The effect of antioxidant supplementation on dysmenorrhea and endometriosis-associated painful symptoms: a systematic review and meta-analysis of randomized clinical trials

Endometriosis is a chronic inflammatory gynecological condition characterized by the presence and growth of tis - sues similar to the endometrium outside the uterine cavity. It affects at least 10% of women of reproductive age [1]. Furthermore, it frequently causes pelvic pain, dysmenorrhea, dyspareunia, and infertility, impairing patients’ quality of life [2]. Endometriosis is a multifaceted, symptomatic, pathobio - logical, multisystem, and heterogeneous disease. Based on the phenotype, it can be categorized into superficial perito - neal lesions, ovarian endometriomas, and deep-infiltrating endometriosis [3,4]. When addressing general endometriosis The effect of antioxidant supplementation on dysmenorrhea and endometriosis-associated painful symptoms: a systematic review and meta-analysis of randomized clinical trials Saeed Baradwan, MD 1 , Abdulrahim Gari, MD 2,3 , Hussein Sabban, MD 1,4 , Majed Saeed Alshahrani, MD 5 , Khalid Khadawardi, MD 2 , Ibtihal Abdulaziz Bukhari, MD 6 , Abdullah Alyousef, MD 7 , Ahmed Abu-Zaid, MD, PhD 8 Department of Obstetrics and Gynecology, 1 King Faisal Specialist Hospital and Research Center, Jeddah, 2 College of Medicine, Umm Al-Qura Univer- sity, Makkah, 3 Al Salama Hospital, Jeddah, 4 Faculty of Medicine at Rabigh, King Abdulaziz University, Rabigh, 5 Faculty of Medicine, Najran University, Najran, 6 College of Medicine, Princess Nourah bint Abdulrahman University, Riyadh, 7 King Abdullah bin Abdulaziz University Hospital, Riyadh, 8 Col- lege of Medicine, Alfaisal University, Riyadh, Saudi Arabia This study aimed to review randomized controlled trials (RCTs) investigating the effects of dietary antioxidant supple- ments on the severity of endometriosis-related pain symptoms. The PubMed/Medline, Scopus, and Web of Science databases were searched until April 2022. Additionally, we manually searched the reference lists. Endpoints were summarized as standardized mean difference (SMD) with 95% confidence intervals (CIs) in a random-effects model. The I 2 statistic was used to assess heterogeneity. Ten RCTs were included in this meta-analysis. Overall, 10 studies were related to dysmenorrhea, four to dyspareunia, and four to pelvic pain. Antioxidants significantly reduced dysmenor- rhea (SMD, -0.48; 95% CI, -0.82 to -0.13; I 2 =75.14%). In a subgroup analysis, a significant reduction of dysmenorrhea was observed only in a subset of trials that administered vitamin D (SMD, -0.59; 95% CI, -1.13 to -0.06; I 2 =69.59%) and melatonin (SMD, -1.40; 95% CI, -2.47 to -0.32; I 2 =79.15%). Meta-analysis results also suggested that antioxidant sup- plementation significantly improved pelvic pain (SMD, -1.51; 95% CI, -2.74 to -0.29; I 2 =93.96%), although they seem not to have a significant beneficial impact on the severity of dyspareunia. Dietary antioxidant supplementation seems to beneficially impact the severity of endometriosis-related dysmenorrhea (with an emphasis on vitamin D and mela - tonin) and pelvic pain. However, due to the relatively small sample size and high heterogeneity, the findings should be interpreted cautiously, and the importance of further well-designed clinical studies cannot be overstated.

Antioxidant; Endometriosis; Dysmenorrhea; Dyspareunia; Pelvic pain Articles published in Obstet Gynecol Sci are open-access, distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons. org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright © 2024 Korean Society of Obstetrics and Gynecology Review Article Obstet Gynecol Sci 2024;67(2):186-198 https://doi.org/10.5468/ogs.23210 eISSN 2287-8580 Received: 2023.08.25. Revised: 2023.10.11. Accepted: 2023.11.09. Corresponding author: Ahmed Abu-Zaid, MD, PhD Department of Obstetrics and Gynecology, College of Medicine, Alfaisal University, Al Zahrawi Street, Riyadh 11533, Saudi Arabia E-mail: [email protected] https://orcid.org/0000-0003-2286-2181 www.ogscience.org 187 Saeed Baradwan, et al. Antioxidants & gynecologic-related pain management, three therapeutic modalities are commonly available: I) medicinal treatment (e.g., painkillers, nonsteroi - dal anti-inflammatory drugs, combined oral contraceptives, progestins, and gonadotropin-releasing hormone analogs); II) surgery (conservative or definitive); and III) assisted repro - ductive technologies (such as in vitro fertilization and intra - cytoplasmic sperm injection). It is crucial to emphasize the timely administration of pain management, as therapeutic inertia portends the development of central sensitization (au- tonomous) [5-7]. Interestingly, reduction in oxidative stress is also a crucial alternative for endometriosis management. Inflammation leads to an increased production of reactive oxygen species (ROS), which play a fundamental role in the proliferation of endometriotic cells as well as in the develop- ment, persistence, and progression of the disease [8]. Dietary micro- and macro-nutrients and dietary factors are pivotal in controlling chronic diseases [9,10]. Dietary antioxidants exert beneficial neutralizing effects against free radicals and ROS produced by endometriotic cells. They demonstrate anti- inflammatory properties [11,12] and pro-apoptotic and anti- angiogenic actions and generally have a favorable safety pro - file. Therefore, one could speculate that they may effectively reduce pain-inducing factors and improve endometriosis- associated clinical symptoms [13-15]. A growing body of literature has explored the impact of different dietary antioxidants on endometriosis-related pain [15]. Antioxidant vitamins successfully reduce the intensity of dysmenorrhea, ameliorate dyspareunia and pelvic pain, and improve the quality of life in patients with endometriosis. Consequently, therapy involving antioxidant vitamins may be considered an alternative treatment approach, independently or in conjunction with other methods, to alleviate endometri - osis-related pain [16]. Zheng et al. [16] showed that supple - mentation with vitamin E improved endometriosis-related pelvic pain, whereas supplementation with vitamin D did not. Some systematic reviews also reported no significant ef - fects of vitamin D on dysmenorrhea or non-cyclic pelvic pain [17]. Nevertheless, no systematic reviews or meta-analyses have comprehensively summarized the effects of dietary antioxidants on endometriosis and dysmenorrhea. To bridge this gap, we conducted a comprehensive systematic review and meta-analysis of randomized controlled trials (RCTs). We aimed to investigate the duration response and the impact of administering various dietary antioxidant supplements, including vitamins D, C, E, and A, melatonin, curcumin, omega-3 fatty acids, resveratrol, zinc, copper, chromium, and selenium, separately or in different combinations, on the severity of endometriosis-associated painful symptoms. The symptoms evaluated include dysmenorrhea, dyspareunia, and chronic pelvic pain in women of reproductive age.

This systematic review and meta-analysis of RCTs was perfor- med according to the guidelines of the preferred reporting items for systematic reviews and meta-analyses statement and the current recommendations of the Cochrane Collabo- ration. 1. Search strategy and study selection We searched electronic research databases, including Sco - pus, Web of Science (Science and Social Science Citation Index), and PubMed/Medline, from their inception until April 2022. In addition, we manually searched the reference lists and citations of the eight identified articles using Google Scholar. We also contacted authors who had published in this area to ensure we did not miss any relevant publications. Search terms were set by the authors and adapted for use in other databases. There were no restrictions on the language or publication dates. Document with incomplete data or the author could not be reached was discarded. The search stra - tegy is described in detail in Supplementary Table 1. Two re- viewers (S.B. and A.G.) independently screened all identified records for potentially eligible studies after reading all titles and abstracts. The final inclusion criteria were determined af - ter reading the full texts strictly. Disagreements were resolved through discussion with a third reviewer (A.A.). 2. Eligibility criteria The eligibility criteria of the studies were formulated ac - cording to the participants, interventions, comparisons, outcomes, and study design criteria. I) Participants: women with clinically and/or histologically confirmed endometriosis; II) intervention: supplementation with antioxidants (vitamins D, C, E, and A, melatonin, curcumin, omega-3 fatty acids, resveratrol, zinc, copper, chromium, and selenium, separately or in different combinations); III) comparators: antioxidant versus no treatment, antioxidant versus placebo; IV) out - comes: severity of dysmenorrhea, dyspareunia and/or chronic www.ogscience.org188 Vol. 67, No. 2, 2024 pelvic pain assessed by any pain assessment scale/tool; and V) study design: a clinical randomized controlled study. Stu - dies meeting any of the following criteria were excluded: I) pain caused by reasons other than endometriosis; II) non-ran- domized clinical trials (e.g., conference abstracts, repeated publications, animal experiments, case reports, and reviews); and III) studies without available data for analysis. 3. Data extraction and assessment of the quality of included studies Two reviewers (H.S. and M.S.A.) independently extracted the following data from the included trials: participant characteri - stics (e.g., age, diagnostic method, body mass index, and pa- rity), study characteristics (e.g., first author name, publication year, region, study design, sample size, intervention type, and intervention characteristics), and study outcomes. The corres - ponding authors were contacted for additional information. Two reviewers (H.S. and M.S.A.) assessed the methodological quality of the included trials according to the Cochrane Han - dbook for Systematic Reviews of Interventions version 5.1.0 (Cochrane, London, England). Disagreements were resolved through discussion with a third reviewer (K.K.). Each trial was evaluated for seven items: random sequence generation, concealed allocation, blinding of participants and personnel (performance bias), blinding of outcome assessment (detecti- on bias), incomplete outcome data (attrition bias), selective reporting (reporting bias), and other biases; each item was rated as “high risk”, “low risk”, or “unclear”. 4. Statistical analysis Stata version 16 (Stata Corporation, College Station, TX, USA) was used for the statistical analyses. All continuous Fig. 1. The flow diagram of literature search and selection of studies. RCT, randomized controlled trials. Records identified through database searching (n=134) Additional records identified through other sources (n=8) Records after duplicates removed (n=109) Records screened (n=109) Records excluded (n=91) Full-text articles excluded, with reasons (n=8) Have no relevant variables (5) Combination with other treatments (2) Not RCT (1) Full-text articles assessed for eligibility (n=18) Studies included in qualitative synthesis (n=10) Studies included in quantitative synthesis (meta-analysis) (n=10) IdentificationScreeningEligibilityIncluded www.ogscience.org 189 Saeed Baradwan, et al. Antioxidants & gynecologic-related pain Table 1. The main characteristics of the included studies Study Country Sample size (final analysis) Antioxidant type Dosage Duration (weeks) Age Endometriosis stage Pain type Pain assessment

Intervention Placebo Mean SD Mean SD Sesti et al. (2007) [26] Italy 145 Mix - 24 29.1 3.9 31.1 4.1 III and I ↔ Dysmenorrhea VAS score ↔ Dyspareunia ↓ Pelvic pain Lasco et al. (2012) [21] Italy 40 Vitamin D 300,000 IU 8 NM NM NM NM NM ↓ Dysmenorrhea VAS score Schwertner et al. (2013) [25] Brazil 40 Melatonin 10 mg 8 36.76 6.4 37.63 5.5 All stages ↓ Dysmenorrhea VAS score Mendes da Silva et al. (2017) [23] Brazil 44 Resveratrol 40 mg 6 35.4 7.1 32.4 7 NM ↔ Dysmenorrhea VAS score Almassinokiani et al. (2016) [19] Iran 40 Vitamin D 50,000 IU/ weekly 12 30.84 5.79 28.95 4.71 All stages ↔ Dysmenorrhea VAS score ↔ Pelvic pain Hoseinalizadeh and Cahichian (2018) [20] Iran 40 Melatonin 5 mg 8 NM NM NM NM III and IV ↓ Dysmenorrhea VAS score ↓ Pelvic pain Abokhrais et al. (2020) [18] UK 30 Omega-3 1,000 mg/ twice a day 8 35.43 8.57 36.08 9.59 All stages ↔ Dysmenorrhea -Pain ↔ Dyspareunia -Catastrophizing -Questionnaire Nodler et al. (2020) [24] USA 42 Omega-3 2,000 IU/day 24 20.01 2.7 20.1 3.5 All stages except stage III ↔ Dysmenorrhea VAS score Amini et al. (2021) [13] Iran 60 Mix (C and E) - C, 1,000 mg/ day 8 35.7 5.71 38.03 6.47 I to III ↔ Dysmenorrhea VAS score ↓ Dyspareunia - E, 800 IU/day ↓ Pelvic pain Mehdizadehkashi et al. (2021) [22] Iran 60 Vitamin D 50,000 IU/each 2 weeks 12 34.8 7.1 35.6 7 NM ↓ Dysmenorrhoea VAS score ↔ Dyspareunia ↓, this symbol is a sign of decreasing variables in the intervention group; ↔, this sign indicates no difference between the two groups. SD, standard deviation; VAS, visual analog scale; IU, international unit; NM, not mentioned. www.ogscience.org190 Vol. 67, No. 2, 2024 variables were pooled by standard mean differences (SMDs) with 95% confidence intervals (CIs). Heterogeneity was eva- luated using the Higgins’ I 2 statistic. I 2 statistics of 0-25%, 25-50%, 50-75%, and >75% were suggestive of very low, low, moderate, and high heterogeneity, respectively. We used a random-effects model to calculate individual study SMD and corresponding 95% CIs. In addition, a chi-square test for heterogeneity was performed, and the P -values were presented. Exploration of the causes of heterogeneity was planned using variations in the antioxidant type and durati - on of the intervention. We performed sensitivity analyses to evaluate the robustness of pooled estimations after exclusion of every single trial through the “Leave-one-out method”. Additionally, we assessed the risk of publication bias across studies using counter-funnel plots of the outcomes. We used the GRADEpro GDT software (Evidence Prime, Hamilton, ON, USA) to formally assess the quality of evidence for sele - cted outcomes. We evaluated the outcomes considering the following five criteria: risk of bias, inconsistency, indirectness, imprecision, and publication bias, and the level of evidence was graded as very low, low, moderate, or high. The certain - ty of the evidence evaluation is presented in Supplementary Table 2.

1. Study selection Initially, 134 records were identified through a database sear- ch, and eight additional records were identified through ot - her sources. All studies were then imported into EndNote X9 software (Thomson Reuters, Philadelphia, PA, USA), 25 dupli - cates were removed, and 91 studies were removed after the title and abstract screening process; thus, 18 studies were screened for eligibility in more detail. Finally, after detailed title, abstract, and full-text evaluations, 10 [13,18-26] RCTs were included in this systematic review and meta-analysis. A flow diagram of the complete literature search and selection of studies is shown in Fig. 1. 2. Study characteristics The ethnicity of the study population varied among the stu - dies: Iran (n=4), Brazil (n=2), Italy (n=2), Britain (n=1), and the United States (n=1). All the included trials, except one, were published in English between 2007 and 2021. A total of 541 women aged 20-40 years were examined, with indivi - dual study sample sizes ranging from 30 to 145 participants. Endometriosis was confirmed by histopathology in all trials. Seven trials reported the endometriosis stage in the patients. The characteristics of the included trials are summarized in Table 1. A summary of the risk of bias demonstrated that the methodological quality of these trials was relatively desirable (Fig. 2). All participants in each trial were randomly allocated to groups using an adequate allocation procedure. The ran - domization was unclear in both trials. Six trials stated that the allocation was concealed, one trial stated that it was unclear, and three trials were at high risk. Half of the trials used pa - tient masking, and the blinding of participants and personnel was unclear in four trials. Thus, only one trial had a high risk of performance bias. Four trials reported appropriate blinding of the outcome assessment, whereas blinding was unclear Fig. 2. The summary of risk of bias assessments. Abokhrais, 2020 Almassinokiani, 2016 Amini, 2021 Hoseinalizadeh, 2018 Lasco, 2012 Mehdizadehkashi, 2021 Mendes da silva, 2017 Nodler, 2020 Schwertner, 2013 Sesti, 2007 Random sequence generation (selection bias) Allocation concealment (selection bias) Blinding of participants and personnel (performance bias) Blinding of outcome assessment (detection bias) Incomplete outcome data (attrition bias) Selective reporting (reporting bias) Other bias www.ogscience.org 191 Saeed Baradwan, et al. Antioxidants & gynecologic-related pain Fig. 3. Forest plot of the effect of antioxidants on dysmenorrhea pain. N, number; SD, standard deviation; CI, confidence interval; FA, fatty acid. Treatment Control Hedges's g Weight (%)Study N Mean SD N Mean SD with 95% CI Vitamin D Mehdizadehkashi, 2021 30 -3.2 1.75 30 -1.9 2.71 -0.56 (-1.07, -0.05) 9.79 Nodler, 2020 27 -1.5 3.01 22 -1.6 2.94 0.03 (-0.52, 0.59) 9.43 Almassinokiani, 2016 19 -5.27 2.48 20 -3.69 2.94 -0.57 (-1.20, 0.06) 8.83 Lasco, 2012 20 -2.35 1.75 20 0.1 1.76 -1.37 (-2.05, -0.69) 8.43 Heterogeneity: τ 2 =0.20, I 2 =69.59%, H 2 =3.29 -0.59 (-1.13, -0.06) Test of θi=θj: Q(3)=9.87, P=0.02 Omega-3 FAs Nodler, 2020 20 -0.7 3.15 22 -1.6 2.94 0.29 (-0.31, 0.89) 9.07 Abokhrais, 2020 14 0.04 1.88 13 -0.67 2.47 0.32 (-0.42, 1.05) 7.97 Heterogeneity: τ 2 =0.00, I 2 =0.00%, H 2 =1.00 0.30 (-0.16, 0.76) Test of θi=θj: Q(1)=0.00, P=0.96 Resveratrol Mendes da Silva, 2017 22 -2.5 2.43 22 -1.5 3.13 -0.35 (-0.94, 0.23) 9.18 Heterogeneity: τ 2 =0.00, I 2 =0.00%, H 2 =0.00 -0.35 (-0.94, 0.23) Test of θi=θj: Q(0)=-0.00, P=0.00 Melatonin Hoseinalizadeh, 2018 20 -3.85 1.37 20 -1.54 0.89 -1.96 (-2.70, -1.22) 7.91 Schwertner, 2013 20 -3.741 2.21 20 1.98 1.75 -0.87 (-1.50, -0.23) 8.76 Heterogeneity: τ 2 =0.47, I 2 =79.15%, H 2 =4.80 -1.40 (-2.47, -0.32) Test of θi=θj: Q(1)=4.80, P=0.03 Combination Sesti, 2007 35 -1.7 1.05 110 -1.5 1.25 -0.17 (-0.54, 0.21) 10.80 Amini, 2021 30 -3.3 5.63 30 -1.95 3.11 -0.29 (-0.80, 0.21) 9.84 Heterogeneity: τ 2 =0.00, I 2 =0.00%, H 2 =1.00 -0.21 (-0.51, 0.09) Test of θi=θj: Q(1)=0.16, P=0.69 -0.48 (-0.82, -0.13) Overall Heterogeneity: τ 2 =0.26, I 2 =75.14%, H 2 =4.02 Test of θi=θj: Q(10)=40.23, P=0.00 Test of group differences: Qb(4)=11.52, P=0.02 Random-effects DerSimonian-Laird model -1 0 12-3 Fig. 4. Subgroup forest plot of the effect of antioxidants on dysmenorrhea pain. N, number; SD, standard deviation; CI, confidence inter - val. Treatment Control Hedges's g Weight (%)Study N Mean SD N Mean SD with 95% CI ≤8 weeks Mendes da Silva, 2017 22 -2.5 2.43 22 -1.5 3.13 -0.35 (-0.94, 0.23) 9.18 Amini, 2021 30 -3.3 5.63 30 -1.95 3.11 -0.29 (-0.80, 0.21) 9.84 Almassinokiani, 2016 19 -5.27 2.48 20 -3.69 2.94 -0.57 (-1.20, 0.06) 8.83 Hoseinalizadeh, 2018 20 -3.85 1.37 20 -1.54 0.89 -1.96 (-2.70, -1.22) 7.91 Schwertner, 2013 20 -3.741 2.21 20 -1.98 1.75 -0.87 (-1.50, -0.23) 8.76 Lasco, 2012 20 -2.35 1.75 20 0.1 1.76 -1.37 (-2.05, -0.69) 8.43 Abokhrais, 2020 14 0.04 1.88 13 -0.67 2.47 0.32 (-0.42, 1.05) 7.97 Heterogeneity: τ 2 =0.35, I 2 =77.16%, H 2 =4.38 -0.71 (-1.22, -0.21) Test of θi=θj: Q(6)=26.27, P=0.00 >8 weeks Mehdizadehkashi, 2021 30 -3.2 1.75 30 -1.9 2.71 -0.56 (-1.07, -0.05) 9.79 Nodler, 2020 27 -1.5 3.01 22 -1.6 2.94 0.03 (-0.52, 0.59) 9.43 Nodler, 2020 20 -0.7 3.15 22 -1.6 2.94 0.29 (-0.31, 0.89) 9.07 Sesti, 2007 35 -1.7 1.05 110 -1.5 1.25 -0.17 (-0.54, 0.21) 10.80 Heterogeneity: τ 2 =0.04, I 2 =40.31%, H 2 =1.68 -0.13 (-0.45, 0.20) Test of θi=θj: Q(3)=5.03, P=0.17 -0.48 (-0.82, -0.13) Overall Heterogeneity: τ 2 =0.26, I 2 =75.14%, H 2 =4.02 Test of θi=θj: Q(10)=40.23, P=0.00 Test of group differences: Qb(1)=3.69, P=0.05 Random-effects DerSimonian-Laird model -1 0 12-3 www.ogscience.org192 Vol. 67, No. 2, 2024 in five trials. Only one trial had a high risk of detection bias. Half the trials provided an intention-to-treat analysis. Three trials were conducted for each protocol. Two trials had a high risk of attrition bias. All the trials were judged to have a low risk of reporting bias. Finally, three trials were judged as unclear for other biases. All trials recorded sufficient data on dysmenorrhea; however, five of these trials provided insuf - ficient data on pelvic pain, and four studies did not provide sufficient information on dyspareunia. 3. The effect of antioxidants on dysmenorrhea Pooling results from 10 trials that compared the effect of an- tioxidants versus placebo in terms of reducing dysmenorrhea suggested that antioxidants significantly reduced dysmenorr - hea (SMD, -0.48; 95% CI, -0.82 to -0.13; P <0.001) (Fig. 3). High heterogeneity was detected between trials (P <0.001; I 2 =75.14%); however, there was no indication of publication bias affecting these findings (Egger’s regression intercept, -4.77; 95% CI, -12.17 to 2.62; P =0.178). In a subgroup analysis, a significant difference was observed in the subset of trials that compared the effect of various antioxidants on dysmenorrhea: vitamin D, omega-3 fatty acids, resve - ratrol, melatonin, and a combination of antioxidants (Chi 2 , 11.52; degree of differentiation, 4; P =0.02). There was a significant reduction in dysmenorrhea in the subset of trials that administered vitamin D (SMD, -0.59; 95% CI, -1.13 to -0.06; P=0.02; I 2 =69.59%), and those that administered melatonin (SMD, -1.40; 95% CI, -2.47 to -0.32; P =0.03; I 2 =79.15%; Fig. 3). No significant reduction in the severity of dysmenorrhea was observed in the subset of trials that administered omega-3 fatty acids (SMD, 0.30; 95% CI, -0.16 to 0.76; P =0.96; I 2 =0%), resveratrol (SMD, -0.35; 95% CI, -0.94 to 0.23), or combination of antioxidants (SMD, -0.21; 95% CI, -0.51 to 0.09; P =0.69; I 2 =0%; Fig. 3). A significant reduction in dysmenorrhea was observed in the subset of trials that administered antioxidant supplementation for ≤8 weeks (SMD, -0.71; 95% CI, -1.22 to -0.21; P <0.001; I 2 =77.16%). Conversely, no significant reduction was obser - ved in the subset of trials with antioxidant supplementation longer than 8 weeks (SMD, -0.13; 95% CI, -0.45 to 0.20; P=0.17; I 2 = 40.31%; Fig. 4). The sensitivity analysis indicated that the exclusion of any individual trial did not significantly alter the overall results of the meta-analysis, revealing the high stability of the results (Supplementary Fig. 1). A funnel plot depicts a triangular region centered on the pooled SMD, in which 95% CI of study findings should fall if there is no publication bias and no heterogeneity in the underlying true effects (Supplementary Fig. 2). Fig. 6. Forest plot of the effect of antioxidants on pelvic pain. N, number; SD, standard deviation; CI, confidence interval; REML, restricted maximum likelihood. Treatment Control Hedges's g Weight (%)Study N Mean SD N Mean SD with 95% CI Sesti, 2007 35 -3.8 0.98 110 -1.8 1.23 -1.69 (-2.12, -1.27) 25.93 Amini, 2021 30 -5.38 2.41 30 0.17 1.73 -2.61 (-3.30, -1.93) 24.71 Almassinokiani, 2016 19 -3.21 2.98 20 -4.14 3.56 0.28 (-0.34, 0.89) 25.06 Hoseinalizadeh, 2018 20 -4.0 1.38 20 -1.57 0.89 -2.05 (-2.81, -1.30) 24.30 Overall -1.51 (-2.74, -0.29) Heterogeneity: τ 2 =1.47, I 2 =93.96%, H 2 =16.56 Test of θi=θj: Q(3)=44.69, P=0.00 Test of θ=0: z=-2.42, P=0.02 Random-effects REML model -1 0 12-3 Fig. 5. Forest plot of the effect of antioxidants on dyspareunia pain. N, number; SD, standard deviation; CI, confidence interval. Treatment Control Hedges's g Weight (%)Study N Mean SD N Mean SD with 95% CI Mehdizadehkashi, 2021 30 -2.3 2.46 30 -2.1 2.55 -0.08 (-0.58, 0.42) 25.57 Sesti, 2007 35 -2.2 1.15 110 -2.0 1.2 -0.17 (-0.55, 0.21) 26.40 Amini et al, 2021 30 -5.08 2.48 30 -0.26 2.08 -2.08 ( -2.70, -1.46) 24.56 Abokhrais et al, 2020 14 -0.03 2.37 13 -1.23 3.03 0.43 (-0.31, 1.17) 23.48 Overall -0.47 (-1.40, 0.45) Heterogeneity: τ 2 =0.81, I 2 =91.61%, H 2 =11.92 Test of θi=θj: Q(3)=35.76, P=0.00 Test of θ=0: z=-1.00, P=0.32 Random-effects DerSimonian-Laird model -1 0 12-3 www.ogscience.org 193 Saeed Baradwan, et al. Antioxidants & gynecologic-related pain 4. The effect of antioxidants on dyspareunia Total effectiveness was reported in four trials. Meta-analysis

suggested that there was no significant difference in terms of dyspareunia levels when comparing antioxidant supplementation and placebo (SMD, -0.47; 95% CI, -1.40 to 0.45; P=0.32; Fig. 5). High heterogeneity was detected between trials (I 2 = 91.61%; P<0.001). The sensitivity analysis indicated that excluding any individual trial did not signifi - cantly alter the overall results of the meta-analysis, revealing the high stability of the results (Supplementary Fig. 3). 5. The effect of antioxidants on pelvic pain Pelvic pain was recorded in four trials. Meta-analysis results suggested that antioxidant supplementation significantly improved endometriosis-associated pelvic pain (SMD, -1.51; 95% CI, -2.74 to -0.29; P =0.01; Fig. 6). High heterogeneity was detected between trials (I 2 =93.96%; P<0.001). Sensitivi- ty analysis showed that the pooled SMD varied considerably with the omission of three trials; in particular, the exclusion of the study by Sesti et al. [26], Amini et al. [13], and Hosei - nalizadeh and Cahichian. [20] which accounted for approxi - mately 25.93%, 24.71%, and 24.30% of all weights in the meta-analysis, resulted in a pooled SMD (95% CI) of -1.45 (-3.20 to 0.29), -1.15 (-2.56 to 0.26), and -1.34 (-3.00 to 0.32), respectively (Supplementary Fig. 4). 6. Quality of evidence We were moderately confident in the outcomes of dysme - norrhea because of the uncertainty regarding the risk of bias. We had very little confidence in the outcomes of dyspareunia and pelvic pain because of some uncertainty regarding the risk of bias, inconsistency (high heterogeneity), and imprecisi - on (non-significant results) (Supplementary Table 2).

Numerous studies have assessed the effectiveness of dietary and supplemental antioxidants for managing different types of pain in women with endometriosis. Previous systematic reviews and meta-analyses have reported inconsistent results. In line with our findings, systematic reviews support the positive effects of antioxidants in improving endometriosis- associated pain [16]. However, systematic reviews and meta- analyses have indicated that certain antioxidant vitamins, such as vitamin D, may not efficiently alleviate pain in pa - tients [17]. Dysmenorrhea, dyspareunia, and chronic pelvic pain stand out as the most prevalent issues in women of re - productive age [27,28]. Our findings suggest that dietary and supplemental antioxidant intake is associated with a notable reduction in dysmenorrhea and chronic pelvic pain. However, our meta-analysis did not reveal a significant effect of anti - oxidant intake on dyspareunia in women with endometriosis. Dietary antioxidants such as vitamin D, omega-3 fatty acids, and melatonin can have an interactive impact on decreasing cellular damage induced by oxidative stress and ROS [29-31]. Dietary antioxidants can neutralize the ROS and oxidative damage associated with pain in endometriosis. The involvement of oxidative stress and related markers in the initiation and progression of endometriotic complications has been suggested in experimental studies [32]. Cultivated endometrial stromal cells were treated with antioxidants and oxidative stress factors. The application of antioxidant agents resulted in a dose-dependent suppression of cell growth. In contrast, control cells treated with oxidative stress factors ex - hibited increased endometrial stromal growth [33]. The most accepted mechanism for this effect is an antioxidant system that removes free radicals and functions through superoxide dismutases that remove the superoxide anion and glutathi - one peroxidase, which then removes hydrogen peroxide. In women with endometriosis, there is a diminished function of the antioxidant system activity [34]. It has been shown that the levels of most oxidative stress parameters are markedly enhanced in women with endome - triosis compared with controls, and this is one of the main factors contributing to pain in these patients [35,36]. Several recent studies have shown that oxidative stress is positively associated with the migration and proliferation of endome - trial cells in the peritoneal cavity, thereby increasing the prob- ability of endometriosis and infertility [32,37]. The correlation between ROS generation and endometriosis is verified and widely investigated [38]. Moreover, it has been demonstrated that ROS and their reactive oxygen precursors are essential in the progression of pain in several etiologies [39]. Therefore, control of ROS leads to pain relief in patients with endome - triosis, and it has been widely reported that dietary antioxi - dant supplements can effectively decrease ROS levels [40,41]. It was also observed that the increased ROS and enhanced proliferative potential in endometriotic cells were related to the full stimulation and elevated levels of phosphorylated www.ogscience.org194 Vol. 67, No. 2, 2024 endoplasmic reticulum kinase (ERK), as previously detected in tumor cells [42]. In addition, enhanced ERK phosphorylation has recently been reported in stromal cells of women with endometriosis [43]. Furthermore, there are links between ROS generation, ERK activation, and endometriotic cell prolif- eration [44]. It was also demonstrated that ERK activation is in response to different pro-inflammatory factors such as tu- mor necrosis factor-α and interleukin-1ß [45,46]. These two pro-inflammatory markers are enhanced in the endometrium of women with endometriosis [47,48]. Pro-inflammatory markers have a pivotal role in the progression and develop - ment of pain in patients with endometriosis [49,50]. In ad - dition, dietary antioxidant supplements have been shown to regulate pro-inflammatory cytokines efficiently [51]. Recent studies have also shown increased concentrations of other oxidative stress parameters in women with primary dysmenorrhea. Malondialdehyde (MDA) is an index of lipid peroxides [52]. MDA concentrations were higher in women with endometriosis than in healthy controls [53]. Lipid per - oxide levels were higher in peritoneal fluid samples from women with endometriosis than in healthy controls [34]. Therefore, a decrease in MDA levels may help control cell proliferation in endometriosis, and it has been demonstrated that dietary antioxidants such as vitamin D and omega-3 fatty acids can decrease MDA levels [29,54]. Recent investigations have reported the elevated level of iron in different parts of the peritoneal cavity of women with endometriosis, including endometriotic lesions, mac - rophages, and peritoneal fluid, which basically proposes dysfunction of iron homeostasis in the peritoneal environ - ment among this group of patients [55,56]. In women with endometriosis, elevated iron levels may result from the lysis of pelvic erythrocytes [57]. Retrograde menstruation leads to severe hemolysis of erythrocytes accompanied by a disrup - tive or overwhelmed peritoneal elimination system, which enhances iron levels in the peritoneal environment, leading to the progression and growth of endometrial cells [58,59]. Iron overload might have several cytotoxic effects because it reduces the equilibrium between free radical generation and the antioxidant system, which can cause oxidative stress [60]. Our subgroup analysis indicated that vitamin D and mela - tonin were more effective in decreasing dysmenorrhea in patients with endometriosis. It has been demonstrated that vitamin D [61] and melatonin can efficiently decrease iron toxicity complications [62,63]. The effectiveness of vitamin D in reducing dysmenorrhea in patients with endometriosis is thought to be mechanistically linked to its anti-inflammatory and immunomodulatory prop - erties [64]. Endometriosis is characterized by endometrial- like tissue outside the uterus, causing inflammation and pain during menstruation [65,66]. Vitamin D receptors exist in various cells, including those involved in immune responses and inflammation [67]. By binding to these receptors, vita - min D may regulate the production of inflammatory media - tors such as cytokines and modulate immune system activity [68]. This regulatory effect is believed to mitigate the height- ened inflammatory response associated with endometriosis and subsequently reduce the severity of dysmenorrhea [69]. Although the precise mechanisms are still under investiga - tion, emerging research suggests that maintaining optimal vitamin D levels may play a role in managing the pain and inflammation associated with endometriosis-related dysmen - orrhea. On the other hand, melatonin, primarily recognized as a regulator of the sleep-wake cycle, also exhibits potent antioxidant properties [70]. Melatonin supplementation may be beneficial in the context of endometriosis-related dysmen - orrhea, where oxidative stress is pivotal to exacerbating pain and inflammation [25]. Endometriosis is associated with in - creased ROS production, contributing to tissue damage and increased pain sensitivity [8,71]. Melatonin functions as a free radical scavenger and mitigates oxidative stress by neu - tralizing harmful molecules [72]. Therefore, the antioxidant effect of melatonin holds promise in reducing the severity of dysmenorrhea in patients with endometriosis [73]. By allevi - ating the oxidative burden, melatonin may contribute to the modulation of inflammatory responses and a subsequent de - crease in pain perception, offering a supplementary avenue for managing the discomfort associated with endometriosis- related menstrual pain [74]. Additionally, in the same context, vitamins C and E, similar to vitamin D and melatonin, are recognized for their role in regulating iron levels, preventing excess, or addressing deficiency. This regulation aids in reducing oxidative stress because these vitamins function as antioxidants. However, perhaps due to the limited number of included studies that assessed the effectiveness of vitamins C and E, coupled with heterogeneous findings, we could not identify significant re - sults for these vitamins. Therefore, further large-scale studies are required. www.ogscience.org 195 Saeed Baradwan, et al. Antioxidants & gynecologic-related pain 1. Strengths and limitations of the current review This systematic review and meta-analysis has several limita - tions. The small number of included trials makes it difficult to draw a definitive conclusion regarding the effect of an - tioxidant supplements on dyspareunia in patients with en - dometriosis; therefore, these results should be interpreted cautiously. In addition, there are not enough trials in the lit - erature to cover all dietary antioxidant supplements that may effectively reduce endometriosis-associated pain symptoms. Another limiting factor in the findings of this meta-analysis was the heterogeneity of the study populations and the types of antioxidants included in the trials. Some researchers failed to provide sufficient information on the selection crite - ria and data regarding where the trials were performed. Dietary antioxidant supplementation seemed to have a beneficial effect on the severity of endometriosis-related dysmenorrhea (with an emphasis on vitamin D and melato - nin) and pelvic pain. In contrast, no significant reduction in dyspareunia was observed. However, the obtained findings should be interpreted cautiously owing to the relatively small sample size and high heterogeneity between studies, and the importance of further well-designed clinical studies cannot be overstated. Conflict of interest All authors have no conflict of interest to declare. Ethical approval Not applicable. Patient consent Not applicable. Funding information None.

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