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Cryopreservation of human spermatozoa using penetrating cryoprotectant-free vitrification medium exhibits altered proteomic profile compared to conventional rapid freezing method

ABSTRACT: Vitrification is not considered as ideal method for cryopreservation of human spermatozoa due to the presence of high concentrations of penetrating cryoprotectants in the vitrification medium. Several studies in the literature suggest that the outcome of semen cryopreservation is superior when cryopreserved using conventional rapid freezing protocol compared to vitrification. The present study was aimed at assessing the functional and proteomic changes in the human spermatozoa cryopreserved in vitrification medium without penetrating cryoprotectant. Leftover ejaculates (n = 71) from men visiting the andrology laboratory for routine semen analysis were used. The liquefied semen samples were cryopreserved using rapid freezing and vitrification for a minimum of seven days. Post-thaw motility, mitochondrial function, DNA integrity, and acrosome intactness of spermatozoa were similar between the cryoprotectant-free vitrification and rapid freezing methods. The head morphology of spermatozoa cryopreserved by the vitrification method was better preserved compared to rapid freezing method. Proteomic analysis led to the identification of a total of 4378 proteins in spermatozoa. However, differential proteomic profile observed in spermatozoa cryopreserved with vitrification and conventional rapid freezing methods compared to spermatozoa from the ejaculate indicates the possible differences in the functional competence of the spermatozoa cryopreserved using these two protocols. Even though vitrification of spermatozoa in medium without penetrating cryoprotectant holds promise as an attractive option owing to the simplicity and rapidity of the protocol, further studies are necessary to understand the safety of the protocol and pregnancy outcome upon using the vitrified-thawed spermatozoa for assisted conception and assisted reproductive technology.

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ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Keshava Prasad T. S.

LAB HEAD: Dr. T. S. Keshava Prasad

PROVIDER: PXD057601 | Pride | 2025-07-22

REPOSITORIES: Pride

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Project description:Background: The cryopreservation of semen is crucial for conserving genetic diversity and reproductive success in endangered species like the Siberian sturgeon. However, this process can cause cryodamage, affecting quality and protein profile of spermatozoa. While cryoprotectants like dimethyl sulfoxide (DMSO) and methanol (MeOH) facilitate post-thaw motility recovery, DMSO-preserved spermatozoa exhibit reduced fertilizing ability. This study investigates how DMSO and MeOH impact the proteome of Siberian sturgeon spermatozoa and examines semen quality parameters. Two complementary approaches of quantitative proteomics, liquid chromatography-mass spectrometry (LC-MS) and two-dimensional difference in gel electrophoresis (2D-DIGE), were used to analyze the proteomic profiles of fresh and cryopreserved spermatozoa, as well as the extracellular medium (EM; n=7 for each group). Results: Cryopreservation led to a decline in motility parameters (MOT, VCL, PROG) and viability, along with an increase in ROS levels, membrane fluidity, and acrosome damage. Despite similar quality parameters between DMSO and MeOH-preserved sperm, DMSO-preserved sperm showed dramatically lower fertilization success (6.2% vs 51.2%). A total of 224 and 118 differentially abundant proteins in spermatozoa cryopreserved with MeOH and DMSO, respectively, were identified compared to fresh samples, with 342 and 363 proteins released into the EM. The most affected proteins by cryopreservation included H2A, CABYR, PYGM, ENO3, DBI and LTA4H. Additionally, 36 and 39 uniquely altered sperm-leakage proteins were identified for MeOH and DMSO cryopreserved samples, respectively. Bioinformatic analysis showed that MeOH-specific proteins were related to chromosomal structure and mitochondrial functionality, while DMSO-specific proteins were mainly involved in acrosome reaction, zona pellucida binding, flagella structure, and nuclear pore organization. These proteins are potentially involved in sturgeon sperm fertilizing ability. The expression of six proteins was verified by western blot analysis. Conclusions: This study provides the first comprehensive proteomic characterization of Siberian sturgeon spermatozoa after cryopreservation with DMSO and MeOH, revealing insights into proteomic changes that affect fertilizing ability and aiding conservation efforts for this endangered species.

			Action	DRS
	Cryopreseravation_DIA_result.tsv	Tabular
	Fresh_DIA_R1.raw	Raw
	Fresh_DIA_R2.raw	Raw
	Fresh_DIA_R3.raw	Raw
	Rapid_freezing_DIA_R1.raw	Raw

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Cryopreservation of human spermatozoa using penetrating cryoprotectant-free vitrification medium exhibits altered proteomic profile compared to conventional rapid freezing method

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