Vitrification of mouse pronuclear oocytes with no direct liquid nitrogen contact

doi:10.1016/S1472-6483(10)60982-0

Reproductive BioMedicine Online

Volume 12, Issue 1, 2006, Pages 66-69

https://doi.org/10.1016/S1472-6483(10)60982-0 Get rights and content

Abstract

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.

Section snippets

Dr Mark Larman obtained his PhD, entitled ‘Calcium and MAP kinase regulation during the cell cycle’, in 2000 from the University of Newcastle upon Tyne, UK. He continued his interest in fertilization and the cell cycle by becoming part of the team that discovered the sperm protein that triggers oocyte activation (phospholipase C zeta). To apply and further his knowledge of mammalian fertilization and embryo development, he joined the research team at the Colorado Centre for Reproductive

References (34)

A Bielanski et al.
Microbial contamination of embryos and semen during long term banking in liquid nitrogen
Cryobiology
(2003)
A Bielanski et al.
Viral contamination of embryos cryopreserved in liquid nitrogen
Cryobiology
(2000)
C Chen
Pregnancy after human oocyte cryopreservation
Lancet
(1986)
T Falcone et al.
Ovarian function preservation in the cancer patient
Fertility and Sterility
(2004)
DK Gardner et al.
Changing the start temperature and cooling rate in a slow-freezing protocol increases human blastocyst viability
Fertility and Sterility
(2003)
AE Hawkins et al.
Hepatitis B nucleotide sequence analysis: linking an outbreak of acute hepatitis B to contamination of a cryopreservation tank
Journal of Virology Methods
(1996)
M Kasai et al.
Cryopreservation of animal and human embryos by vitirification
Reproductive BioMedicine Online
(2004)
KP Katayama et al.
High survival rate of vitrified human oocytes results in clinical pregnancy
Fertility and Sterility
(2003)
LL Kuleshova et al.
Vitrification can be more favorable than slow cooling
Fertility and Sterility
(2002)
M Lane et al.
Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique
Fertility and Sterility
(1999)

GJ Morris

The origin, ultrastructure, and microbiology of the sediment accumulating in liquid nitrogen storage vessels

Cryobiology

(2005)

TL Nedambale et al.

Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification

Theriogenology

(2004)

S Paynter

A rational approach to oocyte cryopreservation

Reproductive BioMedicine Online

(2005)

S Pickering et al.

Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte

Fertility and Sterility

(1990)

LL Veeck

Does the developmental stage at freeze impact on clinical results post-thaw?

Reproductive BioMedicine Online

(2003)

TK Yoon et al.

Live births after vitrification of oocytes in a stimulated in vitro fertilization–embryo transfer program: freezing immature oocytes

Fertility and Sterility

(2003)

GN Charles et al.

Transmission of papovavirus by cryotherapy applicator

Journal of the American Medicine Association

(1971)

Cited by (32)

Vitrification of oocytes, embryos and blastocysts
2012, Best Practice and Research: Clinical Obstetrics and Gynaecology
Citation Excerpt :
With recent improvements in past decades, vitrification has become the most reliable strategy because it is technically simple, and can lead to high survival and implantation rates. To induce vitrification in LN2, or super cooled air,8 the solution must contain a high concentration of CPA. This approach simplifies the cooling process, because embryos can be rapidly cooled directly in LN2.
In assisted reproductive technology, cryopreservation of human oocytes and embryos has been significantly improved by refined slow-cooling and the new vitrification method. The slow-cooling method requires a programmed cryo-machine, and usually takes several hours. It is, however, difficult to eliminate injuries resulting from ice formation completely. Vitrification has become a reliable strategy because it is simple, can lead to high survival rates and viability, and has better clinical outcome. Vitrification transforms cells into an amorphous glassy state inside and outside the vitrified cell with ultra-rapid cooling and warming steps by plunging the oocytes and embryos into liquid nitrogen, instead of ice-crystal formation. Over the past decade, several advances in vitrification technologies have improved clinical efficiency and outcome. In this chapter, we focus on vitrification technologies for cryopreservation in human assisted reproductive technology.
Current results with slow freezing and vitrification of the human oocyte
2011, Reproductive BioMedicine Online
Citation Excerpt :
While under experimental conditions, cross-contamination of cryopreserved reproductive cells has been reported (Bielanski et al., 2000), a recent review of this topic indicates that the actual contamination issue with open systems is not a practical concern when working under standard conditions used for assisted reproduction treatment and OCP (Pomeroy et al., 2010). Strategies to reduce any potential risk of contamination with open vitrification systems could include use of filtered or irradiated liquid nitrogen during the vitrification and/or storage process (Nagy et al., 2009a,b; Parmegiani et al., 2010) or vitrification and storage using nitrogen vapour versus liquid nitrogen (Cobo et al., 2010b; Eum et al., 2009; Larman et al., 2006). However, such approaches must balance the unproven risk of contamination under normal working conditions versus the risk of factors such as temperature fluctuations that could cause damage (Pomeroy et al., 2010).
The past decade has witnessed renewed interest in human oocyte cryopreservation (OCP). This article reviews the two general methods used for OCP, slow freezing and vitrification, compares the outcomes associated with each technique and discusses the factors that might influence success with OCP (such as oocyte selection or day of transfer). Based on available data, OCP offers a reliable, reproducible method for preservation of the female gamete and will find increasing application in assisted reproductive technology.
Oocyte cryopreservation can provide a number of advantages to couples undergoing assisted reproduction or to women interested in fertility preservation. Two methods, slow freezing and vitrification, have been used successfully for oocyte cryopreservation. This article reviews and compares these methods, and discusses various factors that can impact upon success of oocyte cryopreservation.
Vitrification of mouse embryos with super-cooled air
2011, Fertility and Sterility
Citation Excerpt :
It is worth noting that to date there have been no reports of human infection attributed to the use of contaminated cryostored germplasm; however, experimental conditions have shown that the potential still exists (19, 20). A proof-of-principle study by Larman et al. (21) demonstrated that the cryoloop can be used in a noncontact method by super-cooling the air inside the cryovial. Unfortunately, cryovials are not leak-proof during long-term storage in liquid nitrogen.
To develop a closed vitrification device (i.e., one that requires no direct contact with liquid nitrogen) for successful cryostorage of embryos.
Prospective laboratory research study.
University-based research laboratory.
F1 mice and mouse embryos.
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.
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.
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.
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.
Vitrification of human oocytes and different development stages of embryos: An overview
2010, Middle East Fertility Society Journal
Citation Excerpt :
A small nylon loop was attached to a holder and equipped with a container (Fig. 2). Using this tool, safe cryopreservation can be achieved even in the vapour of liquid nitrogen (38). Kuwayama et al. compared between the open system (cryotop) and the closed system (cryotip) over 13,000 embryos at different stages and suggested that cryotop is an efficient and reliable way to vitrify cleavage embryos, blastocysts and oocytes in daily practice (36).
Cryopreservation of oocytes and embryos leads to increased cumulative pregnancy rates along with restored costs of artificial reproductive technology (ART), minimizing the risks of ovarian hyper stimulation syndrome (OHSS) through reducing number of stimulated cycles, reducing the risks of multiple pregnancy, allowing patients undergoing chemotherapy and radiotherapy to preserve their fertility and giving hope to those with genetic diseases as turner syndrome and premature ovarian failure or both.
Vitrification of oocytes and embryos has reduced the idea of embryo selection because of the high viability rate and it is an area of active research justifying optimism for improved results in cryopreservation.
IVM and gene expression of sheep cumulus - oocyte complexes following different methods of vitrification
2010, Reproductive BioMedicine Online
Citation Excerpt :
Mammalian oocyte vitrification was first performed in 1958 (Sherman and Lin, 1958). Since then, many researchers have vitrified the oocytes of different species such as mouse (Larman et al., 2006), cow (Morato et al., 2008), sheep (Antinori et al., 2007; Bogliolo et al., 2007; Cobo et al., 2008) and human (Antinori et al., 2007) using different devices. In most vitrification techniques, the size of the oocyte-containing droplet is relatively large and its cooling rate may be lower than optimal because of the flotation on liquid nitrogen before submerging.
To determine the optimal vitrification conditions for sheep cumulus–oocyte complexes (COC), good-quality isolated COC were randomly divided into non-vitrified control, conventional straw, cryotop and solid-surface vitrification groups. In the conventional and cryotop methods, the vitrified COC were respectively loaded by conventional straw and cryotop, whereas in the solid-surface group, the vitrified COC were loaded by cryotop and then cooled before plunging in liquid nitrogen. The results indicated that the mean percentage of viability of vitrified–warmed COC was higher in both cryotop groups than that of the conventional group (83.84 ± 2.85 and 78.56 ± 1.72 versus 63.43 ± 1.48%, P < 0.05). In the cryotop group, although the mean percentage of oocyte maturation was similar to that in the control group (48.81 ± 3.09 versus 51.94 ± 3.01%), it was significantly higher than the other vitrification groups (48.81 ± 3.09 versus 36.60 ± 1.69 and 6.09 ± 2.51%, respectively, P < 0.05). However, the expression of maturation genes (GDF9, BMP15) was retarded after vitrification. Among the vitrification groups, the cryotop group had better expression. BMPRII was also expressed highly in the control, whereas ALK5 was similar in all groups. In conclusion, direct cryotop, when compared with other vitrification methods, seemed to be safe and could increase the viability, post-warming maturation and maturation-gene expression rates of sheep COC.
Aseptic vitrification of blastocysts from infertile patients, egg donors and after IVM
2009, Reproductive BioMedicine Online
Citation Excerpt :
In view of the issues raised by this directive, it was imperative to revise this study centre’s ultra-rapid vitrification procedure (Vanderzwalmen et al., 2002, 2003) and to develop a new vitrification technique ensuring total protection and isolation of the sample from the liquid nitrogen during the cooling procedure as well as during long-term storage. Hermetically closed containers (closed systems) have already been developed to vitrify mice and human oocytes and embryos (Kuleshova and Shaw, 2000; Walker et al., 2004; Isachenko et al., 2005, 2007; Kuwayama et al., 2005; Larman et al., 2006; Stachecki et al., 2008). Kuwayama et al. (2005) developed the Cryotip device, allowing isolation of biological material from liquid nitrogen.
During embryo vitrification, it is advisable that cooling and storage should occur in a carrier device in which there is complete separation of the embryos from liquid nitrogen to ensure asepsis. The consequence of a reduction in the cooling rate resulting from the heat-insulating barrier aseptic devices has to be counteracted by gradually increasing intracellular concentrations of cryoprotectants without inducing a toxic effect. Blastocysts originating from couples with male and/or female factor infertility (group 1) or from oocyte donors (group 2) or from in-vitro matured oocytes (group 3) were gradually exposed to increasing concentrations of dimethylsulphoxide/ethylene glycol (5/5%, 10/10% and 20/20%) before aseptic vitrification using a specially designed carrier (VitriSafe), a modification of the open hemi-straw plug device. A total of 120 aseptic vitrification/warming cycles were performed in group 1, 91 in group 2 and 22 in group 3. Survival rates before embryo transfer, ongoing pregnancy and implantation rates were as follows: for group 1, 73, 43 and 26%; for group 2, 88, 53 and 34%; and for group 3, 69, 50 and 38%, respectively. In spite of reduced cooling rates due to aseptic vitrification conditions, a three-step exposure to cryoprotectant solutions protects the embryos effectively from cryo-injuries and guaranties high survival rates.

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ArticleVitrification of mouse pronuclear oocytes with no direct liquid nitrogen contact

Abstract

Section snippets

Cryobiology

Cryobiology

Lancet

Fertility and Sterility

Fertility and Sterility

Journal of Virology Methods

Reproductive BioMedicine Online

Fertility and Sterility

Fertility and Sterility

Fertility and Sterility

Cryobiology

Theriogenology

Reproductive BioMedicine Online

Fertility and Sterility

Reproductive BioMedicine Online

Fertility and Sterility

Transmission of papovavirus by cryotherapy applicator

Journal of the American Medicine Association

Article
Vitrification of mouse pronuclear oocytes with no direct liquid nitrogen contact