M.G. Larman

A randomized controlled study of human Day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation†

BACKGROUND The aim of this study was to compare two methods of cryopreservation for the cleavage-stage human embryo: slow freezing and vitrification. METHODS A total of 466 Day 3 embryos, donated with consent, underwent cryopreservation by either slow freezing in straws or vitrification using the cryoloop. The vitrification procedure did not include dimethyl sulfoxide, but rather employed ethylene glycol and 1,2-propanediol as the cryoprotectants. Survival, embryonic metabolism and subsequent development to the blastocyst were used to determine the efficacy of the two procedures. RESULTS Significantly, more embryos survived the vitrification procedure (222/234, 94.8%) than slow freezing (206/232, 88.7%; P < 0.05). Consistent with this observation, pyruvate uptake was significantly greater in the vitrification group, reflecting a higher metabolic rate. Development to the blastocyst was also higher following vitrification (134/222, 60.3%) than following freezing (106/206, 49.5%; P < 0.05). In a separate cohort of 73 patients who had their supernumerary embryos cyropreserved with vitrification, the resulting implantation rate and clinical pregnancy rate were 30 and 49%, respectively. CONCLUSIONS Analysis of metabolism revealed that vitrification had less impact on the metabolic rate of the embryo than freezing, which was reflected in higher survival rate and subsequent development in vitro. Excellent pregnancy outcomes followed the warming and transfer of vitrified cleavage-stage embryos. These data provide further evidence that vitrification imparts less trauma to cells and is, therefore, a more effective means of cryopreserving the human embryo than conventional slow freezing. Clinicaltrials.gov identifier: NCT00608010.

Maintenance of the meiotic spindle during vitrification in human and mouse oocytes

Vitrification appears to be a viable method for the cryopreservation of human metaphase II (MII) oocytes, but concerns regarding the concentration of cryoprotectants used during vitrification have been raised. In an attempt to circumvent this potential problem, the majority of protocols are carried out at room temperature. Exposing oocytes to temperatures below 37°C, however, leads to rapid microtubule depolymerization. Polarized light microscopy was used to measure meiotic spindle retardance following exposure to cryoprotectants and vitrification in human and mouse oocytes. To quantify the extent of depolymerization, spindle retardance was determined before and after each treatment. Exposure to vitrification and warming solutions at room temperature (21–22°C) caused the spindle of mouse MII oocytes to depolymerize. In contrast, no measurable changes in the meiotic spindle were detected by maintaining the temperature at 37°C during the exposure regimen. By carrying out the entire vitrification and warming procedure at 37°C, the spindle was also unaffected. Comparable results were obtained with vitrification of human MII oocytes at 37°C. Analysis of sibling human oocytes demonstrated that slow freezing, in contrast to vitrification, was unable to preserve the meiotic spindle. Using a vitrification protocol employing 37°C impacts negligibly on the meiotic spindle. Thus, fertilization can proceed without having to await spindle reformation.

Sex-related physiology of the preimplantation embryo

Male and female preimplantation mammalian embryos differ not only in their chromosomal complement, but in their proteome and subsequent metabolome. This phenomenon is due to a finite period during preimplantation development when both X chromosomes are active, between embryonic genome activation and X chromosome inactivation, around the blastocyst stage. Consequently, prior to implantation male and female embryos exhibit differences in their cellular phenotype. Manifestations of such differences include altered total activity of specific X-linked enzymes and the metabolic pathways they regulate. Subsequently, one would expect to be able to determine differences in the rate of consumption and utilization of specific nutrients between male and female embryos. Data to date on animal models support this, with sex-specific differences in glucose and amino acid utilization being reported for the mouse and cow blastocysts. Such differences in metabolic phenotype may logically be involved in the reported differences in growth rates between preimplantation embryos of different sex. As the fields of proteomics and metabolomics are being increasingly applied to human assisted conception it is prudent to consider how such technologies may be applied to identify sex differences in the human embryo. Such data would have implications far beyond current invasive technologies used to identify the sex of an embryo conceived in vitro for the diagnosis of X-linked diseases.

Vitrification of mouse pronuclear oocytes with no direct liquid nitrogen contact

The ability to routinely cryopreserve human oocytes and embryos represents a significant advancement in the field of assisted reproductive technology. Although the method of slow freezing is commonly employed, research on the alternative technique of vitrification is promising. Vitrification involves incubation of the cell in a cryoprotectant rich solution, which permits a glass-like state to occur almost instantaneously in liquid nitrogen. A number of different techniques have been invented for holding oocytes and embryos in the cryoprotectant solution during rapid vitrification and subsequent storage. Most of these involve direct contact with liquid nitrogen. Recently, concerns have been raised regarding the sterility of such a method and the potential of viral contamination from the liquid nitrogen. The present study shows that the previously reported Cryoloop method can be used to vitrify and store embryos without direct liquid nitrogen contact (during vitrification and storage). When such vitrified embryos are warmed, they are capable of subsequent development comparable with non-vitrified embryos.

Analysis of global gene expression following mouse blastocyst cryopreservation

BACKGROUND The aim of this study was to examine the effect of the cryopreservation procedure (slow freezing or vitrification) and cryoprotectants (1,2-propanediol or dimethylsulphoxide) on mouse blastocyst gene expression. METHODS Cultured mouse blastocysts were cryopreserved with different protocols. Following thawing/warming, total RNA from re-expanded blastocysts was isolated, amplified and then analyzed using mouse whole-genome microarrays. RESULTS Compared with non-cryopresevered control blastocysts, gene expression was only significantly altered by slow freezing. Slow freezing affected the expression of 115 genes (P < 0.05). Of these, 100 genes exhibited down-regulation and 15 genes were up-regulated. Gene ontology revealed that the majority of these genes are involved in protein metabolism, transcription, cell organization, signal transduction, intracellular transport, macromolecule biosynthesis and development. Neither of the vitrification treatment groups showed statistically different gene expression from the non-cryopreserved control embryos. Hierarchical cluster analysis, did however, reveal that vitrification using 1,2-propanediol could result in a gene expression profile closest to that of non-cryopreserved blastocysts. CONCLUSIONS Investigating the effects of cryopreservation on cellular biology, such as gene expression, is fundamental to improving techniques and protocols. This study demonstrates that of the cryopreservation regimens employed, slow freezing induced the most changes in gene expression compared with controls.

Vitrification of mouse embryos with super-cooled air

OBJECTIVE To develop a closed vitrification device (i.e., one that requires no direct contact with liquid nitrogen) for successful cryostorage of embryos. DESIGN Prospective laboratory research study. SETTING University-based research laboratory. ANIMAL(S) F1 mice and mouse embryos. INTERVENTION(S) Mouse embryos were vitrified using two methods and compared with nonvitrified controls. Embryos were vitrified on a device by either [1] presealing it within a straw before plunging into liquid nitrogen or [2] placing the straw into liquid nitrogen so that the air inside the straw is super-cooled before inserting the device holding the embryos. MAIN OUTCOME MEASURE(S) Survival, subsequent embryo development, and cell number were determined. Embryos were also cryopreserved for 12 months to assess long-term storage. Synchronized ETs were performed to compare viability with nonvitrified control embryos. RESULT(S) All embryos survived with both techniques. Day-4 and -5 embryo development was comparable between the two vitrification methods. Use of the presealing method resulted in a significantly lower mean cell number than the postsealing method and control. Long-term storage did not affect subsequent embryo development or cell number. The implantation and fetal development rates of embryos vitrified with super-cooled air were comparable to those for nonvitrified control embryos. CONCLUSION(S) These data demonstrate that a closed vitrification device (Rapid-i), which does not require direct liquid nitrogen contact for vitrification, is appropriate for vitrification and long-term storage of mouse embryos.

Ultrarapid Vitrification of Mouse Oocytes and Embryos

Cryopreservation facilitates long-term storage of gametes and embryos for numerous purposes. For example, cryobanking of unique mouse strains, particularly transgenic mice, offers important protection of valuable genetics. It also provides a practical solution for facilities trying to house large numbers of research animals or those looking to relocate without the risk of introducing an animal-derived pathogen. Furthermore, cryopreservation is currently being used for fertility preservation both in humans and as a safeguard for endangered animals. Ultrarapid vitrification offers an elegant, quick, and very reliable method for cryopreservation of mouse oocytes and embryos. Furthermore, research into the effects on mouse oocyte and embryo physiology has indicated that ultrarapid vitrification is superior to conventional slow freezing. High survival rates, embryo development, and viability are routinely achieved with the ultrarapid vitrification method described in this chapter.

MAINTENANCE OF THE MEIOTIC SPINDLE DURING VITRIFICATION IN HUMAN AND MOUSE OOCYTES

Vitrification appears to be a viable method for the cryopreservation of human metaphase II (MII) oocytes, but concerns regarding the concentration of cryoprotectants used during vitrification have been raised. In an attempt to circumvent this potential problem, the majority of protocols are carried out at room temperature. Exposing oocytes to temperatures below 37 degrees C, however, leads to rapid microtubule depolymerization. Polarized light microscopy was used to measure meiotic spindle retardance following exposure to cryoprotectants and vitrification in human and mouse oocytes. To quantify the extent of depolymerization, spindle retardance was determined before and after each treatment. Exposure to vitrification and warming solutions at room temperature (21-22 degrees C) caused the spindle of mouse MII oocytes to depolymerize. In contrast, no measurable changes in the meiotic spindle were detected by maintaining the temperature at 37 degrees C during the exposure regimen. By carrying out the entire vitrification and warming procedure at 37 degrees C, the spindle was also unaffected. Comparable results were obtained with vitrification of human MII oocytes at 37 degrees C. Analysis of sibling human oocytes demonstrated that slow freezing, in contrast to vitrification, was unable to preserve the meiotic spindle. Using a vitrification protocol employing 37 degrees C impacts negligibly on the meiotic spindle. Thus, fertilization can proceed without having to await spindle reformation.