Risk of Contamination of Gametes and Embryos during Cryopreservation and Measures to Prevent Cross-Contamination

Abstract

The introduction and widespread application of vitrification are one of the most important achievements in human assisted reproduction techniques (ART) of the past decade despite controversy and unclarified issues, mostly related to concerns about disease transmission. Guidance documents published by US Food and Drug Administration, which focused on the safety of tissue/organ donations during Zika virus spread in 2016, as well as some reports of virus, bacteria, and fungi survival to cryogenic temperatures, highlighted the need for a review of the way how potentially infectious material is handled and stored in ART-related procedures. It was experimentally demonstrated that cross-contamination between liquid nitrogen (LN₂) and embryos may occur when infectious agents are present in LN₂ and oocytes/embryos are not protected by a hermetically sealed device. Thus, this review summarizes pertinent data and opinions regarding the potential hazard of infectious transmission through cryopreserved and banked reproductive cells and tissues in LN₂. Special attention is given to the survival of pathogens in LN₂, the risk of cross-contamination, vitrification methods, sterility of LN₂, and the risks associated with the use of straws, cryovials, and storage dewars.

Figure 1

Illustration showing the process of cross-contamination in germinal tissue storage. (a) Container with “pure” LN₂, without microorganisms. (b) Contaminated samples inserted into the container resulting in LN₂ contamination. (c) Samples without microorganisms inserted in the contaminated container. (d) Contamination of samples that were not contaminated.

Figure 2

Illustration showing the open vitrification and warming system and the risk it offers for the germplasm samples. (a) Cryopreservation straw with the vitrified embryo. (b) Immersion of straw in contaminated LN₂. (c) Straw stored in LN₂ container. (d) Warming of the straw with the contaminated sample. (e) Contaminated germplasm.

Figure 3

Illustration showing the closed vitrification and warming system and its low risk for germplasm samples. (a) Cryopreservation straw with the vitrified embryo. (b) The straw is covered and sealed prior to contact with LN₂. (c) Embryo vitrification in the closed system. (d) Straw stored in LN₂ container. (e) Straw cover is removed prior to warming, avoiding contact of the germplasm sample with the microorganisms. (f) Contaminant-free germplasm.

Figure 4

(a) Culture dish showing contamination with S. minor in programmable LN₂ freezer. (b) Contamination from the vapor phase of a dry shipper, which was stocked with LN₂ contaminated with S. minor.

Figure 5

Comparison between new and used cryogenic storage dewars. (a) New dewar, without sediment. (b) After some time of usage, accumulation of sediments occurs agglomerating microorganisms in the bottom of the container.

Figure 6

Sequence of images showing how the Esther system works. (a) Suspension of the Esther to remove its cap. (b) Insertion of non-Esther samples without it remaining in the LN₂ container exposed to the environment. (c) Close of Esther. (d) Esther immersion (with as samples) in the LN₂ cylinder. (e) Image showing Esther's full format.

Figure 7

Sequence of images showing the different methods of vitrification in the “closed” system. (a) Straw-in-straw. (b) Cryotop—Kitazato. (c) Cryotip—Irvine Scientific. (d) Cryopette—ORIGIO. (e) CVM™ CryoLogic Vitrification Method. (f) Cryoloop.