LEVEL OF COENZYME NAD+ IN FOLLICULAR FLUIDAS A PREDICTOR OF FEMALE FERTILITY

The work aims to study the level and redox status of nicotinamide coenzymes in follicular fluid in women of reproductive age in various age categories.
Material and methods. The study was conducted on the basis of the ART clinic "Family" in Ufa. 65 patients aged 23 to 50 years were examined (average age 32.3±3.6). The main group consisted of 45 patients of reproductive age from infertile couples. The comparison group included 20 fertile women who applied to the same clinic for a preventive examination. To measure the concentration of NAD+ in follicular fluid, a colorimetric method (NAD/NADH Cell-Based Assay Kit, Cayman Chemical) was used on a 96-well flatbed analyzer capable of measuring optical density at 450 nm. 100 µl of follicular fluid supernatant was used for analysis. The redox potential (NAD/NADH ratio) was determined using the SigmaAldrich test system.
Results. At a NAD+ level in the range of 56-76 nmol/mg of follicular fluid protein, oocyte fertility was diagnosed. At concentrations NAD+ below this range, infertility is detected. The developed method is recommended to be used to assess female fertility in clinical gynecology and reproductology as an effective additional method for predicting the development of female infertility of unclear etiology.
Conclusion. The method provides an opportunity to increase the accuracy, specificity and simplify the diagnosis of infertility by evaluating the redox potential in oocytes by determining the level of NAD+, which makes it possible to identify violations of redox processes, including those associated with mitochondrial pathology, DNA repair, regulation of apoptosis and early aging of eggs.

1. Raperport C., Desai J., Qureshi D. [et al.] The definition of unexplained infertility: A systematic review. BJOG. 2023. (in Engl) doi: 10.1111/1471-0528.17697.

2. Faddy M.J. Follicle dynamics during ovarian ageing. Mol.Cell. Endocrinol. 2002;163(1–2):43-48. (in Engl)

3. Gleicher N., Kushnir V.A., D.F. Albertini [et al.] Improvements in IVF in women of advanced age. J. Endocrinol. 2016; 230(1):F1–F6. (in Engl)

4. Yang Q., Cong L., Wang Y. [et al.] Increasing ovarian NAD+ levels improve mitochondrial functions and reverse ovarian aging. Free Radic Biol Med. 2020;156:1-10. (in Engl)

5. Yang Z., Liu S., Pan X. Research progress on mitochondrial damage and repairing in oocytes: A review. Mitochondrion. 2024;75:101845. (in Engl)

6. Litvitsky P.F., Galimov K.Sh., Gromenko Yu.Yu. [et al.] The role of sperm mitochondria in the occurrence and development of male infertility. Pathological physiology and experimental therapy. 2022;66(2):72-79. (in Russ)

7. Zhang W., Wu F. Effects of adverse fertility-related factors on mitochondrial DNA in the oocyte: a comprehensive review. Reprod. Biol. Endocrinol.2023;21(1):27. (in Engl)

8. Canto C., Menzies K.J., Auwerx J. NAD(+) metabolism and the control of energy homeostasis: a balancing act between mitochondria and the nucleus. Cell Metabol. 2015;22(1):31–53. (in Engl)

9. Kasapoglu,I., E. Seli Mitochondrial dysfunction and ovarian aging. Endocrinology. 2020;161(2):bqaa001. (in Engl)

10. Galimov S.N., Galimova E.F., Gromenko J.Y. [et al.] The level of secondary messengers and the redox state of NAD+/NADH are associated with sperm quality in infertility. Journal of Reproductive Immunology. 2021;148:103383. (in Russ)

11. Li H., Wang H., Xu J. [et al.] Nicotinamide riboside supplementation ameliorated post-ovulatory oocyte quality decline. Reproduction. 2022;165(1):103-111. (in Engl)

12. Bertoldo M.J., Listijono D.R., Ho W.J. [et al]. NAD(+) repletion rescues female fertility during reproductive aging.Cell Rep. 2020;30(6):1670–1681.e7. (in Engl)

13. Sposob diagnostiki zhenskoj fertil'nosti (Method for diagnosing female fertility); patent № 2807923 Ros. Federacija; zajavl, 28.02.2023; opubl. 21.11.2023;33:8. (in Russ)

14. Waddell J., Khatoon R., Kristian T. Cellular and Mitochondrial NAD Homeostasis in Health and Disease. Cells. 2023;12(9):1329. (in Engl)

15. Yang Q., Chen W., Cong L. [et al.] NADase CD38 is a key determinant of ovarian aging. Nat. Aging. 2024;4(1):110-128. (in Engl)

16. Gromenko Yu.Yu., Galimova E.F., Gromenko D.D. [et al.]. Activation of oocytes: fundamental and clinical aspects. Issues of gynecology, obstetrics and perinatology. 2020;19(5):77-85. (in Russ)

17. Kobayashi H., Yoshimoto C., Matsubara S. [et al.] Altered Energy Metabolism, Mitochondrial Dysfunction, and Redox Imbalance Influencing Reproductive Performance in Granulosa Cells and Oocyte During Aging. Reprod Sci. 2023. (in Engl) doi: 10.1007/s43032-023-01394-7.

18. Galimov Sh.N., Bozhedomov V.A., Galimova E.F. [et al.]. Muzhskoe besplodie: molekuljarnye i immunologicheskie aspekty (Male infertility: molecular and immunological aspects). Moskva, 2020:208. (in Russ)

19. Sun Z., Wu H., Lian F. [et al.] Study of human follicular fluid metabolomics, study of follicle development and quality eggs. Chromatography. 2017;80:901-909. (in Engl)

20. Sposob prognozirovanija muzhskogo besplodija na osnove analiza fragmentacii DNK spermatozoidov (A method for predicting male infertility based on analysis of sperm DNA fragmentation); patent № 2804207 Ros. Federacija; zajavl. 27.04.2023; opubl. 26.09.2023. (in Russ)

21. Luti S., Fiaschi T., Magherini F. [et al.]. The relationship between the metabolic and lipid profile in the follicular fluid of women undergoing in vitro fertilization. Mol. reproduction. dev. 2020;87:986-997. (in Engl)

22. Galimov Sh.N., Akhmetov R.M., Galimova E.F. [et al.]. Molecular aspects of the influence of the Speroton complex on male fertility in idiopathic infertility. Urology. 2017;2:88-92. (in Russ)