Bacterial and fungal contamination risks in human oocyte and embryo cryopreservation: open versus closed vitrification systems

Cross-contamination by bacteria may not occur in cryopreserved oocytes/embryos that are stored in either open or closed storage devices. Microorganisms survive in liquid nitrogen, so storage containers should be cleaned periodically.


Inmaculada Molina, Ph.D., Miquel Mari, B.Sc., Juan Vicente Martínez, B.Sc., Edurne Novella-Maestre, Ph.D., Nuria Pellicer, M.D., Javier Pemán, M.D.



To study the contamination risk in open and closed vitrification devices for oocyte/embryo cryopreservation by evaluating the contaminants present (bacteria and fungi) in the thaw medium and in liquid nitrogen (LN) storage containers.


Retrospective study.


Human reproduction unit.




Retrospective study of vitrification device safety and LN sterility performed from July to October 2014.

Main Outcome Measure(s)

From each bank container, both open and closed vitrification devices, devitrification media and LN in the containers and as supplied by the company were evaluated for contaminants. An automated system and the corresponding susceptibility to antibiotics were used for bacteria identification. Fungus detection was performed by evaluating the colony morphology and their microscopic characteristics.


No bacteria or fungi were observed in any of the devitrification media regardless of the type of device used, nor in the LN supplied by the company. No fungi were observed in any of the LN samples tested.Stenotrophomonas maltophilia and Bacillus spp. were found in all oocyte/embryo bank LN containers. There was no relationship between the number of samples or the time that each container had been used and the presence of microbiologic contaminants in the LN. At the container's bottom, Acinetobacter lwoffii,Alcaligenes faecalis ssp. faecalis, and Sphingomonas paucimobilis were found.


Bacteria cross-contamination may not occur in oocyte/embryo banking in either open or closed storage devices. However, microorganisms can survive in LN. The bacteria cross-contamination risk is no greater for open than for closed containers. Storage containers should be cleaned periodically owing to the risk of lost straws or small particles of contaminated material.

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Go to the profile of Lodovico Parmegiani
over 6 years ago
To the editor. I read with interest the article by Molina et al. in vol. 106 N° 1 / July 2016 of this journal in which the authors suggested that cryostorage containers “should be emptied and cleaned periodically because of the risk of lost straws or small particles of contaminated material” and that “periodic chemical sterilization of liquid nitrogen (LN2) containers is recommended for oocyte and embryo banking”(1). Regarding procedures for decontamination I would like to suggest that the easiest way to sterilize the inner walls and the liquid nitrogen inside tanks is by ultraviolet (UV) radiation. As is common practice in today’s IVF labs (2- 5), by using specifically designed devices available on the open market which ensure sterilization and certification of LN2 and N vapours (6). These devices also allow the sanification of dewars containing the LN2. The simplicity of this technology permits the decontamination of the storage containers with temporary removal of specimens but not requiring that dewars are emptied of LN2. Thus, it is possible to perform a periodic decontamination of cryostorage containers via UV radiation simply and without the use of chemicals. Thank you for the opportunity to clarify this point. Lodovico Parmegiani M.Sc.; Ph.D. GynePro Medical Centers Bologna, ITALY E-mail: DISCLOSURE STATEMENT L. Parmegiani holds an international patent (PCT/IB2009/007801: Device and method for sterilizing liquid nitrogen by ultraviolet radiation). REFERENCES 1. Molina I, Mari M, Martínez JV, Novella-Maestre E, Pellicer N, Pemán J. Bacterial and fungal contamination risks in human oocyte and embryo cryopreservation: open versus closed vitrification systems. Fertil Steril 2016;106:127-132 2. Parmegiani L, Cognigni GE, Bernardi S, Cuomo S, Ciampaglia W, Infante FE, Tabarelli de Fatis C, Arnone A, Maccarini AM, Filicori M, Efficiency of aseptic open vitrification and hermetical cryostorage of human oocytes. Reprod Biomed Online 2011; 23: 505–512. 3. Parmegiani L, Accorsi A, Bernardi S, Arnone A, Cognigni GE, Filicori M, A reliable procedure for decontamination before thawing of human specimens cryostored in liquid nitrogen: three washes with sterile liquid nitrogen (SLN2). Fertil Steril 2012; 98: 870–875. 4. 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 2010; 94: 1525-1528. 5. Parmegiani L, Cognigni GE, Filicori M. Ultra-violet sterilization of liquid nitrogen prior to vitrification. Hum Reprod 2009; 24:11. 6. Parmegiani L., Troilo E., Arnone A., Maccarini A.M., Rastellini A., Lanzilotti S., Bernardi S. Aseptic procedures for vitrification, warming and cryostorage. Current Trends in Clinical Embryology 2014; 1: 17- 22 . doi: 10.11138/cce/2014.1.1.017