A coordinated relationship exists between thyroid hormone levels, oxidative stress, and inflammation. Thyroid hormones may act as oxidants on their own and may cause DNA damage ¹⁰. In hyperthyroidism, an increase in free radical levels and oxidative stress are observed in various tissues ^11,12. Moreover, it has been reported that oxidative stress and inflammation are closely related, and oxidative stress stimulates inflammation (10). In addition, many inflammatory chemokines and cytokine levels are increased in hyperthyroidism ¹³. Carvacrol (2-methyl-5-(1-methylethyl)- phenol) is a monoterpenic phenol found in many aromatic plants ¹⁴. Carvacrol, with its rich hydroxyl groups, instills confidence in its therapeutic potential, offering anti-inflammatory, antioxidant, antitumour, antibacterial, antifungal, analgesic, antiparasitic and antigenotoxic effects ^14-16. It is also reported that carvacrol decreases proinflammatory cytokines and lipid peroxidation levels and increases total glutathione levels ¹⁷.

This study aimed to investigate the effects of carvacrol on ovarian damage caused by hyperthyroidism. To this end, the effects of carvacrol on ovaries in terms of inflammation, oxidative stress, and ovarian reserves in hyperthyroidism were investigated.

Design and Sample Collection: The sample size calculation was performed using G*Power software version 3.1, which determined a minimum requirement of 35 rats. This calculation was based on a medium effect size of 0.42, with an alpha value of 0.05 and a power of 0.80. Thirty-five female Wistar albino rats (250±25 g, 6-8 weeks old) were randomly divided into five groups of seven animals each: control (C), vehicle (V), hyperthyroidism (HT), carvacrol (CAR), hyperthyroidism+carvacrol (HT+CAR). For the hyperthyroidism model, L-thyroxine (levothyroxine) (0.3 mg/kg/day p.o) was dissolved in saline and administered by intragastric gavage for 25 days ^9,18,19. Carvacrol (100 mg/kg/day p.o) was dissolved in 25% DMSO and administered by gavage for 14 days starting on day 11 of the study ²⁰.

At the end of the study, animals were sacrificed under xylazine-ketamine anaesthesia and blood samples were collected from the intracardiac ventricles. Ovaries and uteri samples were collected and stored until analyses were performed.

Thyroxine (T4) and Anti-Mullerian Hormone (AMH) Analyses: Blood samples were centrifuged (3000 rpm, 5 min), and sera were collected (21). Hormonal analyses were performed on blood sera. Serum free thyroxine (T4) levels were measured by electrochemiluminescence immunoassay (Atellica IM, Siemens Healthineers) and expressed as ng/dL.

Ovary and Uterus Oxidative Stress Analysis (MDA and GSH): For oxidative stress analyses, ovaries (right and left) and uteruses were homogenised using Tris-buffered saline (pH 7.4) and then centrifuged (4000 rpm 60 min), and supernatants were collected ²². Malondialdehyde (MDA) and glutathione (GSH) analyses were performed to determine the oxidant-antioxidant status of the ovary and uterus. Oxidant status was evaluated by measuring MDA levels at 532 nm as a biomarker of lipid peroxidation level ²³. Glutathione levels were measured spectrometrically at 412 nm to evaluate the antioxidant status (24). In addition, total ovarian and uterine protein concentrations were measured using a BCA protein assay kit (Takara Bio. Inc., Japan). MDA and GSH levels were expressed as nmol/gr protein.

Ovarian Inflammatory Cytokine Analyses (TNF-α and IL-6): For each animal, the right and left ovaries were pooled and homogenized after reconstitution with phosphate-buffered saline (PBS) (pH 7.4) at a ratio of (1/10, weight/volume). The homogenate was centrifuged at 5000 x g for 5 minutes, and the supernatant was collected and measured. Tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) levels were measured using enzyme-linked immunosorbent assay (ELISA) kits. In addition, total protein concentrations in the samples were measured using a BCA protein assay kit (Takara Bio. Inc., Japan). Results were expressed as pg/mg protein.

Statistical Analysis: Data were statistically analyzed in the study. Parametric test assumptions were applied before significance tests for all variables were obtained. The variables were analyzed using the Shapiro-Wilk test for normality and the Levene test for homogeneity. Then, a one-way analysis of variance (ANOVA) was used to control the statistical difference between the variables. Duncan's test was used as a post hoc test for the variables when the difference between the groups was significant. All statistical analyses were analyzed with a minimum margin of error of 5%. The IBM SPSS 23 was used for all statistical analyses. All results are expressed as mean ± standard error of the mean (SEM).

The AMH levels of the groups (C, V, HT, CAR and HT+CAR) were 1.572±0.067, 1.560±0.061, 1.724±0.040, 1.575±0.035, 1.718±0.041 ng/mL, respectively. The highest AMH levels were observed in HT and HT+CAR groups. In addition, V, HT, CAR, and HT+CAR groups were similar to the control group (p=0.045). The AMH levels of the groups are shown in Figure 1b.

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Figure 1: a. Serum-free T4 levels b. Serum AMH levels. Values are expressed as Mean±Standard error of the mean. The letters shown in the columns indicate statistical differences. There is a statistical difference between different letters

Ovarian MDA and GSH Analyses: The ovarian MDA levels of the groups (C, V, HT, CAR and HT+CAR) were measured as 3.792±0.141, 3.836±0.099, 5.431±0.319, 3.696±0.151, 4.434±0.271 nmol/g protein, respectively. The highest MDA levels were observed in the HT group. In addition, while V, CAR and HT+CAR were similar to the control group, the HT group differed from the control group (p<0.001). MDA levels of the groups are shown in Figure 2a.

The ovarian GSH levels of the groups (C, V, HT, CAR and HT+CAR) were measured as 6.900±0.175, 6.994±0.386, 5.900±0.232, 7.014±0.181, 6.367±0.154 nmol/g protein, respectively. The lowest GSH levels were observed in the HT group. In addition, while V, CAR and HT+CAR were similar to the control group, the HT group differed from the control group (p=0.009). GSH levels of the groups are shown in Figure 2b.

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Figure 2: a. Ovarian MDA levels b. Ovarian GSH levels. Values are expressed as mean±standard error of the mean. The letters in the columns indicate statistical differences. There is a statistical difference between different letters

Uterus MDA and GSH Analyses: The uterine MDA levels of the groups (C, V, HT, CAR and HT+CAR) were measured as 2.517±0.117, 2.635±0.182, 3.818±0.186, 2.742±0.111, 3.318±0.152 nmol/gr protein, respectively. The highest MDA levels were found in the HT group. In addition, MDA levels were similar to the CAR control group but statistically different from the HT+CAR control group (p<0.001). The uterine MDA levels of the groups are shown in Figure 3a.

The uterine GSH levels of the groups (C, V, HT, CAR and HT+CAR) were measured to be 12.334±0.294, 12.309±0.360, 9.942±0.274, 12.487±0.206, 11.507±0.217 nmol/g protein, respectively. The lowest GSH levels were found in the HT group. In addition, while V, CAR and HT+CAR were similar to the control group, HT was statistically different from the control group (p<0.001). The uterine GSH levels of the groups are shown in Figure 3b.

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Figure 3: a. Uterine MDA levels b. Uterine GSH levels. Values were expressed as Mean±Standard error of the mean. The letters shown in the columns indicate statistical differences. There is a statistical difference between different letters

Ovarian Inflammatory Cytokine Analyses (TNF-α and IL-6): TNF-α levels (C, V, HT, CAR and HT+CAR) were measured as 14.370±0.742, 14.428±1.050, 19.931±0.682, 14.890±0.742, 16.252±0.649 pg/mg protein, respectively. The highest ovarian TNF-α levels were observed in the HT group. In addition, V, CAR, and HT+CAR groups were similar to the control, whereas HT differed from the control (p<0.001). The TNF-α levels of the groups are shown in Figure 4a.

IL-6 levels (C, V, HT, CAR and HT+CAR) were measured as 7.879±0.847, 8.317±0.606, 14.411±0.664, 6.908±0.624, 10.352±0.824 pg/mg protein, respectively. The highest ovarian IL-6 levels were observed in the HT group. In addition, while V and CAR groups were similar to the control, HT and HT+CAR groups differed from the control (p<0.001). IL-6 levels of the groups are shown in Figure 4b.

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Figure 4: a. Ovary TNF-α levels b. Ovary IL-6 levels. Values are expressed as Mean±Standard error of the mean. The letters shown in the columns indicate statistical differences. There is a statistical difference between different letters

Increases in AMH levels were observed in thyroid autoimmune girls, and this was associated with the activation of inactive primordial follicles. It was stated that increased AMH may be a short-term reaction against autoimmunity ³⁰. In other studies associating thyroid hormones with AMH, low TSH levels were associated with high AMH expression ^31,32. A study reported that the number of primordial, primary and secondary follicles increased in hyperthyroid rats while the size of primary and secondary follicles decreased ³³. In addition, hyperthyroidism increases the number of secondary and tertiary follicles and decreases atretic follicles ³⁴. In this study, it is thought that the increases in AMH levels in the hyperthyroidism group, even if not statistically significant, may have short-term effects and may be due to the effects of hyperthyroidism on ovarian metabolism.

Although the female reproductive system ages faster than the rest of the body, the quality and number of oocytes gradually decline, and ovarian reserves diminish with age ³⁵. Oxidative stress and inflammatory processes accelerate ovarian ageing ³⁶. A study of follicular fluid in women reported that oxidative stress and inflammation were associated with decreased ovarian reserve ³⁷. In addition, hyperthyroidism is known to stimulate oxidative stress ^19,38. For example, free radical levels are known to be elevated in patients with Graves' disease ¹¹. A study in rats reported that hyperthyroidism caused oxidative stress in the ovaries ⁹. Increases in MDA levels are observed in hyperthyroidism ³⁹. This study observed statistical increases in MDA levels in the ovary and uterus in the hyperthyroidism group, consistent with previous studies. GSH is known to be one of the biological antioxidants against oxidative damage ⁴⁰. In a study performed in rats, it was reported that GSH levels in various tissues decreased in hyperthyroidism ¹¹. This study observed statistically significant decreases in ovarian and uterine GSH levels in the hyperthyroidism group. There is a close relationship between thyroid diseases and inflammatory mediators, and inflammatory cytokine levels increase in many thyroid diseases ¹³. For example, in a study conducted in mice, increases in TNF-α and IL-6 levels and decreases in anti-inflammatory levels, such as IL-10, were observed in hyperthyroidism ⁴¹. Similarly, in this study, it was observed that there were statistically significant increases in TNF-α and IL-6 levels in the hyperthyroidism group.

Carvacrol may have beneficial effects on female reproductive activity by affecting reproductive hormones in females ^42,43. Carvacrol administration was observed to increase estrogen levels in rats receiving radiation therapy ⁴³. A study correlating carvacrol and ovarian reserve reported that carvacrol had a stimulating effect on ovarian reserve ²⁰. Similarly, in another study conducted in rats, decreased AMH levels caused by radiation were restored to normal levels by carvacrol administration ⁴³. However, in this study, it was observed that carvacrol administration did not cause a statistically significant difference in the ovarian reserves of both treated and healthy animals.

Carvacrol is a bioactive substance with antioxidant and anti-inflammatory effects ^14,44. A study has shown that carvacrol has antioxidant activity in radiation-induced ovarian damage ⁴³. It has been reported that carvacrol treatment in rats causes antioxidant effects by reducing MDA and TOS and increasing TAS and total glutathione in ovarian ischemia and reperfusion damage ⁴⁵. Similarly, carvacrol treatment in cisplatin-induced ovarian damage causes decreases in ovarian MDA levels and increases in total glutathione levels ²⁰. In this study, similar to previous studies, although there was no statistically significant difference, carvacrol application was observed to have an antioxidant effect by reducing MDA and increasing GSH. It has also been shown that carvacrol causes anti-inflammatory effects by inhibiting inflammatory cytokine levels ⁴⁵. In one study, carvacrol was observed to reduce the levels of many inflammatory cytokines in ovarian ischemia-reperfusion injury in a dose-dependent manner ⁴⁶. Similarly, in another study, carvacrol treatment in ovarian injury reduced the levels of inflammatory cytokines such as NF-kB, TNF-α, IL-1b and IL-6 ²⁰. Similarly, in this study, carvacrol administration was observed to statistically reduce the increases in inflammatory cytokine levels caused by hyperthyroidism.

In conclusion, carvacrol, used in hyperthyroidism, has been found to have promising beneficial effects through antioxidant and anti-inflammatory mechanisms. However, future studies investigating different treatment doses and clinical studies would help reveal the long-term effects of carvacrol.

1) Silva JF, Ocarino NM, Serakides R. Thyroid hormones and female reproduction. Biol Reprod 2018; 99: 907-921.

2) Ding Y, Tian Y, Guo M, et al. Regulation of glucose transport by thyroid hormone in rat ovary. Cell Tissue Res 2016; 366: 455-466.

3) Mintziori G, Kita M, Duntas L, Goulis DG. Consequences of hyperthyroidism in male and female fertility: pathophysiology and current management. J Endocrinol Invest 2016; 39: 849-853.

4) Kobayashi N, Orisaka M, Cao M, et al. Growth differentiation factor-9 mediates follicle-stimulating hormone-thyroid hormone interaction in the regulation of rat preantral follicular development. Endocrinology 2009; 150: 5566-5574.

5) Zhang C, Wang X, Wang Z, Niu W, et al. Effect of different culture systems and 3, 5, 30 triiodothyronine / folliclestimulating hormone on preantral follicle development in mice. PLoS One 2013; 86: 19-47.

6) Ramprasad M, Bhattacharyya SS, Bhattacharyya A. Thyroid disorders in pregnancy. Indian J Endocrinol Metab 2012; 16: 167-170.

7) Krassas GE. Thyroid disease and female reproduction. Fertil Steril 2000; 74: 1063-1070.

8) Akande EO, Hockaday TD. Plasma oestrogen and luteinizing hormone concentrations in thyrotoxic menstrual disturbance. Proc R Soc Med 1972; 65: 789-790.

9) Kaplan S, Türk A, Aydın H, Erten M, Kırıcı P. Vitamin D improves oxidative stress and histopathological damage in rat ovaries caused by hyperthyroidism. J Obstet Gynaecol Res 2021; 47: 3551-3560.

10) Mancini A, Di Segni C, Raimondo S, et al. Thyroid hormones, oxidative stress, and inflammation. Mediators Inflamm 2016; 2016: 6757154.

11) Mohamadin AM, Hammad LN, El-Bab MF, Abdel Gawad HS. Attenuation of oxidative stress in plasma and tissues of rats with experimentally induced hyperthyroidism by caffeic acid phenylethyl ester. Basic Clin Pharmacol Toxicol 2007; 100: 84-90.

12) Wang C, Crapo LM. The epidemiology of thyroid disease and implications for screening. Endocrinol Metab Clin North Am 1997; 26: 189-218.

13) Ferrari SM, Ruffilli I, Elia G, et al. Chemokines in hyperthyroidism. J Clin Transl Endocrinol 2019; 16: 100196.

14) Suntres ZE, Coccimiglio J, Alipour M. The bioactivity and toxicological actions of carvacrol. Crit Rev Food Sci Nutr 2015; 55: 304-318.

15) Shoorei H, Khaki A, Khaki AA, et al. The ameliorative effect of carvacrol on oxidative stress and germ cell apoptosis in testicular tissue of adult diabetic rats. Biomed Pharmacother 2019; 111: 568-578.

16) De Vincenzi M, Stammati A, De Vincenzi A, Silano M. Constituents of aromatic plants: Carvacrol. Fitoterapia 2004; 75: 801-804.

17) Ezz-Eldin YM, Aboseif AA, Khalaf MM. Potential anti-inflammatory and immunomodulatory effects of carvacrol against ovalbumin-induced asthma in rats. Life Sci 2020; 242: 117222.

18) Deng J, Zhao R, Zhang Z, Wang J. Changes in vasoreactivity of rat large- and medium-sized arteries induced by hyperthyroidism. Exp Toxicol Pathol 2010; 62: 317-322.

19) Mogulkoc R, Baltaci AK, Oztekin E, Aydin L, Sivrikaya A. Melatonin prevents oxidant damage in various tissues of rats with hyperthyroidism. Life Sci 2006; 79: 311-315.

20) Dinc K, Ozyurt R, Coban TA, et al. The effect of carvacrol on the proinflammatory cytokines, histology, and fertility outcome of cisplatin-related ovarian change in a rat model. Taiwan J Obstet Gynecol 2023; 62: 256-263.

21) Akköse M, Çınar EM, Yazlık MO, et al. Serum pregnancy-associated glycoprotein profiles during early gestation in Karya and Konya Merino sheep. Vet Med Sci 2024; 10: 1345.

22) Arkali G, Aksakal M, Kaya ŞÖ. Protective effects of carvacrol against diabetes-induced reproductive damage in male rats: Modulation of Nrf2/HO-1 signalling pathway and inhibition of Nf-kB-mediated testicular apoptosis and inflammation. Andrologia 2021; 53: 13899.

23) Placer ZA, Cushman LL, Johnson BC. Estimation of product of lipid peroxidation (malonyl dialdehyde) in biochemical systems. Anal Biochem 1966; 16: 359-364.

24) Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem 1968; 25: 192-205.

25) Doufas AG, Mastorakos G. The hypothalamic-pituitary-thyroid axis and the female reproductive system. Ann N Y Acad Sci 2000; 900: 65-76.

26) Al-Saaidi JA, Al-Bedary JK. Gonadotropin profile in experimentally induced hypothyroid and hyperthyroid cyclic female rats. Iraqi J Vet Sci 2022; 36: 745-751.

27) Fedail JS, Zheng K, Wei Q, Kong L, Shi F. Roles of thyroid hormones in follicular development in the ovary of neonatal and immature rats. Endocrine 2014; 46: 594-604.

28) La Marca A, Sighinolfi G, Radi D, et al. Anti-Mullerian hormone (AMH) as a predictive marker in assisted reproductive technology (ART). Hum Reprod Update 2010; 16: 113-130.

29) Cedars MI. Evaluation of female fertility-AMH and ovarian reserve testing. J Clin Endocrinol Metab 2022; 107: 1510-1519.

30) Hasegawa Y, Kitahara Y, Osuka S, et al. Effect of hypothyroidism and thyroid autoimmunity on the ovarian reserve: A systematic review and meta-analysis. Reprod Med Biol 2021; 21: 12427.

31) Kuroda K, Uchida T, Nagai S, et al. Elevated serum thyroid-stimulating hormone is associated with decreased anti-Müllerian hormone in infertile women of reproductive age. J Assist Reprod Genet 2015; 32: 243-247.

32) Weghofer A, Barad DH, Darmon S, Kushnir VA, Gleicher N. What affects functional ovarian reserve, thyroid function or thyroid autoimmunity? Reprod Biol Endocrinol 2016; 14: 26.

33) Mahmud T, Khan QU, Saad S. The interplay between hyperthyroidism and ovarian cytoarchitecture in albino rats. Cureus 2021; 13: 14517.

34) Serakides R, Nunes VA, Nascimento EFD, Ribeiro AFC, Silva CMD. Foliculogênese e esteroidogênese ovarianas em ratas adultas hipertireóideas. Arch Endoc Metab 2001; 45: 258-264.

35) Colella M, Cuomo D, Giacco A, et al. Thyroid hormones and functional ovarian reserve: Systemic vs peripheral dysfunctions. J Clin Med 2020; 9: 1679.

36) Park SU, Walsh L, Berkowitz KM. Mechanisms of ovarian aging. Reproduction 2021; 162: 19-33.

37) Huang Y, Cheng Y, Zhang M, et al. Oxidative stress and inflammatory markers in ovarian follicular fluid of women with diminished ovarian reserve during in vitro fertilization. J Ovarian Res 2023; 16: 206.

38) Videla LA. Energy metabolism, thyroid calorigenesis, and oxidative stress: functional and cytotoxic consequences. Redox Report 2000; 5: 265-275.

39) Khinteel Jabbar N, Al-Abady ZN, Jasib Thaaban Almzail A, Al-Athary RAH. Relationship between menopause and complications of hyperthyroidism in female patients in Iraq. Arch Razi Inst 2022; 77(4): 1481-1489.

40) Di Mascio P, Murphy ME, Sies H. Antioxidant defense systems: the role of carotenoids, tocopherols, and thiols. Am J Clin Nutr 1991; 53: 194-200.

41) Ye Z, Wu H, Chen X, et al. Puerarin inhibits inflammation and oxidative stress in female BALB/c mouse models of Graves' disease. Transl Pediatr 2024; 13: 38-51.

42) Trabace L, Zotti M, Morgese MG, et al. Estrous cycle affects the neurochemical and neurobehavioral profile of carvacrol-treated female rats. Toxicol Appl Pharmacol 2011; 255: 169-175.

43) Mahran YF, Badr AM, Aldosari A, Bin-Zaid R, Alotaibi HN. Carvacrol and thymol modulate the cross-talk between TNF-α and IGF-1 signaling in radiotherapy-induced ovarian failure. Oxid Med Cell Longev 2019; 2019: 3173745.

44) Sharifi-Rad M, Varoni EM, Iriti M, et al. Carvacrol and human health: A comprehensive review. Phytother Res 2018; 32: 1675-1687.

45) Aristatile B, Al-Assaf AH, Pugalendi KV. Carvacrol suppresses the expression of inflammatory marker genes in D-galactosamine-hepatotoxic rats. Asian Pac J Trop Med 2013; 6: 205-211.

46) Sahin N, Delibas IB, Unal Isaoglu BS, et al. The effect of carvacrol on oxido-inflammatory ovarian injury and infertility induced by ischemia-reperfusion in rats. Clin Exp Obstet Gynecol 2022; 49: 058.