ENDOMETRIOSIS AND OVARIAN AGING: A PILOT STUDY EXPLORING OXIDATIVE STRESS AND DNA DAMAGE MARKERS

Purpose To assess whether specific markers of oxidative stress and DNA damage are expressed at higher levels in primordial follicles of patients with endometriomas compared to age-matched controls.

A retrospective pilot cohort study was conducted using ovarian tissue from patients who underwent unilateral or bilateral salpingo-oophorectomy for symptomatic endometriomas, with controls undergoing an identical procedure for other benign gynecologic conditions. Immunohistochemistry (IHC) was used to assess percent positivity in primordial fol- licles for 4-hydroxy-2-nonenal (4-HNE) as a marker of oxidative stress; 8-Oxoguanine (8-Oxo) and phosphorylated H2AX (γH2AX) for DNA damage. Chi-square tests were used for categorical variables and Student t-tests for continuous variables, with p < 0.05 considered significant.

Twenty-nine patients were included (8 controls, 21 with endometriomas). Age (39.4 ± 5.1 vs. 37.1 ± 4.6 years, p = 0.269), BMI (33.6 ± 10.6 vs. 29.5 ± 7.8 kg/m2, p = 0.310) were similar between groups. Racial distribution, nulliparity, hormonal therapy use, and smoking status were also comparable. The average endometrioma size was 6.6 ± 2.8 cm. All molecular markers showed higher percent positivity in the endometrioma group compared to the control group. γH2AX demonstrated a statistically significant increase (63.6% vs. 50.1%, p = 0.033). 8-Oxo (67.5% vs. 55.8%, p = 0.070) and 4-HNE (65.1% vs. 61.8%, p = 0.168) were also elevated in the endometrioma group but did not reach statistical significance.

Patients with endometriomas demonstrated increased follicular DNA damage, with elevated γH2AX expression, supporting the notion that endometriomas may impair follicular quality through DNA damage mechanisms.

Endometrioma · Primordial follicle · Oxidative stress · DNA damage · Ovarian aging

Endometriosis is a chronic inflammatory condition affect- ing 1 in 10 reproductive-aged women [1]. While sometimes asymptomatic, 25–50% of infertile women have endome- triosis, making them 6 to 8 times more likely to have the disease than fertile women [1 , 2]. Among women with chronic pelvic pain, prevalence ranges from 30 to 80% [1 , 2]. The inflammation can affect all pelvic organs, causing adhesions and anatomical distortion that can lead to pain but also infertility with decreased ovarian reserve or implanta- tion failure [2 –4]. Endometriomas, also known as ovarian endometrial cysts, are the most common subtype, affecting 44% of women worldwide and have been linked to decreased ovarian reserve and accelerated ovarian aging [5]. Despite extensive research, a clear causal link between endometriosis and infertility remains elusive [1 ]. A * Luce A. Kassi [email protected] * Kathryn Grive [email protected] Sanjana Konda [email protected] Joyce Ou [email protected] Julia McAdams [email protected] Robin Cram [email protected] May-Tal Sauerbrun-Cutler [email protected] 1 Women and Infants Hospital of Rhode Island, Providence, United States 2 Warren Alpert Medical School of Brown University, Providence, United States Journal of Assisted Reproduction and Genetics meta-analysis reported lower anti-Müllerian hormone (AMH) levels in endometrioma patients compared to those with other benign ovarian cysts [3 ]. A longitudinal study also found an accelerated AMH decline in women with endometriomas compared to healthy controls [6 ]. Prior studies of endometriosis patients showed lower primordial follicle counts, proposing a mechanism similar to prema - ture ovarian insufficiency, though the exact cause remains unclear [3, 7, 8]. Multiple factors such as inflammation resulting in fibro- sis, oxidative stress, and DNA damage affect cellular regula- tion and folliculogenesis [5, 9–11]. One key reactive oxygen species (ROS) marker is 4-hydroxy-2-nonenal (4-HNE), a lipid-derived aldehyde formed by the oxidation of polyun- saturated fatty acid [12]. 4-HNE is a highly reactive and potent ROS and has been linked to several chronic diseases and inflammatory pathways [12]. Its effects range from dis- rupting calcium homeostasis to causing lethal cytotoxicity and DNA damage [12]. Despite its role in inflammatory pro- cesses, there are limited studies examining the potential role of 4-HNE in endometriosis. In another study, the ovarian cortex surrounding endo- metriomas showed excessive oxidative stress with increased 8-Oxoguanine (8-Oxo) expression [5]. Like 4-HNE, 8-Oxo is involved in ROS-related diseases including premature aging and cancer [13– 16]. It accumulates through ROS overload, inducing mutations often seen in cancers with DNA deficient repair mechanisms [13]. Studies examining 8-Oxo in endometriosis also remain limited [5 , 17]. Oxida- tive stress markers like 8-Oxo can also cause DNA damage and potentially affect primordial follicle activation through the Forkhead box O3 (FOXO3) pathway [18]. These findings make 8-Oxo a unique marker as its expression represents both oxidative stress but also the presence of DNA damage. Gamma-H2AX (γH2AX) is a well-established marker of DNA damage and represents a phosphorylated variant of the H2A protein family, a core component of the histone octamer within nucleosomes. Following the occurrence of double-stranded DNA breaks, histone H2AX undergoes rapid phosphorylation to form γH2AX, which accumulates and forms foci at the sites of damage [19– 22]. In ovarian tissue, these γH2AX foci can be detected within primordial follicles using immunofluorescence staining, providing an

means of assessing for follicular DNA damage [20, 21]. Despite its relevance as a biomarker of cellular stress and injury, data on γH2AX expression in patients with endometriosis also remain limited, highlighting the need for further investigation. Although prior studies have evaluated oxidative stress within ovarian tissue affected by endometriosis, limited data exist regarding the impact on primordial follicles, which is the follicular pool most directly associated with ovarian reserve and the reproductive lifespan. Understanding molecular injury within primordial follicles may provide important mechanistic insight into accelerated ovarian aging associated with endometriomas. Therefore, the objective of this study was to evaluate markers of oxidative stress and DNA damage within primordial follicles in ovarian tissue adjacent to endometriomas. Based on these findings, we also hypothesized that primordial follicles from patients with endometriomas would have increased DNA damage and oxidative stress compared to healthy controls.

Study design This was a pilot retrospective cohort study at a single aca- demic institution. The cohort included patients aged 18 to 45 years old who had undergone a unilateral or bilateral salpingo-oophorectomy between June 2006 and June 2024. Patients were identified through electronic medical records using diagnostic (ICD-9/ICD-10 – International Classifica- tion of Diseases) and surgical codes (CPT—Current Pro- cedural Terminology code). Patients were excluded if they had undergone chemotherapy or radiation, had a diagnosis of ovarian cancer or any prior malignancy, BRCA 1 and 2 mutation carriers, prior chemotherapy or radiation exposure, testosterone exposure, postmenopausal status and any con- current benign ovarian pathology (Fig.  1). Patient informa- tion was collected by chart review and included age, body mass index (BMI), race and ethnicity, obstetric history, smoking history, use of exogenous hormones, and speci- men pathology results. Data was recorded and stored on the Care New England REDCap secure online database v10.0.4. Primary antibodies The primary antibodies of choice for oxidative stress were 4-HNE (Thermo Fisher #MA5-27,570), with 8-Oxo (Sigma #MAB3560), and γH2AX (Sigma #07–164-25UG), serving as markers for DNA damage. Ubiquitin Carboxyl-terminal Hydrolase L1 (UCHL1) (Proteintech # 14,730–1-AP), an oocyte-specific marker, was also used to confirm the iden- tification of primordial follicles during IHC analysis [36]. Immunofluorescence Ovary sections of 70 subjects were obtained from Women & Infants Pathology paraffin-embedded, fixed in 1:10 for - malin, and sectioned at 5 μm onto glass slides. To stain, sections were deparaffinized and hydrated. Sections were washed in SafeClear (3 × 5 min), 100% EtOH (3 × 5 min), 95% EtOH (2 × 5 min), 70% EtOH (1 × 5 min), and water (1 × 5 min). Sections were heated for 20 min in 1% sodium Journal of Assisted Reproduction and Genetics citrate antigen-retrieval buffer, cooled for 20 min, and washed 2 × 5 min in 1 × phosphate-buffered saline with 0.1% Triton X (PBST). Tissue sections were blocked in PBST (Fisher Sci- entific), 1% bovine serum albumin (Sigma), 3% goat serum (Sigma) for 1 h. Sections were outlined in hydrophobic marker and incubated with primary antibodies against 4-HNE, 8-Oxo, γH2aX, and Ubiquitin Carboxyl-terminal Hydrolase L1 (UCHL-1) diluted at 1:100 overnight at 4 °C. The next day, the sections were then washed 3 × 5 min in PBST and incubated with secondary antibodies (mouse or rabbit) against γH2aX, 8-Oxo and 4-HNE in a dark for 1 h at room temperature. Sec- tions were then stained with 4',6-diamidino-2-phenylindole (DAPI) to visualize nuclei, mounted and analyzed on Zeiss Axio Imager inverted epifluorescence microscope (Fig.  2). Oocytes of primordial follicles were considered either positive or negative for the specific antibody. Immunohistochemical staining was initially performed on three slides per subject for each antibody. If analysis of these slides identified fewer than ten follicles, additional slides from the remaining available ovarian tissue were stained, up to a maximum of five total slides per antibody. Subjects with fewer than ten follicles iden- tified across all stained slides were excluded from the analysis; specifically, 9 of 17 subjects in the control group and 32 of 53 subjects in the endometrioma group were excluded (Fig.  1). Fig. 1 Flowchart of patient selection for the study. Among 950 initial patients who underwent oophorectomy, 70 met inclusion criteria for review. A total of 880 patients were excluded due to ovarian cancer, BRCA1/2 status, concurrent ovarian cyst, benign ovarian cyst, his- tory of chemoradiation, or postmenopausal status. Of the 70 eligible patients, 17 were classified as controls and 53 had histopathology- confirmed endometriomas. Patients with fewer than 10 identifiable primordial follicles on histologic evaluation were further excluded (9 controls and 32 endometrioma cases), yielding a final cohort of 8 control patients and 21 patients with endometriomas Journal of Assisted Reproduction and Genetics After exclusions based on primordial follicle identification and tissue availability, 29 subjects remained for the final analy- sis, including 8 controls and 21 subjects in the endometrioma group (Fig. 1). Statistical analysis For each marker (4-HNE, 8-Oxo, yH2AX), the primary out- come was percent positivity, defined as the number of posi- tively stained oocytes within primordial follicles divided by the total number of follicles per subject. Primordial follicles were identified based on established morphological criteria, includ- ing a single layer of flattened granulosa cells surrounding an oocyte without an antral space. Only follicles meeting these criteria were included in the analysis. UCHL-1, an oocyte- specific marker, was also used in conjunction with hematoxy- lin and eosin (H&E) stained slides to confirm primordial fol- licule identification and improve classification accuracy. IHC analysis was performed independently by two examiners, with one blinded to the group allocation. Inter-observer variability did not exceed 10%, and the average of the two measurements was used for analysis. Statistical analysis was performed in GraphPad Prism. Smoking status and use of exogenous hor- mones was analyzed by Chi Square test. Student’s t-test and a p-value < 0.05 were used.

Study population Based on diagnostic and surgical codes, 950 patients were initially identified during the time period (Fig.  1, Supplemental Table 1). After extensive chart review, 70 patients met inclusion criteria. A total of 880 patients were excluded due to the diagnosis of ovarian cancer, BRCA 1 and 2 mutation carriers, the presence of concurrent ovar - ian cysts, non-endometrioma benign ovarian cysts, a prior history of chemoradiation, or postmenopausal status. Of the 70 eligible patients, 17 were classified as controls and 53 had histopathology-confirmed endometriomas. Patients with fewer than 10 identifiable primordial follicles on histologic evaluation were further excluded (9 controls and 32 endometrioma cases), yielding a final cohort of 8 control patients and 21 patients with endometriomas (Fig.  1). Baseline demographic and clinical character - istics were similar between groups (Table  1). The mean age (37.1 ± 4.6 vs. 39.4 ± 5.1 years, p = 0.269), and BMI (29.5 ± 7.8 vs. 33.6 ± 10.6 kg/m2, p = 0.310) were compa- rable between patients with endometriomas and controls. Racial and ethnic distribution among the two groups was Fig. 2 Schematic overview of the immunohistochemical (IHC) stain- ing process to evaluate oxidative stress and DNA damage markers in ovarian tissue. Formalin-fixed, paraffin-embedded ovarian sec- tions were mounted on glass slides, stained with primary antibodies (4-HNE, 8-Oxo, γH2AX, UCHL-1), and detected using fluorescent secondary antibodies. Stained slides were visualized using a Zeiss Axio Imager inverted epifluorescence microscope Journal of Assisted Reproduction and Genetics also not significant (p = ns), with the majority identifying as White in both groups (Table  1). Nulliparity was more common among patients with endometriomas (38.1%) than controls (12.5%), although this difference did not reach statistical significance (p = 0.372). The use of current hor - monal therapy was similar between the groups (9.5% vs. 12.5%, p > 0.999). Smoking status distributions were also comparable, with most individuals reporting never smok - ing (p = 0.662). Among patients with endometriomas, the mean endometrioma size was 6.6 ± 2.8 cm (Fig.  3). Oxidative stress In IHC analyses of oxidative stress within oocytes of pri- mordial follicles, 4-HNE staining demonstrated a higher percent positivity in the endometrioma group compared to the control group; though this difference did not reach sta- tistical significance (65.1% vs. 61.8%; p = 0.168) (Fig.  4; 7A; Table  2). DNA damage IHC assessment of DNA damage with 8-Oxo staining within oocytes of primordial follicles showed a higher Table 1 Baseline demographic and clinical characteristics of patients with endometriomas compared to controls. Values are presented as mean ± standard deviation or number (percentage). Statistical comparisons were performed using Student t-tests with a p value set at 0.05 Characteristics Control (N = 8) Endometriosis (N = 21) P-value Age (years), mean (± SD) 39.4 (± 5.1) 37.1 (± 4.6) 0.269 BMI (kg/m2), mean (± SD) 33.6 (± 10.6) 29.5 (± 7.8) 0.310 Race/Ethnicity, n (%) 0.490 White/Caucasian 7 (87.5%) 17 (81.0%) Black/African American 1 (12.5%) 1 (4.5%) Hispanic/Latino 0 1 (4.5%) Asian 0 0 Not documented 0 2 (10.0%) Nulliparous, n (%) 1 (12.5%) 9 (38.1%) 0.372 Current hormonal therapy, n (%) > 0.999 Yes 1 (12.5%) 2 (9.5%) No 7 (85.5%) 19 (90.5%) Smoking status, n (%) 0.662 Never smoker 4 (57.1%) 12 (57.1%) Current or previous smoker 3 (42.7%) 8 (38.1%) Not documented 1 (14.3%) 1 (4.8%) Size of endometrioma (cm), mean (± SD) – 6.6 (± 2.8) Fig. 3 Immunohistochemical (IHC) images demonstrating an example of concurrent staining of 4-HNE with UCHL-1 for appropriate identifica- tion of primordial follicles during analysis using a Zeiss Axio Imager inverted epifluorescence microscope. Green circle = primordial follicles Table 2 Comparison of oxidative stress and DNA damage marker expression in primordial follicles in the control vs. endometrioma groups. Values represent the percentage of follicles demonstrating positive staining for each marker (4-HNE, 8-Oxo, γH2AX). P-values derived from two-tailed statistical testing; p < 0.05 considered signifi- cant Variables Control (N = 8) Endometri- oma (N = 21) P-value Primordial Follicles Oxidative Stress Marker 4-HNE (% positivity) 61.8% 65.1% 0.168 Primordial Follicles DNA Damage Marker(s) 8-Oxo (% positivity) 55.8% 67.5% 0.070 y-H2Ax (% positivity) 50.1% 63.6% *0.0331 Journal of Assisted Reproduction and Genetics percent positivity in the endometrioma group relative to the control group but that difference did not reach sta - tistical significance (67.5% vs 55.8%, p = 0.070) (Figs.  5, 7B; Table  2). Lastly, immunohistochemical assessment of γH2aX showed statistically significant higher percent positivity within oocytes of primordial follicles of patients in the endometrioma group compared to the control group (63.6% vs. 50.1%, p = 0.0331) (Figs.  6, 7C; Table  2).

This pilot study demonstrates that primordial follicles from patients with endometriomas expressed higher lev - els of DNA damage compared to age-matched controls. Specifically, γH2AX expression, an indicator of double- stranded DNA breaks, was significantly elevated in the endometrioma group. Although 4-HNE and 8-Oxo also Fig. 4 Immunohistochemical (IHC) images demonstrating the expression of 4-hydroxynonenal (4-HNE) in primordial follicles of control (top row) vs. endome- trioma (endo) subjects (bottom row) using a Zeiss Axio Imager inverted epifluorescence micro- scope. Green circle = positive primordial follicle with 4-HNE. White circle = primordial fol- licle without detectable 4-HNE levels Fig. 5 Immunohistochemical (IHC) images demonstrating the expression of 8-Oxoguanine (8-Oxo) in primordial follicles of control (top row) vs. endome- trioma (endo) subjects (bottom row) using a Zeiss Axio Imager inverted epifluorescence micro- scope. Green circle = positive primordial follicle with 8-Oxo. White circle = primordial fol- licle without detectable 8-Oxo levels Journal of Assisted Reproduction and Genetics showed higher percent positivity in the endometrioma group, which would also suggest increased oxidative stress, these trends did not reach statistical significance. These findings suggest that the presence of an endome- trioma could induce a local microenvironment that may compromise follicular integrity, though given the lack of statistical power related to the overall sample size, these

should be interpreted with caution. In a previous study, impaired oocyte quality in patients with endometriosis was associated with increased level of 8-Oxo [23]. Additionally, 8-Oxo expression has been shown to be significantly higher in the ovarian cortex adjacent to an endometrioma compared with the cortex surrounding other benign ovarian cysts, such as der - moid and serous cysts [17]. These findings suggest that ovarian cortex adjacent to endometriomas may be more severely affected by oxidative stress than cortex surround- ing other benign ovarian lesions [17]. The ovarian tissue adjacent to endometriomas can also be altered morpho - logically and also lead to a decrease in follicular density and abnormal follicular development compared to ovar - ian cortex of patients without the disease or with other benign cysts [24, 25]. From a fertility perspective, higher 8-Oxo levels in granulosa cells have also been associated Fig. 6 Immunohistochemical (IHC) images demonstrating the expression of Gamma- H2AX (γH2AX) in primordial follicles of control (top row) vs. endometrioma (endo) subjects (bottom row) using a Zeiss Axio Imager inverted epifluorescence microscope. Red circle = posi- tive primordial follicle with γH2AX. White circle = primor- dial follicle without detectable γH2AX levels Fig. 7 A  Percentage of primordial follicles demonstrating 4-HNE percent positivity in the control vs. endometrioma group. Bars repre- sent mean (± SD) with the corresponding p-value (p = 0.1677). B Per - centage of primordial follicles demonstrating 8-Oxoguanine (8-Oxo) percent positivity in the control vs. endometrioma group. Bars repre- sent mean (± SD) with the corresponding p-value (p = 0.0701). C Per - centage of primordial follicles demonstrating yH2AX percent positiv- ity in the control vs. endometioma group. Bars represent mean (± SD) with the corresponding p-value (p = 0.0331) Journal of Assisted Reproduction and Genetics with lower fertilization rates and reduced embryo qual - ity, further supporting the negative effect on reproductive outcomes [ 23]. Although we did not evaluate granulosa cells in our study, 8-Oxo levels did not reach statistical significance (p = 0.070), likely due to the limited number of age-matched controls; however, this association may have reached significance with a larger sample size. In our study, 4-HNE expression, which is a potent ROS and a product of lipid peroxidation, also had the smallest dif- ference and was comparable between the endometrioma and control. First, aging could explain this finding. The effects of lipid peroxidation derived aldehydes, such as 4-HNE, might be further enhanced with aging, as their concentra- tions in cells and biological fluids increase with age [26]. Clinically, 4-HNE has been associated with aging-related disorders such as neurodegeneration, cancer, inflamma- tion and osteopenia [26]. Specific to ovarian aging, prior animal models using mouse ovaries have demonstrated a statistically significant increase in both 4-HNE and 8-Oxo immunostaining in ovarian interstitial cells and follicles with advancing age [27]. In another mouse study, exposure of ger- minal vesicle (GV) oocytes to 4-HNE resulted in impaired meiotic completion, increased spindle abnormalities, chro- mosome misalignment, and aneuploidy [28]. We speculate that age-related baseline accumulation of 4-HNE may have been similar between cohorts, thereby attenuating detectable differences in 4-HNE expression. Small older studies look - ing at levels of 4-HNE and other lipid peroxidation derivates in serum or peritoneal fluid of patients with endometriosis did not find a difference compared to controls or patients with other diagnosis of infertility [29, 30]. Additional larger studies are still needed to better understand the primary role of ROS by lipid peroxidation and impaired ovarian function in endometriosis. Lastly, the significant increase in γH2AX observed in our study supports evidence of increased DNA damage within primordial follicles of patients with endometriomas. Such damage may affect the dormant follicular pool and poten - tially accelerate follicular depletion, contributing to ovar - ian aging. These findings are consistent with prior work in which mouse oocytes exposed to follicular fluid in vitro from patients with endometriosis demonstrated a significant increase in γH2AX foci, indicating higher levels of DNA damage compared to oocytes cultured in follicular fluid from patients without endometriosis [31]. Notably, the authors also reported that while all oocytes exposed to endometrio- sis follicular fluid exhibited increased DNA damage, not all demonstrated elevated ROS levels, suggesting that γH2AX may be more sensitive than direct ROS markers. Consistent with this observation, among the DNA damage and oxida- tive stress markers evaluated in our study, γH2AX was the only marker to reach statistical significance despite our lim- ited sample size. The clinical implications of these findings could be impactful. Clinically, endometriomas are consistently asso- ciated with reduced or accelerated decline in AMH levels, lower antral follicle counts, and poorer ovarian response dur- ing assisted reproductive technologies [3 , 6–8]. Our study provides additional evidence of the potential link between these clinical observations and molecular findings within the follicular pool. Such damage may promote premature follicle activation, atresia, or impaired developmental com- petence, contributing to accelerated ovarian aging [3 , 5]. These results align with emerging models in which inflam- mation-induced oxidative stress dysregulates folliculogen- esis through pathways involved in primordial follicle activa- tion, including FOXO3 [3 , 5]. This gene is a key regulator of primordial follicle quiescence and is controlled by the PI3K–PTEN–AKT signaling pathway [ 3, 5, 18, 32–34]. Hyperactivation of this pathway has been observed in ova- ries affected by endometriomas. [ 5, 18] A previous study also suggested that diminished ovarian reserve in endome- triosis occurred concomitantly with increased DNA dam- age and compromised double stranded break (DSB) repair mechanism; as indicated by increased levels of γH2AX, low mRNA expression of BRCA1 (a key regulator of DNA DSB repair mechanism) and Rad51 (a critical gene involved in oocyte resistance to apoptosis) [35]. The authors concluded that these findings could be indicative of impaired DNA DSB repair and ultimately, attributable to follicular damage [35]. Similarly in our study, increased γH2AX expression was observed in the endometrioma group. In the context of follicular depletion, impaired repair mechanism and cellular stress signaling could potentially influence PI3K/ AKT–FOXO3 pathways and contribute to premature primor- dial follicle activation and depletion. Although these sign- aling pathways were not directly evaluated in the present study, the observed increase in DNA damage within primor- dial follicles could represent a link between these molecular signaling and the accelerated ovarian aging associated with endometriosis. This study has several limitations. First, it is a pilot study with a relatively small sample size, particularly within the control group. As a consequence, there is limited statistical power. Even though baseline characteristics were similar between groups, the retrospective design of the study also restricts control of potential confounders, such as accu- rate hormonal treatment history. Thus, residual confound- ing cannot be excluded, as other factors (smoking, parity, disease severity and laterality) may independently affect ovarian biology and therefore could have contributed to the observed findings. Our analysis was limited to ovarian endo- metriomas, and the findings may not be generalized to other forms of endometriosis. The cohort may also represent more severe disease, as all patients underwent oophorectomies. In addition, the high exclusion rate due to inadequate follicle Journal of Assisted Reproduction and Genetics density or suboptimal tissue samples may have introduced selection bias. However, these exclusions were necessary to ensure reliable identification and standardized assessment of primordial follicles across samples. Finally, the absence of clinical ovarian reserve markers, such as AMH and antral follicle count (AFC), prevented correlation of our molecular findings with clinically relevant measures of ovarian reserve. Overall, given the exploratory and pilot nature of this study, the findings should be interpreted cautiously and considered hypothesis-generating until validation in larger prospective studies. Integrating molecular findings with clinical mark - ers (e.g., AMH and AFC) in future studies could be critical, as it would have a more meaningful impact during patient counseling. Clinically, it may also inform counseling regard- ing fertility potential and timing of fertility preservation in patients at risk for diminished ovarian function. Despite these limitations, this study has several nota- ble strengths. We utilized well-established and validated immunohistochemical markers of oxidative stress and DNA damage, including 8-Oxo, 4-HNE, and γH2AX, to evaluate molecular injury within primordial follicles. Although these markers do not directly assess signaling pathway activation, their increased expression provides biologically relevant evi- dence supporting possible follicular injury in ovarian tissue adjacent to endometriomas. To minimize assessment bias, a second blinded examiner was assigned to all IHC analy - sis. Controls with comparable demographic and clinical characteristics were also included. Strict inclusion criteria limited our control group size but reduced potential con- founders, including exclusion of concurrent benign ovarian cysts. Importantly, while prior studies have explored oxida- tive stress in endometriosis, the specific focus on primor - dial follicles is a key strength of this study. The dormant primordial follicle pool is a direct representation of ovarian reserve but most importantly, of the reproductive lifespan potential. To our knowledge, this is the first study to investi- gate these molecular markers within primordial follicles of patients with endometriomas compared with controls. Thus, the novelty of this study lies in its evaluation of oxidative stress and DNA damage within primordial follicles to further elucidate potential mechanisms underlying endometriosis- associated ovarian aging.

In conclusion, a gap continues to exist in the understanding of endometriosis-related ovarian aging. While additional studies with larger cohorts are needed, these results com- plement existing clinical and molecular evidence suggesting that endometriomas may accelerate ovarian aging through increased DNA damage. Identifying biomarkers of ovarian aging may guide counseling and therapeutic strategies aimed at preserving reproductive potential in this population. Supplementary Information The online version contains supplemen- tary material available at https:// doi. org/ 10. 1007/ s10815- 026- 03990-3.

This work was presented and published as an

at the American Society for Reproductive Medicine (ASRM) Annual Meeting, October 2025. The authors would like to thank the Program in Women’s Oncology of Women and Infants Hospital. The authors also thank the Freiman and James laboratories for their helpful feedback on this project. Author contributions All original writing of the manuscript was done by LK, SK and KJG. Manuscript editing was performed by LK, MS, and KJG. Intellectual development of the project was done by LK, JO, and KJG. Experimental design was performed by LK and KJG. Experi- ments for the manuscript were carried out by LK. Clinical analysis and guidance were performed by LK and KJG. Experimental analysis for the manuscript was performed by LK, SK and KJG. Project administra- tion was provided by JM and RC. Data availability Raw data can be made available upon request. Declarations Consent to participate All subjects provided freely given, informed consent for their participation in this study. Consent for publication All subjects provided freely given, informed consent for their data to be published in this study. Competing interest This was a pilot retrospective cohort study at a single academic institution. This study was performed in accordance with the ethical standards of the institutional research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The Women and Infants Hospital Insti- tutional Review Board approved this study (protocol #2203963–1) in accordance with all federal and local requirements. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and repro- duction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party

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