Aseptic capillary vitrification of human spermatozoa: Cryoprotectant-free vs. cryoprotectant-included technologies

Wang, Mengying; Todorov, Plamen; Isachenko, Evgenia; Rahimi, Gohar; Wang, Wanxue; Brandenstein, Melanie von; Kumar, Pradeep; Mallmann, Peter; Isachenko, Vladimir

doi:10.1016/j.cryobiol.2021.01.006

Cited by 7 publications

(6 citation statements)

References 46 publications

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“…As described in our previously published protocol, cryoprotectant-free vitrification was also performed in capillaries [ 4 ]. Briefly, ten μL spermatozoa suspension of each treatment group was aspirated in capillaries (Gynemed GmbH & Co. KG, Lensahn, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…It presents an explanation in physiological parameters for why cryoprotectant-free vitrification for some human ejaculates is better than conventional slow freezing and vitrification with cryoprotectants. Spermatozoa vitrification is superior to conventional slow freezing, in total motility and progressive motility, Mitochondrial Membrane Potential (MMP), DNA fragmentation, acrosome, morphology, and cytoskeleton [ 4 , 5 , 6 ].…”

Section: Introductionmentioning

confidence: 99%

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Cryoprotectants-Free Vitrification and Conventional Freezing of Human Spermatozoa: A Comparative Transcript Profiling

Wang

Todorov

Wang

et al. 2022

IJMS

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Introduction: Spermatozoa cryopreservation is an important technique to preserve fertility for males. This study aimed at exploring the stability of epigenetics information in human spermatozoa, manipulated by two different technologies, freezing and vitrification. Methods: Spermatozoa samples were distributed into three groups: 1. Fresh spermatozoa (control group), 2. Frozen spermatozoa, 3. Vitrified spermatozoa. Epigenetic differences of fresh and cryopreserved spermatozoa were evaluated using high-throughput RNA sequencing. Results: Differentially expressed genes (DEGs) in frozen (1103 genes) and vitrified (333 genes) spermatozoa were evaluated. The bioinformatical analysis identified 8 and 15 significant pathways in groups of frozen and vitrified spermatozoa, respectively. The majority of these pathways are most relevant to immune and infectious diseases. The DEGs of the fertilization process are not detected during vitrification. The freezing process induces more down-regulation of genes and is relevant to apoptosis changes and immune response. Conclusion: Cryopreservation of human spermatozoa is an epigenetically safe method for male fertility preservation. Cryoprotectant-free vitrification can induce more minor biological changes in human spermatozoa, in comparison with conventional freezing.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Cryoprotectants-Free Vitrification and Conventional Freezing of Human Spermatozoa: A Comparative Transcript Profiling

Wang

Todorov

Wang

et al. 2022

IJMS

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“…The detailed protocol of cryopreservation is published [9,23,35,36]. The package for cryoprotectant-free vitrified spermatozoa (group 2) was used in form of 600 µm capillaries from hydrophobic material (Vitromed GmbH, Jena, Germany).…”

Section: Experimental Design and Cryopreservationmentioning

confidence: 99%

“…Then, a micropipettor (Eppendorf, Hamburg, Germany) was used to aspirate 10 µL of spermatozoa suspension and dropped into a 35×10 mm Falcon Petri dish (Life Sciences, Durham, NC, USA). The capillaries one by one were filled with 10 µL of spermatozoa suspension by aspiration using an IVF pipette controller (Medical Technology GmbH, Bruckberg, Germany) [23]. Next, the capillary was put into a one-end-sealed straw (Medical Technology GmbH, Bruckberg, Germany) prepared by a heat-sealer.…”

Section: Experimental Design and Cryopreservationmentioning

confidence: 99%

“…Vitrification of spermatozoa is a technology that involves no ice formation in the cooling down and warming process, with less injury to the samples [19][20][21]. CPAs used for ART in most labs are permeable, including dimethyl sulfoxide (DMSO), ethylene glycol (EG), and glycerol [22,23]. In recent years, non-permeable cryoprotectants such as sucrose or other agents have played a role in osmotic balance [24,25].…”

Section: Introductionmentioning

confidence: 99%

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Epigenetic Alterations in Cryopreserved Human Spermatozoa: Suspected Potential Functional Defects

Wang

Todorov

Cheng

et al. 2022

Cells

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Background: Gene set enrichment analysis (GSEA) was conducted on raw data, and alternative splicing (AS) events were found after mRNA sequencing of human spermatozoa. In this study, we aimed to compare unknown micro-epigenetics alternations in fresh and cryopreserved spermatozoa to evaluate the effectivity of cryopreservation protocols. Methods: Spermatozoa were divided into three groups: fresh spermatozoa (group 1), cryoprotectant-free vitrified spermatozoa (group 2), and conventionally frozen spermatozoa (group 3). Nine RNA samples (three replicates in each group) were detected and were used for library preparation with an Illumina compatible kit and sequencing by the Illumina platform. Results: Three Gene Ontology (GO) terms were found to be enriched in vitrified spermatozoa compared with fresh spermatozoa: mitochondrial tRNA aminoacylation, ATP-dependent microtubule motor activity, and male meiotic nuclear division. In alternative splicing analysis, a number of unknown AS events were found, including functional gene exon skipping (SE), alternative 5′ splice sites (A5SS), alternative 3′ splice sites (A3SS), mutually exclusive exon (MXE), and retained intron (RI). Conclusions: Cryopreservation of spermatozoa from some patients can agitate epigenetic instability, including increased alternative splicing events and changes in crucial mitochondrial functional activities. For fertilization of oocytes, for such patients, it is recommended to use fresh spermatozoa whenever possible; cryopreservation of sperm is recommended to be used only in uncontested situations.

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