Introduction
Vinylcyclohexene diepoxide (VCD) is an ovotoxic catabolite of 4-vinylcyclohexene (VCH), an intermediate employed in the manufacturing of flame retardants, pesticides, adhesives and synthetic rubber (Cao et al., Reference Cao, Liu, Lu, Lv, Leung, Du, Wang, Xiong, Su, Li, Chen, Ma and Chan2020; Li et al., Reference Li, Li, Song, Lei, Zhou, Zhang, Sun, Zhang and Zhang2024) VCD, utilized commercially within industrial environments, is categorized as a carcinogenic substance in accordance with the Hazardous Substances Data Bank (HSDB) (Sen Halicioglu et al., Reference Sen Halicioglu, Saadat and Tuglu2021). Research conducted by the National Toxicology Program (NTP) on the carcinogenic potential of VCD has indicated that, aside from its carcinogenic effects, it also exhibits ovotoxic effects (National Toxicology Program, Reference Program1986).
VCD has been demonstrated to augment degeneration within ovarian tissue in rats by promoting oxidative stress via lipid peroxidation and hydrogen peroxide formation (Sen Halicioglu et al., Reference Sen Halicioglu, Saadat and Tuglu2021). Reactive oxygen species (ROS) induce alterations in cellular membrane permeability, activate pro-apoptotic signalling molecules and consequently lead to cellular apoptosis (Song et al., Reference Song, Qiu, Li, Sun and Chen2023). Exposure to VCD results in an increase in intracellular ROS, and oxidative stress induces DNA damage and apoptosis in granulosa cells and oocytes (Zhou et al., Reference Zhou, Jin, Wang, Li, Yang, Wu and Chen2023).
A study conducted by Mark-Kappeler et al., demonstrates that VCD exerts its effects directly through the KITLG/KIT pathway, rather than non-specifically influencing multiple signalling pathways involved in follicle viability or growth. The same study indicates that the direct targeting of KIT by VCD may contribute to its physiologically specific effect (Mark-Kappeler et al., Reference Mark-Kappeler, Sen, Lukefahr, McKee, Sipes, Konhilas and Hoyer2011). Another study has also corroborated the essential role of KIT and its signalling pathway in the activation and survival of primordial follicles (PMFs), in addition to its involvement in VCD-induced ovotoxicity (Kappeler et al., Reference Kappeler, Sipes and Hoyer2011).
Springer et al., observed that VCD results in oocyte destruction, especially within small preantral follicles, and proposed that modifications in protein synthesis may constitute a contributing mechanism. Considering the limited inhibition of overall protein synthesis, it is probable that VCD influences the expression of particular genes encoding a small subset of proteins within these follicles (Springer et al., Reference Springer, Flaws, Sipes and Hoyer1996a, Roosa et al., Reference Roosa, Mukai and Place2015)
Due to its pronounced ovotoxic effect, VCD is extensively utilized to establish animal models of menopause. This model circumvents the need for surgical intervention and results in menopause-like effects, such as a gradual decrease in estrogen (E2) and the retention of the ovarian tissue’s capacity to produce androgens – effects that cannot be replicated by the surgically castrated model. Additionally, it enables differentiation between the effects of ageing and ovarian hormone decline and can induce an irreversible apoptotic mechanism that closely resembles the follicular atresia process observed in women (Kao et al., Reference Kao, Sipes and Hoyer1999; Zhang et al., Reference Zhang, Yan, Yang, Ma, Li, Wang, Pan and Sun2016; Pestana-Oliveira et al., Reference Pestana-Oliveira, Carolino, Kalil, Mota Leite, Dalpogeto, De Paula, Collister and Anselmo-Franci2021; Craig et al., Reference Craig, Keating, Flaws and Brooks2026)
The term ovarian reserve denotes the reservoir of PMFs established during fetal development; approximately 85% of these are lost prior to birth. The number of follicles persistently diminishes throughout the reproductive lifespan, with approximately 450 follicles reaching ovulation, while the remaining follicles undergo atresia (Houeis et al., Reference Houeis, Donnez and Dolman2025). The decline in female fertility with advancing age is a natural physiological process associated with a decrease in both the quantity and quality of oocytes – referred to as diminished ovarian reserve (DOR). Premature ovarian insufficiency (POI) is characterized by menopausal symptoms manifesting prior to the age of 40, indicating early ovarian dysfunction (Mao et al., Reference Mao, Yang, Wang and Cao2026).
Oocytes develop within a relatively hypoxic environment in the ovarian cortex and are exposed to minimal levels of ROS owing to their quiescent metabolic state. The accumulation of elevated ROS levels in oocytes has been linked to the pathogenesis of primary ovarian insufficiency (POI) (Tiosano et al., Reference Tiosano, Mears and Buchner2019). Plasma ROS levels have been demonstrated to be markedly elevated in patients with POI relative to control subjects (Venkatesh et al., Reference Venkatesh, Kumar, Sharma, Kriplani, Ammini, Talwar, Agarwal and Dada2010).
One of the most prevalent oxidation products in genomic DNA is the 8-oxo-7,8-dihydroguanine (8-oxoG) base lesion, which serves as one of the most reliable biomarkers of oxidative stress. 8-OxoG exhibits mutagenic properties. The corresponding enzyme that specifically recognizes and repairs 8-oxoG and its open-ring derivative, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), is known as 8-oxoguanine DNA glycosylase 1 (OGG1) (Wang et al., Reference Wang, Li, Qiao, Xue, Zheng, Chen, Zeng, Liu, Boldogh and Ba2018). OGG1 is a DNA glycosylase enzyme possessing apurinic/apyrimidinic (AP) site lyase activity that facilitates the removal of ROS-induced 8-oxoG. Through the process of base excision repair, 8-oxoG is identified and excised, followed by the recruitment of other proteins to finalize the repair. The elimination of 8-oxoG is essential to prevent genomic instability and to ensure accurate gene transmission across generations. OGG1 plays a regulatory role in maintaining homeostasis via various pathways, including DNA repair, oxidative stress response, inflammatory response, fibrosis, mitophagy, apoptosis, autophagy and energy metabolism (Visnes et al., Reference Visnes, Cázares-Körner, Hao, Wallner, Masuyer, Loseva, Mortusewicz, Wiita, Sarno, Manoilov, Astorga-Wells, Jemth, Pan, Sanjiv, Karsten, Gokturk, Grube, Homan, Hanna, Paulin, Pham, Rasti, Berglund, von Nicolai, Benitez-Buelga, Koolmeister, Ivanic, Iliev, Scobie, Krokan, Baranczewski, Artursson, Altun, Jensen, Kalderén, Ba, Zubarev, Stenmark, Boldogh and Helleday2018; Zhao et al., Reference Zhao, Zhu, Shi and Wu2022).
The inhibition of OGG1 has been demonstrated to induce oxidative stress in the granulosa cell line. However, in certain cell types, this inhibition appears not to exert a significant influence on ROS. Consequently, it is hypothesized that the impact of OGG1 on oxidative stress may be contingent upon the specific cell type (Xia et al., Reference Xia, Wu, Wu and Yang2022).
DNA damage induced by oxidative stress has been identified as a significant cause of cell death, which is aetiologically associated with ischaemic injury and degenerative modifications. However, the role of OGG1-BER in oxidative stress-induced cellular apoptosis remains inadequately explored (Wang et al., Reference Wang, Li, Qiao, Xue, Zheng, Chen, Zeng, Liu, Boldogh and Ba2018). In addition to targeting DNA, oxidative stress can also impact proteins such as OGG1. Under conditions of oxidative stress, the DNA repair function of OGG1 is diminished. OGG1 can restore its repair activity once redox balance is reestablished re-established (Zhao et al., Reference Zhao, Zhu, Shi and Wu2022).
In a particular study, plasma OGG-1 levels in women with DOR were observed to be elevated in comparison to the general population (Xia et al., Reference Xia, Wang, Zou, Jin, Wu and Yang2023). In a different study, diminished expression of OGG1 was shown to decrease cell death through the mediation of the PARP1 molecule. It has been established that PARP-dependent cell death contributes to the pathophysiology associated with reduced ovarian reserve (Batnasan et al., Reference Batnasan, Xie, Zhang and Li2020; Stringer et al., Reference Stringer, Alesi, Winship and Hutt2023). The results of these studies suggest that increased levels of OGG-1 are correlated with a reduced ovarian reserve.
In our study, building upon the established relationship between DOR and OGG-1 identified in prior research, our objective is to elucidate the correlation among the toxicity induced in VCD-treated rats, the ovarian follicle count and the levels of OGG-1.
Materials and methods
Animals
A total of twenty-four female Wistar rats, aged between 6 and 8 weeks, were procured from TECHCERT Laboratory Animals (Ankara). They were maintained in a controlled environment at a temperature of 22 ± 2°C, adhering to a twelve-hour dark/light cycle, with unrestricted access to food and water. Ethical approval for all experimental procedures was granted by the TECHCERT Laboratory Animals Local Ethics Committee, under the ethics committee number EKN.0042/2024/REV.01 and the decision number KN.0042/2024.
This study was supported by the University of Health Sciences Scientific Research Projects Coordination Unit with project number 2025/014.
VCD administration
Following a one-week acclimatization period, the subjects were randomly assigned to three groups. The VCD group was administered intraperitoneal (IP) injections of VCD (Sigma–Aldrich, Saint Louis, MO) at a dosage of 160 mg/kg/day over a span of 15 days. VCD was dissolved in DMSO and administered in a volume of 0.1 ml. The Sham group received an equivalent volume of DMSO via IP injection. To mitigate peritoneal irritation caused by DMSO, it was diluted with saline at a dose corresponding to 160 mg/kg (Figure 1). The control group did not receive any injections. During VCD treatment, the weight of each animal was measured on alternate days to determine the appropriate VCD dose. To examine the effects of VCD on estrous cyclicity, vaginal smears were collected daily using cotton swabs for 5 consecutive days (between days 71 and 75 post-injection) and stained with cresyl violet for microscopic evaluation.
Experimental design and timeline.
Collection of ovarian and blood specimens
The rats were sacrificed on day 60 following 15 days of injections conducted during the diestrus cycle. An anaesthetic mixture comprising 90 mg/kg ketamine and 10 mg/kg xylazine was employed. While under deep anaesthesia, intracardiac blood was collected, after which euthanasia was performed via cervical dislocation. Once the blood had coagulated, it was centrifuged at 4000 rpm for 10 minutes; the resultant serum was transferred to Eppendorf tubes and stored at −80°C until subsequent ELISA analyses. Following dissection of the ovaries, the right ovary of each rat was fixed in 10% formaldehyde solution for histochemical examination. The left ovaries were stored at −80°C for ELISA analyses.
Histochemistry
The ovaries, immersed in a 10% formaldehyde solution for histological examination, were processed through standard histological procedures and stained using Hematoxylin & Eosin. Follicles within the stained ovaries were classified and quantified according to the Pedersen classification. Based on this classification, PMFs consist of a single layer of flattened epithelial cells surrounding the oocyte; in primary follicles, the flattened cells transition to cuboidal form but remain in a single layer; in secondary follicles, cuboidal granulosa cells organize into multiple layers; and in tertiary follicles, an antral cavity has developed.
Follicles containing a degenerate oocyte with pyknotic nuclei or vacuolated cells in the granulosa cells were classified and counted as atretic.
Sections of 8 µm thickness were included in the follicle count using 1-in-10 sampling. Only follicles in which the nucleus could be distinguished were counted. The resulting counts were then multiplied by 10 to obtain the final follicle count (Tilly et al., Reference Tilly2003; Gül et al., Reference Gül, Gül, Otlu, Erdemli and Gözükara Bağ2024).
Immunohistochemistry
Sections with a thickness of 4 µm were prepared for immunohistochemical (IHC) staining, employing a 1-in-40 sampling methodology. Randomly selected sections from the slides ensured unbiased sampling from each animal. The staining procedure utilized an OGG-1 primary antibody (Thermo Fisher OGG1 Polyclonal EA, Cat No: PA131402), an immunohistochemistry staining kit (Thermo Fisher UltraVision Detection System Large Volume Anti Polyvalent HRP, Ref: TP-125-HL), and DAB chromogen (Thermo Fisher UltraVision Detection System Large Volume DAB Substrate System, Ref: TA-125-HD). Counterstaining was conducted using Mayer’s haematoxylin. One section from the VCD group was stained by excluding the incubation with the primary antibody during the protocol to serve as a negative control. The areas stained brown with DAB were evaluated.
ELISA
ELISA analyses were performed in accordance with the kit procedures. The sandwich ELISA technique was used. Rat FSH ELISA Kit (Catalog No: ABT3021Ra), Rat AMH ELISA Kit (Catalog No: ABT3376Ra) and Rat OGG1 ELISA Kit (Catalog No: ABT2136Ra) were used. Estradiol measurement was performed using the immunoassay method on a Cobas 801 analyzer.
Serum samples were used in all ELISA kits; only the OGG1 kit was run with both serum and tissue samples.
Statistical analyses
Statistical analyses were conducted utilizing IBM SPSS Statistics, Version 25.0. The assessment of data distribution normality was performed via the Shapiro–Wilk test. For data conforming to parametric test assumptions, mean and standard deviation values were computed. One-Way ANOVA was employed for comparisons, with Tukey’s HSD test used for pairwise evaluations. For data not exhibiting normal distribution, medians, as well as minimum and maximum values, were determined. The Kruskal–Wallis test facilitated group comparisons, whilst the Mann–Whitney U test was used for pairwise assessments among non-parametric datasets. To mitigate the risk of Type I errors due to multiple comparisons, a Bonferroni correction was applied, establishing the significance threshold at P < 0.0167.
Results
Evaluation of vaginal cytology over a 5-day monitoring period revealed significant differences in the duration of the diestrus phase among the groups (Kruskal–Wallis test, P < 0.05). Post-hoc pairwise comparisons with Bonferroni correction demonstrated that rats in the VCD-treated group spent significantly more days in the diestrus phase compared to both the Control group (adjusted P = 0.029) and the Sham group (adjusted P = 0.029). No statistical difference was observed between the Control and Sham groups (adjusted P = 1.000). This prolongation of the diestrus phase confirms functional estrous cycle disruption and anovulatory impairment induced by VCD administration at day 60 post-treatment.
In the histopathological examination of sections taken from the ovaries, clusters of PMFs were particularly notable in the Sham and control groups (Figure 2A). In the VCD group, PMFs were seldom observed, and numerous atretic follicles were noted (Figure 2B). In addition to these findings, the prevalence of haemorrhagic areas in the VCD group was striking.
Representative micrographs of ovarian sections stained with Hematoxylin & Eosin (H&E). A. Primordial follicles within the circle and a primary follicle indicated by an arrow in the Sham group (x20, scale bar = 50 µm). B. Atretic follicles (black asterisks) in the VCD group (x20, scale bar = 50 µm).
The quantity of PMFs was observed to be reduced in the VCD group in comparison to both the control and Sham groups (P < 0.001). Furthermore, a statistically significant difference was present between the control and Sham groups (P < 0.01), with the overall variation among the groups was statistically significant (Figure 3A). Primary and secondary follicle counts were also lower in the VCD group compared to the Sham and control groups. The differences between groups were statistically significant for both follicle types (P < 0.001 and P < 0.01) (Figure 3B and C).
Effects of VCD administration on ovarian follicle counts. A. Primordial, B. primary, C. secondary and D. antral follicle counts in Control, Sham and VCD groups. Data are presented as mean ± standard error (SE). (**P < 0,01; ***P < 0,001) (Control n = 8; Sham n = 8; VCD n = 8).
Antral follicles included counted tertiary follicles and preovulatory follicles. When follicle counts were compared, no significant difference was found between groups (P > 0.05) (Figure 3D). When examining atretic follicle counts, it was observed that the number of atretic follicles in the VCD group was greater than that in the Sham and control groups, and this difference was determined to be statistically significant (P < 0.01) (Figure 4). There were no significantly differences between Sham and control groups about atretic follicles (P = 0.676).
Distribution of atretic follicle counts by group (SE: standard error) (***P < 0,001) (Control n = 8; Sham n = 8; VCD n = 8).
Serum estradiol (E2) measurement was conducted utilizing the immunoassay technique. Although estrogen levels appeared marginally lower in the VCD group, no statistically significant difference was identified between the groups. Furthermore, there were no differences between Sham and control group’s serum estradiol levels (P > 0.05). Anti-Müllerian hormone (AMH) and follicle-stimulating hormone (FSH) levels were measured from serum samples employing the enzyme-linked immunosorbent assay (ELISA) method. No significant differences were observed between the groups for either hormone (P > 0.05).
Serum OGG-1 levels were quantified utilizing the ELISA technique. The levels of serum OGG1 were observed to be elevated in the VCD group relative to the other groups; however, this difference was statistically significant solely between the VCD and Sham groups (P < 0.05). No statistically significant difference was observed between the control and VCD groups, nor between the control and sham groups (P > 0.05) (Figure 5A).
OGG1 levels in serum and ovarian tissue measured by ELISA. A. Box-plot representation of serum OGG1 levels across groups. The horizontal line within the box indicates the median value. B. OGG1 tissue levels presented as mean ± standard error (SE). (*P < 0,05) (Control n = 8; Sham n = 8; VCD n = 8).
Tissue homogenates prepared from the ovaries indicated that OGG1 levels, as measured by ELISA, were substantially higher in the VCD group in comparison to the Sham and control groups (P < 0.016) (Figure 5B). In tissue sections immunohistochemically stained for OGG-1, OGG-1 positivity was observed to be higher in the VCD group compared to the control and Sham groups (Figure 6A,B,C).
OGG1 immunohistochemical staining in ovarian sections. Representative micrographs showing OGG1 immunoreactivity in the A. VCD group (scale bar = 500 µm), B. Control group (scale bar = 750 µm) and C. Sham group (scale bar = 500 µm). (*P < 0,05).
Discussion
Rats are among the most used experimental animals for modelling the female genital system. Rodents do not naturally undergo menopause and have a considerably shorter menstrual cycle compared to humans. In rats, follicle development up to postnatal (PN) day 7 is independent of pituitary gonadotropins. During the early infantile period (PN days 8–15), atypical antral follicles develop, and these follicles are sensitive to gonadotropins. During the late infantile period (PND 15–20), antral follicles shed their atypical morphology and undergo maturation. In the peripubertal period (PND 33–37), a bimodal fluctuation pattern of LH commences, accompanied by an elevation in FSH levels. The stages spanning PN days 36–48 are indicative of the pubertal phase (Picut et al., Reference Picut, Dixon, Simons, Stump, Parker and Remick2015). There are no precise data on the duration required for follicles to mature from primordial to antral stages in rats; however, autoradiographic studies in mice utilizing zona pellucida labelling have demonstrated that a primary follicle requires approximately 5–6 weeks prior to ovulation (Zheng et al., Reference Zheng, Zhang and Liu2014).
In our study, rats in the pubertal period were utilized. Following 15 days of VCD administration, the subjects were monitored for a duration of 60 days. This interval encompasses approximately 10–12 ovarian cycles in rats, thereby offering adequate opportunity to observe variations in ovarian cycle parameters. In rats, folliculogenesis from PMF activation to ovulation lasts an average of 60 days. Specifically, the growth from the primordial to the secondary follicle stage takes approximately 30 days, while the transition from a secondary to an antral follicle requires an average of 28 days. Finally, an additional 2 to 3 days are needed for an antral follicle to develop into a preovulatory follicle. (McGee et al., Reference McGee and Hsueh2000)
Various methods are also available for creating POI or DOR models. Ovariectomy has been employed to establish a menopause model; however, it results in an abrupt cessation of female sex hormones and fails to replicate the gradual hormonal transition observed in ovarian ageing. One chemical approach involves the administration of cyclophosphamide, which is not selective for primordial and primary follicles and depletes the entire ovarian reserve rapidly. The most appropriate method for modelling DOR or menopause is considered to be VCD administration. VCD is selective for the atresia of primordial and primary follicles and can mimic both the hormonal transition period and endocrine changes. However, it may take a long time for hormonal changes to become apparent with VCD administration. VCD can also be administered subcutaneously, but in approximately 90% of studies it has been administered intraperitoneally, generally for 2–3 weeks, most commonly at doses of 80 and 160 mg/kg (Kao et al., Reference Kao, Sipes and Hoyer1999; Roosa et al., Reference Roosa, Mukai and Place2015; Zhang et al., Reference Zhang, Yan, Yang, Ma, Li, Wang, Pan and Sun2016; Pestana-Oliveira et al., Reference Pestana-Oliveira, Carolino, Kalil, Mota Leite, Dalpogeto, De Paula, Collister and Anselmo-Franci2021; Craig et al., Reference Craig, Keating, Flaws and Brooks2026). In our study, IP injections of 160 mg/kg were administered for 15 days to rats aged 6–8 weeks.
VCD is recognized for exerting an atretic effect on primordial and primary follicles. It diminishes the follicular reserve through direct binding to the c-KIT receptor within the follicles, thereby activating the pro-apoptotic Bcl-2 and MAPK pathways (Özel et al., Reference Özel, Kiray, Göker, Aydemir and Mıcılı2020). VCD must be administered for a certain period to exert its atretic properties. One study has shown that a single dose of VCD has a protective effect on follicles (Borman et al., Reference Borman, VanDePol, Kao, Thompson, Sipes and Hoyer1999). In the literature, 2-week administration protocols have generally been preferred. A significant increase in atretic PMFs is observed around day 12 (Kao et al., Reference Kao, Sipes and Hoyer1999).
In a dose study conducted in rats, VCD administered at 80 mg/kg was reported to cause a greater reduction in secondary follicle count compared to VCD administered at 160 mg/kg (Muhammad et al., Reference Muhammad, Goode, Kock, Arifin, Cline, Adams, Hoyer, Christian, Isom, Kaplan and Appt2009). It has been demonstrated that the administration of VCD for a duration of 6–8 days does not exert a significant effect on follicular development. However, a notable decline in primordial and primary follicles becomes apparent starting from day 15 onwards (Springer et al., Reference Springer, McAsey, Flaws, Tilly, Sipes and Hoyer1996b). One study demonstrated that following 15 days of VCD administration, a reduction of approximately 50–70% was observed in small follicles, while no decrease occurred in secondary and antral follicle counts (Devine et al., Reference Devine, Sipes and Hoyer2001).
In our study, primordial, primary and secondary follicles decreased in the VCD-treated group compared to the other groups, while the number of atretic follicles increased. No difference was observed between groups in antral and tertiary follicle counts. This was interpreted, in line with other studies, as an indicator of VCD’s selectivity among follicle types.
In our study, AMH levels were observed to be lower exclusively in the VCD group relative to the control group, whereas FSH levels were found to be elevated solely in the VCD group in comparison to the Sham group. E2 values were lower in the VCD group compared to the other groups; however, none of these differences in hormone levels were statistically significant. Research has indicated that a prolonged period may be necessary before a fully established picture of POI becomes evident. The appropriate markers to identify this early stage of POI have not yet been established. Patients at this stage may present to the clinic for various reasons, including menstrual irregularities, unexplained infertility or failure of IVF. In this condition, commonly known as occult POI, hormone levels such as FSH and E2 are maintained within normal ranges (Shestakova et al., Reference Shestakova, Radzinsky and Khamoshina2016). Occult POI may develop insidiously before the onset of oligo/amenorrhoea and might remain unnoticed until patients seek medical consultation for infertility. Furthermore, there is no established AMH value for the diagnosis of occult POI (Guzel et al., Reference Guzel, Aba, Yakin and Oktem2017). When reviewed in light of the literature, the hormonal picture obtained from our experimental results appears consistent with occult POI.
It is evident that hormonal changes observed following VCD administration differ significantly across various studies. Elevated FSH levels relative to controls are often anticipated in models of POI or menopause; however, the variation in FSH levels among adult rats is notable. Early alterations in FSH are observed in adult rats aged 2–3 months, whereas the duration required for FSH changes may be extended in younger rats. Although some studies have indicated a substantial increase in FSH beginning from day 5, the majority of research suggests that a period of 100 to 120 days is necessary to observe a significant rise in FSH levels (Ekapatria et al., Reference Ekapatria, Permadi, Tobing, Dhamayanti and Mose2022; Mayer et al., Reference Mayer, Pearsall, Christian, Devine, Payne, McCuskey, Marion, Sipes and Hoyer2002). In our study, consistent with the literature, no significant difference in FSH was observed in rats sacrificed on day 60 after VCD administration.
Although alterations in E2 are detected earlier in mice, a reduction in E2 levels is observed at a later stage in rats. Two studies have indicated a substantial decline in serum E2 levels at days 30 and 85 following VCD administration in rats. Conversely, other investigations assessing E2 levels at days 40, 55 and 70 after VCD administration reported no statistically significant differences (Mashimo et al., Reference Mashimo, Oshida, Oka, Kawabata, Takasu, Nihei, Kojima, Kanemura and Murata2025). Carolina et al., demonstrated that estrogen changes in VCD-treated rats were transient, with estrogen unaffected at days 70, 80 and 100, but estrogen levels changed at day 90 (Carolino et al., Reference Carolino, Barros, Kalil and Anselmo-Franci2019). However, there are numerous studies in which no change in estrogen levels was recorded at all (Thompson et al., Reference Thompson, Sipes, Greenstein and Hoyer2002; Reis et al. Reference Reis, Pestana-Oliveira, Leite, Lima, Brandão, Graeff, Del-Ben and Anselmo-Franci2014). The absence of a significant difference in E2 levels between groups in our study is consistent with many rat studies in the literature.
Although less than 60 days had elapsed following VCD administration, numerous studies demonstrate a decline in AMH levels; however, other studies indicate that AMH levels do not change despite 15 days of VCD treatment in rats (Zhao et al. Reference Zhao, Lian and Wang2017; Ozatik et al., Reference Ozatik, Ozatik, Yigitaslan, Kaygısız and Erol2020). In our study, no significant difference was found between groups for AMH levels. AMH is a hormone secreted by antral follicles. Since no significant difference was found between groups regarding the number of antral follicles in our study, consistent results were obtained for both AMH and antral follicles.
VCD additionally promotes follicular atresia, primarily by inducing oxidative stress. Özel F. et al., demonstrated that VCD administration elevated oxidative stress markers in ovarian tissue (Özel et al., Reference Özel, Kiray, Göker, Aydemir and Mıcılı2020). VCD exerts its destructive effects not only directly on oocytes but also through granulosa cells. Oxidative stress also stimulates the inflammatory response in the body. VCD has been shown to increase inflammatory markers alongside oxidative stress (Abolaji et al., Reference Abolaji, Adedara, Abajingin, Fatunmibi, Ladipo and Farombi2016; Sen Halicioglu et al., Reference Sen Halicioglu, Saadat and Tuglu2021; Li et al., Reference Li, Li, Song, Lei, Zhou, Zhang, Sun, Zhang and Zhang2024)
In a study, women with DOR were observed to possess higher serum OGG-1 levels compared to the control group (Xia et al., Reference Xia, Wang, Zou, Jin, Wu and Yang2023). However, this height has not been validated for the over tissue. The microenvironment of oocytes or the molecular level in any tissue does not necessarily correlate with serum levels. In our investigation, the OGG-1 levels were assessed through IHC methods. It was observed that, in the rat group subjected to VCD, which exhibited a reduction in ovarian reserve, OGG-1 positivity was higher relative to the control and sham groups. This height has also been confirmed by the ELISA method.
In the analysis of serum OGG-1 levels, it was observed that OGG-1 levels in the VCD group were significantly higher solely in comparison to the sham group (P < 0.05), while no statistical difference was identified between the control group and other groups. This pattern can be elucidated by the systemic pharmacological properties of dimethyl sulfoxide (DMSO), used as the vehicle agent. DMSO is a potent free-radical scavenger and anti-oxidative compound at low doses (Di Giorgio et al., Reference Di Giorgio, Hou, Zhao, Zhang, Lyeth and Russell2008), (Bulama et al., Reference Bulama, Nasiru, Bello, Abbas, Nasiru, Saidu and Suleman2022). Given that serum OGG-1 reflects global systemic oxidative stress rather than isolated ovarian tissue damage, the reduced baseline serum OGG-1 levels in the Sham group (compared to untreated Controls) align directly with DMSO’s anti-oxidative buffering action. Conversely, despite the co-administration of DMSO in the VCD group, serum OGG-1 levels significantly elevated above the Sham baseline. This confirms that VCD-induced oxidative damage surpassed the protective threshold of DMSO. The absence of a statistically significant difference between the VCD and untreated Control groups is likely attributable to this vehicle-mediated mitigation of systemic oxidative stress. Therefore, while tissue-level OGG-1 remains a robust marker of localized follicular damage, serum OGG-1 trends must be interpreted within the context of systemic antioxidant dynamics.
Although the differences between the Sham and Control groups did not achieve statistical significance (P > 0.05), a observable trend of improved baseline parameters was noted in the Sham group, including slightly higher primordial, primary, and secondary follicle counts and reduced OGG-1 levels. We hypothesize that this trend arises from two synergizing mechanisms. First, the systemic antioxidant effect of DMSO may prevent physiological follicular atresia by scavening ambient ROS, thereby preserving early-stage follicles. Second, repeated IP injections induce mild local abdominal micro-inflammation and mechanical stimulus around the ovarian stroma. Localized inflammatory cascades can stimulate microvascular recruitment, growth factor release and transiently enhance early folliculogenesis. These factors combined likely provided a subtle protective microenvironment in the Sham group. (Chang, Reference Chang2023), (Duffy et al., Reference Duffy, Ko, Jo, Brannstrom and Curry2019)
In the rat ovary, the transition from a PMF to a fully mature preovulatory follicle takes approximately 60 days (with primordial-to-secondary growth requiring ∼30 days, secondary-to-antral transition taking ∼28 days, and final preovulatory maturation requiring 2–3 days). McGee et al., Reference McGee and Hsueh2000) At the 60-day post-induction assessment, serum E2, AMH and FSH levels exhibited no significant differences among groups (P > 0.05). However, our evaluation of vaginal cytology demonstrated severe estrous cycle irregularities and persistent diestrus arrest in VCD-treated rats compared to control groups. This functional disturbance, combined with the histological depletion of primordial and growing follicle pools, confirms that 60 days post-VCD treatment captures the precise transition into the ‘Occult POI’ stage – where endocrine feedback mechanisms remain transiently compensated despite structural and functional ovarian reserve decline.
Conclusion
Our study demonstrated that exposure to VCD, in conjunction with the resultant reduction in ovarian reserve, elevates the expression level of OGG-1 in ovarian tissue. Serum level analyses revealed a significant difference between the VCD and Sham groups, whereas no significant difference was observed between the VCD and control groups. We hypothesize that OGG-1 may be unable to exert its anti-apoptotic effect due to the excessive ROS caused by VCD, or it may be triggering follicle depletion via PARP. Therefore, OGG-1 could serve as an indicator for predicting primordial and primary follicle reserves that are not biochemically detectable. However, since the OGG-1 protein is not ovary-specific and can be influenced by numerous parameters, its serum level may not consistently correlate with protein expression in the tissue.
In conclusion, the findings demonstrate that VCD-induced follicular damage activates DNA repair mechanisms at the tissue level via OGG-1. The ability to estimate the existing ovarian reserve before the onset of clinical symptoms is essential for determining both diagnostic and therapeutic approaches. Accordingly, considering that OGG-1 displays multiple isoforms specific to various organelles or tissues, it is our belief that additional research focusing on isoforms potentially more specific to the ovary could be beneficial in guiding diagnostic and therapeutic approaches.
Acknowledgement
None.
Funding
This work was supported by the University of Health Sciences Scientific Research Projects Coordination Unit with project number 2025/014.
Competing interests
The authors declare none.
Ethical standards
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals.
