Vitrification Protocol Research Articles

Effects of different sucrose concentrations on vitrified porcine preantral follicles: Qualitative and quantitative analysis

The aim of the present study was to perform a qualitative and quantitative analysis of the effect of different sucrose concentrations combined with ethylene glycol in the preservation of vitrified porcine preantral follicles. Fragments of ovarian cortex were vitrified in cryotubes containing 200 μl of the vitrification solution (30% Ethylene Glycol; 20% Fetal Bovine Serum; 0 M–0.25 M – 0.75 M or 1 M sucrose) and stored in liquid nitrogen for a week. Histological analysis showed that after vitrification the number of normal follicles decreased compared to the fresh tissue (control). The percentage of normal primordial follicles was sucrose dose dependent. The percentage of normal primary follicles was similar in 0 M or 0.25 M sucrose, while higher concentrations (0.75 M and 1 M) increased significantly the percentage of abnormal follicles (p < 0.05). Morphometric analysis showed a statistically significant reduction in the total area of primordial follicles with 0.75 M sucrose and a significant increase in the cytoplasmic area of primordial follicles with 0 M sucrose (p < 0.05). The qualitative and the quantitative analysis appear to be a complementary tool when choosing a vitrification protocol. For our cryopreservation system - vitrification of ovarian cortex slices in cryotubes-the best vitrification medium was TCM 199-Hepes with 30% de ethylene glycol, 20% of Fetal Bovine Serum and 0 or 0.25 M sucrose. The present study shows that the use of high sucrose concentrations in the vitrification solution has a deleterious effect on the preservation of porcine preantral follicles contained in ovarian tissue. Consequently, its use at 0.75 M or 1 M wouldn't be recommended.

First successful vitrification of salmonid ovarian tissue

Due to a lack of cryopreservation protocols for fish eggs and embryos, alternative techniques which will enable storage of female genetic resources are crucial for future development of reproduction management in conservation biology and aquaculture. Experiments were conducted to develop an optimal vitrification protocol for cryopreservation of brown trout Salmo trutta juvenile ovarian tissue. Needle immersed vitrification (NIV) method was used where ovaries were pinned on an acupuncture needle, passaged through equilibration and vitrification solutions containing different combinations and concentrations of methanol (MeOH), propylene glycol (PG) and dimethyl sulfoxide (Me2SO) and subsequently plunged into liquid nitrogen. Vitrification solutions containing equal cryoprotectant concentrations (3M Me2SO and 3M PG) yielded the highest oogonia survival rates (up to 40%) and qualitatively and quantitatively unaltered perinucleolar follicles. The method developed for brown trout could be applied to the conservation of female genetic resources of other salmonid species, including endangered and endemic species or populations.

In Vitro Cryopreservation of Date Palm Caulogenic Meristems.

Cryopreservation is the technology of choice not only for plant genetic resource preservation but also for virus eradication and for the efficient management of large-scale micropropagation. In this chapter, we describe three cryopreservation protocols (standard vitrification, droplet vitrification, and encapsulation vitrification) for date palm highly proliferating meristems that are initiated from vitro-cultures using plant growth regulator-free MS medium. The positive impact of sucrose preculture and cold hardening treatments on survival rates is significant. Regeneration rates obtained with standard vitrification, encapsulation-vitrification, and droplet-vitrification protocols can reach 30, 40, and 70%, respectively. All regenerated plants from non-cryopreserved or cryopreserved explants don't show morphological variation by maintaining genetic integrity without adverse effect of cryogenic treatment. Cryopreservation of date palm vitro-cultures enables commercial tissue culture laboratories to move to large-scale propagation from cryopreserved cell lines producing true-to-type plants after clonal field-testing trials. When comparing the cost of cryostorage and in-field conservation of date palm cultivars, tissue cryopreservation is the most cost-effective. Moreover, many of the risks linked to field conservation like erosion due to climatic, edaphic, and phytopathologic constraints are circumvented.

Chapter 14 Establishing an Oocyte Cryobank Network.

Improvements in oocyte cryopreservation has prompted wider acceptance of this technology leading to its use for several reasons. Notably, in addition to elective and medically driven reasons for oocyte cryostorage for fertility preservation, donor oocyte cryobanking is beginning to gain traction, potentially replacing fresh oocyte donation in assisted reproduction. Donor "egg banking," while not totally analogous to donor sperm banking, does provide strong benefits in terms of scheduling flexibility and improved clinical efficiencies, while providing a wider immediate inventory choice. The development of a successful cryobank "network" and subsequent growth into a full-access donor egg bank are only possible through adoption of a series of key steps involving establishment of a repeatable vitrification protocol with a strong clinical record, incorporation of a comprehensive database and quality management system, and strict control over the logistics of inventory and shipping and receiving, to establish a flawless chain from donor to recipient. Confidence will grow in this potentially difficult process over time.

Ethylene glycol and glycerol loading and unloading in porcine meniscal tissue

The development of a long-term storage method for meniscus, a complex tissue of the knee prone to injury, would improve the procedure and outcomes of meniscus transplantation. Cryopreservation uses cryoprotective agents (CPAs) including ethylene glycol (EG) and glycerol to preserve a variety of live tissues, and understanding of the CPA permeation kinetics will be critical in designing a vitrification protocol for meniscus.The purpose of this preliminary study was to understand the loading and unloading behaviours of EG and glycerol in meniscus by observing their efflux. For the main experiment, lateral and medial porcine menisci were incubated with CPA for 24 h at three temperatures (i.e., 4, 22, and 37 °C). Then, the menisci were immersed in 25 ml of X-VIVO™10 and CPA efflux was recorded by monitoring the molality of two consecutive washout solutions at different time points. In a subsequent experiment, menisci were incubated in the CPA solutions for 48 h at 22 °C, and the results were compared to those obtained at 22 °C in the main experiment.Results showed a rapid efflux of CPA from meniscus at the beginning of each wash. With increasing temperature, the amount of CPA efflux (and hence loading) increased. Using 24 h incubation, EG loaded the menisci more completely than glycerol. But after 48 h of incubation, both EG and glycerol achieved approximately the same degree of meniscus loading.This study provides preliminary data that will facilitate future design of experiments aimed at development of meniscus permeation studies.

48 EFFECT OF DIMETHYL SULFOXIDE- OR GLYCEROL-BASED VITRIFICATION PROTOCOLS ON THE DNA METHYLATION OF BOVINE CUMULUS-OOCYTE COMPLEXES

Although vitrification is becoming increasingly common for oocyte preservation, there has been recent evidence that some cryoprotectants may alter DNA methylation and so result in decreased oocyte developmental competence and abnormal embryonic development post-warming. The objective of this study was to determine the effect of dimethyl sulfoxide (DMSO)- or glycerol-based vitrification protocols on DNA methylation in bovine cumulus-oocyte complexes (COC). The vitrification protocols evaluated included a combination of ethylene glycol (EG) with either DMSO or glycerol (GLY). Cumulus-oocyte complexes were first exposed to equilibration solution (ES) consisting of 7.5% DMSO or GLY and 7.5% EG for 9 min at room temperature (RT) before being transferred to vitrification solution (VS) containing 15% DMSO or GLY, 15% EG, and 0.5 M sucrose. While in VS, 3 to 4 COC were loaded into an open system vitrification device (Cryolock®, BioTech Inc.) and plunged into LN within 1 min. For warming, COC were exposed to dilution solution 1 consisting of 0.5 M sucrose (37°C) for 2.5 min and to dilution solution 2 consisting of 0.25 M sucrose for 2.5 min (RT). Base media for all solution was PBS supplemented with 20% FBS. Cumulus-oocyte complexes (n = 137) were collected from crossbred cows by ovum pick-up on 3 separate days (repetitions), with half of the oocytes each day being matured to metaphase II (MII) stage before vitrification and the other half vitrified as germinal vesicle (GV) COC. The resulting treatments were DMSO GV v. Glycerol GV and DMSO MII v. Glycerol MII. Fresh COC (GV and MII stage, respectively) were utilised as controls. DNA methylation analysis of oocytes was performed through fluorescent staining using a primary antibody anti-5 mC (1:1000) and secondary antibody Alexa Fluor 488 (1:500). Fluorescence intensity of samples was measured using ImageJ software (NIH, Bethesda, MD, USA). Data were analysed through ANOVA with post hoc Tukey’s test. No differences between the vitrification groups or controls were found when analysing DNA methylation of GV stage COC (P = 0.1825). Metaphase II stage COC showed statistical difference between groups (P &lt; 0.001) where glycerol-treated MII oocytes exhibited higher DNA methylation compared with DMSO MII and fresh MII COC. Results of the experiment showed that the glycerol-based vitrification protocol causes alteration of DNA methylation of bovine metaphase II cumulus-oocyte complexes, and as such may not be an optimal choice in mature oocyte vitrification protocols.

93 BLASTOCYST BISECTION TO MULTIPLY BIOPSIED AND VITRIFIED BOVINE EMBRYOS

Dairy cattle breeding schemes increasingly integrate embryo-based genomic selection to accelerate genetic gain. In contrast to the single offspring produced with conventional animal-based genomic selection, multifactorial IVF between elite parents increases genotypes for selection. Genetically superior embryos are identified from biopsies, and only those with the desired genotypes are transferred. To manage the logistics of such schemes, and enable seasonally born progeny, the cryo-preservation of embryos after biopsy and before embryo transfer is critical. Here, we compare 2 methods of cryo-preserving biopsied Day 7 blastocysts and report results from bisecting blastocysts to increase the number of selected embryos for transfer. Abattoir-sourced oocytes were matured in vitro and fertilized with sperm from a single sire. Embryos were cultured for 7 days in a modified Synthetic Oviduct Fluid medium. Approximately 15 cells were biopsied from the mural trophectoderm of grade 1 and 2 blastocysts in Embryo Hold medium minus BSA, using a micro-surgical blade (Bioniche Animal Health, Athens, GA, USA). Following biopsy, each blastocyst was cultured in Embryo Hold with 3 mg mL−1 BSA for ~2 h at 38.5°C to allow for re-expansion. In Experiment 1, embryos were randomly assigned to 1 of 2 cryo-preservation treatments: conventional slow freezing or the Cryologic vitrification method (CVM). Slow freezing entailed freezing in 1.5 M ethylene glycol and 0.1 M sucrose. The CVM involved a 2-step vitrification protocol, with 15% of both ethylene glycol and dimethyl sulphoxide in the final solution comprising Embryo Hold, 20% FCS, 1 M sucrose, and 0.1 mM Ficoll (GE Healthcare). Selected embryos were thawed/warmed and transferred in pairs to the uterine horn ipsilateral to the corpus luteum of each synchronized recipient heifer. In Experiment 2, each biopsied blastocyst was individually vitrified using CVM. Following warming, blastocysts were bisected into approximately equal halves. After ~2 h recovery, pairs of demi-embryos were transferred to recipients categorized with either normal (&gt;2.5, &lt;7 ng mL−1) or low (≥2, &lt;2.5 ng mL−1) plasma progesterone concentrations on Day 5 after oestrus. Embryo survival in both experiments was monitored by ultrasonography of fetal heartbeats up to Day 65 of gestation. Statistical significance was determined using Fisher’s exact test. In Experiment 1, embryo survival on Day 65 was significantly greater with CVM than slow freezing (25/54 = 46% v. 9/54 = 17%; P = 0.002). In Experiment 2, there was no significant difference in the number of fetuses as a percentage of original blastocysts, regardless of normal versus low progesterone levels (13/22 = 59% v. 4/9 = 44%, respectively). In conclusion, vitrification is superior for cryo-preserving biopsied blastocysts, possibly reducing cryo-damage compared with conventional slow freezing, and achieves rates of in vivo development similar to fresh IVF embryos. Embryo bisection potentially provides only a modest increase in the probability of generating a calf from each valuable, genomically selected embryo. Improving embryo competency and other methods of multiplication may maximize this likelihood.

40 THE EFFECT OF EXPOSURE TIME ON TOXICITY OF VITRIFICATION SOLUTION ON PORCINE CUMULUS–OOCYTE COMPLEXES BEFORE IN VITRO MATURATION

Although offspring have been produced from porcine cumulus-oocyte complexes (COC) vitrified at the immature stage (Somfai et al. 2014 PLoS One 9, e97731), embryo development rates have remained low. Numerous vitrification protocols are reported with a wide variation in the applied exposure time to the vitrification solution. Because cryoprotectants in the vitrification solution can be detrimental to the oocytes and their subsequent development, it is important to verify the effect of their exposure time to the COC. In this study, we compared the development of a control group with 3 toxicity control (TC) groups in which COC were exposed to the vitrification solution for 30 s, 1 min, or 1.5 min (TC1, TC2, and TC3, respectively) at 38.5°C. Before exposure, the COC were rinsed and equilibrated in 7 µg mL−1 cytochalasin B. The equilibration solution consisted of 2% (vol/vol) ethylene glycol + 2% (vol/vol) propylene glycol and the vitrification solution contained 17.5% (vol/vol) ethylene glycol + 17.5% (vol/vol) propylene glycol, 50 mg mL−1 polyvinylpyrrolidone and 0.3 M sucrose. The COC were not exposed to liquid nitrogen. After washing in a warming solution of 0.4 M sucrose at 42°C, COC were washed in a sucrose gradient from 0.2 to 0.0 M. Subsequently, the COC were subjected to in vitro maturation in porcine oocyte medium. During the first 20 h of in vitro maturation, the porcine oocyte medium was supplemented with 10 IU mL−1 eCG, 10 IU mL−1 hCG, 1 mM dibutyryl cAMP, and 10 ng mL−1 epidermal growth factor. Then, the medium was replaced with dibutyryl cAMP-free porcine oocyte medium for an additional 28 h. After in vitro maturation, oocytes were parthenogenetically activated (Day 0) and cultured for 7 days in porcine zygote medium. Survival, nuclear maturation, cleavage, and blastocysts rates (Days 6 and 7) were assessed. All parameters were statistically analysed by binary logistic regression. Only the survival rate of TC3 was significantly lower than that of the control group (89.2 v. 95.6%). Exposure to cryoprotectants significantly decreased maturation rates in TC1, TC2, and TC3 compared with the control (72.6%, 75.2%, 76.3% v. 86.1%). Cleavage rates were significantly lower in TC2 and TC3 than that in the control (82.8% and 81.7% v. 92.9%). Concerning blastocyst rates on Day 6 and Day 7 of in vitro culture, only TC1 could reach the same level as the control, expressed on the total number of activated oocytes (54.6% v. 67.7%, and 64.0% v. 72.9%, respectively) as well as expressed on the cleaved oocytes (61.4% v. 72.4% and 72.0% v. 78.0%, respectively). Consequently, despite the reduced maturation rate, TC1 provides the same quantity of blastocysts from matured oocytes as the control. In conclusion, exposure to the vitrification solution for longer than 30 s has toxic effects on COC and therefore is not recommended for vitrification. R. Appeltant is an International Research Fellow of the JSPS Japan (P15402).

Cryopreservation of Jerusalem artichoke cultivars using an improved droplet-vitrification method

Jerusalem artichoke (Helianthus tuberosus L.) cultivars are conserved in genebanks for use in breeding and horticultural research programs. Jerusalem artichoke collections are particularly vulnerable to environmental and biological threats because they are often maintained in the field. These field collections could be securely conserved in genebanks if improved cryopreservation methods were available. This work used four Jersualem artichoke cultivars (‘Shudi’, ‘M6’, ‘Stampede’, and ‘Relikt’) to improve upon an existing procedure. Four steps were optimized and the resulting procedure is as follows: preculture excised shoot tips (2–3 mm) in liquid MS medium supplemented with 0.4 M sucrose for 3 days, osmoprotect shoot tips in loading solution for 30 min, dehydrate with plant vitrification solution 2 for 15 min before rapid cooling in liquid nitrogen, store in liquid nitrogen, rapidly rewarm in MS liquid medium containing 1.2 M sucrose, and recover on MS medium supplemented with 0.1 mg L−1 GA3 for 3–5 days in the dark and then on the same medium for 4–6 weeks in the light (14 h light/10 h dark). After cryopreservation, Jerusalem artichoke cultivar ‘Shudi’ had the highest survival (93%) and regrowth (83%) percentages. Cultivars ‘M6’, ‘Stampede’, and ‘Relikt’ achieved survival and regrowth percentages ranging from 44 to 72%, and 37–53%, respectively. No genetic changes, as assessed by using simple sequence repeat markers, were detected in plants regenerated after LN exposure in Jerusalem artichoke cultivar ‘Shudi’. Differential scanning calorimetry analyses were used to investigate the thermal activities of the tissues during the cryopreservation process and it was determined that loading with 2.0 M sucrose and 0.4 M sucrose dehydrated the shoot tips prior to treatment with PVS2. Histological observations revealed that the optimized droplet vitrification protocol caused minimal cellular damage within the meristem cells of the shoot tips.

Optimization of human somatic cells cryopreservation protocols by polyethylene glycols

Using of polyethylene glycol in optimization of human somatic cells of cryopreservation protocols was analyzed. Low molecular weight PEG as vitrification solution supplement exhibited a high cooling speed and provided cell survival in 200 % comparing with control. It allows recommending the use of low molecular weight PEG in vitrification environment for effective cell vitrification protocols.

Expression of the T85A mutant of zebrafish aquaporin 3b improves post-thaw survival of cryopreserved early mammalian embryos.

While vitrification has become the method of choice for preservation of human oocytes and embryos, cryopreservation of complex tissues and of large yolk-containing cells, remains largely unsuccessful. One critical step in such instances is appropriate permeation while avoiding potentially toxic concentrations of cryoprotectants. Permeation of water and small non-charged solutes, such as those used as cryoprotectants, occurs largely through membrane channel proteins termed aquaporins (AQPs). Substitution of a Thr by an Ala residue in the pore-forming motif of the zebrafish (Dario rerio) Aqp3b paralog resulted in a mutant (DrAqp3b-T85A) that when expressed in Xenopus or porcine oocytes increased their permeability to ethylene glycol at pH 7.5 and 8.5. The main objective of this study was to test whether ectopic expression of DrAqp3b-T85A also conferred higher resistance to cryoinjury. For this, DrAqp3b-T85A + eGFP (reporter) cRNA, or eGFP cRNA alone, was microinjected into in vivo fertilized 1-cell mouse zygotes. Following culture to the 2-cell stage, appropriate membrane expression of DrAqp3b-T85A was confirmed by immunofluorescence microscopy using a primary specific antibody directed against the C-terminus of DrAqp3b. Microinjected 2-cell embryos were then cryopreserved using a fast-freezing rate and low concentration (1.5 M) of ethylene glycol in order to highlight any benefits from DrAqp3b-T85A expression. Notably, post-thaw survival rates were higher (P<0.05) for T85A-eGFP-injected than for -uninjected or eGFP-injected embryos (73±7.3 vs. 28±7.3 or 14±6.7, respectively). We propose that ectopic expression of mutant AQPs may provide an avenue to improve cryopreservation results of large cells and tissues in which current vitrification protocols yield low survival.

Minimal volume vitrification of epididymal spermatozoa results in successful in vitro fertilization and embryo development in mice.

This study compared three cryopreservation protocols on sperm functions, IVF outcomes, and embryo development. Epididymal spermatozoa cryopreserved using slow-cooling (18% w/v raffinose, RS-C) were compared with spermatozoa vitrified using 0.25 M sucrose (SV) or 18% w/v raffinose (RV). The motility, vitality, and DNA damage (TUNEL assay) of fresh control (FC) spermatozoa were compared with post-thawed or warmed RS-C, RV, and SV samples. Mouse oocytes (n = 267) were randomly assigned into three groups for insemination: RV (n = 102), RS-C (n = 86), and FC (n = 79). The number and the proportion of two-cell embryos and blastocysts from each treatment were assessed. Sperm motility (P < 0.01) and vitality (P < 0.05) were significantly reduced after vitrification compared with slow-cooled spermatozoa. However, DNA fragmentation was significantly reduced in spermatozoa vitrified using sucrose (15 ± 1.8% [SV] vs 26 ± 2.8% [RV] and 27 ± 1.2% [RS-C]; P < 0.01). Although the number of two-cell embryos produced by RS-C, RV, and FC spermatozoa was not significantly different, the number of blastocysts produced from two-cell embryos using RV spermatozoa was significantly higher than FC spermatozoa (P = 0.0053). This simple, small volume vitrification protocol and standard insemination method allows successful embryo production from small numbers of epididymal spermatozoa and may be applied clinically to circumvent the need for ICSI, which has the disadvantage of bypassing sperm selection.

Comparison of apoptosis pathway following the use of two protocols for vitrification of immature mouse testicular tissue

Our objective was to evaluate the apoptosis incidence in immature mouse testicular tissue after two different protocols of vitrification and short-term culture. Testes of 7-day-old Naval Medical Research Institute mice were isolated and distributed into control and vitrification groups. In vitrification 1 group, testes were vitrified using a combination of ethylene glycol and DMSO in three steps, and in vitrification 2 group, testes were vitrified using a combination of ethylene glycol and sucrose in five steps. Then, fresh and vitrified-warmed testis fragments were cultured for 20 hours. Morphology, cell viability, apoptosis incidence, and apoptosis gene expression (BAX, BCL2, Caspase 3, Fas, Fas ligand, p53) were evaluated at 0, 3, and 20 hours of culture by light microscopy, flow cytometry, and real-time polymerase chain reaction, respectively. Significant decrease of early apoptosis (annexin V+/PI− cells in vitrification 1 and 2 groups at 0 hours of culture, 37.34 ± 0.91 and 30.72 ± 2.2, and at 20 hours of culture, 1.46 ± 0.28 and 0.76 ± 0.11, respectively), increase of late apoptosis (annexin V+/PI+ cells in vitrification 1 group at 0 hours of culture, 14.46 ± 0.86, and at 20 hours of culture, 37.18 ± 2.34), and BAX/BCL-2 ratio (in vitrification 1 and 2 groups at 0 hours of culture, 7.31 ± 0.31 and 6.83 ± 1.38, and at 20 hours of culture, 24.08 ± 4.32 and 9.35 ± 1.91, respectively) were observed in vitrification groups during culture period. Caspase 3 expression was significantly decreased in all groups after 3 hours of culture (in control, vitrification 1, and vitrification 2 groups at 0 hours of culture, 1.00 ± 0.0, 1.56 ± 0.09, and 0.79 ± 0.06, and at 20 hours of culture, 0.37 ± 0.0, 0.96 ± 0.10, and 0.12 ± 0.03, respectively). Expression of p53 was significantly lower in vitrification 1 (0.32 ± 0.02) and control (0.50 ± 0.03) groups in 20 hours of culture as compared with vitrification 2 (0.88 ± 0.14) group. Fas (in vitrification 1 and 2 groups at 0 hours of culture, 2.29 ± 0.23 and 1.14 ± 0.15, and at 20 hours of culture, 12.43 ± 0.46 and 6.7 ± 0.48, respectively) and Fas Ligand (in vitrification 1 and 2 groups at 0 hours of culture, 1.2 ± 0.28 and 5.24 ± 0.32, and at 20 hours of culture, 21.75 ± 2.00 and 25.82 ± 2.15, respectively) expressions significantly increased in vitrification groups after 20 hours of culture. Although both vitrification protocols cause cell death via apoptotic and necrotic pathway, it seems that vitrification 1 protocol induces cell death more via apoptotic pathway than via necrosis. The apoptosis incidence after vitrification may have occurred independent of p53.

A droplet-vitrification protocol enabled cryopreservation of doubled haploid explants of Malus x domestica Borkh. ‘Golden Delicious’

High throughput cryopreservation is a key step in the development of an apple t-DNA mutant library. The aim of this study was to develop an easy, efficient and rapid droplet vitrification protocol to cryopreserve doubled haploids of Malus x domestica Borkh. ‘Golden Delicious’. The doubled haploid genotype proved to be less tolerant to cryopreservation than ‘Golden Delicious’. Apices improved the quality of regeneration and the facility of dissection compared to meristems. About 50% of the explants treated with PVS2 for 40min survived after immersion in liquid nitrogen, and 10% showed sustainable development. Phytotoxicity was observed mostly after immersion in the vitrification solution. Hyperhydricity was regularly observed in these experiments and decreased final efficiency. The use of meta-topolin instead of 6-benzylaminopurine (BAP) reduced hyperhydricity by almost 50%. We demonstrated that doubled haploid apple apices can be cryopreserved, but further improvements are needed before this technique can be used at high throughput.

Pregnancy and birth outcomes following fresh or vitrified embryo transfer according to blastocyst morphology and expansion stage, and culturing strategy for delayed development

How do live birth rates (LBRs), following fresh and vitrified/warmed embryo transfer, compare according to morphological grade, developmental stage and culturing strategy of human blastocysts in vitro? Equivalent LBRs were obtained after fresh embryo transfer and after vitrified/warmed embryo transfer of blastocysts of top or non-top quality, while vitrification after prolonged embryo culture of blastocysts with delayed development had a positive impact on LBR. Blastocyst morphology correlates with clinical outcome; however, few data are available on vitrified/warmed embryo transfer using non-top quality blastocysts. The aim of this study was to determine clinical outcomes of non-top quality blastocysts and blastocysts with delayed development that underwent vitrified/warmed embryo transfer. This retrospective, single-centre study (conducted January 2009 to June 2013) compared 1010 fresh embryo transfer and 1270 vitrified/warmed embryo transfer of blastocysts originating from the same stimulation cycle. Furthermore, 636 fresh embryo transfers and 304 vitrified/warmed embryo transfer after delayed expansion or blastulation in the same period were also analysed. Clinical outcomes after fresh and vitrified/warmed embryo transfer according to blastocyst morphology were compared in both groups. Similar LBRs after fresh embryo transfer or after vitrified/warmed embryo transfer of top or non-top quality blastocysts were observed. A statistically significant improvement in clinical outcomes was obtained after vitrified/warmed embryo transfer of Day 5 embryos with delayed expansion or blastulation when applying prolonged culture. Our study suggests that vitrification of non-top quality blastocysts as well as delayed cavitating and blastulating Day 5 embryos should be considered in autologous IVF cycles. Given that the present retrospective study used aseptic vitrification of blastocysts, the results, particularly the survival rates, may not be fully applicable to other vitrification protocols. The retrospective nature of the study has to be mentioned. Restriction of vitrification to top quality blastocysts may result in discarding potentially viable embryos. This study was not externally funded. There are no conflicts of interest to declare.

Toxicity of a vitrification protocol for equine immature oocytes

Toxicity of a vitrification protocol for equine immature oocytes

Vitrified-warmed embryo transfer is associated with mean higher singleton birth weight compared to fresh embryo transfer

ObjectiveTo test for differences in birth weight between singletons born after IVF with fresh embryo transfer vs. vitrified-warmed 2PN embryo transfer (vitrification protocol). DesignRetrospective analysis of 464 singleton live births after IVF or ICSI during a 12 year period. SettingUniversity hospital. InterventionsFresh embryo transfer, vitrified-warmed 2PN embryo transfer (vitrification protocol). Main outcome measuresBirth weight standardized as a z-score, adjusting for gestational week at delivery and fetal sex. As a reference, birth weight means from regular deliveries from the same hospital were used. Multivariate regression analysis was used to investigate the relationship between the dependent variable z-score (fetal birth weight) and the independent predictor variables maternal age, weight, height, body mass index, RDS prophylaxis, transfer protocol, number of embryos transferred, indication for IVF treatment and sperm quality. ResultsThe mean z-score was significantly lower after fresh transfer (−0.11±92) as compared to vitrification transfer (0.72±83) (p<0.001). Multivariate regression analysis indicated that only maternal height and maternal body mass index, but not type of cryopreservation protocol, was a significant predictor of birth weight. ConclusionsIn this analysis focusing on 2PN oocytes, vitrified-warmed embryo transfer is associated with mean higher birth weight compared to fresh embryo transfer. Maternal height and body mass index are significant confounders of fetal birth weight and need to be taken into account when studying birth weight differences between ART protocols.

Development of sperm vitrification protocols for freshwater fish (Eurasian perch, Perca fluviatilis) and marine fish (European eel, Anguilla anguilla)

Vitrification was successfully applied to the sperm of two fish species, the freshwater Eurasian perch (Perca fluviatilis) and marine European eel (Anguilla anguilla). Sperm was collected, diluted in species-specific non-activating media and cryoprotectants and vitrified by plunging directly into liquid nitrogen without pre-cooling in its vapor. Progressive motility of fresh and vitrified-thawed sperm was evaluated with computer-assisted sperm analysis (CASA). Additional sperm quality parameters such as sperm head morphometry parameters (in case of European eel) and fertilizing capacity (in case of Eurasian perch) were carried out to test the effectiveness of vitrification. The vitrification method for Eurasian perch sperm resulting the highest post-thaw motility (14±1.6%) was as follows: 1:5 dilution ratio, Tanaka extender, 30% cryoprotectant (15% methanol+15% propylene-glycol), cooling device: Cryotop, 2μl droplets, and for European eel sperm: dilution ratio 1:1, with 40% cryoprotectant (20% MeOH and 20% PG), and 10% FBS, cooling device: Cryotop, with 2μl of sperm suspension. Viable embryos were produced by fertilization with vitrified Eurasian perch sperm (neurulation: 2.54±1.67%). According to the ASMA analysis, no significant decrease in head area and perimeter of vitrified European eel spermatozoa were found when compared to fresh spermatozoa.

Assessment of the effect of different vitrification solutions on human ovarian tissue after short-term xenotransplantation onto the chick embryo chorioallantoic membrane.

Chemotherapy can lead to the loss of fertility and premature ovarian failure in young women who suffer from malignant diseases. Freezing ovarian tissue by vitrification allows for the preservation of a large number of follicles prior to treatment, yet no established protocols have been optimized with respect to the vitrification solution. The aim of the present study was to evaluate the early stage of human ovarian tissue xenotransplantated onto the chick embryo chorioallantoic membrane after vitrification, and to determine the effect of different vitrification solutions on ovarian tissue quality-as defined by morphology and viability of follicles, neovascularization, cell proliferation, and apoptosis. Each vitrification protocol had a different impact on ovarian tissue at the early transplantation stage; one process using the lowest concentrations of ethylene glycol and dimethylsulfoxide, plus sucrose, demonstrated a moderate advantage compared to the other protocols. We also demonstrated that the chorioallantoic membrane model can be a useful alternative for short-term xenotransplantation studies of angiogenesis into human ovarian cortical tissue.

Étude prospective comparant l’efficacité de la vitrification ovocytaire en système fermé ou en système ouvert

ObjectivesOocyte vitrification using an open device is thought to be a source of microbiological and chemical contaminations that can be avoided using a closed device. The principal purpose of this study was to compare the two vitrification protocols: closed and open system. The secondary aim was to study the effects of the storage in the vapor phase of nitrogen (VPN) on oocytes vitrified using an open system and to compare it to those of a storage in liquid nitrogen (LN). MethodsForty-four patients have been included in our study between November 2014 and May 2015. Two hundred and fourteen oocytes have been vitrified at germinal vesicle (GV), metaphase I (0PB) and metaphase II (1PB) stages. We vitrified 96 oocytes (59 GV/37 0PB) using a closed vitrification device and 118 oocytes (57 GV/31 0PB/30 1PB) using an open device. The vitrified oocytes were then stored either in LN or in VPN. The main outcome measures were the survival rate after warming (SR), meiosis resumption rate (MRR) and maturation rate (MR). ResultsThe global post-thaw SR was significantly higher for oocytes vitrified using an open system (93.2%) compared to those vitrified using a closed one (64.5%; P<0.001). On the contrary, there was no significant difference in terms of global MRR and MR (82.1% vs. 87.5% and 60.7% vs. 61.2% using closed and open system respectively). The SR, MRR and the MR were not significantly different when vitrified oocytes were stored in VPN or LN (91.6, 83.8, 64.5% vs. 93.9, 89.8, 59.1% respectively). ConclusionTaking into account the limits of our protocol, the open vitrification system remains the more efficient system. The use of sterile liquid nitrogen for oocyte vitrification and the subsequent storage in vapor phase of nitrogen could minimize the hypothetical risks of biological and chemical contaminations.