Introduction
Postoperative pelvic adhesions are a frequent complication of endometriosis surgery and contribute to chronic pain, infertility, and bowel obstruction. Surgical trauma initiates a wound-healing cascade that can result in the formation of fibrous bands between peritoneal surfaces [1]. The use of biochemical barriers such as hyaluronic acid (HA) has previously been proposed as an effective strategy for reducing postoperative adhesions. HA forms a temporary physical layer between healing peritoneal surfaces, thereby preventing direct tissue apposition and adhesion formation. HA gel has been shown to significantly decrease the incidence and severity of pelvic adhesions following gynecologic surgery, particularly surgery for endometriosis [2,3]. More recently, natural bioactive gels such as human amniotic membrane gel (HAG) have attracted attention because of their dual function: they not only serve as physical barriers but also deliver anti-inflammatory and regenerative signals to the surgical site. These amniotic-derived products are rich in growth factors, extracellular matrix proteins, HA, and immunomodulatory cytokines that support tissue repair and modulate inflammation [4,5]. In preclinical studies, they have been shown to reduce fibrosis, downregulate inflammatory pathways such as transforming growth factor-β (TGF-β), and suppress proinflammatory cytokines including interleukin 6 (IL-6) and IL-1β [6-8].
Endometriosis is characterized by a proinflammatory peritoneal environment, with IL-6 and IL-1β identified as key mediators of disease progression and adhesion formation. Women with endometriosis have significantly elevated levels of IL-6 and IL-1β in serum and peritoneal fluid compared with disease-free controls [9]. IL-6, in particular, correlates with endometriosis severity and has been implicated in lesion proliferation and survival, especially through its soluble receptor, which enhances IL-6 signaling [10]. Surgical injury further amplifies these cytokines. Experimental models have shown that early postoperative surges in IL-6 and IL-1β are strongly associated with the extent of subsequent adhesions [11,12]. IL-1β promotes adhesion formation by reducing fibrinolysis; it induces plasminogen activator inhibitor-1, leading to persistence of fibrin scaffolds, and it stimulates mesothelial cells to undergo profibrotic changes [13]. IL-6, often induced downstream of IL-1β and tumor necrosis factor-α, further drives fibrogenesis by triggering mesothelial-to-myofibroblast transition and altering the expression of matrix-remodeling enzymes, thereby reinforcing adhesion development [13]. Given the central role of IL-6 and IL-1β, anti-inflammatory interventions targeting these cytokines may markedly influence postoperative outcomes. Indeed, blocking IL-1β activity in animal models reduces peritoneal adhesion formation [14]. Likewise, neutralization of IL-6 signaling with an anti-IL-6 receptor antibody has been shown to abrogate neutrophil recruitment and significantly diminish adhesion bands in a murine surgery model [15]. These findings underscore that dampening the IL-6/IL-1β-driven inflammatory cascade after surgery may reduce adhesion formation and fibrosis and potentially lower endometriosis recurrence. Collectively, these insights provide a strong rationale for therapies that combine physical anti-adhesion barriers such as HA gel with strategies that modulate IL-6 and IL-1β, with the aim of improving healing and long-term outcomes in patients with endometriosis.
Methods
1. Materials and animals
This experimental study was conducted in adult, nonpregnant female Wistar albino rats weighing 220 to 250 g. The animals were housed in standard cages at the Center for Experimental Studies, Shahid Beheshti University of Medical Sciences, under controlled environmental conditions (temperature, 22±2 °C; 12-hour light/12-hour dark cycle) with free access to food and water. Amniotic membrane allograft gel was obtained from Lifescell (International Bioimplant of Borna Co.). Commercial enzyme-linked immunosorbent assay (ELISA) kits were obtained from Karmania Pars Gene Company.
All procedures were approved by the Research and Ethics Committee of Iran University of Medical Sciences, Tehran, Iran (IR.IUMS.REC.1399.726), and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 80-23, revised 1996). After an adaptation period and before the first laparotomy, each rat received a subcutaneous injection of 2.0 mg/kg estradiol to support the establishment of endometriotic implants.
2. Induction of endometriosis
At the time of the first laparotomy, the rats were in the estrus phase, during which estrogen levels are naturally elevated and facilitate implantation and survival of endometrial fragments. This timing is consistent with established protocols for endometriosis induction in rodent models. In line with previous studies demonstrating that exogenous estradiol enhances endometriosis development [16], intramuscular estradiol was also administered to further improve lesion establishment. Importantly, the ovaries were preserved to maintain endogenous hormonal cycles, thereby better simulating the hormonal milieu of endometriosis. Thus, the combination of natural estrus, exogenous estradiol, and ovarian preservation optimized the conditions for reliable induction of peritoneal endometriosis [16,17]. Anesthesia was induced with intramuscular ketamine hydrochloride (60 mg/kg) and xylazine hydrochloride (10 mg/kg) administered in the same syringe. Before surgery, tail vein blood samples were collected to measure baseline serum IL-6 and IL-1β levels.
A vertical midline abdominal incision of approximately 3 cm was made, and the two uterine horns were exposed. The right uterine horn was ligated with a 4-0 silk suture, and a 1-cm segment was excised and opened longitudinally. Sterile phosphate-buffered saline was used to keep the tissue moist.
The uterine segment was then divided into three equal parts. All three fragments were transplanted onto the inner surface of the right abdominal wall, with the endometrial surface facing the peritoneum. The serosal surface was positioned against the peritoneal surface and fixed with 4–0 nonabsorbable silk sutures (Figure 1). During the procedure, the surgical field was frequently moistened with normal saline to prevent tissue drying. Before closure of the abdominal wall, 2 mL of saline was instilled into the peritoneal cavity.
The abdominal wall was closed in layers using simple interrupted 2–0 polyglactin 910 sutures. After surgery, the animals were housed individually and monitored for 3 weeks. They received 50 µg/kg intramuscular depot estradiol twice weekly during this period. Body weight and general health were monitored regularly, and no mortality occurred during the study.
3. HAG application
At the end of the third week, all animals underwent a second laparotomy to verify the viability of the endometriotic implants by histopathological assessment (Figure 2). In all animals, the implants were measured in two dimensions (length and width, in mm) using a caliper, photographed with a digital camera, and documented in the study records [18]. Immediately thereafter, 44 rats with established endometriotic implants were randomly assigned to two groups: group A (HAG group, n=22) and group B (normal saline group, n=22). Randomization was performed immediately before the intervention, ensuring that only animals with successful model induction were included.
In group A, sterile HAG (International Bioimplant of Borna Co., Tehran, Iran) was instilled into the abdominal cavity during laparotomy, and the abdominal wall was closed with simple 2–0 polyglactin 910 sutures. In group B, normal saline was instilled into the abdominal cavity during laparotomy according to protocols similar to those used in previous experimental studies [17,19].
4. Implant measurements and histopathology
Two weeks after HAG or saline application (i.e., 5 weeks after induction of endometriosis), blood samples were again obtained from the tail vein under light sedation to assess serum interleukin levels. The rats were then humanely euthanized using nitrous oxide. During the final laparotomy, adhesions were evaluated in each group using Hoffmann’s quantitative score and Lauder’s qualitative grading system. A researcher blinded to group allocation measured the width and length of each implant with a caliper. Endometrial implants were excised and fixed in formaldehyde for routine histopathological examination.
Both the biochemist and the histopathologist were blinded to treatment allocation. Data on lesion size, adhesion severity, and gross appearance were recorded using a structured checklist, and standardized photographs were taken at each laparotomy to document adhesion formation and implant morphology. The histological diagnosis of endometriosis was based on the presence of endometrial glands and stroma, with epithelial lining and luminal formation within the ectopic implants [17]. In addition, the presence of fibrosis, abscess formation, and adipose tissue reaction was recorded as part of the pathological evaluation.
5. Cytokine assays
Serum levels of IL-6 and IL-1β were measured using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturers’ instructions. IL-6 and IL-1β concentrations were determined using commercially available ELISA kits.
6. Statistical analysis
All analyses were performed using SPSS ver. 24 (IBM Co.). Continuous variables were assessed for normality using the Kolmogorov–Smirnov test. Repeated-measures analysis of variance was used to evaluate temporal trends across the three measurement points and their interaction with treatment group. Greenhouse–Geisser corrections were applied when the assumption of sphericity was violated. For comparisons between the two groups at individual time points (baseline, week 3, and post-treatment), the Mann–Whitney U test was used because cytokine levels and lesion measurements did not fully meet parametric assumptions. The Wilcoxon signed-rank test was used to assess within-group changes over time (pre- vs. post-treatment). Adhesion severity scores (Hoffmann and Lauder) were also compared between groups using the Mann–Whitney U test. All categorical histopathological findings were analyzed using the chi-square test or Fisher’s exact test, as appropriate. Results were reported as mean±standard deviation for continuous variables and frequency (percentage) for categorical variables. A two-sided p<0.05 was considered statistically significant.
Results
1. Surgical outcomes and adhesion severity
All 44 animals completed the study and were included in the analysis. At baseline, the two groups were comparable in lesion size and cytokine profiles, confirming that randomization produced well-balanced groups. Endometriotic implants initially appeared as typical vascularized or cystic nodules surrounded by filmy or inflammatory adhesions.
By the end of the experiment, the difference between groups had become both visually and statistically evident. Lesions in the HAG-treated animals had shrunk dramatically, with post-intervention lesion size decreasing to approximately 7 mm², in contrast to the control group, in which lesions remained large, averaging approximately 81 mm² (p<0.001). The overall reduction in lesion size from baseline was also markedly greater in the HAG group (approximately 80 mm²) than in the control group (approximately 9 mm²). As shown in Table 1, this improvement remained significant within animals over time, whereas the control group showed no meaningful regression.
Adhesion scores showed a similar pattern. Both Hoffmann and Lauder scores were substantially lower in rats receiving HAG, with mean Hoffmann scores of approximately 2.5 versus 6.4 in the control group and mean Lauder scores of 1.0 versus 2.9, respectively (both p<0.001). Figure 1 illustrates these macroscopic differences. Overall, the surgical findings indicate a consistent and robust therapeutic effect of HAG on both lesion size and adhesion severity.
2. Histopathological findings
Histological evaluation reinforced the macroscopic findings. Only a minority of HAG-treated rats showed evidence of persistent endometriosis (36%), whereas nearly all animals in the control group remained positive for active lesions (95.5%; p<0.001). The pattern of tissue changes also differed meaningfully between groups. Fibrosis and abscess formation—markers of chronic or ongoing inflammatory activity—were frequent in control animals, whereas these features were much less common in animals exposed to HAG. In contrast, adipose tissue reaction, which is more consistent with tissue repair and remodeling, predominated in the HAG group. These distributions are summarized in Table 2.
Figure 2A shows the histological appearance of endometriotic implants immediately after confirmation of endometriosis during the second laparotomy. Figure 2B, 2C show histological sections of implants 2 weeks after HAG treatment (Figure 2B) and normal saline treatment in the control group (Figure 2C), highlighting the differences in tissue response between treated and untreated animals. In Figure 2B, following HAG treatment, the endometriotic epithelium is absent, and only residual fibrotic tissue remains.
3. Serum cytokine levels
Changes in systemic cytokine levels closely mirrored the surgical and histological outcomes. At baseline, IL-6 and IL-1β levels were similar between groups, indicating no pre-existing differences. Following treatment, the trajectories diverged sharply. IL-6 levels decreased steadily in HAG-treated animals, falling to approximately 1.24 pg/mL, whereas levels increased in the control group, reaching approximately 2.65 pg/mL at the same time point (p<0.001). A highly significant time-by-group interaction confirmed that the pattern of IL-6 change differed fundamentally between the two groups.
IL-1β showed a similar pattern. In the HAG group, IL-1β decreased to approximately 1.38 pg/mL, whereas in control animals it increased sharply to more than 3.2 pg/mL (p<0.001). The linear trend over time was strong and highly significant for both cytokines, reflecting a sustained anti-inflammatory response to HAG. These patterns correspond closely to the macroscopic regression of lesions and the histological shift toward healing. The cytokine trajectories are illustrated in Figure 3, and the full numerical data are summarized in Table 3.
Discussion
This study demonstrates that intraperitoneal HAG significantly attenuated endometriosis progression and postoperative adhesions in a rat model. Animals treated with HAG had markedly smaller endometriotic lesions and substantially lower adhesion severity, based on Hoffmann and Lauder scores, than saline-treated controls. These findings are consistent with prior evidence that barrier gels can reduce pelvic adhesions. A recent meta-analysis of gynecologic surgery showed that HA gel significantly decreases overall adhesion formation [2]. Likewise, Moazezi et al. [20] reported that a human amnion-derived jelly applied after laparotomy resulted in lower adhesion scores and histologically milder adhesions than those observed in untreated rats. Our study extends these observations by demonstrating concurrent modulation of inflammatory pathways in an endometriosis-specific context.
A plausible explanation for the benefits of HAG is its combination of barrier function and intrinsic anti-inflammatory properties. By physically separating injured peritoneal surfaces, the gel likely helped prevent the initial fibrin bridging that leads to adhesions, similar to the action of other anti-adhesion barriers [21]. Beyond this mechanical role, the amniotic membrane is known to release bioactive factors that suppress inflammation and fibrosis. In a mouse surgery model, fragmented amniotic membrane significantly reduced postoperative adhesion incidence and fibrosis [22]. Consistent with these observations, HAG-treated rats in our study showed a lower incidence of fibrotic tissue and abscess formation on histological examination, suggesting that HAG attenuated the intense inflammatory response that can drive scar formation. Instead, we observed an adipose tissue-rich reaction at lesion sites in the HAG group, which may indicate a more regenerative and less fibrotic healing process. This interpretation is consistent with reports that the amniotic membrane can promote constructive healing while limiting excessive scarring [22]. Furthermore, the amniotic membrane contains antiproteases and antimicrobial peptides [23,24], which may explain the absence of abscesses in HAG-treated animals.
The most striking immunological finding was the divergent cytokine trajectory between groups: HAG-treated rats showed declining serum IL-6 and IL-1β levels over time, whereas control rats exhibited increasing levels of both cytokines. This interaction suggests that HAG actively blunted the postoperative inflammatory surge. The significance of this cytokine modulation is substantial, as IL-6 and IL-1β are key drivers of both adhesion formation and the chronic inflammatory state associated with endometriosis. IL-6, in particular, has been identified as an upstream mediator of peritoneal fibrosis: surgical injury triggers IL-6 release, which in turn induces profibrotic mediators such as TGF-β1, thereby promoting collagen deposition and dense adhesions [24]. Consistent with this mechanism, neutralization of IL-6 signaling has been shown to protect against adhesions. Uyama et al. [15] demonstrated that an anti-IL-6 receptor antibody markedly reduced neutrophil influx and adhesion scores in a mouse adhesion model. In the context of endometriosis, elevated IL-6 in lesion microenvironments is thought to promote the survival and invasiveness of ectopic endometrial cells, thereby contributing to disease persistence [25]. Notably, El-Zayadi et al. [26] reported that blocking IL-6 receptors in an experimental endometriosis model significantly reduced lesion size relative to untreated controls. Our finding that HAG treatment led to a decrease in IL-6 over time, whereas IL-6 increased in untreated rats, mirrors the effects of those IL-6-targeted interventions and underscores the cytokine-modulating capacity of HAG.
IL-1β is another proinflammatory cytokine that was reduced by HAG, with important implications for adhesion pathology. IL-1β has long been implicated in postoperative adhesion formation because it stimulates mesothelial and inflammatory cells to release plasminogen activator inhibitors, thereby suppressing fibrin clearance and facilitating the formation of permanent fibrous bands [13]. Neutralization of IL-1 in animal models results in significantly fewer adhesions, highlighting its role as a pro-adhesive mediator [13]. In endometriosis, IL-1β is abundantly expressed and appears to exacerbate the chronic inflammatory milieu. For example, Peng et al. [27] found that IL-1β directly upregulated nerve growth factor in endometriotic tissue, thereby driving local neurogenesis and pain, including deep dyspareunia, in affected women. By reducing IL-1β levels, HAG may therefore interrupt a vicious cycle of inflammation, nerve sensitization, and fibrosis that underlies endometriosis progression.
Numerous studies have demonstrated that human amniotic membrane (HAM) can reduce tissue injury, inhibit fibrosis, and prevent postoperative adhesions through multiple molecular pathways. In particular, HAM may inhibit fibroblast proliferation and collagen accumulation through the TGF-β/Smad pathway, which plays a central role in fibrogenesis [28]. In addition, modulation of the nuclear factor-κB and signal transducer and activator of transcription 3 pathways has been associated with reduced levels of proinflammatory cytokines such as IL-6 and IL-1β, thereby promoting tissue repair [29]. Moreover, regulation of matrix metalloproteinase 2 (MMP-2) and MMP-9 activity may help control extracellular matrix degradation in a manner that prevents fibrotic adhesion formation [4]. HAM may also inhibit abnormal neoangiogenesis through effects on vascular endothelial growth factor, which could help reduce the persistence of endometriotic lesions [30]. These molecular and cellular characteristics suggest that amniotic products such as amniotic membrane gel may have not only a preventive role but also a therapeutic effect on chronic lesions such as endometriosis.
From a translational perspective, these findings suggest that HAG may serve as a valuable adjunct in endometriosis surgery by targeting both the anatomical and inflammatory contributors to postoperative complications. Given its favorable safety profile in other clinical settings, HAG’s dual role as a physical barrier and anti-inflammatory agent may help reduce lesion recurrence and adhesion-related outcomes such as chronic pelvic pain and infertility.
This study has several limitations. First, although the animal model is widely used, it does not fully replicate the cyclical hormonal environment or the complex immunological features of human endometriosis. Second, although adhesion scores and cytokine levels were assessed, functional outcomes such as pain behavior and fertility were not evaluated. Third, additional cytokines beyond IL-6 and IL-1β could not be measured because of limited financial resources, which may have restricted a more comprehensive characterization of the inflammatory response. Although inclusion of a comparator anti-adhesion agent would have strengthened the analysis, no gold-standard product currently exists for this specific application. Therefore, the study was designed to assess the efficacy of HAG as a standalone intervention. Finally, practical concerns such as product consistency and cost require further investigation.
In conclusion, HAG reduced lesion size, adhesions, and proinflammatory cytokine levels (IL-6 and IL-1β) in this experimental model. These findings support its potential to modulate both inflammatory and fibrotic pathways in endometriosis. Further long-term and clinical studies are warranted to confirm these benefits and to explore the integration of HAG into standard surgical care.
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