 2× pFSH, and 2× rhFSH. After 22 to 24h, fertilization (Day 0) was carried out using female sexed-sorted semen selected with a mini single-continuous 80% layer (PureSperm, Nidacon International AB, Mölndal, Sweden) and diluted to 1×106 sperm mL−1. The SOF-FERT medium was supplemented with fructose (90µg mL−1), penicillamine (3µg mL−1), hypotaurine (11µg mL−1), and heparin (20µg mL−1). After 18h, presumptive zygotes were denuded and cultured under low oxygen tension in groups of 15 to 20 in 50-µL drops of SOF-BSA for 7 days. Also, 2% FBS was added post-fertilization on Day 3.5. Expanded blastocysts were selected based on IETS standards at Day 6.5 to 7 of culture. Only grade 1 expanded blastocysts were vitrified (Cryotop, Kitazato, Tokyo, Japan). Vitrification medium was 15% (vol/vol) ethylene glycol+propylene glycol. Vitrified embryos were thawed in a solution of H199+20% FBS and 0.25M sucrose at 39°C. Thawed embryos were cultured in SOF-BSA+10% FBS under cumulus/granulosa cell monolayer co-culture. Embryo assessment involved post-thaw survival (0h), re-expansion and development progress (24-48h), and hatching of the zona pellucida (72h). A minimum of 4 replicates were performed. Data were analysed using a generalized linear mixed model with logit-link binomial distribution. Media treatment differences were determined using Fisher’s least significant difference test with the Bonferroni correction (α-level=0.05). The FSH origin affected cleavage and embryo development rate but not cryotolerance (Table 1). The results support previous research on low dose versus high dose rhFSH effectiveness and interspecies interaction of FSH on follicular receptors. Table 1.Cleavage, embryo development rate, and cryotolerance of FSH of various origins

22 Vitrification of bovine embryo using antifreeze polyamino acid

Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound and a polyamino acid with a known functional resemblance to antifreeze proteins. We previously reported that CPLL is an effective cryoprotectant for bovine cells, sperm, and slow-frozen embryos. In this study, we investigated CPLL as a cryoprotectant for vitrified bovine embryos. We developed bovine embryos in vitro and vitrified them at the blastocyst stage. Embryos were equilibrated (3min) and vitrified (1min). Vitrified embryos were cryopreserved in LN (Cryotop® device; Kitazato Corp., Tokyo, Japan) for at least 1 week, thawed with a 0.3M sucrose warming solution, and then cultured in a basal medium (Gibco® medium 199, Grand Island, NY, USA; supplemented with 100µM 2-mercaptoethanol, 10% fetal bovine serum, and antibiotics) at 38.5°C in a humidified atmosphere (5% CO2, 5% O2, 90% N2). We evaluated the embryos morphologically for survival and hatched rate at 0, 24, 48, and 72h post-thawing. In control, the equilibration solution (ES) consisted of 7.5% (vol/vol) dimethyl sulfoxide (DMSO) and 7.5% (vol/vol) ethylene glycol, and the vitrification solution (VS) consisted of 16.5% (vol/vol) DMSO and 16.5% (vol/vol) ethylene glycol and 0.5M sucrose. In this study, CPLL was added to ES and VS at various concentrations instead of DMSO. The CPLL was added at 16.5, 11.0, 5.5, and 2.2% (wt/vol) to VS; respectively, these solutions were named P16.5, P11.0, P5.5, and P2.2. The ES was used 45% CPLL of VS each. Embryos underwent the above procedure concurrently, with testing replicated at least 3 times. We evaluated 88, 34, 38, 44, and 28 embryos with each solution (control, P16.5, P11.0, P5.5, and P2.2, respectively). Results were analysed statistically with a chi-square test and residual analysis, regarding P&amp;lt;0.05 as significant. Survival rates were significantly greater in P11.0 at 24h post-thawing (55.7% v. 89.5%; P&amp;lt;0.05) and in P11.0 and P5.5 at 48h post-thawing (47.7% v. 78.9% and 47.7% v. 79.5%, respectively; P&amp;lt;0.05) relative to controls but showed no significant differences at 0h post-thawing. Hatched rates were significantly greater in P11.0 and P5.5 through 72h post-thawing relative to controls (44.7% v. 22.7% and 52.3% v. 22.7%, respectively; P&amp;lt;0.05). The CPLL improved post-thawing embryo survival and hatched rates when applied during vitrification, thus demonstrating cryoprotective effectiveness. We conclude that CPLL acts as a low-toxicity cryoprotectant for vitrified bovine embryos, and our results are consistent with previous reports of protective CPLL effects for cells and cell membranes.

28 Effect of deuterium oxide on bovine oocyte cryotolerance

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

Effect of type and concentration of cryoprotectant on post-thaw survivability of vitrified porcine follicular oocytes

A total of 950 and 510 oocytes with two or more cumulus cell layers adhered to zona pellucida obtained by aspiration from follicles (2–8 mm dia) of 315 and 135 abattoir porcine ovaries, respectively, were utilized in the study to find the effect of cryoprotectant and concentration on post-thaw survivability of porcine follicular oocytes. Vitrified post-thaw oocytes with intact zona pellucida and vitelline membrane, normal spherical shape and dark and evenly granulated cytoplasm under a stereo-zoom microscope were considered as viable. Out of the 10 cryoprotectant treatments i.e. ethylene glycol (EG), propylene glycol (PG), dimethyl sulphoxide (DMSO), glycerol (GL), EG + PG, EG + DMSO, EG + GL, PG + DMSO, PG + GL and DMSO + GL used for vitrification of oocytes at a concentration of 35%, the post-thaw survivability rate was the highest in EG +DMSO followed by EG + PG group which had significantly higher mean post-thaw survived oocytes as compared to GL, PG + DMSO, PG + GL and DMSO + GL groups. Exposing the oocytes to 30, 35 and 40% of EG + DMSO and EG + PG yielded no significant difference in post-thaw survivability rate of vitrified oocytes, although the highest value was obtained with 35%. It was concluded that 35 to 40% EG + DMSO yielded efficient vitrification of porcine oocytes.

Evaluation of cryopreservation efficiency on embryo growth after exposing them in intracellular cryoprotectant ethylene glycol or glycerol and replacing them by trehalose

The present study was conducted to see whether toxicity could be reduced by replacing some of the penetrating cryoprotectant (intracellular cryoprotectant) with a disaccharide as well as finding out the optimum equilibration time in a vitrification solution before rapid cooling. Goat ovaries (1,685) were aspirated and 1,761 culturable oocytes (COC) were recovered. The recovered COCs were matured in maturation medium and evaluated on the basis of cumulus expansion. The matured oocytes were coincubated with fresh semen capacitated in TALP media, for 18 h. The inseminated oocytes were further cultured in mSOF for in vitro embryo development. The cleavage rate was 11.76% and the development rate of embryos to 4–8 and 8–16 cells morulae was 55.6% and 42.4%, respectively. In vitro produced goat embryos (132), 4–16 cell stages, were used for cryopreservation using different protocols. In protocol- 1, 2, 3 and 4, there was no survivability of embryos. In protocol- 5, 6, 7 and 8, the survivability of embryo after freezing was 10, 25, 35.7 and 72.2%, respectively. In protocol- 8, the percentage of live embryos was significantly higher than other protocols. Also it was found that the survivability rate of embryos in protocol 6 and 7 were significantly higher as compared to protocol 1, 2, 3, 4 and 5. However, non-significant differences were found among protocol 1, 2, 3, 4 and 5, and between 6 and 7. From our results, it can be concluded that cryopreservation using 20% ethylene glycol and 0.9 M trehalose for 30 min gives significantly higher post thaw (72.2%) survivability of embryos.

Zooming in on Cryopreservation of hiPSCs and Neural Derivatives: A Dual-Center Study Using Adherent Vitrification.

Human induced pluripotent stem cells (hiPSCs) are an important tool for research and regenerative medicine, but their efficient cryopreservation remains a major challenge. The current gold standard is slow‐rate freezing of dissociated colonies in suspension, but low recovery rates limit immediate post‐thawing applicability. We tested whether ultrafast cooling by adherent vitrification improves post‐thawing survival in a selection of hiPSCs and small molecule neural precursor cells (smNPCs) from Parkinson's disease and controls. In a dual‐center study, we compared the results by immunocytochemistry (ICC), fluorescence‐activated cell sorting analysis, and RNA‐sequencing (RNA‐seq). Adherent vitrification was achieved in the so‐called TWIST substrate, a device combining cultivation, vitrification, storage, and post‐thawing cultivation. Adherent vitrification resulted in preserved confluency and significantly higher cell numbers, and viability at day 1 after thawing, while results were not significantly different at day 4 after thawing. RNA‐seq and ICC of hiPSCs revealed no change in gene expression and pluripotency markers, indicating that physical damage of slow‐rate freezing disrupts cellular membranes. Scanning electron microscopy showed preserved colony integrity by adherent vitrification. Experiments using smNPCs demonstrated that adherent vitrification is also applicable to neural derivatives of hiPSCs. Our data suggest that, compared to the state‐of‐the‐art slow‐rate freezing in suspension, adherent vitrification is an improved cryopreservation technique for hiPSCs and derivatives. stem cells translational medicine 2019;8:247&259

Optimizing sperm cryopreservation and recovery (OSCAR)

The objective of this study is to compare three common sperm freezing methods and two common thawing methods to one another to determine if there is a superior method to enhance post-thaw sperm survival. Prospective cohort study. Twelve discarded fresh semen samples were obtained with patient consent. Each fresh sample was washed with mHTF (LifeGlobal, Guilford, CT) and centrifuged for 15 minutes at 400G. The pellet was resuspended in a 3:1 ratio of mHTF to Artic™ Sperm Cryopreservation Medium (Irvine Scientific, Santa Ana, CA). Motility was evaluated manually and each participant’s sample was divided into six aliquots containing 0.5mL of specimen. Two aliquots of each specimen underwent one of 3 different freezing methods: (a) plunge into liquid nitrogen (LN2), (b) suspension in LN2 vapor for 15 minutes followed by plunge into LN2 or (c) suspension in LN2 vapor for 1 hour followed by plunge into liquid nitrogen. Each of the freezing methods was subject to two different thaw methods: (a) 37°C dry bath for 20 minutes followed by 40 minutes at room temperature or (b) room temperature (RT) for 1 hour. Following recovery, a second evaluation of motility was performed for each aliquot as a measure of sperm viability (percent motility versus initial). Analysis by two way repeated measures ANOVA was utilized with a p-value of 0.05 to determine significance. The mean motility for plunge sperm thawed at RT and 37°C were 22.9 ± 0.04% and 20.3 ± 0.04%, respectively. The mean motility for 15 minutes vapor then plunge thawed at RT and 37°C were 31.7 ± 0.06% and 31.5 ± 0.07%, respectively. For 1 hour in vapor then plunge, the mean motility at RT and 37°C were 43.8 ± 0.07% and 48.1 ± 0.06%, respectively. Survival rates between all freezing methods varied significantly, with greater recovery noted in the 1 hour vapor phase when compared both plunge (p<0.001) and 15 minutes in vapor (p=0.005). The shorter vapor phase of 15 minutes also demonstrated statistically greater viability compared to plunge (p = 0.027). Thawing methods did not significantly affect sperm recovery. The recovery of motile sperm varied directly with the length of cooling prior to plunge in LN2 with an hour suspension in vapor resulting in greatest motility compared to the initial sample. Recovery was not dependent on the thaw method used, with either of the two methods producing similar results for a given cryopreservation method. Interestingly, it was also noted that samples plunged directly from room temperature into LN2 retained appreciable, if diminished viability

Effects of different diluents, cryoprotective agents, and freezing rates on sperm cryopreservation in Epinephelus akaara

We investigated various factors, including cryoprotective agents (CPAs), diluents, and freezing rates, to develop an optimal cryopreservation protocol for Epinephelus akaara sperm. In experiments using 10% dimethyl sulfoxide (DMSO), glycerol, and methanol with various diluents, 10% DMSO and 300 mM glucose yielded the highest post-thaw sperm motility. The combination of 10% glycerol and 300 mM sucrose yielded significantly higher post-thaw sperm motility than did combinations using other diluents. Glycerol and DMSO at a concentration of 10% as CPAs with 300 mM glucose as the diluent resulted in the highest MSR and sperm activity index (SAI). An investigation to determine the effects of glycerol and DMSO concentrations on post-thaw sperm survival rate revealed no significant differences among 5, 10, 15, and 20% concentrations of either CPA. In assessing the effects of CPA concentration on the fertilization rate, the 10% concentration yielded the highest fertilization rate (81.4 ± 4.3%) in DMSO, whereas 15% was the optimal concentration for glycerol (fertilization rate = 66.7 ± 6.1%). The hatching rate was also highest in 10% DMSO (40.1 ± 0.4%) and in 15% glycerol (27.8 ± 2.3%). In conclusion, the optimal rates of post-thaw sperm motility, fertilization, and hatching were achieved when E. akaara sperm were cryopreserved in a diluent of 300 mM glucose with 10% DMSO as the CPA at a freezing rate of −5 °C/min. We therefore recommend this protocol for the cryopreservation of E. akaara sperm.

Impact of l-carnitine on lipid content and post thaw survivability of buffalo embryos produced in vitro

The aim of the present study was to see the impact of L-Carnitine (LC) on lipid biosynthesis and metabolism of buffalo embryos, and post thaw blastocyst survivability. In vitro fertilized (IVF) embryos were derived from slaughterhouse derived COCs and cultured in different doses of LC i.e. 0, 1 mM, 1.5 mM, 2 mM starting at 48 h post IVF. Blastocyst rate was significantly (p < 0.05) higher in 1.5 mM group than control and 1.0 mM group. Lipid content was measured indirectly by fluorescent intensity of lipid droplets after Nile red staining, and it was lower (p < 0.05) in treated than control groups. CPT1B, DGAT2 and DGAT1 mRNA expression was up regulated (p < 0.05) while AMPKg1 expression was down regulated in 1.5 mM and 2 mM groups compared to other groups (p < 0.05). mRNA expression of GLUT1, OCT4 and IFN-tau was higher (P < 0.05) in 1.5 mM group than the control group. Expression of BAX was down regulated at 1.5 mM LC. Blastocyts were vitrified by a modified OPS method and post thaw survivability of blastocysts was higher (P < 0.05) in 1.5 mM LC than other groups. In post thaw blastocysts, mRNA expression of GLUT1, OCT4 and IFN-tau was higher (P < 0.05) in 1.5 mM than other groups. Thus, it can be concluded that supplementation of l-carnitine (1.5 mM) in embryo culture media improved the quality of buffalo embryo production and post thaw blastocysts survivability by reducing fatty acid synthesis, enhancing fatty acid metabolism, and reducing lipid droplet formation.

High post-thaw survival of ram sperm after partial freeze-drying.

Recrystallization damages occur when a frozen sample is held at high subzero temperatures and when the warming process is too slow. In this work, ram semen diluted in two different concentrations of sugar solutions (Lyo A consisted of 0.4M sorbitol and 0.25M trehalose, and the second, Lyo B composed of 0.26M sorbitol and 0.165M trehalose) in egg yolk and Tris medium were compared after freezing 10μL samples to: (1) - 10, - 25, and - 35°C and thawing. (2)Freezing to - 10 and - 25°C, holding for 1h and then thawing, and (3)freezing to - 10 and - 25°C and drying for 1h at these temperatures at a vacuum of 80mTorr, prior thawing. For drying, we used a new freeze-drying apparatus (Darya, FertileSafe, Israel) having a condensation temperature below - 110°C and a vacuum pressure of 10-100mTorr that is reached in less than 10s. Results showed that samples in Lyo B solution frozen at - 25°C had significantly higher sperm motility in partially freeze-dried samples than frozen samples (46.6 ± 2.8% vs 1.2 ± 2.5%, P < 0.001). Moreover, partially dried samples in Lyo B showed higher motility than Lyo A at - 25°C (46.6 ± 2.8% vs 35 ± 4%). Cryomicroscopy and low-temperature/low-pressure environmental scanning electronic microscope demonstrated that the amount of the ice crystals present in partially dried samples was lower than in the frozen samples. Holding the sperm at high subzero temperatures is necessary for the primary drying of cells during the freeze-drying process. Rapid freeze-drying can be achieved using this new device, which enables to reduce recrystallization damages.