{"paper_id":"581bc6c9-b217-4b98-a8d7-706029b4866d","body_text":"© 2026 THE KOREAN SOCIETY FOR REPRODUCTIVE MEDICINE www.eCERM.org 1\nThis is an Open Access article distributed under the terms of the Creative Commons Attribu-\ntion Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits \nunrestricted non-commercial use, distribution, and reproduction in any medium, provided the \noriginal work is properly cited.\nInvestigating the effect of human amniotic \nmembrane gel to minimize adhesions in \nendometriosis: findings from an experimental \nstudy using a rat model\nRoya Derakhshan\n1\n, Abolfazl Mehdizadehkashi\n1,2\n, Babak Sabet\n3,4\n, Shahla Chaichian\n1\n, Arash Mohazzab\n5\n, Behrang Kazeminezhad\n6\n,  \nTayyebeh Nazeran\n7\n, Mohammad Abbas Sheikholeslami\n8\n, Seyed Ali Ziai\n8\n, Samaneh Rokhgireh\n1\n, Banafsheh Nikfar\n9\n,  \nMarefat Ghaffari Novin\n10\nFor further information on the authors’ affiliations, see Additional information.\nORIGINAL ARTICLE\nhttps://doi.org/10.5653/cerm.2025.09110\npISSN 2233-8233 · eISSN 2233-8241\nClin Exp Reprod Med [Epub ahead of print]\nReceived: December 25, 2025 ∙ Revised: February 27, 2026 ∙ Accepted: March 9, \n2026 \nCorresponding author: Shahla Chaichian\nEndometriosis Research Center, Rasool-e Akram Hospital, Iran University of \nMedical Sciences, Niyayesh St., Sattarkhan Ave., Tehran, Iran\nTel: +982166509283 Fax: +982188989690 E-mail: shchaichian@gmail.com\n*Iran University of Medical Sciences has been supported as part of the research \ncost.\nObjective: Postoperative adhesions and inflammation remain major challenges following endometriosis surgery. Human amniotic mem-\nbrane gel (HAG) has emerged as a potential therapeutic strategy with both anti-adhesive and anti-inflammatory properties. This study aimed \nto evaluate the efficacy of intraperitoneal HAG in reducing adhesion severity, lesion persistence, and inflammatory cytokine levels in an ex-\nperimental rat model of endometriosis. \nMethods: Forty-four adult female Wistar rats underwent surgical induction of endometriosis through autotransplantation of uterine horn \ntissue. After 3 weeks, the animals were randomized into two groups (n=22 each): one receiving intraperitoneal HAG and the other receiving \nnormal saline during a second laparotomy. Adhesion severity was evaluated using the Hoffmann and Lauder scoring systems. Two weeks af-\nter the intervention, endometriotic lesion size was measured. Serum levels of interleukin (IL)-6 and IL-1β were assessed at three time points. \nHistopathological examination was conducted to evaluate the presence of endometriotic tissue, fibrosis, abscess formation, and adipose tis-\nsue remodeling. \nResults: Rats treated with HAG showed a significant reduction in lesion size (6.8 mm² vs. 81 mm², p<0.001) and significantly lower adhesion \nscores (p<0.001). Persistent endometriosis was observed in only 36% of animals in the HAG group compared with 95.5% in the control group \n(p<0.001). Histological analysis demonstrated fewer fibrotic and purulent reactions and a shift toward adipose tissue remodeling in the \nHAG-treated animals. Serum IL-6 and IL-1β levels decreased significantly in the HAG group, whereas both cytokines increased in the control \ngroup (p<0.001 for both). \nConclusion: HAG significantly improved macroscopic, histological, and biochemical outcomes in this experimental model. These findings \nsuggest that HAG may serve as a biologically active adjunct for reducing postoperative adhesion formation and inflammation in endometrio-\nsis.\nKeywords: Amniotic membrane; Animal models; Endometriosis; Inflammation mediators; Postoperative complications; Tissue adhesions\nIntroduction\nPostoperative pelvic adhesions are a frequent complication of en-\ndometriosis surgery and contribute to chronic pain, infertility, and \nbowel obstruction. Surgical trauma initiates a wound-healing cas-\ncade that can result in the formation of fibrous bands between peri-\ntoneal surfaces [1]. The use of biochemical barriers such as hyaluron-\nic acid (HA) has previously been proposed as an effective strategy for \nreducing postoperative adhesions. HA forms a temporary physical \n\nMethods\n1. Materials and animals\nThis experimental study was conducted in adult, nonpregnant fe-\nmale Wistar albino rats weighing 220 to 250 g. The animals were \nhoused in standard cages at the Center for Experimental Studies, \nShahid Beheshti University of Medical Sciences, under controlled en-\nvironmental conditions (temperature, 22±2 °C; 12-hour light/12-\nhour dark cycle) with free access to food and water. Amniotic mem-\nbrane allograft gel was obtained from Lifescell (International Bioim-\nplant of Borna Co.). Commercial enzyme-linked immunosorbent as-\nsay (ELISA) kits were obtained from Karmania Pars Gene Company.\nAll procedures were approved by the Research and Ethics Com-\nmittee of Iran University of Medical Sciences, Tehran, Iran (IR.IUMS.\nREC.1399.726), and were conducted in accordance with the Guide \nfor the Care and Use of Laboratory Animals (National Institutes of \nHealth Publication No. 80-23, revised 1996). After an adaptation peri-\nod and before the first laparotomy, each rat received a subcutaneous \ninjection of 2.0 mg/kg estradiol to support the establishment of en-\ndometriotic implants.\n2. Induction of endometriosis\nAt the time of the first laparotomy, the rats were in the estrus \nphase, during which estrogen levels are naturally elevated and facili-\ntate implantation and survival of endometrial fragments. This timing \nis consistent with established protocols for endometriosis induction \nin rodent models. In line with previous studies demonstrating that \nexogenous estradiol enhances endometriosis development [16], in-\ntramuscular estradiol was also administered to further improve le-\nsion establishment. Importantly, the ovaries were preserved to main-\ntain endogenous hormonal cycles, thereby better simulating the \nhormonal milieu of endometriosis. Thus, the combination of natural \nestrus, exogenous estradiol, and ovarian preservation optimized the \nconditions for reliable induction of peritoneal endometriosis [16,17]. \nAnesthesia was induced with intramuscular ketamine hydrochloride \n(60 mg/kg) and xylazine hydrochloride (10 mg/kg) administered in \nthe same syringe. Before surgery, tail vein blood samples were col-\nlected to measure baseline serum IL-6 and IL-1β levels.\nA vertical midline abdominal incision of approximately 3 cm was \nmade, and the two uterine horns were exposed. The right uterine \nhorn was ligated with a 4-0 silk suture, and a 1-cm segment was ex-\ncised and opened longitudinally. Sterile phosphate-buffered saline \nwas used to keep the tissue moist.\nThe uterine segment was then divided into three equal parts. All \nthree fragments were transplanted onto the inner surface of the \nright abdominal wall, with the endometrial surface facing the perito-\nneum. The serosal surface was positioned against the peritoneal sur-\nlayer between healing peritoneal surfaces, thereby preventing direct \ntissue apposition and adhesion formation. HA gel has been shown to \nsignificantly decrease the incidence and severity of pelvic adhesions \nfollowing gynecologic surgery, particularly surgery for endometriosis \n[2,3]. More recently, natural bioactive gels such as human amniotic \nmembrane gel (HAG) have attracted attention because of their dual \nfunction: they not only serve as physical barriers but also deliver an-\nti-inflammatory and regenerative signals to the surgical site. These \namniotic-derived products are rich in growth factors, extracellular \nmatrix proteins, HA, and immunomodulatory cytokines that support \ntissue repair and modulate inflammation [4,5]. In preclinical studies, \nthey have been shown to reduce fibrosis, downregulate inflammato-\nry pathways such as transforming growth factor-β (TGF-β), and sup-\npress proinflammatory cytokines including interleukin 6 (IL-6) and IL-\n1β [6-8].\nEndometriosis is characterized by a proinflammatory peritoneal \nenvironment, with IL-6 and IL-1β identified as key mediators of dis-\nease progression and adhesion formation. Women with endometrio-\nsis have significantly elevated levels of IL-6 and IL-1β in serum and \nperitoneal fluid compared with disease-free controls [9]. IL-6, in par-\nticular, correlates with endometriosis severity and has been implicat-\ned in lesion proliferation and survival, especially through its soluble \nreceptor, which enhances IL-6 signaling [10]. Surgical injury further \namplifies these cytokines. Experimental models have shown that \nearly postoperative surges in IL-6 and IL-1β are strongly associated \nwith the extent of subsequent adhesions [11,12]. IL-1β promotes ad-\nhesion formation by reducing fibrinolysis; it induces plasminogen \nactivator inhibitor-1, leading to persistence of fibrin scaffolds, and it \nstimulates mesothelial cells to undergo profibrotic changes [13]. IL-6, \noften induced downstream of IL-1β and tumor necrosis factor-α, fur-\nther drives fibrogenesis by triggering mesothelial-to-myofibroblast \ntransition and altering the expression of matrix-remodeling en -\nzymes, thereby reinforcing adhesion development [13]. Given the \ncentral role of IL-6 and IL-1β, anti-inflammatory interventions target-\ning these cytokines may markedly influence postoperative out -\ncomes. Indeed, blocking IL-1β activity in animal models reduces \nperitoneal adhesion formation [14]. Likewise, neutralization of IL-6 \nsignaling with an anti-IL-6 receptor antibody has been shown to ab-\nrogate neutrophil recruitment and significantly diminish adhesion \nbands in a murine surgery model [ 15]. These findings underscore \nthat dampening the IL-6/IL-1β-driven inflammatory cascade after \nsurgery may reduce adhesion formation and fibrosis and potentially \nlower endometriosis recurrence. Collectively, these insights provide a \nstrong rationale for therapies that combine physical anti-adhesion \nbarriers such as HA gel with strategies that modulate IL-6 and IL-1β, \nwith the aim of improving healing and long-term outcomes in pa-\ntients with endometriosis.\nhttps://doi.org/10.5653/cerm.2025.091102\nClin Exp Reprod Med [Epub ahead of print]\n\nface and fixed with 4–0 nonabsorbable silk sutures (Figure 1). During \nthe procedure, the surgical field was frequently moistened with nor-\nmal saline to prevent tissue drying. Before closure of the abdominal \nwall, 2 mL of saline was instilled into the peritoneal cavity.\nThe abdominal wall was closed in layers using simple interrupted \n2–0 polyglactin 910 sutures. After surgery, the animals were housed \nindividually and monitored for 3 weeks. They received 50 µg/kg in-\ntramuscular depot estradiol twice weekly during this period. Body \nweight and general health were monitored regularly, and no mortal-\nity occurred during the study.\n3. HAG application\nAt the end of the third week, all animals underwent a second lapa-\nrotomy to verify the viability of the endometriotic implants by histo-\npathological assessment (Figure 2). In all animals, the implants were \nmeasured in two dimensions (length and width, in mm) using a cali-\nFigure 1. Surgical procedure and assessment of animal model of endometriosis. (A) Induction of endometriosis (the white arrow shows \nthe left uterine horn). (B) Endometriotic implant before intervention. (C) Adhesion severity and size of endometriotic implant in the human \namniotic membrane gel group. (D) Adhesion severity and size of endometriotic implant in the control group.\nFigure 2. Pathological views of ectopic endometriosis. (A) After induction of endometriosis, (B) after human amniotic membrane gel \napplication, and (C) control group. Hematoxylin and eosin staining; original magnification: A, ×100; B and C, ×40.\nwww.eCERM.org 3\nR Derakhshan et al. HAG and endometriosis adhesions\nAA\nCC\nBB\nDD\nAA BB CC\n\nper, photographed with a digital camera, and documented in the \nstudy records [18]. Immediately thereafter, 44 rats with established \nendometriotic implants were randomly assigned to two groups: \ngroup A (HAG group, n=22) and group B (normal saline group, \nn=22). Randomization was performed immediately before the inter-\nvention, ensuring that only animals with successful model induction \nwere included.\nIn group A, sterile HAG (International Bioimplant of Borna Co., Teh-\nran, Iran) was instilled into the abdominal cavity during laparotomy, \nand the abdominal wall was closed with simple 2–0 polyglactin 910 \nsutures. In group B, normal saline was instilled into the abdominal \ncavity during laparotomy according to protocols similar to those \nused in previous experimental studies [17,19].\n4. Implant measurements and histopathology\nTwo weeks after HAG or saline application (i.e., 5 weeks after in-\nduction of endometriosis), blood samples were again obtained from \nthe tail vein under light sedation to assess serum interleukin levels. \nThe rats were then humanely euthanized using nitrous oxide. During \nthe final laparotomy, adhesions were evaluated in each group using \nHoffmann’s quantitative score and Lauder’s qualitative grading sys-\ntem. A researcher blinded to group allocation measured the width \nand length of each implant with a caliper. Endometrial implants \nwere excised and fixed in formaldehyde for routine histopathological \nexamination.\nBoth the biochemist and the histopathologist were blinded to \ntreatment allocation. Data on lesion size, adhesion severity, and \ngross appearance were recorded using a structured checklist, and \nstandardized photographs were taken at each laparotomy to docu-\nment adhesion formation and implant morphology. The histological \ndiagnosis of endometriosis was based on the presence of endome-\ntrial glands and stroma, with epithelial lining and luminal formation \nwithin the ectopic implants [17]. In addition, the presence of fibrosis, \nabscess formation, and adipose tissue reaction was recorded as part \nof the pathological evaluation.\n5. Cytokine assays\nSerum levels of IL-6 and IL-1β were measured using enzyme-linked \nimmunosorbent assay (ELISA) kits according to the manufacturers’ \ninstructions. IL-6 and IL-1β concentrations were determined using \ncommercially available ELISA kits.\n6. Statistical analysis\nAll analyses were performed using SPSS ver. 24 (IBM Co.). Continu-\nous variables were assessed for normality using the Kolmogorov–\nSmirnov test. Repeated-measures analysis of variance was used to \nevaluate temporal trends across the three measurement points and \ntheir interaction with treatment group. Greenhouse–Geisser correc-\ntions were applied when the assumption of sphericity was violated. \nFor comparisons between the two groups at individual time points \n(baseline, week 3, and post-treatment), the Mann–Whitney U test \nwas used because cytokine levels and lesion measurements did not \nfully meet parametric assumptions. The Wilcoxon signed-rank test \nwas used to assess within-group changes over time (pre- vs. \npost-treatment). Adhesion severity scores (Hoffmann and Lauder) \nwere also compared between groups using the Mann–Whitney U \ntest. All categorical histopathological findings were analyzed using \nthe chi-square test or Fisher’s exact test, as appropriate. Results were \nreported as mean±standard deviation for continuous variables and \nfrequency (percentage) for categorical variables. A two-sided p<0.05 \nwas considered statistically significant.\nResults\n1. Surgical outcomes and adhesion severity\nAll 44 animals completed the study and were included in the anal-\nysis. At baseline, the two groups were comparable in lesion size and \ncytokine profiles, confirming that randomization produced well-bal-\nanced groups. Endometriotic implants initially appeared as typical \nvascularized or cystic nodules surrounded by filmy or inflammatory \nadhesions.\nBy the end of the experiment, the difference between groups had \nbecome both visually and statistically evident. Lesions in the \nHAG-treated animals had shrunk dramatically, with post-interven-\ntion lesion size decreasing to approximately 7 mm², in contrast to the \ncontrol group, in which lesions remained large, averaging approxi-\nmately 81 mm² (p<0.001). The overall reduction in lesion size from \nbaseline was also markedly greater in the HAG group (approximately \n80 mm²) than in the control group (approximately 9 mm²). As shown \nin Table 1, this improvement remained significant within animals \nover time, whereas the control group showed no meaningful regres-\nsion.\nAdhesion scores showed a similar pattern. Both Hoffmann and \nLauder scores were substantially lower in rats receiving HAG, with \nmean Hoffmann scores of approximately 2.5 versus 6.4 in the control \ngroup and mean Lauder scores of 1.0 versus 2.9, respectively (both \np<0.001). Figure 1 illustrates these macroscopic differences. Overall, \nthe surgical findings indicate a consistent and robust therapeutic ef-\nfect of HAG on both lesion size and adhesion severity.\n2. Histopathological findings\nHistological evaluation reinforced the macroscopic findings. Only \na minority of HAG-treated rats showed evidence of persistent endo-\nmetriosis (36%), whereas nearly all animals in the control group re-\nhttps://doi.org/10.5653/cerm.2025.091104\nClin Exp Reprod Med [Epub ahead of print]\n\nmained positive for active lesions (95.5%; p<0.001). The pattern of \ntissue changes also differed meaningfully between groups. Fibrosis \nand abscess formation—markers of chronic or ongoing inflammato-\nry activity—were frequent in control animals, whereas these features \nwere much less common in animals exposed to HAG. In contrast, ad-\nipose tissue reaction, which is more consistent with tissue repair and \nremodeling, predominated in the HAG group. These distributions are \nsummarized in Table 2.\nFigure 2A shows the histological appearance of endometriotic im-\nplants immediately after confirmation of endometriosis during the \nsecond laparotomy. Figure 2B, 2C show histological sections of im-\nplants 2 weeks after HAG treatment (Figure 2B) and normal saline \ntreatment in the control group (Figure 2C), highlighting the differ-\nences in tissue response between treated and untreated animals. In \nFigure 2B, following HAG treatment, the endometriotic epithelium is \nabsent, and only residual fibrotic tissue remains.\n3. Serum cytokine levels\nChanges in systemic cytokine levels closely mirrored the surgical \nand histological outcomes. At baseline, IL-6 and IL-1β levels were \nsimilar between groups, indicating no pre-existing differences. Fol-\nlowing treatment, the trajectories diverged sharply. IL-6 levels de-\ncreased steadily in HAG-treated animals, falling to approximately \n1.24 pg/mL, whereas levels increased in the control group, reaching \napproximately 2.65 pg/mL at the same time point (p<0.001). A high-\nly significant time-by-group interaction confirmed that the pattern \nof IL-6 change differed fundamentally between the two groups.\nIL-1β showed a similar pattern. In the HAG group, IL-1β decreased \nto approximately 1.38 pg/mL, whereas in control animals it increased \nsharply to more than 3.2 pg/mL (p<0.001). The linear trend over time \nwas strong and highly significant for both cytokines, reflecting a sus-\ntained anti-inflammatory response to HAG. These patterns corre-\nspond closely to the macroscopic regression of lesions and the histo-\nlogical shift toward healing. The cytokine trajectories are illustrated \nin Figure 3, and the full numerical data are summarized in Table 3.\nDiscussion\nThis study demonstrates that intraperitoneal HAG significantly at-\ntenuated endometriosis progression and postoperative adhesions in \na rat model. Animals treated with HAG had markedly smaller endo-\nmetriotic lesions and substantially lower adhesion severity, based on \nHoffmann and Lauder scores, than saline-treated controls. These \nfindings are consistent with prior evidence that barrier gels can re-\nduce pelvic adhesions. A recent meta-analysis of gynecologic surgery \nshowed that HA gel significantly decreases overall adhesion forma-\ntion [2]. Likewise, Moazezi et al. [20] reported that a human amni-\non-derived jelly applied after laparotomy resulted in lower adhesion \nscores and histologically milder adhesions than those observed in \nuntreated rats. Our study extends these observations by demonstrat-\nTable 1. Pre- and post-intervention surgical characteristics of lesions in two groups\nGroup  HAG Control Mann–Whitney p-value\nHoffmann adhesion score 3 (0 to 5) 6 (4 to 10) < 0.001\nLauder adhesion score 1 (0 to 2) 2 (1 to 5) < 0.001\nSize difference (mm\n2\n) 81 (28 to 158) 8 (–36 to 52) < 0.001\nSize: Pre-intervention (mm\n2\n)\na)\n85 (36 to 162) 85 (36 to 162) 0.777\nSize: Post-intervention (mm\n2\n)\na)\n4 (4 to 27) 75 (33 to 132) < 0.001\nWithin-group changes p-value < 0.001 0.083\nValues are presented as median (range). Within-group changes were assessed using the Wilcoxon signed-rank test.\nHAG, human amniotic membrane gel.\na)\nWithin-group changes were assessed using the Wilcoxon signed-rank test.\nTable 2. Serum cytokine levels pre- and post-intervention in two study groups\nCytokine Time point HAG Control Mann–Whitney p-value p-value for trend: comparison between groups\na)\nIL-6 Baseline 1.57 ± 0.51 1.63 ± 0.56 0.705\nBefore intervention 1.77 ± 0.16 1.85 ± 0.32 0.168 < 0.001\nPost-intervention 1.24 ± 0.12 2.65 ± 0.072 < 0.001\nIL-1β Baseline 1.93 ± 0.33 1.92 ± 0.32 0.858\nBefore intervention 2.01 ± 0.29 2.05 ± 0.22 0.373 < 0.001\nPost-intervention 1.38 ± 0.01 3.28 ± 0.11 < 0.001\nValues are presented as mean±standard deviation.\nHAG, human amniotic membrane gel; IL, interleukin.\na)\nTrend p-values reflect within-subject linear change from baseline to post-intervention (repeated-measures analysis of variance).\nwww.eCERM.org 5\nR Derakhshan et al. HAG and endometriosis adhesions\n\ning concurrent modulation of inflammatory pathways in an endo-\nmetriosis-specific context.\nA plausible explanation for the benefits of HAG is its combination \nof barrier function and intrinsic anti-inflammatory properties. By \nphysically separating injured peritoneal surfaces, the gel likely \nhelped prevent the initial fibrin bridging that leads to adhesions, \nsimilar to the action of other anti-adhesion barriers [21]. Beyond this \nmechanical role, the amniotic membrane is known to release bioac-\ntive factors that suppress inflammation and fibrosis. In a mouse sur-\ngery model, fragmented amniotic membrane significantly reduced \npostoperative adhesion incidence and fibrosis [22]. Consistent with \nthese observations, HAG-treated rats in our study showed a lower in-\ncidence of fibrotic tissue and abscess formation on histological ex-\namination, suggesting that HAG attenuated the intense inflammato-\nry response that can drive scar formation. Instead, we observed an \nadipose tissue-rich reaction at lesion sites in the HAG group, which \nmay indicate a more regenerative and less fibrotic healing process. \nThis interpretation is consistent with reports that the amniotic mem-\nbrane can promote constructive healing while limiting excessive \nscarring [22]. Furthermore, the amniotic membrane contains anti-\nproteases and antimicrobial peptides [23,24], which may explain the \nabsence of abscesses in HAG-treated animals.\nThe most striking immunological finding was the divergent cyto-\nkine trajectory between groups: HAG-treated rats showed declining \nserum IL-6 and IL-1β levels over time, whereas control rats exhibited \nincreasing levels of both cytokines. This interaction suggests that \nHAG actively blunted the postoperative inflammatory surge. The sig-\nnificance of this cytokine modulation is substantial, as IL-6 and IL-1β \nare key drivers of both adhesion formation and the chronic inflam-\nmatory state associated with endometriosis. IL-6, in particular, has \nbeen identified as an upstream mediator of peritoneal fibrosis: surgi-\ncal injury triggers IL-6 release, which in turn induces profibrotic me-\ndiators such as TGF-β1, thereby promoting collagen deposition and \ndense adhesions [24]. Consistent with this mechanism, neutraliza-\ntion of IL-6 signaling has been shown to protect against adhesions. \nUyama et al. [15] demonstrated that an anti-IL-6 receptor antibody \nmarkedly reduced neutrophil influx and adhesion scores in a mouse \nadhesion model. In the context of endometriosis, elevated IL-6 in le-\nsion microenvironments is thought to promote the survival and in-\nvasiveness of ectopic endometrial cells, thereby contributing to dis-\nease persistence [25]. Notably, El-Zayadi et al. [ 26] reported that \nblocking IL-6 receptors in an experimental endometriosis model sig-\nnificantly reduced lesion size relative to untreated controls. Our find-\ning that HAG treatment led to a decrease in IL-6 over time, whereas \nIL-6 increased in untreated rats, mirrors the effects of those IL-6-tar-\ngeted interventions and underscores the cytokine-modulating ca-\npacity of HAG.\nIL-1β is another proinflammatory cytokine that was reduced by \nHAG, with important implications for adhesion pathology. IL-1β has \nFigure 3. Cytokine changes during the study period between study groups. (A) Serum interleukin 6 (IL-6) levels over time in the human \namniotic membrane gel (HAG) and control groups. (B) Serum IL-1β levels over time in the HAG and control groups. Data are presented as \nmean±standard deviation (SD).\nTable 3. Histopathologic assessment 3 weeks post-intervention in \ntwo study groups\nHistopathological features HAG Control p-value\nPresence of endometriosis 8 (36.4) 21 (95.5) < 0.001\nOther pathological findings\n Fibrosis 4 (18.2) 7 (31.8) < 0.001\n Abscess 3 (13.6) 13 (59.1)\n Adipose tissue reaction 15 (68.2) 2 (9.1)\nValues are presented as number (%).\nHAG, human amniotic membrane gel.\nIL-6 levels over time IL-1β  levels over time\nHAG\nControl\nHAG\nControl\nMean±SD\nBaseline BaselineBefore intervention  \ntime point\nBefore intervention  \ntime point\nPost-intervention Post-intervention\n2.75\n2.50\n2.25\n2.00\n1.75\n1.50\n1.25\n1.00\n3.25\n3.00\n2.75\n2.50\n2.25\n2.00\n1.75\n1.50\nMean±SD\nhttps://doi.org/10.5653/cerm.2025.091106\nClin Exp Reprod Med [Epub ahead of print]\nAA BB\n\nlong been implicated in postoperative adhesion formation because \nit stimulates mesothelial and inflammatory cells to release plasmino-\ngen activator inhibitors, thereby suppressing fibrin clearance and fa-\ncilitating the formation of permanent fibrous bands [13]. Neutraliza-\ntion of IL-1 in animal models results in significantly fewer adhesions, \nhighlighting its role as a pro-adhesive mediator [13]. In endometrio-\nsis, IL-1β is abundantly expressed and appears to exacerbate the \nchronic inflammatory milieu. For example, Peng et al. [27] found that \nIL-1β directly upregulated nerve growth factor in endometriotic tis-\nsue, thereby driving local neurogenesis and pain, including deep \ndyspareunia, in affected women. By reducing IL-1β levels, HAG may \ntherefore interrupt a vicious cycle of inflammation, nerve sensitiza-\ntion, and fibrosis that underlies endometriosis progression.\nNumerous studies have demonstrated that human amniotic \nmembrane (HAM) can reduce tissue injury, inhibit fibrosis, and pre-\nvent postoperative adhesions through multiple molecular pathways. \nIn particular, HAM may inhibit fibroblast proliferation and collagen \naccumulation through the TGF-β/Smad pathway, which plays a cen-\ntral role in fibrogenesis [28]. In addition, modulation of the nuclear \nfactor-κB and signal transducer and activator of transcription 3 path-\nways has been associated with reduced levels of proinflammatory \ncytokines such as IL-6 and IL-1β, thereby promoting tissue repair [29]. \nMoreover, regulation of matrix metalloproteinase 2 (MMP-2) and \nMMP-9 activity may help control extracellular matrix degradation in \na manner that prevents fibrotic adhesion formation [4]. HAM may \nalso inhibit abnormal neoangiogenesis through effects on vascular \nendothelial growth factor, which could help reduce the persistence \nof endometriotic lesions [30]. These molecular and cellular character-\nistics suggest that amniotic products such as amniotic membrane \ngel may have not only a preventive role but also a therapeutic effect \non chronic lesions such as endometriosis.\nFrom a translational perspective, these findings suggest that HAG \nmay serve as a valuable adjunct in endometriosis surgery by target-\ning both the anatomical and inflammatory contributors to postoper-\native complications. Given its favorable safety profile in other clinical \nsettings, HAG’s dual role as a physical barrier and anti-inflammatory \nagent may help reduce lesion recurrence and adhesion-related out-\ncomes such as chronic pelvic pain and infertility.\nThis study has several limitations. First, although the animal model \nis widely used, it does not fully replicate the cyclical hormonal envi-\nronment or the complex immunological features of human endome-\ntriosis. Second, although adhesion scores and cytokine levels were \nassessed, functional outcomes such as pain behavior and fertility \nwere not evaluated. Third, additional cytokines beyond IL-6 and IL-1β \ncould not be measured because of limited financial resources, which \nmay have restricted a more comprehensive characterization of the \ninflammatory response. Although inclusion of a comparator anti-ad-\nhesion agent would have strengthened the analysis, no gold-stan-\ndard product currently exists for this specific application. Therefore, \nthe study was designed to assess the efficacy of HAG as a standalone \nintervention. Finally, practical concerns such as product consistency \nand cost require further investigation.\nIn conclusion, HAG reduced lesion size, adhesions, and proinflam-\nmatory cytokine levels (IL-6 and IL-1β) in this experimental model. \nThese findings support its potential to modulate both inflammatory \nand fibrotic pathways in endometriosis. Further long-term and clini-\ncal studies are warranted to confirm these benefits and to explore \nthe integration of HAG into standard surgical care.\nAdditional information\n1\n Endometriosis Research Center, Iran University of Medical Sciences, \nTehran, Iran\n2\nIranian Scientific Society of Minimally Invasive Gynecology, Tehran, Iran\n3\n Department of Surgery, Faculty of Medicine, Shahid Beheshti University \nof Medical Sciences, Tehran, Iran\n4\nSmart University of Medical Sciences, Tehran, Iran\n5\n Department of Epidemiology, School of Public Health, Iran University of \nMedical Sciences, Tehran, Iran\n6\n Department of Pathology, Clinical Research Development Center, \nShahid Modarres Educational Hospital, Shahid Beheshti University of \nMedical Sciences, Tehran, Iran\n7\n University of British Columbia, Vancouver General Hospital, and BC \nCancer. British Columbia’s Gynecological Cancer Research Team \n(OVCARE), Vancouver, BC, Canada\n8\n Department of Pharmacology, Faculty of Medicine, Shahid Beheshti \nUniversity of Medical Sciences, Tehran, Iran\n9\n Pars Advanced and Minimally Invasive Medical Manner Research Center, \nPars Hospital, Iran University of Medical Sciences, Tehran, Iran\n10\n Department of Biology and Anatomical Sciences, School of Medicine, \nShahid Beheshti University of Medical Sciences, Tehran, Iran\nConflict of interest\nNo potential conflict of interest relevant to this article was reported.\nAcknowledgments\nThe authors thank the Endometriosis Research Center, Iran Univer-\nsity of Medical Sciences.\nORCID\nRoya Derakhshan https://orcid.org/0000-0001-7425-3412\nShahla Chaichian https://orcid.org/0000-0001-5772-8711\nAuthor contributions\nConceptualization: RD, SC. Methodology: RD, AM (Abolfazl Meh-\nwww.eCERM.org 7\nR Derakhshan et al. HAG and endometriosis adhesions\n\ndizadehkashi), SC, AM (Arash Mohazzab). Formal analysis: AM (Arash \nMohazzab). Data curation: RD, BS, MGN. Funding acquisition: AM \n(Abolfazl Mehdizadehkashi). Project administration: AM (Abolfazl \nMehdizadehkashi), SC. Investigation: RD, BS, MGN. Validation: AM \n(Arash Mohazzab), SR. Writing–original draft: RD, SC, AM (Arash Mo-\nhazzab). Writing–review and editing: RD, AM (Abolfazl Mehdizadeh-\nkashi), BS, SC, AM (Arash Mohazzab), BK, TN, MAS, SAZ, SR, BN, MGN.\nApproval of final manuscript: RD, AM (Abolfazl Mehdizadehkashi), \nBS, SC, AM (Arash Mohazzab), BK, TN, MAS, SAZ, SR, BN, MGN.\nReferences\n1. Ghobrial S, Ott J, Parry JP . An overview of postoperative intraab-\ndominal adhesions and their role on female infertility: a narrative \nreview. J Clin Med 2023;12:2263.\n2. Mazloomi M, Mohazzab A, Tahermanesh K, Fakehi M, Saeedzaran-\ndi M, Kooshari Z, et al. 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