{"paper_id":"60935f3a-6d12-4b0d-b463-d04c7ad672b7","body_text":"Vol.:(0123456789)\nJournal of Assisted Reproduction and Genetics \nhttps://doi.org/10.1007/s10815-026-03990-3\nRESEARCH\nEndometriosis and ovarian aging: a pilot study exploring oxidative \nstress and DNA damage markers\nLuce A. Kassi1 · Sanjana Konda2 · Joyce Ou1,2 · Julia McAdams1 · Robin Cram1 · May‑Tal Sauerbrun‑Cutler1,2 · \nKathryn Grive1,2\nReceived: 11 March 2026 / Accepted: 29 July 2026 \n© The Author(s) 2026\nAbstract\nPurpose To assess whether specific markers of oxidative stress and DNA damage are expressed at higher levels in primordial \nfollicles of patients with endometriomas compared to age-matched controls.\nMethods A retrospective pilot cohort study was conducted using ovarian tissue from patients who underwent unilateral \nor bilateral salpingo-oophorectomy for symptomatic endometriomas, with controls undergoing an identical procedure for \nother benign gynecologic conditions. Immunohistochemistry (IHC) was used to assess percent positivity in primordial fol-\nlicles for 4-hydroxy-2-nonenal (4-HNE) as a marker of oxidative stress; 8-Oxoguanine (8-Oxo) and phosphorylated H2AX \n(γH2AX) for DNA damage. Chi-square tests were used for categorical variables and Student t-tests for continuous variables, \nwith p < 0.05 considered significant.\nResults Twenty-nine patients were included (8 controls, 21 with endometriomas). Age (39.4 ± 5.1 vs. 37.1 ± 4.6 years, \np = 0.269), BMI (33.6 ± 10.6 vs. 29.5 ± 7.8 kg/m2, p = 0.310) were similar between groups. Racial distribution, nulliparity, \nhormonal therapy use, and smoking status were also comparable. The average endometrioma size was 6.6 ± 2.8 cm. All \nmolecular markers showed higher percent positivity in the endometrioma group compared to the control group. γH2AX \ndemonstrated 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 \n(65.1% vs. 61.8%, p = 0.168) were also elevated in the endometrioma group but did not reach statistical significance.\nConclusion Patients with endometriomas demonstrated increased follicular DNA damage, with elevated γH2AX expression, \nsupporting the notion that endometriomas may impair follicular quality through DNA damage mechanisms.\nKeywords Endometrioma · Primordial follicle · Oxidative stress · DNA damage · Ovarian aging\nIntroduction\nEndometriosis is a chronic inflammatory condition affect-\ning 1 in 10 reproductive-aged women [1]. While sometimes \nasymptomatic, 25–50% of infertile women have endome-\ntriosis, making them 6 to 8 times more likely to have the \ndisease than fertile women [1 , 2]. Among women with \nchronic pelvic pain, prevalence ranges from 30 to 80% [1 , \n2]. The inflammation can affect all pelvic organs, causing \nadhesions and anatomical distortion that can lead to pain but \nalso infertility with decreased ovarian reserve or implanta-\ntion failure [2 –4]. Endometriomas, also known as ovarian \nendometrial cysts, are the most common subtype, affecting \n44% of women worldwide and have been linked to decreased \novarian reserve and accelerated ovarian aging [5].\nDespite extensive research, a clear causal link between \nendometriosis and infertility remains elusive [1 ]. A \n * Luce A. Kassi \n lkassi@wihri.org\n * Kathryn Grive \n kgrive@wihri.org\n Sanjana Konda \n sanjana_konda@brown.edu\n Joyce Ou \n jou@wihri.org\n Julia McAdams \n juliamcadams17@gmail.com\n Robin Cram \n rcram@wihri.org\n May-Tal Sauerbrun-Cutler \n msauerbruncutle@carene.org\n1 Women and Infants Hospital of Rhode Island, Providence, \nUnited States\n2 Warren Alpert Medical School of Brown University, \nProvidence, United States\n\n Journal of Assisted Reproduction and Genetics\nmeta-analysis reported lower anti-Müllerian hormone \n(AMH) levels in endometrioma patients compared to those \nwith other benign ovarian cysts [3 ]. A longitudinal study \nalso found an accelerated AMH decline in women with \nendometriomas compared to healthy controls [6 ]. Prior \nstudies of endometriosis patients showed lower primordial \nfollicle counts, proposing a mechanism similar to prema -\nture ovarian insufficiency, though the exact cause remains \nunclear [3, 7, 8].\nMultiple factors such as inflammation resulting in fibro-\nsis, oxidative stress, and DNA damage affect cellular regula-\ntion and folliculogenesis [5, 9–11]. One key reactive oxygen \nspecies (ROS) marker is 4-hydroxy-2-nonenal (4-HNE), a \nlipid-derived aldehyde formed by the oxidation of polyun-\nsaturated fatty acid [12]. 4-HNE is a highly reactive and \npotent ROS and has been linked to several chronic diseases \nand inflammatory pathways [12]. Its effects range from dis-\nrupting calcium homeostasis to causing lethal cytotoxicity \nand DNA damage [12]. Despite its role in inflammatory pro-\ncesses, there are limited studies examining the potential role \nof 4-HNE in endometriosis.\nIn another study, the ovarian cortex surrounding endo-\nmetriomas showed excessive oxidative stress with increased \n8-Oxoguanine (8-Oxo) expression [5]. Like 4-HNE, 8-Oxo \nis involved in ROS-related diseases including premature \naging and cancer [13– 16]. It accumulates through ROS \noverload, inducing mutations often seen in cancers with \nDNA deficient repair mechanisms [13]. Studies examining \n8-Oxo in endometriosis also remain limited [5 , 17]. Oxida-\ntive stress markers like 8-Oxo can also cause DNA damage \nand potentially affect primordial follicle activation through \nthe Forkhead box O3 (FOXO3) pathway [18]. These findings \nmake 8-Oxo a unique marker as its expression represents \nboth oxidative stress but also the presence of DNA damage.\nGamma-H2AX (γH2AX) is a well-established marker of \nDNA damage and represents a phosphorylated variant of \nthe H2A protein family, a core component of the histone \noctamer within nucleosomes. Following the occurrence of \ndouble-stranded DNA breaks, histone H2AX undergoes \nrapid phosphorylation to form γH2AX, which accumulates \nand forms foci at the sites of damage [19– 22]. In ovarian \ntissue, these γH2AX foci can be detected within primordial \nfollicles using immunofluorescence staining, providing an \nobjective means of assessing for follicular DNA damage \n[20, 21]. Despite its relevance as a biomarker of cellular \nstress and injury, data on γH2AX expression in patients with \nendometriosis also remain limited, highlighting the need for \nfurther investigation.\nAlthough prior studies have evaluated oxidative stress \nwithin ovarian tissue affected by endometriosis, limited \ndata exist regarding the impact on primordial follicles, \nwhich is the follicular pool most directly associated with \novarian reserve and the reproductive lifespan. Understanding \nmolecular injury within primordial follicles may provide \nimportant mechanistic insight into accelerated ovarian aging \nassociated with endometriomas. Therefore, the objective of \nthis study was to evaluate markers of oxidative stress and \nDNA damage within primordial follicles in ovarian tissue \nadjacent to endometriomas. Based on these findings, we \nalso hypothesized that primordial follicles from patients \nwith endometriomas would have increased DNA damage \nand oxidative stress compared to healthy controls.\nMaterials and methods\nStudy design\nThis was a pilot retrospective cohort study at a single aca-\ndemic institution. The cohort included patients aged 18 to \n45 years old who had undergone a unilateral or bilateral \nsalpingo-oophorectomy between June 2006 and June 2024. \nPatients were identified through electronic medical records \nusing diagnostic (ICD-9/ICD-10 – International Classifica-\ntion of Diseases) and surgical codes (CPT—Current Pro-\ncedural Terminology code). Patients were excluded if they \nhad undergone chemotherapy or radiation, had a diagnosis \nof ovarian cancer or any prior malignancy, BRCA 1 and 2 \nmutation carriers, prior chemotherapy or radiation exposure, \ntestosterone exposure, postmenopausal status and any con-\ncurrent benign ovarian pathology (Fig.  1). Patient informa-\ntion was collected by chart review and included age, body \nmass index (BMI), race and ethnicity, obstetric history, \nsmoking history, use of exogenous hormones, and speci-\nmen pathology results. Data was recorded and stored on the \nCare New England REDCap secure online database v10.0.4.\nPrimary antibodies\nThe primary antibodies of choice for oxidative stress were \n4-HNE (Thermo Fisher #MA5-27,570), with 8-Oxo (Sigma \n#MAB3560), and γH2AX (Sigma #07–164-25UG), serving \nas markers for DNA damage. Ubiquitin Carboxyl-terminal \nHydrolase L1 (UCHL1) (Proteintech # 14,730–1-AP), an \noocyte-specific marker, was also used to confirm the iden-\ntification of primordial follicles during IHC analysis [36].\nImmunofluorescence\nOvary sections of 70 subjects were obtained from Women \n& Infants Pathology paraffin-embedded, fixed in 1:10 for -\nmalin, and sectioned at 5 μm onto glass slides. To stain, \nsections were deparaffinized and hydrated. Sections were \nwashed in SafeClear (3 × 5 min), 100% EtOH (3 × 5 min), \n95% EtOH (2 × 5 min), 70% EtOH (1 × 5 min), and water \n(1 × 5 min). Sections were heated for 20 min in 1% sodium \n\nJournal of Assisted Reproduction and Genetics \ncitrate antigen-retrieval buffer, cooled for 20 min, and washed \n2 × 5 min in 1 × phosphate-buffered saline with 0.1% Triton X \n(PBST). Tissue sections were blocked in PBST (Fisher Sci-\nentific), 1% bovine serum albumin (Sigma), 3% goat serum \n(Sigma) for 1 h. Sections were outlined in hydrophobic marker \nand incubated with primary antibodies against 4-HNE, 8-Oxo, \nγH2aX, and Ubiquitin Carboxyl-terminal Hydrolase L1 \n(UCHL-1) diluted at 1:100 overnight at 4 °C. The next day, the \nsections were then washed 3 × 5 min in PBST and incubated \nwith secondary antibodies (mouse or rabbit) against γH2aX, \n8-Oxo and 4-HNE in a dark for 1 h at room temperature. Sec-\ntions were then stained with 4',6-diamidino-2-phenylindole \n(DAPI) to visualize nuclei, mounted and analyzed on Zeiss \nAxio Imager inverted epifluorescence microscope (Fig.  2). \nOocytes of primordial follicles were considered either positive \nor negative for the specific antibody. Immunohistochemical \nstaining was initially performed on three slides per subject for \neach antibody. If analysis of these slides identified fewer than \nten follicles, additional slides from the remaining available \novarian tissue were stained, up to a maximum of five total \nslides per antibody. Subjects with fewer than ten follicles iden-\ntified across all stained slides were excluded from the analysis; \nspecifically, 9 of 17 subjects in the control group and 32 of 53 \nsubjects in the endometrioma group were excluded (Fig.  1). \nFig. 1  Flowchart of patient selection for the study. Among 950 initial \npatients who underwent oophorectomy, 70 met inclusion criteria for \nreview. A total of 880 patients were excluded due to ovarian cancer, \nBRCA1/2 status, concurrent ovarian cyst, benign ovarian cyst, his-\ntory of chemoradiation, or postmenopausal status. Of the 70 eligible \npatients, 17 were classified as controls and 53 had histopathology-\nconfirmed endometriomas. Patients with fewer than 10 identifiable \nprimordial follicles on histologic evaluation were further excluded \n(9 controls and 32 endometrioma cases), yielding a final cohort of 8 \ncontrol patients and 21 patients with endometriomas\n\n Journal of Assisted Reproduction and Genetics\nAfter exclusions based on primordial follicle identification and \ntissue availability, 29 subjects remained for the final analy-\nsis, including 8 controls and 21 subjects in the endometrioma \ngroup (Fig. 1).\nStatistical analysis\nFor each marker (4-HNE, 8-Oxo, yH2AX), the primary out-\ncome was percent positivity, defined as the number of posi-\ntively stained oocytes within primordial follicles divided by the \ntotal number of follicles per subject. Primordial follicles were \nidentified based on established morphological criteria, includ-\ning a single layer of flattened granulosa cells surrounding an \noocyte without an antral space. Only follicles meeting these \ncriteria were included in the analysis. UCHL-1, an oocyte-\nspecific marker, was also used in conjunction with hematoxy-\nlin and eosin (H&E) stained slides to confirm primordial fol-\nlicule identification and improve classification accuracy. IHC \nanalysis was performed independently by two examiners, with \none blinded to the group allocation. Inter-observer variability \ndid not exceed 10%, and the average of the two measurements \nwas used for analysis. Statistical analysis was performed in \nGraphPad Prism. Smoking status and use of exogenous hor-\nmones was analyzed by Chi Square test. Student’s t-test and a \np-value < 0.05 were used.\nResults\nStudy population\nBased on diagnostic and surgical codes, 950 patients \nwere initially identified during the time period (Fig.  1, \nSupplemental Table 1). After extensive chart review, 70 \npatients met inclusion criteria. A total of 880 patients were \nexcluded due to the diagnosis of ovarian cancer, BRCA 1 \nand 2 mutation carriers, the presence of concurrent ovar -\nian cysts, non-endometrioma benign ovarian cysts, a prior \nhistory of chemoradiation, or postmenopausal status. Of \nthe 70 eligible patients, 17 were classified as controls and \n53 had histopathology-confirmed endometriomas. Patients \nwith fewer than 10 identifiable primordial follicles on \nhistologic evaluation were further excluded (9 controls \nand 32 endometrioma cases), yielding a final cohort of \n8 control patients and 21 patients with endometriomas \n(Fig.  1). Baseline demographic and clinical character -\nistics were similar between groups (Table  1). The mean \nage (37.1 ± 4.6 vs. 39.4 ± 5.1 years, p = 0.269), and BMI \n(29.5 ± 7.8 vs. 33.6 ± 10.6 kg/m2, p = 0.310) were compa-\nrable between patients with endometriomas and controls. \nRacial and ethnic distribution among the two groups was \nFig. 2  Schematic overview of the immunohistochemical (IHC) stain-\ning process to evaluate oxidative stress and DNA damage markers \nin ovarian tissue. Formalin-fixed, paraffin-embedded ovarian sec-\ntions were mounted on glass slides, stained with primary antibodies \n(4-HNE, 8-Oxo, γH2AX, UCHL-1), and detected using fluorescent \nsecondary antibodies. Stained slides were visualized using a Zeiss \nAxio Imager inverted epifluorescence microscope\n\nJournal of Assisted Reproduction and Genetics \nalso not significant (p = ns), with the majority identifying \nas White in both groups (Table  1). Nulliparity was more \ncommon among patients with endometriomas (38.1%) than \ncontrols (12.5%), although this difference did not reach \nstatistical significance (p  = 0.372). The use of current hor -\nmonal therapy was similar between the groups (9.5% vs. \n12.5%, p > 0.999). Smoking status distributions were also \ncomparable, with most individuals reporting never smok -\ning (p = 0.662). Among patients with endometriomas, the \nmean endometrioma size was 6.6 ± 2.8 cm (Fig.  3).\nOxidative stress\nIn IHC analyses of oxidative stress within oocytes of pri-\nmordial follicles, 4-HNE staining demonstrated a higher \npercent positivity in the endometrioma group compared to \nthe control group; though this difference did not reach sta-\ntistical significance (65.1% vs. 61.8%; p  = 0.168) (Fig.  4; \n7A; Table  2).\nDNA damage\nIHC assessment of DNA damage with 8-Oxo staining \nwithin oocytes of primordial follicles showed a higher \nTable 1  Baseline demographic \nand clinical characteristics of \npatients with endometriomas \ncompared to controls. \nValues are presented as \nmean ± standard deviation or \nnumber (percentage). Statistical \ncomparisons were performed \nusing Student t-tests with a p \nvalue set at 0.05\nCharacteristics Control (N = 8) Endometriosis (N = 21) P-value\nAge (years), mean (± SD) 39.4 (± 5.1) 37.1 (± 4.6) 0.269\nBMI (kg/m2), mean (± SD) 33.6 (± 10.6) 29.5 (± 7.8) 0.310\nRace/Ethnicity, n (%) 0.490\nWhite/Caucasian 7 (87.5%) 17 (81.0%)\nBlack/African American 1 (12.5%) 1 (4.5%)\nHispanic/Latino 0 1 (4.5%)\nAsian 0 0\nNot documented 0 2 (10.0%)\nNulliparous, n (%) 1 (12.5%) 9 (38.1%) 0.372\nCurrent hormonal therapy, n (%)  > 0.999\nYes 1 (12.5%) 2 (9.5%)\nNo 7 (85.5%) 19 (90.5%)\nSmoking status, n (%) 0.662\nNever smoker 4 (57.1%) 12 (57.1%)\nCurrent or previous smoker 3 (42.7%) 8 (38.1%)\nNot documented 1 (14.3%) 1 (4.8%)\nSize of endometrioma (cm), mean (± SD) – 6.6 (± 2.8)\nFig. 3  Immunohistochemical (IHC) images demonstrating an example of concurrent staining of 4-HNE with UCHL-1 for appropriate identifica-\ntion of primordial follicles during analysis using a Zeiss Axio Imager inverted epifluorescence microscope. Green circle = primordial follicles\nTable 2  Comparison of oxidative stress and DNA damage marker \nexpression in primordial follicles in the control vs. endometrioma \ngroups. Values represent the percentage of follicles demonstrating \npositive staining for each marker (4-HNE, 8-Oxo, γH2AX). P-values \nderived from two-tailed statistical testing; p < 0.05 considered signifi-\ncant\nVariables Control (N = 8) Endometri-\noma (N = 21)\nP-value\nPrimordial Follicles Oxidative Stress Marker\n4-HNE (% positivity) 61.8% 65.1% 0.168\nPrimordial Follicles DNA Damage Marker(s)\n8-Oxo (% positivity) 55.8% 67.5% 0.070\ny-H2Ax (% positivity) 50.1% 63.6% *0.0331\n\n Journal of Assisted Reproduction and Genetics\npercent positivity in the endometrioma group relative to \nthe control group but that difference did not reach sta -\ntistical significance (67.5% vs 55.8%, p  = 0.070) (Figs.  5, \n7B; Table  2). Lastly, immunohistochemical assessment \nof γH2aX showed statistically significant higher percent \npositivity within oocytes of primordial follicles of patients \nin the endometrioma group compared to the control group \n(63.6% vs. 50.1%, p = 0.0331) (Figs.  6, 7C; Table  2).\nDiscussion\nThis pilot study demonstrates that primordial follicles \nfrom patients with endometriomas expressed higher lev -\nels of DNA damage compared to age-matched controls. \nSpecifically, γH2AX expression, an indicator of double-\nstranded DNA breaks, was significantly elevated in the \nendometrioma group. Although 4-HNE and 8-Oxo also \nFig. 4  Immunohistochemical \n(IHC) images demonstrating the \nexpression of 4-hydroxynonenal \n(4-HNE) in primordial follicles \nof control (top row) vs. endome-\ntrioma (endo) subjects (bottom \nrow) using a Zeiss Axio Imager \ninverted epifluorescence micro-\nscope. Green circle = positive \nprimordial follicle with 4-HNE. \nWhite circle = primordial fol-\nlicle without detectable 4-HNE \nlevels\nFig. 5  Immunohistochemical \n(IHC) images demonstrating \nthe expression of 8-Oxoguanine \n(8-Oxo) in primordial follicles \nof control (top row) vs. endome-\ntrioma (endo) subjects (bottom \nrow) using a Zeiss Axio Imager \ninverted epifluorescence micro-\nscope. Green circle = positive \nprimordial follicle with 8-Oxo. \nWhite circle = primordial fol-\nlicle without detectable 8-Oxo \nlevels\n\n\nJournal of Assisted Reproduction and Genetics \nshowed higher percent positivity in the endometrioma \ngroup, which would also suggest increased oxidative \nstress, these trends did not reach statistical significance. \nThese findings suggest that the presence of an endome-\ntrioma could induce a local microenvironment that may \ncompromise follicular integrity, though given the lack of \nstatistical power related to the overall sample size, these \nresults should be interpreted with caution.\nIn a previous study, impaired oocyte quality in patients \nwith endometriosis was associated with increased level \nof 8-Oxo [23]. Additionally, 8-Oxo expression has been \nshown to be significantly higher in the ovarian cortex \nadjacent to an endometrioma compared with the cortex \nsurrounding other benign ovarian cysts, such as der -\nmoid and serous cysts [17]. These findings suggest that \novarian cortex adjacent to endometriomas may be more \nseverely affected by oxidative stress than cortex surround-\ning other benign ovarian lesions [17]. The ovarian tissue \nadjacent to endometriomas can also be altered morpho -\nlogically and also lead to a decrease in follicular density \nand abnormal follicular development compared to ovar -\nian cortex of patients without the disease or with other \nbenign cysts [24, 25]. From a fertility perspective, higher \n8-Oxo levels in granulosa cells have also been associated \nFig. 6  Immunohistochemical \n(IHC) images demonstrating \nthe expression of Gamma-\nH2AX (γH2AX) in primordial \nfollicles of control (top row) vs. \nendometrioma (endo) subjects \n(bottom row) using a Zeiss Axio \nImager inverted epifluorescence \nmicroscope. Red circle = posi-\ntive primordial follicle with \nγH2AX. White circle = primor-\ndial follicle without detectable \nγH2AX levels\nFig. 7  A  Percentage of primordial follicles demonstrating 4-HNE \npercent positivity in the control vs. endometrioma group. Bars repre-\nsent mean (± SD) with the corresponding p-value (p = 0.1677). B Per -\ncentage of primordial follicles demonstrating 8-Oxoguanine (8-Oxo) \npercent positivity in the control vs. endometrioma group. Bars repre-\nsent mean (± SD) with the corresponding p-value (p = 0.0701). C Per -\ncentage of primordial follicles demonstrating yH2AX percent positiv-\nity in the control vs. endometioma group. Bars represent mean (± SD) \nwith the corresponding p-value (p = 0.0331)\n\n Journal of Assisted Reproduction and Genetics\nwith lower fertilization rates and reduced embryo qual -\nity, further supporting the negative effect on reproductive \noutcomes [ 23]. Although we did not evaluate granulosa \ncells in our study, 8-Oxo levels did not reach statistical \nsignificance (p  = 0.070), likely due to the limited number \nof age-matched controls; however, this association may \nhave reached significance with a larger sample size.\nIn our study, 4-HNE expression, which is a potent ROS \nand a product of lipid peroxidation, also had the smallest dif-\nference and was comparable between the endometrioma and \ncontrol. First, aging could explain this finding. The effects \nof lipid peroxidation derived aldehydes, such as 4-HNE, \nmight be further enhanced with aging, as their concentra-\ntions in cells and biological fluids increase with age [26]. \nClinically, 4-HNE has been associated with aging-related \ndisorders such as neurodegeneration, cancer, inflamma-\ntion and osteopenia [26]. Specific to ovarian aging, prior \nanimal models using mouse ovaries have demonstrated a \nstatistically significant increase in both 4-HNE and 8-Oxo \nimmunostaining in ovarian interstitial cells and follicles with \nadvancing age [27]. In another mouse study, exposure of ger-\nminal vesicle (GV) oocytes to 4-HNE resulted in impaired \nmeiotic completion, increased spindle abnormalities, chro-\nmosome misalignment, and aneuploidy [28]. We speculate \nthat age-related baseline accumulation of 4-HNE may have \nbeen similar between cohorts, thereby attenuating detectable \ndifferences in 4-HNE expression. Small older studies look -\ning at levels of 4-HNE and other lipid peroxidation derivates \nin serum or peritoneal fluid of patients with endometriosis \ndid not find a difference compared to controls or patients \nwith other diagnosis of infertility [29, 30]. Additional larger \nstudies are still needed to better understand the primary role \nof ROS by lipid peroxidation and impaired ovarian function \nin endometriosis.\nLastly, the significant increase in γH2AX observed in our \nstudy supports evidence of increased DNA damage within \nprimordial follicles of patients with endometriomas. Such \ndamage may affect the dormant follicular pool and poten -\ntially accelerate follicular depletion, contributing to ovar -\nian aging. These findings are consistent with prior work \nin which mouse oocytes exposed to follicular fluid in vitro \nfrom patients with endometriosis demonstrated a significant \nincrease in γH2AX foci, indicating higher levels of DNA \ndamage compared to oocytes cultured in follicular fluid from \npatients without endometriosis [31]. Notably, the authors \nalso reported that while all oocytes exposed to endometrio-\nsis follicular fluid exhibited increased DNA damage, not all \ndemonstrated elevated ROS levels, suggesting that γH2AX \nmay be more sensitive than direct ROS markers. Consistent \nwith this observation, among the DNA damage and oxida-\ntive stress markers evaluated in our study, γH2AX was the \nonly marker to reach statistical significance despite our lim-\nited sample size.\nThe clinical implications of these findings could be \nimpactful. Clinically, endometriomas are consistently asso-\nciated with reduced or accelerated decline in AMH levels, \nlower antral follicle counts, and poorer ovarian response dur-\ning assisted reproductive technologies [3 , 6–8]. Our study \nprovides additional evidence of the potential link between \nthese clinical observations and molecular findings within \nthe follicular pool. Such damage may promote premature \nfollicle activation, atresia, or impaired developmental com-\npetence, contributing to accelerated ovarian aging [3 , 5]. \nThese results align with emerging models in which inflam-\nmation-induced oxidative stress dysregulates folliculogen-\nesis through pathways involved in primordial follicle activa-\ntion, including FOXO3 [3 , 5]. This gene is a key regulator \nof primordial follicle quiescence and is controlled by the \nPI3K–PTEN–AKT signaling pathway [ 3, 5, 18, 32–34]. \nHyperactivation of this pathway has been observed in ova-\nries affected by endometriomas. [ 5, 18] A previous study \nalso suggested that diminished ovarian reserve in endome-\ntriosis occurred concomitantly with increased DNA dam-\nage and compromised double stranded break (DSB) repair \nmechanism; as indicated by increased levels of γH2AX, low \nmRNA expression of BRCA1 (a key regulator of DNA DSB \nrepair mechanism) and Rad51 (a critical gene involved in \noocyte resistance to apoptosis) [35]. The authors concluded \nthat these findings could be indicative of impaired DNA \nDSB repair and ultimately, attributable to follicular damage \n[35]. Similarly in our study, increased γH2AX expression \nwas observed in the endometrioma group. In the context \nof follicular depletion, impaired repair mechanism and \ncellular stress signaling could potentially influence PI3K/\nAKT–FOXO3 pathways and contribute to premature primor-\ndial follicle activation and depletion. Although these sign-\naling pathways were not directly evaluated in the present \nstudy, the observed increase in DNA damage within primor-\ndial follicles could represent a link between these molecular \nsignaling and the accelerated ovarian aging associated with \nendometriosis.\nThis study has several limitations. First, it is a pilot study \nwith a relatively small sample size, particularly within the \ncontrol group. As a consequence, there is limited statistical \npower. Even though baseline characteristics were similar \nbetween groups, the retrospective design of the study also \nrestricts control of potential confounders, such as accu-\nrate hormonal treatment history. Thus, residual confound-\ning cannot be excluded, as other factors (smoking, parity, \ndisease severity and laterality) may independently affect \novarian biology and therefore could have contributed to the \nobserved findings. Our analysis was limited to ovarian endo-\nmetriomas, and the findings may not be generalized to other \nforms of endometriosis. The cohort may also represent more \nsevere disease, as all patients underwent oophorectomies. In \naddition, the high exclusion rate due to inadequate follicle \n\nJournal of Assisted Reproduction and Genetics \ndensity or suboptimal tissue samples may have introduced \nselection bias. However, these exclusions were necessary to \nensure reliable identification and standardized assessment \nof primordial follicles across samples. Finally, the absence \nof clinical ovarian reserve markers, such as AMH and antral \nfollicle count (AFC), prevented correlation of our molecular \nfindings with clinically relevant measures of ovarian reserve. \nOverall, given the exploratory and pilot nature of this study, \nthe findings should be interpreted cautiously and considered \nhypothesis-generating until validation in larger prospective \nstudies. Integrating molecular findings with clinical mark -\ners (e.g., AMH and AFC) in future studies could be critical, \nas it would have a more meaningful impact during patient \ncounseling. Clinically, it may also inform counseling regard-\ning fertility potential and timing of fertility preservation in \npatients at risk for diminished ovarian function.\nDespite these limitations, this study has several nota-\nble strengths. We utilized well-established and validated \nimmunohistochemical markers of oxidative stress and DNA \ndamage, including 8-Oxo, 4-HNE, and γH2AX, to evaluate \nmolecular injury within primordial follicles. Although these \nmarkers do not directly assess signaling pathway activation, \ntheir increased expression provides biologically relevant evi-\ndence supporting possible follicular injury in ovarian tissue \nadjacent to endometriomas. To minimize assessment bias, \na second blinded examiner was assigned to all IHC analy -\nsis. Controls with comparable demographic and clinical \ncharacteristics were also included. Strict inclusion criteria \nlimited our control group size but reduced potential con-\nfounders, including exclusion of concurrent benign ovarian \ncysts. Importantly, while prior studies have explored oxida-\ntive stress in endometriosis, the specific focus on primor -\ndial follicles is a key strength of this study. The dormant \nprimordial follicle pool is a direct representation of ovarian \nreserve but most importantly, of the reproductive lifespan \npotential. To our knowledge, this is the first study to investi-\ngate these molecular markers within primordial follicles of \npatients with endometriomas compared with controls. Thus, \nthe novelty of this study lies in its evaluation of oxidative \nstress and DNA damage within primordial follicles to further \nelucidate potential mechanisms underlying endometriosis-\nassociated ovarian aging.\nConclusion\nIn conclusion, a gap continues to exist in the understanding \nof endometriosis-related ovarian aging. While additional \nstudies with larger cohorts are needed, these results com-\nplement existing clinical and molecular evidence suggesting \nthat endometriomas may accelerate ovarian aging through \nincreased DNA damage. Identifying biomarkers of ovarian \naging may guide counseling and therapeutic strategies aimed \nat preserving reproductive potential in this population.\nSupplementary Information The online version contains supplemen-\ntary material available at https:// doi. org/ 10. 1007/ s10815- 026- 03990-3.\nAcknowledgements This work was presented and published as an \nabstract at the American Society for Reproductive Medicine (ASRM) \nAnnual Meeting, October 2025. The authors would like to thank the \nProgram in Women’s Oncology of Women and Infants Hospital. The \nauthors also thank the Freiman and James laboratories for their helpful \nfeedback on this project.\nAuthor contributions All original writing of the manuscript was done \nby LK, SK and KJG. Manuscript editing was performed by LK, MS, \nand KJG. Intellectual development of the project was done by LK, JO, \nand KJG. Experimental design was performed by LK and KJG. Experi-\nments for the manuscript were carried out by LK. Clinical analysis and \nguidance were performed by LK and KJG. Experimental analysis for \nthe manuscript was performed by LK, SK and KJG. Project administra-\ntion was provided by JM and RC.\nData availability Raw data can be made available upon request.\nDeclarations \nConsent to participate All subjects provided freely given, informed \nconsent for their participation in this study.\nConsent for publication All subjects provided freely given, informed \nconsent for their data to be published in this study.\nCompeting interest This was a pilot retrospective cohort study at a \nsingle academic institution. This study was performed in accordance \nwith the ethical standards of the institutional research committee and \nwith the 1964 Declaration of Helsinki and its later amendments or \ncomparable ethical standards. The Women and Infants Hospital Insti-\ntutional Review Board approved this study (protocol #2203963–1) in \naccordance with all federal and local requirements.\nOpen Access This article is licensed under a Creative Commons \nAttribution-NonCommercial-NoDerivatives 4.0 International License, \nwhich permits any non-commercial use, sharing, distribution and repro-\nduction in any medium or format, as long as you give appropriate credit \nto the original author(s) and the source, provide a link to the Creative \nCommons licence, and indicate if you modified the licensed material. \nYou do not have permission under this licence to share adapted material \nderived from this article or parts of it. The images or other third party \nmaterial in this article are included in the article’s Creative Commons \nlicence, unless indicated otherwise in a credit line to the material. 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