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Evaluation of microRNA expression profiles in human sperm frozen using permeable cryoprotectant-free droplet vitrification and conventional methods.

Author(s): Zhang LX, Mao J, Zhou YD, Mao GY, Guo RF, Ge HS, Chen X

Publication: Asian J Androl, 2024, Vol. 26, Page 366-376

PubMed ID: 38738948 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared motility parameters, plasma membrane integrity, mitochondrial membrane potential, DNA fragmentation index, ultrastructure and miRNA expression among case-matched sperm specimens that were fresh, conventionally frozen, or frozen by droplet vitrification. Semen was collected from 43 male volunteers (21-41 years of age; diagnosis was not specified) following 2-7 days of sexual abstinence. Semen was centrifuged at 300 g for 15 min, and sperm were washed in Cooper Surgical washing medium, centrifuged at 200 g for 10 min and resuspended in Cooper Surgical washing medium. For droplet vitrification and freeze-thawing, sperm was mixed with 0.5 mol/L sucrose solution containing serum albumin and “trace amounts” of cryopreservation solution and then 30 uL was dropped into liquid nitrogen, where drops were stored for 1 week before two to three drops were thawed (one at a time) in 37°C washing media. Conventional freezing entailed mixing sperm dropwise with ORIGIO A/S freezing media until a 1:1 ratio was achieved, storage at room temperature for 10 min, freezing in liquid nitrogen vapor, storage in liquid nitrogen for 1 week, thawing for 3 min in a 25°C water bath with agitation, centrifugation at 200 g for 10 min, resuspension in sperm washing media and incubation at 37°C for 15 min. Sperm from 10 volunteers were assessed for motility parameters (total motility, TM; progressive motility, PM; average path velocity, VAP; curvilinear velocity , VCL; linearity; straight line velocity , VSL; and beat cross frequency, BCF) using a sperm analyzing system in four microscopic areas with >200 spermatazoa each. Plasma membrane integrity was analyzed in the sperm of 10 volunteers (same as motility) by propidium iodide flow cytometry of 10,000 spermatazoa.  DNA fragmentation index and mitochondrial membrane potential were determined in sperm from 10 volunteers (same as motility and membrane integrity) using the BRED LIfeScience Sperm Chromatin Dispersion Kit and the Beyotime Biotechnology flow cytometry Mitochondrial Membrane Potential Assay Kit with JC-1, respectively. Sperm acrosome integrity was evaluated in the sperm of 8 volunteers using the BRED Life Science Induced Acrosome Reaction Detection Kit. Sperm ultrastructure was analyzed in specimens from six volunteers by transmission (TEM) and scanning electron microscopy (SEM). To allow for miRNASeq analysis, sperm from nineteen volunteers were removed from nonsperm cells by density gradient centrifugation before freezing. Upon thawing, sperm for miRNA analysis was resuspended in fertilization media, RNA was extracted using the HiPure Universal RNA Mini Kit and quantified by Qubit fluorometer. Sequencing libraries were constructed using the VAHTS Total RNA-seq Library Kit and sequenced on a NovaSeq instrument. Reads were aligned to miRbase and differential expression was evaluated using DESeq software. Differential expression of select miRNAs (miR-132-3p, miR-223-3p, miR-375-3p, miR-4516, miR-363-3p, miR-516-5p, let-7b-3p, miR-34b-5p, and U6) was verified by real-time PCR.

   Summary of Findings:

   Compared to fresh sperm, post-thaw conventionally frozen (CF) and droplet vitrification frozen (DF) sperm exhibited lower total motility (P<0.0001, both), progressive motility (P<0.0001, both), curvilinear velocity (P<0.0001, both), straight line velocity (P<0.001 and P<0.05, respectively), and average path velocity (P<0.0001 and P<0.001, respectively); decreased plasma membrane integrity (P<0.0001, both), mitochondrial membrane potential (P<0.0001, both) and acrosome-intact sperm (P<0.0001, both); and DF sperm had higher beat cross frequency (P<0.05).  Relative to CF sperm, DF sperm had higher curvilinear velocity (P<0.001), straight line velocity (P<0.05), and average path velocity (P<0.01), but comparable total motility, progressive motility, beat cross frequency, plasma membrane integrity, mitochondrial membrane potential and acrosome-intact sperm. The sperm linearity and DNA fragmentation index did not differ significantly among fresh, CF and DF sperm.  The authors show SEM images of CF and DF sperm with structural damage to the head, neck and tail but the frequency of this damage was not reported. The authors show TEM images of damaged sperm, but state that there were no specific damages attributed to either freezing method.

   Compared to fresh sperm, CF sperm had higher levels of 11 miRNAs and lower levels of 8 miRNAs, and DF sperm had higher levels of 25 miRNAs and lower levels of 3 miRNAs. Eight miRNAs were differentially expressed between fresh and DF and fresh and CF sperm.  DF sperm had higher levels of 7 miRNAs and lower levels of 2 miRNAs than CF sperm; two of these miRNAs were also differentially expressed between DF and fresh sperm, and one was also differentially expressed between CF and fresh sperm. Importantly, differential expression by sequencing was confirmed by real-time PCR for all miRNAs investigated (four miRNAs in CF versus fresh sperm, six miRNAs in DF versus fresh sperm and three miRNAs in DF versus CF sperm). Pathway analysis revealed that DF sperm was enriched for miRNAs involved in “DNA-binding transcription activity and elements” relative to fresh and CF sperm. Importantly, five of the eight miRNAs differentially expressed in both CF and DF sperm relative to fresh sperm have previously been implicated in early embryonic development, but many of the changes would not be anticipated to negatively impact embryonic development. The authors conclude that differences between CF and DF sperm were minimal, suggesting DF is a suitable alternative to CF.

   Biospecimens

   • Bodily Fluid - Semen

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Light microscopy
   DNA	Next generation sequencing
   Morphology	Light microscopy
   Morphology	Electron microscopy
   Morphology	Flow cytometry
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Cooling or freezing method/ rate	Liquid nitrogen vapor Droplet vitrification
   Biospecimen Preservation	Type of fixation/preservation	Snap frozen None (fresh)

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