Cryopreservation induces changes in the expression of noncoding RNAs in semen: a mini-review

Semen cryopreservation is an important approach for preserving male fertility, and thawed semen is often used in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) procedures. This process has been used for decades and seems both safe and efficient, despite causing cryodamage to spermatozoa, including the impairment of sperm functions, with decreased viability and motility being the main indicators of poor sperm quality post-thawing. Although an increase in sperm DNA fragmentation is common during cryopreservation, data on how cryopreservation affects sperm at the molecular level are scarce. There are especially limited data on the influence of sperm cryopreservation on spermatozoa non-coding RNA (ncRNA) stability and expression. To date, only three publications have explored this issue in human sperm samples, and there have been only a few more studies in animals, including mouse, bull, ram, boar and giant panda. The results of studies on human and animal semen indicate that cryopreservation affects ncRNA expression, which could crucially affect fertilization and embryonic development. Moreover, a study employing boar spermatozoa further showed that alterations in ncRNA expression also depend on the sperm cryopreservation protocol used, and a study employing human spermatozoa showed that microRNA (miRNA) expression changes during cryostorage. Therefore, the effects of cryopreservation on ncRNA expression in spermatozoa should be studied more thoroughly, mostly because sperm cryopreservation is important for medically assisted reproduction. Furthermore, despite the wide usage of sperm cryopreservation, how cryopreservation changes sperm at the molecular level and whether these changes have any effect on future generations have not been determined.

Sperm quality; Sperm cryopreservation; Sperm freezing; MicroRNAs; Non-coding RNAs

[1] Grin L, Girsh E, Harlev A. Male fertility preservation—methods, indications and challenges. Andrologia. 2021; 53: e13635.

[2] Borate GM, Meshram A. Cryopreservation of sperm: a review. Cureus. 2022; 14: e31402.

[3] Ozimic S, Ban-Frangez H, Stimpfel M. Sperm cryopreservation today: approaches, efficiency, and pitfalls. Current Issues in Molecular Biology. 2023; 45: 4716–4734.

[4] Gómez-Torres MJ, Medrano L, Romero A, Fernández-Colom PJ, Aizpurúa J. Effectiveness of human spermatozoa biomarkers as indicators of structural damage during cryopreservation. Cryobiology. 2017; 78: 90–94.

[5] Le MT, Nguyen TTT, Nguyen TT, Nguyen TV, Nguyen TAT, Nguyen QHV, et al. Does conventional freezing affect sperm DNA fragmentation? Clinical and Experimental Reproductive Medicine. 2019; 46: 67–75.

[6] Gouhier C, Pons-Rejraji H, Dollet S, Chaput L, Bourgne C, Berger M, et al. Freezing does not alter sperm telomere length despite increasing DNA oxidation and fragmentation. Genes. 2023; 14: 1039.

[7] Du C, Tuo Y. Correlation of DNA fragments with routine semen parameters and lifestyle and their impact on assisted reproductive outcomes. Revista Internacional De Andrología. 2023; 21: 100337.

[8] Ďuračka M, Benko F, Tvrdá E. Molecular markers: a new paradigm in the prediction of sperm freezability. International Journal of Molecular Sciences. 2023; 24: 3379.

[9] Tomic M, Bolha L, Pizem J, Ban-Frangez H, Vrtacnik-Bokal E, Stimpfel M. Association between sperm morphology and altered sperm microRNA expression. Biology. 2022; 11: 1671.

[10] Vallet-Buisan M, Mecca R, Jones C, Coward K, Yeste M. Contribution of semen to early embryo development: fertilization and beyond. Human Reproduction Update. 2023; 29: 395–433.

[11] Li H, Li L, Lin C, Hu M, Liu X, Wang L, et al. Decreased miR-149 expression in sperm is correlated with the quality of early embryonic development in conventional in vitro fertilization. Reproductive Toxicology. 2021; 101: 28–32.

[12] Yeh LY, Lee RK, Lin MH, Huang CH, Li SH. Correlation between sperm micro ribonucleic Acid-34b and -34c levels and clinical outcomes of intracytoplasmic sperm injection in men with male factor infertility. International Journal of Molecular Sciences. 2022; 23: 12381.

[13] Conflitti AC, Cicolani G, Buonacquisto A, Pallotti F, Faja F, Bianchini S, et al. Sperm DNA fragmentation and sperm-borne miRNAs: molecular biomarkers of embryo development? International Journal of Molecular Sciences. 2023; 24: 1007.

[14] Cui L, Fang L, Zhuang L, Shi B, Lin CP, Ye Y. Sperm-borne microRNA-34c regulates maternal mRNA degradation and preimplantation embryonic development in mice. Reproductive Biology and Endocrinology. 2023; 21: 40.

[15] Ezzati M, Shanehbandi D, Bahramzadeh B, Hamdi K, Pashaiasl M. Investigation of molecular cryopreservation, fertility potential and microRNA-mediated apoptosis in oligoasthenoteratozoospermia men. Cell and Tissue Banking. 2021; 22: 123–135.

[16] Xu X, Li W, Zhang L, Ji Y, Qin J, Wang L, et al. Effect of sperm cryopreservation on miRNA expression and early embryonic development. Frontiers in Cell and Developmental Biology. 2021; 9: 749486.

[17] Huang C, Ji XR, Huang ZH, Liu Q, Wang RJ, Fan LQ, et al. Long-term storage modifies the microRNA expression profile of cryopreserved human semen. To be published in Biomolecules & Biomedicine. 2023. [Preprint].

[18] Zhang Y, Zeng C, He L, Ding L, Tang K, Peng W. Selection of endogenous reference microRNA genes for quantitative reverse transcription polymerase chain reaction studies of boar spermatozoa cryopreservation. Theriogenology. 2015; 83: 634–641.

[19] Zhang Y, Dai D, Chang Y, Li Y, Zhang M, Zhou G, et al. Cryopreservation of boar sperm induces differential microRNAs expression. Cryobiology. 2017; 76: 24–33.

[20] Dai DH, Qazi IH, Ran MX, Liang K, Zhang Y, Zhang M, et al. Exploration of miRNA and mRNA profiles in fresh and frozen-thawed boar sperm by transcriptome and small RNA sequencing. International Journal of Molecular Sciences. 2019; 20: 802.

[21] Pedrosa AC, Andrade Torres M, Vilela Alkmin D, Pinzon JEP, Kitamura Martins SMM, Coelho da Silveira J, et al. Spermatozoa and seminal plasma small extracellular vesicles miRNAs as biomarkers of boar semen cryotolerance. Theriogenology. 2021; 174: 60–72.

[22] Ran MX, Zhou YM, Liang K, Wang WC, Zhang Y, Zhang M, et al. Comparative analysis of MicroRNA and mRNA profiles of sperm with different freeze tolerance capacities in Boar (Susscrofa) and Giant Panda (Ailuropodamelanoleuca). Biomolecules. 2019; 9: 432.

[23] Ran MX, Li Y, Zhang Y, Liang K, Ren YN, Zhang M, et al. Transcriptome sequencing reveals the differentially expressed lncRNAs and mRNAs involved in cryoinjuries in frozen-thawed Giant Panda (Ailuropoda melanoleuca) Sperm. International Journal of Molecular Sciences. 2018; 19: 3066.

[24] Salas-Huetos A, Ribas-Maynou J, Mateo-Otero Y, Tamargo C, Llavanera M, Yeste M. Expression of miR-138 in cryopreserved bovine sperm is related to their fertility potential. Journal of Animal Science and Biotechnology. 2023; 14: 129.

[25] Shangguan A, Zhou H, Sun W, Ding R, Li X, Liu J, et al. Cryopreservation induces alterations of miRNA and mRNA fragment profiles of bull sperm. Frontiers in Genetics. 2020; 11: 419.

[26] Keles E, Malama E, Bozukova S, Siuda M, Wyck S, Witschi U, et al. The micro-RNA content of unsorted cryopreserved bovine sperm and its relation to the fertility of sperm after sex-sorting. BMC Genomics. 2021; 22: 30.

[27] Güngör BH, Tektemur A, Arkali G, Dayan Cinkara S, Acisu TC, Koca RH, et al. Effect of freeze-thawing process on lipid peroxidation, miRNAs, ion channels, apoptosis and global DNA methylation in ram spermatozoa. Reproduction, Fertility, and Development. 2021; 33: 747–759.

[28] Fagerlind M, Stålhammar H, Olsson B, Klinga-Levan K. Expression of miRNAs in bull spermatozoa correlates with fertility rates. Reproduction in Domestic Animals. 2015; 50: 587–594.

[29] Capra E, Turri F, Lazzari B, Cremonesi P, Gliozzi TM, Fojadelli I, et al. Small RNA sequencing of cryopreserved semen from single bull revealed altered miRNAs and piRNAs expression between high- and low-motile sperm populations. BMC Genomics. 2017; 18: 14.