hysteroscopic adhesiolysis is a type of plastic surgery performed on the uterine cavity under direct hysteroscopic visualization to resect scar tissue and restore anatomical morphology. Notably, endometrial damage is extensive in severe cases of IUA, therefore, surgery emphasizes effective protection of the residual endometrium. However, the residual endometrium area is less than 1/3 the area of the uterine cavity, causing a large wound after surgical resection[1]. Besides, due to electrical intervention during the operation, the high-frequency electric action of the electrode inevitably produces a particular electrical heating effect on healthy endometrial tissue and uterine muscle wall tissue, simultaneously separating and removing scar tissue in the uterine cavity[4]. Nevertheless, the role of high-frequency electric hysteroscopy in separation and correction surgery for severe IUA is still significantly important. Moreover, the unique anatomical morphology of the uterine cavity is a factor in wound exudation after hysteroscopic adhesiolysis surgery and in the action of cytokines on surgical wounds. It is currently agreed that wound exudates contain many repair and adhesion-related factors. Partial cytokines might activate the proliferation of mesenchymal cells (mainly fibroblasts), thereby causing large amounts of fibrinogen, fibronectin, and other substances deposited to form fibrin scaffold-covered wounds resulting in the accumulation of excess ECM. Inflammatory granulation tissue and fibrous scars would be formed, which cause re-adhesion. This is in agreement with previous clinical results, which showed a re-adhesion rate of 62.5% after the implementation of hysteroscopic adhesiolysis for severe IUA[2]. Meanwhile, some other factors play a vital role in the repair of the endometrium and incision thereby restoring the normal function and receptivity of the endometrium.
Like other surgical wounds, uterine wounds after hysteroscopic adhesiolysis exude high levels of cytokines due to electrothermal effects. Among these cytokines, IL-1β, TNF-α, and VEGF regulate tissue damage and repair[10]. Notably, TNF-α and VEGF are the central cytokines secreted in the early stages of inflammation. Their expression is closely related to the formation of new capillaries and the prevention of wound infections. In contrast, excessive cytokine exudation might induce excessive accumulation of ECM and promote granulation tissue and fibrous scar formation. The high expression of TNF-α in wound exudates release high levels of VEGF, thereby triggering fibroblast aggregation and an increase in ECM synthesis[11]. Moreover, TNF-α causes unbalanced ECM synthesis and degradation by inhibiting the secretion of matrix metalloproteinase-9 (MMP-9) resulting in the accumulation of ECM and promotion of fibrous tissue hyperplasia as well as scar formation[12]. In this study, the two aforementioned cytokines reached their peaks on the second day and decreased significantly on the seventh day after surgery. However, compared to the control group, the concentration of postoperative cytokines in the study group was significantly lower.
Macrophages secrete IL-1β facilitated by TNF-α or itself activating TNFR and IL-1R1, respectively. Here, the duration taken by IL-1β concentration to reach its peak was longer than the above two cytokines[13]. Besides promoting repair of endometrial tissue and maintaining a balance of immune activity in the uterine cavity, it was found that IL-1β promotes a healthy intrauterine environment and normal receptivity of the endometrium[14]. Although its concentration in the study group was lower than the control group due to the inhibition of TNF-α and VEGF in the early stage, IL-1β promotes self-secretion through the IL-1β/IL-1R1 pathway. Moreover, its level in the study group was similar to that of the control group during the endometrial repair period 7 days after operation. According to statistical trends, IL-1β in the study group can be maintained for a prolonged duration to promote the endometrial repair process. Therefore, effective postoperative inhibition of adhesion-related factor exudation after hysteroscopic adhesiolysis is essential for reducing the re-adhesion rate and improving surgical outcomes.
Being widely used in the medical fields, the freeze-dried amnion contains a high level of cytokines and cytokine receptors, which promote the repair of damaged tissues. In animal studies, freeze-dried amnion graft repaired eyelid corneal injury in rabbits by inhibiting the expression of TNF-ɑ and VEGF in the cornea. Further studies found that the freeze-dried amnion inhibits the expression of TNF and VEGF in injured tissues, accelerates apoptosis of inflammatory cells, and reduces the inflammatory response. At the same time, freeze-dried amnion graft can promote the migration and adhesion of corneal epithelial cells and prevent their apoptosis. Also, it has been used in the treatment of IUA due to its unique features including its ability to reduce the release of adhesion factors and promote damage repair. The advantage of freeze-dried amnion graft on intimalization after hysteroscopic adhesiolysis might be the reason it improves the reproductive prognosis. Our clinical study, which involved postoperative hysteroscopic re-examination, improvements in menstrual flow and postoperative follow-up for an average of 14.6 months during pregnancy, found that the increase in the postoperative re-adhesion score and menstrual flow was significantly different than the score and menstrual flow before treatment, and the pregnancy rate also increased[15]. This indicates that freeze-dried amnion graft can effectively improve the endometrial status by preventing the occurrence of re-adhesions[16].
Therefore, freeze-dried amnion graft can be better attached to wounds in the uterine cavity to exert their functions. While the balloon supports the anterior and posterior walls of the uterus in the uterine cavity, being a foreign body in the wound, it also stimulates the production of inflammatory cytokines. The freeze-dried amnion blocks the direct contact between the rubber balloon and the wound, thereby exerting their functions, which include promoting anti-inflammatory and anti-fibrotic responses as well as scar suppression. A study by Orhue et al. [17] argued that after hysteroscopic adhesiolysis, the anterior and posterior walls of the uterus separate for at least 7 days before the wound can heal, whereas the function of biological barrier in the freeze-dried amnion persists for at least 21 days to maintain a continuous effect in the uterine cavity. Furthermore, Amer et al.[18] used a balloon combined with freeze-dried amnion graft to treat severe IUA. Then, hysteroscopic exploration was performed 2 months after the surgery and the original scar tissue was obtained for examination. The basal cells of the endometrium were observed under an electron microscope and were also found on the degenerated freeze-dried amnion graft, implying that the freeze-dried amnion graft continue to function even with the removal of the balloon 7 days after surgery. Moreover, epithelial and mesenchymal cells derived from freeze-dried amnion have been demonstrated to survive for several weeks, a period sufficient to prevent a new cycle of fibrosis and scar healing.
Whilst acknowledging the present corresponding clinical studies on the application of freeze-dried amnion after hysteroscopic adhesiolysis, there is no consistent theoretical foundation. In this study, we found that freeze-dried amnion graft can effectively reduce wound exudation at the uterine cavity, regulate the increase of acute inflammation-related factors (TNF-α and VEGF), and maintain a relative concentration of IL-1β in the endometrial repair phase. Also, according to our follow-up results, we found a significant decrease in postoperative AFS and postoperative adhesion reformation rate in patients using freeze-dried amnion graft compared to the control group. Thus, we believe that the freeze-dried amnion graft may improve patient outcomes by altering intrauterine cytokine concentrations.
However, it is worth mentioning that the freeze-dried amnion currently used clinically has limitations including, their morphological dimensions which do not adapt to the morphology of the uterine cavity thus limit their clinical application. Nevertheless, this study lays a foundation for the use of freeze-dried amnion graft in the prevention and intervention of re-adhesion in severe cases of IUA. As a consequence, this provides novel strategies for the prevention of postoperative uterine wound re-adhesion after hysteroscopic adhesiolysis surgery. However, the efficacy of freeze-dried amnion in surgeries for severe IUA merits further validation by prospective, multicentre, and large sample size studies.
According to our clinical outcome, freeze-dried amnion graft can effectively reduce the postoperative AFS and reduce the postoperative re-adhesion rate. The duration of Folly’s balloon placement is an important clinical consideration. Meanwhile, balloon placement for too long can lead to compression and necrosis of the endometrial tissues, while too short a period of time is not effective in preventing reattachment formation. And according to our study, removal of the balloon 7 days after surgery was found to be an optimal time point to effectively prevent postoperative adhesions. The time point for removal of the balloon after placement of the freeze-dried amnion graft could be considered to be appropriately advanced, thus reducing the necrosis of the surrounding normal endometrial tissue that may result from compression.
In conclusion, our findings show that freeze-dried amnion graft reduces the amount of uterine exudates after hysteroscopic adhesiolysis and shortens the duration of exudation. Furthermore, we discovered that the freeze-dried amnion regulates the concentrations of the adhesion-associated factors including TNF-α, VEGF, and repair associated factors (IL-1β) in uterine exudates thereby restoring relatively low levels of these factors within a limited time.