Combined Analysis of the Transcriptome, Proteome and Metabolome in Human Cryopreserved Sperm

Abstract

Purpose This study aimed to identify the altered pathways and genes associated with freezing damage in human sperm during cryopreservation by multiomics analysis. Materials and Methods Fifteen fresh human semen samples were collected for transcriptomic analysis, and another 5 fresh human semen samples were obtained for metabolomic analysis. For each semen sample, 1 mL was cryopreserved, and another 1 mL was left untreated for paired design. The results were then combined with previously published proteomic results to identify key genes/pathways. Results Cryopreservation significantly reduced sperm motility and mitochondrial structure. Transcriptomic analysis revealed altered mitochondrial function, including changes in tRNA-methyltransferase activity and adenosine tri-phosphate/adenosine di-phosphate transmembrane transporter activity. Metabolomic analysis showed that the citrate cycle in mitochondria was significantly altered. Combining transcriptomic, proteomic, and metabolomic analyses revealed 346 genes that were altered in at least two omics analyses. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that metabolic pathways were significantly altered and strongly associated with mitochondria. Five genes were altered in all three omics analyses: COL11A1 , COL18A1 , LPCAT3 , NME1 , and NNT . Conclusions Five genes were identified by multiomics analysis in human cryopreserved sperm. These genes might have specific functions in cryopreservation. Explorations of the functions of these genes will be helpful for sperm cryopreservation and sperm motility improvement or even for reproduction in the future.

A Pilot Analysis of Whole Transcriptome of Human Cryopreserved Sperm

“…The number of decreased transcripts in cryopreserved spermatozoa was higher than those increased compared to fresh samples. These results agree with previous studies that identified a lower abundance of transcripts in frozen and vitrified sperm than fresh samples [18,19].…”

“…Mitochondrial dysfunctions in sperm of different species caused by cryopreservation have been reported [6]. Also, the recent multiomics analysis performed by Fu et al [19] found that the mitochondria in cryopreserved sperm had an abnormal ultrastructure, which indicated mitochondrial dysfunction. The presence of structural and functional alterations in mitochondria from asthenozoospermic subjects confirms the important role played by these organelles in the energetic maintenance of sperm motility [30,31].…”

“…As a novelty of this study compared to previous omics analyses of cryopreserved human spermatozoa [18,19], we identified a reduction in transcripts involved in early embryo development, known to be affected by DNA damage after human sperm cryopreservation [20]. In particular, we found low levels of several transcripts involved in cell differentiation (CCIN, CPNE9, GLRX2, HEMGN, KRTDAP, MEA1, ODF2, SYAP1, SPATA6, SPATA19, SPMIP6, TCF4, TXNDC2) and early embryo development (CNN1, EPB41L2, GPI, IFT172, KRTDAP, MICAL2, NF2, NRDC, PHACTR1, POPDC3, ROBO1, SPRR2D, TIAM2, TNNI3).…”

“…In a comparison of transcript profiles in fresh, frozen, and vitrified human spermatozoa, Wang et al found that the conventional freezing process induces more transcript alterations than vitrification and most of these pathways are relevant for apoptosis and immune response [18]. Very recently, a multiomics analysis confirmed that conventional cryopreservation significantly reduced sperm motility and mitochondrial structure [19].…”

“…All over the world, there is a huge amount of semen samples cryopreserved after slow cooling, and laboratories keep freezing like this. To date, studies published on the transcriptomic profile of fresh and conventional frozen human sperm have not recruited a large number of donors (4 by Wang et al [18] and 15 by Fu et al [19]). Considering these factors, we believe there is still room to analyze other normozoospermic donors using high-throughput technology.…”