N-acetylcysteine as an Investigational Molecular Modulator in Endometriosis: A Narrative Review

Background: Endometriosis is an inflammatory condition featuring persistent oxidative stress. Usual hormonal therapy and surgery can be limited because they introduce contraceptive effects. Aim: This review considers the potential of N - acetylcysteine (NAC) as a candidate non -hormonal molecule that could influence endometriosis, while considering the preliminary and methodological weaknesses of the available data. Materials and Methods: A structured PubMed-MEDLINE search (January 2010 – May 2026) was performed to identify the role of N -acetylcysteine (NAC) in endometriosis. A comparative approach was used to summarize the methodological validity of preclinical animal studies and clinical trials and to assess study quality. Results: Evidence from mechanistic studies and experimental models indicates NAC as a candidate factor associated with molecular changes in tissue and lesion regression in preclinical models. Clinical research reports pain relief and a reduction in endometrioma size; however, the results are affected by small sample sizes, lack of randomization and placebo control, or mixed antioxidant formulations. Conclusions: Current knowledge is insufficient to support NAC as a conventional treatment for endometriosis. Limitations stemming from the heavy reliance on preclinical models and the confounding design of clinical trials hinder understanding of NAC’s role. Further research needs to focus on randomized controlled trials on NAC alone. Showing the disease -modifying potential of NAC requires long-term outcomes.

endometriosis, n-acetylcysteine (NAC), redox signaling modulation, proliferation-to-differentiation switch 1. INTRODUCTION

Endometriosis represents an estrogen -dependent chronic illness characterized by the proliferation of endometrium -like lesions and/or stroma beyond the endometrium and myometrium (Tomassetti et al., 2021). The pathogenesis of this disease involves chronic inflammation, in which reactive oxygen species (ROS) production exceeds antioxidant defenses, leading to oxidative stress (Clower et al., Medical Science To Cite: Rakuś M, Łukasz K, Marzec J, Nowakowski M, Musioł A, Gwałt P. N- acetylcysteine as an Investigational Molecular Modulator in Endometriosis: A Narrative Review. Medical Science 2026; 30: e112ms3921 doi: https://doi.org/10.54905/disssi.v30i172.e112ms3921 Authors’ Affiliation: 1Medical University of Warsaw; ul. Żwirki i Wigury 61, 02-091 Warsaw, Poland ⃰ Corresponding author: Magdalena Rakuś, Medical University of Warsaw; ul. Żwirki i Wigury 61, 02-091 Warsaw, Poland, E-mail: [email protected]; phone number: +48883147626 Contact list: Magdalena Rakuś: [email protected] Krzysztof Łukasz: [email protected] Jakub Marzec: [email protected] Michał Nowakowski: [email protected] Aleksandra Musioł: [email protected] Paweł Gwałt: [email protected] ORCID List: Magdalena Rakuś 0009-0008-9301-6680 Krzysztof Łukasz 0009-0002-3438-1967 Jakub Marzec 0009-0003-4096-543X Michał Nowakowski 0009-0004-9113-4780 Aleksandra Musioł 0009-0000-0495-2146 Paweł Gwałt 0009-0005-3163-9696 Peer-Review History Received: 07 May 2026 Reviewed & Revised: 15/May/2026 to 16/June/2026 Accepted: 21 June 2026 Published: 29 June 2026 Peer-review Method External peer-review was done through double-blind method. Medical Science pISSN 2321–7359; eISSN 2321–7367 © The Author(s) 2026. Open Access. This article is licensed under a Creative Commons Attribution License 4.0 (CC BY 4.0)., which permits use, sharing, adaptation, distribution and reproduction 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 license, and indicate if changes were made. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. DISCOVERY SCIENTIFIC SOCIETY REVIEW | OPEN ACCESS Medical Science 30, e112ms3921 (2026) 2 of 11 2022). Endometriosis occurs in women of reproductive age all around the world, with a prevalence rate of around 10%. It is kn own to impact health through symptoms of chronic pelvic pain, dysmenorrhea, dyspareunia, dyschezia, dysuria, and infertility (Zonder van et al., 2020). This pathology negatively impacts psychological health and is associated with disruption of the education and emp loyment opportunities (Facchin et al., 2026). Current Clinical Guidelines and Standard of Care At the moment, endometriosis cannot be completely cured (As-Sanie et al., 2025). Treatment strategy includes symptom control and the prevention of recurrence. According to the ESHRE guidelines of 2022, a combination of hormonal contraceptives is recommended for managing pain, while progestins act as second -line medications for controlling pain and promoting fertility. GnRH agonists and antagonists should be used after other treatment options fail. Another treatment pillar involves surgery with a predilection towards lesions excision and cystectomy, with post -surgical hormone suppression being necessary unless pregnancy is sought by a woman (Becker et al., 2022). It is essential to emphasize that N -acetylcysteine (NAC) described in the current review is not a part of any treatment protocol and is not recommended as for now. Unmet Medical Need and Treatment Gap A serious problem associated with the majority of currently available hormonal treatment options is the fact that the mechanism behind these medicines consists of blocking ovulation. As a consequence, these methods are contraception methods and cannot be administered to those women who wish to conceive (Taylor et al., 2021). The recurrence rate is high when a patient does not r eceive hormone suppression after surgery (Veth et al., 2024). Moreover, some surgical approaches have been shown to deplete ovarian reserve (Li et al., 2026). Thus, a medical gap arises due to the necessity of non -hormonal drug development that will stop disease progression while not affecting reproductive capabilities. Research Objective The current narrative review evaluates the potential of N-acetylcysteine (NAC) as a model for experimental research into the molecular regulation of endometriosis. Specifically, the aim of the present research aims to discuss the opportunities for developing a non- hormonal model that facilitates stabilization of the disease progression without affecting hormones. At the same time, given that the majority of scientific work addressing the problem in question consists of in vitro and preclinical research, this review focuses on this important issue. 2. REVIEW METHODS Search Strategy To get a picture of how NAC could affect endometriosis, a thorough review of the PubMed/MEDLINE database was performed. The timeline for the literature search was set from January 2010 to May 2026. The process relied on a combination of Medical Subj ect Headings (MeSH) terms: (“endometriosis” OR “endometrioma”) AND (“N-acetylcysteine” OR “NAC” OR “N-Acetyl cysteine”). Selection Criteria The inclusion criteria targeted research analyzing NAC as an intervention in the endometriosis model system or as an important factor to be considered in studies involving the disease. As a consequence of a gap in current therapies, parameters of interest inc luded oxidative environmental changes, alterations in lesion size, pain scores, and reproductive performance. Studies investigating monotherapy were chosen to isolate the modulatory effect of NAC directly; however, multicomponent antioxidative studies were included as well to examine the antioxidative capabilities of the compound. During the initial screening stage, reviews and m eta- analyses were excluded. During the eligibility assessment, papers related to other pathologies, treatments, hormonal manipula tions, and other unrelated matters were excluded, resulting in the selection of 14 papers for the review. For presentation purposes and to ensure methodological transparency, a PRISMA flowchart was created (Figure 1). REVIEW | OPEN ACCESS Medical Science 30, e112ms3921 (2026) 3 of 11 Figure 1. PRISMA flow chart Data Extraction and Synthesis Narrative synthesis was applied. Given the exploratory character of current research and the heterogeneity of approaches used in relevant papers, a critical analysis of methodology was conducted for each study. The synthesis focused on quality, methodolo gical aspects, and risk of bias in preclinical models and clinical trials to isolate NAC's molecular efficacy. 3. RESULTS Study Findings: Murine Model A possible “proliferation -to-differentiation switch” mechanism was first tested in a group of 40 mice, in which NAC administration reduced endometrioma mass by 60% compared with untreated controls. The regression was observed due to the antiproliferative effect, characterized by a decrease in the proliferation marker Ki -67, from 1.88±0.28% in the untreated animals to 0.87±0.11% ( P < 0.01). It seems that NAC induced differentiation and maturation processes, as reflected by E -cadherin localization at cell junctions rather than in the cytoplasm, with a 67% increase in the junctional protein compared with controls. This effect corresponded to the lower motility and invasiveness of the endometriotic cells. Moreover, the treatment reduced cyclooxygenase -2 (Cox2) mRNA expression and inhibited matrix metalloproteinase-9 (MMP-9) activity by more than 60%. Thus, NAC targets inflammation and invasive processes characteristic of this pathology. The described findings provide the basis for this mechanism, but they are still only the first stage of ex perimental studies, since it is necessary to account for the specific microenvironment of human organisms when translating findings from animal experiments into practice (Pittaluga et al., 2010). REVIEW | OPEN ACCESS Medical Science 30, e112ms3921 (2026) 4 of 11 Clinical Observations: Results in Non-Randomized Populations The efficacy of N -acetylcysteine (NAC) therapy in the clinical practice of endometriosis among 92 patients, 47 treated and 45 controls without the drug administration, has been evaluated using ultrasound diagnosis of endometriomas. To optimize pharmacokinetics , a “pulse therapy” was utilized: NAC was given three times per day at 600 mg, resulting in an overall intake of 1.8 g/day for th ree days a week, followed by a four -day medication-free period. During three months of observations, a minimal 1.5 mm reduction in cyst size in treated patients was observed, compared with a 6.6 mm increase in untreated patients (P = 0.001). While the discussed study is useful to transfer the findings from laboratory animals to the clinical setting, its methodology bears certain restrictions due to the relatively low number of patients ( n = 92) involved in the research process, as well as due to the rather brief observation period (3 months), which is not sufficient to determine the durability of the effects observed. The investigation reported 24 operations canceled because of symptom remission: dysmenorrhea (55%, P = 0.001), dyspareunia (50%, P = 0.027), and chronic pelvic pain (59%, P = 0.015). Without a placebo control, it is impossible to distinguish the drug's physiological effect from placebo-related changes in subjective complaints. A morphologically differentiated epithelium and higher expression levels of junctional proteins like E -cadherin and β-catenin have been identified in treated lesions. A simultaneous decrease in the COX-2 expression has also been established. It could explain cyst regression via the hypothesized mechanism, which includes lower Prostaglandin -E2 (PGE2) production, thereby decreasing the stimulation of aromatase activity and estrogen formation. The immunohistochemical approach to confirm the identified biochemical mechanisms has been carried out by the authors on 4 cyst samples taken from the operated patients (laparoscopy) to avoid poss ible confounding factors. The described research findings are prone to selection bias, since the assignment of patients to the con trol and treatment groups depended on their willingness to undergo NAC administration. In addition, the study's one -site design limits its external validity (Porpora et al., 2013). Symptom Evaluation: Prospective Single-Arm Data Prospective research involving 120 female subjects demonstrated the clinical efficacy of the 3 -day NAC pulse therapy protocol. It was reported that there was a decrease in physical lesions, as the mean cyst diameter went down from 36. 5 ± 25.4 mm to 33.0 ± 23.5 mm during three months (P < 0.001). Nevertheless, given the significant Standard Deviation and the lack of a control group, one can see the preliminary nature of the results. The number of patients involved in the research does not represent an adequate sample size considering the widespread occurrence and different types of the disease. The period of three months seems to be too short to evaluate the long -term effects of such a chronic and recurrent disorder. Concurrent changes include both lowering serum cancer antigen (CA125) (45.5 U/mL to 35.6 U/mL; P = 0.001) and body mass index (BMI) of the participants (22.2 to 21.2 kg/m 2). Those changes seem to reflect the anti-inflammatory properties of NAC rather than lesion-specific effects and cannot be attributed solely to the drug. Management of clinical symptoms has resulted in lower Visual Analog Scale (VAS) scores for dysmenorrhea (6.9 to 4.8; P < 0.0001) and chronic pelvic pain (7.2 to 5.7; P < 0.001), as well as decreased need for NSAID usage. A single -arm design does not allow evaluation of the intervention's efficacy relative to the control group, making the conclusions questionable. In addition, th ere is a high level of uncertainty related to reproduction outcomes, as only 39 out of 52 patients conceived spontaneously within six month s. Since the researchers have noted a decrease in dyspareunia, it is quite challenging to differentiate between cellular receptiveness and behavioral changes, as increased sexual activity might have been the consequence of less pain. Therefore, biological efficacy requires verification in future RCTs (Anastasi et al., 2023). IFN-γ Synergy and Programmed Organelle Failure: In Vitro Mechanisms An in vitro approach aimed at elucidating the molecular mechanisms underlying the proposed molecular switch, using human endometriotic ( 12Z) and endometrial ( HESC) stromal cell lines. Consequently, these outcomes cannot be translated clinically and require validation in human -derived tissue. Therefore, the results of this work can only be validated clinically for human tissue samples and cannot yet be applied clinically. This research revealed that NAC’s activity is dependent on the immune milieu, demonstrating a cooperative effect with Interferon -gamma ( IFN-γ) in inhibiting cell proliferation. While they each inhibited cell proliferation, their combination led to the production of antiproliferative effects by inducing mitochondrial dysfunction and ER stress, as evidenced by elevated levels of p -IRE1-α. A combined analysis strategy, comprising xCELLigence (real-time cell impedance analyses) assays, flow cytometry, immunofluorescence staining, western blotting, and metabolomics analysis, gave a detailed understanding of the molecular processes driving this disturbance of energy pathways in cells. REVIEW | OPEN ACCESS Medical Science 30, e112ms3921 (2026) 5 of 11 The data revealed that it leads to cell starvation and organellar dysfunction, where p -IRE1-α acts as a mediator of this process, which makes it a possible therapeutic or prognostic target. Despite these advances, this study was limited by the use of immo rtalized cell lines, such as 12Z and HESC, which exhibit characteristics similar to cancerous cells and may therefore lead to increased metabolic changes. Additionally, NAC did not exert any effects on cytokine -induced cell proliferation, like IL -6, or TNF -α, showing that its therapeutic utility may be limited to specific inflammatory profiles rather than serving as a universal intervention (Karakoç et al., 2025). Systemic Immunomodulation and Regression of Endometriotic Lesions The in vivo efficacy of systemic N -acetylcysteine (NAC) was compared with current pharmaceutical options in a randomized experiment in rats using surgically induced endometriosis. The findings indicated that NAC administration after 3 weeks reduc ed the average size of endometrial ectopic lesions (61.2±57.9 mm 2 to 30.5±21.9 mm 2; P = 0.043). While this treatment showed competitive performance to leuprolide acetate, causing a reduction in lesion size (44.3±43.5 mm 2 to 19.6±20.0 mm 2; P = 0,008) as well as amifostine (30.8±16.4 mm2 to 18.7±15 mm2; P = 0.012), no changes were observed in the control group. Apart from lesion structural diminishment, a decrease in levels of TNF-α was detected both in systemic (41.7±9.9 pg/mL to 17.25±10.1 pg/mL; P = 0.01) and peritoneal fluid (37.5±15.3 pg/mL to 19.2±13.1 pg/mL; P = 0.06) within NAC -, amifostine - and leuprolide -treated groups. Although this experiment exhibited promising results, there are limitations to its generalizability. First, rodents with surgically induced endometriosis do not replicate the complex immune pathology seen in humans, and second, since the treatment lasted only three weeks, there is a probability of endometriosis recurrence, or withdrawal of improvements in chronic clinical signs of pelvic pain and infertility (Onalan et al., 2014). Modulation of Autophagy in Endometriotic Lesions Another piece of literature shows that N -acetylcysteine (NAC) regulates defective autophagy in ectopic endometrium via inhibiting reactive oxygen species (ROS). Using a surgically induced rat model (n = 40), investigators discovered that the endometriosis-associated inflammatory and oxidative environment elevates ROS, which stimulates the expression of certain autophagy proteins, such as microtubule-associated protein light chain 3 (LC3), and Beclin -1. Injection of the peritoneum with NAC (200 mg/kg) for 21 days reduced oxidative stress and subsequently inhibited both LC3 fluorescence and Beclin -1 protein expression. This effect of NAC is related to interference with the connection between ROS and autophagy. It helps avoid ongoing oxidative damage and supports c ell survival strategies, thereby leading to lesion proliferation. However, the use of the surgically induced rat model is limited by its lack of translational potential, because suturing uterine tissue cannot repeat the multifactorial pathogenesis of endometriosis in hu mans. Additionally, the small number of animals per subgroup ( n = 10) shows variability, and the relatively short treatment period makes it challenging to extrapolate results (Lu et al., 2020). 3D Spheroid Modeling and Glutathione Exhaustion under NAC Interventions Modern in vitro studies have moved to 3D modeling to more accurately simulate metabolic stress. As part of this research, 3D tissue - like models were created from biopsies of human endometriosis lesions, 12Z cells, and Ishikawa cells as the control culture. They underwent an oxidative challenge with a low concentration of hydrogen peroxide (H2O2) to assess the limitations of pure NAC therapy. According to the findings, ectopic 12Z tissue is prone to redox-induced stress; thus, its total intracellular glutathione content decreased by 72% under acute oxidative conditions. When used as a molecular modulator, NAC hindered lipid peroxidation and reduced malondialdehyde (MDA) levels, which had increased threefold, back to baseline. Additionally, cytoplasmic proteins were resist ant to oxidation due to NAC intervention; hence, there was no protein carbonylation in the test group, whereas there was a fourfold increase in protein carbonyl content. On the other hand, NAC could not reverse oxidative DNA fragmentation and genomic damage. Such a

implies that iron -dependent DNA damage caused by endometrial hemorrhage cannot be targeted by a conventional radical scavenger such as NAC and that it occurs through alternative biochemical pathways. As for these models, although tissue viability is preserved, they can only be considered complete once metabolic manipulation s beyond antioxidant monotherapy are undertaken. From the methodological point of view, major limitations should be considered. For example, in vitro testing uses immortalized cell lines rather than primary human tissues, which cannot imitate all aspects of the physiological processes occurring in living endometriotic tissue in vivo. Another limitation includes the use of cancerous Ishikawa cells to model oxidative conditions. Finally, it should be pointed out that iron -driven biochemical reactions resulting from erythrocyte hemolysis in human endometriomas are not accounted for in this traditional model (Coelho et al., 2026). REVIEW | OPEN ACCESS Medical Science 30, e112ms3921 (2026) 6 of 11 NAC in Multicomponent Supplementation While the individual effects of NAC are the primary topic of the review at hand, the complexity of endometriosis is reflected by studies exploring multicomponent antioxidant supplementation. Research done with mouse models and endometriotic endothelial cells demonstrates the ability of the three -component preparation containing NAC, alpha -lipoic acid, and bromelain to function as a molecular modulator influencing the activity of endometriotic cells in specific ways. Together, this mixture brings about an increase in programmed cell death through caspase-3 and -7 activation and the downregulation of inflammatory factors such as VCAM-1 in lesion- derived endometriotic cells, without affecting normal myometrial/uterine cells. Clinical trials confirm these results with a decrease in cyst size and fewer cysts seen in the treated patients. Nonetheless, this

is based on the impact of the entire multicomponent formula rather than that of NAC only. There are some limitations of the in vivo study to be noted here. For one, SCID mice were used for experiments, which means that their adaptive immune system is compromised and does not fully represent the interaction between inflammation and other factors that are present in human endometriosis. In addition, a 21 -day-long intervention period cannot serve as an approximation for the multiyear -long development of the disease, which is why cyst prevention cannot be confirmed (Agostinis et al., 2015). In the case of a multicenter clinical study conducted on the basis of the LEAP trial, a cohort of 346 female patients with endometriosis-related pelvic pain was investigated, where the subset included 45 women interested in pregnancy and not taking any hormonal medications. During a six -month follow-up protocol, the analysis showed decreased levels of pain scores according to VAS (6.66 ±1.81 to 3.14 ±2.22; P < 0.001). The study also showed significant changes associated with a decrease in the number of patients having high pain index values (from 40.2% at baseline to 3.6% at study conclusion; P < 0.001). In addition, the frequency of consumption of standard analgesics was reduced, as the need for rescue NSAID became lower (86.4% to 37.4%; P < 0,001). From the standpoint of methodology, as applicable for this review, it should be highlighted that the LEAP trial did not involve only t he NAC medication as a monotherapy. Instead, participants received the mixture containing the NAC dose of 600 mg per day with 200 mg of alpha-lipoic acid (LA), 25 mg of bromelain (Br), and 10 mg of zinc. Even though the data about 27 cases of spontaneous pregnancies shows potential for fertility enhancement in combination with pain control, the design of the experiment does not allow us to prove any therapeutic effects of NAC specifically (Lete et al., 2018). Other research focused on evaluating the synergism between NAC and natural polyphenolic agents (curcumin and quercetin). In a twofold approach, scientists used both the in vitro model based on endometrial cell cultures and the in vivo model involving 33 female subjects diagnosed with endometriosis. Treatment with antioxidants in the combined form during 60 days contributed toward improved pain indices, including reduction in dysmenorrhea (Numeric Rating Scale NRS 6.1 to 2.8; P < 0.01), chronic pelvic pain (NRS 5.7 to 2.1; P < 0.01), and dyspareunia (NRS 5.3 to 2.5; P < 0.01) and less consumption of NSAIDs. However, the use of the mixture still hinders the isolation of the impact of NAC treatment. Moreover, the small sample size limits further generalization due to li mited information on recurrence over several months (Fadin et al., 2020). Overall, these experiments confound existing evidence bec ause NAC cannot be identified as the factor responsible for the beneficial effects. Follicular Redox Modulation In addition to modulating redox activity in endometriotic tissue, more comprehensive molecular effects have been studied in reproductive tissues. As has been shown, NAC promotes the nuclear translocation of nuclear factor erythroid 2 -related factor 2 (Nrf2), thereby triggering the transcription of several antioxidant enzymes in mouse oocytes (Fan et al., 2022). NAC was used to asse ss its effects on embryonic development and meiotic spindle integrity in oocytes from cows incubated with follicular fluid from patients with mild endometriosis characterized by increased oxidative stress (Giorgi et al., 2016; 2021). Nevertheless, these data warrant detailed consideration, as oxidative stress is likely a downstream effect of the main pathogenesis of endometriosis (hormonal). The ex ternal validity of these studies is poor; therefore, they can be interpreted only as explorations, as there is still insufficient ev idence for clinical use. As mentioned above, NAC is considered as an intervention for improving oocyte quality. Therefore, a double -blind RCT of 25 infertile women with stage III/IV endometriosis was conducted to explore this issue further. In the absence of statistical si gnificance, these clinical results must be interpreted as preliminary given the small sample size. The study showed an improvement in Tot al Antioxidant Capacity (TAC) upon administration of 1200 mg/day of NAC for six weeks ( P = 0.031). Moreover, cellular analysis of GCs confirmed a defensive mechanism through downregulation of BAX and CASP-3, responsible for apoptosis, and upregulation of BCL2 (P < 0.05). There was also a lower percentage of defunct oocytes (4.9%) versus the placebo group (6.1%). Although this study pro vides REVIEW | OPEN ACCESS Medical Science 30, e112ms3921 (2026) 7 of 11 an approach to exploring the redox-mediated oocyte protection mechanism, there is insufficient evidence for this biological shift, as the sample size of 25 participants is too small (Heshmati et al., 2026). 4. DISCUSSION The literature on NAC in endometriosis varies, ranging from experimental laboratory data to clinical research. The research o n in vitro models has reported interesting NAC’s coaction with IFN -γ, involving stress -related organelle signaling (Karakoç et al., 2025). However, experimental works have certain limitations. More precisely, most experiments use immortalized 12Z and HESC cell lines, together with cancer -like control cells that cannot imitate complicated, inflammatory conditions typical of the pelvic area and do not involve iron metabolism pathways (Karakoç et al., 2025; Coelho et al., 2026). This limits their external validity and ability to replicate chronic and progressive features inherent to human endometriosis. Preclinical animal studies involving rodents have shown both lesion regression and decreased cytokine levels. Although the re sults may seem positive, they cannot be generalized to clinical benefits due to the use of surgically induced implants, an acute fo rm of damage rather than a progressive inflammation that defines endometriosis. These studies also use a small subgroup, a very sho rt time of drug administration, and no specific human clinical outcomes, making it difficult to infer the possible durable effectiven ess or safety of the therapy (Pittaluga et al., 2010; Onalan et al., 2014; Lu et al., 2020; Agostinis et al., 2015). Clinical evidence gathered via observational studies, as well as prospective trials involving single -arm analyses and multicenter studies, faces additional challenges because NAC is often included in multi -component drugs containing LA, Br, and zinc. Multiple substances make it impossible to identify the actual effects of NAC, hence limiting the possibility of attributing beneficial effects observed in patients to it (Agostinis et al., 2015; Lete et al., 2018; Fadin et al., 2020). Moreover, the research on this topic features many

in methodology, like the use of non -randomized study designs, a lack of placebo controls, insufficient numbers of participants, and very limited follow -ups, which do not allow the assessment of lesion regression, alleviation of pain or any other reported change in the long term (Porpora et al., 2013; Anastasi et al., 2023). Overall, the current state of knowledge, ranging from cellular processes to clinical findings in humans, remains highly explo ratory. The reliance on artificially created cells, experimentally operated animals, and unclear multi -component compositions in clinical settings precludes establishing NAC as the standard. To make the comparison more transparent, a table 1 has been provided. In addition, the exploratory nature of the findings is evident in reproductive research. While animal experiments suggest benefi ts of NAC on egg quality, human tissue samples were not used in this case (Fan et al., 2022; Giorgi et al., 2016; 2021). Pilot human trials in this area have suffered from low power and a lack of clear results (Heshmati et al. 2026). Table 1. Overview of preclinical and clinical studies investigating NAC in endometriosis. Author Study design Sample size Intervention Main Outcomes Principal Limitations Pittaluga et al., 2010 Experimental murine study n = 40 mice NAC oral administration vs. untreated controls 60% reduction in lesion mass; Ki-67 decrease (52% to 23%); repositioning of junctional E-cadherin; downregulation of Cox2 and MMP-9. Animal model: limited translational relevance to human tissue. Porpora et al., 2013 Single-center observational cohort n = 92 women (47 NAC- treated; and 45 control group) NAC oral “pulse therapy” (1.8 g/day, 3 days/week) for 3 months Mean cyst reduction of 1.5 mm (vs 6.6 mm increase in controls P = 0.001); relief in dysmenorrhea (55%, P = 0.001), dyspareunia (50%, P = 0.027), chronic pelvic pain (59%, P = 0.015). Non-randomized design; single-center design: limited external validity; small cohort; short follow-up period; lack of placebo control. Anastasi et al., Prospective n = 120 NAC oral “pulse Reduction in VAS score: Non-randomized, REVIEW | OPEN ACCESS Medical Science 30, e112ms3921 (2026) 8 of 11 2023 single-arm cohort women therapy” (1.8 g/day, 3 days/week) for 3 months dysmenorrhea (6.9 to 4.8; P < 0.0001), chronic pelvic pain (7.2 to 5.7; P < 0.001); decrease in cyst size (36.5 to 33.0 mm, P < 0.001); reported spontaneous pregnancies. single-arm design: absence of control group; small cohort; brief follow-up period; incoherence in metabolic shifts (BMI); behavioral confounding of reproductive data. Karakoç et al., 2025 In vitro experimental study Human cell lines (12Z/ HESC) Addition to culture medium (NAC combined with IFN-) Synergistic induction of mitochondrial dysfunction and ER stress via p-IRE1-; suppression of energy pathways causing cellular starvation. Modeling gap using immortalized cell lines: exaggerated metabolic shifts; ineffective against IL-6 or TNF- driven proliferation. Onalan et al., 2014 Experimental in vivo animal study n = 40 female rats NAC vs. Leuprolide acetate and amifostine Macroscopic regression of endometriotic implant surface area (61.2±57.9 mm2 to 30.5±21.9 mm2; P = 0.043); decrease in TNF- systemic (41.7±9.9 pg/ml to 17.25±10.1 pg/ml; P = 0.01) and local peritoneal fluid (37.5± 15.3 pg/ml to 19.2±13.1 pg/ml; P =0.06). Surgically induced model does not replicate spontaneous human disease pathogenesis; acute 3- week treatment limits long-term relevance; lacks human clinical endpoints. Lu et al., 2020 Experimental in vivo animal study n = 50 female rats NAC (21 days) vs. Catalase and Control Reduced Local ROS levels; downregulated autophagy markers (LC3 and Beclin-1) in ectopic lesions. Surgically induced animal model does not mimic human pathogenesis; small sample size per subgroup. Coelho et al., 2026 In vitro 2D and 3D spheroid experimental study Human cell lines (12Z vs. Ishikawa) Pretreatment with isolated NAC (5mM in 2D / 1.25mM in 3D) prior to controlled H2O2 oxidative challenge NAC restored cell viability and prevented lipid peroxidation and protein carbonylation; 3D modeling captured a 72% depletion of the total glutathione pool; NAC failed to mitigate oxidative DNA fragmentation. Low external validity: in vitro architecture using immortalized cells lacking multicellular and inflammatory complexity of in vivo lesions; adenocarcinoma origin of the comparator line introduces cancer- specific redox adaptations; lacks iron- driven pathways. REVIEW | OPEN ACCESS Medical Science 30, e112ms3921 (2026) 9 of 11 Agostinis et al., 2015 Experimental in vitro / in vivo murine study n = 16 SCID mice and human endothelial cell lines (EECs/UtME Cs) Oral administration of combined mixture (NAC 250 m + LA 125 mg + Br 12.5 mg / day) vs. untreated controls Lower number and smaller size of cysts in vivo; synergistic downregulation of VCAM-1 expression in EECs; selective induction of apoptosis via caspases 3 and 7 activation. Low external validity: preclinical animal model using SCID mice lacking functional adaptive immunity; extremely brief 21-day intervention timeframe; therapeutic success depends on multi-ingredient synergy. Lete et al., 2018 Multicenter, open-label, non- comparative clinical trial n = 346 women Oral intake of a combination tablet containing 600 mg NAC, 200 mg LA, 25 mg Br, 10 mg Zinc/day for 6 months Significant drop in mean VAS scores (6.66 to 3.14; P < 0.001); a reduction in the proportion of patients suffering from severe pain (40.2% to 3.6%; P < 0.001); reduction in rescue NSAIDs reliance from 86.4% to 37.4% (P < 0.001); 27 spontaneous pregnancies. Open-label, non- comparative trial; architecture lacking placebo control arm; low specificity: fixed multicomponent formulation makes it impossible to isolate or verify the therapeutic effect of the NAC module. Fadin et al., 2020 Experimental in vitro and in vivo medical trial In vitro (- estradiol- induced endometrial cells) and n = 33 women NAC+ Tumeric + Quercetin for 60 days In vitro: Downregulated pro-inflammatory cytokines (IL-6, TNF-) and induced apoptosis. In vivo: Reduced dysmenorrhea (NRS 6.1 to 2.8; P < 0.01), chronic pelvic pain (NRS 5.7 to2.1; P < 0.01) and dyspareunia (NRS 5.3 to 2.5; P < 0.01) as well as a decrease in NSAID reliance. Small sample size; short follow-up duration; lack of a randomized placebo control group; low specificity: the isolation of NAC’s effect is not possible 5. CONCLUSION Despite the present research on NAC in the context of endometriosis, the existing translational gap cannot be ignored, with a simple experimentation design being used and clinical research being structurally invalid. It uses immortalized cells in vitro as well as animals that underwent surgery to create similar conditions. Most importantly, the existing data from clinical experiments are compro mised by the simultaneous use of several components of treatment. Observational studies, in addition, suffer from relatively small sample sizes, a lack of randomization, and inadequate follow -up. The placebo effect can easily influence the results of such studies, leading to high bias. The only solution here would be to move on to conducting RCTs in which NAC is used as the sole intervention. Acknowledgments There are no acknowledgments to disclose. REVIEW | OPEN ACCESS Medical Science 30, e112ms3921 (2026) 10 of 11 Authors’ Contributions Conceptualization: Magdalena Rakuś Methodology: Magdalena Rakuś, Krzysztof Łukasz, Jakub Marzec, Michał Nowakowski, Aleksandra Musioł, Paweł Gwałt Resources: Magdalena Rakuś, Krzysztof Łukasz, Jakub Marzec, Michał Nowakowski, Aleksandra Musioł, Paweł Gwałt Investigation: Magdalena Rakuś, Krzysztof Łukasz, Jakub Marzec, Michał Nowakowski, Aleksandra Musioł, Paweł Gwałt Writing-rough preparation: Magdalena Rakuś, Krzysztof Łukasz, Jakub Marzec, Michał Nowakowski, Aleksandra Musioł, Paweł Gwałt Writing-editing: Magdalena Rakuś, Krzysztof Łukasz, Jakub Marzec, Michał Nowakowski, Aleksandra Musioł, Paweł Gwałt Supervision: Magdalena Rakuś All authors have read and agreed to the published version of the manuscript. Informed consent Not applicable. Ethical approval Not applicable. This article does not contain any studies with human participants or animals performed by any of the authors. Funding This research did not receive any external funding like specific grant from funding agencies in the public, commercial, or nonprofit sectors. Conflict of interest The authors declare that they have no conflicts of interest, competing financial interests or personal relationships that could have influenced the work reported in this paper. Data and materials availability All data associated with this study will be available based on the reasonable request to corresponding author.

1. Agostinis C, Zorzet S, De Leo R, Zauli G, De Seta F, Bulla R. The combination of N -acetyl cysteine, alpha -lipoic acid, and bromelain shows high anti -inflammatory properties in novel in vivo and in vitro models of endometriosis. Mediators Inflamm 2015;2015:918089. doi: 10.1155/2015/918089. 2. Anastasi E, Scaramuzzino S, Viscardi MF, Viggiani V, Piccioni MG, Cacciamani L, Merlino L, Angeloni A, Muzii L, Porpora MG. Efficacy of N -Acetylcysteine on Endometriosis -Related Pain, Size Reduction of Ovarian Endometriomas, and Fertility Outcomes. Int J Environ Res Public Health 2023;20(6):4686. doi: 10.3390/ijerph20064686. 3. As-Sanie S, Mackenzie SC, Morrison L, Schrepf A, Zondervan KT, Horne AW, Missmer SA. Endometriosis: A Review. JAMA 2025;334(1):64-78. doi: 10.1001/jama.2025.2975. 4. Becker CM, Bokor A, Heikinheimo O, Horne A, Jansen F, Kiesel L, King K, Kvaskoff M, Nap A, Petersen K, Saridogan E, Tomassetti C, van Hanegem N, Vulliemoz N, Vermeulen N; ESHRE Endometriosis Guideline Group. ESHRE guideline: endometriosis. Hum Reprod Open 2022;2022(2):hoac009. doi: 10.1093/hropen/hoac009. 5. Clower L, Fleshman T, Geldenhuys WJ, Santanam N. Targeting Oxidative Stress Involved in Endometriosis and Its Pain. Biomolecules 2022;12(8):1055. doi: 10.3390/biom12081 055. 6. Coelho JA, Gomes KS, Cerchiaro G. Differential Antioxidant Capacities of Human Endometriotic and Endometrial Cell Models Under H2O2 Exposure. Int J Mol Sci 2026;27(9):4131. doi: 10.3390/ijms27094131. 7. Facchin F, Vercellini P, Somigliana E. The impact of endometriosis on educational and professional pathways: a cross-sectional online study. BMC Psychol 2026;14(1):303. doi: 10.1186/s40359-026-04112-5. 8. Fadin M, Nicoletti MC, Pellizzato M, Accardi M, Baietti MG, Fratter A. Effectiveness of the integration of quercetin, turmeric, and N -acetylcysteine in reducing inflammation and pain associated with endometriosis. In -vitro and in -vivo REVIEW | OPEN ACCESS Medical Science 30, e112ms3921 (2026) 11 of 11 studies. Minerva Ginecol 2020;72(5):285 -291. doi: 10.23736/S00 26-4784.20.04615-8. 9. Fan L, Guan F, Ma Y, Zhang Y, Li L, Sun Y, Cao C, Du H, He M. N -Acetylcysteine improves oocyte quality through modulating the Nrf2 signaling pathway to ameliorate oxidative stress caused by repeated controlled ovarian hyperstimulation. Reprod Fertil Dev 2022;34(10):736 -750. doi: 10.1071/RD22020. 10. Giorgi VS, Da Broi MG, Paz CC, Ferriani RA, Navarro PA. N- Acetyl-Cysteine and l -Carnitine Prevent Meiotic Oocyte Damage Induced by Follicular Fluid From Infertile Women With Mild Endometriosis. Reprod Sci 2016;23(3):342 -51. doi: 10.1177/1933719115602772. 11. Giorgi VSI, Ferriani RA, Navarro PA. Follicular Fluid from Infertile Women with Mild Endometriosis Impairs In Vitro Bovine Embryo Development: Potential Role of Oxidative Stress. Rev Bras Ginecol Obstet 2021;43(2):119 -125. doi: 10.1055/s-0040-1718443. 12. Heshmati ZS, Amiri -Yekta A, Khosravifar M, Akbarian F, Moini A, Eftekhari -Yazdi P, Hafezi M, Afsharian P. Administration of N-acetylcysteine influence the expression of apoptotic genes in the granulosa cells of infertile women diagnosed with endometriosis. Sci Rep 2026;16(1):7961. doi: 10.1038/s41598-025-34202-0. 13. Karakoç E, Halaçlı SO, Hanelçi RH, Ayhan S, Eylem CC, Nemutlu E, Atilla P. N -acetylcysteine stimulates organelle malfunction in endometriotic cells via IFN -gamma signaling. Sci Rep 2025;15(1):15120. doi: 10.1038/s41598-025-00195-z. 14. Lete I, Mendoza N, de la Viuda E, Carmona F. Effectiveness of an antioxidant preparation with N -acetyl cysteine, alpha lipoic acid and bromelain in the treatment of endometriosis - associated pelvic pain: LEAP study. Eur J Obstet Gynecol Reprod Biol 2018;228:221 -224. doi: 10.1016/j.ejogrb.2018.07.00 2. 15. Li Y, Gong Y, Jiang H, Ji M. Impact of endometriotic cystectomy on ovarian reserve function and ovulation induction outcomes in women with endometriosis undergoing assisted reproductive technology. Front Endocrinol (Lausanne) 2026;16:1687765. doi: 10.3389/fendo. 2025.1687765. Erratum in: Front Endocrinol (Lausanne) 2026; 17:1806799. doi: 10.3389/fendo.2026.1806799. 16. Lu H, Hu H, Yang Y, Li S. The inhibition of reactive oxygen species (ROS) by antioxidants inhibits the release of an autophagy marker in ectopic endometrial cells. Taiwan J Obstet Gynecol 2020;59(2):256 -261. doi: 10.1016/j.tjog.2020.01. 014. 17. Onalan G, Gulumser C, Mulayim B, Dagdeviren A, Zeyneloglu H. Effects of amifostine on endometriosis, comparison with N -acetyl cysteine, and leuprolide as a new treatment alternative: a randomized controlled trial. Arch Gynecol Obstet 2014;289(1):193 -200. doi: 10.1007/s00404 -013- 2963-0. 18. Pittaluga E, Costa G, Krasnowska E, Brunelli R, Lundeberg T, Porpora MG, Santucci D, Parasassi T. More than antioxidant: N-acetyl-L-cysteine in a murine model of endometriosis. Fertil Steril 2010;94(7):2905-8. doi: 10.1016/j.fertnstert.2010.06.038. 19. Porpora MG, Brunelli R, Costa G, Imperiale L, Krasnowska EK, Lundeberg T, Nofroni I, Piccioni MG, Pittaluga E, Ticino A, Parasassi T. A promise in the treatment of endometriosis: an observational cohort study on ovarian endometrioma reduction by N -acetylcysteine. Evid Based Complement Alternat Med 2013;2013:240702. doi: 10.1155/2013/240702. 20. Taylor HS, Kotlyar AM, Flores VA. Endometriosis is a chronic systemic disease: clinical challenges and novel innovations. Lancet 2021;397(10276):839 -852. doi: 10.1016/S0140 -6736(21)00 389-5. 21. Tomassetti C, Johnson NP, Petrozza J, Abrao MS, Einarsson JI, Horne AW, Lee TTM, Missmer S, Vermeulen N, Zondervan KT, Grimbizis G, De Wilde RL. An international terminology for endometriosis, 2021. Hum Reprod Open 2021(4):hoab029. doi: 10.1093/hropen/hoab029. 22. Veth VB, Keukens A, Reijs A, Bongers MY, Mijatovic V, Coppus SFPJ, Maas JWM. Recurrence after surgery for endometrioma: a systematic review and meta -analyses. Fertil Steril 2024;122(6):1079-1093. doi: 10.1016/j.fertnstert.2024.07.03 3. 23. Zondervan KT, Becker CM, Missmer SA. Endometriosis. N Engl J Med 2020;382(13):1244 -1256. doi: 10.1056/NEJMra1810 764.