Journal of Reproduction & Infertility

Journal of Reproduction & Infertility

The Effect of Serine Proteases and Their Inhibitors on In Vitro Fertilization Outcomes

Authors
Liudmila Spirina 1
Olga Akbasheva 1
Denis Dyakov 1
Ksenia Kudisova 1
Ekaterina Romanova 1
Kira Sidorenkova 2
Ilya Petrov 2
Iuliia Samoilova 3
Evgeny Merkulov 1
Diana Shilova 1
Marina Stakheeva 1
1 Department of Biochemistry and Molecular Biology, Siberian State Medical University, Tomsk, Russian Federation
2 Department of Obstetrics and Gynecology, Siberian State Medical University, Tomsk, Russian Federation
3 Department of Pediatrics with a Course in Endocrinology, Siberian State Medical University, Tomsk, Russian Federation
Abstract
Background: Proteolysis imbalance can result in infertility. The purpose of the current study was to investigate the function of kininogen, neutrophil elastase, trypsin-like proteases, α1-protease inhibitor (α1-PI), and α2-macroglobulin (α2-MG) in blood, saliva, and follicular fluid as an indicator of the success of in vitro fertilization (IVF). Methods: In general, twenty-one women undergoing an IVF cycle, as well as a control group consisting of ten healthy women, were included. The proteinase activity was measured by the rate of hydrolysis of specific substrates. The proteinase inhibitor activity was determined by the intensity of trypsin inhibition. The analysis was conducted using R software (version 4.4.1). The significance level was set at p<0.05. Results: In women with infertility, blood activities of neutrophil elastase, α1-PI, and α2-MG were elevated, whereas kallikrein and prekallikrein activities were decreased, compared with controls. In follicular fluid, neutrophil elastase activity increased 3.4-fold, α1-PI 1.8-fold, and α2-MG 5.6-fold, whereas prekallikrein decreased by 85%, compared with women who had been infertile for fewer than five years (p<0.05). During pregnancy, kallikrein and α1- PI levels in follicular fluid were low, and unsuccessful IVF was associated with higher trypsin-like protease activity and an increased kallikrein/α1- PI ratio. Conclusion: High elastase- and trypsin-like proteinase activity, along with elevated α1- PI and α2- MG levels in blood was associated with IVF benefits. Salivary kininogenase and α2-MG can be early prognostic indicators for IVF outcomes.
Keywords
Follicular fluid
In vitro fertilization
Saliva
Serine proteases
α1
protease inhibitor
α2
macroglobulin
  1. World Health Organization. Infertility prevalence estimates, 1990–2021. Geneva: World Health Orga-nization; 2023. 79 p. Available from: https://www. who.int/publications/i/item/978920068315?utm_source=chatgpt.com
  2. Russian Association of Human Reproduction. ART register: report for 2021. Saint Petersburg: Russian Association of Human Reproduction; 2023. 29 p. Available from: https://www.rahr.ru/d_registr_ otchet/RegistrVRT_2021.pdf
  3. Franasiak JM, Alecsandru D, Forman EJ, Gemmell LC, Goldberg JM, Llarena N, et al. A review of the pathophysiology of recurrent implantation failure. Fertil Steril. 2021;117(3):1436-48.
  4. Strizhakov AN, Ignatko IV, Davydov AI. Obstetri-cs: textbook. Moscow: GEOTAR-Media; 2020. 1072 p.
  5. Gutiérrez JA, Gómez I, Chiarello DI, Salsoso R, Klein AD, Guzmán-Gutiérrez E, Toledo F, Sobrevia L. Role of proteases in dysfunctional placental vas-cular remodelling in preeclampsia. Biochim Bio-phys Acta Mol Basis Dis. 2020;1866(2):165448.
  6. Strizhova NV, Dzhaguga VD. [Significance of the kallikrein-kinin system of the blood in obstetrics and gynecology]. Akush Ginekol (Mosk). 1988;(6):5-8. Russian.
  7. Schuster R, Motola-Kalay N, Baranovski BM, Bar L, Tov N, Stein M, et al. Distinct anti-inflammatory properties of alpha1-antitrypsin and corticosteroids reveal unique underlying mechanisms of action. Cell Immunol. 2020;356:104177.
  8. de Serres F, Blanco I. Role of alpha-1 antitrypsin in human health and disease. J Intern Med. 2014 Oct; 276(4):311-35.
  9. Lagrange J, Lecompte T, Knopp T, Lacolley P, Regnault V. Alpha-2-macroglobulin in hemostasis and thrombosis: An underestimated old double-edged sword. J Thromb Haemost. 2022;20(4):806-15.
  10. Ogloblina OG, Platonova LV, Paskhina TS. Meas-urement of trypsin- and elastase-like proteinases of polymorphonuclear leukocytes and the level of their acid-stable inhibitors in human bronchial se-cretions: methodological recommendations. Mos-cow; 1984. 14 p.
  11. Paskhina TS, Krinskaya AV. Determination of kal-likrein activity in human serum/plasma. In: Sovre-mennye metody v biokhimii [Modern methods in biochemistry]. 1977. p. 70-2.
  12. Nartikova VF, Paskhina TS. [Uniform method for determining the alpha 1-antitrypsin and alpha 2-macroglobulin activity in human blood serum (plasma)]. Vopr Med Khim. 1979;25(4):494-9. Russian.
  13. R Core Team. Documentation for package ‘stats’ version 4.4.1. Vienna: R Foundation for Statistical Computing; 2024. Available from: https://search.r-project.org/R/refmans/stats/html/00Index.html
  14. Yakovleva N, Saprykina D, Vasiljeva E, Bettikher O, Godzoeva A, Kazantseva T, Zazerskaya I. Matrix metalloproteinase -12: A marker of pre-eclampsia? Placenta. 2022;129:36-42.
  15. Orlov AV, Kudinova EI. Etiopathogenetic aspects of the development of placental insufficiency and fetal growth restriction. J Fundam Med Biol. 2016; (4):5-10.
  16. Jugniot N, Voisin P, Bentaher A, Mellet P. Neu-trophil Elastase Activity Imaging: Recent Appro-aches in the Design and Applications of Activity-Based Probes and Substrate-Based Probes. Con-trast Media Mol Imaging. 2019;2019:7417192.
  17. Vorobjeva NV. Neutrophil Extracellular Traps: New Aspects. Moscow Univ Biol Sci Bull. 2020; 75(4):173-88.
  18. Yang C, Keshavjee S, Liu M. Alpha-1 Antitrypsin for COVID-19 Treatment: Dual Role in Antiviral Infection and Anti-Inflammation. Front Pharmacol. 2020;11:615398.
  19. Ozeri E, Mizrahi M, Shahaf G, Lewis EC. α-1 antitrypsin promotes semimature, IL-10-producing and readily migrating tolerogenic dendritic cells. J Immunol. 2012;189(1):146-53.
  20. Vandooren J, Itoh Y. Alpha-2-Macroglobulin in Inflammation, Immunity and Infections. Front Immunol. 2021;12:803244.
  21. Kim KM, Chung KW, Jeong HO, Lee B, Kim DH, Park JW, et al. MMP2-A2M interaction increases ECM accumulation in aged rat kidney and its modulation by calorie restriction. Oncotarget. 2017;9(5):5588-99.
  22. Widegren H, Erjefält J, Korsgren M, Andersson M, Greiff L. Effects of intranasal TNFalpha on gra-nulocyte recruitment and activity in healthy sub-jects and patients with allergic rhinitis. Respir Res. 2008;9(1):15.
  23. Meyer P, Andersson M, Persson CG, Greiff L. Steroid-sensitive indices of airway inflammation in children with seasonal allergic rhinitis. Pediatr Allergy Immunol. 2003;14(1):60-5.
  24. Harwood SL, Nielsen NS, Diep K, Jensen KT, Nielsen PK, Yamamoto K, et al. Development of selective protease inhibitors via engineering of the bait region of human α2-macroglobulin. J Biol Chem. 2021;297(1):100879.
  25. Seitz R, Gürtler L, Schramm W. Thromboinflam-mation in COVID-19: Can α2 -macroglobulin help to control the fire? J Thromb Haemost. 2021;19(2):351-4.
  26. Ireland JL, Jimenez-Krassel F, Winn ME, Burns DS, Ireland JJ. Evidence for autocrine or paracrine roles of alpha2-macroglobulin in regulation of es-tradiol production by granulosa cells and development of dominant follicles. Endocrinology. 2004; 145(6):2784-94.
  27. Gao S, De Geyter C, Kossowska K, Zhang H. FSH stimulates the expression of the ADAMTS-16 protease in mature human ovarian follicles. Mol Hum Reprod. 2007;13(7):465-71.
  28. Wasiluk A, Dabrowska M, Jaworski S, Prokopo-wicz J. [Levels of alpha-2-macroglobulin in blood serum of women giving birth to hypotrophic and eutrophic newborns]. Przegl Lek. 2000;57(12):717-9. Polish.
  29. Bahabayi A, Yang N, Xu T, Xue Y, Ma L, Gu X, et al. Expression of matrix metalloproteinase-2,-7,-9 in serum during pregnancy in patients with pre-eclampsia: a prospective study. Int J Environ Res Public Health. 2022;19(21):14500.
  30. Fischer U, Birkenmeier G, Horn LC. Localization of alpha(2)-macroglobulin receptor/low-density li-poprotein receptor in third-trimester human pla-centas: a preliminary immunohistochemical study. Gynecol Obstet Invest. 2001;52(1):22-5.
  31. Markina LA, Zorina VN, Shramko SV, Zorina RM, Arkhipova SV, Bazhenova LG. [The serum levels of inflammatory reactants in women with inflammatory diseases of the uterine appendages, who participate in the in-vitro fertilization]. Klin Lab Diagn. 2008;(2):15-7. Russian.
  32. Goettig P, Brandstetter H, Magdolen V. Surface loops of trypsin-like serine proteases as deter-minants of function. Biochimie. 2019;166:52-76.
  33. Oncul S, Afshar-Kharghan V. The interaction between the complement system and hemostatic factors. Curr Opin Hematol. 2020;27(5):341-52.
  34. Ferguson TEG, Reihill JA, Martin SL, Walker B. Novel inhibitors and activity-based probes targe-ting serine proteases. Front Chem. 2022;10:1006618.
  35. Ossovskaya VS, Bunnett NW. Protease-activated receptors: contribution to physiology and disease. Physiol Rev. 2004;84(2):579-621.
  36. Motta G, Juliano L, Chagas JR. Human plasma kallikrein: roles in coagulation, fibrinolysis, in-flammation pathways, and beyond. Front Physiol. 2023;14:1188816.
  37. Bekassy Z, Lopatko Fagerström I, Bader M, Karp-man D. Crosstalk between the renin-angiotensin, complement and kallikrein-kinin systems in in-flammation. Nat Rev Immunol. 2022;22(7):411-28.
  38. Ivanov I, Verhamme IM, Sun MF, Mohammed B, Cheng Q, Matafonov A, et al. Protease activity in single-chain prekallikrein. Blood. 2020;135(8):558-67.
  39. Pampalakis G, Zingkou E, Panagiotidis C, Sotiro-poulou G. Kallikreins emerge as new regulators of viral infections. Cell Mol Life Sci. 2021;78(21-22):6735-44.
  40. Clements J, Mukhtar A, Yan S, Holland A. Kallikreins and kinins in inflammatory-like events in the reproductive tract. Pharmacol Res. 1997;35 (6):537-40.
  41. Strizhova NV, Dzhaguga VD. [The significance of the kallikrein-kinin system of blood in obstetrics and gynecology]. Akush Ginekol (Mosk). 1988;(6):5-8. Russian.
  42. Yarovaya GA, Neshkova AE. Kallikrein-kinin system: past and present research (on the 90th anniversary of the discovery of the system). Russ J Bioorg Chem. 2015;41(3):245-59.
  43. Ignatieva RE, Gustovarova TA, Babich EN, Kry-ukovsky AS. Violation of vascular endothelial function in women with premature ovarian insuf-ficiency. Bull Smolensk State Med Acad. 2016;15(3):93-100.
  44. Dabi Y, Suisse S, Puchar A, Delbos L, Poilblanc M, Descamps P, et al. Endometriosis-associated in-fertility diagnosis based on saliva microRNA signatures. Reprod Biomed Online. 2023;46(1):138-49.
  45. Wróbel M, Zuzanna Z, Ołdak Ł, Kalicka A, Mańka G, Kiecka M, et al. Evaluation of Proteasome and Immunoproteasome Levels in Plasma and Perito-neal Fluid in Patients with Endometriosis. Int J Mol Sci. 2023;24(18):14363.
  46. Collodel G, Gambera L, Stendardi A, Nerucci F, Signorini C, Pisani C, et al. Follicular Fluid Com-ponents in Reduced Ovarian Reserve, Endo-metriosis, and Idiopathic Infertility. Int J Mol Sci. 2023;24(3):2589.
  47. Ilika VV, Garvasiuk OV, Dogolich OІ, Iryna BV. The features of limited proteolysis in placental fibrinoid in combination with inflammation and iron deficiency anemia of pregnant women. Wiad Lek. 2023;76(5 pt 1):1022-8.
  48. Fang Y, Jingjing F, Tiantain C, Huanhuan X, Qiaohua H. Impact of the number of previous embryo implantation failures on IVF/ICSI-ET pregnancy outcomes in patients younger than 40 years: a retrospective cohort study. Front Endo-crinol (Lausanne). 2023;14:1243402.
  49. Cimadomo D, Capalbo A, Dovere L, Tacconi L, Soscia D, Giancani A, et al. Leave the past behind: women's reproductive history shows no association with blastocysts' euploidy and limited association with live birth rates after euploid embryo transfers. Hum Reprod. 2021;36(4):929-40.
Journal of Reproduction & Infertility
Volume 27, Issue 1
Winter 2026
Pages 57-69