Oocyte Vitrification Protocol Research Articles

#386 : Warming Vitrified Oocytes in a Fraction of the Current Required Time Results in Superior Survival Rates

Background and Aims: Oocyte vitrification and warming is very time consuming. Oocyte vitrification protocols are different from that of embryos or blastocysts, while warming protocols allow all to be warmed the same. For blastocysts, frequently only one device is warmed per patient; however, numerous devices are required in oocyte warming. We aim to evaluate whether vitrified oocytes can successfully survive a warming protocol involving only Thawing Solution (TS) with a total elapsed warming time of a mere 90 seconds. Methods: 58 vitrified donor oocytes (Treatment Group) were prospectively warmed in January 2023. 71 vitrified donor oocytes (Control Group) warmed in 2022 were retrospectively analyzed. Both groups were vitrified in Kitazato or Irvine Scientific Vit Kit with identical protocols (elapsed time approximately 17-minutes). In both groups the TS was equilibrated to 37-degrees for a minimum of 2-hours. The Control Group was warmed in Irvine Scientific Vit Kit (1-minute TS, 3-minute DS, 8-minute WS) for a total warming time of 12-minutes. Warming for the Treatment Group was in Irvine Scientific Vit Kit Warm-NX, TS only, for 90-seconds. Results: After Chi-square analysis, [Formula: see text] value < 0.05 reflected significance. The Control Group warmed with 90.1% survival. The Treatment Group resulted in 98.3% survival, ([Formula: see text] -value = 0.056). Survival at 3-hours post warm in the Control Group was 100%. At this point for the Control Group, ICSI was performed, and the oocytes placed back into the incubator for resulting embryo culture. For the Treatment Group, oocyte survival was re-assessed at 1-hour and 24-hours, and 100% survival was maintained over the course of 24-hour incubation, with little-to-no visible cryo-damage. Conclusions: Vitrified oocytes exhibit outstanding survival when warmed with a highly modified and considerably faster protocol than current practice. The extended oocyte survival of at least 24-hours shows a very favorable outcome for the accelerated warming protocol and gives credibility to the survival assessment.

Embryology outcomes after oocyte vitrification with super-cooled slush nitrogen are similar to outcomes with conventional liquid nitrogen: a randomized controlled trial

To determine whether the use of slush nitrogen (SN), a super-cooled form of nitrogen with a temperature from -207 to -210 °C, can improve oocyte survival after vitrification and warming compared with conventional liquid nitrogen (LN). Randomized controlled trial. Academic-affiliated private practice. A total of 556 metaphase II oocytes from 32 oocyte donor cycles were included. Donor oocytes were block randomized to undergo vitrification with either SN or LN. Vitrification was followed by warming, fertilization with donor sperm, embryo culture to the blastocyst stage, and preimplantation genetic testing for aneuploidy via trophectoderm biopsy with targeted next-generation sequencing. The primary outcome was oocyte survival after vitrification and warming. Secondary outcomes included rates of fertilization, usable blastocyst formation, and whole chromosome aneuploidy. Half of the metaphase II oocytes (n = 278) were randomized to undergo vitrification with SN, whereas the other half (n=278) were randomized to undergo vitrification with LN. There were no statistically significant differences noted in oocyte survival rate (85.3% vs. 86.3%), fertilization rate (84.0% vs. 80.0%), rate of usable blastocyst formation (54.3% vs. 55.7%), or rate of whole chromosome aneuploidy (9.4% vs. 11.7%) between the SN and LN arms, respectively. The implementation of an SN oocyte vitrification protocol resulted in similar embryology outcomes compared with LN. The use of SN did not lead to any demonstrable improvement in oocyte survival after vitrification and warming. NCT04342364.

Impact of equilibration duration during oocyte vitrification protocol: preliminary results of a prospective observational study

Impact of equilibration duration during oocyte vitrification protocol: preliminary results of a prospective observational study

An efficiency comparison of different in vitro fertilization methods: IVF, ICSI, and PICSI for embryo development to the blastocyst stage from vitrified porcine immature oocytes

BackgroundMost studies carried out to evaluate recovery and development after porcine oocyte vitrification, reported better rates when cryopreserved in embryonic development stages or zygotes, but not in immature oocytes. For this reason, many studies are performed to improve immature oocyte vitrification protocols testing the use of different cryoprotectant concentrations, cooling devices, incubation times; but only a few of them have evaluated which fertilization procedure enhances blastocyst rates in vitrified oocytes. Therefore, this study was aimed to evaluate: 1) if the sperm selection with hyaluronic acid (HA) or polyvinylpyrrolidone (PVP) before injection could play a key role in increasing fertilization and blastocyst formation and 2) the embryo developmental ability and blastocyst production of porcine immature oocytes retrieved after vitrification-warming and co-cultured with granulosa cells during IVM, using different fertilization techniques: in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and conventional ICSI with hyaluronic acid (HA) sperm selection, known as physiological intracytoplasmic sperm injection (PICSI) and.ResultsSperm selected with HA-PICSI displayed a higher percentage of live/acrosome reacted status compared to those in control and exposed to PVP. Higher dead/acrosome reacted rates were obtained after PVP exposure compared to control and HA. In oocytes, viability significantly decreased after IVM in vitrified oocytes. Besides, IVM rates were not different between control denuded oocytes cultured with granulosa cells (DO-GC) and vitrified oocytes. Regarding fertilization parameters, IVF showed higher percentages of total fertilization rate than those obtained by ICSI and PICSI. However, results demonstrate that PICSI fertilization increased the blastocysts formation rate in control DO-GC and vitrified oocytes compared to IVF and ICSI.ConclusionsTo achieve high blastocyst formation rates from vitrified GV oocytes, it is recommended that sperm should be selected with HA instead of PVP before injection since high viability and acrosome reaction rates were obtained. Also, PICSI fertilization was the best method to produce higher blastocyst rates compared to the IVF and ICSI procedures.

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 < 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.

Ultrastructural and molecular changes after oocyte vitrification protocol

Kuwayama (2007) describe the vitrification method for oocytes and embryo cryopreservation. The aim of the present study was to compare ultrastructural and molecular changes that are present in oocytes from egg donors after vitrification in compare with fresh oocytes. Prospective and comparative cohort study. One hundred remanent oocytes from egg donation cycles were included in this study (2012-2013). Oocytes were selected from donors that had at least one previous donation cycle. Half of the oocytes were vitrified (V) by Cryotop method and the other half was evaluated in fresh (F). All oocytes were vitrified/thawed by the same operator. Ten oocytes of each group (V and F) were randomly selected for transmission electron microscopy in order to assess mitochondrial ultrastructural analysis. The rest of the oocytes were evaluated by immunocytochemistry for: DNA fragmentation (TUNEL), microtubules and metaphase plate organization (Tubulin antibodies and Hoechst). The survival rate after vitrification was 96% (CI95% 86.3-99.5). The comparison of the assessed variables are shown in table 1. The percentage of the quantified variables seems to be higher in the vitrified oocytes group, however, the differences were not statistically significant. Mitochondrial alterations, evidenced by the inner plasma membrane dilation and electron-dense appearance, are associated with the activation of apoptotic mechanisms. In both groups, most of the oocytes that showed chromosomal alignment anomalies had DNA damage at the same time.Tabled 1Fresh vs. Vitrified/Thawed oocytesFresh oocytesVit/Thawed oocytesp valueDNA fragmentation8.6%15.2%0.64Mitochondrial alterations13.3%20%0.62Metaphase plate disarrangements5.7%12.1%0.61Parthenogenetic activation0%3%0.31 Open table in a new tab Regarding the ultrastructural and molecular changes assessed in this study, no significant differences were detected between fresh or vitrified/thawed oocytes. Thus, in our experience, the vitrification technique appears to be safe, confirming the previous reports by other authors.

Risque génotoxique et ovocytes : principes de toxicologie génétique et applications

The aim of genotoxicity tests in germ cells is to assess the impact of exposure to environmental mutagens that may represent a risk for the fertility or for the offspring of exposed subject. The comet assay on mature mouse oocytes is a simple, reproductive and rapid test to study primary DNA damage in oocytes. This test is used to complete toxicology assays applied in first line to somatic cells, and could find many applications in reproductive toxicology to study impact of environmental factors on female germ cells. We describe a practical application of comet assay in reproductive biology to assess the genotoxicity of cryoprotectants used at high concentrations in oocyte vitrification protocols. This test allowed us to demonstrate that dimethylsulfoxide and ethylene glycol are non-genotoxic for the mouse oocytes and led us to hypothesize a genotoxic effect of 1,2-propanediol (PrOH) at high concentrations after having observed induction of significant DNA damage on CHO cell line and on mouse oocytes.

Genotoxicity assessment of mouse oocytes by comet assay before vitrification and after warming with three vitrification protocols

To assess the genotoxicity of three oocyte vitrification protocols. Murine assay. Biogenotoxicology research laboratory. CD1 female mice. Three mouse oocyte groups were exposed to three commercialized human oocyte vitrification protocols. Protocols 1 and 2 contained dimethyl sulfoxide and ethylene glycol (EG), and protocol 3 contained EG and 1,2-propanediol (PrOH). DNA damage was first evaluated by comet assay after oocyte exposure to the three different equilibration and vitrification solutions. Comet assay was also performed after full vitrification and warming procedure and compared with a negative control group (oocytes stored in medium culture only) and a positive control group (oocytes exposed to hydrogen peroxide just before comet assay). DNA damage was quantified as Olive tail moment (OTM). Statistical analysis consisted of a Shapiro-Wilk test. Then, median protocol OTM was compared with the negative control group with the Mann-Whitney U test. The difference was considered to be statistically significant if the P value was <.05. In both parts of our study, protocols 1 and 2 did not induce significant DNA damage, whereas protocol 3 induced statistically higher DNA damage compared with the negative control group. Vitrification protocols containing PrOH induced significant DNA damage on mouse oocytes, both before cooling and after warming. Therefore, for the moment, we prefer vitrification techniques without PrOH while we await more studies on PrOH toxicity and long-term evaluation.

Vitrification as a method for genome resource banking oocytes from the endangered Tasmanian devil (Sarcophilus harrisii)

Populations of Australia's largest terrestrial marsupial carnivore, the Tasmanian devil (Sarcophilus harrisii), are rapidly declining in the wild due to Tasmanian Devil Facial Tumour Disease (TDFTD). One tool which can reduce the loss of genetic diversity is genome resource banking. This study examines the application of an oocyte vitrification protocol, initially developed in a model marsupial carnivore, to the endangered Tasmanian devil. Ovarian tissue was transported to the laboratory on ice from Tasmania which took up to 48 h. Individual granulosa oocyte complexes (GOC) were isolated enzymatically and the viability of oocytes from primary GOC was assessed immediately following isolation or after exposure to cold shock, vitrification and thawing media without exposure to liquid nitrogen or the full vitrification and thawing process. There was no decline in oocyte viability following cold shock or exposure to the vitrification and thawing media. Following the full vitrification and thawing process there was a decline in oocyte viability (chi(2)=20.0, P<0.001) but approximately 70% of oocytes remained viable. This study provides further evidence that oocyte vitrification is a promising strategy for genome resource banking in carnivorous marsupials and suggests that it should be considered in conservation plans for the survival of the iconic Tasmanian devil.

Application of different oocyte vitrification protocols, laser assisted zona slitting, and temperature on post-warming survival of bovine oocyte

Application of different oocyte vitrification protocols, laser assisted zona slitting, and temperature on post-warming survival of bovine oocyte

Comparison of two different cryoprotectant formulations for oocyte vitrification

Studies have suggested that using a combination of cryoprotectants (CPA) at lower concentrations can lower the toxicity of vitrification solutions and yield better overall results. In this investigation we compared oocyte vitrification protocols with either ethylene glycol (EG) alone or else in combination with dimethylsulfoxide (DMSO).

Cryopreservation of Cattle Oocytes: Effects of Meiotic Stage, Cycloheximide Treatment, and Vitrification Procedure

Different parameters likely to influence the survival of bovine oocytes after a vitrification procedure were evaluated: oocyte meiotic stage, cycloheximide treatment at the beginning or the end of maturation, and three vitrification procedures using conventional straws, open pulled straws (OPS), or microdrops. For each procedure a mixture of cryoprotectants (25% ethylene glycol and 25% glycerol) was used. After the oocytes were warmed and subjected toin vitromaturation and fertilization, the number that developed into blastocysts was determined. Results show that cryoprotectant exposure reduced embryo development and that cycloheximide treatment had no beneficial effect on oocytes vitrified in conventional straws. Among the three vitrification procedures, only the OPS method yielded blastocysts (approximately 3% of vitrified oocytes) irrespective of their initial meiotic stage. This result highlights the major influence of the cooling rate in an oocyte vitrification protocol.

Changes in membrane integrity, cytoskeletal structure, and developmental potential of murine oocytes after vitrification in ethylene glycol.

A systematic approach was taken to assess and optimize a protocol for intracellular vitrification by introducing high concentrations of the cryoprotectant agent (CPA) ethylene glycol (EG) into unfertilized murine oocytes. The effects of EG on membrane integrity, microfilament organization, and developmental potential were evaluated. During exposure to 0.5-2 M EG, oocytes showed maximum shrinkage to 55.5% of the isotonic volume within the first minute and reexpanded to their initial volume within 15 min. Transferral of oocytes to higher concentrations of EG (4-8 M EG) for 1-5 min after 15 min of equilibration at 2 M EG was tolerated well. Microfilament organization appeared normal after this equilibration period. During prolonged exposure (> 5 min) to high concentrations of EG (> 4 M), membrane blebs were noticed on the surface of the cells, and microfilament distribution was disturbed. After treatment with 6 M EG and vitrification with 6 M EG + f2p40.5 M sucrose, there were no significant differences in development to the two-cell and blastocyst stages between CPA-treated, vitrified, and control oocytes. These results indicate that EG is an effective CPA for mouse oocyte vitrification protocols without any observed compromise in morphology and developmental functions.