{"paper_id":"c8ca034e-928b-4f4a-bd7f-926cbbeb3cd1","body_text":"Endometriosis is a common gynaecological reproductive age disorder characterized by\nthe ectopic presence of endometrial-like tissue (gland and/or stroma). The familiar\nclassic symptoms of endometriosis are pain and infertility. Endometriosis is also\nseen in women aged 12–80 years (average 28 years). 1 , 2  The prevalence of endometriosis\nis assumed to be more than 10% in reproductive-age women. A high prevalence ( 20% to\n90%) of the disease has been reported in women with a history of pelvic pain\n(chronic pelvic pain, dysmenorrhea and dyspareunia) and infertility. The economic\nburden of endometriosis equals diseases, such as diabetes, Crohn’s and rheumatoid arthritis. \n 1\nEndometriosis lesions were divided into three groups: peritoneal, ovarian and deep\ninfiltrating, based on the number, location and appearance. Endometriosis is an\noestrogen-dependent disease with three theories regarding its pathogenesis: ectopic\nendometrial tissue implantation, coelomic metaplasia and induction theory. No single\ntheory can explain the situation of endometriosis in all cases. 1 , 3  Over the past 20 years,\nnumerous studies on the pathogenesis and pathophysiology of endometriosis have\nenhanced our knowledge of the role of steroid hormones, genetics, environment,\nimmune system, peripheral and central nervous system, inflammatory mediators and\noxidative stress in the establishment, progress/regression, signs, symptoms and\ncomplications are associated with the disease. 4 , 5\nEndometriotic cysts create a toxic environment for ovarian tissue, including high\nlevels of proteolytic enzymes, inflammatory cytokines and reactive oxygen species.\nAs a result, increased oxidative stress levels cause oocyte apoptosis and\nconsequently reduced fertility. \n 3 \n  Activating macrophages and neutrophils in response to ectopic endometrial\ntissue and retrograde menstruation increases oxidative stress levels in women with\nendometriosis. Immune cells produce reactive oxygen species. Therefore, in oxidative\nstress conditions, it is crucial to have enough neutralizing antioxidants to prevent\ndamage to the immune cells themselves. \n 6\nThe antioxidants eliminate the overproduction of ROS (reactive oxygen species) during\noxidative stress and thus have a protective role in the body. Imbalance in the\nantioxidant defence system is associated with reproductive disorders such as\noestrous cycle defects, impaired follicogenesis, follicular atresia and\nendometriosis, which may cause adverse effects on fertility and reproductive\nphysiology. 7 – 9  The results of\na study by Bhardwaj and Saraf \n 10 \n  showed a negative correlation between the frequency of apoptosis and the\nactivity of antioxidant enzymes. In patients with endometriosis, oxidative stress is\nresponsible for local tissue destruction and aggressive disease. \n 11\nVitamin C prevents germ cell apoptosis by reducing oxidative stress and scavenging\nfree radicals as a potent water-soluble antioxidant. 7 , 12  Reducing oxidative stress in\nthe peritoneal cavity can prevent the onset and recurrence of endometriosis. Vitamin\nC has effective anti-inflammatory, anti-angiogenic and immune stimulator roles,\nwhich are influential factors in preventing the development of endometriosis. \n 13\nAlthough endometriosis is non-malignant and not marked by uncontrolled lesion growth,\nit shares similar features with cancer. Among those features are increasing pelvic\nand distal cysts, resistance to apoptosis and invasion of other tissues with\nsubsequent damage to the target organs are among those features. \n 14\nEndometriosis has been reported to be associated with increased risk factors that are\nrelated to several types of cancer. Recently, it has been estimated that 20% of\novarian and deep endometriosis lesions contain cancer-causing gene alterations.\nCancer risk assessment of women with endometriosis is crucial in screening,\nprevention and disease management. According to meta-analysis studies, women with\nendometriosis are more likely to develop ovarian, endometrial and thyroid cancers,\nand endometriosis itself appears to be a risk factor for ovarian cancer. 14 – 16\nBased on ethical considerations and the risk and burden of experimental research on\nhuman patients, animal models have been used to develop non-invasive diagnostic\nmethods, classification systems, novel therapeutic approaches and even prevention\nmethods in managing endometriosis. Using rodent models is cost-effective, easily\naccessible and provides a way to examine multiple aspects of the disease. \n 2\nSince endometriosis is a very complex disease with a high impact on women’s quality\nof life, the need for a broader range of medical treatments is essential. Studies\nshow that common pharmacological treatments for the disease encompass hormonal\nagents that cause fertility problems, while newer therapies focus on oxidative\nstress responses. Due to the prevalence of endometriosis and the limited drug\ntherapies, there is an increasing need for further research and development of\nnon-hormonal drugs to treat endometriosis. Based on this background, we investigated\nthe therapeutic effect of vitamin C on endometrial lesions and fecundity disorders\nof experimentally induced ovarian endometriosis in a mouse model. A few studies have\ninvestigated the effect of vitamin C on endometriosis before, but they have used the\nperitoneal induction method, which makes this study a novelty.\n\nA double-blind placebo-controlled randomized experimental study was conducted on\nfourteen mature, virgin female NMRI mice (25–35 g, 6–8 weeks) provided by Kurdistan\nMedical Sciences University’s experimental Animal Center (Sanandaj, Iran). The mice\nwere housed in polypropylene cages (3 per cage) in a well-ventilated room and under\nthe standard condition (12‑h light and 12‑h dark periods, the temperature range of\n22–25°C, the humidity of 55%–60%). The mice had ad libitum access to standard dry\npellets and water throughout the study. They were maintained under standard\nconditions to observe their health conditions before the experiment for one week.\nOur study design was based on the requirements of ARRIVE (Animal Research: Reporting\nof In Vivo Experiments).\nThe following formula (comparison between two groups in animal study) was used for\nthe calculation of sample size: \n 17\nSample size = 2 SD 2 ( Z α 2 + Z β ) 2 / d 2\nFrom previous studies, standard deviation = 5.2, Z a/2  = 1.96 (from Z\ntable) at type 1 error of 0.05, Z β  = 0.842 (from Z table) at 80% power,\neffect size = difference between mean values from previous studies = 7.7; sample\nsize was calculated = 6. For 10% attrition, seven mice per group were chosen.\nThe oestrous cycle (including pro-oestrus, oestrus, metoestrus and dioestrus) was\nassessed by observing vaginal smears, according to protocol paper. \n 18 \n  All procedures, including surgery, endometriosis induction and tissue\ncollection, were performed at the oestrous phase.\nThe mice were administrated 50 mg/kg ketamine and 7 mg/kg xylazine\nintraperitoneally for anaesthesia. The endometriosis induction surgery was\nperformed in the aseptic situation according to protocol paper. \n 18 \n  After shaving and disinfecting the surgical field, a vertical incision\n(1.5–2 cm) was briefly made below the left kidney. An approximate 1-cm segment\nof the middle part of the left uterine horn was clamped with 5-0 vicryl sutures\n(Ethicon, Denmark) to the utero-tubal and uterocervical junction excised. The\nuterine tissue was placed in a petri dish containing PBS (phosphate-buffered\nsaline) supplemented with penicillin (100 U/mL) and streptomycin (100 mg/mL) and\nsplit longitudinally with the blade of scissors. Using a 3 mm punch biopsy\n(Sklar, Tru-Unch, India), the tissue was divided into two 3×3 mm 2 \nsections. Each piece of the uterine horn was implanted to one of the ovary sides\nusing a single suture with 6-0 black silk and 6-0 blue nylon. The implants were\nwashed with 0.5–1 ml PBS supplemented with penicillin and streptomycin to\nprevent adhesions and dryness. The abdomen wall (peritoneum and fascia) and the\nskin were closed by suturing with 5-0 separate vicryl and 5-0 nylon,\nrespectively. After the operations and recovery, the mice were situated\nindividually, and 0.2 mg/kg buprenorphine was administrated subcutaneously to\nrelieve the pain.\nFour weeks after the induction of endometriosis, a second surgery was directed to\nassess the endometriotic implants. Each implant’s volume was calculated by\nmeasuring the maximum diameter of each side to one-tenth of a millimetre with a\nVernier calliper. The volume was calculated using the ellipsoid formula (V\nmm 3  = 0.52 × A × B × C, where A: width, B: length and C: height). \n 19 \n  The implants were photographed, and their clinical adhesion and size were\nmeasured ( Figure\n1 ).\nMeasurement of an implant (length, width and height) using the calliper\nat the second surgery (4 weeks after induction of endometriosis).\nThe implants were successfully developed in all the mice. All environmental and\nanimal preparation stages were executed with the first surgery’s procedure\n(anaesthesia, cleaning, left lower back incision, suturing and recovery\nsteps).\nThree days after the second surgery, the mice were randomly divided into two\nintervention groups of control (placebo) and treatment with seven mice in each\ngroup. The treatment group was given 50 mg/kg (0.5 mL) vitamin C (500 mg Nature\nMade, American, Health Code Certificate 310009077035) every two days orally for\nfour weeks. The control group was given a 0.5 mL mix of water and starch. The\ngiven dosages of vitamin C were calculated based on the human dose equivalent as\nfollows:\nAED (animal equivalent dose) (mg/kg) = Human dose (mg/kg)× K m \n(Kunming mice) ratio\nK m  ratio for mice = 12.3, human dose = 2 mg/kg (120 mg/d)\nAED (Animal equivalent dose) = 24.6/kg/dIs almost equal to 50 mg/kg every\n2 days\nThe researcher was blinded to the groups at all stages.\nIn the oestrus phase, the mice were sacrificed using isoflurane asphyxiation and\nwere dissected. The endometriosis lesions were evaluated and scored based on\nmacroscopic findings ( Table 1 ). The endometriotic implants were excised and weighed\naccurately.\nScoring macroscopic and microscopic evaluations.\nCollected samples (the ovary, the remaining implant and the uterine horn) were\nfixed in a 10% formalin solution. Specimens were dehydrated in a graded series\nof ethanol rinses, cleared in xylene, embedded in paraffin and cut into\n4-micrometre sections (three sections for each specimen). The sections were\ndeparaffinized by xylene and then rehydrated using ethanol and decreasing\ndegree. The sections were stained with haematoxylin–eosin (HE) and trichrome\n(Masson’s stain).\nMacroscopic Assessment: Endometriotic implants (the size, volume and weight of\nendometriotic implants, growth score and adhesion score); \n 20 \n  microscopic Assessment: The ovarian tissue (the number of follicles,\ncorpus luteum and atretic follicles) and endometriotic lesion (histologic and\ntrichrome fibrosis scores) ( Table 1  and  Figures 2 – 4 ). 21 – 23  The follicles, corpus\nluteum and atretic follicles on each three ovary sections were counted blindly\ntwice with a two-week interval by an investigator (pathologist). The final\nresult for each ovary was achieved by the average slide count of the two\nobservations.\nOvarian tissue stained with H&E ((a): atretic follicle, (b): antral\nfollicle and (c): corpus luteum) (4 weeks after treatment, control\ngroup).\nImplant stained with H&E, 4 weeks after treatment: (a) The epithelial\nlayers are well maintained and remain (control group); (b) endometriotic\nimplant with defective epithelium after vitamin C treatment (treatment\ngroup).\nComparison of the degree of fibrosis between the two groups by Masson’s\nstaining. (4 weeks after treatment, (a) control group, (b) treatment\ngroup).\nDefinition of atretic follicles: The follicle is characterized by a fragmented\negg, rupture of the plasma membrane and the shedding of granulosa cells into the\nantrum.\nThe Stata (version 13) and GraphPad Prism (version 8) were used for the\nstatistical analysis and graph generations. The Shapiro–Wilk test, measured\ndispersion, central tendency and histogram graph were used to check the\ncontinuous variable’s normality.\nThe obtained results were expressed as median and quadrant and the effect size\n(i.e. mean difference, standardized mean difference and mean ratio). The mean\nvolume and scores of the extent and adhesion of the endometriotic lesions and\nfecundity status of the two groups (7 mice in each group) were compared.\nThe metric variables were compared by t-test, one-way ANOVA test and Bonferroni\npost hoc tests. Further, the non-metric variables were examined by the\nKruskal–Wallis and Mann–Whitney U-tests.\nThe study groups evaluated metric (preceding and after the medical treatment) and\nnon-metric variables, respectively, by repeated-measures ANOVA and the\ngeneralized estimating equation model. p values of < 0.05 were considered\nsignificant.\nThis experimental study was carried out in an animal laboratory, and its approval\nwas received from the Ethics Committee of Shahid Beheshti University of Medical\nSciences, Tehran, Iran (ethics code IR.SBMU.PHARMACY.REC.1399.136). All animal\nexperiments were performed under the Animals (Scientific Procedures) Act 1986\nAmendment Regulations 2012. \n 24\n\nThe pre-treatment volume (p = 0.521), growth score (p = 0.259), adhesion extent\nscore (p = 0.129) and adhesion severity score (p = 0.069) of endometriotic\nimplants were similar between the two groups. The overall result of fitting the\nmodel GEE (generalized estimating equation model) showed that post-treatment\nimplant volume, growth score, adhesion extent score and adhesion severity score\nof the treatment group (vitamin C) were significantly lower than those of the\ncontrol group (placebo) (p < 0.0001) ( Figures 5 – 7 ).\nEvaluation and measurement of endometriotic lesions 4 weeks after\ntreatment in the control group (placebo).\nEvaluation and measurement of endometriotic lesions 4 weeks after\ntreatment in the treatment group (vitamin C).\nThe comparison chart of implant volume (mm3), growth score, adhesion\nextent and severity before and after treatment (control and\ntreatment).\nThe difference between the median weight of endometriotic implants,\nepithelialization of implant tissue and trichrome fibrosis scores in the two\ngroups (treatment and control) were statistically significant (p < 0.05;\n Figure 8 ,  Table 2 ). The mean\nratio in this test shows that the weight of endometriotic implants (77%),\nepithelialization of implant tissue score (33%) and trichrome fibrosis scores\n(16%) in the treatment group have decreased compared with the control group.\nComparison charts of implant weights, epithelialization and fibrosis\nscores in two groups (4 weeks after treatment).\n*p < 0.05, **p < 0.01.\nComparison of implant weights, epithelialization of implant tissue score,\ntrichrome fibrosis scores in two groups (7 in each group).\nstatistically significant.\nThe statistical test results concerning fecundity changes showed that the number\nof follicles was significantly increased, and atretic follicles number were\ndecreased considerably after vitamin C therapy (p < 0.05). Although corpus\nluteum number seemed to be more preserved in specimens from the treatment group,\nthere was no statistical significance between the histological scores of the\ngroups ( Table 3 ;\n Figure 9 ). The\nsample weighted mean for the standardized mean difference of follicle number and\natretic follicle number was 0.88 (0.84–0.93) and 0.54 (0.17–1.78), large and\nmedium, respectively. The mean ratio in this test shows that the weight of the\ncorpus luteum number (15%) in the treatment group has increased compared with\nthe control group.\nComparison of follicle, atretic follicle number and corpus luteum number\nin two groups (7 in each group).\nSMD: standardized mean difference.\nstatistically significant.\nComparison charts of follicle, atretic follicle and corpus luteum number\nin two groups (4 weeks after treatment).\n**p < 0.01, ***p < 0.001.\n\nEndometriosis is a complicated disease, and no animal model can fully demonstrate\nhuman conditions. This study is the first one evaluating the effect of vitamin C\ntherapy on ovarian implant growing in an experimental endometriosis model. In the\ncurrent study, the treatment group showed significant decreases in the lesion’s\nvolume, weight, adhesion, extent, severity and histopathologic scores\n(epithelialization of implant tissue score and trichrome fibrosis Scores). In\ncontrast to the treatment group, the implants volumes and weights continued to\nincrease during the study in the control group.\nIn comparing the two groups of vitamin C treatment and placebo before treatment in\nterms of volume, intensity, extent, adhesion score and growth score of implants, no\nsignificant difference was observed between the two groups, which indicated a\nsimilar condition between the two groups before treatment.\nBut the result of fitting the generalized estimating equation (GEE) model in terms of\ncomparison of volume, intensity, proliferation and adhesion score and growth score\nof implants in the two groups of vitamin C treatment and placebo after treatment,\naccording to the pre-treatment results, the reduction of the mentioned cases after\nthe treatment with vitamin C compared to the placebo group showed that it was\nstatistically significant in all cases.\nOne study has reported that the endometriotic implants in rat models in all treatment\ngroups (antioxidant herbs supplementation) were lower than the control group. \n 25\nThe study results of Erten et al. \n 4 \n  showed a significant difference in the volume of the implants before and\nafter the treatment with vitamin C. In one study, 1,200 units of vitamin E and 1,000\nmg of vitamin C were prescribed for eight weeks before endometriosis surgery. That\nstudy showed a 43% reduction in pain compared to placebo recipients. \n 26 \n  Durak et al. investigated the effect of vitamin C at doses of 0.5, 1.25 and\n2.5 mg on induced endometriosis cysts in rats. The weight and volume of cysts\ntreated at a dose of 2.5 mg were reduced significantly. \n 6\nIn agreement with our results, Yavuz et al. \n 27 \n  revealed that antioxidant treatment had significantly reduced the\nhistological score compared to the control group. In a study by Bakacak et al., \n 28 \n  significant decreases in the treatment group’s implant volume and\nhistopathologic scores were observed, unlike the control group. Tissue fibrosis\nresults from a chronic inflammation following tissue damage and invasion thought to\nbe associated with an inadequate immune response. \n 29\nIt was well estimated that by allowing free radicals to remove a hydrogen atom from\nthe antioxidant molecule instead of polyunsaturated fatty acids, vitamins C and E\neffectively enhanced SOD, GST and CAT activity within granulosa cells, so, breaking\nfree radical chain reactions and producing relatively inactive radicals. \n 30\nAlthough it was a difference between the type of induction of endometriosis in the\npresent study (ovarian induction) and other studies (peritoneal and subcutaneous\ninduction), similar results are observed regarding the effect of vitamin C on\nreducing the volume, intensity, proliferation, adhesion and growth score of\nimplants.\nFollicles are a key component of reproduction biology in terms of development and function. \n 31 \n  In this study, to investigate the effect of treatment on fertility, the\nnumber of follicles, corpus luteum and atretic follicles were compared in the two\ngroups of vitamin C treatment and placebo treatment. The number of follicles and\ncorpus luteum in the vitamin C treatment group increased compared to the placebo\ngroup. This difference in the number of follicles was statistically significant.\nThe number of atretic follicles in the vitamin C treatment group showed a\nstatistically significant decrease compared to the placebo group. One study has\nfound that treatment with antioxidants (microginone and African antada root extract)\nreduced the number of atretic follicles and increased follicle development, puberty\nand ovulation. \n 32\nCummings and Metcalf \n 33 \n  observed that the mice with surgically induced endometriosis did not exhibit\nthe severe fertility reduction seen in women with endometriosis.\nSurgically induced endometriosis in mice conduces fecundity dysfunction, anomalous\noocyte quality, embryo growth and early lost pregnancy. Balancing between reactive\noxygen species and antioxidants is essential for oocyte maturation. Researchers have\nshown that insufficient or excessive levels of reactive oxygen species in\nendometriosis have adverse effects on oocyte and embryo development, a lower\nimplantation rate, and pregnancy outcomes. 34 , 35  It is in line with the\npresent study’s results that the treatment group had significantly higher follicles\nand decreased atretic follicles than the control group. This study validates the\nwork of Moon et al., \n 36 \n  who found more luteinized unruptured follicles and fewer follicles in Endo\nrats compared to the controls. Recently, the number of ovulated oocytes does not\ndecrease in endometriosis-induced mice, but the quality of the oocytes and the\nnumber of embryos decreases. \n 37 \n  The type of endometriosis induction can be one of the reasons for the\ndifferences in the results of studies.\nA novel, long-term treatment of endometriosis, must affect the disease initiation and\nprogression stages, including proinflammatory environment, increased angiogenesis,\nresistance to apoptosis, structural and epigenetic changes, and oxidative stress,\nwith minimum adverse effects on fertility.\nThere is an urgent need for a new treatment that eliminates the lesions without any\nside effects. Our findings showed that vitamin C affects multiple fundamental\nprocesses in the pathogenesis of endometriosis, which may help treat this common\ngynaecological disorder. Furthermore, vitamin C significantly inhibited the growth\nand cyst formation of endometriotic lesions compared to vehicle-treated controls.\nAll these findings imply that vitamin C may play a protective or therapeutic role in\nendometriosis.\nThere were several limitations to the present study. It was an experimental study\nwith a small number of mice per group. The second limitation of this study was the\nlack of using various doses in groups. Although this study’s overall results show\nthat vitamin C has an excellent effect on endometriosis, further investigation is\nneeded to assess its potential as an alternative therapy for endometriosis. The\nthird limitation is that the current study does not compare the effects of a\nwell-known antioxidant agent or a gonadotropin-releasing hormone agonist (GnRH\nagonist), which are proven therapies used in humans.\n\nAs a result, we may imply that vitamin C has a significant effect on reducing the\ninduction and growth of endometrial implants, improving the fecundity function of\novaries, and consequently prevention of endometriosis-associated cancers. Further\nresearch is needed to improve targeted interventions aimed at preventing and\ntreating human endometriosis. Various observations from studies support the role of\noxidative stress in the development and progression of endometriosis. This evidence\nmay pave the way for evaluating treatment approaches targeting oxidative imbalance:\nOxidative stress can be the key to treatment and ultimately prevent endometriosis.\nIn particular, clinical trial studies will help better explain the effect of\nantioxidants as potential treatments for endometriosis in the future.\n\nClick here for additional data file.\nSupplemental material, sj-docx-1-whe-10.1177_17455057221096218 for The\neffectiveness of antioxidant therapy (vitamin C) in an experimentally induced\nmouse model of ovarian endometriosis by Hayedeh Hoorsan, Masoumeh Simbar,\nFahimeh Ramezani Tehrani, Fardin Fathi, Nariman Mosaffa, Hedyeh Riazi, Loghman\nAkradi, Sherko Nasseri and Shayan Bazrafkan in Women’s Health","source_license":"CC0","license_restricted":false}