Experimental Evidence Reveals Both Cross-Infection and Cross-Contamination Risk of Embryo Storage in Liquid Nitrogen Biobanks

Abstract

In recent decades, gamete and embryo cryopreservation have become routine procedures in livestock and human assisted reproduction. However, the safe storage of germplasm and the prevention of disease transmission continue to be potential hazards of disease transmission through embryo transfer. This study aimed to demonstrate the potential risk of cross-infection of embryos from contaminated liquid nitrogen, and cross-contamination of sterile liquid nitrogen from infected embryos in naked and closed devices. Additionally, we examined the effects of antibiotic-free media on culture development of infected embryos. The study was a laboratory-based analysis using rabbit as a model. Two experiments were performed to evaluate both cross-infection (liquid nitrogen to embryos) and cross-contamination (embryos to liquid nitrogen) of artificially inoculated Salmonella Typhimurium, Staphylococcus aureus, Enterobacter aerogenes, and Aspergillus brasiliensis. Rapid cooling through vitrification was conducted on rabbit embryos, stored for a year, thawed, and cultured. In vivo produced late morulae–early blastocyst stages (72 h) embryos were used (n = 480). Embryos were cultured for 1 h in solutions with and without pathogens. Then, the embryos were vitrified and stored in naked and closed devices for one year in two liquid nitrogen biobanks (one pathogen-free and the other artificially contaminated). Embryos were warmed and cultured for a further 48 h, assessing the development and the presence of microorganism (chromogenic media, scanning electron microscopy). Embryos stored in naked devices in artificially contaminated liquid nitrogen became infected (12.5%), while none of the embryos stored in closed devices were infected. Meanwhile, storage of artificially infected embryos incurred liquid nitrogen biobank contamination (100%). Observations by scanning electron microscopy revealed that all the microorganisms were caught in the surface of embryos after the vitrification-thawed procedure. Nevertheless, embryos cultured in antibiotics and antimycotic medium developed to the hatched blastocyst stage, while artificially infected embryos cultured in antibiotic-free medium failed to develop. In conclusion, our findings support that both cross-contamination and cross-infection during embryo storage in liquid nitrogen biobanks are plausible. So, to ensure biosafety for the cryogenic storage, closed systems that avoid direct contact with liquid nitrogen must be used. Moreover, it seems essential to provide best practice guidelines for the cryogenic preservation and storage of gametes and embryos, to define appropriate quality and risk management procedures.

Candida contamination of embryos in the ART laboratory: a case report and literature review

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The Impact of SARS-CoV-2 on Sperm Cryostorage, Theoretical or Real Risk?

“…Although no case exists within the human IVF community of cross-contamination during the course of cryostorage, research has demonstrated that cross-contamination does pose a real risk [ 45 , 46 ]. If we accept that the SARS-CoV-2 virus can be detected in the semen and the virus is resistant to liquid nitrogen, as is the case with many other viruses, maybe additional specific measures should be followed to minimize the presence of the virus during sperm cryopreservation and any possible cross-contamination effect.…”

Application of Nanoparticles and Melatonin for Cryopreservation of Gametes and Embryos

“…Despite the similar success rates in open IVF using frozen oocytes and fresh oocytes, asepsis-related problems have been observed due to cross-contamination between vitrificated oocyte samples and microorganisms in liquid nitrogen [ 87 , 88 , 89 ]. To solve this issue and minimize surrounding contaminants, liquid nitrogen was sterilized using UV light, or additional pretreatment was applied [ 89 , 90 ]. However, due to the difficulty of implementing a perfectly aseptic system, in-straw vitrification was developed.…”