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      Aseptic Technology for Cryoprotectant-Free Vitrification of Human Spermatozoa by Direct Dropping into Clean Liquid Air: Apoptosis, Necrosis, Motility, and Viability

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          Abstract

          This study aimed to compare the quality of human spermatozoa vitrified by direct plunging into liquid nitrogen vs. liquid air. Spermatozoa were divided into three groups: fresh spermatozoa (Group F) were used as a control. Spermatozoa suspension (20  μl) was vitrified in open granules by direct dropping into liquid nitrogen (Group LN) or clean liquid air (Group LA). After warming at 37°C, the progressive motility rate of Group F was reduced from 65.9 ± 2.5% to 34.0 ± 1.9% (Group LN) and 38.1 ± 2.3% (Group LA), respectively (P 1-2,3 < 0.05). The reductions in viability were 65.6 ± 2.2%, 29.0 ± 1.8%, and 36.6 ± 2.6% for Groups F, LN, and LA, respectively (P 1-2,3 < 0.05). Comparing spermatozoa vitrified in liquid nitrogen vs. liquid air, no significant differences were detected in motility (34.0 ± 1.9% vs. 38.1 ± 2.3%), viability (29.0 ± 1.8% vs. 36.6 ± 2.6%), early apoptosis (13.8 ± 1.5% vs. 14.3 ± 1.8%), late apoptosis (45.5 ± 1.8% vs. 43.7 ± 2.2%), and necrosis (19.5 ± 2.0% vs. 15.0 ± 1.8%; p > 0.01 for all respective differences). There was a statistical tendency for increasing rates of “progressive motility” and “viability” and decreasing rates of “apoptosis” and “necrosis” when comparing spermatozoa vitrified in liquid air vs. liquid nitrogen. It is concluded that cryoprotectant-free vitrification by the direct dropping of human spermatozoa in a clean cooling agent (liquid air) is a good alternative to the use of nonsterile liquid nitrogen and can be used to cool cells while minimising the risk of microbial contamination.

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          Most cited references41

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          Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART

          Cryopreservation of human spermatozoa—introduced in the 1960's—has been recognized as an efficient procedure for management of male fertility before therapy for malignant diseases, vasectomy or surgical infertility treatments, to store donor and partner spermatozoa before assisted reproduction treatments and to ensure the recovery of a small number of spermatozoa in severe male factor infertility. Despite the usefulness of it, cryopreservation may lead to deleterious changes of sperm structure and function: while the effects of cryopreservation on cells are well documented, to date there is no agreement in the literature on whether or not cryopreservation affects sperm chromatin integrity or on the use of a unique and functional protocol for the freezing-thawing procedure. Therefore, sperm cryopreservation is an important component of fertility management and much of its successful application seems to affect the reproductive outcome of assisted reproduction technologies (ART): appropriate use of cryoprotectants before and sperm selection technologies after cryopreservation seem to have the greatest impact on preventing DNA fragmentation, thus improving sperm cryosurvival rates.
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            Microbial contamination of embryos and semen during long term banking in liquid nitrogen.

            We report on microbial contamination of embryos and semen cryopreserved in sealed plastic straws and stored for 6-35 years in liquid nitrogen. There were 32 bacterial and 1 fungal species identified from randomly drawn liquid nitrogen, frozen semen, and embryos samples stored in 8 commercial and 8 research facility liquid nitrogen (LN) tanks. The identified bacteria represented commensal or environmental microorganisms and some, such as Escherichia coli, were potential or opportunistic pathogens for humans and animals. Stenotrophomonas maltophilia was the most common contaminant identified from the samples and was further shown to significantly suppress fertilization and embryonic development in vitro. Analysis of the strains by pulsed field gel electrophoresis revealed restriction patterns with no relatedness indicating that there was no apparent cross-contamination of S. maltophilia strains between the germplasm and liquid nitrogen samples. In addition, no transmission of bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BHV-1) from infected semen and embryos straws to clean germplasm stored in the same LN tanks or LN was detected.
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              Freeze-drying of live virus vaccines: A review

              Freeze-drying is the preferred method for stabilizing live, attenuated virus vaccines. After decades of research on several aspects of the process like the stabilization and destabilization mechanisms of the live, attenuated viruses during freeze-drying, the optimal formulation components and process settings are still matter of research. The molecular complexity of live, attenuated viruses, the multiple destabilization pathways and the lack of analytical techniques allowing the measurement of physicochemical changes in the antigen's structure during and after freeze-drying mean that they form a particular lyophilization challenge. The purpose of this review is to overview the available information on the development of the freeze-drying process of live, attenuated virus vaccines, herewith focusing on the freezing and drying stresses the viruses can undergo during processing as well as on the mechanisms and strategies (formulation and process) that are used to stabilize them during freeze-drying.
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                Author and article information

                Contributors
                Journal
                Biomed Res Int
                Biomed Res Int
                BMRI
                BioMed Research International
                Hindawi
                2314-6133
                2314-6141
                2020
                24 January 2020
                : 2020
                : 2934315
                Affiliations
                1Research Group for Reproductive Medicine and IVF-Laboratory, Department of Obstetrics and Genecology, Cologne University, Kerpener Str. 34, 50931 Cologne, Germany
                2Institute of Biology and Immunology of Reproduction, Tzarigradsko Shosse 73, 1113 Sofia, Bulgaria
                3Stem Cell Center, University of California at San Diego, La Jolla, CA 92037, USA
                Author notes

                Academic Editor: José L. Campos

                Author information
                https://orcid.org/0000-0003-2542-3408
                https://orcid.org/0000-0001-9251-0494
                https://orcid.org/0000-0002-3674-543X
                Article
                10.1155/2020/2934315
                7003260
                213f4ac2-d1f5-46f1-9884-b0f4aa3528b8
                Copyright © 2020 Mengying Wang et al.

                This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 5 September 2019
                : 4 December 2019
                : 16 December 2019
                Funding
                Funded by: Alexander von Humboldt-Stiftung
                Categories
                Research Article

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