Methodological aspects of extracellular DNA isolation from antrum ovarian follicles of Sus scrofa domesticus

Assessment of the quality of animal donor oocytes and their competence for fertilization is an important task in reproductive biotechnology. Extracellular DNA is one of the promising noninvasive biomarkers that can be used for the above purposes. This indicator is actively being investigated in human-oriented medicine, but its significance in assessing the female gametes of productive animals remains poorly understood. The present study analyzes the content of cDNA in pig follicular fluid aspirated from antral ovarian follicles (diameter 3mm, 3-5mm, < 5mm), analyzes the effect of various centrifugation modes on its isolation, and evaluates the viability of granulosa cells (proportion of cells with pycnotic nuclei). The level of cells in a state of pyknosis was determined by the Tarkovsky method, which included hypotonic treatment with sodium citrate, fixation with an acetic-methanol mixture, and microscopic analysis after Romanovsky–Giemse staining. It was found that the concentration of cDNA in the follicle fluid varies depending on their diameter and mode of centrifugation. With an increase in the acceleration of centrifugation, a decrease in the amount of cDNA and an increase in the proportion of cells with pycnotic nuclei were observed, which indicates their damage. The centrifugation mode (603.72 g for 5 minutes) was identified, providing the maximum level of cDNA content with a minimum number of pycnotic cells. In addition, the data obtained is consistent with the hypothesis that cDNA can serve as an indicator of cellular stress, which is especially important when assessing the quality of biomaterials in vitro. The presented results emphasize the importance of choosing the parameters of centrifugation for the effective isolation of сDNA to further study it as a potential biomarker of oocyte quality and a promising predictive indicator of fertility in productive animals.

1. Safronova N.A., Kalinina E.A., Donnikov A.E., Burmenskaya O.V., Makarova N.P., Kulakova E.V., Alieva K.U. Prospects for studying cumulus cell markers to assess the quality of oocytes and embryos in assisted reproductive technology programs // Obstetrics and Gynecology. – 2015. – № 12. (In Russ.)

2. Stanislavovich T. I., Kuzmina T. I., Molchanov A. V. Assessment of the Destructive Processes of Chromatin in Granular Cells of Ovarian Follicles in Cows and the Functional Status of the Oocyte // Agrarian Bulletin of the Urals. 2019. No. 12 (191). Pp. 60‒64. DOI: 10.32417/1997-4868-2019- 191-12-60-64. (In Russ.)

3. Gaponenko A.A., Mityurina E.V., Frankevich V.E. The metabolic profile of follicular fluid as a marker of oocyte quality // Obstetrics and Gynecology. – 2021. – No. 11. – pp. 26-31. DOI:10.18565/aig.2021.11.26-31. (In Russ.)

4. Scalici E., Mulet T., Ferrier Hoa A., Gala A., Lu V., Anahori T. et al. Circulating nuclear acids and infertility. Gynecol Obstetric Fertil. 2015; DOI:10.18565/aig.2021.11.26-31. (in French)

5. Andreeva E.A., Honina N.A., Demchenko E.N., Gavrilova E.D., Pasman N.M., Kozlov V.A., Chernykh E.R. Free DNA in follicular fluid in women with different indicators of ovarian function. Bulletin of Siberian medicine. 2019; 18 (2): 16–23. DOI: 10.20538/1682-0363-2019-2-16-23 (In Russ.)

6. De Borre M, Che H, Yu Q, et al. Cellfree DNA methylome analysis for early preeclampsia prediction. Nat Med. 2023;29 (9):2206-15. DOI:10.1038/s41591-023- 02510-5 (in English)

7. Adil, M., Kolarova, T.R., Doebley, AL. et al. Preeclampsia risk prediction from prenatal cell-free DNA screening. Nat Med (2025). DOI:10.1038/s41591-025-03509-w (in English)

8. Stroun M, Lyautey J, Olson-Sand A, Anker P. 2001. About the possible origin and mechanism of circulating DNA. Apoptosis and active DNA release. Clin Chim Acta. 313(1–2):139–142. DOI:10.1016/s0009-8981(01)00665-9.15 (in English)

9. Aucamp J, Bronkhorst AJ, Badenhorst CPS, Pretorius PJ. 2018. The diverse origins of circulating cell-free DNA in the human body: a critical re-evaluation of the literature. Biol Rev Camb Philos Soc. 93(3):1649, 168. DOI: 10.1111/BRV.12413. (in English)

10. Fernández-Domínguez, I. J., ManzoMerino, J., Taja-Chayeb, L., DueñasGonzález, A., Pérez-Cárdenas, E., amp; Trejo-Becerril, C. The role of extracellular DNA (exDNA) in cellular processes. Cancer Biology; Therapy, 22(4), 267–278. Fernández-Domínguez, I. J., Manzo-Merino, J., Taja-Chayeb, L., Dueñas-González, A., Pérez-Cárdenas, E., amp; Trejo- Becerril, C. (2021). The role of extracellular DNA (exDNA) in cellular processes. Cancer Biology amp; Therapy, 22(4), 267–278. DOI:10.1080/15384047.2021.1890319 (in English)

11. Keeling PJ, Palmer JD. 2008. Horizontal gene transfer in eukaryotic evolution. Nat Rev Genet. 9(8):605–618. DOI: 10.1038/ nrg2386. (in English)

12. Kustanovich A, Schwartz R, Peretz T, Grinshpun A. Life and death of circulating cell-free DNA. Cancer Biol Ther. 2019;20 (8):1057–1067. DOI:10.1080/15384047.2019.1598759. (in English)

13. Kostyuk S. V. The role of extracellular DNA in the functional activity of the human genome // Bulletin of the Russian Academy of Sciences. - 2010. — Vol.80, No. 9. — pp. 746-752. (In Russ.)

14. McCarthy C. G., Wenceslau C. F., Goulopoulou S., Ogbi S., Baban B., Sullivan J. C., et al. (2015). Circulating mitochondrial DNA and Toll-like receptor 9 are associated with vascular dysfunction in spontaneously hypertensive rats. Cardiovasc. Res. 107 119– 130. DOI: 10.1093/cvr/cvv137 (in English)

15. Rykova E, Morozkin E, Ponomaryova A, Loseva E, Zaporozhchenko I, Cherdyntseva N, Vlassov V, Laktionov P. 2012. Cellfree and cell-bound circulating nucleic acid complexes: mechanisms of generation, concentration and content. Expert Opin Biol Ther. 12(Suppl sup1):S141–S153. DOI:10.1517/14712598.2012.673577. (in English)

16. Thierry AR, El Messaoudi S, Gahan PB, Anker P, Stroun M. 2016. Origins, structures, and functions of circulating DNA in oncology. Cancer Metastasis Rev. 35(3):347 –376. DOI:10.1007/s10555-016-9629-x. (in English)

17. Wang W, Kong P, Ma G, Li L, Zhu J, Xia T, Xie H, Zhou W, Wang S. 2017. Characterization of the release and biological significance of cell-free DNA from breast cancer cell lines. Oncotarget. 8(26):43180– 43191. DOI: 10.18632/oncotarget.17858. (in English)

18. Sato Y, Matoba R, Kato K. 2019. Recent advances in liquid biopsy in precision oncology research. Biol Pharm Bull. 42(3):337– 342. DOI: 10.1248/bpb.b18-00804.13 (in English)

19. McCarthy C. G., Wenceslau C. F., Goulopoulou S., Ogbi S., Baban B., Sullivan J. C., et al. (2015). Circulating mitochondrial DNA and Toll-like receptor 9 are associated with vascular dysfunction in spontaneously hypertensive rats. Cardiovasc. Res. 107 119– 130. DOI: 10.1093/cvr/cvv137 (in English)

20. Lowes H., Pyle A., Santibanez-Koref M., Hudson G. (2020). Circulating cell-free mitochondrial DNA levels in Parkinson’s disease are influenced by treatment. Mol. Neurodegener. 15:10. DOI: 10.1186/s13024-020-00362-y (in English)

21. Vakrakou A. G., Boiu S., Ziakas P. D., Xingi E., Boleti H., Manoussakis M. N. (2018). Systemic activation of NLRP3 inflammasome in patients with severe primary Sjögren’s syndrome fueled by inflammagenic DNA accumulations. J. Autoimmun. 91 23 –33. DOI:10.1016/j.jaut.2018.02.010 (in English)

22. Scalici E., Mulet T., Ferrier Hoa A., Gala A., Lu V., Anahori T. et al. Circulating nuclear acids and infertility. Gynecol Obstetric Fertil. 2015; 43:593-8. DOI: 10.1016/j.gyobfe.2015.07.016. (in English)

23. Traver S, Scalici E, Mullet T, Molinari N, Vincens C, Anahory T, Hamamah S. Cellfree DNA in Human Follicular Microenvironment: New Prognostic Biomarker to Predict in vitro Fertilization Outcomes PLoS ONE. 2015;10(8):e0136172. DOI: 10.1371/journal.pone.0136172. (in English)

24. Guan Y., Zhang W., Wang X., Cai P., Jia Q., Zhao W. Cell-free DNA induced apoptosis of granulosa cells by oxidative stress. Clin. Chim. Acta. 2017; 473: 213–217. DOI: 10.1016/j.cca.2016.11.023. (in English)

25. Ichikawa K., Shibahara H., Shirasuna K., Kuwayama T., Iwata H. Cell-free DNA content in follicular fluid: a marker for the developmental ability of porcine oocytes. Reproductive Medicine and Biology. 2019. Vol. 19. P. 95-103. DOI: 10.1002/rmb2.12309. (in English)

26. Tarkowski A. K. An air-drying method for chromosome preparation from mouse eggs. (1966) Cytogenetics, nr. 5, 3, P. 394- 400. DOI:10.1159/000129914 (in English)

27. Woodruff TK, Shea LD. The role of the extracellular matrix in ovarian follicle development. Reprod Sci. 2007 Dec;14(8 Suppl):6-10. DOI: 10.1177/1933719107309818. (in English)

28. Liu Z, Li F, Xue J, Wang M, Lai S, Bao H, He S. Esculentoside A rescues granulosa cell apoptosis and folliculogenesis in mice with premature ovarian failure. Aging (Albany NY). 2020 Aug 5;12(17):16951- 16962. DOI: 10.18632/aging.103609. Epub 2020 Aug 5. (in English)

29. Xie J, Xu X, Liu S. Intercellular communication in the cumulus-oocyte complex during folliculogenesis: A review. Front Cell Dev Biol. 2023 Jan 19; 11:1087612. DOI: 10.3389/fcell.2023.1087612. (in English)

30. Avalos PN, Wong LL, Forsthoefel DJ. Extracellular vesicles promote proliferation in an animal model of regeneration. bioRxiv [Preprint]. 2024 Mar 27:2024.03.22.586206. DOI: 10.1101/2024.03.22.586206. (in English)

31. Chaves MA, Ferst JG, Fiorenza MF, Vit FF, da Silveira JC. The Influence of Ovarian-Derived Extracellular Vesicles in Reproduction. Adv Anat Embryol Cell Biol. 2025 Jan 1. DOI: 10.1007/102_2024_9. (in English)