Vapor-phase Nitrogen System Superior to Traditional Liquid Nitrogen for Storing Vitrified Human Oocytes

Any therapeutic intervention in reproductive biology must ensure complete protection of biological materials against a wide range of microorganisms. Liquid nitrogen (LN) used for cryostorage collects particulate contaminants from the atmosphere or container surface over a period of time, which could be a potential source of pathogenic microbes, including herpes, papova, hepatitis, and vesicular stomatitis virus. A new study suggests that storage of vitrified oocytes using the vapor-phase nitrogen systems serve as a safer alternative to the traditional LN storage tank, as it avoids the risk of cross-contamination, apart from retaining the developmental potential of these oocytes. The findings of the prospective randomized trial have been published in the latest issue of Fertility and Sterility.

Ana Cobo and colleagues, from the IVI-Universidad de Valencia, Spain, conducted the study on 44 oocyte donors and 46 recipients at a private infertility center to assess the efficacy of long-duration storage of vitrified oocytes in vapor-phase nitrogen. The traditional LN freezer and vapor-phase nitrogen system were randomly used for the storage of oocytes vitrified using the Cryotop method. The surviving oocytes were later donated and evaluated for fertilization potential, embryo development, and clinical outcomes. The study results are listed in the table below.

Outcomes LN Storage Tank (%) Vapor-phase Nitrogen System (%)
Survival rates 94.5 95.3
Fertilization rates 71.7 73.1
Cleavage rates on day 2 94.7 95.6
Cleavage rates on day 3 79.9 84.5
Blastocyst formation 53.9 54.7
Implantation rates 33.7 40.5
Clinical pregnancy rates 53.3 58.1
Ongoing pregnancy rates 46.6 48.8

Based on the study findings, it was found that storing vitrified oocytes in vapor-phase nitrogen systems reduces the risk of vitrified sample contamination that occur commonly from direct LN contact, while maintaining the developmental potential of the oocytes into competent embryos.

In recent years, several studies have provided information on various mechanisms of controlling cross-contamination. A study by Isachenko et al (Human Reproduction, 2005) suggested that the storage of human pronuclear oocytes, using open-pulled straws located inside hermetically closed containers, ensures complete sealing in order to minimize the risk of cross-contamination. A more recent study by Parmegiani et al (Fertility and Sterility, 2009) reported that the rapid sterilization of LN was achievable with the use of ultraviolet irradiation, thereby making it possible for oocyte vitrification in open carriers.

A recent report by the Practice Committee of the American Society for Reproductive Medicine and the Society for Assisted Reproductive Technology (Fertility and Sterility, 2008) recommended the use of separate sample containers for specimens infected with microorganisms such as HIV, in order to avoid significant cross-contamination. In addition, the following measures were reported to reduce cross-contamination of the cryopreserved samples stored in LN:
• Storing specimens in the vapor phase of nitrogen in place of LN
• Stocking samples in containers manufactured to resist freezing temperatures and thawing cycles
• Using double bagging or sealing methods in order to eliminate direct contact of the storage tanks with LN
• Employing techniques such as “sperm washing” before semen cryopreservation in order to reduce the viral load.


1. Cobo A, Romero JL, Pérez S, de Los Santos MJ, Meseguer M, Remohí J. Storage of human oocytes in the vapor phase of nitrogen. Fertil Steril. 2010 Feb 4. [Epub ahead of print]

2. Isachenko V, Montag M, Isachenko E, et al. Aseptic technology of vitrification of human pronuclear oocytes using open-pulled straws. Hum Reprod. 2005 Feb;20(2):492-6.

3. Parmegiani L, Accorsi A, Cognigni GE, Bernardi S, Troilo E, Filicori M. Sterilization of liquid nitrogen with ultraviolet irradiation for safe vitrification of human oocytes or embryos. Fertil Steril. 2009 Jul 8. [Epub ahead of print]

4. Practice Committee of the American Society for Reproductive Medicine. ASRM Practice Committee response to Rybak and Lieman: elective self-donation of oocytes. Fertil Steril. 2009 Nov;92(5):1513-4.

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