Cryopreservation Of Preimplantation Embryos Research Articles

Cryopreservation increases accumulation of exogenous stearic acid in mouse embryos

Cryopreservation of preimplantation embryos is a widely used technique, but this procedure might impact the subsequent embryo development. The effect of slow freezing and vitrification on the lipid metabolism in preimplantation mammalian embryos is not well studied. In this work, we applied Raman spectroscopy of isotopically labeled molecules to address the effects of cryopreservation on fatty acid accumulation in mouse embryos. Embryos after slow freezing or vitrification were cultured for 20 h in a medium supplemented with bovine serum albumin saturated with deuterated stearic acid (dSA). After this period the concentration of dSA estimated from Raman spectra of frozen-thawed and vitrified-warmed embryos at the morula stage was almost twice higher compared to non-cryopreserved morulas. At the same time, frozen-thawed and vitrified-warmed 4-cell embryos did not demonstrate any difference in the level of stearic acid uptake from non-cryopreserved embryos of the same stage. After an additional 24 h culture, cryopreserved and non-cryopreserved embryos demonstrated similar dSA uptake.

Age-related Yields of Mouse Sperm and Oocytes

Title: Genetically modified animals, in particular mice, are unique mutants with a major scientific potential. Cryopreservation of pre-implantation embryos or of spermatozoa is a common approach to protect those lines against loss or to archive them. Prerequisite to take a mutant line out of a breeding nucleus is the cryopreservation of a sufficient number of samples and a powerful quality assessment. Background: Several common protocols to cryopreserve spermatozoa and subsequently to forward these to an in vitro-fertilization under standardized conditions are published using donors of a strictly limited age-span. In practice, several limitations happen frequently for those approaches, e.g. the availability of genetically modified males, in particular if a line is in an experimental use or the mutation leads to restrictions. Female donors are often received from external sources and are subsequently not available before weaning. They are subject of several delays to induce an early superovulation. The aim of this report is to study the suitable age span of sperm and oocyte donors. Methods and Findings: A total of 441 GM sperm donors and the production of wild type oocytes with the genetic backgrounds BALB/c, C57BL/6N, and DBA/2 were investigated. The quality was assessed with simple, but purposive scores. Polynomial statistical analyses investigating increasing and decreasing yields show that the age of sperm-donors can be extended to ten months; the suitable age of oocyte donors is strain specific up to twelve weeks. Conclusions: The extended age spans demonstrated allow a more flexible use of donors and contribute subsequently to the 3Rs.

The role of diet and housing-temperature in the production of genetically modified mouse embryos and their developmental capacity after cryopreservation

Mutant mouse lines are unique models with an enormous scientific potential. Cryopreservation of preimplantation embryos or of spermatozoa is a common approach to save those lines. The breeding of a line can be discontinued if sufficient specimens have been cryopreserved. Prerequisites to economically cryopreserve embryos are high yields of embryos prepared from donors and a high recovery rate after revitalization. Diets for laboratory animals are often produced from phytoestrogen-containing soy; the present study shows that feeding the donor animals with a phytoestrogen-poor diet is more efficient compared to a phytoestrogen-containing, soy-based diet. Additionally, a uterotrophic bioassay indicating the estrogenic role of compounds showed a significant increase of the relative uterus size of females fed with a phytoestrogen-rich diet. The role of the housing-temperature was investigated, too, showing that a housing-temperature of 24 °C results in the best embryo yields. The production of two-cell embryos is more economic than the production of eight-cell embryos. Investigating the recovery rate of frozen/thawed embryos, a very high recovery rate was determined when both, two- and eight-cell embryos were thawed. However, the capacity to develop to the next embryonic stage in vitro was dramatically reduced when two-cell embryos were compared to eight-cell embryos. After embryo transfer, the sex ratio became uneven and more males were delivered. This effect might be due to the procedures to which animals and embryos were subjected. These data show that many parameters can influence the production of animals when using (frozen/thawed) embryos. These parameters need continuous surveillance.

Stability of cryopreserved samples of mutant mice.

Genetically modified animals are unique models with enormous scientific potential. Cryopreservation of pre-implantation embryos or of spermatozoa is a common approach to save those lines. The breeding of a line can be discontinued if a sufficient number of samples have been cryopreserved. To maintain the opportunity to recover a line, it is mandatory to assess the quality of the cryopreserved samples and to assure safe long-term storage conditions. Here, we investigated the revitalization rate of cryopreserved pre-implantation embryos stored in-house up to 158 months, of imported (and shipped) embryos, and of embryos received after in vitro fertilization. The storage period did not affect the revitalization rate, whereas the recovery of imported embryos was significantly reduced, possibly due to shipment conditions. The genotypes of genetically modified pups received following embryo-transfer were slightly smaller than expected by Mendelian laws. Intensive investigations of the hygienic state of the cryopreserved samples and the equipment used never showed microbiological contamination of a sample within a cryo-tube. However, environmental organisms were found frequently in the permanent freezers and dry shippers used. Since such contamination cannot be completely excluded and an embryo-transfer might not lead in all cases to a secure rederivation, foster mothers and revitalized pups should be housed in an intermediate facility and their health assessed before introducing them into the target facility.

Improved assessment of frozen/thawed mouse spermatozoa using fluorescence microscopy

Genetically modified (GM) animals are unique mutants with an enormous scientific potential. Cryopreservation of pre-implantation embryos or spermatozoa is a common approach for protecting these lines from being lost or to store them in a repository. A mutant line can be taken out of a breeding nucleus only if sufficient numbers of samples with an appropriate level of quality are cryopreserved. The quality of different donors within the same mouse line might be heterogeneous and the cryopreservation procedure might also be error-prone. However, only limited amounts of material are available for analysis. To improve the monitoring of frozen/thawed spermatozoa, commonly used in vitro fertilization (IVF) followed by embryo transfer were replaced with animal-free techniques. Major factors for assessing spermatozoa quality (i.e., density, viability, motility, and morphology) were evaluated by fluorescence microscopy. For this, a live/dead cell staining protocol requiring only small amounts of material was created. Membrane integrity was then examined as major parameter closely correlated with successful IVF. These complex analyses allow us to monitor frozen/thawed spermatozoa from GM mice using a relatively simple staining procedure. This approach leads to a reduction of animal experiments and contributes to the 3R principles (replacement, reduction and refinement of animal experiments).

Cryopreservation of human gametes: Establishment of gamete banks and cryopreservation of preimplantation embryos in assisted reproduction programs

Cryopreservation of human gametes: Establishment of gamete banks and cryopreservation of preimplantation embryos in assisted reproduction programs

Cryopreservation of oocyte and ovarian tissue.

Cryopreservation of reproductive cells (i.e., oocytes, spermatozoa) and tissues (i.e., ovarian and testicular tissue) is a developing technology that has tremendous implications for rapid advancement of biomedical research in general. Since the early 1980s, advances have been made in establishing optimal conditions for in vitro oocyte maturation, fertilization, and culture of resulting embryos. These in vitro systems have contributed significantly to the utilization of these cells and tissues after thawing and have made it possible to evaluate protocols designed to cryopreserve such biomaterials more effectively. Although cryopreservation of preimplantation embryos from various species including mouse, human, and farm animals has been successful, cryopreservation of oocytes from most mammalian species has been more challenging due to their extreme sensitivity to suboptimal conditions during the cryopreservation process. Cryopreservation on mouse oocytes have been well documented and have resulted in greater success than studies with other mammalian species. Ovarian tissue cryopreservation and transplantation techniques have recently received much scientific and public attention due to their great potential use in human infertility treatment, in safeguarding the reproductive potential of the endangered species, and in genome banking of genetically important lab animal strains. A review of past and current research in the field of oocyte and ovarian tissue cryopreservation and transplantation and discussion of possible strategies for oocyte and ovarian tissue banking are provided.

Human preimplantation embryo cryopreservation: selected aspects

This report describes the results of cryopreserving human preimplantation zygotes and cleaved embryos (2-4 cells) in our in-vitro fertilization programme. Cryopreserved zygotes and cleaved embryos resulted in similar post-thaw survival rates (74.8 versus 70.9%). Pregnancy rates per retrieval cycle (RC) and embryos transferred per pregnancy for frozen-thawed zygotes versus frozen-thawed cleaved embryos were 21.8 versus 11.5% (P less than 0.2) and 12.6 versus 17.5 (P less than 0.2), respectively. Pregnancy rates increased significantly for both fresh (P less than 0.0005) and frozen-thawed (P less than 0.05) embryos as the number of embryos replaced per transfer increased from one to three or more. Frozen-thawed embryos resulted in multiple implantation rates per transfer of 25 compared to 6.4% (P less than 0.1) for fresh embryos when two embryos were replaced. Pregnancy rates were reduced for fresh (P less than 0.05) and frozen-thawed (P less than 0.1) embryos obtained from patient retrieval cycle numbers greater than 3. The method of follicular stimulation during the retrieval cycle did not affect frozen-thawed embryo survival rates. There was no difference in pregnancy rates from frozen-thawed embryos replaced during natural or clomiphene citrate transfer cycles. Patients with cryopreserved embryos had cumulative pregnancy rates of 37.1% (66/178) compared to 23.5% (110/468) (P less than 0.01) for patients with no embryos cryopreserved; cryopreservation of preimplantation embryos is a reliable therapeutic procedure that enhances achievement of pregnancy through in-vitro fertilization.